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Patent 2386199 Summary

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(12) Patent Application: (11) CA 2386199
(54) English Title: ENDOZEPINE-LIKE POLYPEPTIDES AND POLYNUCLEOTIDES ENCODING SAME
(54) French Title: POLYPEPTIDES DE TYPE ENDOZEPINE ET POLYNUCLEOTIDES CODANT CES DERNIERS
Status: Dead
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 15/12 (2006.01)
  • A61K 38/17 (2006.01)
  • C07K 14/47 (2006.01)
  • C12Q 1/68 (2006.01)
  • G01N 33/50 (2006.01)
  • G01N 33/53 (2006.01)
  • A61K 38/00 (2006.01)
(72) Inventors :
  • PRAYAGA, SUDHIRDAS K. (United States of America)
  • SHIMKETS, RICHARD A. (United States of America)
  • MAJUMDER, KUMUD (United States of America)
  • EISEN, ANDREW (United States of America)
  • VERNET, CORINE (United States of America)
  • SPADERNA, STEVEN K. (United States of America)
(73) Owners :
  • CURAGEN CORPORATION (United States of America)
(71) Applicants :
  • CURAGEN CORPORATION (United States of America)
(74) Agent: RIDOUT & MAYBEE LLP
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 2000-10-05
(87) Open to Public Inspection: 2001-04-12
Examination requested: 2005-08-24
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/US2000/041077
(87) International Publication Number: WO2001/025436
(85) National Entry: 2002-03-28

(30) Application Priority Data:
Application No. Country/Territory Date
60/157,786 United States of America 1999-10-05
60/227,072 United States of America 2000-08-22
09/679,460 United States of America 2000-10-04
60/164,164 United States of America 1999-11-09
60/174,505 United States of America 2000-01-04
60/183,859 United States of America 2000-02-22
60/190,740 United States of America 2000-03-20
60/191,133 United States of America 2000-03-22
60/206,006 United States of America 2000-05-19
60/215,684 United States of America 2000-06-30
60/219,490 United States of America 2000-07-20

Abstracts

English Abstract




Disclosed herein are human nucleic acid sequences which encode endozepine-like
polypeptides. Also disclosed are polypeptides encoded by these nucleic acid
sequences, and antibodies which immunospecifically bind to the polypeptide, as
well as derivatives, variants, mutants, or fragments of the aforementioned
polypeptide, polynucleotide, or antibody. The invention further discloses
therapeutic, diagnostic and research methods for diagnosis, treatment, and
prevention of disorders involving this novel human endozepine-like nucleic
acid and protein.


French Abstract

La présente invention concerne de nouvelles séquences d'acide nucléique d'origine humaine qui codent des polypeptides de type endozépine ; des polypeptides codés par ces séquences d'acide nucléique et des anticorps qui se lient de manière immunospécifique au polypeptide, ainsi que des dérivés, des variants, des mutants ou des fragments dudit polypeptide, dudit polynucléotide ou dudit anticorps. On décrit également des procédés de thérapie, de diagnostic et de recherche permettant de diagnostiquer, traiter et prévenir des maladies associées à ce nouvel acide nucléique de type endozépine et à la protéine d'origine humaine.

Claims

Note: Claims are shown in the official language in which they were submitted.





WHAT IS CLAIMED IS:
1. An isolated polypeptide comprising an amino acid sequence selected from the
group
consisting of:
(a) a mature form of an amino acid sequence selected from the group consisting
of SEQ
ID NO:2, 4, 6, 8, 10, 15, i 6, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32,
33, 35 to 45
inclusive, 47, and 49;
(b) a variant of a mature form of an amino acid sequence selected from the
group
consisting of SEQ ID NO:2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24,
26, 27, 29,
30, 32, 33, 35 to 45 inclusive, 47, and 49, wherein one or more amino acid
residues in
said variant differs from the amino acid sequence of said mature form,
provided that
said variant differs in no more than 15% of the amino acid residues from the
amino
acid sequence of said mature form;
(c) an amino acid sequence selected from the group consisting of SEQ ID NO:2,
4, 6, 8,
10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 45
inclusive, 47, and
49; and
(d) a variant of an amino acid sequence selected from the group consisting of
SEQ ID
NO:2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33,
35 to 45
inclusive, 47, and 49, wherein one or more amino acid residues in said variant
differs
from the amino acid sequence of said mature form, provided that said variant
differs in
no more than 15% of amino acid residues from said amino acid sequence.
2 The polypeptide of claim 1, wherein said polypeptide comprises the amino
acid
sequence of a naturally-occurring allelic variant of an amino acid sequence
selected from the
group consisting of SEQ ID NO:2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23,
24, 26, 27, 29, 30,
32, 33, 35 to 45 inclusive, 47, and 49.
3. The polypeptide of claim 2, wherein said allelic variant comprises an
amino acid
sequence that is the translation of a nucleic acid sequence differing by a
single nucleotide from
a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, 3,
5, 7, 9, 22, 25,
28, 31, 34, 46, and 48.
4. The polypeptide of claim 1, wherein the amino acid sequence of said variant
comprises
a conservative amino acid substitution.
125




5. An isolated nucleic acid molecule comprising a nucleic acid sequence
encoding a
polypeptide comprising an amino acid sequence selected from the group
consisting of:
(a) a mature form of an amino acid sequence selected from the group consisting
of SEQ
ID NO:2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32,
33, 35 to 45
inclusive, 47, and 49;
(b) a variant of a mature form of an amino acid sequence selected from the
group
consisting of SEQ ID NO:2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24,
26, 27, 29,
30, 32, 33, 35 to 45 inclusive, 47, and 49, wherein one or more amino acid
residues in
said variant differs from the amino acid sequence of said mature form,
provided that
said variant differs in no more than 15% of the amino acid residues from the
amino
acid sequence of said mature form;
(c) an amino acid sequence selected from the group consisting of SEQ ID NO:2,
4, 6, 8,
10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 45
inclusive, 47, and
49;
(d) a variant of an amino acid sequence selected from the group consisting of
SEQ ID
NO:2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33,
35 to 45
inclusive, 47, and 49, wherein one or more amino acid residues in said variant
differs
from the amino acid sequence of said mature form, provided that said variant
differs in
no more than 15% of amino acid residues from said amino acid sequence;
(e) a nucleic acid fragment encoding at least a portion of a polypeptide
comprising an
amino acid sequence chosen from the group consisting of SEQ ID NO: 2, 4, 6, 8,
10,
15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 45
inclusive, 47, and 49,
or a variant of said polypeptide, wherein one or more amino acid residues in
said
variant differs from the amino acid sequence of said mature form, provided
that said
variant differs in no more than 15% of amino acid residues from said amino
acid
sequence; and
(f) a nucleic acid molecule comprising the complement of (a), (b), (c), (d) or
(e).
6. The nucleic acid molecule of claim S, wherein the nucleic acid molecule
comprises the
nucleotide sequence of a naturally-occurring allelic nucleic acid variant.
7. The nucleic acid molecule of claim S, wherein the nucleic acid molecule
encodes a
polypeptide comprising the amino acid sequence of a naturally-occurring
polypeptide variant.
126




8. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule
differs by a
single nucleotide from a nucleic acid sequence selected from the group
consisting of SEQ ID
NO:1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46, and 48.
9. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule
comprises a
nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence selected from the group consisting of SEQ ID NO:1,
3, 5, 7, 9,
22, 25, 28, 31, 34, 46, and 48;
(b) a nucleotide sequence differing by one or more nucleotides from a
nucleotide sequence
selected from the group consisting of SEQ ID NO:I, 3, 5, 7, 9, 22, 25, 28, 31,
34, 46, and 48,
provided that no more than 20% of the nucleotides differ from said nucleotide
sequence;
(c) a nucleic acid fragment of (a); and
(d) a nucleic acid fragment of (b).
10. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule
hybridizes
under stringent conditions to a nucleotide sequence chosen from the group
consisting of SEQ
ID NO:1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46, and 48, or a complement of said
nucleotide
sequence.
11. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule
comprises a
nucleotide sequence selected from the group consisting of:
(a) a first nucleotide sequence comprising a coding sequence differing by one
or more
nucleotide sequences from a coding sequence encoding said amino acid sequence,
provided that no more than 20% of the nucleotides in the coding sequence in
said first
nucleotide sequence differ from said coding sequence;
(b) an isolated second polynucleotide that is a complement of the first
polynucleotide; and
(c) a nucleic acid fragment of (a) or (b).
12. A vector comprising the nucleic acid molecule of claim 11.
13. The vector of claim 12, further comprising a promoter operably-linked to
said nucleic
acid molecule.
14. A cell comprising the vector of claim 12.
127




15. An antibody that binds immunospecifically to the polypeptide of claim 1.
16. The antibody of claim 15, wherein said antibody is a monoclonal antibody.
17. The antibody of claim 15, wherein the antibody is a humanized antibody.
18. A method for determining the presence or amount of the polypeptide of
claim 1 in a
sample, the method comprising:
(a) providing the sample;
(b) contacting the sample with an antibody that binds immunospecifically to
the
polypeptide; and
(c) determining the presence or amount of antibody bound to said polypeptide,
thereby determining the presence or amount of polypeptide in said sample.
19. A method for determining the presence or amount of the nucleic acid
molecule of
claim 5 in a sample, the method comprising:
(a) providing the sample;
(b) contacting the sample with a probe that binds to said nucleic acid
molecule; and
(c) determining the presence or amount of the probe bound to said nucleic acid
molecule,
thereby determining the presence or amount of the nucleic acid molecule in
said sample.
20. The method of claim 19 wherein presence or amount of the nucleic acid
molecule is
used as a marker for cell or tissue type.
21. The method of claim 20 wherein the cell or tissue type is cancerous.
22. A method of identifying an agent that binds to a polypeptide of claim 1,
the method
comprising:
(a) contacting said polypeptide with said agent; and
(b) determining whether said agent binds to said polypeptide.
23. The method of claim 22 wherein the agent is a cellular receptor or a
downstream
effector.
128




24. A method for identifying an agent that modulates the expression or
activity of the
polypeptide of claim 1, the method comprising:
(a) providing a cell expressing said polypeptide;
(b) contacting the cell with said agent, and
(c) determining whether the agent modulates expression or activity of said
polypeptide,
whereby an alteration in expression or activity of said peptide indicates said
agent modulates
expression or activity of said polypeptide.
25. A method for modulating the activity of the polypeptide of claim 1, the
method
comprising contacting a cell sample expressing the polypeptide of said claim
with a compound
that binds to said polypeptide in an amount sufficient to modulate the
activity of the
polypeptide.
26. A method of treating or preventing a ENDOX-associated disorder, said
method
comprising administering to a subject in which such treatment or prevention is
desired the
polypeptide of claim 1 in an amount sufficient to treat or prevent said ENDOX-
associated
disorder in said subject.
27. The method of claim 26 wherein the disorder is selected from the group
consisting of
diabetes, metabolic disturbances associated with obesity, the metabolic
syndrome X, anorexia,
wasting disorders associated with chronic diseases, cancers, cancer-associated
cachexia, and
dyslipidemia.
28. The method of claim 26 wherein the disorder is related to organismal
energy
metabolism that effect adipose stores, muscle mass, insulin secretion, glucose
utilization and
serum lipid levels including triglycerides and cholesterol
29. The method of claim 26, wherein said subject is a human.
30. A method of treating or preventing a ENDOX-associated disorder, said
method
comprising administering to a subject in which such treatment or prevention is
desired the
nucleic acid of claim 5 in an amount sufficient to treat or prevent said ENDOX-
associated
disorder in said subject.
129




31. The method of claim 30 wherein the disorder is selected from the group
consisting of
diabetes, metabolic disturbances associated with obesity, the metabolic
syndrome X, anorexia,
wasting disorders associated with chronic diseases, cancers, cancer-associated
cachexia, and
dyslipidemia.
32. The method of claim 30 wherein the disorder is related to organismal
energy
metabolism that effects adipose stores, muscle mass, insulin secretion,
glucose utilization and
serum lipid levels including, triglycerides and cholesterol
32. The method of claim 30, wherein said subject is a human.
34. A method of treating or preventing a ENDOX-associated disorder, said
method
comprising administering to a subject in which such treatment or prevention is
desired the
antibody of claim 15 in an amount sufficient to treat or prevent said ENDOX-
associated
disorder in said subject
35. The method of claim 34 wherein the disorder is selected from the group
consisting of
diabetes, metabolic disturbances associated with obesity, the metabolic
syndrome X, anorexia,
wasting disorders associated with chronic diseases, cancers, cancer-associated
cachexia, and
dyslipidemia.
36. The method of claim 34 wherein the disorder is related to organismal
energy
metabolism that effects adipose stores, muscle mass, insulin secretion,
glucose utilization and
serum lipid levels including, triglycerides and cholesterol
37. The method of claim 34, wherein the subject is a human.
38. A pharmaceutical composition comprising the polypeptide of claim 1 and a
pharmaceutically-acceptable carrier.
39. A pharmaceutical composition comprising the nucleic acid molecule of claim
5 and a
pharmaceutically-acceptable carrier.
130




40. A pharmaceutical composition comprising the antibody of claim 15 and a
pharmaceutically-acceptable carrier.
41. A kit comprising in one or more containers, the pharmaceutical composition
of claim
38.
42. A kit comprising in one or more containers, the pharmaceutical composition
of claim
39.
43. A kit comprising in one or more containers, the pharmaceutical composition
of claim
40.
44. A method for determining the presence of or predisposition to a disease
associated with
altered levels of the polypeptide of claim 1 in a first mammalian subject, the
method
comprising:
(a) measuring the level of expression of the polypeptide in a sample from the
first
mammalian subject; and
(b) comparing the amount of said polypeptide in the sample of step (a) to the
amount of
the polypeptide present in a control sample from a second mammalian subject
known
not to have, or not to be predisposed to, said disease;
wherein an alteration in the expression level of the polypeptide in the first
subject as compared
to the control sample indicates the presence of or predisposition to said
disease.
45. The method of claim 44 wherein the predisposition is to cancers.
46. A method for determining the presence of or predisposition to a disease
associated with
altered levels of the nucleic acid molecule of claim 5 in a first mammalian
subject, the method
comprising:
(a) measuring the amount of the nucleic acid in a sample from the first
mammalian
subject; and
(b) comparing the amount of said nucleic acid in the sample of step (a) to the
amount of
the nucleic acid present in a control sample from a second mammalian subject
known
not to have or not be predisposed to, the disease;
131




wherein an alteration in the level of the nucleic acid in the first subject as
compared to the
control sample indicates the presence of or predisposition to the disease.
47. The method of claim 46 wherein the predisposition is to cancers.
48. A method of treating a pathological state in a mammal, the method
comprising
administering to the mammal a polypeptide in an amount that is sufficient to
alleviate the
pathological state, wherein the polypeptide is a polypeptide having an amino
acid sequence at
least 95% identical to a polypeptide comprising an amino acid sequence of at
least one of SEQ
ID NO:2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32,
33, 35 to 45
inclusive, 47, and 49, or a biologically active fragment thereof.
49. A method of treating a pathological state in a mammal, the method
comprising
administering to the mammal the antibody of claim 15 in an amount sufficient
to alleviate the
pathological state.
132

Description

Note: Descriptions are shown in the official language in which they were submitted.



CA 02386199 2002-03-28
WO 01/25436 PCT/US00/41077
ENDOZEPINE-LIKE POLYPEPTIDES AND POLYNUCLEOTIDES
ENCODING SAME
FIELD OF THE INVENTION
The invention generally relates to nucleic acids and polypeptides encoded
therefrom.
More specifically, the invention relates to nucleic acids encoding endozepine-
like polypeptides
as well as vectors, host cells, antibodies, and recombinant methods for
producing these
nucleic acids and polypeptides.
BACKGROUND OF THE INVENTION
In the developed world there are over 250 million individuals who suffer from
disorders associated with nutritional excess. Obesity, Type II diabetes, and
the metabolic
syndrome X have reached epidemic proportions. The problems associated with
these
disorders such as hyperlipidemia, hypertension, vascular disease, stroke and
end-organ
damage exact a huge financial burden to afflicted individuals and society at
large. Thus, new
pharmacologic approaches to regulate energy metabolism would be of significant
medical
benefit. The present invention contains a novel family of endozepine
polypeptides that offer
new therapeutic opportunities to regulate metabolism through their modulation
of insulin
secretion, insulin sensitivity, glucose and fatty acid utilization and other
endocrine functions.
Endozepines are a family of proteins whose members have been reported to have
diverse biological effects. These effects can include modulation of gamma-
aminobutyric acid
(GABA) receptors, insulin homeostasis, and regulation of mitochondrial
steriodogenesis.
Modulation of GABA receptors, which are present in brain, is thought to
modulate
pathological anxiety.
Members of the endozepine family include diazepam binding inhibitor (DBI).
DBI, or
a derivative of DBI, is thought to down-regulate the effects of GABA. DBI is
also reported to
inhibit both the early and the late phases of glucose-induced insulin release
from the isolated
perfused rat pancreas.
Several mammalian DBI polypeptides have been described. Mammalian DBIs tend to
be highly conserved at their carboxy termini. A human DBI polypeptide of
approximately 11
kilodaltons (kD) has been described. This polypeptide has been shown to
displace (3-
carbolines and benzodiazepines bound to brain membrane fractions in vitro.


CA 02386199 2002-03-28
WO 01/25436 PCT/US00/41077
DBI polypeptides have been reported to be present in both brain and non-brain
tissue,
including gut. It has been proposed that DBI may belong to a new family of gut
polypeptides
that inhibit glucose-mediated insulin release by hormonal, neurocrine
mechanisms, or both.
S SUMMARY OF THE INVENTION
The invention is based in part upon the discovery of novel nucleic acid
sequences
encoding polypeptides related to known endozepines. Nucleic acids encoding
endozepine-like
polypeptides and derivatives and fragments thereof, will hereinafter be
collectively designated
as "ENDOX".
In one aspect, the invention provides an isolated ENDOX nucleic acid molecule
encoding an ENDOX polypeptide that includes a nucleic acid sequence that has
identity to the
nucleic acid sequence of human endozepine mRNA. In some embodiments, the ENDOX
nucleic acid molecule can hybridize under stringent conditions to a nucleic
acid sequence
complementary to a nucleic acid molecule that includes a protein-coding
sequence of an
endozepine nucleic acid sequence. The invention also includes an isolated
nucleic acid that
encodes an ENDOX polypeptide, or a fragment, homolog, analog or derivative
thereof. For
example, the nucleic acid can encode a polypeptide at least 85% identical to a
polypeptide
comprising the amino acid sequences of SEQ ID N0:2, 4, 6, 8, 10, 15, 16, 17,
18, 19, 20, 21,
23, 24, 26, 27, 29, 30, 32, 33, 35 to 45 inclusive, 47, and 49. The nucleic
acid can be, for
example, a genomic DNA fragment or a cDNA molecule that includes the nucleic
acid
sequence of any of SEQ ID NO: l, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46, and 48.
Also included in the invention is an oligonucleotide, e.g., an oligonucleotide
which
includes at least 6 contiguous nucleotides of an ENDOX nucleic acid (e.g., SEQ
ID NO: 1, 3,
5, 7, 9, 22, 25, 28, 31, 34, 46,~and 48) or a complement of said
oligonucleotide.
Also included in the invention are substantially purified ENDOX polypeptides
(SEQ
ID N0:2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32,
33, 35 to 45
inclusive, 47, and 49). In some embodiments, the ENDOX polypeptides include an
amino
acid sequence that is substantially identical to the amino acid sequence of
human endozepine
polypeptide. '
The invention also features antibodies that immunoselectively-binds to ENDOX
polypeptides.
In another aspect, the invention includes pharmaceutical compositions which
include
therapeutically- or prophylactically-effective amounts of a therapeutic and a
pharmaceutically-
2


CA 02386199 2002-03-28
WO 01/25436 PCT/US00/41077
acceptable carrier. The therapeutic can be, e.g., an ENDOX nucleic acid, an
ENDOX
polypeptide, or an antibody specific for an ENDOX polypeptide. In a further
aspect, the
invention includes, in one or more containers, a therapeutically- or
prophylactically-effective
amount of this pharmaceutical composition.
In a further aspect, the invention includes a method of producing a
polypeptide by
culturing a cell that includes an ENDOX nucleic acid, under conditions
allowing for
expression of the ENDOX polypeptide encoded by the DNA. If desired, the ENDOX
polypeptide can then be recovered.
In another aspect, the invention includes a method of detecting the presence
of an
ENDOX polypeptide in a sample. In the method, a sample is contacted with a
compound that
selectively binds to the polypeptide under conditions allowing for formation
of a complex
between the polypeptide and the compound. The complex is detected, if present,
thereby
identifying the ENDOX polypeptide within the sample.
The invention also includes methods to identify specific cell or tissue types
based on
their expression of ENDOX.
Also included in the invention is a method of detecting the presence of an
ENDOX
nucleic acid molecule in a sample by contacting the sample with an ENDOX
nucleic acid
probe or primer, and detecting whether the nucleic acid probe or primer bound
to an ENDOX
nucleic acid molecule in the sample.
In a further aspect, the invention provides a method for modulating the
activity of an
ENDOX polypeptide by contacting a cell sample that includes the ENDOX
polypeptide with a
compound that binds to the ENDOX polypeptide in an amount sufficient to
modulate the
activity of said polypeptide. The compound can be, e.g., a small molecule,
such as a nucleic
acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other
organic (carbon
containing) or inorganic molecule, as further described herein.
Also within the scope of the invention is the use of a Therapeutic in the
manufacture of
a medicament for treating or preventing disorders or syndromes including,
e.g., metabolic
disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated
cachexia, and the
various dyslipidemias. The Therapeutic can be, e.g., an ENDOX nucleic acid, an
ENDOX
polypeptide, or an ENDOX-specific antibody, or biologically-active derivatives
or fragments
thereof.


CA 02386199 2002-03-28
WO 01/25436 PCT/US00/41077
The invention further includes a method for screening for a modulator of
disorders or
syndromes including, e.g., metabolic disorders, diabetes, obesity, infectious
disease, anorexia,
cancer-associated cachexia, and the various dyslipidemias. The method includes
contacting a
test compound with an ENDOX polypeptide and determining if the test compound
binds to
said ENDOX polypeptide. Binding of the test compound to the ENDOX polypeptide
indicates
the test compound is a modulator of activity, or of latency or predisposition
to the
aforementioned disorders or syndromes.
Also within the scope of the invention is a method for screening for a
modulator of
activity, or of latency or predisposition to an disorders or syndromes
including, e.g., metabolic
disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated
cachexia, and the
various dyslipidemias, by administering a test compound to a test animal at
increased risk for
the aforementioned disorders or syndromes. The test animal expresses a
recombinant
polypeptide encoded by an ENDOX nucleic acid. Expression or activity of ENDOX
polypeptide is then measured in the test animal, as is expression or activity
of the protein in a
control animal which recombinantly-expresses ENDOX polypeptide and is not at
increased
risk for the disorder or syndrome. Next, the expression of ENDOX polypeptide
in both the
test animal and the control animal is compared. A change in the activity of
ENDOX
polypeptide in the test animal relative to the control animal indicates the
test compound is a
modulator of latency of the disorder or syndrome.
In yet another aspect, the invention includes a method for determining the
presence of
or predisposition to a disease associated with altered levels of an ENDOX
polypeptide, an
ENDOX nucleic acid, or both, in a subject (e.g., a human subject). The method
includes
measuring the amount of the ENDOX polypeptide in a test sample from the
subject and
comparing the amount of the polypeptide in the test sample to the amount of
the ENDOX
polypeptide present in a control sample. An alteration in the level of the
ENDOX polypeptide
in the test sample as compared to the control sample indicates the presence of
or predisposition
to a disease in the subject. Preferably, the predisposition includes, e.g.,
metabolic disorders,
diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia,
and the various
dyslipidemias. Also, the expression levels of the new endozepines of the
invention can be used
in a method to screen for various cancers.
In a further aspect, the invention includes a method of treating or preventing
a
pathological condition associated with a disorder in a mammal by administering
to the subject
an ENDOX polypeptide, an ENDOX nucleic acid, or an ENDOX-specific antibody to
a
4


CA 02386199 2002-03-28
WO 01/25436 PCT/US00/41077
subject (e.g., a human subject), in an amount sufficient to alleviate or
prevent the pathological
condition. In preferred embodiments, the disorder, includes, e.g., metabolic
disorders,
diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia,
and the various
dyslipidemias..
In a further aspect, the invention includes a method to alter global energy
metabolism
or weight loss by altering in serum cholesterol, lipids, glucose and insulin.
In yet another aspect, the invention includes a method to modulate weight loss
due to
specific adipose deposit reduction, muscle mass increases associated with some
medical
treatments, or modulation of lipid volume in some adipocytes
In yet a further aspect, the invention can be used in methods to influence
appetite,
absorption of nutrients and the disposition of metabolic substrates in both a
positive and
negative fashion. The invention can also be used in the treatment of diabetes,
metabolic
disturbances associated with obesity, the metabolic syndrome X as well as
anorexia and
wasting disorders associated with chronic diseases and various cancers by
modulating
metabolism.
In yet another aspect, the invention can be used in a method to identity the
cellular
receptors and downstream effectors of the invention by any one of a number of
techniques
commonly employed in the art. These include but are not limited to the two-
hybrid system,
affinity purification, co-precipitation with antibodies or other specific-
interacting molecules.
Unless otherwise defined, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary skill in the art to which
the invention
belongs. Although methods and materials similar or equivalent to those
described herein can
be used in the practice or testing of the invention, suitable methods and
materials are described
below. All publications, patent applications, patents, and other references
mentioned herein
are incorporated by reference in their entirety. In case of conflict, the
present specification,
including definitions, will control. In addition, the materials, methods, and
examples are
illustrative purposes only, and not intended to be limiting in any manner.
Other features and
advantages of the invention will be apparent from the following detailed
description and
claims.


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BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows 6 photomicrographs deoicting mesenteric adipose deposits in mice
in response
to treatment.
FIG. 2 shows PCR products of endozepine coding sequences.
DETAILED DESCRIPTION OF THE INVENTION
The invention is based, in part, upon the discovery of novel nucleic acid
sequences that
encode polypeptides related to previously described polypeptides in the
endozepine protein
family. Nucleic acids encoding endozepine-like polypeptides based on the
discovered
sequences are referred to individually as ENDO1, END02, END03, END04, ENDOS,
END06, END07, END08, END09, and ENDO10. The nucleic acids, and their encoded
polypeptides, are collectively designated herein as "ENDOX".
ENDOl
An ENDO1 nucleic acid of the invention comprises nucleic acid sequences shown
in
1 S SEQ ID NOs: 1, 11, 13, 14, 46, and 48. SEQ ID NOs: 1 l, 13, and 14 can be
combined to
provide the nucleotide sequence of SEQ ID NO:1. SEQ ID NO:1 may not be a
complete
coding sequence as it does not have a starting methioine, and it has no stop
codon. SEQ ID
NOs: 46 and 48 both encompass nucleotide sequences which encode an amino acid
sequence
which is also encoded by SEQ ID NO:1. Each of these nucleotide sequences and
the amino
acids which they encode are described in detail before.
An ENDOI nucleic acid of the invention includes the nucleic acid sequence
shown in
Table 1 (SEQ ID NO:11). The sequence is related to an expression sequence tag
(EST) from a
previously described human cDNA clone, with database accession number
AA877351. The
AA877351 EST is reported to be similar to a nucleic acid encoding "diazepam
binding
inhibitor-like S".
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Table 1
ACCGCCTCCACCACCCCATGTGCCAAGTGGAGTTCGAGCTGCGCGGCCCTCAAGCAGCTGAAG
GGTCCCGTGAGCGATCAGGAGAAGCTGCTGGTCTACGGCTTGTACAAACAGGCCACCCAGGGC
GACTGCGACATCCCCGGCCCTCCGGCCTCAGACGTGAGAGCCAGGGCCAAGTGGGAGGCTTGG
AGCGCGAACAAAGGGGCGTCCAAGATGGACGCCATGAGGGGCTACGCGGCCAAAGTGGAGGAG
CTGACGAAGAAGGAA (SEQ ID N0:11)
The nucleic acid sequence disclosed in Table 1 includes an open reading frame
("ORF") beginning at position 1. The ORF encodes a polypeptide sequence of 89
amino acid
residues. The sequence of this encoded polypeptide is presented in Table 2
(SEQ ID N0:12).
The translated protein is related (Identities 62/81; 76%) and Positives =
70/81; 86% ) to
diazepam binding inhibitor-like 5(SWISSPROT-ACC:009035) from mouse. Also, the
translated protein is related (Identities = 89/89 (100%), Positives = 89/89
(100%)) to Homo
Sapiens endozepine-like protein type 2 mutant (GENBANK-
ID:AF229804~acc:AF229804).
Table 2
TASTTPCAKWSSSCAALKQLKGPVSDQEKLLVYGLYKQATQGDCDIPGPPASDVRARAKWEAW
SANKGASKMDAMRGYAAKVEELTKKE (SEQ ID N0:12)
The polypeptide encoded by the ORF present in SEQ ID NO:11 does not contain an
amino terminal methionine residue. Therefore, the disclosed nucleic acid
sequence may be a
portion of an open reading frame encoding a larger gene product. The larger
gene product
may include, for example, at least a signal peptide that is cleaved during
protein processing.
To identify additional sequences encoding an ENDO1 nucleic acid, the disclosed
ENDO1 sequence (SEQ ID NO:11) was used to design primers for use in PCR
reactions to
isolate additional sequences encoding the ENDO1 polypeptide shown in Table 2.
The
amplified sequences were cloned and sequenced. The sequences of two products
were named
18517852-2 (SEQ ID N0:13) and 118517852-3 (SEQ ID N0:14). These sequences are
shown in Tables 3 and 4, respectively.
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Table 3
TCTTCTTCGTCAGCTCCTCCACTTTGGCCGCGTAGCCCCTCATGGCGTCCATCTTGGACGCCCCTTTGTTCGCGCT
CCAAGCCTCCCACTTGGCCCTGGCTCTCACGTCTGAGGCCGGAGGGCCGGGGATGTCGCAGTCGCCCTGGGTGGCC
S TGTTTGTACAAGCCGTAGACCAGCAGCTTCTCCTGATCGCTCACGGGACCCTTCAGCTGCTTGAGGGCCGCAAAGC
TCGAACTCCACTTGGCACATGGGGTGGTGGAGGCGGTCCCTGGTGCTAGAAGCTGGAGGTGGAGAGTTGGAGTGGC
TGTTACTACTCGATCTCAGGGGGAGGAGACAGGCACGCGATGTTTGTGTTTTGTCAAGCACAGATTGCAAGCTCGG
GGTCCAGCGTAAACCCCACCATGTTTGGGCTCACACGGCGCATTTTCTGGGGAGGACCAGCCGTCAAAAAGCGTCT
AGGATCCGGAACGCTGCTGTCTGGA (SEQ ID N0:13)
Table 4
GCGTCCATCTTGGACGCCCCTTTGTTCGCGCTCCAAGCCTCCCACTTGGCCCTGGCTCTCACGTCTGAGGCCGGAG
IS
GGCCGGGGATGTCGCAGTCGCCCTGGGTGGCCTGTTTGTACAAGCCGTAGACCAGCAGCTTCTCCTGATCGCTCAC
GGGACCCTTCAGCTGCTTGAGGGCCGCGCAGCTCGAACTCCACTTGGCACATGGGGTGGTGGAGGCGGTCCCTGGT
GCTAGAAGCTGGAGGTGGAGAGTTGGAGTGGCTGTTACTACTCGC (SEQ ID N0:14)
The sequences shown in Tables l, 3, and 4 can be combined to provide the
nucleotide
sequence shown in Table S (SEQ ID NO:1).
Table S
GAT CGA GTA GTA ACA GCC ACT CCA ACT CTC CAC CTC CAG CTT CTA GCA CCA GGG ACC
GCC TCC
2S ACC ACC CCA TGT GCC AAG TGG AGT TCG AGC TXT GCG GCC CTC AAG CAG CTG AAG GGT
CCC GTG
AGC GAT CAG GAG AAG CTG CTG GTC TAC GGC TTG TAC AAA CAG GCC ACC CAG GGC GAC
TGC GAC
ATC CCC GGC CCT CCG GCC TCA GAC GTG AGA GCC AGG GCC AAG TGG GAG GCT TGG AGC
GCG AAC
AAA GGG GCG TCC AAG ATG GAC GCC ATG AGG GGC TAC GCG GCC AAA GTG GAG GAG CTG
ACG AAG
AAG (SEQ ID N0:1)
The nucleotide residue denoted by "X" can be T or G , i.e., in various
embodiments an
ENDO1 nucleic acid of the invention includes a T at the position denoted by X
in the nucleic
acid sequence. In other embodiments, an ENDO1 nucleic acid sequence of the
invention
includes a G at the position denoted by X in the disclosed nucleotide
sequence.
The polypeptide encoded by the ORF in SEQ ID NO: l does not contain an amino
terminal methionine residue. Therefore, the disclosed nucleic acid sequence
may be a portion
of an open reading frame encoding a larger gene product. To identify
additional sequences
encoding an ENDO1 nucleic acid, nucleic acid database searches were conducted.
Two new
ENDOI sequences have now been indentified and are shown in Table SA. SEQ ID
N0:46 was
compiled from a previously described human clone with GenBank AccNo AL121672
(SEQ
ID N0:46). SEQ ID N0:48 was compiled from a previously described human clone
with
GenBank clone AC02S743 (SEQ ID N0:48).
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Table SA
>AL121672 GENSCAN~redicted CDS S 687 by
ATGGGAGACGCAGGAGCCACGGCGGCCGCGCTTAGGCCTGCTCACAACCTCCGCCCGGCCCCGCCCACAG
CCTCCGCCGCGCACGCGCAGTCCTCACGAACGAGCGCGCCAAGCGCACAGCGCCGCCTTCCGGCAGAGCC
S CTCCCACCAGCCCTCAGCACCAGGGACCGCCTCCACCACCCCATGTGCCAAGTGGAGTTCGAGCTGCGCG
GCCCTCAAGCAGCTGAAGGGTCCCGTGAGCGATCAGGAGAAGCTGCTGGTCTACGGCTTGTACAAACAGG
CCACCCAGGGCGACTGCGACATCCCCGGCCCTCCGGCCTCAGACGTGAGAGCCAGGGCCAAGTGGGAGGC
TTGGAGCGCGAACAAAGGGGCGTCCAAGATGGACGCCATGAGGGGCTACGCGGCCAAAGTGGAGGAGCTG
ACGAAGAAGGAAGTGGGGGGCGTGGAGCGCGAACAAAGGGGCGTGCAAGATGGACGCCATGAGGGGCTAC
1O GCGGCCAAAGTGGAGGAGCTGACGAAGAAGGAAGGGCGTCCAAGATGGACGCCATGAGGGGCTACGCGGC
CAAAGTGGAGGAGCTGACGAAGAAGGAAGTGGGGGGCGTGGAGCGCGAACAAAGGGGCGTCCAAGATGGA
CGCCATGAGGGGCTACGCGGCCAGAGTGAGGAGATGAGGAAGAAGGAGGCTGGCTGA (SEQ ID N0:46)
>AC02S743 GENSCAN~redicted CDS 7 S76 by
IS ATGGGAGACGCAGGAGCCACGGCGGCCGCGCTTAGGCCTGCTCACAACCTCCGCCCGGCCCCGCCCACAG
CCTCCGCCGCGCACGCCAGTCCTCACGAACGAGCGCGCCAAGCAAGCCGCGCCTTCCGGCAGAGCCCTCC
CACCAGCCCTCAGCTTCTAGCACCAGGGACCGCCTCCACCACCCCATGTGCCAAGTGGAGTTCGAGCTGC
GCGGCCCTCAAGCAGCTGAAGGGTCCCGTGAGCGATCAGGAGAAGCTGCTGGTCTACGGCTTGTACAAAC
AGGCCACCCAGGGCGACTGCGACATCCCCGGCCCTCCGGCCTCAGACGTGAGAGCCAGGGCCAAGTGGGA
2O GGCTTGGAGCGCGAAAAAAGGGGCGTCCAAGATGGACGCCATGAGGGGCTACGCGGCCAAAGTGGAGGAG
CTGACGAAGAAGGAAGTGGGGGGCGTGGAGCGCGAACAAAGGGGCGTGCAAGATGGACGCCATGAGGGGC
TACGCGGCCAAAGTGGAGGAGCTGACGAAGAAGGAAGTGGGGGGCGTGGAGCGCGAACAAAGGGGCGTCC
AAGATGGACGCCATGA (SEQ ID N0:48)
2S The nucleic acid shown in Table S (SEQ ID NO:1) encodes a polypeptide
having the
amino acid sequence shown in Table 6 (SEQ ID N0:2).
Table 6
DRVVTATPTLHLQLLAPGTASTTPCAKWSSSXAALKQLKGPVSDQEKLLVYGLYKQATQGDCD
IPGPPASDVRARAKWEAWSANKGASKMDAMRGYAAKVEELTKKE(SEQ ID N0:2)
The nucleic acids shown in Table SA (SEQ ID N0:46 and 48) encode polypeptides
having the amino acid sequences shown in Table 6A (SEQ ID N0:47 and 49,
respectively).
Table 6A
>AL121672 GENSCAN~redicted_peptide S 228 as
MGDAGATAAALRPAHNLRPAPPTASAAHAQSSRTSAPSAQRRLPAEPSHQPSAPGTASTTPCAKWSSSCA
ALKQLKGPVSDQEKLLVYGLYKQATQGDCDIPGPPASDVRARAKWEAWSANKGASKMDAMRGYAAKVEEL
TKKEVGGVEREQRGVQDGRHEGLRGQSGGADEEGRASKMDAMRGYAAKVEELTKKEVGGVEREQRGVQDG
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RHEGLRGQSEEMRKKEAG(SEQ ID N0:47)
>AC025743 GENSCAN-predicted~eptide 7-191 as
MGDAGATAAALRPAHNLRPAPPTASAAHASPHERARQASRAFRQSPPTSPQLLAPGTASTTPCAKWSSSC
S AALKQLKGPVSDQEKLLVYGLYKQATQGDCDIPGPPASDVRARAKWEAWSAKKGASKMDAMRGYAAKVEE
LTKKEVGGVEREQRGVQDGRHEGLRGQSGGADEEGSGGRGARTKGRPRWTP (SEQ ID N0:49)
The amino acid residue denoted by "X" can be C or F, i.e., in various
embodiments a
polypeptide of the invention will include a C at the position denoted by X in
the amino acid
sequence. In other embodiments, an ENDO1 polypeptide of the invention will
include an F at
the position denoted by X in the recited amino acid sequence.
An ENDO1 nucleic acid of the invention can include a nucleic acid encoding the
polypeptide of SEQ ID N0:2,12, 47, or 49, e.g., the ENDO1 nucleic acid can
include the
nucleic acid sequence of SEQ ID NO:1, 11, 46, or 48. The invention also
includes a mutant or
variant nucleic acid any of whose bases may be changed from the corresponding
base shown
in Table 1 or Table 5. In some embodiments, the ENDO1 nucleic acid encodes a
protein that
maintains its endozepine-like activities and physiological functions, or a
fragment of such a
nucleic acid. The invention further includes nucleic acids whose sequences are
complementary to those just described, including nucleic acid fragments that
are
complementary to any of the nucleic acids just described. The invention
additionally includes
nucleic acids or nucleic acid fragments, or complements thereto, whose
structures include
chemical modifications. Such modifications include, but are not limited to:
modified bases,
and nucleic acids whose sugar phosphate backbones are modified or derivatized.
These
modifications are carried out at least in part to enhance the chemical
stability of the modified
nucleic acid, such that they may be used, for example, as antisense binding
nucleic acids in
therapeutic applications in a subject.
An ENDO1 polypeptide of the invention can include the amino acid sequence of
SEQ
ID NO: 2,12, 47, or 49. The invention also includes a mutant or variant
protein any of whose
residues may be changed from the corresponding residue shown in SEQ ID
N0:2,12, 47, or
49, while still encoding a protein that maintains its endozepine-like
activities and
physiological functions, or a functional fragment thereof such as the
following active peptide
to


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(SEQ ID N0:15)
Metabolism-Regulating Peptide #6 (MRP-6) Sequence:
QATQGDCD1PGPPASDVRAR (SEQ ID N0:15)
A multiple sequence alignment of various embodiments of the ENDO1 polypeptide
(Table 6B) displays their relationship to one another.
Table 6B
AL121672 GENSCAN~redicted~ep 1 ~ ~ -SS~TSAP~AQR~LPAEP~ H ~PS59
AC023743 GENSCAN~redicted~ep 1 i ~ ~ SPHEy.ARQA~RAF~QSP P T~ P ~LL 60
AL121672 GENSCAN~redicted~ep 60 ~ ~ ' ~ 119
MRP-6 5 ~ ~ ~ ~ ~ 64
AC025743 GENSCAN-predicted~ep 61 ~ ~ ~ ~ ~ ~ ~ ' ~ ~ '~ ' ti ~ i~ 120
AL121672_GENSCAN~redicted~ep 120 ~i ~ ~ ~ ~ R~S~K~M 179
MRP-6 65 i ~ ~ - - - . - - 89
AC025743 GENSCAN-predicted~ep 12l ~Ki ~ ~ ~ - S GG'1G 180
AL121672 GENSCAN~redicted~ep 180
DAMR(~YAAKVEELTKKEVGGVEREQRGVQDGRHEGLRGQSEEMRKKEAG 228
MRP -6 *** ***
AC023743 GENSCAN~redicted~ep 181 ARTK~RPRWTP-----------------------------------
--- 191
The invention further encompasses antibodies and antibody fragments, such as
Fab or (Fan)z,
that bind immunospecifically to the ENDO1 polypeptide, and derivatives and
fragments,
thereof.
An ENDO1 sequence is useful for detecting specific types of tissue. For
example when
a panel of tissue is assayed for expression, ENDO1 is highly expressed in
liver and endothelial
cells. Also, high expression of ENDO1 is a marker for multiple types of
cancer.
An ENDO1 sequence is also useful to modulate global energy metabolism or
weight
by altering serum glucose or adipose.level
An ENDO1 sequence is also useful in a method to identity the cellular
receptors and
downstream effectors of the invention by any one of a number of techniques
commonly
employed in the art.
An ENDO1 sequence is useful in the treatment of diabetes, metabolic
disturbances
associated with obesity, the metabolic syndrome X as well as anorexia and
wasting disorders
associated with chronic diseases and various cancers by modulating metabolism.
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END02
An END02 nucleic acid of the invention includes the nucleic acid sequence
shown in
Table 7 (SEQ ID N0:3). An ORF, as well as putative untranslated regions
upstream from the
initiation codon and downstream from the stop codon of the ORF, is present in
the nucleotide
sequence disclosed in Table 7. Untranslated nucleotides are shown by
underlining. The start
and stop codons of the ORF are shown in bold letters
Table 7
GTATAAGACATACAGAAGGAATGCCTGGAGAGCAGCAACAGCCCAGCTGCGGCCACCATGTCC
CTGCAGGCTGATTTTGACATGGTCACAGAAGATGTGAGGAAGCTGAAAACAAGACCAGATGAT
GAAGAACTGAAAGAACTTTATGGGCTTTACAAACAAGCTGTAATTGGAAACATTAATATTGAG
TGTTCAGAAATGCTAGAATTAAAAGGCAAGGCCAAATGGGAAGCACAGAACCCCCAAAAAGGA
TTGTCAGAGGAAGATATGATGCGTGCCTTTATTTCTAAAGCCGAAGAGCTGATAGAAAAATAT
GGAATTTAGAATAAAGCATATGATAAATTTTCCTTT (SEQ ID N0:3)
The nucleic acid sequence of Table 7 has 218 of 294 bases (74%) identical and
positive
to a 470 nucleotide Rana ridibunda diazepam-binding inhibitor (DBI) mRNA
(GENBANK-
ID: RRU09205~acc:U09205). A BLASTN identify search comparing regions of the
sequence
disclosed in Table 7 to the Rana ridbunda DPI mRNA is shown in Table 8.
Regions of the
disclosed sequence is are presented as "Query" sequences, and the Rana
ridbunda DPI mRNA
(SEQ ID NO:11) sequences are presented as the "Subject sequences".
Table 8
Query: 46 GCTGCGGCCACCATGTCCCTGCAGGCTGATTTTGACATGGTCA-CAGAAGATGTGAGGAA
104
IIII I IIIIIIII I IIIII IIIIIIIIII I II III IIIII I III
3O Sbjct: 1 GCTGAATCAACCATGTCACCCCAGGCAGATTTTGACAAAG-CAGCAGGGGATGTAAAGAA 59
Query: 105 GCTGAAAACAAGACCAGATGATGAAGAACTGAAAGAACTTTATGGGCTTTACAAACAAGC
164
IIIIIIIII IIII III II IIIIIII) (1111 II II II IIIII II I
3S Sbjct: 60 ATTGAAAACAAAACCAACTGACGATGAACTGAAGGAACTGTACGGACTCTACAAGCAGTC
119
Query: 165 TGTAATTGGAAACATTAATATTGAGTGTTCAGAAATGCTAGAATTAAAAGGCAAGGCCAA
224
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IIII IIII IIIII 111111 I I IIIIIIII I II IIIIIIIIIII
Sbjct: 120 CACTGTTGGGGACATAAATATAGAGTGTCCTGGCATGCTAGATCTGAAGGGCAAGGCCAA
179
S Query: 225 ATGGGAAGCACAGAACCCCCAAAAAGGATTGTCAGAGGAAGATATGATGCGTGCCTTTAT
284
IIIII III IIIII I IIIII IIIII IIIIIIII IIII I II I I I
Sbjct: 180 GTGGGACGCATGGAACCTAAAGAAAGGCTTGTCTAAGGAAGATGCGATGAGCGCTTATGT
239
Query: 285 TTCTAAAGCCGAAGAGCTGATAGAAAAATATGGAATTTAGAATAAAG-CATATGAT 339
IIIIIIIIII I IIIIIIIIIIIIIIIIIIII I II I II I I IIIII
Sbjct: 240 TTCTAAAGCCCATGAGCTGATAGAAAAATATGGCCTGTA-AC-AAGGTCGCATGAT 293
1 S The ORF encodes a polypeptide of 8S amino acids (SEQ ID N0:4). The amino
acid
sequence of this polypeptide is shown in Table 9 ( S EQ I D NO : 4 ) .
Table 9
MSLQADFDMVTEDVRKLKTRPDDEELKELYGLYKQAVIGNINIECSEMLELKGKAKWEAQNPQ
KGLSEEDMMRAFISKAEELIEKYGI (SEQ ID N0:4)
The polypeptide sequence disclosed in Table 9 is related to a previously
described
duck diazepam binding inhibitor polypeptide. This relationship is shown in
Table 10. The
amino acid sequence shown in Table 9 (SEQ ID N0:4) has 60 of 85 amino acid
residues (70
%) identical to, and 72 of 85 residues (84%) positive with, the 103 amino acid
residue acyl-
coA-binding protein (ACBP) (diazepam binding inhibitor)(DBI) (endozepine) (EP)
from Anas
platyrhynchos (domestic duck) (ptnr: SWISSPROT-ACC:P45882). Regions of the
polypeptide sequnce shown in Table 9 are presented as the "Query" sequence.
Regions of the
duck polypeptide sequence are shown as the "Sbct" sequences.
Table 10
Query: 67 QADFDMVTEDVRKLKTRPDDEELKELYGLYKQAVIGNINIECSEMLELKGKAKWEAQNPQ
246
IIIII I+I+111111 IIIIIIIII IIII +i+11111 II+IIIIIIIII I +
Sbjct: 19 QADFDEAAEEVKKLKTRPTDEELKELYGFYKQATVGDINIECPGMLDLKGKAKWEAWNLK 78
Query: 247 KGLSEEDMMRAFISKAEELIEKYGI 321
II+I+II I I+1111+ ++11111
Sbjct: 79 KGISKEDAMNAYISKAKTMVEKYGI 103
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A multiple sequence alignment between the amino acids of SEQ ID N0:4 and
various
acyl coA binding polypeptides is illustrated in Table 11. Shown is an
alignment between the
amino acid sequence of Table 7 ("ACBP Novel"), porcine acyl-coA binding
protein
(SWISSPROT locus ACBP PIG, accession No. P12026) ("ACBP PIG"), bovine acyl-coA
binding protein (SWISSPROT locus ACBP BOVIN, accession no.
P07107)("ACBP BOVIN"), and human acyl-coA binding protein (SWISSPROT locus
ACBP HUMAN, accession no. P07108)("ACBP_HUMAN"). Regions of perfect homology
are shown in black. Regions with conservative amino acid substitutions are
shown in gray.
Non-conservative amino acid substitutions are presented without shading.
Table 11
ACBP PIG - -
ACBP_BCVIN - -
ACBP_HUMAN --
ACBP Novel MS
ACBP PIG G
ACBP_BCVIN
ACBP_HUMAN .
ACBP Novel P
An END02 nucleic acid of the invention encoding a endozepine-like protein
includes
the nucleic acid encoding a polypeptide that includes the amino acid sequence
of SEQ ID
N0:4, e.g., a nucleic acid whose sequence is provided in SEQ ID N0:3. The
invention also
includes a fragment of SEQ ID N0:3, or a mutant or variant nucleic acid any of
whose bases
may be changed from the corresponding base shown in SEQ ID N0:3. In some
embodiments,
the mutant or variant nucleic acid encodes a protein that maintains its
endozepine-like
activities and physiological functions, or a fragment of such a nucleic acid.
The invention
further includes nucleic acids whose sequences are complementary to those just
described,
including nucleic acid fragments that are complementary to any of the nucleic
acids just
described. The invention additionally includes nucleic acids or nucleic acid
fragments, or
complements thereto, whose structures include chemical modifications. Such
modifications
include, but are not limited to, modified bases, and nucleic acids whose sugar
phosphate
backbones are modified or derivatized. These modifications are carned out at
least in part to
enhance the chemical stability of the modified nucleic acid, such that they
may be used, for
example, as antisense binding nucleic acids in therapeutic applications in a
subject.
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An END02 polypeptide according to the invention includes a polypeptide
comprising
the an amino acid sequence shown in Table 9 (SEQ ID N0:4). The invention also
includes a
mutant or variant protein any of whose residues may be changed from the
corresponding
residue shown in Table 8, while still encoding a protein that maintains its
endozepine-like
activities and physiological functions, or a functional fragment thereof such
as the following
active peptide (SEQ ID NO: 16)
Metabolism-Regulating Peptide #S (MRP-5) Sequence:
QAVIGNII~IIECSEMLELKGK (SEQ ID NO: 16).
The invention further encompasses antibodies and antibody fragments, such as
Fab or
(Fab)2, that bind immunospecifically to the END02 polypeptide, and derivatives
and fragments,
thereof.
An END02 sequence is useful for detecting specific types of tissue. For
example when
a panel of tissue is assayed for expression, END02 is highly expressed in
brain and pancreas
cells. Also, high expression of END02 is a marker for colon and lung cancer.
An END02 sequence is also useful to modulate global energy metabolism or
weight
by altering serum glucose or adipose level.
An END02 sequence is also useful in a method to identity the cellular
receptors and
downstream effectors of the invention by any one of a number of techniques
commonly
employed in the art.
An END02 sequence is useful in the treatment of diabetes, metabolic
disturbances
associated with obesity, the metabolic syndrome X as well as anorexia and
wasting disorders
associated with chronic diseases and various cancers by modulating metabolism.
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END03
An END03 nucleic acid of the invention includes the nucleic acid sequence
shown in
Table 12 (SEQ ID N0:5). An ORF, as well as putative untranslated regions
upstream from
the initiation codon and downstream from the stop codon of the ORF, are shown
in Table 12
by underlining. The start and stop codons of the ORF are shown in bold
letters. The ORF
begins with an atg initiation codon at nucleotides 86-88 and ends with a tga
codon at
nucleotide 403-405. Putative untranslated regions upstream from the initiation
codon and
downstream from the stop codon are shown in Table 12 by underlining, whereas
the start and
stop codons are shown in bold letters.
Table 12
GCTCACACCTGTAATCCCAGCATTTGGGAGGCCAAGGCAGGCAGATTATGTGAGGTCAAGAGT
TCCAGACCAGCTGTCCAACATGGCAAAACCCATCTCCACTAAAAATACAAA.AATTAGCCGGCA
TGGGTGGCATGCAGCTGTAATCACAGCTGCTCGGGAGGCTGAGGCGGAGAATCACTTGAGCTG
GGAAGAAAAAAAAAAAAAAAAAAGATGTGCAGGTATTAAGCACTTTAAGACCAAGCCAGCAGA
TGATGAGATGCGGTTCCTTTACGGCCACTACAAACGAGCGACTGTAGGCAACATAAAGACAGA
ACGGCCAGGGATGGTGGACTTCAAGGGCAAAGCCAAGTGGGATCCCTGGAATTTAGTGAAAGG
GGCTGCCAGGGAAGATCCCATGAAAGCTAAAGCTTACGTCAAAAA.AGTAGAAGAGTTAAAGAA
AAAATTCAGAATACGAGAGACTGGAATTGTTGCCAGCCATGCCTTTGTCCTAAACTGAGACAA
TGCCTTGTTTTTTCTACACTGTGGATGGTGGGAACTGATGGAAAGAATCAGCTAACCCATC
(SEQ ID N0:5)
The disclosed END03 nucleic acid sequence (SEQ ID N0:5) has 168 of 199 bases
(84%) identical to a sequence on human chromosome 16 incorporated into
bacterial artificial
chromosome 462618 (LANL) (GENBANK-ID: AC005736~acc:AC005736).
The ORF identified in Table 12 encodes a polypeptide sequence of 83 residues
(SEQ
ID N0:6), which is presented in Table 13
Table 13
MAKPISTKNTKISRHGWHAAVITAAREAEAENHLSWEEKKKKKRCAGIKHFKTKPADDEMRFL
YGHYKRATVGNIKTERPGMVDFKGKAKWDPWNLVKGAAREDPMKAKAYVKKVEELKKKFRIRE
TGIVASHAFVLN (SEQ ID N0:6)
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The amino acid sequence of the polypeptide sequence disclosed in Table 13 (SEQ
ID
N0:6) has 55 of 82 amino acid residues (67%) identical to, and 66 of 82
residues (80%)
positive with, the 86 amino acid residue bovine acyl-coA-binding protein
(ACBP) (diazepam
binding inhibitor)(DBI) (endozepine) (EP) (ptnr: SWISSPROT-ACC: P07107). A
comparison
of these sequences is presented in Table 14. Regions of the polypeptide of
Table 13 are
presented as the "Query" sequence, and regions of the bovine ACBP sequence are
presented as
the "Sbjct" sequence.
Table 14
Query: 209 KRCAGIKHFKTKPADDEMRFLYGHYKRATVGNIKTERPGMVDFKGKAKWDPWNLVKGAAR
388
I +1I 111111+II I+I III+1111+I 111111+IIIIIIIII II +1I ++
IS Sbjct: 7 KAAEEVKHLKTKPADEEMLFIYSHYKQATVGDINTERPGMLDFKGKAKWDAWNELKGTSK 66
Query: 389 EDPMKAKAYVKKVEELKKKFRI 454
II III I+ IIIIIIII+ I
Sbjct: 67 EDAMKA--YIDKVEELKKKYGI 86
The amino acid sequence of the polypeptide sequence disclosed in Table 13 (SEQ
ID
N0:6) has 57 of 91 amino acid residues (62%) identical to, and 72 of 91
residues (79%)
positive with 91 amino acid residues of Human diazepam binding inhibitor (DBI)
(gb: GENBANK-ID:HUMDBI~ acc:M 14200)
A comparison of these sequences is presented in Table 14A. Regions of the
polypeptide of Table 13 are presented as the "Query" sequence, and regions of
the Human
diazepam binding inhibitor (DBI) sequence are presented as the "Sbjct"
sequence.
Table 14A
>gb:GENBANK-ID:HUMDBIIacc:M14200 Human diazepam binding inhibitor (DBI)
mRNA, complete cds - Homo Sapiens, 556 bp.
Length = 556
Plus Strand HSPs:
Score = 310 (109.1 bits), Expect = 3.4e-26, P = 3.4e-26
Identities = 57/91 (62~), Positives = 72/91 (79~), Frame = +2
Query: 38 EKKKKKRCAGIKHFKTKPADDEMRFLYGHYKRATVGNIKTERPGMVDFKGKAKWDPWNLV
97
I + +I ++I IIII+I+II I+11111+1111+I 111111+II 111111 II +
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Sbjct: 77 EAEFEKAAEEVRHLKTKPSDEEMLFIYGHYKQATVGDINTERPGMLDFTGKAKWDAWNEL
256
Query: 98 KGAAREDPMKAKAYVKKVEELKKKFRIRETG 128
Sbjct: 257 KGTSKEDAMKA--YINKVEELKKKYGI*ETG 343
A multiple sequence alignment illustrating the relatedness of the polypeptide
disclosed
in Table 13 to bovine and human Acyl co-A binding proteins is presented in
Table 15 as a
ClustalW analysis. Compared are the polypeptide of Table 12 ("DBI novel"),
bovine acyl
coA-binding protein (SWISSPROT locus ACBP BOVIN, accession P07107)
("ACBP bovin"), and ACBP human SWISSPROT locus ACBP HUMAN, accession -
P07108 ("ACBP Human"). Regions of perfect homology are shown in black. Regions
with
conservative amino acid substitutions are shown in gray. Non-conservative
amino acid
substitutions are presented without shading.
Table 15
DBI novel MAKP I STKNTKI SRHGWHAAV I TAAREAE NH L S KKKKKRCAGeIF
ACBP BCVIN __________________________ . p _______
ACBP HUMAN ___________________________ ~ ~ _______ ~S~
DBI novel R L R N K ' ~~ ~P L~! A~ PMKA ~K
ACBP BQVIN S ~ " ~ ~ D
ACBP HUMAN w ~ " ~ T ~ ' ~-- ' N
DBI novel FR RETG I VASHAFV L N
ACBP B~VIN -------------
ACBP HUMAN -------------
An END03 nucleic acid of the invention includes a nucleic acid encoding a
polyeptide
comprising SEQ ID N0:6, e.g., a nucleic acid whose sequence is shown in SEQ ID
NO:S.
The invention also includes a fragment of the nucleic acid of SEQ ID NO:S, as
well as a
mutant or variant nucleic acid, any of whose bases may be changed from the
corresponding
base shown in Table 12, while still encoding a protein that maintains its
endozepine-like
activities and physiological functions, or a fragment of such a nucleic acid.
The invention
further includes nucleic acids whose sequences are complementary to those just
described,
including nucleic acid fragments that are complementary to any of the nucleic
acids just
described. The invention additionally includes nucleic acids or nucleic acid
fragments, or
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complements thereto, whose structures include chemical modifications. Such
modifications
include, but are not limited to: modified bases, and nucleic acids whose sugar
phosphate
backbones are modified or derivatized. These modifications are carned out at
least in part to
enhance the chemical stability of the modified nucleic acid, such that they
may be used, for
example, as antisense binding nucleic acids in therapeutic applications in a
subject.
An END03 protein of the invention includes the amino acid sequence shown in
Table
13 (SEQ ID N0:6). The invention also includes a mutant or variant protein any
of whose
residues may be changed from the corresponding residue shown in Table 13,
while still
encoding a protein that maintains its endozepine-like activities and
physiological functions, or
a functional fragment thereof such as the following active peptides
Metabolism-Regulating Peptide #3 (MRP-3, 3s) Sequences (SEQ ID N0:17) and
(SEQ ID N0:18)
RATVGNIKTERPGMVDFKGK (SEQ ID N0:17)
RATVGNIKTERPGMVDFK--(SEQ ID N0:18)
The invention further encompasses antibodies and antibody fragments, such as
Fab or
(Fdb)Z, that bind immunospecifically to the END03 polypeptide, and derivatives
and fragments,
thereof.
An END03 sequence is useful for detecting specific types of tissue. For
example when
a panel of tissue is assayed for expression, END03 is highly expressed in
adipose and skeletal
muscle. Also, high expression of END03 is a marker for breast cancer.
An END03 sequence is also useful to modulate global energy metabolism or
weight
by altering serum insulin or adipose level.
An END03 sequence is also useful in a method to identity the cellular
receptors and
downstream effectors of the invention by any one of a number of techniques
commonly
employed in the art.
An END03 sequence is useful in the treatment of diabetes, metabolic
disturbances
associated with obesity, the metabolic syndrome X as well as anorexia and
wasting disorders
associated with chronic diseases and various cancers by modulating metabolism.
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END04
An END04 nucleic acid of the invention includes the nucleic acid sequence
shown in
Table 16 (SEQ ID N0:7). The sequence shown in Table 16 includes an ORF, as
well as
putative untranslated regions upstream and downstream from the ORF. The ORF
begins with
an atg initiation codon at nucleotides 11-13 and ends with a tga codon at
nucleotides 299-301.
The putative upstream and downstream untranslated regions are shown by
underlining in
Table 16.
Table 16
TTGGTGGTAAATGCTCCTTTTGTTTGTTTGTTTGTTCTTCCTTAAGGCTGATTTTGACAGGGC
TGCAGAAGATGTGAGGAAGCTGAAAGCAAGACCAGATGATGGAGAACTGAAAGAACTCTATGG
GCTTTACAAACAAGCAATAGTTGGAGACATTAATATTGCGTGTCCAGGAATGCTAGATTTAAA
AGGCAAAGCCAAATGGGAAGCATGGAACCTCAAAAAAGGGTTGTCGACGGAAGATGCGACGAG
TGCCTATATTTCTAAAGCAAAGGAGCTGATAGAAAAATACGGAATTTAGAATACAGCA (SEQ
ID N0:7)
The disclosed nucleic acid sequence (SEQ ID N0:7) has 200 of 256 bases (78%)
identical to a Rana ridibunda endozepine mRNA (GENBANK-ID: RRU09205~
acc:U09205).
The relationship between the sequence of Table 16 and the Rana ridibunda
sequence is
presented in Table 17. Regions of the sequence shown in Table 16 are listed as
the "Query"
sequence. Regions of the Rana ridibunda endozepine mRNA sequence are shown as
the
"Subject" sequence.
Table 17
Query: 45 AGGCTGATTTTGACAGGGCTGCAGAAGATGTGAGGAAGCTGAAAGCAAGACCAGATGATG
104
Sbjct: 23 AGGCAGATTTTGACAAAGCAGCAGGGGATGTAAAGAAATTGAAAACAAAACCAACTGACG
sz
Query: 105 GAGAACTGAAAGAACTCTATGGGCTTTACAAACAAGCAATAGTTGGAGACATTAATATTG
164
3S Sbjct: 83 ATGAACTGAAGGAACTGTACGGACTCTACAAGCAGTCCACTGTTGGGGACATAAATATAG
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Query: 165 CGTGTCCAGGAATGCTAGATTTAAAAGGCAAAGCCAAATGGGAAGCATGGAACCTCAAAA
224
111111 II IIIIIIIII I II IIIII IIIII IIIII IIIIIIIIIII II I
Sbjct: 143 AGTGTCCTGGCATGCTAGATCTGAAGGGCAAGGCCAAGTGGGACGCATGGAACCTAAAGA
S 202
Query: 225 AAGGGTTGTCGACGGAAGATGCGACGAGTGCCTATATTTCTAAAGCAAAGGAGCTGATAG
284
IIII IIIII I IIIIIIIIIII III II III IIIIIIIIII I IIIIIIIIII
1O Sbjct: 203 AAGGCTTGTCTAAGGAAGATGCGATGAGCGCTTATGTTTCTAAAGCCCATGAGCTGATAG
262
Query: 285 AAAAATACGGAATTTA 300
1111111 II I II
1S Sbjct: 263 AAAAATATGGCCTGTA 278
The disclosed nucleic acid sequence (SEQ ID N0:7) is also related to a human
diazepam binding inhibitor mRNA (GENBANK-ID:HUMDBI~acc:M14200). The disclosed
20 sequence is identical at 179 of 2S9 residues (69%) to the human diazepam
inhibitor mRNA.
The relationship between the disclosed and the human sequence is presented in
Table 18.
Regions of the sequence shown in Table 16 are listed as the "Query" sequence.
Regions of the
human mRNA sequence are shown as the "Subject" sequence.
2S Table 18
Query: 45 AGGCTGATTTTGACAGGGCTGCAGAAGATGTGAGGAAGCTGAA-AGCAAGACCAGATGAT
103
1111111 IIIII I Ililllll II II III I II II I IIII III III
3O Sbjct: 78 AGGCTGAGTTTGAGAAAGCTGCAGAGGAGGTTAGGCACCTTAAGACCAAG-CCATCGGAT
136
Query: 104 GGAGAACTGAAAGAAC-TCTATGGGCTTTACAAACAAGCAATAGTTGGAGACATTAATAT
3S 162
II II I I 1111111 I IIIIIIIIIIIII II II IIIII IIII
Sbjct: 137 GAGGAGATGCT-GTTCATCTATGGCCACTACAAACAAGCAACTGTGGGCGACATAAATAC
195
Query: 163 TGCGTGTCCAGGAATGCTAGATTTAAAAGGCAAAGCCAAATGGGAAGCATGGAACCTCAA
222
I I II II III I II II I IIIII IIIII IIIII II IIIII
Sbjct: 196 AGAACGGCCCGGGATGTTGGACTTCACGGGCAAGGCCAAGTGGGATGCCTGGAATGAGCT
4S 255
SO Query: 223 AAAAGGGTTGTCGACGGAAGATGCGACGAGTGCCTATATTTCTAAAGCAAAGGAGCTGAT
282
111111 II I IIIIIIIII I II II II II Ilil I I IIIII I
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SbjCl: 256 GAAAGGGACTTCCAAGGAAGATGCCATGAAAGCTTACATCAACAAAGTAGAAGAGCTAA-
314
Query: 283 AGAAAAA-TACGGAATTT-AGA 302
1111111 IIIII II I III
Sbjct: 315 AGAAAAAATACGGGATATGAGA 336
The ORF identified in Table 16 encodes an amino acid sequence of 89 amino
acids
(SEQ ID N0:8). The amino acid sequence of the encoded protein (SEQ ID N0:8) is
shown in
Table 19.
Table 19
MLLLFVCLFFLKADFDRA.AEDVRKLKARPDDGELKELYGLYKQAIVGDINIACPGMLDLKGKA
KWEAWNLKKGLSTEDATSAYISKAKELIEKYGI (SEQ ID N0:8)
A signal peptide is present in the polypeptide sequence shown in Table 19. The
most
likely cleavage site between residues 18 and 19, at the sequence DRA-AE. The
program
PSORT predicts a moderate likelihood of extracellular secretion for the END04
protein.
The polypeptide shown in Table 19 is related to previously described acyl-coA
binding
proteins and diazepam binding inhibitor proteins. Table 20 shows that the
amino acid
sequence of Table 19 has 71 of 89 amino acid residues (79%) identical to, and
78 of 89
residues (87%) positive with, the 103 amino acid residue protein from Anas
platyrhynchos
(ptnr: SWISSPROT-ACC:P45882). Regions of the polypeptide sequence shown in
Table 19
are presented as the "Query" sequence. Regions of the Anas platyrhynchos
sequence are
shown as the "Sbjct" sequence.
Table 20
Query: 35 FFL-KADFDRAAEDVRKLKARPDDGELKELYGLYKQAIVGDINIACPGMLDLKGKAKWEA
211
3o III +1111 III+I+III II I 1111111 IIII 111111 IIIIIIIIIIIIIII
Sbjct: 15 FFLHQADFDEAAEEVKKLKTRPTDEELKELYGFYKQATVGDINIECPGMLDLKGKAKWEA
74
Query: 212 WNLKKGLSTEDATSAYISKAKELIEKYGI 298
3s 111111+I III +1111111 ++11111
SbjCt: 75 WNLKKGISKEDAMNAYISKAKTMVEKYGI 103
Table 20A shows that the amino acid sequence of Table 19 has homology to Rana
ridibunda diazepam-binding inhibitor (DBI). Regions of the polypeptide
sequence shown in
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Table 19 are presented as the "Query" sequence. Regions of the Rana ridibunda
diazepam-
binding inhibitor (DBI)sequence are shown as the "Sbjct" sequence.
Table 20A
>gb:GENBANK-ID:RRU09205~acc:U09205 Rana ridibunda diazepam-binding
inhibitor (DBI) mRNA, complete cds - Rana ridibunda, 470 bp.
Length = 470
Plus Strand HSPs:
Score = 365 (128.5 bits), Expect = 6.0e-32, P = 6.0e-32
Identities = 68/85 (80%), Positives = 76/85 (89%), Frame = +1
Query: 12 KADFDRAAEDVRKLKARPDDGELKELYGLYKQAIVGDINIACPGMLDLKGKAKWEAWNLK
71
+ADFD+AA DV+KLK +P D ELKELYGLYKQ+ VGDINI CPGMLDLKGKAKW+AWNLK
Sbjct: 22 QADFDKAAGDVKKLKTKPTDDELKELYGLYKQSTVGDINIECPGMLDLKGKAKWDAWNLK
201
2O Query: 72 KGLSTEDATSAYISKAKELIEKYGI 96
KGLS EDA SAY+SKA ELIEKYG+
Sbjct: 202 KGLSKEDAMSAYVSKAHELIEKYGL 276
An alignment between the polypeptide sequence shown in Table 18 and
previously described diazepam binding inhibitor or Acyl-coA binding
polypeptides is shown
in Table 21. The amino acid sequence shown in Table 19 is presented as
"ba271m1 A". An
88 amino acid frog acyl co-A binding protein amino acid sequence (PIR-ID:
A57711) is
indicated by "A57711 ACBP Frog.: An 88 amino acid human acyl co-A binding
polypeptide
(PIR-ID: NZHU) sequence is shown by "NZHU_ACBP Human". A 103 amino acid duck
endozepine amino acid sequence (SWISSPROT-ACC: 45882) is indicated by
"P45882 endozepine Duck". Regions with conservative amino acid substitutions
are shown
in gray. Non-conservative amino acid substitutions are presented without
shading.
Table 21
ba271m1 A - - - ----MLLOF~'~CL~FLK a Dn ~ I
A57711 ACHP Frog - - - - - - - - - - - - - - - M S P G ~ t~ S
P45882 Endeaopine Duck MF Q AHLL RGTOT ~S F ~I~H E E n n ~ F
NZHU ACBP Human _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ~S ~ y y E ~ =S5~ LF~I
ba271m1 A
A57711 ACBP Frog
P45882 Endezopine Duck
NZHU ACBP Human
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An END04 nucleic acid of the invention includes a nucleic acid encoding a
polypeptide that includes the amino acid sequence of SEQ ID N0:8. For example,
an END04
nucleic acid can include the sequence disclosed in Table 16 (SEQ ID N0:7). The
invention
alco includes fragments of a nucleic acid encoding a polypeptide that includes
the amino acid
sequence of SEQ ID N0:8. The invention also includes a mutant or variant
nucleic acid any
of whose bases may be changed from the con esponding base shown in Table 16,
while still
encoding a protein that maintains its endozepine-like activities and
physiological functions, or
a fragment of such a nucleic acid. The invention further includes nucleic
acids whose
sequences are complementary to those just described, including nucleic acid
fragments that are
complementary to any of the nucleic acids just described. The invention
additionally includes
nucleic acids or nucleic acid fragments, or complements thereto, whose
structures include
chemical modifications. Such modifications include, but are not limited to:
modified bases,
and nucleic acids whose sugar phosphate backbones are modified or derivatized.
These
modifications are carried out at least in part to enhance the chemical
stability of the modified
nucleic acid, such that they may be used, for example, as antisense binding
nucleic acids in
therapeutic applications in a subject.
An END04 protein of the invention includes the protein whose amino acid
sequence is
shown in Table 19 (SEQ ID N0:8). The invention also includes a mutant or
variant protein
any of whose residues may be changed from the corresponding residue shown in
Table 19,
while still encoding a protein that maintains its endozepine-like activities
and physiological
functions, or a functional fragment thereof such as the following active
peptides
Metabolism-Regulating Peptide #4 (MRP-4,4s) Sequences:
QAIVGDINIACPGMLDLKGK (SEQ ID N0:19)
QAIVGDiNIACPGMLDLK--(SEQ ID N0:20)
The invention further encompasses antibodies and antibody fragments, such as
Fab or
(Fab)2, that bind immunospecifically to the END04 polypeptide, and derivatives
and fragments,
thereof.
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An END04 sequence is useful for detecting specific types of tissue. For
example when
a panel of tissue is assayed for expression, END04 is highly expressed in
hematopoietic
tissue.
An END04 sequence is also useful to modulate global energy metabolism or
weight
by altering serum insulin and glucose.
An END04 sequence is also useful in a method to identity the cellular
receptors and
downstream effectors of the invention by any one of a number of techniques
commonly
employed in the art.
An END04 sequence is useful in the treatment of diabetes, metabolic
disturbances
associated with obesity, the metabolic syndrome X as well as anorexia and
wasting disorders
associated with chronic diseases and various cancers by modulating metabolism.
ENDOS
1 S An END05 nucleic acid according to the invention includes the nucleic acid
sequence
shown in Table 25 (SEQ ID N0:9). An ORF is present in the disclosed sequence,
as well as
putative untranslated regions upstream and downstream of the ORF. The ORF
begins with an
atg initiation codon at nucleotides 7-9 and ends with a tag codon at
nucleotides 265-267. The
putative upstream and downstream untranslated regions are shown by underlining
in Table 22.
Table 22
ACCACCATGGCACTGCAGGCTGAATTCGACAAGGCTGCAGAAGACGTGAGGAAGCTGCCAACA
AGACCAGCAGATAATAAAGAACTGAA.A.AAACTCGATGGACTTTACAAACAAGCTATAATTGGA
GACATTAATATTGAGTATCTGGGAATGCTGGACTTTAAGGGCAAGGCCAA.ATGCGCAGCATGG
ACCCTCCAAAAAAGGTTGTCAAAGGAAGATGCAACGAGTGTCTCTATTTCTAAGGCAAA.AGAG
CCGATAGA<~.A.A.ATAGGACATTTAGAATA ( SEQ ID NO : 9 )
The END05 nucleic acid sequence (SEQ ID N0:9) has 199 of 274 nucleotides (72%)
identical to a Rana ridibunda endozepine mRNA GENBANK-ID: RRU09205~acc:U09205.
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comparison of these nucleotide sequences is shown in Table 23. The sequence
disclosed in
Table 22 is presented as the "Query" sequence, and the Rana ridibunda
endozepine mRNA
sequence is presented as as the "Sbjct" sequence.
Table 23
Query: 2 CCACCATGGCACTGCAGGCTGAATTCGACAAGGCTGCAGAAGACGTGAGGAAGCTGCCAA 61
I 111111 III IIIII II II IIIII II IIII II II I III II II
1O Sbjct: 8 CAACCATGTCACCCCAGGCAGATTTTGACAAAGCAGCAGGGGATGTAAAGAAATTGAAAA 67
Query: 62 CAAGACCAGCAGATAATAAAGAACTGAAAAAACTCGATGGACTTTACAAACAAGCTATAA 121
III IIII I II II IIIIIIII IIII I IIIII IIIII II I I
Sbjct: 68 CAAAACCAACTGACGAT---GAACTGAAGGAACTGTACGGACTCTACAAGCAGTCCACTG 124
Query: 122 TTGGAGACATTAATATTGAGTATCTGGGAATGCTGGACTTTAAGGGCAAGGCCAAATGCG 181
IIII IIIII IIIII IIII II II IIIII II I IIIIIIIIIIIIII II I
SbjCt: 125 TTGGGGACATAAATATAGAGTGTCCTGGCATGCTAGATCTGAAGGGCAAGGCCAAGTGGG 184
2O Query: 182 CAGCATGGACCCTCCAAAAAAGG-TTGTCAAAGGAAGATGCAACGAGTGTCTCTATTTCT 240
1111111 III II IIIII IIIII IIIIIIIIIII I III I I I IIIII
Sbjct: 185 ACGCATGGAACCTA-AAGAAAGGCTTGTCTAAGGAAGATGCGATGAGCGCTTATGTTTCT 243
Query: 241 AAGGCAAAAGAGCCGATAGAAAAATAGGACATTTA 275
II II I IIII IIIIIIIIIIII I I I II
Sbjct: 244 AAAGCCCATGAGCTGATAGAAAAATATGGCCTGTA 278
The ENDOS nucleic acid sequence (SEQ ID N0:9) also has 173 of 262 nucleotides
(66%) identical to a Homo sapiens endozepine mRNA GENBANK-ID:
HUMEDZ~acc:M15887. A comparison of these nucleotide sequences is shown in
Table 24.
The sequence disclosed in Table 22 is presented as the "Query" sequence, and
the human
endozepine mRNA sequence is presented as the "Sbjct" sequence.
Table 24
Query: 1'6 CAGGCTGAATTCGACAAGGCTGCAGAAGACGTGAGGAAGCTGCCAAC-AAGACCAGCAGA 74
IIIIIIII II II II IIIIIIII II II III I II II III III I II
Sbjct: 64 CAGGCTGAGTTTGAGAAAGCTGCAGAGGAGGTTAGGCACCTTAAGACCAAG-CCATCGGA 122
Query: 75 TAATAAAGAA-CTGAAAAAACTCGATGGACTTTACAAACAAGCTATAATTGGAGACATTA 133
I I I II III I II IIII I IIIIIIIIIII I I II IIIII I
4S Sbjct: 123 TGAGGA-GATGCTGTTCAT-CT--ATGGCCACTACAAACAAGCAACTGTGGGCGACATAA 178
Query: 134 ATATTGAGTATCTGGGAATGCTGGACTTTAAGGGCAAGGCCAAATGCGCAGCATGGACCC 193
III II I II III 1111111 I IIIIIIIIIIII II I II IIII
Sbjct: 179 ATACAGAACGGCCCGGGATGTTGGACTTCACGGGCAAGGCCAAGTGGGATGCCTGGAATG 238
Query: 194 TCCAAAAAAGGTTGTCAAAGGAAGATGCAACGAGTGTCTCTATTTCTAAGGCAAAAGAGC 253
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Sbjct: 239 AGCTGAAAGGGACTTCCAAGGAAGATGCCATGAAAGCTTACATCAACAAAGTAGAAGAGC 298
Query: 254 CGATAGAAAAA-TAGGACATTT-AGA 277
Sbjct: 299 TAA-AGAAAAAATACGGGATATGAGA 323
The ORF identified in Table 22 encodes a polypeptide of 86 amino acid residues
(SEQ
ID NO:10). The amino acid sequence of the encoded polypeptide is shown in
Table 25.
Table 25
MALQAEFDKAAEDVRKLPTRPADNKELKKLDGLYKQAIIGDINIEYLGMLDFKGKAKCAAWTL
QKRLSKEDATSVSISKAKEPIEK (SEQ ID NO:10)
The encoded polypeptide (SEQ ID NO:10) shown in Table 25 has 57 of 86 amino
acid
residues (66%) identical to, and 67 of 86 residues (77%) positive with, the 88
amino acid
residue diazepam binding inhibitor protein from Rana ridibunda (ptnr:PIR-
ID:A57711). An
alignment of these sequences is shown in Table 26. A comparison of these amino
acid
sequences is shown in Table 26. The sequence disclosed in Table 25 is
presented as the
"Query" sequence, and the Rana ridibunda endozepine polypeptide sequence is
presented as
the "Sbjct" sequence.
Table 26
Query: 7 MALQAEFDKAAEDVRKLPTRPADNKELKKLDGLYKQAIIGDINIEYLGMLDFKGKAKCAA 186
Sbjct: 1 MSPQADFDKAAGDVKKLKTKPTDD-ELKELYGLYKQSTVGDINIECPGMLDLKGKAKWDA 59
Query: 187 WTLQKRLSKEDATSVSISKAKEPIEK 264
Sbjct: 60 WNLKKGLSKEDAMSAYVSKAHELIEK 85
An alignment showing the relatedness of the polypeptide sequence shown in
Table 25 to previously described endozepine sequences is shown in Table 27.
The 86 amino
acid polypeptide of Table 25 is shown as "citb e1 2540m10 A". The other
endozepine
polypeptide sequences present in the table include 88 amino acid sequence of
frog diazepam
binding inhibitor DBI (PIR-ID:A57711 ) ("A57711 "), a 103 amino acid sequence
duck
polypeptide (SWISSPROT-ACC:P45882) ("P45882 Duck DB1), and an 87amino acid
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human polypeptide (NZHU_Human_DBI). Regions with conservative amino acid
substitutions are shown in gray. Non-conservative amino acid substitutions are
presented
without shading.
Table 27
1~57711_Frog-DBI _ _ _ _ - __ _ _ _ _ _ _ _ _~SP D ~ GK 1('
P45882_Duck-DBI MFQAHLLRGTLTLSFF~H D E E I
NZHU Human DBI ----------- ---_hIIS E E E f~H K S
cilb e1 2540m10 A - - - - - - - - - - - - -~A L E ~ D ~ P R~N
A57711_Frog-DBI L t H L
P45$82_Duck_DBI L E 1E N T~ul~l
NZHU_Human DBI R FT ~ EL IC N E I(I(
cil~ e1 2540m10 A Y L C T Q R ~ T S P - -
Using the PSORT program, it is predicted that the disclosed ENDOS protein
localizes to the cytoplasm with a certainty of 0.6500. In an analysis using
the SIGNALP
program, it is predicted that the ENDOS protein does not possess a signal
peptide.
The invention includes an ENDOS nucleic acid encoding a polypeptide that
includes
the amino acid sequence of SEQ ID NO:10, e.g., a nucleic acid including the
nucleotide
sequence of SEQ ID N0:9, or a fragment thereof. The invention also includes a
mutant or
variant nucleic acid, any one of whose bases may be changed from the
corresponding base, as
illustrated in Table 22, while still encoding a protein which maintains its
endozepine-like
activities and physiological functions, or a fragment of such a nucleic acid.
The invention
further includes nucleic acids whose sequences are complementary to those
previously
described, including nucleic acid fragments that are complementary to any of
the nucleic acids
previously described. The invention additionally includes nucleic acids or
nucleic acid
fragments, or complements thereto, whose structures include chemical
modifications. Such
modifications include, by way of non-limiting example, modified bases, and
nucleic acids
whose sugar-phosphate backbones are modified or derivatized. These
modifications are
carned out, at least in part, to enhance the chemical stability of the
modified nucleic acid, such
that they may be used, e.g., as antisense binding nucleic acids in therapeutic
applications.
An ENDOS protein of the invention includes the polypeptide (SEQ ID NO:10)
whose
sequence is illustrated in Table 25. The invention also includes a mutant or
variant protein any
of whose residues may be changed from the corresponding residue illustrated in
Table 25,
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while still encoding a protein which maintains its endozepine-like activities
and physiological
functions, or a functional fragment thereof such as the following active
peptide
Metabolism-Regulating Peptide #7 (MRP-7) Sequence:
1 QAIIGDINIEYLGMLDFKGK (SEQ ID N0:21)
The invention further encompasses antibodies and antibody fragments, such as
Fab or (Fab)z, which
immunospecifically-bind to the END05 polypeptide, and derivatives and
fragments, thereof.
An ENDOS sequence is useful for detecting specific types of tissue. for
example when
a panel of tissue is assayed for expression, ENDOS is highly expressed in
adipose and
hematopietic tissue.
An ENDOS sequence is also useful to modulate global energy metabolism or
weight
by altering serum cholesterol or glucose.
An ENDOS sequence is also useful in a method to identity the cellular
receptors and
downstream effectors of the invention by any one of a number of techniques
commonly
employed in the art.
An ENDOS sequence is useful in the treatment of diabetes, metabolic
disturbances
associated with obesity, the metabolic syndrome X as well as anorexia and
wasting disorders
associated with chronic diseases and various cancers by modulating metabolism.
END06
An END06 nucleic acid of the invention includes the nucleic acid sequence
shown in
Table 27A (SEQ ID N0:22).
Table 27A
1
GGG
81
AGA
ATGTTCCAGTTTCATGCAGGCTCTTGGGAAAGCTGGTGCTGCTGCTGCCTGATTCCCGCCGACAGACCTTGGGACCG
CCAACACTGGCAGCTGGAGATGGCGGACACGAGATCCGTGCACGAGACTAGGTTTGAGGCGGCCGTGAAGGTGATCC
29


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161


GTTTGCCGAAGAATGGTTCATTCCAGCCAACAAATGAAATGATGCTTAAATTTTATAGCTTCTATAAGCAGGCAACT


GAA


241


S
GGACCCTGTAAACTTTCAAGGCCTGGATTTTGGGATCCTATTGGAAGATATAAATGGGATGCTTGGAGTTCACTGGG


TGA


321


TATGACCAAAGAGGAAGCCATGATTGCATATGTTGAAGAAATGAAAAAGATTATTGAAACTATGCCAATGACTGAGA


AAG


401


TTGAAGAATTGCTGCGTGTCATAGGTCCATTTTATGAAATTGTCGAGGACAAAAAGAGTGGCAGGAGTTCTGATATA


ACC


481


TCAGTCCGACTGGAGAAAATCTCTAAATGTTTAGAAGATCTTGGTAATGTTCTCACTTCTACTCCAAACGCCAAAAC


1 CGT
S


561


TAATGGTAAAGCTGAAAGCAGTGACAGTGGAGCGGAGTCTGAGGAAGAAGAGGCCCAAGAAGAAGTGAAAGGAGCAG


AAC


641


2O
ACAGTGATAATGATAAGAAAATGATGAAGAAGTCAGCAGACCATAAGAATTTGGAAGTCATTGTCACTAATGGCTAT


GAT


721


AAAGATGGCTTTGTTCAGGATATACAGAATGACATTCATGCCAGTTCTTCCCTGAATGGCAGAAGCACTGAAGAAGT


AAA


2S 801


GCCCATTGATGAAAACTTGGGGCAAACTGGAAAATCTGCTGTTTGCATTCACCAAGGTATTAATGATGATCATGTTG


AAG


881


ATGTTACAGGAATTCAGCATTTGACAAGCGATTCAGACAGTGAAGTTTACTGTGATTCTATGGAACAATTTGGACAA


3 GAA
0


961


GAGTCTTTAGACAGCTTTACGTCCAACAATGGACCATTTCAGTATTACTTGGGTGGTCATTCCAGTCAACCCATGGA
A


AA
1041


3S
TTCTGGATTTCGTGAAGATATTCAAGTACCTCCTGGAAATGGCAACATTGGGAATATGCAGGTGGTTGCAGTTGAAG


GAA


1121


AAGGTGAAGTCAAGCATGGAGGAGAAGATGGCAGGAATAACAGCGGAGCACCACACCGGGAGAAGCGAGGCGGAGAA


ACT


40 1201


GACGAATTCTCTAATGTTAGAAGAGGAAGAGGTCATAGGATGCAACACTTGAGCGAAGGAACCAAGGGCCGGCAGGT


GGG


1281


AAGTGGAGGTGATGGGGAGCGCTGGGGCTCCGACAGAGGGTCCCGAGGCAGCCTCAATGAGCAGATCGCCCTCGTGC


45 TGA


1361


TGAGACTGCAGGAGGACATGCAGAATGTCCTTCAGAGACTGCAGAAACTGGAAACGCTGACTGCTGCAAAATCATCA


ACA


1441


SO
TCAACATTGCAGACTGCTCCTCAGCCCACCTCATCTCAGAGACCATCTTGGTGGCCCTTCGAGATGTCTCCTGGTGT


GCT


1521 AACGTTTGCCATCATATGGCCTTTTATTGCACAGTGGTTGGTGTATTTATACTATCAAAGAAGGAGAAGGTAA


(SEQ ID N0:22)


SS
The nucleic acid sequence disclosed in Table 27A includes an open reading
frame
("ORF") beginning at position 1 with start and stop codons indicated in bold.
The ORF
encodes a polypeptide sequence of S30 amino acid residues. The sequence of
this encoded
polypeptide (SEQ ID N0:23)is presented in Table 27B. The homology between the
translated


CA 02386199 2002-03-28
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protein and Bovine endozepine (putativee ligand of benzodiazepine receptor)
related protein
(gb:GENBANK-ID:BOVEDZR~acc:M1S888) is presented in Table 27C.
Table 27B
S
1
MFQFHAGSWESWCCCCLIPADRPWDRGQHWQLEMADTRSVHETRFEAAVKVIQSLPKNGSFQPTNEMMLKFYSFYKg
A_TE


81


1O
GPCKLSRPGFWDPIGRYKWDAWSSLGDMTKEEAMIAWEEMKKIIETMPMTEKVEELLRVIGPFYEIVEDKKSGRSS


DIT


161


SVRLEKISKCLEDLGNVLTSTPNAKTVNGKAESSDSGAESEEEEAQEEVKGAEHSDNDKKMMKKSADHKNLEVIVTN


GYD


1S 241


KDGFVQDIQNDIHASSSLNGRSTEEVKPIDENLGQTGKSAVCIHQGINDDHVEDVTGIQHLTSDSDSEVYCDSMEQF


GQE


321


ESLDSFTSNNGPFQYYLGGHSSQPMENSGFREDIQVPPGNGNIGNMQWAVEGKGEVKHGGEDGRNNSGAPHREKRG


20 GET


401


DEFSNVRRGRGHRMQHLSEGTKGRQVGSGGDGERWGSDRGSRGSLNEQIALVLMRLQEDMQNVLQRLQKLETLTAAK


SST


481 STLQTAPQPTSSQRPSWWPFEMSPGVLTFAIIWPFIAQWLVYLYYQRRRR (SEQ
ID N0:23)


2S


Translated Protein - Frame: 1 - Nucleotide 1 to 1590


Table 27C
>gb:GENBANK-ID:BOVEDZRIacc:M15888 Bovine endozepine (putativee ligand of
30 benzodiazepine receptor) related protein mRNA, complete cds - Bos taurus,
1750 bp.
Length = 1750
3S
Plus Strand HSPs:
Score = 2401 (845.2 bits), Expect = 2.9e-248, P = 2.9e-248
Identities = 448/530 (84is), Positives = 481/530 (90%), Frame = +2
Query: 1 MFQFHAGSWESWCCCC-LIPADRPWDRGQHWQLEMADTRSVHETRFEAAVKVIQSLPKNG
40 59
IIIIIIIIIIIIIIII III 1111111+ I+III IIIIIIIIIIIIIIIIIIIIIII
Sbjct: 68 MFQFHAGSWESWCCCCCLIPGDRPWDRGRRWRLEMRHTRSVHETRFEAAVKVIQSLPKNG
247
4S Query: 60 SFQPTNEMMLKFYSFYKQATEGPCKLSRPGFWDPIGRYKWDAWSSLGDMTKEEAMIAYVE
119
IIIIIIIIIIIIIIIIIIIIIIIIIII+111111+Ilillllllllllllllllllllll
Sbjct: 248 SFQPTNEMMLKFYSFYKQATEGPCKLSKPGFWDPVGRYKWDAWSSLGDMTKEEAMIAYVE
427
SO
Query: 120 EMKKIIETMPMTEKVEELLRVIGPFYEIVEDKKSGRSSDITSVRLEKISKCLEDLGNVLT
179
IIIII+IIIIIIIIIIIII IIIIIIIIIIIIIIIIIII+IIIIIIIIIIIIIIIIIII
31


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Sbjct: 428 EMKKILETMPMTEKVEELLHVIGPFYEIVEDKKSGRSSDLTSVRLEKISKCLEDLGNVLA
607
Query: 180 STPNAKTVNGKAESSDSGAESEEEEAQEEVKGAEHSDNDKKMMKKSADHKNLEVIVTNGY
S 239
IIIIIIIIIIIIIIIIIIIIIIiI III+ I I I+IIIIIIIIIIIIIII+111111
SbjCt: 608 STPNAKTVNGKAESSDSGAESEEEAAQEDPKRPEPRDSDKKMMKKSADHKNLEIIVTNGY
787
1O Query: 240 DKDGFVQDIQNDIHASSSLNGRSTEEVKPIDENLGQTGKSAVCIHQGINDDHVEDVTGIQ
299
III III +1I II I IIIII IIIII +1111 IIII+ I +1I +I IIIII++Iil
Sbjct: 788 DKDSFVQGVQNSIHTSPSLNGRCTEEVKSVDENLEQTGKTWFVHQDVNSDHVEDISGIQ
967
1S
Query: 300 HLTSDSDSEVYCDSMEQFGQEESLDSFTSNNGPFQYYLGGHSSQPMENSGFREDIQVPPG
359
IIIIIIIIIIIIIIIIIIIIIIIII I 111111 IIIII+ III+I+III I +I II
Sbjct: 968 HLTSDSDSEVYCDSMEQFGQEESLDGFISNNGPFSYYLGGNPSQPLESSGFPEAVQGLPG
20 1147
Query: 360 NGNIGNMQWAVEGKGEVKHGGEDGRNNSGAPHREKRGGETDEFSNVRRGRGHRMQHLSE
419
II+ +1I IIIIIIII IIIII +IIIIIIIIII II++1111+IIIIIIIIIIIII
2S Sbjct: 1148 NGSPEDMQGAWEGKGEVKRGGEDGGSNSGAPHREKRAGESEEFSNIRRGRGHRMQHLSE
1327
Query: 420 GTKGRQVGSGGDGERWGSDRGSRGSLNEQIALVLMRLQEDMQNVLQRLQKLETLTAAKSS
479
3o I+IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII III I I+++
Sbjct: 1328 GSKGRQVGSGGDGERWGSDRGSRGSLNEQIALVLMRLQEDMQNVLQRLHKLEMLAASQAK
1507
Query: 480 TSTLQTAPQPTSSQRPSWWPFEMSPGVLTFAIIWPFIAQWLVYLYYQRRRR 530
35 +I III+ IIII IIIIIIIIIIII IIIIIIIIIIIIIII+IIIIIIII
Sbjct: 1508 SSALQTSNQPTSP-RPSWWPFEMSPGALTFAIIWPFIAQWLVHLYYQRRRR 1657
An END06 nucleic acid of the invention can include a nucleic acid encoding the
polypeptide of SEQ ID N0:23, e.g., the END06 nucleic acid can include the
nucleic acid
40 sequence of SEQ ID N0:22. The invention also includes a mutant or variant
nucleic acid any
of whose bases may be changed from the corresponding base shown in Table 27A.
In some
embodiments, the END06 nucleic acid encodes a protein that maintains its
endozepine-like
activities and physiological functions, or a fragment of such a nucleic acid.
The invention
further includes nucleic acids whose sequences are complementary to those just
described,
4S including nucleic acid fragments that are complementary to any of the
nucleic acids just
described. The invention additionally includes nucleic acids or nucleic acid
fragments, or
complements thereto, whose structures include chemical modifications. Such
modifications
include, but are not limited to: modified bases, and nucleic acids whose sugar
phosphate
backbones are modified or derivatized. These modifications are carned out at
least in part to
32


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enhance the chemical stability of the modified nucleic acid, such that they
may be used, for
example, as antisense binding nucleic acids in therapeutic applications in a
subject.
An END06 polypeptide of the invention can include the amino acid sequence of
SEQ
ID N0:23. The invention also includes a mutant or variant protein any of whose
residues may
be changed from the corresponding residue shown in SEQ ID N0:23, while still
encoding a
protein that maintains its endozepine-like activities and physiological
functions, or a functional
fragment thereof, such as the active peptide (SEQ ID N0:24)
Metabolism-Regulating Peptide #8 (MRP-8) Sequence:
QATEGPCKLSRPGFWDP (SEQ ID N0:24)
The invention further encompasses antibodies and antibody fragments, such as
Fab or
(Fab)2, that bind immunospecifically to the END06 polypeptide, and derivatives
and fragments,
thereof.
An END06 sequence is useful for detecting specific types of tissue. For
example when
a panel of tissue is assayed for expression, END06 is highly expressed in
skeletal muscle.
Also, high expression of END06 is a marker for multiple types of cancer.
An END06 sequence is also useful to modulate global energy metabolism or
weight
by altering serum cholesterol and insulin.
An END06 sequence is also useful in a method to identity the cellular
receptors and
downstream effectors of the invention by any one of a number of techniques
commonly
employed in the art.
An END06 sequence is useful in the treatment of diabetes, metabolic
disturbances
associated with obesity, the metabolic syndrome X as well as anorexia and
wasting disorders
associated with chronic diseases and various cancers by modulating metabolism.
END07
An END07 nucleic acid of the invention includes the nucleic acid sequence
shown in
Table 27D (SEQ ID N0:25).
33


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Table 27D
1
S
CCAGTATGTCTCAGGCGTTTGAGAAAGCTGCCAAGGATATTAAGCACCTTGAGACCAAGCCAGCAGATGATGAGAGG
ATG
81
CAA
161
1S
TTCATCTACAGCCGCTGCAAACAAGCGACTGTGCATGACTTAAATACAGAATGGCCCAGGATGTTAGACCTCAAAGG
GGCAAAGCAGGATGCTTGGAATGAGCTGAAAGACACTGCCAAGGAAGATGCTGTGAAAGCTGATATCAACAAAGTAG
AAG
241 AGCGAAATAAAAAATACAGAATATAAGAGATTG
(SEQ ID N0:25)
The nucleic acid sequence disclosed in Table 27D includes an open reading
frame
("ORF") beginning at position 6 with start and stop codons indicated in bold.
The ORF
encodes a polypeptide sequence of 86 amino acid residues. The sequence of this
encoded
polypeptide is presented in Table 27E (SEQ ID N0:26). The homology between the
translated
protein and Bovine endozepine (putative ligand of benzodiazepine receptor)
(gb:GENBANK-
ID:BOVEDZ~acc:M15886) is presented in Table 27F.
Table 27E
2S
1
MSQAFEKAAKDIKHLETKPADDERMFIYSRCKQATVHDLNTEWPRMLDLKGKAKQDAWNELKDTAKEDAVKADINKV
EER
81 NKKYRI (SEQ ID N0:26)
Table 27F
>gb:GENBANK-ID:BOVEDZ~acc:M15886 Bovine endozepine (putative ligand of
benzodiazepine receptor) mRNA, complete cds - Bos taurus, 608 bp.
3S Length = 608
Plus Strand HSPs:
Score = 307 (108.1 bits), Expect = 6.5e-26, P = 6.5e-26
Identities = 62/87 (71~), Positives = 73/87 (83~), Frame = +2
Query: 1 MSQA-FEKAAKDIKHLETKPADDERMFIYSRCKQATVHDLNTEWPRMLDLKGKAKQDAWN
59
4S Sbjct: 125 MSQAEFDKAAEEVKHLKTKPADEEMLFIYSHYKQATVGDINTERPGMLDFKGKAKWDAWN
304
Query: 60 ELKDTAKEDAVKADINKVEERNKKYRI 86
~~~ ~+~~~~+~~ ~+~~~~
34


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SbjCt: 305 ELKGTSKEDAMKAYIDKVEELKKKYGI 385
An END07 nucleic acid of the invention can include a nucleic acid encoding the
polypeptide of SEQ ID N0:26, e.g., the END07 nucleic acid can include the
nucleic acid
sequence of SEQ ID N0:25. The invention also includes a mutant or variant
nucleic acid any
of whose bases may be changed from the corresponding base shown in Table 27D.
In some
embodiments, the END07 nucleic acid encodes a protein that maintains its
endozepine-like
activities and physiological functions, or a fragment of such a nucleic acid.
The invention
further includes nucleic acids whose sequences are complementary to those just
described,
including nucleic acid fragments that are complementary to any of the nucleic
acids just
described. The invention additionally includes nucleic acids or nucleic acid
fragments, or
complements thereto, whose structures include chemical modifications. Such
modifications
include, but are not limited to: modified bases, and nucleic acids whose sugar
phosphate
backbones are modified or derivatized. These modifications are carried out at
least in part to
enhance the chemical stability of the modified nucleic acid, such that they
may be used, for
example, as antisense binding nucleic acids in therapeutic applications in a
subject.
An END07 polypeptide of the invention can include the amino acid sequence of
SEQ
ID N0:26. The invention also includes a mutant or variant protein any of whose
residues may
be changed from the corresponding residue shown in SEQ ID N0:26, while still
encoding a
protein that maintains its endozepine-like activities and physiological
functions, or a functional
fragment thereof such as the following active peptide (SEQ ID N0:27)
Metabolism-Regulating Peptide #9 (MRP-9) Sequence:
QATVHDLNTEWPRMLDLKGK (SEQ ID N0:27)
The invention further encompasses antibodies and antibody fragments, such as
Fab or
(Fab)2, that bind immunospecifically to the END07 polypeptide, and derivatives
and fragments,
thereof.
An END07 sequence is useful for detecting specific types of tissue. For
example when
a panel of tissue is assayed for expression, END07 is highly expressed in
adipose tissue. Also,
high expression of END07 is a marker for liver cancer.


CA 02386199 2002-03-28
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An END07 sequence is also useful to modulate global energy metabolism or
weight
by altering serum cholesterol.
An END07 sequence is also useful in a method to identity the cellular
receptors and
downstream effectors of the invention by any one of a number of techniques
commonly
S employed in the art.
An END07 sequence is useful in the treatment of diabetes, metabolic
disturbances
associated with obesity, the metabolic syndrome X as well as anorexia and
wasting disorders
associated with chronic diseases and various cancers by modulating metabolism.
END08
An END08 nucleic acid of the invention includes the nucleic acid sequence
shown in
Table 27G (SEQ ID N0:28).
Table 27G
1S
1
ATGTGGGGCGACCTCTGGCTCCTCCCGCCTGCCTCTGCCAATCCGGGCACTGGGACAGAGGCTGAGTTTGAGAAAGC
TGC
81
2O
AGAGGAGGTTAGGCACCTTAAGACCAAGCCATCGGATGAGGAGATGCTGTTCATCTATGGCCACTACAAACAAGCAA
CTG
161
TGGGCGACATAAATACAGAACGGCCCGGGATGTTGGACTTCACGGGCAAGGCCAAGTGGGATGCCTGGAATGAGCTG
AAA
2S 241
GGGACTTCCAAGGAAGATGCCATGAAAGCTTACATCAACAAAGTAGAAGAGCTAAAGAAAAAATACGGGATATGA
(SEQ ID N0:28)
The nucleic acid sequence disclosed in Table 27G includes an open reading
frame
30 ("ORF") beginning at position 1 with start and stop codons in bold. The ORF
encodes a
3S
polypeptide sequence of 104 amino acid residues. The sequence of this encoded
polypeptide
is presented in Table 27H (SEQ ID N0:29). The homology between the translated
protein and
Human diazepam binding inhibitor (DBI) (gb:GENBANK-ID:HUMDBI~acc:M14200) is
presented in Table 27I.
Table 27H
36


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1
MWGDLWLLPPASANPGTGTEAEFEKAAEEVRHLKTKPSDEEMLFIYGHYKQATVGDINTERPGMLDFTGKAKWDAWN
ELK
81 GTSKEDAMKAYINKVEELKKKYGI
$ (SEQ ID N0:29)
Table 27I
>gb:GENBANK-ID:HUMDBIIacc:M14200 Human diazepam binding inhibitor (DBI)
mRNA, complete cds - Homo sapiens, 556 bp.
Length = 556
Plus Strand HSPs:
1$ Score = 562 (197.8 bits), Expect = 6.7e-53, P = 6.7e-53
Identities = 104/104 (100%), Positives = 104/104 (100%), Frame = +2
Query: 1 MWGDLWLLPPASANPGTGTEAEFEKAAEEVRHLKTKPSDEEMLFIYGHYKQATVGDINTE
20 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Sbjct: 20 MWGDLWLLPPASANPGTGTEAEFEKAAEEVRHLKTKPSDEEMLFIYGHYKQATVGDINTE
199
Query: 61 RPGMLDFTGKAKWDAWNELKGTSKEDAMKAYINKVEELKKKYGI 104
2s IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIillllllllll
Sbjct: 200 RPGMLDFTGKAKWDAWNELKGTSKEDAMKAYINKVEELKKKYGI 331
An END08 nucleic acid of the invention can include a nucleic acid encoding the
polypeptide of SEQ ID N0:29, e.g., the END08 nucleic acid can include the
nucleic acid
30 sequence of SEQ ID N0:28. The invention also includes a mutant or variant
nucleic acid any
of whose bases may be changed from the corresponding base shown in Table 27G.
In some
embodiments, the END08 nucleic acid encodes a protein that maintains its
endozepine-like
activities and physiological functions, or a fragment of such a nucleic acid.
The invention
further includes nucleic acids whose sequences are complementary to those just
described,
3$ including nucleic acid fragments that are complementary to any of the
nucleic acids just
described. The invention additionally includes nucleic acids or nucleic acid
fragments, or
complements thereto, whose structures include chemical modifications. Such
modifications
include, but are not limited to: modified bases, and nucleic acids whose sugar
phosphate
backbones are modified or derivatized. These modifications are carned out at
least in part to
40 enhance the chemical stability of the modified nucleic acid, such that they
may be used, for
example, as antisense binding nucleic acids in therapeutic applications in a
subject.
An END08 polypeptide of the invention can include the amino acid sequence of
SEQ
ID N0:29. The invention also includes a mutant or variant protein any of whose
residues may
be changed from the corresponding residue shown in SEQ ID N0:29, while still
encoding a
37


CA 02386199 2002-03-28
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protein that maintains its endozepine-like activities and physiological
functions, or a functional
fragment thereof such as the following active peptide (SEQ ID N0:30)
Metabolism-Regulating Peptide #1 (MRP-1) Sequence:
QATVGDINTERPGMLDFTGK
(SEQ ID N0:30)
The invention further encompasses antibodies and antibody fragments, such as
Fab or
(Fdb)2, that bind immunospecifically to the END08 polypeptide, and derivatives
and fragments,
thereof.
An END08 sequence is useful for detecting specific types of tissue. For
example when
a panel of tissue is assayed for expression, END08 is highly expressed in
heart skeletal
muscle, liver and endothelial tissue. Also, high expression of END08 is a
marker for breast
and colon cancers as well as melanoma.
An END08 sequence is also useful to modulate global energy metabolism or
weight
by altering serum cholesterol, insulin, or glucose. The END08 sequence is also
useful to
modulate muscle mass or adipose level.
An END08 sequence is also useful in a method to identity the cellular
receptors and
downstream effectors of the invention by any one of a number of techniques
commonly
employed in the art.
An END08 sequence is useful in the treatment of diabetes, metabolic
disturbances
associated with obesity, the metabolic syndrome X as well as anorexia and
wasting disorders
associated with chronic diseases and various cancers by modulating metabolism.
END09
An END09 nucleic acid of the invention includes the nucleic acid sequence
shown in
Table 27J (SEQ ID N0:31).
Table 27J
38


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WO 01/25436 PCT/US00/41077
ATGAGAGCCAGTCAGAAGGACTTTGAAAATTCAATGAATCAAGTGAAACTCTTGAAAAAGGATCCAGGAAACGAAGT
GAA


81


S
GCTAAAACTCTACGCGCTATATAAGCAGGCCACTGAAGGACCTTGTAACATGCCCAAACCAGGTGTATTTGACTTGA


TCA


161


ACAAGGCCAAATGGGACGCATGGAATGCCCTTGGCAGCCTGCCCAAGGAAGCTGCCAGGCAGAACTATGTGGATTTG


GTG


241


TCCAGTTTGAGTCCTTCATTGGAATCCTCTAGTCAGGTGGAGCCTGGAACAGACAGGAAATCAACTGGGTTTGAAAC


TCT


321


GGTGGTGACCTCCGAAGATGGCATCACAAAGATCATGTTCAACCGGCCCAAAAAGAAAAATGCCATAAACACTGAGA


IS TGT


401


ATCATGAAATTATGCGTGCACTTAAAGCTGCCAGCAAGGATGACTCAATCATCACTGTTTTAACAGGAAATGGTGAC


TAT


481


2O
TACAGTAGTGGGAATGATCTGACTAACTTCACTGATATTCCCCCTGGTGGAGTAGAGGAGAAAGCTAAAAATAATGC


CGT


561


TTTACTGAGGGAATTTGTGGGCTGTTTTATAGATTTTCCTAAGCCTCTGATTGCAGTGGTCAATGGTCCAGCTGTGG


GCA


25 641


TCTCCGTCACCCTCCTTGGGCTATTCGATGCCGTGTATGCATCTGACAGGGCAACATTTCATACACCATTTAGTCAC


CTA


721


GGCCAAAGTCCGGAAGGATGCTCCTCTTACACTTTTCCGAAGATAATGAGCCCAGCCAAGGCAACAGAGATGCTTAT


30 TTT


801


TGGAAAGAAGTTAACAGCGGGAGAGGCATGTGCTCAAGGACTTGTTACTGAAGTTTTCCCTGATAGCACTTTTCAGA


AAG


881


3S
AAGTCTGGACCAGGCTGAAGGCATTTGCAAAGCTTCCCCCAAATGCCTTGAGAATTTCAAAAGAGGTAATCAGGAAA


AGA


961


GAGAGAGAAAAACTACACGCTGTTAATGCTGAAGAATGCAATGTCCTTCAGGGAAGATGGCTATCAGATGAATGCAC


AAA


4O 1041 TGCTGTGGTGAACTTCTTATCCAGAAAATCAAAACTGTGA


(SEQ ID N0:31)


The nucleic acid sequence disclosed in Table 27J includes an open reading
frame
4S ("ORF") beginning at position 1 with start and stop codons in bold. The ORF
encodes a
polypeptide sequence of 3S9 amino acid residues. The sequence of this encoded
polypeptide
is presented in Table 27K (SEQ ID N0:32). The homology between the translated
protein and
Homo sapiens peroxisomal D3,D2-enoyl-CoA isomerase (PECI) (gb:GENBANK-
ID:AF153612~acc:AF1S3612) is presented in Table 27L.
SO
Table 27K
39


CA 02386199 2002-03-28
DLV
81
GDY
161
SHL
241
RKR
WO 01/25436 PCT/US00/41077
MRASQKDFENSMNQVKLLKKDPGNEVKLKLYALYKQATEGPCNMPKPGVFDLINKAKWDAWNALGSLPKEAARQNW
SSLSPSLESSSQVEPGTDRKSTGFETLWTSEDGITKIMFNRPKKKNAINTEMYHEIMRALKAASKDDSIITVLTGN
YSSGNDLTNFTDIPPGGVEEKAKNNAVLLREFVGCFIDFPKPLIAWNGPAVGISVTLLGLFDAVYASDRATFHTPF
GQSPEGCSSYTFPKIMSPAKATEMLIFGKKLTAGEACAQGLVTEVFPDSTFQKEWTRLKAFAKLPPNALRISKEVI
321 EREKLHAVNAEECNVLQGRWLSDECTNAVVNFLSRKSKL
(SEQ ID N0:32)
IS


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Table 27L
>gb:GENBANK-ID:AF153612~acc:AF153612 Homo Sapiens peroxisomal
D3,D2-enoyl-CoA isomerase (PECI) mRNA, complete cds - Homo
sapiens, 1348 bp.
Length = 1348
Plus Strand HSPs:
Score = 1857 (653.7 bits), Expect = 1.6e-190, P = 1.6e-190
Identities = 359/359 (1000 , Positives = 359/359 (1000 , Frame = +1
Query: 1 MRASQKDFENSMNQVKLLKKDPGNEVKLKLYALYKQATEGPCNMPKPGVFDLINKAKWDA
IIIIIIIIIIIII~IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IS Sbjct: 103 MRASQKDFENSMNQVKLLKKDPGNEVKLKLYALYKQATEGPCNMPKPGVFDLINKAKWDA
282
Query: 61 WNALGSLPKEAARQNYVDLVSSLSPSLESSSQVEPGTDRKSTGFETLWTSEDGITKIMF
120
20 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Sbjct: 283 WNALGSLPKEAARQNYVDLVSSLSPSLESSSQVEPGTDRKSTGFETLWTSEDGITKIMF
462
Query: 121 NRPKKKNAINTEMYHEIMRALKAASKDDSIITVLTGNGDYYSSGNDLTNFTDIPPGGVEE
25 180
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Sbjct: 463 NRPKKKNAINTEMYHEIMRALKAASKDDSIITVLTGNGDYYSSGNDLTNFTDIPPGGVEE
642
3O Query: 181 KAKNNAVLLREFVGCFIDFPKPLIAVVNGPAVGISVTLLGLFDAVYASDRATFHTPFSHL


240
III
I
I
IIIIIIII


SbjCt: 643 IIII~IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
I
I
I
KAKNNAVLLREFVGCFIDFPKPLIAVVNGPAVGISVTLLGLFDAVYASDRATFHTPFSHL


822


35


Query: 241 GQSPEGCSSYTFPKIMSPAKATEMLIFGKKLTAGEACAQGLVTEVFPDSTFQKEVWTRLK


300
llllllllllllllllll
~l


SbjCt: 823 IIIIIIIillllllllllllllllllllllllllllll
1l
GQSPEGCSSYTFPKIMSPAKATEMLIFGKKLTAGEACAQGLVTEVFPDSTFQKEVWTRLK


40 loot


Query: 301 AFAKLPPNALRISKEVIRKREREKLHAVNAEECNVLQGRWLSDECTNAVVNFLSRICSKL
359
IIIIiillllllillllllllllllllllliillllllllllilllllllllllllill
4S Sbjct: 1003 AFAKLPPNALRISKEVIRKREREKLHAVNAEECNVLQGRWLSDECTNAWNFLSRKSKL
1179
An END09 nucleic acid of the invention can include a nucleic acid encoding the
polypeptide of SEQ ID N0:32, e.g., the END09 nucleic acid can include the
nucleic acid
50 sequence of SEQ ID N0:31. The invention also includes a mutant or variant
nucleic acid any
of whose bases may be changed from the corresponding base shown in Table 27J.
In some
embodiments, the END09 nucleic acid encodes a protein that maintains its
endozepine-like
activities and physiological functions, or a fragment of such a nucleic acid.
The invention
further includes nucleic acids whose sequences are complementary to those just
described,
41


CA 02386199 2002-03-28
WO 01/25436 PCT/US00/41077
including nucleic acid fragments that are complementary to any of the nucleic
acids just
described. The invention additionally includes nucleic acids or nucleic acid
fragments, or
complements thereto, whose structures include chemical modifications. Such
modifications
include, but are not limited to: modified bases, and nucleic acids whose sugar
phosphate
backbones are modified or derivatized. These modifications are carried out at
least in part to
enhance the chemical stability of the modified nucleic acid, such that they
may be used, for
example, as antisense binding nucleic acids in therapeutic applications in a
subject.
An END09 polypeptide of the invention can include the amino acid sequence of
SEQ
ID N0:32. The invention also includes a mutant or variant protein any of whose
residues may
be changed from the corresponding residue shown in SEQ ID N0:32, while still
encoding a
protein that maintains its endozepine-like activities and physiological
functions, or a functional
fragment thereof such as the following active peptide (SEQ ID N0:33)
Metabolism-Regulating Peptide #2 (MRP-2).Sequence:
QATEGPCNMPKPGVFDLINK
(SEQ ID N0:33)
The invention further encompasses antibodies and antibody fragments, such as
Fab or
(Fab)2, that bind immunospecifically to the END09 polypeptide, and derivatives
and fragments,
thereof.
An END09 sequence is also useful to modulate global energy metabolism or
weight
by altering serum cholesterol, insulin, or glucose.
An END09 sequence is also useful in a method to identity the cellular
receptors and
downstream effectors of the invention by any one of a number of techniques
commonly
employed in the art.
An END09 sequence is useful in the treatment of diabetes, metabolic
disturbances
associated with obesity, the metabolic syndrome X as well as anorexia and
wasting disorders
associated with chronic diseases and various cancers by modulating metabolism.
42


CA 02386199 2002-03-28
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ENDO10
An ENDO10 nucleic acid of the invention includes the nucleic acid sequence
shown in
Table 27M (SEQ ID N0:34).
Table 27M
S
1
TCCTTCCCCCACCCCCGGGGGCCCATCCCGGTGGCGGGCTCCGGAGCTCGGGACTGCTAATTTCAGCGAAACGATTA
AAA


1O 81


GACGCCCCTACAGCTGACGGCACTTTCTCTCCTCCGGCAGGANAGGACGTCCAGCGTACGTCNGCCCGCGCTTCCCC


GCC


161


GGCGCAGAGCAGGCCTCACAGAATCGCACGCCGCTGGCACGCACGCCGCCCCGCCCCCACGGCCCAGCGCCAGCGCG


IS CCC


241


CGCGTCGCACGCATCCCGGCCTCACTGCCCCTCGACTCCTGTTCCGTTGGAGGGGCCTGAGGCGAGCCTGAGCGCGC


TGT


321


2O
TGGCCGGAGGAAGCCGGAGAGACCGGGTCGACTGGGCAGAGCGGCAGAGGGTCGAGGAGCCTGCTCTGCACGCCCAG


GGA


401


GTAGAAGTGGGCAGGGAGCAGGGTCACGTGAGGGAGCGCGCCGCGACTGAGCTTGGGTCCGACTGGAGCTCAGGCTC


GCG


2S 481


ACCCAGACTGGTGGGCCAGGCCTCCAAGCCGGCCTTACACCCAATCCAAGGAGGACAGACCGGACACAGAGGGACGG


AGC


561


GAGCAAGGAGACATGGCTTCATCATTCCTGCCCGCGGGGGCCATCACCGGCGACAGCGGTGGAGAGCTGAGCTCAGG


3O _GGA


641


CGACTCCGGGGAGGTGGAGTTCCCCCATAGCCCTGAGATCGAGGAGACCAGTTGCCTGGCCGAGCTGTTTGAGAAGG
CTG
721
3S
CCGCGCACCTGCAAGGCCTGATTCAGGTGGCCAGCAGGGAGCAGCTCTTGTACCTGTATGCCAGGTACAAACAGGTC
AAA
801
TGG
4O 881
TAC
961
4S AGG
1041
GTTGGAAATTGTAATACTCCTAAACCAAGCTTCTTTGATTTTGAAGGAAAGCAAAAATGGGAAGCTTGGAAAGCACT
TGATTCAAGCCCCAGCCAAGCAATGCAGGAATATATCGCAGTAGTTAAAAAACTAGATCCAGGTTGGAATCCTCAGA
CAGAGAAGAAAGGAAAAGAAGCAAATACAGGTTTTGGTGGGCCAGTTATTAGTTCTCTATATCATGAAGAAACCATC
GAAGAAGACAAAAATATATTTGATTACTGCAGGGAAAACAACATTGACCATATAACCAAAGCCATCAAATCGAAAAA
TGT
1121
SO
GGATGTGAATGTGAAAGATGAAGAGGGTAGGGCTCTACTTCACTGGGCCTGTGATCGAGGACATAAGGAACTAGTCA
CAG
1201
GAG
SS 1281
GGA
1361
TGTTGCTGCAACATAGAGCTGACATTAACTGTCAGGACAATGAAGGCCAAACAGCTCTACATTATGCCTCTGCCTGT
TTTCTGGATATTGTAGAGCTGCTGCTCCAGTCTGGTGCTGACCCCACTCTCCGAGACCAGGATGGCTGCCTGCCAGA
GGTGACAGGCTGCAAAACAGTTTCTTTGGTGCTGCAGCGGCACACAACTGGCAAGGCTTAATCAAAAGACTGGAAAA
C)O CTG
43


CA 02386199 2002-03-28
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1441
CAGTCTGTAATAGCATAAGGCTTCCATTATGAAAGAAAACTACAAAAATAATACTTCTTTTCCACCCGTCTTTGGTA
TGT
1521 ATTGGCTAATAAAATCAGTTCTGTGGAACTGGG
(SEQ ID N0:34)
The nucleic acid sequence disclosed in Table 27M includes an open reading
frame
("ORF") beginning at position 573 with start and stop codons in bold. The ORF
encodes a
polypeptide sequence of 282 amino acid residues. The sequence of this encoded
polypeptide
is presented in Table 27N (SEQ ID N0:35). The homology between the translated
protein
and(Human DBI/ACBP-like protein Patent: US 5734038-A 2 31-MAR-1998) is
presented in
Table 270.
Table 27N
1
_GNC
al
EDK
161
MASSFLPAGAITGDSGGELSSGDDSGEVEFPHSPEIEETSCLAELFEKAAAHLQGLIQVASREQLLYLYARYK VKV
NTPKPSFFDFEGKQKWEAWKALGDSSPSQAMQEYIAWKKLDPGWNPQIPEKKGKEANTGFGGPVISSLYHEETIRE
NIFDYCRENNIDHITKAIKSKNVDVNVKDEEGRALLHWACDRGHKELVTVLLQHRADINCQDNEGQTALHYASACEF
LDI
241 VELLLQSGADPTLRDQDGCLPEEVTGCKTVSLVLQRHTTGKA
(SEQ ID N0:35)
Table 270
35
>gb:GENBANK-ID:I96163~acc:I96163 Sequence 2 from patent US 5734038 -
Unknown., 1123 bp.
Length = 1123
Plus Strand HSPs:
Score = 1340 (471.7 bits), Expect = 1.2e-135, P = 1.2e-135
Identities = 259/282 (91~), Positives = 262/282 (92~), Frame = +1
Query: 1 MASSFLPAGAITGDSGGELSSGDDSGEVEFPHSPEIEETSCLAELFEKAAAHLQGLIQVA
60
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIilill
Sbjct: 121 MASSFLPAGAITGDSGGELSSGDDSGEVEFPHSPEIEETSCLAELFEKAAAHLQGLIQVA
300
4S Query: 61 SREQLLYLYARYKQVKVGNCNTPKPSFFDFEGKQKWEAWKALGDSSPSQAMQEYIAWKK
120
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Sbjct: 301 SREQLLYLYP.RYKQVKVGNCNTPKPSFFDFEGKQKWEAWKALGDSSPSQAMQEYIAWKK
480
44


CA 02386199 2002-03-28
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Query: 121 LDPGWNPQIPEKKGKEANTGFGGPVISSLYHEETIREEDKNIFDYCRENNIDHITKAIKS
180
Iilllllllllll I + I+ + IIIIIIIIIIIIII~II~IIII
S SbjCt: 481 LDPGWNPQIPEKKRKRSKYKVWASY*FSIS*RNH-QGRDKNIFDYCRENNIDHITKAIKS
657
Query: 181 KNVDVNVKDEEGRALLHWACDRGHKELVTVLLQHRADINCQDNEGQTALHYASACEFLDI
240
to IIIIII~IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII~IIIIIIIIIIIIIII
Sbjct: 658 KNVDVNVKDEEGRALLHWACDRGHKELVTVLLQHRADINCQDNEGQTALHYASACEFLDI
837
Query: 241 VELLLQSGADPTLRDQDGCLPEEVTGCKTVSLVLQRHTTGKA 282
15 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
SbjCt: 838 VELLLQSGADPTLRDQDGCLPEEVTGCKTVSLVLQRHTTGKA 963
An ENDO10 nucleic acid of the invention can include a nucleic acid encoding
the
polypeptide of SEQ ID N0:35, e.g., the ENDO10 nucleic acid can include the
nucleic acid
20 sequence of SEQ ID N0:34. The invention also includes a mutant or variant
nucleic acid any
of whose bases may be changed from the corresponding base shown in Table 27M.
In some
embodiments, the ENDO10 nucleic acid encodes a protein that maintains its
endozepine-like
activities and physiological functions, or a fragment of such a nucleic acid.
The invention
further includes nucleic acids whose sequences are complementary to those just
described,
25 including nucleic acid fragments that are complementary to any of the
nucleic acids just
described. The invention additionally includes nucleic acids or nucleic acid
fragments, or
complements thereto, whose structures include chemical modifications. Such
modifications
include, but are not limited to: modified bases, and nucleic acids whose sugar
phosphate
backbones are modified or derivatized. These modifications are carried out at
least in part to
30 enhance the chemical stability of the modified nucleic acid, such that they
may be used, for
example, as antisense binding nucleic acids in therapeutic applications in a
subject.
An ENDO10 polypeptide of the invention can include the amino acid sequence of
SEQ
ID N0:35. The invention also includes a mutant or variant protein any of whose
residues may
be changed from the corresponding residue shown in SEQ ID N0:35, while still
encoding a
35 protein that maintains its endozepine-like activities and physiological
functions, or a functional
fragment thereof such as the following active peptide (SEQ ID N0:36)
Metabolism-Regulating Peptide #10 (MRP-10) Sequence:
1 QVKVGNCNTPKPSFFDFEGK
40 (SEQ ID N0:36)


CA 02386199 2002-03-28
WO 01/25436 PCT/US00/41077
The invention further encompasses antibodies and antibody fragments, such as
Fab or
(Fab)a, that bind immunospecifically to the ENDO10 polypeptide, and
derivatives and
fragments, thereof.
An ENDO10 sequence is also useful to modulate global energy metabolism or
weight
by altering serum insulin or glucose. The ENDO10 sequence is also useful to
modulate muscle
mass or adipose level
An ENDO10 sequence is also useful in a method to identity the cellular
receptors and
downstream effectors of the invention by any one of a number of techniques
commonly
employed in the art.
An ENDO10 sequence is useful in the treatment of diabetes, metabolic
disturbances
associated with obesity, the metabolic syndrome X as well as anorexia and
wasting disorders
associated with chronic diseases and various cancers by modulating metabolism.
ENDOX nucleic acids encode polypeptides that are novel members of the
endozepine
family. The endozepines have been reported to be involved in multiple
biologically importatnt
functions. Related endozepine polypeptides, such as diazepam binding
inhibitor, bind GABA
receptors. Accordingly, the new ENDOX polypeptides, or fragments or variants
thereof, can
be used in, e.g., screening assays to identify new agonists or antagonists for
GABA receptors.
The new ENDOX polypeptides and also be used to modulate GABA receptor
activity.
ENDOX polypeptides, nucleic acids, antibodies, and other compositions
according to
the invention also have utilities based on other known functions of endozepine
family
members. For example, diazepam binding inhibitor, is also known as acyl-CoA
binding
protein (ACBP). Acyl-CoA-binding protein binds to medium- and long-chain acyl-
CoA esters
with high affinity, and may act as an intra-cellular Garner of acyl-CoA
esters. Thus,
The ACBP gene has also been cloned in yeast. The yeast cognate is named acyl-
CoA
binding (ACB) (Rose et al., Proc. Nat. Acad. Sci. (USA) 89: 11287-11291). The
yeast gene
encodes a polypeptide of 87 amino acid residues (including the initiating
methionine), which
is identical in length to the human gene product. The yeast polypeptide is 48%
conserved
with human amino acid residues. The most highly conserved yeast domain was
found to
comprise a total of 7 contiguous amino acid residues which are identical in
all known protein
species from yeast, birds, and mammals. This domain constitutes the
hydrophobic binding site
46


CA 02386199 2002-03-28
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for acyl-CoA esters, and is located within the second helical region of the
molecule. The
presence of such a highly conserved gene in primitive organisms (e.g., yeast)
supports its basic
biological role as an acyl-CoA binding protein and also suggests that many of
the biological
functions attributed to it in higher organisms may result from its ability to
interact with acyl-
CoA.
Various endozepine family members, or derivatives of these polypeptides, have
also
been identified as antibacterial peptides. Examples include cecropin P1 and PR-
39. PR-39 is
a 39 amino acid residue proline- and arginine-rich polypeptide isolated from
the upper part of
pig small intestine. Amino acid sequence analysis in combination with mass
spectrometry
identified two of three of the the peptides as gastric inhibitory polypeptide
(7-42) (GIP(7-42))
and diazepam-binding inhibitor (32-86) (DBI(32-86)), derived from factors
which had been
previously identified. The third polypeptide constituted a previously-unknown
structure,
which was designated peptide 3910, in relation to its molecular mass. All
three polypeptides
demonstrate antibacterial activity against Bacillus megaterium. GIP (7-42)
also shows some
activity against Streptococcus pyogenes and an Escherichia coli mutant with a
defect in its
outer membrane.
A summary of the ENDOX nucleic acid sequences, encoded ENDOX polypeptides, as
well as Sequence Identifier Numbers (SEQ ID NOS) corresponding to various
disclosed
sequences and clones containing these nucleic acids, is shown in Table 28,
below.
Table 28: Disclosed Sequences and Corresponding SEQ ID Numbers of ENDOX
Polypeptides
47


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CA 02386199 2002-03-28
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Sequence ORF start Sequence Sequence
ENDOX Identifier and Identifier Identifier
Number of stop codonsNumber Number of
Disclosed from of Encoded Active
Nucleic correspondingPolypeptide Peptide
Acid Sequencenucleic Sequence
acid


ENDO1 SEQ ID NO:1 1-267 SEQ ID N0:2 SEQ ID NO:15
SEQ ID NO: 1-687 SEQ ID N0:47
46 1-576 SEQ ID N0:49
SEQ ID N0:48


END02 SEQ ID N0:3 58-321 SEQ ID N0:4 SEQ ID N0:16


END03 SEQ ID NO:S 83-496 SEQ ID N0:6 SEQ ID N0:17,18


END04 SEQ ID N0:7 11-298 SEQ ID N0:8 SEQ ID N0:19,20


ENDOS SEQ ID N0:9 7-264 SEQ ID NO:10 SEQ ID N0:21


END06 SEQ ID N0:22 1-1590 SEQ ID N0:23 SEQ ID N0:24


END07 SEQ ID N0:25 6-263 SEQ ID N0:26 SEQ ID N0:27


END08 SEQ ID N0:28 1-312 SEQ ID N0:29 SEQ ID N0:30


END09 SEQ ID N0:31 1-1077 SEQ ID N0:32 SEQ ID N0:33


ENDO10 SEQ ID N0:34 573-1418 SEQ ID N0:35 SEQ ID N0:36


ENDOX Nucleic Acids and Polypeptides
One aspect of the invention pertains to isolated nucleic acid molecules that
encode
ENDOX polypeptides or biologically-active portions thereof. Also included in
the invention
are nucleic acid fragments sufficient for use as hybridization probes to
identify ENDOX-
encoding nucleic acids (e.g., ENDOX mRNAs) and fragments for use as PCR
primers for the
amplification and/or mutation of ENDOX nucleic acid molecules. As used herein,
the term
"nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or
genomic
DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using
nucleotide analogs, and derivatives, fragments and homologs thereof. The
nucleic acid
molecule may be single-stranded or double-stranded, but preferably is
comprised double-
stranded DNA.
An ENDOX nucleic acid can encode a mature ENDOX polypeptide. As used herein, a
"mature" form of a polypeptide or protein disclosed in the present invention
is the product of a
naturally occurring polypeptide or precursor form or proprotein. The naturally
occurring
polypeptide, precursor or proprotein includes, by way of nonlimiting example,
the full length
gene product, encoded by the corresponding gene. Alternatively, it may be
defined as the
polypeptide, precursor or proprotein encoded by an open reading frame
described herein. The
49


CA 02386199 2002-03-28
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product "mature" form arises, again by way of nonlimiting example, as a result
of one or more
naturally occurring processing steps as they may take place within the cell,
or host cell, in
which the gene product arises. Examples of such processing steps leading to a
"mature" form
of a polypeptide or protein include the cleavage of the N-terminal methionine
residue encoded
by the initiation codon of an open reading frame, or the proteolytic cleavage
of a signal
peptide or leader sequence. Thus a mature form arising from a precursor
polypeptide or
protein that has residues 1 to N, where residue 1 is the N-terminal
methionine, would have
residues 2 through N remaining after removal of the N-terminal methionine.
Alternatively, a
mature form arising from a precursor polypeptide or protein having residues 1
to N, in which
an N-terminal signal sequence from residue 1 to residue M is cleaved, would
have the residues
from residue M+1 to residue N remaining. Further as used herein, a "mature"
form of a
polypeptide or protein may arise from a step of post-translational
modification other than a
proteolytic cleavage event. Such additional processes include, by way of non-
limiting
example, glycosylation, myristoylation or phosphorylation. In general, a
mature polypeptide
or protein may result from the operation of only one of these processes, or a
combination of
any of them.
The term "probes", as utilized herein, refers to nucleic acid sequences of
variable
length, preferably between at least about 10 nucleotides (nt), 100 nt, or as
many as
approximately, e.g., 6,000 nt, depending upon the specific use. Probes are
used in the
detection of identical, similar, or complementary nucleic acid sequences.
Longer length
probes are generally obtained from a natural or recombinant source, are highly
specific, and
much slower to hybridize than shorter-length oligomer probes. Probes may be
single- or
double-stranded and designed to have specificity in PCR, membrane-based
hybridization
technologies, or ELISA-like technologies.
The term "isolated" nucleic acid molecule, as utilized herein, is one which is
separated
from other nucleic acid molecules which are present in the natural source of
the nucleic acid.
Preferably, an "isolated" nucleic acid is free of sequences which naturally
flank the nucleic
acid (i.e., sequences located at the 5'- and 3'-termini of the nucleic acid)
in the genomic DNA
of the organism from which the nucleic acid is derived. For example, in
various embodiments,
the isolated ENDOX nucleic acid molecules can contain less than about 5 kb, 4
kb, 3 kb, 2 kb,
1 kb,
0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic
acid molecule in
genomic DNA of the cell/tissue from which the nucleic acid is derived (e.g.,
brain, heart, liver,
spleen, etc.). Moreover, an "isolated" nucleic acid molecule, such as a cDNA
molecule, can


CA 02386199 2002-03-28
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be substantially free of other cellular material or culture medium when
produced by
recombinant techniques, or of chemical precursors or other chemicals when
chemically
synthesized.
A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having
the
S nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46, and
48, or a
complement of this aforementioned nucleotide sequence, can be isolated using
standard
molecular biology techniques and the sequence information provided herein.
Using all or a
portion of the nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 22, 25, 28,
31, 34, 46, and 48
as a hybridization probe, ENDOX molecules can be isolated using standard
hybridization and
cloning techniques (e.g., as described in Sambrook, et al., (eds.), MOLECULAR
CLONING: A
LABORATORY MANUAL 2"d Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor,
NY, 1989; and Ausubel, et al., (eds.), CURRENT PROTOCOLS 11~ MOLECULAR
Blol.oGY, John
Wiley & Sons, New York, NY, 1993.)
A nucleic acid of the invention can be amplified using cDNA, mRNA or
alternatively,
genomic DNA, as a template and appropriate oligonucleotide primers according
to standard
PCR amplification techniques. The nucleic acid so amplified can be cloned into
an
appropriate vector and characterized by DNA sequence analysis. Furthermore,
oligonucleotides corresponding to ENDOX nucleotide sequences can be prepared
by standard
synthetic techniques, e.g., using an automated DNA synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked
nucleotide
residues, which oligonucleotide has a sufficient number of nucleotide bases to
be used in a
PCR reaction. A short oligonucleotide sequence may be based on, or designed
from, a
genomic or cDNA sequence and is used to amplify, confirm, or reveal the
presence of an
identical, similar or complementary DNA or RNA in a particular cell or tissue.
Oligonucleotides comprise portions of a nucleic acid sequence having about 10
nt, 50 nt, or
100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment
of the
invention, an oligonucleotide comprising a nucleic acid molecule less than 100
nt in length
would further comprise at least 6 contiguous nucleotides of SEQ ID NO: 1, 3,
5, 7, 9, 22, 25,
28, 31, 34, 46, and 48, or a complement thereof. Oligonucleotides may be
chemically
synthesized and may also be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention
comprises a
nucleic acid molecule that is a complement of the nucleotide sequence shown in
SEQ ID NO:
1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46, and 48, or a portion of this nucleotide
sequence (e.g., a
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CA 02386199 2002-03-28
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fragment that can be used as a probe or primer or a fragment encoding a
biologically-active
portion of an ENDOX polypeptide). A nucleic acid molecule that is
complementary to the
nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46,
and 48, is one
that is sufficiently complementary to the nucleotide sequence shown in SEQ ID
NO: 1, 3, 5, 7,
9, 22, 25, 28, 31, 34, 46, and 48, that it can hydrogen bond with little or no
mismatches to the
nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46,
and 48, thereby
forming a stable duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen
base
pairing between nucleotides units of a nucleic acid molecule, and the term
"binding" means
the physical or chemical interaction between two polypeptides or compounds or
associated
polypeptides or compounds or combinations thereof. Binding includes ionic, non-
ionic, van
der Waals, hydrophobic interactions, and the like. A physical interaction can
be either direct
or indirect. Indirect interactions may be through or due to the effects of
another polypeptide or
compound. Direct binding refers to interactions that do not take place
through, or due to, the
effect of another polypeptide or compound, but instead are without other
substantial chemical
intermediates.
Fragments provided herein are defined as sequences of at least 6 (contiguous)
nucleic
acids or at least 4 (contiguous) amino acids, a length sufficient to allow for
specific
hybridization in the case of nucleic acids or for specific recognition of an
epitope in the case of
amino acids, respectively, and are at most some portion less than a full
length sequence.
Fragments may be derived from any contiguous portion of a nucleic acid or
amino acid
sequence of choice. Derivatives are nucleic acid sequences or amino acid
sequences formed
from the native compounds either directly or by modification or partial
substitution. Analogs
are nucleic acid sequences or amino acid sequences that have a structure
similar to, but not
identical to, the native compound but differs from it in respect to certain
components or side
chains. Analogs may be synthetic or from a different evolutionary origin and
may have a
similar or opposite metabolic activity compared to wild type. Homologs are
nucleic acid
sequences or amino acid sequences of a particular gene that are derived from
different species.
Derivatives and analogs may be full length or other than full length, if the
derivative or
analog contains a modified nucleic acid or amino acid, as described below.
Derivatives or
analogs of the nucleic acids or proteins of the invention include, but are not
limited to,
molecules comprising regions that are substantially homologous to the nucleic
acids or
proteins of the invention, in various embodiments, by at least about 70%, 80%,
or 95%
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CA 02386199 2002-03-28
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identity (with a preferred identity of 80-95%) over a nucleic acid or amino
acid sequence of
identical size or when compared to an aligned sequence in which the alignment
is done by a
computer homology program known in the art, or whose encoding nucleic acid is
capable of
hybridizing to the complement of a sequence encoding the aforementioned
proteins under
stringent, moderately stringent, or low stringent conditions. See e.g.
Ausubel, et al., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and
below.
A "homologous nucleic acid sequence"'or "homologous amino acid sequence," or
variations thereof, refer to sequences characterized by a homology at the
nucleotide level or
amino acid level as discussed above. Homologous nucleotide sequences encode
those
sequences coding for isoforms of ENDOX polypeptides. Isoforms can be expressed
in
different tissues of the same organism as a result of, for example,
alternative splicing of RNA.
Alternatively, isoforms can be encoded by different genes. In the invention,
homologous
nucleotide sequences include nucleotide sequences encoding for an ENDOX
polypeptide of
species other than humans, including, but not limited to: vertebrates, and
thus can include, e.g.,
frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous
nucleotide
sequences also include, but are not limited to, naturally occurring allelic
variations and
mutations of the nucleotide sequences set forth herein. A homologous
nucleotide sequence
does not, however, include the exact nucleotide sequence encoding human ENDOX
protein.
Homologous nucleic acid sequences include those nucleic acid sequences that
encode
conservative amino acid substitutions (see below) in SEQ ID N0:2, 4, 6, 8, 10,
15, 16, 17, 18,
19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 45 inclusive, 47, and 49, as
well as a
polypeptide possessing ENDOX biological activity. Various biological
activities of the
ENDOX proteins are described below.
An ENDOX polypeptide is encoded by the open reading frame ("ORF") of an ENDOX
nucleic acid. An ORF corresponds to a nucleotide sequence that could
potentially be translated
into a polypeptide. A stretch of nucleic acids comprising an ORF is
uninterrupted by a stop
codon. An ORF that represents the coding sequence for a full protein begins
with an ATG
"start" codon and terminates with one of the three "stop" codons, namely, TAA,
TAG, or
TGA. For the purposes of this invention, an ORF may be any part of a coding
sequence, with
or without a start codon, a stop codon, or both. For an ORF to be considered
as a good
candidate for coding for a bona fide cellular protein, a minimum size
requirement is often set,
e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.
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The nucleotide sequences determined from the cloning of the human ENDOX genes
allows for the generation of probes and primers designed for use in
identifying and/or cloning
ENDOX homologues in other cell types, e.g. from other tissues, as well as
ENDOX
homologues from other vertebrates. The probe/primer typically comprises
substantially
purified oligonucleotide. The oligonucleotide typically comprises a region of
nucleotide
sequence that hybridizes under stringent conditions to at least about 12, 25,
50, 100, 150, 200,
250, 300, 350 or 400 consecutive sense strand nucleotide sequence of SEQ ID
NO: 1, 3, 5, 7,
9, 22, 25, 28, 31, 34, 46, and 48; or an anti-sense strand nucleotide sequence
of SEQ ID NO: 1,
3, 5, 7, 9, 22, 25, 28, 31, 34, 46, and 48; or of a naturally occurring mutant
of SEQ ID NO: 1,
3, S, 7, 9, 22, 25, 28, 31, 34, 46, and 48.
Probes based on the human ENDOX nucleotide sequences can be used to detect
transcripts or genomic sequences encoding the same or homologous proteins. In
various
embodiments, the probe further comprises a label group attached thereto, e.g.
the label group
can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-
factor. Such
1 S probes can be used as a part of a diagnostic test kit for identifying
cells or tissues which mis-
express an ENDOX protein, such as by measuring a level of an ENDOX-encoding
nucleic
acid in a sample of cells from a subject e.g., detecting ENDOX ml2NA levels or
determining
whether a genomic ENDOX gene has been mutated or deleted.
"A polypeptide having a biologically-active portion of an ENDOX polypeptide"
refers
to polypeptides exhibiting activity similar, but not necessarily identical to,
an activity of a
polypeptide of the invention, including mature forms, as measured in a
particular biological
assay, with or without dose dependency. A nucleic acid fragment encoding a
"biologically-
active portion of ENDOX" can be prepared by isolating a portion of SEQ ID NO:
1, 3, 5, 7, 9,
22, 25, 28, 31, 34, 46, and 48, that encodes a polypeptide having an ENDOX
biological
activity (the biological activities of the ENDOX proteins are described
below), expressing the
encoded portion of ENDOX protein (e.g., by recombinant expression in vitro)
and assessing
the activity of the encoded portion of ENDOX.
ENDOX Nucleic Acid and Polypeptide Variants
The invention further encompasses nucleic acid molecules that differ from the
nucleotide sequences shown in SEQ ID NO: 1, 3, 5, 7, 9, 22, 25, 28, 31, 34,
46, and 48, due to
degeneracy of the genetic code and thus encode the same ENDOX proteins as that
encoded by
the nucleotide sequences shown in SEQ ID NO: 1, 3, 5, 7, 9, 22, 25, 28, 31,
34, 46, and 48. In
another embodiment, an isolated nucleic acid molecule of the invention has a
nucleotide
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sequence encoding a protein having an amino acid sequence shown in SEQ ID
N0:2, 4, 6, 8,
10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 45
inclusive, 47, and 49.
In addition to the human ENDOX nucleotide sequences shown in SEQ ID NO: l, 3,
5,
7, 9, 22, 25, 28, 31, 34, 46, and 48, it will be appreciated by those skilled
in the art that DNA
sequence polymorphisms that lead to changes in the amino acid sequences of the
ENDOX
polypeptides may exist within a population (e.g., the human population). Such
genetic
polymorphism in the ENDOX genes may exist among individuals within a
population due to
natural allelic variation. As used herein, the terms "gene" and "recombinant
gene" refer to
nucleic acid molecules comprising an open reading frame (ORF) encoding an
ENDOX
protein, preferably a vertebrate ENDOX protein. Such natural allelic
variations can typically
result in 1-5% variance in the nucleotide sequence of the ENDOX genes. Any and
all such
nucleotide variations and resulting amino acid polymorphisms in the ENDOX
polypeptides,
which are the result of natural allelic variation and that do not alter the
functional activity of
the ENDOX polypeptides, are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding ENDOX proteins from other species,
and
thus that have a nucleotide sequence that differs from the human sequence of
SEQ ID NO: 1,
3, 5, 7, 9, 22, 25, 28, 31, 34, 46, and 48, are intended to be within the
scope of the invention.
Nucleic acid molecules corresponding to natural allelic variants and
homologues of the
ENDOX cDNAs of the invention can be isolated based on their homology to the
human
ENDOX nucleic acids disclosed herein using the human cDNAs, or a portion
thereof, as a
hybridization probe according to standard hybridization techniques under
stringent
hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the
invention is at least 6 nucleotides in length and hybridizes under stringent
conditions to the
nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3,
5, 7, 9, 22, 25,
28, 31, 34, 46, and 48. In another embodiment, the nucleic acid is at least
10, 25, 50, 100, 250,
500, 750, 1000, 1500, or 2000 or more nucleotides in length. In yet another
embodiment, an
isolated nucleic acid molecule of the invention hybridizes to the coding
region. As used
herein, the term "hybridizes under stringent conditions" is intended to
describe conditions for
hybridization and washing under which nucleotide sequences at least 60%
homologous to each
other typically remain hybridized to each other.
Homologs (i.e., nucleic acids encoding ENDOX proteins derived from species
other
than human) or other related sequences (e.g., paralogs) can be obtained by
low, moderate or


CA 02386199 2002-03-28
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high stringency hybridization with all or a portion of the particular human
sequence as a probe
using methods well known in the art for nucleic acid hybridization and
cloning.
As used herein, the phrase "stringent hybridization conditions" refers to
conditions
under which a probe, primer or oligonucleotide will hybridize to its target
sequence, but to no
other sequences. Stringent conditions are sequence-dependent and will be
different in
different circumstances. Longer sequences hybridize specifically at higher
temperatures than
shorter sequences. Generally, stringent conditions are selected to be about
5°C lower than the
thermal melting point (Tm) for the specific sequence at a defined ionic
strength and pH. The
Tm is the temperature (under defined ionic strength, pH and nucleic acid
concentration) at
which 50% of the probes complementary to the target sequence hybridize to the
target
sequence at equilibrium. Since the target sequences are generally present at
excess, at Tm,
50% of the probes are occupied at equilibrium. Typically, stringent conditions
will be those in
which the salt concentration is less than about 1.0 M sodium ion, typically
about 0.01 to 1.0 M
sodium ion (or other salts) at
pH 7.0 to 8.3 and the temperature is at least about 30°C for short
probes, primers or
oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for
longer probes, primers and
oligonucleotides. Stringent conditions may also be achieved with the addition
of destabilizing
agents, such as formamide.
Stringent conditions are known to those skilled in the art and can be found in
Ausubel,
et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons,
N.Y.
(1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at
least about 65%,
70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain
hybridized to each other. A non-limiting example of stringent hybridization
conditions are
hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH
7.5), 1 mM
EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm
DNA
at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at
50°C. An isolated
nucleic acid molecule of the invention that hybridizes under stringent
conditions to the
sequences of SEQ ID NO: l, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46, and 48,
corresponds to a
naturally-occurring nucleic acid molecule. As used herein, a "naturally-
occurnng" nucleic
acid molecule refers to an RNA or DNA molecule having a nucleotide sequence
that occurs in
nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the
nucleic
acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9,
22, 25, 28, 31,
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34, 46, and 48, or fragments, analogs or derivatives thereof, under conditions
of moderate
stringency is provided. A non-limiting example of moderate stringency
hybridization
conditions are hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and
100 mg/ml
denatured salmon sperm DNA at 55°C, followed by one or more washes in
1X SSC, 0.1%
SDS at 37°C. Other conditions of moderate stringency that may be used
are well-known
within the art. See, e.g., Ausubel ,et al. (eds.), 1993, CURRENT PROTOCOLS IN
MOLECULAR
BIOLOGY, john Wiley & Sons, NY, and Kriegler, 1990; GENE TRANSFER AND
EXPRESSION, A
LABORATORY MANUAL, StOCktOri Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid
molecule
comprising the nucleotide sequences of SEQ ID NO: 1, 3, 5, 7, 9, 22, 25, 28,
31, 34, 46, and
48, or fragments, analogs or derivatives thereof, under conditions of low
stringency, is
provided. A non-limiting example of low stringency hybridization conditions
are
hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA,
0.02%
PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10%
(wt/vol)
dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25
mM Tris-HCl (pH
7.4), 5 mM EDTA, and 0.1 % SDS at 50°C. Other conditions of low
stringency that may be
used are well known in the art (e.g., as employed for cross-species
hybridizations). See, e.g.,
Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John
Wiley &
Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY
MANUAL,
Stockton Press, NY; Shilo and Weinberg, 1981. Proc Natl Acad Sci USA 78: 6789-
6792.
Conservative Mutations
In addition to naturally-occurnng allelic variants of ENDOX sequences that may
exist
in the population, the skilled artisan will further appreciate that changes
can be introduced by
mutation into the nucleotide sequences of SEQ ID NO: 1, 3, 5, 7, 9, 22, 25,
28, 31, 34, 46, and
48, thereby leading to changes in the amino acid sequences of the encoded
ENDOX proteins,
without altering the functional ability of said ENDOX proteins. For example,
nucleotide
substitutions leading to amino acid substitutions at "non-essential" amino
acid residues can be
made in the sequence of SEQ ID N0:2, 4, 6, 8, 10~ 15, 16, 17, 18, 19, 20, 21,
23, 24, 26, 27,
29, 30, 32, 33, 35 to 45 inclusive, 47, and 49. A "non-essential" amino acid
residue is a
residue that can be altered from the wild-type sequences of the ENDOX proteins
without
altering their biological activity, whereas an "essential" amino acid residue
is required for such
biological activity. For example, amino acid residues that are conserved among
the ENDOX
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proteins of the invention are predicted to be particularly non-amenable to
alteration. Amino
acids for which conservative substitutions can be made are well-known within
the art.
Another aspect of the invention pertains to nucleic acid molecules encoding
ENDOX
proteins that contain changes in amino acid residues that are not essential
for activity. Such
S ENDOX proteins differ in amino acid sequence from SEQ ID N0:2, 4, 6, 8, 10,
15, 16, 17, 18,
19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 45 inclusive, 47, and 49,
yet retain biological
activity. In one embodiment, the isolated nucleic acid molecule comprises a
nucleotide
sequence encoding a protein, wherein the protein comprises an amino acid
sequence at least
about 45% homologous to the amino acid sequences of SEQ ID N0:2, 4, 6, 8, 10,
15, 16, 17,
18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 45 inclusive, 47, and
49. Preferably, the
protein encoded by the nucleic acid molecule is at least about 60% homologous
to SEQ ID
N0:2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33,
35 to 45 inclusive,
47, and 49; more preferably at least about 70% homologous to SEQ ID N0:2, 4,
6, 8, 10, 15,
16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 45 inclusive,
47, and 49; still more
preferably at least about 80% homologous to SEQ ID N0:2, 4, 6, 8, 10, 15, 16,
17, 18, 19, 20,
21, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 45 inclusive, 47, and 49; even more
preferably at least
about 90% homologous to SEQ ID N0:2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21,
23, 24, 26, 27,
29, 30, 32, 33, 35 to 45 inclusive, 47, and 49; and most preferably at least
about 95%
homologous to SEQ ID N0:2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24,
26, 27, 29, 30, 32,
33, 35 to 45 inclusive, 47, and 49.
An isolated nucleic acid molecule encoding an ENDOX protein homologous to the
protein of SEQ ID N0:2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26,
27, 29, 30, 32, 33,
35 to 45 inclusive, 47, and 49, can be created by introducing one or more
nucleotide
substitutions, additions or deletions into the nucleotide sequence of SEQ ID
NO: 1, 3, 5, 7, 9,
22, 25, 28, 31, 34, 46, and 48, such that one or more amino acid
substitutions, additions or
deletions are introduced into the encoded protein.
Mutations can be introduced into SEQ ID N0:2, 4, 6, 8, 10, 15, 16, 17, 18, 19,
20, 21,
23, 24, 26, 27, 29, 30, 32, 33, 35 to 45 inclusive, 47, and 49, by standard
techniques, such as
site-directed mutagenesis and PCR-mediated mutagenesis. Preferably,
conservative amino
acid substitutions are made at one or more predicted, non-essential amino acid
residues. A
"conservative amino acid substitution" is one in which the amino acid residue
is replaced with
an amino acid residue having a similar side chain. Families of amino acid
residues having
similar side chains have been defined within the art. These families include
amino acids with
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basic side chains (e.g., lysine, arginine, histidine), acidic side chains
(e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine,
threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,
leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine,
valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,
tryptophan,
histidine). Thus, a predicted non-essential amino acid residue in the ENDOX
protein is
replaced with another amino acid residue from the same side chain family.
Alternatively, in
another embodiment, mutations can be introduced randomly along all or part of
an ENDOX
coding sequence, such as by saturation mutagenesis, and the resultant mutants
can be screened
for ENDOX biological activity to identify mutants that retain activity.
Following mutagenesis
of SEQ ID N0:2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29,
30, 32, 33, 35 to 45
inclusive, 47, and 49, the encoded protein can be expressed by any recombinant
technology
known in the art and the activity of the protein can be determined.
In one embodiment, a mutant ENDOX protein can be assayed for (i) the ability
to form
protein:protein interactions with other ENDOX proteins, other cell-surface
proteins, or
biologically-active portions thereof, (ii) complex formation between a mutant
ENDOX protein
and an ENDOX ligand; or (iii) the ability of a mutant ENDOX protein to bind to
an
intracellular target protein or biologically-active portion thereof; (e.g.
avidin proteins).
In yet another embodiment, a mutant ENDOX protein can be assayed for the
ability to
regulate a specific biological function (e.g., regulation of insulin release).
Antisense Nucleic Acids
Another aspect of the invention pertains to isolated antisense nucleic acid
molecules
that are hybridizable to or complementary to the nucleic acid molecule
comprising the
nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46, and
48, or fragments,
analogs or derivatives thereof. An "antisense" nucleic acid comprises a
nucleotide sequence
that is complementary to a "sense" nucleic acid encoding a protein (e.g.,
complementary to the
coding strand of a double-stranded cDNA molecule or complementary to an mRNA
sequence). In specific aspects, antisense nucleic acid molecules are provided
that comprise a
sequence complementary to at least about 10, 25, 50, 100, 250 or 500
nucleotides or an entire
ENDOX coding strand, or to only a portion thereof. Nucleic acid molecules
encoding
fragments, homologs, derivatives and analogs of an ENDOX protein of SEQ ID
N0:2, 4, 6, 8,
10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 45
inclusive, 47, and 49; or
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antisense nucleic acids complementary to an ENDOX nucleic acid sequence of SEQ
ID NO: 1,
3, 5, 7, 9, 22, 25, 28, 31, 34, 46, and 48, are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a
"coding
region" of the coding strand of a nucleotide sequence encoding an ENDOX
protein. The term
"coding region" refers to the region of the nucleotide sequence comprising
codons which are
translated into amino acid residues. In another embodiment, the antisense
nucleic acid
molecule is antisense to a "noncoding region" of the coding strand of a
nucleotide sequence
encoding the ENDOX protein. The term "noncoding region" refers to 5' and 3'
sequences
which flank the coding region that are not translated into amino acids (i.e.,
also referred to as
S' and 3' untranslated regions).
Given the coding strand sequences encoding the ENDOX protein disclosed herein,
antisense nucleic acids of the invention can be designed according to the
rules of Watson and
Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be
complementary
to the entire coding region of ENDOX mRNA, but more preferably is an
oligonucleotide that
is antisense to only a portion of the coding or noncoding region of ENDOX
mRNA. For
example, the antisense oligonucleotide can be complementary to the region
surrounding the
translation start site of ENDOX mRNA. An antisense oligonucleotide can be, for
example,
about 5, 10, 15, 20, 25, 30, 35, 40, 45 or SO nucleotides in length. An
antisense nucleic acid of
the invention can be constructed using chemical synthesis or enzymatic
ligation reactions
using procedures known in the art. For example, an antisense nucleic acid
(e.g., an antisense
oligonucleotide) can be chemically synthesized using naturally-occurring
nucleotides or
variously modified nucleotides designed to increase the biological stability
of the molecules or
to increase the physical stability of the duplex formed between the antisense
and sense nucleic
acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides
can be used).
Examples of modified nucleotides that can be used to generate the antisense
nucleic
acid include: S-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-
carboxymethylaminomethyl-
2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-
galactosylqueosine,
inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-
dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-
adenine,
7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine,
pseudouracil,


CA 02386199 2002-03-28
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queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, S-
methyluracil,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-
thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
Alternatively, the
antisense nucleic acid can be produced biologically using an expression vector
into which a
nucleic acid has been subcloned in an antisense orientation (i.e., RNA
transcribed from the
inserted nucleic acid will be of an antisense orientation to a target nucleic
acid of interest,
described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically
administered to a
subject or generated in situ such that they hybridize with or bind to cellular
mRNA and/or
genomic DNA encoding an ENDOX protein to thereby inhibit expression of the
protein (e.g.,
by inhibiting transcription and/or translation). The hybridization can be by
conventional
nucleotide complementarity to form a stable duplex, or, for example, in the
case of an
antisense nucleic acid molecule that binds to DNA duplexes, through specific
interactions in
the major groove of the double helix. An example of a route of administration
of antisense
nucleic acid molecules of the invention includes direct injection at a tissue
site. Alternatively,
antisense nucleic acid molecules can be modified to target selected cells and
then administered
systemically. For example, for systemic administration, antisense molecules
can be modified
such that they specifically bind to receptors or antigens expressed on a
selected cell surface
(e.g., by linking the antisense nucleic acid molecules to peptides or
antibodies that bind to cell
surface receptors or antigens). The antisense nucleic acid molecules can also
be delivered to
cells using the vectors described herein. To achieve sufficient nucleic acid
molecules, vector
constructs in which the antisense nucleic acid molecule is placed under the
control of a strong
pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the
invention is an
a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms
specific
double-stranded hybrids with complementary RNA in which, contrary to the usual
~i-units, the
strands run parallel to each other. See, e.g., Gaultier, et al., 1987. Nucl.
Acids Res. 15:
6625-6641. The antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (see, e.g., moue, et al. 1987. Nucl. Acids Res. 15:
6131-6148) or a
chimeric RNA-DNA analogue (see, e.g., moue, et al., 1987. FEBS Lett. 215: 327-
330.
Ribozymes and PNA Moieties
Nucleic acid modifications include, by way of non-limiting example, modified
bases,
and nucleic acids whose sugar phosphate backbones are modified or derivatized.
These
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modifications are carried out at least in part to enhance the chemical
stability of the modified
nucleic acid, such that they may be used, for example, as antisense binding
nucleic acids in
therapeutic applications in a subject.
In one embodiment, an antisense nucleic acid of the invention is a ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity that are
capable of
cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described
in
Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically
cleave
ENDOX mRNA transcripts to thereby inhibit translation of ENDOX mRNA. A
ribozyme
having specificity for an ENDOX-encoding nucleic acid can be designed based
upon the
nucleotide sequence of an ENDOX cDNA disclosed herein (i.e., SEQ ID NO: 1, 3,
5, 7, 9, 22,
25, 28, 31, 34, 46, and 48). For example, a derivative of a Tetrahymena L-19
IVS RNA can
be constructed in which the nucleotide sequence of the active site is
complementary to the
nucleotide sequence to be cleaved in an ENDOX-encoding mRNA. See, e.g., U.S.
Patent
4,987,071 to Cech, et al. and U.S. Patent 5,116,742 to Cech, et al. ENDOX mRNA
can also
be used to select a catalytic RNA having a specific ribonuclease activity from
a pool of RNA
molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.
Alternatively, ENDOX gene expression can be inhibited by targeting nucleotide
sequences complementary to the regulatory region of the ENDOX nucleic acid
(e.g., the
ENDOX promoter andlor enhancers) to form triple helical structures that
prevent transcription
of the ENDOX gene in target cells. See, e.g., Helene, 1991. Anticancer Drug
Des. 6: 569-84;
Helene, et al. 1992. Ann. N. Y. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays
14: 807-15.
In various embodiments, the ENDOX nucleic acids can be modified at the base
moiety,
sugar moiety or phosphate backbone to improve, e.g., the stability,
hybridization, or solubility
of the molecule. For example, the deoxyribose phosphate backbone of the
nucleic acids can
be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al., 1996.
Bioorg Med
Chem 4: 5-23. As used herein, the terms "peptide nucleic acids" or "PNAs"
refer to nucleic
acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is
replaced by
a pseudopeptide backbone and only the four natural nucleobases are retained.
The neutral
backbone of PNAs has been shown to allow for specific hybridization to DNA and
RNA under
conditions of low ionic strength. The synthesis of PNA oligomers can be
performed using
standard solid phase peptide synthesis protocols as described in Hyrup, et
al., 1996. supra;
Perry-O'Keefe, et al., 1996. Proc. Natl. Acad. Sci. USA 93: 14670-14675.
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PNAs of ENDOX can be used in therapeutic and diagnostic applications. For
example,
PNAs can be used as antisense or antigene agents for sequence-specific
modulation of gene
expression by, e.g., inducing transcription or translation arrest or
inhibiting replication. PNAs
of ENDOX can also be used, for example, in the analysis of single base pair
mutations in a
S gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes
when used in
combination with other enzymes, e.g., Sl nucleases (see, Hyrup, et al.,
1996.supra); or as
probes or primers for DNA sequence and hybridization (see, Hyrup, et al.,
1996, supra;
Perry-O'Keefe, et al., 1996. supra).
In another embodiment, PNAs of ENDOX can be modified, e.g., to enhance their
stability or cellular uptake, by attaching lipophilic or other helper groups
to PNA, by the
formation of PNA-DNA chimeras, or by the use of liposomes or other techniques
of drug
delivery known in the art. For example, PNA-DNA chimeras of ENDOX can be
generated
that may combine the advantageous properties of PNA and DNA. Such chimeras
allow DNA
recognition enzymes (e.g., RNase H and DNA polymerises) to interact with the
DNA portion
while the PNA portion would provide high binding affinity and specificity. PNA-
DNA
chimeras can be linked using linkers of appropriate lengths selected in terms
of base stacking,
number of bonds between the nucleobases, and orientation (see, Hyrup, etal.,
1996. supra).
The synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et
al., 1996.
supra and Finn, et al., 1996. Nucl Acids Res 24: 3357-3363. For example, a DNA
chain can
be synthesized on a solid support using standard phosphoramidite coupling
chemistry, and
modified nucleoside analogs, e.g., S'-(4-methoxytrityl)amino-5'-deoxy-
thymidine
phosphoramidite, can be used between the PNA and the 5' end of DNA. See, e.g.,
Mag, et al.,
1989. Nucl Acid Res 17: 5973-5988. PNA monomers are then coupled in a stepwise
manner
to produce a chimeric molecule with a S' PNA segment and a 3' DNA segment.
See, e.g.,
Finn, et al.; 1996. supra. Alternatively, chimeric molecules can be
synthesized with a 5' DNA
segment and a 3' PNA segment. See, e.g., Petersen, et al., 1975. Bioorg. Med.
Chem. Lett. 5:
1119-11124.
In other embodiments, the oligonucleotide may include other appended groups
such as
peptides (e.g., for targeting host cell receptors in vivo), or agents
facilitating transport across
the cell membrane (see, e.g., Letsinger, et al., 1989. Proc. Natl. Acad.~Sci.
U.S.A. 86:
6553-6556; Lemaitre, et al., 1987. Proc. Natl. Acid. Sci. 84: 648-652; PCT
Publication No.
W088/09810) or the blood-brain .barner (see, e.g., PCT Publication No. WO
89/10134). In
addition, oligonucleotides can be modified with hybridization triggered
cleavage agents (see,
e.g., Krol, et al., 1988. BioTechnigues 6:958-976) or intercalating agents
(see, e.g., Zon, 1988.
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Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to
another
molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a
transport agent, a
hybridization-triggered cleavage agent, and the like.
ENDOX Polypeptides
A polypeptide according to the invention includes a polypeptide including the
amino
acid sequence of ENDOX polypeptides whose sequences are provided in SEQ ID
N0:2, 4, 6,
8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 45
inclusive, 47, and 49.
The invention also includes a mutant or variant protein any of whose residues
may be changed
from the corresponding residues shown in SEQ ID N0:2, 4, 6, 8, 10, 15, 16, 17,
18, 19, 20, 21,
23, 24, 26, 27, 29, 30, 32, 33, 35 to 45 inclusive, 47, and 49, while still
encoding a protein that
maintains its ENDOX activities and physiological functions, or a functional
fragment thereof.
In general, an ENDOX variant that preserves ENDOX-like function includes any
variant in which residues at a particular position in the sequence have been
substituted by
other amino acids, and further include the possibility of inserting an
additional residue or
residues between two residues of the parent protein as well as the possibility
of deleting one or
more residues from the parent sequence. Any amino acid substitution,
insertion, or deletion is
encompassed by the invention. In favorable circumstances, the substitution is
a conservative
substitution as defined above.
One aspect of the invention pertains to isolated ENDOX proteins, and
biologically-
active portions thereof, or derivatives, fragments, analogs or homologs
thereof. Also provided
are polypeptide fragments suitable for use as immunogens to raise anti-ENDOX
antibodies. In
one embodiment, native ENDOX proteins can be isolated from cells or tissue
sources by an
appropriate purification scheme using standard protein purification
techniques. In another
embodiment, ENDOX proteins are produced by recombinant DNA techniques.
Alternative to
recombinant expression, an ENDOX protein or polypeptide can be synthesized
chemically
using standard peptide synthesis techniques.
An "isolated" or "purified" polypeptide or protein or biologically-active
portion thereof
is substantially free of cellular material or other contaminating proteins
from the cell or tissue
source from which the ENDOX protein is derived, or substantially free from
chemical
precursors or other chemicals when chemically synthesized. The language
"substantially free
of cellular material" includes preparations of ENDOX proteins in which the
protein is
separated from cellular components of the cells from which it is isolated or
recombinantly-
produced. In one embodiment, the language ."substantially free of cellular
material" includes
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preparations of ENDOX proteins having less than about 30% (by dry weight) of
non-ENDOX
proteins (also referred to herein as a "contaminating protein"), more
preferably less than about
20% of non-ENDOX proteins, still more preferably less than about 10% of non-
ENDOX
proteins, and most preferably less than about 5% of non-ENDOX proteins. When
the ENDOX
S protein or biologically-active portion thereof is recombinantly-produced, it
is also preferably
substantially free of culture medium, i.e., culture medium represents less
than about 20%,
more preferably less than about 10%, and most preferably less than about S% of
the volume of
the ENDOX protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes
preparations of ENDOX proteins in which the protein is separated from chemical
precursors or
other chemicals that are involved in the synthesis of the protein. In one
embodiment, the
language "substantially free of chemical precursors or other chemicals"
includes preparations
of ENDOX proteins having less than about 30% (by dry weight) of chemical
precursors or
non-ENDOX chemicals, more preferably less than about 20% chemical precursors
or
non-ENDOX chemicals, still more preferably less than about 10% chemical
precursors or
non-ENDOX chemicals, and most preferably less than about 5% chemical
precursors or
non-ENDOX chemicals.
Biologically-active portions of ENDOX proteins include peptides comprising
amino
acid sequences sufficiently homologous to or derived from the amino acid
sequences of the
ENDOX proteins (e.g., the amino acid sequence shown in SEQ ID N0:2, 4, 6, 8,
10, 1 S, 16,
17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 45 inclusive, 47,
and 49) that include
fewer amino acids than the full-length ENDOX proteins, and exhibit at least
one activity of an
ENDOX protein. Typically, biologically-active portions comprise a domain or
motif with at
least one activity of the ENDOX protein. A biologically-active portion of an
ENDOX protein
can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid
residues in
length.
Moreover, other biologically-active portions, in which other regions of the
protein are
deleted, can be prepared by recombinant techniques and evaluated for one or
more of the
functional activities of a native ENDOX protein.
In an embodiment, the ENDOX protein has an amino acid sequence shown in SEQ ID
N0:2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33,
35 to 45 inclusive,
47, and 49. In other embodiments, the ENDOX protein is substantially
homologous to SEQ
ID N0:2, 4, 6, 8, 10, 1 S, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32,
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inclusive, 47, and 49, and retains the functional activity of the protein of
SEQ ID N0:2, 4, 6,
8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 45
inclusive, 47, and 49,
yet differs in amino acid sequence due to natural allelic variation or
mutagenesis, as described
in detail, below. Accordingly, in another embodiment, the ENDOX protein is a
protein that
comprises an amino acid sequence at least about 45% homologous to the amino
acid sequence
of SEQ ID N0:2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29,
30, 32, 33, 35 to 45
inclusive, 47, and 49 and retains the functional activity of the ENDOX
proteins of SEQ ID
N0:2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33,
35 to 45 inclusive,
47, and 49.
Determining Homology Between Two or More Sequences
To determine the percent homology of two amino acid sequences or of two
nucleic
acids, the sequences are aligned for optimal comparison purposes (e.g., gaps
can be introduced
in the sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a
second amino or nucleic acid sequence). The amino acid residues or nucleotides
at
corresponding amino acid positions or nucleotide positions are then compared.
When a
position in the first sequence is occupied by the same amino acid residue or
nucleotide as the
corresponding position in the second sequence, then the molecules are
homologous at that
position (i. e., as used herein amino acid or nucleic acid "homology" is
equivalent to amino
acid or nucleic acid "identity").
The nucleic acid sequence homology may be determined as the degree of identity
between two sequences. The homology may be determined using computer programs
known
in the art, such as GAP software provided in the GCG program package. See,
Needleman and
Wunsch, 1970. JMoI Biol 48: 443-453. Using GCG GAP software with the following
settings
for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP
extension penalty
of 0.3, the coding region of the analogous nucleic acid sequences referred to
above exhibits a
degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%,
or 99%, with
the CDS (encoding) part of the DNA sequence shown in SEQ ID NO: 1, 3, 5, 7, 9,
22, 25, 28,
31, 34, 46, and 48.
The term "sequence identity" refers to the degree to which two polynucleotide
or
polypeptide sequences are identical on a residue-by-residue basis over a
particular region of
comparison. The term "percentage of sequence identity" is calculated by
comparing two
optimally aligned sequences over that region of comparison, determining the
number of
positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I,
in the case of
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nucleic acids) occurs in both sequences to yield the number of matched
positions, dividing the
number of matched positions by the total number of positions in the region of
comparison (i. e.,
the window size), and multiplying the result by 100 to yield the percentage of
sequence
identity. The term "substantial identity" as used herein denotes a
characteristic of a
polynucleotide sequence, wherein the polynucleotide comprises a sequence that
has at least 80
percent sequence identity, preferably at least 85 percent identity and often
90 to 95 percent
sequence identity, more usually at least 99 percent sequence identity as
compared to a
reference sequence over a comparison region.
Chimeric and Fusion Proteins
The invention also provides ENDOX chimeric or fusion proteins. As used herein,
an
ENDOX "chimeric protein" or "fusion protein" comprises an ENDOX polypeptide
operatively-linked to a non-ENDOX polypeptide. An "ENDOX polypeptide"'refers
to a
polypeptide having an amino acid sequence corresponding to an ENDOX protein
(SEQ ID
N0:2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33,
35 to 45 inclusive,
47, and 49), whereas a "non-ENDOX polypeptide" refers to a polypeptide having
an amino
acid sequence corresponding to a protein that is not substantially homologous
to the ENDOX
protein, e.g., a protein that is different from the ENDOX protein and that is
derived from the
same or a different organism. Within an ENDOX fusion protein the ENDOX
polypeptide can
correspond to all or a portion of an ENDOX protein. In one embodiment, an
ENDOX fusion
protein comprises at least one biologically-active portion of an ENDOX
protein. In another
embodiment, an ENDOX fusion protein comprises at least two biologically-active
portions of
an ENDOX protein. In yet another embodiment, an ENDOX fusion protein comprises
at least
three biologically-active portions of an ENDOX protein. Within the fusion
protein, the term
"operatively-linked" is intended to indicate that the ENDOX polypeptide and
the non-ENDOX
polypeptide are fused in-frame with one another. The non-ENDOX polypeptide can
be fused
to the N-terminus or C-terminus of the ENDOX polypeptide.
In one embodiment, the fusion protein is a GST-ENDOX fusion protein in which
the
ENDOX sequences are fused to the C-terminus of the GST (glutathione S-
transferase)
sequences. Such fusion proteins can facilitate the purification of recombinant
ENDOX
polypeptides.
In another embodiment, the fusion protein is an ENDOX protein containing a
heterologous signal sequence at its N-terminus. In certain host cells (e.g.,
mammalian host
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cells), expression and/or secretion of ENDOX can be increased through use of a
heterologous
signal sequence.
In yet another embodiment, the fusion protein is an ENDOX-immunoglobulin
fusion
protein in which the ENDOX sequences are fused to sequences derived from a
member of the
~ immunoglobulin protein family. The ENDOX-immunoglobulin fusion proteins of
the
invention can be incorporated into pharmaceutical compositions and
administered to a subject
to inhibit an interaction between an ENDOX ligand and an ENDOX protein on the
surface of a
cell, to thereby suppress ENDOX-mediated signal transduction in vivo. The
ENDOX-
immunoglobulin fusion proteins can be used to affect the bioavailability of an
ENDOX
cognate ligand. Inhibition of the ENDOX ligand/ENDOX interaction may be useful
therapeutically for both the treatment of proliferative and differentiative
disorders, as well as
modulating (e.g. promoting or inhibiting) cell survival. Moreover, the
ENDOX-immunoglobulin fusion proteins of the invention can be used as
immunogens to
produce anti-ENDOX antibodies in a subject, to purify ENDOX ligands, and in
screening
assays to identify molecules that inhibit the interaction of ENDOX with an
ENDOX ligand.
An ENDOX chimeric or fusion protein of the invention can be produced by
standard
recombinant DNA techniques. For example, DNA fragments coding for the
different
polypeptide sequences are ligated together in-frame in accordance with
conventional
techniques, e.g., by employing blunt-ended or stagger-ended termini for
ligation, restriction
enzyme digestion to provide for appropriate termini, filling-in of cohesive
ends as appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and enzymatic
ligation. In
another embodiment, the fusion gene can be synthesized by conventional
techniques including
automated DNA synthesizers. Alternatively, PCR amplification of gene fragments
can be
carried out using anchor primers that give rise to complementary overhangs
between two
consecutive gene fragments that can subsequently be annealed and reamplified
to generate a
chimeric gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN
MOLECULAR
BtoLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are
commercially
available that already encode a fusion moiety (e.g., a GST polypeptide). An
ENDOX-encoding nucleic acid can be cloned into such an expression vector such
that the
fusion moiety is linked in-frame to the ENDOX protein.
ENDOX Agonists arid Antagonists
The invention also pertains to variants of the ENDOX proteins that function as
either
ENDOX agonists (i.e., mimetics) or as ENDOX antagonists. Variants of the ENDOX
protein
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can be generated by mutagenesis (e.g., discrete point' mutation or truncation
of the ENDOX
protein). An agonist of the ENDOX protein can retain substantially the same,
or a subset of,
the biological activities of the naturally occurnng form of the ENDOX protein.
An antagonist
of the ENDOX protein can inhibit one or more of the activities of the
naturally occurring form
of the ENDOX protein by, for example, competitively binding to a downstream or
upstream
member of a cellular signaling cascade which includes the ENDOX protein. Thus,
specific
biological effects can be elicited by treatment with a variant of limited
function. In one
embodiment, treatment of a subject with a variant having a subset of the
biological activities of
the naturally occurnng form of the protein has fewer side effects in a subject
relative to
treatment with the naturally occurring form of the ENDOX proteins.
Variants of the ENDOX proteins that function as either ENDOX agonists (i.e.,
mimetics) or as ENDOX antagonists can be identified by screening combinatorial
libraries of
mutants (e.g., truncation mutants) of the ENDOX proteins for ENDOX protein
agonist or
antagonist activity. In one embodiment, a variegated library of ENDOX variants
is generated
by combinatorial mutagenesis at the nucleic acid level and is encoded by a
variegated gene
library. A variegated library of ENDOX variants can be produced by, for
example,
enzymatically ligating a mixture of synthetic oligonucleotides into gene
sequences such that a
degenerate set of potential ENDOX sequences is expressible as individual
polypeptides, or
alternatively, as a set of larger fusion proteins (e.g., for phage display)
containing the set of
ENDOX sequences therein. There are a variety of methods which can be used to
produce
libraries of potential ENDOX variants from a degenerate oligonucleotide
sequence. Chemical
synthesis of a degenerate gene sequence can be performed in an automatic DNA
synthesizer,
and the synthetic gene then ligated into an appropriate expression vector. Use
of a degenerate
set of genes allows for the provision, in one mixture, of all of the sequences
encoding the
desired set of potential ENDOX sequences. Methods for synthesizing degenerate
oligonucleotides are well-known within the art. See, e.g., Narang, 1983.
Tetrahedron 39: 3;
Itakura, et al., 1984. Annu. Rev. Biochem. 53: 323; Itakura, et al., 1984.
Science 198: 1056;
Ike, et al., 1983. Nucl. Acids Res. 11: 477.
Polypeptide Libraries
In addition, libraries of fragments of the ENDOX protein coding sequences can
be used
to generate a variegated population of ENDOX fragments for screening and
subsequent
selection of variants of an ENDOX protein. In one embodiment, a library of
coding sequence
fragments can be generated by treating a double stranded PCR fragment of an
ENDOX coding
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sequence with a nuclease under conditions wherein nicking occurs only about
once per
molecule, denaturing the double stranded DNA, renaturing the DNA to form
double-stranded
DNA that can include sense/antisense pairs from different nicked products,
removing single
stranded portions from reformed duplexes by treatment with S, nuclease, and
ligating the
resulting fragment library into an expression vector. By this method,
expression libraries can
be derived which encodes N-terminal and internal fragments of various sizes of
the ENDOX
proteins.
Various techniques are known in the art for screening gene products of
combinatorial
libraries made by point mutations or truncation, and for screening cDNA
libraries for gene
products having a selected property. Such techniques are adaptable for rapid
screening of the
gene libraries generated by the combinatorial mutagenesis of ENDOX proteins.
The most
widely used techniques, which are amenable to high throughput analysis, for
screening large
gene libraries typically include cloning the gene library into replicable
expression vectors,
transforming appropriate cells with the resulting library of vectors, and
expressing the
combinatorial genes under conditions in which detection of a desired activity
facilitates
isolation of the vector encoding the gene whose product was detected.
Recursive ensemble
mutagenesis (REM), a new technique that enhances the frequency of functional
mutants in the
libraries, can be used in combination with the screening assays to identify
ENDOX variants.
See, e.g., Arkin and Yourvan, 1992. Proc. Natl. Acad. Sci. USA 89: 7811-7815;
Delgrave, et
al., 1993. Protein Engineering 6:327-331.
Anti-ENDOX Antibodies
The invention encompasses antibodies and antibody fragments, such as Fab or
(Fab)2,
that bind immunospecifically to any of the ENDOX polypeptides of said
invention.
An isolated ENDOX protein, or a portion or fragment thereof, can be used as an
immunogen to generate antibodies that bind to ENDOX polypeptides using
standard
techniques for polyclonal and monoclonal antibody preparation. The full-length
ENDOX
proteins can be used or, alternatively, the invention provides antigenic
peptide fragments of
ENDOX proteins for use as immunogens. The antigenic ENDOX peptides comprises
at least
4 amino acid residues of the amino acid sequence shown in SEQ ID N0:2, 4, 6,
8, 10, 15, 16,
17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 45 inclusive, 47,
and 49, and
encompasses an epitope of ENDOX such that an antibody raised against the
peptide forms a
specific immune complex with ENDOX. Preferably, the antigenic peptide
comprises at least
6, 8, 10, 15, 20, or 30 amino acid residues. Longer antigenic peptides are
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preferable over shorter antigenic peptides, depending on use and according to
methods well
known to someone skilled in the art.
In certain embodiments of the invention, at least one epitope encompassed by
the
antigenic peptide is a region of ENDOX that is located on the surface of the
protein (e.g., a
hydrophilic region). As a means for targeting antibody production, hydropathy
plots showing
regions of hydrophilicity and hydrophobicity may be generated by any method
well known in
the art, including, for example, the Kyte Doolittle or the Hopp Woods methods,
either with or
without Fourier transformation (see, e.g., Hopp and Woods, 1981. Proc. Nat.
Acad. Sci. USA
78: 3824-3828; Kyte and Doolittle, 1982. J. Mol. Biol. 157: 105-142, each
incorporated herein
by reference in their entirety).
As disclosed herein, ENDOX protein sequences of SEQ ID N0:2, 4, 6, 8, 10, or
derivatives, fragments, analogs or homologs thereof, may be utilized as
immunogens in the
generation of antibodies that immunospecifically-bind these protein
components. The term
"antibody" as used herein refers to immunoglobulin molecules and
immunologically-active
portions of immunoglobulin molecules, i. e., molecules that contain an antigen
binding site that
specifically-binds (immunoreacts with) an antigen, such as ENDOX. Such
antibodies include,
but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab
and F~ab')2 fragments,
and an Fab expression library. In a specific embodiment, antibodies to human
ENDOX
proteins are disclosed. Various procedures known within the art may be used
for the
production of polyclonal or monoclonal antibodies to an ENDOX protein sequence
of SEQ ID
N0:2, 4, 6, 8, 10, or a derivative, fragment, analog or homolog thereof. Some
of these
proteins are discussed below.
For the production of polyclonal antibodies, various suitable host animals
(e.g., rabbit,
goat, mouse or other mammal) may be immunized by injection with the native
protein, or a
synthetic variant thereof, or a derivative of the foregoing. An appropriate
immunogenic
preparation can contain, for example, recombinantly-expressed ENDOX protein or
a
chemically-synthesized ENDOX polypeptide. The preparation can further include
an
adjuvant. Various adjuvants used to increase the immunological response
include, but are not
limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum
hydroxide),
surface active substances (e.g., lysolecithin, pluronic polyols, polyanions,
peptides, oil
emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-
Guerin and
Corynebacterium parvum, or similar immunostimulatory agents. If desired, the
antibody
molecules directed against ENDOX can be isolated from the mammal (e.g., from
the blood)
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and further purified by well known techniques, such as protein A
chromatography to obtain
the IgG fraction.
The term "monoclonal antibody" or "monoclonal antibody composition", as used
herein, refers to a population of antibody molecules that contain only one
species of an antigen
binding site capable of immunoreacting with a particular epitope of ENDOX. A
monoclonal
antibody composition thus typically displays a single binding affinity for a
particular ENDOX
protein with which it immunoreacts. For preparation of monoclonal antibodies
directed
towards a particular ENDOX protein, or derivatives, fragments, analogs or
homologs thereof,
any technique that provides for the production of antibody molecules by
continuous cell line
culture may be utilized. Such techniques include, but are not limited to, the
hybridoma
technique (see, e.g., Kohler & Milstein, 1975. Nature 256: 495-497); the
trioma technique; the
human B-cell hybridoma technique (see, e.g., Kozbor, et al., 1983. Immunol.
Today 4: 72) and
the EBV hybridoma technique to produce human monoclonal antibodies (see, e.g.,
Cole, et al.,
1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-
96).
Human monoclonal antibodies may be utilized in the practice of the invention
and may be
produced by using human hybridomas (see, e.g., Cote, et al., 1983. Proc Natl
Acad Sci USA
80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in
vitro (see, e.g.,
Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.
Liss, Inc.,
pp. 77-96). Each of the above citations is incorporated herein by reference in
their entirety.
According to the invention, techniques can be adapted for the production of
single-chain antibodies specific to an ENDOX protein (see, e.g., U.S. Patent
No. 4,946,778).
In addition, methods can be adapted for the construction of Fab expression
libraries (see, e.g.,
Huse, et al., 1989. Science 246: 1275-1281) to allow rapid and effective
identification of
monoclonal Fab fragments with the desired specificity for an ENDOX protein or
derivatives,
fragments, analogs or homologs thereof. Non-human antibodies can be
"humanized" by
techniques well known in the art. See, e.g., U.S. Patent No. 5,225,539.
Antibody fragments
that contain the idiotypes to an ENDOX protein may be produced by techniques
known in the
art including, but not limited to: (i) an F~ab')z fragment produced by pepsin
digestion of an
antibody molecule; (ii) an Fab fragment generated by reducing the disulfide
bridges of an F~ab')z
fragment; (iii) an Fab fragment generated by the treatment of the antibody
molecule with
papain and a reducing agent; and (iv) F,, fragments.
Additionally, recombinant anti-ENDOX antibodies, such as chimeric and
humanized
monoclonal antibodies, comprising both human and non-human portions, which can
be made
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using standard recombinant DNA techniques, are within the scope of the
invention. Such
chimeric and humanized monoclonal antibodies can be produced by recombinant
DNA
techniques known in the art, for example using methods described in
International Application
No. PCT/US86/02269; European Patent Application No. 184,187; European Patent
Application No. 171,496; European Patent Application No. 173,494; PCT
International
Publication No. WO 86/01533; U.S. Patent No. 4,816,567; U.S. Pat. No.
5,225,539; European
Patent Application No. 125,023; Better, et al., 1988. Science 240: 1041-1043;
Liu, et al., 1987.
Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu, et al., 1987. J. Immunol. 139:
3521-3526; Sun,
et al., 1987. Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura, et al., 1987.
Cancer Res. 47:
999-1005; Wood, et al., 1985. Nature 314 :446-449; Shaw, et al., 1988. J.
Natl. Cancer Inst.
80: 1553-1559); Mornson(1985) Science 229:1202-f207; Oi, et al. (1986)
BioTechniques
4:214; Jones, et al., 1986. Nature 321: 552-525; Verhoeyan, et al., 1988.
Science 239: 1534;
and Beidler, et al., 1988. J. Immunol. 141: 4053-4060. Each of the above
citations are
incorporated herein by reference in their entirety.
In one embodiment, methods for the screening of antibodies that possess the
desired
specificity include, but are not limited to, enzyme-linked immunosorbent assay
(ELISA) and
other immunologically-mediated techniques known within the art. In a specific
embodiment,
selection of antibodies that are specific to a particular domain of an ENDOX
protein is
facilitated by generation of hybridomas that bind to the fragment of an ENDOX
protein
possessing such a domain. Thus, antibodies that are specific for a desired
domain within an
ENDOX protein, or derivatives, fragments, analogs or homologs thereof, are
also provided
herein.
Anti-ENDOX antibodies may be used in methods known within the art relating to
the
localization and/or quantitation of an ENDOX protein (e.g., for use in
measuring levels of the
ENDOX protein within appropriate physiological samples, for use in diagnostic
methods, for
use in imaging the protein, and the like). In a given embodiment, antibodies
for ENDOX
proteins, or derivatives, fragments, analogs or homologs thereof, that contain
the antibody
derived binding domain, are utilized as pharmacologically-active compounds
(hereinafter
"Therapeutics").
An anti-ENDOX antibody (e.g., monoclonal antibody) can be used to isolate an
ENDOX polypeptide by standard techniques, such as affinity chromatography or
immunoprecipitation. An anti-ENDOX antibody can facilitate the purification of
natural
ENDOX polypeptide from cells and of recombinantly-produced ENDOX polypeptide
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expressed in host cells. Moreover, an anti-ENDOX antibody can be used to
detect ENDOX
protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate
the abundance and
pattern of expression of the ENDOX protein. Anti-ENDOX antibodies can be used
diagnostically to monitor protein levels in tissue as part of a clinical
testing procedure, e.g., to,
for example, determine the efficacy of a given treatment regimen. Detection
can be facilitated
by coupling (i.e., physically linking) the antibody to a detectable substance.
Examples of
detectable substances include various enzymes, prosthetic groups, fluorescent
materials,
luminescent materials, bioluminescent materials, and radioactive materials.
Examples of
suitable enzymes include horseradish peroxidase, alkaline phosphatase, (3-
galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin; examples of suitable fluorescent
materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent
material includes
luminol; examples of bioluminescent materials include luciferase, luciferin,
and aequorin, and
examples of suitable radioactive material include lzsh 1311, 3sS or 3H.
ENDOX Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression
vectors,
containing a nucleic acid encoding an ENDOX protein, or derivatives,
fragments, analogs or
homologs thereof. As used herein, the term "vector" refers to a nucleic acid
molecule capable
of transporting another nucleic acid to which it has been linked. One type of
vector is a
"plasmid", which refers to a circular double stranded DNA loop into which
additional DNA
segments can be ligated. Another type of vector is a viral vector, wherein
additional DNA
segments can be ligated into the viral genome. Certain vectors are capable of
autonomous
replication in a host cell into which they are introduced (e.g., bacterial
vectors having a
bacterial origin of replication and episomal mammalian vectors). Other vectors
(e.g.,
non-episomal mammalian vectors) are integrated into the genome of a host cell
upon
introduction into the host cell, and thereby are replicated along with the
host genome.
Moreover, certain vectors are capable of directing the expression of genes to
which they are
operatively-linked. Such vectors are referred to herein as "expression
vectors". In general,
expression vectors of utility in recombinant DNA techniques are often in the
form of plasmids.
In the present specification, "plasmid" and "vector" can be used
interchangeably as the
plasmid is the most commonly used form of vector. However, the invention is
intended to
include such other forms of expression vectors, such as viral vectors (e.g.,
replication defective
retroviruses, adenoviruses and adeno-associated viruses), which serve
equivalent functions.
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The recombinant expression vectors of the invention comprise a nucleic acid of
the
invention in a form suitable for expression of the nucleic acid in a host
cell, which means that
the recombinant expression vectors include one or more regulatory sequences,
selected on the
basis of the host cells to be used for expression, that is operatively-linked
to the nucleic acid
S sequence to be expressed. Within a recombinant expression vector, "operably-
linked" is
intended to mean that the nucleotide sequence of interest is linked to the
regulatory
sequences) in a manner that allows for expression of the nucleotide sequence
(e.g., in an in
vitro transcription/translation system or in a host cell when the vector is
introduced into the
host cell).
The term "regulatory sequence" is intended to includes promoters, enhancers
and other
expression control elements (e.g., polyadenylation signals). Such regulatory
sequences are
described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences
include
those that direct constitutive expression of a nucleotide sequence in many
types of host cell
and those that direct expression of the nucleotide sequence only in certain
host cells (e.g.,
tissue-specific regulatory sequences). It will be appreciated by those skilled
in the art that the
design of the expression vector can depend on such factors as the choice of
the host cell to be
transformed, the level of expression of protein desired, etc. The expression
vectors of the
invention can be introduced into host cells to thereby produce proteins or
peptides, including
fusion proteins or peptides, encoded by nucleic acids as described herein
(e.g., ENDOX
proteins, mutant forms of ENDOX proteins, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for
expression of
ENDOX proteins in prokaryotic or eukaryotic cells. For example, ENDOX proteins
can be
expressed in bacterial cells such as Escherichia coli, insect cells (using
baculovirus expression
vectors) yeast cells or mammalian cells. Suitable host cells are discussed
further in Goeddel,
GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San
Diego, Calif. (1990). Alternatively, the recombinant expression vector can be
transcribed and
translated in vitro, for example using T7 promoter regulatory sequences and T7
polymerise.
Expression of proteins in prokaryotes is most often carned out in Escherichia
coli with
vectors containing constitutive or inducible promoters directing the
expression of either fusion
or non-fusion proteins. Fusion vectors add a number of amino acids to a
protein encoded
therein, usually to the amino terminus of the recombinant protein. Such fusion
vectors
typically serve three purposes: (i) to increase expression of recombinant
protein; (ii) to


CA 02386199 2002-03-28
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increase the solubility of the recombinant protein; and (iii) to aid in the
purification of the
recombinant protein by acting as a ligand in affinity purification. Often, in
fusion expression
vectors, a proteolytic cleavage site is introduced at the junction of the
fusion moiety and the
recombinant protein to enable separation of the recombinant protein from the
fusion moiety
subsequent to purification of the fusion protein. Such enzymes, and their
cognate recognition
sequences, include Factor Xa, thrombin and enterokinase. Typical fusion
expression vectors
include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40),
pMAL
(New England Biolabs, Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N.J.)
that fuse
glutathione S-transferase (GST), maltose E binding protein, or protein A,
respectively, to the
target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include
pTrc
(Amrann et al., (1988) Gene 69:301-315) and pET 1 1d (Studier et al., GENE
ExPRESSION
TECHNOLOGY: METHODS 1N ENZYMOLOGY 185, Academic Press, San Diego, Calif.
(1990)
60-89).
One strategy to maximize recombinant protein expression in E. coli is to
express the
protein in a host bacteria with an impaired capacity to proteolytically cleave
the recombinant
protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY
185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to
alter the
nucleic acid sequence of the nucleic acid to be inserted into an expression
vector so that the
individual codons for each amino acid are those preferentially utilized in E.
coli (see, e.g.,
Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of
nucleic acid sequences
of the invention can be carned out by standard DNA synthesis techniques.
In another embodiment, the ENDOX expression vector is a yeast expression
vector.
Examples of vectors for expression in yeast Saccharomyces cerivisae include
pYepSecl
(Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz,
1982. Cell 30:
933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen
Corporation,
San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
Alternatively, ENDOX can be expressed in insect cells using baculovirus
expression
vectors. Baculovirus vectors available for expression of proteins in cultured
insect cells (e.g.,
SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:
2156-2165) and the
pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in
mammalian
cells using a mammalian expression vector. Examples of mammalian expression
vectors
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include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufinan, et al.,
1987. EMBO
J. 6: 187-195). When used in mammalian cells, the expression vector's control
functions are
often provided by viral regulatory elements. For example, commonly used
promoters are
derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For
other suitable
expression systems for both prokaryotic and eukaryotic cells see, e.,g.,
Chapters 16 and 17 of
Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1989.
In another embodiment, the recombinant mammalian expression vector is capable
of
directing expression of the nucleic acid preferentially in a particular cell
type (e.g.,
tissue-specific regulatory elements are used to express the nucleic acid).
Tissue-specific
regulatory elements are known in the art. Non-limiting examples of suitable
tissue-specific
promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987.
Genes Dev. 1:
268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol.
43:
235-275), in particular promoters of T cell receptors (Winoto and Baltimore,
1989. EMBO J.
8: 729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740;
Queen and
Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the
neurofilament
promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477),
pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and
mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316
and European
Application Publication No. 264,166). Developmentally-regulated promoters are
also
encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science
249: 374-379)
and the a-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-
546).
The invention further provides a recombinant expression vector comprising a
DNA
molecule of the invention cloned into the expression vector in an antisense
orientation. That
is, the DNA molecule is operatively-linked to a regulatory sequence in a
manner that allows
for expression (by transcription of the DNA molecule) of an RNA molecule that
is antisense to
ENDOX mRNA. Regulatory sequences operatively linked to a nucleic acid cloned
in the
antisense orientation can be chosen that direct the continuous expression of
the antisense RNA
molecule in a variety of cell types, for instance viral promoters and/or
enhancers, or regulatory
sequences can be chosen that direct constitutive, tissue specific or cell type
specific expression
of antisense RNA. The antisense expression vector can be in the form of a
recombinant
plasmid, phagemid or attenuated virus in which antisense nucleic acids are
produced under the
control of a high efficiency regulatory region, the activity of which can be
determined by the
cell type into which the vector is introduced. For a discussion of the
regulation of gene
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expression using antisense genes see, e.g., Weintraub, et al., "Antisense RNA
as a molecular
tool for genetic analysis," Reviews-Trends in Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a
recombinant
expression vector of the invention has been introduced. The terms "host cell"
and
"recombinant host cell" are used interchangeably herein. It is understood that
such terms refer
not only to the particular subject cell but also to the progeny or potential
progeny of such a
cell. Because certain modifications may occur in succeeding generations due to
either
mutation or environmental influences, such progeny may not, in fact, be
identical to the parent
cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, ENDOX
protein
can be expressed in bacterial cells such as E. coli, insect cells, yeast or
mammalian cells (such
as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells
are known to
those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional
transformation or transfection techniques. As used herein, the terms
"transformation" and
"transfection" are intended to refer to a variety of art-recognized techniques
for introducing
foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate
or calcium
chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or
electroporation.
Suitable methods for transforming or transfecting host cells can be found in
Sambrook, et al.
(MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor
Laboratory,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and
other laboratory
manuals.
For stable transfection of mammalian cells, it is known that, depending upon
the
expression vector and transfection technique used, only a small fraction of
cells may integrate
the foreign DNA into their genome. In order to identify and select these
integrants, a gene that
encodes a selectable marker (e.g., resistance to antibiotics) is generally
introduced into the host
cells along with the gene of interest. Various selectable markers include
those that confer
resistance to drugs, such as 6418, hygromycin and methotrexate. Nucleic acid
encoding a
selectable marker can be introduced into a host cell on the same vector as
that encoding
ENDOX or can be introduced on a separate vector. Cells stably transfected with
the
introduced nucleic acid can be identified by drug selection (e.g., cells that
have incorporated
the selectable marker gene will survive, while the other cells die).
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A host cell of the invention, such as a prokaryotic or eukaryotic host cell in
culture, can
be used to produce (i.e., express) ENDOX protein. Accordingly, the invention
further
provides methods for producing ENDOX protein using the host cells of the
invention. In one
embodiment, the method comprises culturing the host cell of invention (into
which a
recombinant expression vector encoding ENDOX protein has been introduced) in a
suitable
medium such that ENDOX protein is produced. In another embodiment, the method
further
comprises isolating ENDOX protein from the medium or the host cell.
Transgenic ENDOX Animals
The host cells of the invention can also be used to produce non-human
transgenic
animals. For example, in one embodiment, a host cell of the invention is a
fertilized oocyte or
an embryonic stem cell into which ENDOX protein-coding sequences have been
introduced.
Such host cells can then be used to create non-human transgenic animals in
which exogenous
ENDOX sequences have been introduced into their genome or homologous
recombinant
animals in which endogenous ENDOX sequences have been altered. Such animals
are useful
for studying the function and/or activity of ENDOX protein and for identifying
and/or
evaluating modulators of ENDOX protein activity. As used herein, a "transgenic
animal" is a
non-human animal, preferably a mammal, more preferably a rodent such as a rat
or mouse, in
which one or more of the cells of the animal includes a transgene. Other
examples of
transgenic animals include non-human primates, sheep, dogs, cows, goats,
chickens,
amphibians, etc. A transgene is exogenous DNA that is integrated into the
genome of a cell
from which a transgenic animal develops and that remains in the genome of the
mature animal,
thereby directing the expression of an encoded gene product in one or more
cell types or
tissues of the transgenic animal. As used herein, a "homologous recombinant
animal" is a
non-human animal, preferably a mammal, more preferably a mouse, in which an
endogenous
ENDOX gene has been altered by homologous recombination between the endogenous
gene
and an exogenous DNA molecule introduced into a cell of the animal, e.g., an
embryonic cell
of the animal, prior to development of the animal.
A transgenic animal of the invention can be created by introducing ENDOX-
encoding
nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by
microinjection, retroviral
infection) and allowing the oocyte to develop in a pseudopregnant female
foster animal. The
human ENDOX cDNA sequences of SEQ ID NO: 1, 3, 5, 7, 9, 22, 25, 28, 31, 34,
46, and 48,
can be introduced as a transgene into the genome of a non-human animal.
Alternatively, a
non-human homologue of the human ENDOX gene, such as a mouse ENDOX gene, can
be
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isolated based on hybridization to the human ENDOX cDNA (described further
supra) and
used as a transgene. Intronic sequences and polyadenylation signals can also
be included in
the transgene to increase the efficiency of expression of the transgene. A
tissue-specific
regulatory sequences) can be operably-linked to the ENDOX transgene to direct
expression of
ENDOX protein to particular cells. Methods for generating transgenic animals
via embryo
manipulation and microinjection, particularly animals such as mice, have
become conventional
in the art and are described, for example, in U.S. Patent Nos. 4,736,866;
4,870,009; and
4,873,191; and Hogan, 1986. In: MANIPULATING THE MousE EMBRYO, Cold Spring
Harbor
Laboratory Press, Cold Spring Harbor, N.Y. Similar methods are used for
production of other
transgenic animals. A transgenic founder animal can be identified based upon
the presence of
the ENDOX transgene in its genome and/or expression of ENDOX mRNA in tissues
or cells
of the animals. A transgenic founder animal can then be used to breed
additional animals
carrying the transgene. Moreover, transgenic animals carrying a transgene-
encoding ENDOX
protein can further be bred to other transgenic animals carrying other
transgenes.
1 S To create a homologous recombinant animal, a vector is prepared which
contains at
least a portion of an ENDOX gene into which a deletion, addition or
substitution has been
introduced to thereby alter, e.g., functionally disrupt, the ENDOX gene. The
ENDOX gene
can be a human gene (e.g., the cDNA of SEQ ID NO:1, 3, 5, 7, 9, 22, 25, 28,
31, 34, 46, and
48), but more preferably, is a non-human homologue of a human ENDOX gene. For
example,
a mouse homologue of human ENDOX gene of SEQ ID NO:1, 3, 5, 7, 9, 22, 25, 28,
31, 34,
46, and 48, can be used to construct a homologous recombination vector
suitable for altering
an endogenous ENDOX gene in the mouse genome. In one embodiment, the vector is
designed such that, upon homologous recombination, the endogenous ENDOX gene
is
functionally disrupted (i.e., no longer encodes a functional protein; also
referred to as a "knock
out" vector).
Alternatively, the vector can be designed such that, upon homologous
recombination,
the endogenous ENDOX gene is mutated or otherwise altered but still encodes
functional
protein (e.g., the upstream regulatory region can be altered to thereby alter
the expression of
the endogenous ENDOX protein). In the homologous recombination vector, the
altered
portion of the ENDOX gene is flanked at its 5'- and 3'-termini by additional
nucleic acid of the
ENDOX gene to allow for homologous recombination to occur between the
exogenous
ENDOX gene carried by the vector and an endogenous ENDOX gene in an embryonic
stem
cell. The additional flanking ENDOX nucleic acid is of sufficient length for
successful
homologous recombination with the endogenous gene. Typically, several
kilobases of
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flanking DNA (both at the 5'- and 3'-termini) are included in the vector. See,
e.g., Thomas, et
al., 1987. Cell 51: 503 for a description of homologous recombination vectors.
The vector is
ten introduced into an embryonic stem cell line (e.g., by electroporation) and
cells in which the
introduced ENDOX gene has homologously-recombined with the endogenous ENDOX
gene
are selected. See, e.g., Li, et al., 1992. Cell 69: 915.
The selected cells are then injected into a blastocyst of an animal (e.g., a
mouse) to
form aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCINOMAS AND
EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp.
113-152.
A chimeric embryo can then be implanted into a suitable pseudopregnant female
foster animal
and the embryo brought to term. Progeny harboring the homologously-recombined
DNA in
their germ cells can be used to breed animals in which all cells of the animal
contain the
homologously-recombined DNA by germline transmission of the transgene. Methods
for
constructing homologous recombination vectors and homologous recombinant
animals are
described further in Bradley, 1991. Curr. Opin. Biotechnol. 2: 823-829; PCT
International
Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that
contain
selected systems that allow for regulated expression of the transgene. One
example of such a
system is the cre/loxP recombinase system of bacteriophage P1. For a
description of the
cre/loxP recombinase system, See, e.g., Lakso, et al., 1992. Proc. Natl. Acad.
Sci. USA 89:
6232-6236. Another example of a recombinase system is the FLP recombinase
system of
Saccharomyces cerevisiae. See, O'Gorman, et al., 1991. Science 251:1351-1355.
If a cre/loxP
recombinase system is used to regulate expression of the transgene, animals
containing
transgenes encoding both the Cre recombinase and a selected protein are
required. Such
animals can be provided through the construction of "double" transgenic
animals, e.g., by
mating two transgenic animals, one containing a transgene encoding a selected
protein and the
other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be
produced
according to the methods described in Wilmut, et al., 1997. Nature 385: 810-
813. In brief, a
cell (e.g., a somatic cell) from the transgenic animal can be isolated and
induced to exit the
growth cycle and enter Go phase. The quiescent cell can then be fused, e.g.,
through the use of
electrical pulses, to an enucleated oocyte from an animal of the same species
from which the
quiescent cell is isolated. The reconstructed oocyte is then cultured such
that it develops to
morula or blastocyte and then transferred to pseudopregnant female foster
animal. The
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offspring borne of this female foster animal will be a clone of the animal
from which the cell
(e.g., the somatic cell) is isolated.
Pharmaceutical Compositions
The ENDOX nucleic acid molecules, ENDOX proteins, and anti-ENDOX antibodies
(also referred to herein as "active compounds") of the invention, and
derivatives, fragments,
analogs and homologs thereof, can be incorporated into pharmaceutical
compositions suitable
for administration. Such compositions typically comprise the nucleic acid
molecule, protein,
or antibody and a pharmaceutically acceptable carrier. .As used herein,
"pharmaceutically
acceptable carrier" is intended to include any and all solvents, dispersion
media, coatings,
antibacterial and antifungal agents, isotonic and absorption delaying agents,
and the like,
compatible with pharmaceutical administration. Suitable carriers are described
in the most
recent edition of Remington's Pharmaceutical Sciences, a standard reference
text in the field,
which is incorporated herein by reference. Preferred examples of such carriers
or diluents
include, but are not limited to, water, saline, forger's solutions, dextrose
solution, and 5%
human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may
also be
used. The use of such media and agents for pharmaceutically active substances
is well known
in the art. Except insofar as any conventional media or agent is incompatible
with the active
compound, use thereof in the compositions is contemplated. Supplementary
active
compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible
with its
intended route of administration. Examples of routes of administration include
parenteral,
e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (i.e., topical),
transmucosal, and rectal administration. Solutions or suspensions used for
parenteral,
intradermal, or subcutaneous application can include the following components:
a sterile
diluent such as water for injection, saline solution, fixed oils, polyethylene
glycols, glycerine,
propylene glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate;
chelating agents such
as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates
or phosphates,
and agents for the adjustment of tonicity such as sodium chloride or dextrose.
The pH can be
adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral
preparation can be enclosed in ampoules, disposable syringes or multiple dose
vials made of
glass or plastic.
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Pharmaceutical compositions suitable for injectable use include sterile
aqueous
solutions (where water soluble) or dispersions and sterile powders for the
extemporaneous
preparation of sterile injectable solutions or dispersion. For intravenous
administration,
suitable carriers include physiological saline, bacteriostatic water,
Cremophor ELTM (BASF,
Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the
composition must be
sterile and should be fluid to the extent that easy syringeability exists. It
must be stable under
the conditions of manufacture and storage and must be preserved against the
contaminating
action of microorganisms such as bacteria and fungi. The carrier can be a
solvent or
dispersion medium containing, for example, water, ethanol, polyol (for
example, glycerol,
propylene glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof.
The proper fluidity can be maintained, for example, by the use of a coating
such as lecithin, by
the maintenance of the required particle size in the case of dispersion and by
the use of
surfactants. Prevention of the action of microorganisms can be achieved by
various
antibacterial and antifungal agents, for example, parabens, chlorobutanol,
phenol, ascorbic
acid, thimerosal, and the like. In many cases, it will be preferable to
include isotonic agents,
for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride
in the
composition. Prolonged absorption of the injectable compositions can be
brought about by
including in the composition an agent which delays absorption, for example,
aluminum
monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound (e.g.,
an ENDOX protein or anti-ENDOX antibody) in the required amount in an
appropriate solvent
with one or a combination of ingredients enumerated above, as required,
followed by filtered
sterilization. Generally, dispersions are prepared by incorporating the active
compound into a
sterile vehicle that contains a basic dispersion medium and the required other
ingredients from
those enumerated above. In the case of sterile powders for the preparation of
sterile injectable
solutions, methods of preparation are vacuum drying and freeze-drying that
yields a powder of
the active ingredient plus any additional desired ingredient from a previously
sterile-filtered
solution thereof.
Oral compositions generally include an inert diluent or an edible carrier.
They can be
enclosed in gelatin capsules or compressed into tablets. For the purpose of
oral therapeutic
administration, the active compound can be incorporated with excipients and
used in the form
of tablets, troches, or capsules. Oral compositions can also be prepared using
a fluid Garner
for use as a mouthwash, wherein the compound in the fluid Garner is applied
orally and
swished and expectorated or swallowed. Pharmaceutically compatible binding
agents, and/or
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adjuvant materials can be included as part of the composition. The tablets,
pills, capsules,
troches and the like can contain any of the following ingredients, or
compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth or
gelatin; an excipient
such as starch or lactose, a disintegrating agent such as alginic acid,
Primogel, or corn starch; a
lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal
silicon dioxide; a
sweetening agent such as sucrose or saccharin; or a flavoring agent such as
peppermint,
methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of
an
aerosol spray from pressured container or dispenser which contains a suitable
propellant, e.g.,
a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For
transmucosal or transdermal administration, penetrants appropriate to the
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art, and
include, for example, for transmucosal administration, detergents, bile salts,
and fusidic acid
derivatives. Transmucosal administration can be accomplished through the use
of nasal sprays
or suppositories. For transdermal administration, the active compounds are
formulated into
ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with
conventional suppository bases such as cocoa butter and other glycerides) or
retention enemas
for rectal delivery.
In one embodiment, the active compounds are prepared with Garners that will
protect
the compound against rapid elimination from the body, such as a controlled
release
formulation, including implants and microencapsulated delivery systems.
Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides,
polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for
preparation of
such formulations will be apparent to those skilled in the art. The materials
can also be
obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
Liposomal
suspensions (including liposomes targeted to infected cells with monoclonal
antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers. These can
be prepared
according to methods known to those skilled in the art, for example, as
described in U.S.
Patent No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in
dosage
unit form for ease of administration and uniformity of dosage. Dosage unit
form as used
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WO 01/25436 PCT/US00/41077
herein refers to physically discrete units suited as unitary dosages for the
subject to be treated;
each unit containing a predetermined quantity of active compound calculated to
produce the
desired therapeutic effect in association with the required pharmaceutical
carrier. The
specification for the dosage unit forms of the invention are dictated by and
directly dependent
on the unique characteristics of the active compound and the particular
therapeutic effect to be
achieved, and the limitations inherent in the art of compounding such an
active compound for
the treatment of individuals.
The nucleic acid molecules of the invention can be inserted into vectors and
used as
gene therapy vectors. Gene therapy vectors can be delivered to a subject by,
for example,
intravenous injection, local administration (see, e.g., U.S. Patent No.
5,328,470) or by
stereotactic injection (see, e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci.
USA 91: 3054-3057).
The pharmaceutical preparation of the gene therapy vector can include the gene
therapy vector
in an acceptable diluent, or can comprise a slow release matrix in which the
gene delivery
vehicle is imbedded. Alternatively, where the complete gene delivery vector
can be produced
intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical
preparation can
include one or more cells that produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or
dispenser
together with instructions for administration.
Screening and Detection Methods
The isolated nucleic acid molecules of the invention can be used to express
ENDOX
protein (e.g., via a recombinant expression vector in a host cell in gene
therapy applications),
to detect ENDOX mRNA (e.g., in a biological sample) or a genetic lesion in an
ENDOX gene,
and to modulate ENDOX activity, as described further, below. In addition, the
ENDOX
proteins can be used to screen drugs or compounds that modulate the ENDOX
protein activity
or expression as well as to treat disorders characterized by insufficient or
excessive production
of ENDOX protein or production of ENDOX protein forms that have decreased or
aberrant
activity compared to ENDOX wild-type protein (e.g.; diabetes (regulates
insulin release);
obesity (binds and transport lipids); metabolic disturbances associated with
obesity, the
metabolic syndrome X as well as anorexia and wasting disorders associated with
chronic
diseases and various cancers, and infectious disease(possesses anti-microbial
activity) and the
various dyslipidemias. In addition, the anti-ENDOX antibodies of the invention
can be used
to detect and isolate ENDOX proteins and modulate ENDOX activity. In yet a
further aspect,
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the invention can be used in methods to influence appetite, absorption of
nutrients and the
disposition of metabolic substrates in both a positive and negative fashion.
The invention further pertains to novel agents identified by the screening
assays
described herein and uses thereof for treatments as described, supra.
Screening Assays
The invention provides a method (also referred to herein as a "screening
assay") for
identifying modulators, i.e., candidate or test compounds or agents (e.g.,
peptides,
peptidomimetics, small molecules or other drugs) that bind to ENDOX proteins
or have a
stimulatory or inhibitory effect on, e.g., ENDOX protein expression or ENDOX
protein
activity. The invention also includes compounds identified in the screening
assays described
herein.
In one embodiment, the invention provides assays for screening candidate or
test
compounds which bind to or modulate the activity of the membrane-bound form of
an
ENDOX protein or polypeptide or biologically-active portion thereof. The test
compounds of
the invention can be obtained using any of the numerous approaches in
combinatorial library
methods known in the art, including: biological libraries; spatially
addressable parallel solid
phase or solution phase libraries; synthetic library methods requiring
deconvolution; the
"one-bead one-compound" library method; and synthetic library methods using
affinity
chromatography selection. The biological library approach is limited to
peptide libraries,
while the other four approaches are applicable to peptide, non-peptide
oligomer or small
molecule libraries of compounds. See, e.g., Lam, 1997. Anticancer Drug Design
12: 145.
A "small molecule" as used herein, is meant to refer to a composition that has
a
molecular weight of less than about 5 kD and most preferably less than about 4
kD. Small
molecules can be, e.g., nucleic acids, peptides, polypeptides,
peptidomimetics, carbohydrates,
lipids or other organic or inorganic molecules. Libraries of chemical and/or
biological
mixtures, such as fungal, bacterial, or algal extracts, are known in the art
and can be screened
with any of the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in
the art,
for example in: DeWitt, et al., 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909;
Erb, et al., 1994.
Proc. Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med.
Chem. 37: 2678;
Cho, et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem. Int.
Ed. Engl. 33:
2059; Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop,
et al., 1994. J.
Med. Chem. 37: 1233.
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Libraries of compounds may be presented in solution (e.g., Houghten, 1992.
Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on
chips (Fodor,
1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409),
spores (Ladner,
U.S. Patent 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl. Acad. Sci.
USA 89:
1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin,
1990. Science
249: 404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 6378-
6382; Felici, 1991.
J. Mol. Biol. 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).
In one embodiment, an assay is a cell-based assay in which a cell which
expresses a
membrane-bound form of ENDOX protein, or a biologically-active portion
thereof, on the cell
surface is contacted with a test compound and the ability of the test compound
to bind to an
ENDOX protein determined. The cell, for example, can of mammalian origin or a
yeast cell.
Determining the ability of the test compound to bind to the ENDOX protein can
be
accomplished, for example, by coupling the test compound with a radioisotope
or enzymatic
label such that binding of the test compound to the ENDOX protein or
biologically-active
portion thereof can be determined by detecting the labeled compound in a
complex. For
example, test compounds can be labeled with lzsh 3sS~ ~4C, or 3H, either
directly or indirectly,
and the radioisotope detected by direct counting of radioemission or by
scintillation counting.
Alternatively, test compounds can be enzymatically-labeled with, for example,
horseradish
peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label
detected by
determination of conversion of an appropriate substrate to product. In one
embodiment, the
assay comprises contacting a cell which expresses a membrane-bound form of
ENDOX
protein, or a biologically-active portion thereof, on the cell surface with a
known compound
which binds ENDOX to form an assay mixture, contacting the assay mixture with
a test
compound, and determining the ability of the test compound to interact with an
ENDOX
protein, wherein determining the ability of the test compound to interact with
an ENDOX
protein comprises determining the ability of the test compound to
preferentially bind to
ENDOX protein or a biologically-active portion thereof as compared to the
known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a
cell
expressing a membrane-bound form of ENDOX protein, or a biologically-active
portion
thereof, on the cell surface with a test compound and determining the ability
of the test
compound to modulate (e.g., stimulate or inhibit) the activity of the ENDOX
protein or
biologically-active portion thereof. Determining the ability of the test
compound to modulate
the activity of ENDOX or a biologically-active portion thereof can be
accomplished, for
example, by determining the ability of the ENDOX protein to bind to or
interact with an
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ENDOX target molecule. As used herein, a "target molecule" is a molecule with
which an
ENDOX protein binds or interacts in nature, for example, a molecule on the
surface of a cell
which expresses an ENDOX interacting protein, a molecule on the surface of a
second cell, a
molecule in the extracellular milieu, a molecule associated with the internal
surface of a cell
membrane or a cytoplasmic molecule. An ENDOX target molecule can be a non-
ENDOX
molecule or an ENDOX protein or polypeptide of the invention . In one
embodiment, an
ENDOX target molecule is a component of a signal transduction pathway that
facilitates
transduction of an extracellular signal (e.g. a signal generated by binding of
a compound to a
membrane-bound ENDOX molecule) through the cell membrane and into the cell.
The target,
for example, can be a second intercellular protein that has catalytic activity
or a protein that
facilitates the association of downstream signaling molecules with ENDOX.
Determining the ability of the ENDOX protein to bind to or interact with an
ENDOX
target molecule can be accomplished by one of the methods described above for
determining
direct binding. In one embodiment, determining the ability of the ENDOX
protein to bind to or
interact with an ENDOX target molecule can be accomplished by determining the
activity of
the target molecule. For example, the activity of the target molecule can be
determined by
detecting induction of a cellular second messenger of the target (i.e.
intracellular Caz+,
diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the
target an appropriate
substrate, detecting the induction of a reporter gene (comprising an ENDOX-
responsive
regulatory element operatively linked to a nucleic acid encoding a detectable
marker, e.g.,
luciferase), or detecting a cellular response, for example, cell survival,
cellular differentiation,
or cell proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay
comprising
contacting an ENDOX protein or biologically-active portion thereof with a test
compound and
determining the ability of the test compound to bind to the ENDOX protein or
biologically-
active portion thereof. Binding of the test compound to the ENDOX protein can
be
determined either directly or indirectly as described above. In one such
embodiment, the assay
comprises contacting the ENDOX protein or biologically-active portion thereof
with a known
compound which binds ENDOX to form an assay mixture, contacting the assay
mixture with a
test compound, and determining the ability of the test compound to interact
with an ENDOX
protein, wherein determining the ability of the test compound to interact with
an ENDOX
protein comprises determining the ability of the test compound to
preferentially bind to
ENDOX or biologically-active portion thereof as compared to the known
compound.
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In still another embodiment, an assay is a cell-free assay comprising
contacting
ENDOX protein or biologically-active portion thereof with a test compound and
determining
the ability of the test compound to modulate (e.g. stimulate or inhibit) the
activity of the
ENDOX protein or biologically-active portion thereof. Determining the ability
of the test
compound to modulate the activity of ENDOX can be accomplished, for example,
by
determining the ability of the ENDOX protein to bind to an ENDOX target
molecule by one of
the methods described above for determining direct binding. In an alternative
embodiment,
determining the ability of the test compound to modulate the activity of ENDOX
protein can
be accomplished by determining the ability of the ENDOX protein further
modulate an
ENDOX target molecule. For example, the catalytic/enzymatic activity of the
target molecule
on an appropriate substrate can be determined as described, supra.
In yet another embodiment, the cell-free assay comprises contacting the ENDOX
protein or biologically-active portion thereof with a known compound which
binds ENDOX
protein to form an assay mixture, contacting the assay mixture with a test
compound, and
1 S determining the ability of the test compound to interact with an ENDOX
protein, wherein
determining the ability of the test compound to interact with an ENDOX protein
comprises
determining the ability of the ENDOX protein to preferentially bind to or
modulate the activity
of an ENDOX target molecule.
The cell-free assays of the invention are amenable to use of both the soluble
form or
the membrane-bound form of ENDOX protein. In the case of cell-free assays
comprising the
membrane-bound form of ENDOX protein, it may be desirable to utilize a
solubilizing agent
such that the membrane-bound form of ENDOX protein is maintained in solution.
Examples
of such solubilizing agents include non-ionic detergents such as n-
octylglucoside,
n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,
decanoyl-N-methylglucamide, Triton~ X-100, Triton~ X-114, Thesit~,
Isotridecypoly(ethylene glycol ether)", N-dodecyl--N,N-dimethyl-3-ammonio-1-
propane
sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS),
or
3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).
In more than one embodiment of the above assay methods of the invention, it
may be
desirable to immobilize either ENDOX protein or its target molecule to
facilitate separation of
complexed from uncomplexed forms of one or both of the proteins, as well as to
accommodate
automation of the assay. Binding of a test compound to ENDOX protein, or
interaction of
ENDOX protein with a target molecule in the presence and absence of a
candidate compound,
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can be accomplished in any vessel suitable for containing the reactants.
Examples of such
vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In
one embodiment, a
fusion protein can be provided that adds a domain that allows one or both of
the proteins to be
bound to a matrix. For example, GST-ENDOX fusion proteins or GST-target fusion
proteins
can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,
MO) or
glutathione derivatized microtiter plates, that are then combined with the
test compound or the
test compound and either the non-adsorbed target protein or ENDOX protein, and
the mixture
is incubated under conditions conducive to complex formation (e.g., at
physiological
conditions for salt and pH). Following incubation, the beads or microtiter
plate wells are
washed to remove any unbound components, the matrix immobilized in the case of
beads,
complex determined either directly or indirectly, for example, as described,
supra.
Alternatively, the complexes can be dissociated from the matrix, and the level
of ENDOX
protein binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the
screening assays of the invention. For example, either the ENDOX protein or
its target
molecule can be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated
ENDOX protein or target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well-known within the art (e.g.,
biotinylation kit,
Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well
plates (Pierce Chemical). Alternatively, antibodies reactive with ENDOX
protein or target
molecules, but which do not interfere with binding of the ENDOX protein to its
target
molecule, can be derivatized to the wells of the plate, and unbound target or
ENDOX protein
trapped in the wells by antibody conjugation. Methods for detecting such
complexes, in
addition to those described above for the GST-immobilized complexes, include
immunodetection of complexes using antibodies reactive with the ENDOX protein
or target
molecule, as well as enzyme-linked assays that rely on detecting an enzymatic
activity
associated with'the ENDOX protein or target molecule.
In another embodiment, modulators of ENDOX protein expression are identified
in a
method wherein a cell is contacted with a candidate compound and the
expression of ENDOX
mRNA or protein in the cell is determined. The level of expression of ENDOX
mRNA or
protein in the presence of the candidate compound is compared to the level of
expression of
ENDOX mRNA or protein in the absence of the candidate compound. The candidate
compound can then be identified as a modulator of ENDOX mRNA or protein
expression
based upon this comparison. For example, when expression of ENDOX mRNA or
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CA 02386199 2002-03-28
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greater (i.e., statistically significantly greater) in the presence of the
candidate compound than
in its absence, the candidate compound is identified as a stimulator of ENDOX
mRNA or
protein expression. Alternatively, when expression of ENDOX mRNA or protein is
less
(statistically significantly less) in the presence of the candidate compound
than in its absence,
the candidate compound is identified as an inhibitor of ENDOX mRNA or protein
expression.
The level of ENDOX mRNA or protein expression in the cells can be determined
by methods
described herein for detecting ENDOX mRNA or protein.
In yet another aspect of the invention, the ENDOX proteins can be used as
"bait
proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent
No. 5,283,317;
Zervos, et al., 1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. Chem.
268: 12046-12054;
Bartel, et al., 1993. Biotechniques 14: 920-924; Iwabuchi, et al., 1993.
Oncogene 8:
1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or
interact with
ENDOX ("ENDOX-binding proteins" or "ENDOX-by") and modulate ENDOX activity.
Such
ENDOX-binding proteins are also likely to be involved in the propagation of
signals by the
ENDOX proteins as, for example, upstream or downstream elements of the ENDOX
pathway.
The two-hybrid system is based on the modular nature of most transcription
factors,
which consist of separable DNA-binding and activation domains. Briefly, the
assay utilizes
two different DNA constructs. In one construct, the gene that codes for ENDOX
is fused to a
gene encoding the DNA binding domain of a known transcription factor (e.g.,
GAL-4). In the
other construct, a DNA sequence, from a library of DNA sequences, that encodes
an
unidentified protein ("prey" or "sample") is fused to a gene that codes for
the activation
domain of the known transcription factor. If the "bait" and the "prey"
proteins are able to
interact, in vivo, forming an ENDOX-dependent complex, the DNA-binding and
activation
domains of the transcription factor are brought into close proximity. This
proximity allows
transcription of a reporter gene (e.g., LacZ) that is operably linked to a
transcriptional
regulatory site responsive to the transcription factor. Expression of the
reporter gene can be
detected and cell colonies containing the functional transcription factor can
be isolated and
used to obtain the cloned gene that encodes the protein which interacts with
ENDOX.
The invention further pertains to novel agents identified by the
aforementioned
screening assays and uses thereof for treatments as described herein.
Detection Assays
Portions or fragments of the cDNA sequences identified herein (and the
corresponding
complete gene sequences) can be used in numerous ways as polynucleotide
reagents. By way
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of example, and not of limitation, these sequences can be used to: (i) map
their respective
genes on a chromosome; and, thus, locate gene regions associated with genetic
disease; (ii)
identify an individual from a minute biological sample (tissue typing); and
(iii) aid in forensic
identification of a biological sample. Some of these applications are
described in the
subsections, below.
Chromosome Mapping
Once the sequence (or a portion of the sequence) of a gene has been isolated,
this
sequence can be used to map the location of the gene on a chromosome. This
process is called
chromosome mapping. Accordingly, portions or fragments of the ENDOX sequences,
SEQ
ID NO: 1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46, and 48, or fragments or
derivatives thereof, can be
used to map the location of the ENDOX genes, respectively, on a chromosome.
The mapping
of the ENDOX sequences to chromosomes is an important first step in
correlating these
sequences with genes associated with disease.
Briefly, ENDOX genes can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 by in length) from the ENDOX sequences. Computer analysis of
the
ENDOX, sequences can be used to rapidly select primers that do not span more
than one exon
in the genomic DNA, thus complicating the amplification process. These primers
can then be
used for PCR screening of somatic cell hybrids containing individual human
chromosomes.
Only those hybrids containing the human gene corresponding to the ENDOX
sequences will
yield an amplified fragment.
Somatic cell hybrids are prepared by fusing somatic cells from different
mammals
(e.g., human and mouse cells). As hybrids of human and mouse cells grow and
divide, they
gradually lose human chromosomes in random order, but retain the mouse
chromosomes. By
using media in which mouse cells cannot grow, because they lack a particular
enzyme, but in
which human cells can, the one human chromosome that contains the gene
encoding the
needed enzyme will be retained. By using various media, panels of hybrid cell
lines can be
established. Each cell line in a panel contains either a single human
chromosome or a small
number of human chromosomes, and a full set of mouse chromosomes, allowing
easy
mapping of individual genes to specific human chromosomes. See, e.g.,
D'Eustachio, et al.,
1983. Science 220: 919-924. Somatic cell hybrids containing only fragments of
human
chromosomes can also be produced by using human chromosomes with
translocations and
deletions.
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PCR mapping of somatic cell hybrids is a rapid procedure for assigning a
particular
sequence to a particular chromosome. Three or more sequences can be assigned
per day using
a single thermal cycler. Using the ENDOX sequences to design oligonucleotide
primers, sub-
localization can be achieved with panels of fragments from specific
chromosomes.
Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase
chromosomal spread can further be used to provide a precise chromosomal
location in one
step. Chromosome spreads can be made using cells whose division has been
blocked in
metaphase by a chemical like colcemid that disrupts the mitotic spindle. The
chromosomes
can be treated briefly with trypsin, and then stained with Giemsa. A pattern
of light and dark
bands develops on each chromosome, so that the chromosomes can be identified
individually.
The FISH technique can be used with a DNA sequence as short as 500 or 600
bases.
However, clones larger than 1,000 bases have a higher likelihood of binding to
a unique
chromosomal location with sufficient signal intensity for simple detection.
Preferably 1,000
bases, and more preferably 2,000 bases, will suffice to get good results at a
reasonable amount
of time. For a review of this technique, see, Verma, et al., HUMAN
CHROMOSOMES: A
MANUAL of BASIC TECHNIQUES (Pergamon Press, New York 1988).
Reagents for chromosome mapping can be used individually to mark a single
chromosome or a single site on that chromosome, or panels of reagents can be
used for
marking multiple sites and/or multiple chromosomes. Reagents corresponding to
noncoding
regions of the genes actually are preferred for mapping purposes. Coding
sequences are more
likely to be conserved within gene families, thus increasing the chance of
cross hybridizations
during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the
physical
position of the sequence on the chromosome can be correlated with genetic map
data. Such
data are found, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN, available on-
line
through Johns Hopkins University Welch Medical Library). The relationship
between genes
and disease, mapped to the same chromosomal region, can then be identified
through linkage
analysis (co-inheritance of physically adjacent genes), described in, e.g.,
Egeland, et al., 1987.
Nature, 325: 783-787.
Moreover, differences in the DNA sequences between individuals affected and
unaffected with a disease associated with the ENDOX gene, can be determined.
If a mutation
is observed in some or all of the affected individuals but not in any
unaffected individuals,
then the mutation is likely to be the causative agent of the particular
disease. Comparison of
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affected and unaffected individuals generally involves first looking for
structural alterations in
the chromosomes, such as deletions or translocations that are visible from
chromosome
spreads or detectable using PCR based on that DNA sequence. Ultimately,
complete
sequencing of genes from several individuals can be performed to confirm the
presence of a
mutation and to distinguish mutations from polymorphisms.
Tissue Typing
The ENDOX sequences of the invention can also be used to identify individuals
from
minute biological samples. In this technique, an individual's genomic DNA is
digested with
one or more restriction enzymes, and probed on a Southern blot to yield unique
bands for
identification. The sequences of the invention are useful as additional DNA
markers for RFLP
("restriction fragment length polymorphisms," described in U.S. Patent No.
5,272,057).
Furthermore, the sequences of the invention can be used to provide an
alternative
technique that determines the actual base-by-base DNA sequence of selected
portions of an
individual's genome. Thus, the ENDOX sequences described herein can be used to
prepare
two PCR primers from the S'- and 3'-termini of the sequences. These primers
can then be used
to amplify an individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this
manner,
can provide unique individual identifications, as each individual will have a
unique set of such
DNA sequences due to allelic differences. The sequences of the invention can
be used to
obtain such identification sequences from individuals and from tissue. The
ENDOX
sequences of the invention uniquely represent portions of the human genome.
Allelic variation
occurs to some degree in the coding regions of these sequences, and to a
greater degree in the
noncoding regions. It is estimated that allelic variation between individual
humans occurs
with a frequency of about once per each 500 bases. Much of the allelic
variation is due to
single nucleotide polymorphisms (SNPs), which include restriction fragment
length
polymorphisms (RFLPs).
Each of the sequences described herein can, to some degree, be used as a
standard
against which DNA from an individual can be compared for identification
purposes. Because
greater numbers of polymorphisms occur in the noncoding regions, fewer
sequences are
necessary to differentiate individuals. The noncoding sequences can
comfortably provide
positive individual identification with a panel of perhaps 10 to 1,000 primers
that each yield a
noncoding amplified sequence of 100 bases. If predicted coding sequences, such
as those in
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SEQ ID NO:1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46, and 48, are used, a more
appropriate number
of primers for positive individual identification would be 500-2,000.
Predictive Medicine
The invention also pertains to the field of predictive medicine in which
diagnostic
S assays, prognostic assays, pharmacogenomics, and monitoring clinical trials
are used for
prognostic (predictive) purposes to thereby treat an individual
prophylactically. Accordingly,
one aspect of the invention relates to diagnostic assays for determining ENDOX
protein and/or
nucleic acid expression as well as ENDOX activity, in the context of a
biological sample (e.g.,
blood, serum, cells, tissue) to thereby determine whether an individual is
afflicted with a
disease or disorder, or is at risk of developing a disorder, associated with
aberrant ENDOX
expression or activity. The disorders include metabolic disorders, diabetes,
obesity, infectious
disease, anorexia, cancer-associated cachexia, , and the various
dyslipidemias., metabolic
disturbances associated with obesity, the metabolic syndrome X and wasting
disorders
associated with chronic diseases and various cancers. The invention also
provides for
prognostic (or predictive) assays for determining whether an individual is at
risk of developing
a disorder associated with ENDOX protein, nucleic acid expression or activity.
For example,
mutations in an ENDOX gene can be assayed in a biological sample. Such assays
can be used
for prognostic or predictive purpose to thereby prophylactically treat an
individual prior to the
onset of a disorder characterized by or associated with ENDOX protein, nucleic
acid
expression, or biological activity.
Another aspect of the invention provides methods for determining ENDOX
protein,
nucleic acid expression or activity in an individual to thereby select
appropriate therapeutic or
prophylactic agents for that individual (referred to herein as
"pharmacogenomics").
Pharmacogenomics allows for the selection of agents (e.g., drugs) for
therapeutic or
prophylactic treatment of an individual based on the genotype of the
individual (e.g., the
genotype of the individual examined to determine the ability of the individual
to respond to a
particular agent.)
Yet another aspect of the invention pertains to monitoring the influence of
agents (e.g.,
drugs, compounds) on the expression or activity of ENDOX in clinical trials.
These and other agents are described in further detail in the following
sections.


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Diagnostic Assays
An exemplary method for detecting the presence or absence of ENDOX in a
biological
sample involves obtaining a biological sample from a test subject and
contacting the biological
sample with a compound or an agent capable of detecting ENDOX protein or
nucleic acid
(e.g., mRNA, genomic DNA) that encodes ENDOX protein such that the presence of
ENDOX
is detected in the biological sample. An agent for detecting ENDOX mRNA or
genomic DNA
is a labeled nucleic acid probe capable of hybridizing to ENDOX mRNA or
genomic DNA.
The nucleic acid probe can be, for example, a full-length ENDOX nucleic acid,
such as the
nucleic acid of SEQ ID NO: 1, 3, S, 7, 9, 22, 25, 28, 31, 34, 46, and 48, or a
portion thereof,
such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides
in length and
sufficient to specifically hybridize under stringent conditions to ENDOX mRNA
or genomic
DNA. Other suitable probes for use in the diagnostic assays of the invention
are described
herein.
An agent for detecting ENDOX protein is an antibody capable of binding to
ENDOX
protein, preferably an antibody with a detectable label. Antibodies can be
polyclonal, or more
preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab
or F(ab')2) can be
used. The term "labeled", with regard to the probe or antibody, is intended to
encompass
direct labeling of the probe or antibody by coupling (i.e., physically
linking) a detectable
substance to the probe or antibody, as well as indirect labeling of the probe
or antibody by
reactivity with another reagent that is directly labeled. Examples of indirect
labeling include
detection of a primary antibody using a fluorescently-labeled secondary
antibody and
end-labeling of a DNA probe with biotin such that it can be detected with
fluorescently-
labeled streptavidin. The term "biological sample" is intended to include
tissues, cells and
biological fluids isolated from a subject, as well as tissues, cells and
fluids present within a
subject. That is, the detection method of the invention can be used to detect
ENDOX mRNA,
protein, or genomic DNA in a biological sample in vitro as well as in vivo.
For example, in
vitro techniques for detection of ENDOX mRNA include Northern hybridizations
and in situ
hybridizations. In vitro techniques for detection of ENDOX protein include
enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and
immunofluorescence. In vitro techniques for detection of ENDOX genomic DNA
include
Southern hybridizations. Furthermore, in vivo techniques for detection of
ENDOX protein
include introducing into a subject a labeled anti-ENDOX antibody. For example,
the antibody
can be labeled with a radioactive marker whose presence and location in a
subject can be
detected by standard imaging techniques.
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In one embodiment, the biological sample contains protein molecules from the
test
subject. Alternatively, the biological sample can contain mRNA molecules from
the test
subject or genomic DNA molecules from the test subject. A preferred biological
sample is a
peripheral blood leukocyte sample isolated by conventional means from a
subject.
In another embodiment, the methods further involve obtaining a control
biological
sample from a control subject, contacting the control sample with a compound
or agent
capable of detecting ENDOX protein, mRNA, or genomic DNA, such that the
presence of
ENDOX protein, mRNA or genomic DNA is detected in the biological sample, and
comparing
the presence of ENDOX protein, mRNA or genomic DNA in the control sample with
the
presence of ENDOX protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of ENDOX in a
biological sample. For example, the kit can comprise: a labeled compound or
agent capable of
detecting ENDOX protein or mRNA in a biological sample; means for determining
the
amount of ENDOX in the sample; and means for comparing the amount of ENDOX in
the
sample with a standard. The compound or agent can be packaged in a suitable
container. The
kit can further comprise instructions for using the kit to detect ENDOX
protein or nucleic acid.
Prognostic Assays
The diagnostic methods described herein can furthermore be utilized to
identify
subjects having or at risk of developing a disease or disorder associated with
aberrant ENDOX
expression or activity. For example, the assays described herein, such as the
preceding
diagnostic assays or the following assays, can be utilized to identify a
subject having or at risk
of developing a disorder associated with ENDOX protein, nucleic acid
expression or activity.
Alternatively, the prognostic assays can be utilized to identify a subject
having or at risk for
developing a disease or disorder. Thus, the" invention provides a method for
identifying a
disease or disorder associated with aberrant ENDOX expression or activity in
which a test
sample is obtained from a subject and ENDOX protein or nucleic acid (e.g.,
mRNA, genomic
DNA) is detected, wherein the presence of ENDOX protein or nucleic acid is
diagnostic for a
subject having or at risk of developing a disease or disorder associated with
aberrant ENDOX
expression or activity. As used herein, a "test sample" refers to a biological
sample obtained
from a subject of interest. For example, a test sample can be a biological
fluid (e.g., serum),
cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine
whether
a subject can be administered an agent (e.g., an agonist, antagonist,
peptidomimetic, protein,
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peptide, nucleic acid, small molecule, or other drug candidate) to treat a
disease or disorder
associated with aberrant ENDOX expression or activity. For example, such
methods can be
used to determine whether a subject can be effectively treated with an agent
for a disorder.
Thus, the invention provides methods for determining whether a subject can be
effectively
treated with an agent for a disorder associated with aberrant ENDOX expression
or activity in
which a test sample is obtained and ENDOX protein or nucleic acid is detected
(e.g., wherein
the presence of ENDOX protein or nucleic acid is diagnostic for a subject that
can be
administered the agent to treat a disorder associated with aberrant ENDOX
expression or
activity).
The methods of the invention can also be used to detect genetic lesions in an
ENDOX
gene, thereby determining if a subject with the lesioned gene is at risk for a
disorder
characterized by aberrant cell proliferation and/or differentiation. In
various embodiments, the
methods include detecting, in a sample of cells from the subject, the presence
or absence of a
genetic lesion characterized by at least one of an alteration affecting the
integrity of a gene
1 S encoding an ENDOX-protein, or the misexpression of the ENDOX gene. For
example, such
genetic lesions can be detected by ascertaining the existence of at least one
o~ (i) a deletion of
one or more nucleotides from an ENDOX gene; (ii) an addition of one or more
nucleotides to
an ENDOX gene; (iii) a substitution of one or more nucleotides of an ENDOX
gene, (iv) a
chromosomal rearrangement of an ENDOX gene; (v) an alteration in the level of
a messenger
RNA transcript of an ENDOX gene, (vi) aberrant modification of an ENDOX gene,
such as of
the methylation pattern of the genomic DNA, (vii) the presence of a non-wild-
type splicing
pattern of a messenger RNA transcript of an ENDOX gene, (viii) a non-wild-type
level of an
ENDOX protein, (ix) allelic loss of an ENDOX gene, and (x) inappropriate post-
translational
modification of an ENDOX protein. As described herein, there are a large
number of assay
techniques known in the art which can be used for detecting lesions in an
ENDOX gene. A
preferred biological sample is a peripheral blood leukocyte sample isolated by
conventional
means from a subject. However, any biological sample containing nucleated
cells may be
used, including, for example, buccal mucosal cells.
In certain embodiments, detection of the lesion involves the use of a
probe/primer in a
polymerise chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and
4,683,202), such
as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction
(LCR) (see, e.g.,
Landegran, et al., 1988. Science 241: 1077-1080; and Nakazawa, et al., 1994.
Proc. Natl.
Acid. Sci. USA 91: 360-364), the latter of which can be particularly useful
for detecting point
mutations in the ENDOX-gene (see, Abravaya, et al., 1995. Nucl. Acids Res. 23:
675-682).
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This method can include the steps of collecting a sample of cells from a
patient, isolating
nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample,
contacting the
nucleic acid sample with one or more primers that specifically hybridize to an
ENDOX gene
under conditions such that hybridization and amplification of the ENDOX gene
(if present)
occurs, and detecting the presence or absence of an amplification product, or
detecting the size
of the amplification product and comparing the length to a control sample. It
is anticipated
that PCR and/or LCR may be desirable to use as a preliminary amplification
step in
conjunction with any of the techniques used for detecting mutations described
herein.
Alternative amplification methods include: self sustained sequence replication
(see,
Guatelli, et al., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878),
trariscriptional amplification
system (see, Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177);
Q(3 Replicase
(see, Lizardi, et al, 1988. BioTechnology 6: 1197), or any other nucleic acid
amplification
method, followed ~by the detection of the amplified molecules using techniques
well known to
those of skill in the art. These detection schemes are especially useful for
the detection of
nucleic acid molecules if such molecules are present in very low numbers.
In an alternative embodiment, mutations in an ENDOX gene from a sample cell
can be
identified by alterations in restriction enzyme cleavage patterns. For
example, sample and
control DNA is isolated, amplified (optionally), digested with one or more
restriction
endonucleases, and fragment length sizes are determined by gel electrophoresis
and compared.
Differences in fragment length sizes between sample and control DNA indicates
mutations in
the sample DNA. Moreover, the use of sequence specific ribozymes (see, e.g.,
U.S. Patent No.
5,493,531) can be used to score for the presence of specific mutations by
development or loss
of a ribozyme cleavage site.
In other embodiments, genetic mutations in ENDOX can be identified by
hybridizing a
sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays
containing
hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et al.,
1996. Human
Mutation 7: 244-255; Kozal, et al., 1996. Nat. Med. 2: 753-759. For example,
genetic
mutations in ENDOX can be identified in two dimensional arrays containing
light-generated
DNA probes as described in Cronin, et al., supra. Briefly, a first
hybridization array of probes
can be used to scan through long stretches of DNA in a sample and control to
identify base
changes between the sequences by making linear arrays of sequential
overlapping probes.
This step allows the identification of point mutations. This is followed by a
second
hybridization array that allows the characterization of specific mutations by
using smaller,
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CA 02386199 2002-03-28
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specialized probe arrays complementary to all variants or mutations detected.
Each mutation
array is composed of parallel probe sets, one complementary to the wild-type
gene and the
other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in
the art
can be used to directly sequence the ENDOX gene and detect mutations by
comparing the
sequence of the sample ENDOX with the corresponding wild-type (control)
sequence.
Examples of sequencing reactions include those based on techniques developed
by Maxim and
Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl.
Acad. Sci. USA
74: 5463. It is also contemplated that any of a variety of automated
sequencing procedures
can be utilized when performing the diagnostic assays (see, e.g., Naeve, et
al., 1995.
Biotechni9ues 19: 448), including sequencing by mass spectrometry (see, e.g.,
PCT
International Publication No. WO 94/16101; Cohen, et al., 1996. Adv.
Chromatography 36:
127-162; and Griffin, et al., 1993. Appl. Biochem. Biotechnol. 38: 147-159).
Other methods for detecting mutations in the ENDOX gene include methods in
which
protection from cleavage agents is used to detect mismatched bases in RNA/RNA
or
RNA/DNA heteroduplexes. See, e.g., Myers, et al., 1985. Science 230: 1242. In
general, the
art technique of "mismatch cleavage" starts by providing heteroduplexes of
formed by
hybridizing (labeled) RNA or DNA containing the wild-type ENDOX sequence with
potentially mutant RNA or DNA obtained from a tissue sample. The double-
stranded
duplexes are treated with an agent that cleaves single-stranded regions of the
duplex such as
which will exist due to basepair mismatches between the control and sample
strands. For
instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids
treated with
S1 nuclease to enzymatically digesting the mismatched regions. In other
embodiments, either
DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium
tetroxide
and with piperidine in order to digest mismatched regions. ABer digestion of
the mismatched
regions, the resulting material is then separated by size on denaturing
polyacrylamide gels to
determine the site of mutation. See, e.g., Cotton, et al., 1988. Proc. Natl.
Acad. Sci. USA 85:
4397; Saleeba, et al., 1992. Methods Enzymol. 217: 286-295. In an embodiment,
the control
DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or
more
proteins that recognize mismatched base pairs in double-stranded DNA (so
called "DNA
mismatch repair" enzymes) in defined systems for detecting and mapping point
mutations in
ENDOX cDNAs obtained from samples of cells. For example, the mutt enzyme of E.
coli
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CA 02386199 2002-03-28
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cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells
cleaves T
at G/T mismatches. See, e.g., Hsu, et al., 1994. Carcinogenesis 15: 1657-1662.
According to
an exemplary embodiment, a probe based on an ENDOX sequence, e.g., a wild-type
ENDOX
sequence, is hybridized to a cDNA or other DNA product from a test cell(s).
The duplex is
treated with a DNA mismatch repair enzyme, and the cleavage products, if any,
can be
detected from electrophoresis protocols or the like. See, e.g., U.S. Patent
No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to
identify
mutations in ENDOX genes. For example, single strand conformation polymorphism
(SSCP)
may be used to detect differences in electrophoretic mobility between mutant
and wild type
nucleic acids. See, e.g., Orita, et al., 1989. Proc. Natl. Acad. Sci. USA: 86:
2766; Cotton,
1993. Mutat. Res. 285: 125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9: 73-
79.
Single-stranded DNA fragments of sample and control ENDOX nucleic acids will
be
denatured and allowed to renature. The secondary structure of single-stranded
nucleic acids
varies according to sequence, the resulting alteration in electrophoretic
mobility enables the
detection of even a single base change. The DNA fragments may be labeled or
detected with
labeled probes. The sensitivity of the assay may be enhanced by using RNA
(rather than
DNA), in which the secondary structure is more sensitive to a change in
sequence. In one
embodiment, the subject method utilizes heteroduplex analysis to separate
double stranded
heteroduplex molecules on the basis of changes in electrophoretic mobility.
See, e.g., Keen, et
al., 1991. Trends Genet. 7: 5.
In yet another embodiment, the movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed using
denaturing gradient
gel electrophoresis (DGGE). See, e.g., Myers, et al., 1985. Nature 313: 495.
When DGGE is
used as the method of analysis, DNA will be modified to insure that it does
not completely
denature, for example by adding a GC clamp of approximately 40 by of high-
melting GC-rich
DNA by PCR. In a further embodiment, a temperature gradient is used in place
of a
denaturing gradient to identify differences in the mobility of control and
sample DNA. See,
e.g., Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753.
Examples of other techniques for detecting point mutations include, but are
not limited
to, selective oligonucleotide hybridization, selective amplification, or
selective primer
extension. For example, oligonucleotide primers may be prepared in which the
known
mutation is placed centrally and then hybridized to target DNA under
conditions that permit
hybridization only if a perfect match is found. See, e.g., Saiki, et al.,
1986. Nature 324: 163;
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CA 02386199 2002-03-28
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Saiki, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 6230. Such allele specific
oligonucleotides
are hybridized to PCR amplified target DNA or a number of different mutations
when the
oligonucleotides are attached to the hybridizing membrane and hybridized with
labeled target
DNA.
Alternatively, allele specific amplification technology that depends on
selective PCR
amplification may be used in conjunction with the instant invention.
Oligonucleotides used as
primers for specific amplification may carry the mutation of interest in the
center of the
molecule (so that amplification depends on differential hybridization; see,
e.g., Gibbs, et al.,
1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one
primer where,
under appropriate conditions, mismatch can prevent, or reduce polymerase
extension (see, e.g.,
Prossner, 1993. Tibtech. 11: 238). In addition it may be desirable to
introduce a novel
restriction site in the region of the mutation to create cleavage-based
detection. See, e.g.,
Gasparini, et al., 1992. Mol. Cell Probes 6: 1. It is anticipated that in
certain embodiments
amplification may also be performed using Taq ligase for amplification. See,
e.g., Barany,
1991. Proc. Natl. Acad. Sci. USA 88: 189. In such cases, ligation will occur
only if there is a
perfect match at the 3'-terminus of the 5' sequence, making it possible to
detect the presence of
a known mutation at a specific site by looking for the presence or absence of
amplification.
The methods described herein may be performed, for example, by utilizing
pre-packaged diagnostic kits comprising at least one probe nucleic acid or
antibody reagent
described herein, which may be conveniently used, e.g., in clinical settings
to diagnose
patients exhibiting symptoms or family history of a disease or illness
involving an ENDOX
gene.
Furthermore, any cell type or tissue, preferably peripheral blood leukocytes,
in which
ENDOX is expressed may be utilized in the prognostic assays described herein.
However, any
biological sample containing nucleated cells may be used, including, for
example, buccal
mucosal cells.
Pharmacogenomics
Agents, or modulators that have a stimulatory or inhibitory effect on ENDOX
activity
(e.g., ENDOX gene expression), as identified by a screening assay described
herein can be
administered to individuals to treat (prophylactically or therapeutically)
disorders (The
disorders include metabolic disorders, diabetes, obesity, infectious disease,
anorexia, cancer-
associated cachexia, , and the various dyslipidemias., metabolic disturbances
associated with
obesity, the metabolic syndrome X and wasting disorders associated with
chronic diseases
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and various cancers.) In conjunction with such treatment, the pharmacogenomics
(i.e., the
study of the relationship between an individual's genotype and that
individual's response to a
foreign compound or drug) of the individual may be considered. Differences in
metabolism of
therapeutics can lead to severe toxicity or therapeutic failure by altering
the relation between
dose and blood concentration of the pharmacologically active drug. Thus, the
pharmacogenomics of the individual permits the selection of effective agents
(e.g., drugs) for
prophylactic or therapeutic treatments based on a consideration of the
individual's genotype.
Such pharmacogenomics can further be used to determine appropriate dosages and
therapeutic
regimens. Accordingly, the activity of ENDOX protein, expression of ENDOX
nucleic acid, or
mutation content of ENDOX genes in an individual can be determined to thereby
select
appropriate agents) for therapeutic or prophylactic treatment of the
individual.
Pharmacogenomics deals with clinically significant hereditary variations in
the
response to drugs due to altered drug disposition and abnormal action in
affected persons. See
e.g., Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol., 23: 983-985; Linder,
1997. Clin.
Chem., 43: 254-266. In general, two types of pharmacogenetic conditions can be
differentiated. Genetic conditions transmitted as a single factor altering the
way drugs act on
the body (altered drug action) or genetic conditions transmitted as single
factors altering the
way the body acts on drugs (altered drug metabolism). These pharmacogenetic
conditions can
occur either as rare defects or as polymorphisms. For example, glucose-6-
phosphate
dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the
main
clinical complication is hemolysis after ingestion of oxidant drugs (anti-
malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a
major
determinant of both the intensity and duration of drug action. The discovery
of genetic
polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT
2) and
cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to
why
some patients do not obtain the expected drug effects or show exaggerated drug
response and
serious toxicity after taking the standard and safe dose of a drug. These
polymorphisms are
expressed in two phenotypes in the population, the extensive metabolizer (EM)
and poor
metabolizer (PM). The prevalence of PM is different among different
populations. For
example, the gene coding for CYP2D6 is highly polymorphic and several
mutations have been
identified in PM, which all lead to the absence of functional CYP2D6. Poor
metabolizers of
CYP2D6 and CYP2C 19 quite frequently experience exaggerated drug response and
side
effects when they receive standard doses. If a metabolite is the active
therapeutic moiety, PM
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show no therapeutic response, as demonstrated for the analgesic effect of
codeine mediated by
its CYP2D6-formed metabolite morphine. At the other extreme are the so called
ultra-rapid
metabolizers who do not respond to standard doses. Recently, the molecular
basis of
ultra-rapid metabolism has been identified to be due to CYP2D6 gene
amplification.
S Thus, the activity of ENDOX protein, expression of ENDOX nucleic acid, or
mutation
content of ENDOX genes in an individual can be determined to thereby select
appropriate
agents) for therapeutic or prophylactic treatment of the individual. In
addition,
pharmacogenetic studies can be used to apply genotyping of polymorphic alleles
encoding
drug-metabolizing enzymes to the identification of an individual's drug
responsiveness
phenotype. This knowledge, when applied to dosing or drug selection, can avoid
adverse
reactions or therapeutic failure and thus enhance therapeutic or prophylactic
efficiency when
treating a subject with an ENDOX modulator, such as a modulator identified by
one of the
exemplary screening assays described herein.
Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e.g., drugs, compounds) on the expression
or
activity of ENDOX (e.g., the ability to modulate aberrant cell proliferation
and/or
differentiation) can be applied not only in basic drug screening, but also in
clinical trials. For
example, the effectiveness of an agent determined by a screening assay as
described herein to
increase ENDOX gene expression, protein levels; or upregulate ENDOX activity,
can be
monitored in clinical trails of subjects exhibiting decreased ENDOX gene
expression, protein
levels, or downregulated ENDOX activity. Alternatively, the effectiveness of
an agent
determined by a screening assay to decrease ENDOX gene expression, protein
levels, or
downregulate ENDOX activity, can be monitored in clinical trails of subjects
exhibiting
increased ENDOX gene expression, protein levels, or upregulated ENDOX
activity. In such
clinical trials, the expression or activity of ENDOX and, preferably, other
genes that have been
implicated in, for example, a cellular proliferation or immune disorder can be
used as a "read
out" or markers of the immune responsiveness of a particular cell.
By way of example, and not of limitation, genes, including ENDOX, that are
modulated in cells by treatment with an agent (e.g., compound, drug or small
molecule) that
modulates ENDOX activity (e.g., identified in a screening assay as described
herein) can be
identified. Thus, to study the effect of agents on cellular proliferation
disorders, for example,
in a clinical trial, cells can be isolated and RNA prepared and analyzed for
the levels of
expression of ENDOX and other genes implicated in the disorder. The levels of
gene
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expression (i.e., a gene expression pattern) can be quantified by Northern
blot analysis or
RT-PCR, as described herein, or alternatively by measuring the amount of
protein produced,
by one of the methods as described herein, or by measuring the levels of
activity of ENDOX
or other genes. In this manner, the gene expression pattern can serve as a
marker, indicative of
the physiological response of the cells to the agent. Accordingly, this
response state may be
determined before, and at various points during, treatment of the individual
with the agent.
In one embodiment, the invention provides a method for monitoring the
effectiveness
of treatment of a subject with an agent (e.g., an agonist, antagonist,
protein, peptide,
peptidomimetic, nucleic acid, small molecule, or other drug candidate
identified by the
screening assays described herein) comprising the steps of (i) obtaining a pre-
administration
sample from a subject prior to administration of the agent; (ii) detecting the
level of expression
of an ENDOX protein, mRNA, or genomic DNA in the preadministration sample;
(iii)
obtaining one or more post-administration samples from the subject; (iv)
detecting the level of
expression or activity of the ENDOX protein, mRNA, or genomic DNA in the
1 S post-administration samples; (v) comparing the level of expression or
activity of the ENDOX
protein, mRNA, or genomic DNA in the pre-administration sample with the ENDOX
protein,
mRNA, or genomic DNA in the post administration sample or samples; and (vi)
altering the
administration of the agent to the subject accordingly. For example, increased
administration
of the agent may be desirable to increase the expression or activity of ENDOX
to higher levels
than detected, i. e., to increase the effectiveness of the agent.
Alternatively, decreased
administration of the agent may be desirable to decrease expression or
activity of ENDOX to
lower levels than detected, i.e., to decrease the effectiveness of the agent.
Methods of Treatment
The invention provides for both prophylactic and therapeutic methods of
treating a
subject at risk of (or susceptible to) a disorder or having a disorder
associated with aberrant
ENDOX expression or activity. The disorders include metabolic disorders,
diabetes, obesity,
infectious disease, anorexia, cancer-associated cachexia, , and the various
dyslipidemias.,
metabolic disturbances associated with obesity, the metabolic syndrome X and
wasting
disorders associated with chronic diseases and various cancers. These methods
of treatment
will be discussed more fully, below.
Disease and Disorders
Diseases and disorders that are characterized by increased (relative to a
subject not
suffering from the disease or disorder) levels or biological activity may be
treated with
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Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics
that antagonize
activity may be administered in a therapeutic or prophylactic manner.
Therapeutics that may
be utilized include, but are not limited to: (i) an aforementioned peptide, or
analogs,
derivatives, fragments or homologs thereof; (ii) antibodies to an
aforementioned peptide; (iii)
nucleic acids encoding an aforementioned peptide; (iv) administration of
antisense nucleic acid
and nucleic acids that are "dysfunctional" (i. e., due to a heterologous
insertion within the
coding sequences of coding sequences to an aforementioned peptide) that are
utilized to
"knockout" endoggenous function of an aforementioned peptide by homologous
recombination (see, e.g., Capecchi, 1989: Science 244: 1288-1292); or (v)
modulators ( i.e.,
inhibitors, agonists and antagonists, including additional peptide mimetic of
the invention or
antibodies specific to a peptide of the invention) that alter the interaction
between an
aforementioned peptide and its binding partner.
Diseases and disorders that are characterized by decreased (relative to a
subject not
suffering from the disease or disorder) levels or biological activity may be
treated with
1 S Therapeutics that increase (i.e., are agonists to) activity. Therapeutics
that upregulate activity
may be administered in a therapeutic or prophylactic manner. Therapeutics that
may be
utilized include, but are not limited to, an aforementioned peptide, or
analogs, derivatives,
fragments or homologs thereof; or an agonist that increases bioavailability.
Increased or decreased levels can be readily detected by quantifying peptide
and/or
RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and
assaying it in vitro for
RNA or peptide levels, structure and/or activity of the expressed peptides (or
mRNAs of an
aforementioned peptide). Methods that are well-known within the art include,
but are not
limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation
followed by
sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis,
immunocytochemistry, etc.)
and/or hybridization assays to detect expression of mRNAs (e.g., Northern
assays, dot blots, in
situ hybridization, and the like).
Prophylactic Methods
In one aspect, the invention provides a method for preventing, in a subject, a
disease or
condition associated with an aberrant ENDOX expression or activity, by
administering to the
subject an agent that modulates ENDOX expression or at least one ENDOX
activity. Subjects
at risk for a disease that is caused or contributed to by aberrant ENDOX
expression or activity
can be identified by, for example, any or a combination of diagnostic or
prognostic assays as
described herein. Administration of a prophylactic agent can occur prior to
the manifestation
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of symptoms characteristic of the ENDOX aberrancy, such that a disease or
disorder is
prevented or, alternatively, delayed in its progression. Depending upon the
type of ENDOX
aberrancy, for example, an ENDOX agonist or ENDOX antagonist agent can be used
for
treating the subject. The appropriate agent can be determined based on
screening assays
S described herein. The prophylactic methods of the invention are further
discussed in the
following subsections.
Therapeutic Methods
Another aspect of the invention pertains to methods of modulating ENDOX
expression
or activity for therapeutic purposes. The modulatory method of the invention
involves
contacting a cell with an agent that modulates one or more of the activities
of ENDOX protein
activity associated with the cell. An agent that modulates ENDOX protein
activity can be an
agent as described herein, such as a nucleic acid or a protein, a naturally-
occurnng cognate
ligand of an ENDOX protein, a peptide, an ENDOX peptidomimetic, or other small
molecule.
In one embodiment, the agent stimulates one or more ENDOX protein activity.
Examples of
such stimulatory agents include active ENDOX protein and a nucleic acid
molecule encoding
ENDOX that has been introduced into the cell. In another embodiment, the agent
inhibits one
or more ENDOX protein activity. Examples of such inhibitory agents include
antisense
ENDOX nucleic acid molecules and anti-ENDOX antibodies. These modulatory
methods can
be performed in vitro (e.g., by culturing the cell with the agent) or,
alternatively, in vivo (e.g.,
by administering the agent to a subject). As such, the invention provides
methods of treating
an individual afflicted with a disease or disorder characterized by aberrant
expression or
activity of an ENDOX protein or nucleic acid molecule. In one embodiment, the
method
involves administering an agent (e.g., an agent identified by a screening
assay described
herein), or combination of agents that modulates (e.g., up-regulates or down-
regulates)
ENDOX expression or activity. In another embodiment, the method involves
administering an
ENDOX protein or nucleic acid molecule as therapy to compensate for reduced or
aberrant
ENDOX expression or activity.
Stimulation of ENDOX activity is desirable in situations in which ENDOX is
abnormally downregulated and/or in which increased ENDOX activity is likely to
have a
beneficial effect. One example of such a situation is where a subject has a
disorder
characterized by aberrant cell proliferation and/or differentiation (e.g.,
cancer or immune
associated disorders). Another example of such a situation is where the
subject has a
gestational disease (e.g., preclampsia).
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Determination of the Biological Effect of the Therapeutic
In various embodiments of the invention, suitable in vitro or in vivo assays
are
performed to determine the effect of a specific Therapeutic and whether its
administration is
indicated for treatment of the affected tissue.
In various specific embodiments, in vitro assays may be performed with
representative
cells of the types) involved in the patient's disorder, to determine if a
given Therapeutic exerts
the desired effect upon the cell type(s). Compounds for use in therapy may be
tested in
suitable animal model systems including, but not limited to rats, mice,
chicken, cows,
monkeys, rabbits, and the like, prior to testing in human subjects. Similarly,
for in vivo
testing, any of the animal model system known in the art may be used prior to
administration
to human subjects.
Prophylactic and Therapeutic Uses of the Compositions of the Invention
The ENDOX nucleic acids and proteins of the invention are useful in potential
prophylactic and therapeutic applications implicated in a variety of disorders
including, but not
limited to: metabolic disorders, diabetes, obesity, infectious disease,
anorexia, cancer-
associated cachexia, , and the various dyslipidemias., metabolic disturbances
associated with
obesity, the metabolic syndrome X and wasting disorders associated with
chronic diseases
and various cancers.
As an example, a cDNA encoding the ENDOX protein of the invention may be
useful
in gene therapy, and the protein may be useful when administered to a subject
in need thereof.
By way of non-limiting example, the compositions of the invention will have
efficacy for
treatment of patients suffering from: metabolic disorders, diabetes, obesity,
infectious disease,
anorexia, cancer-associated cachexia, , and the various dyslipidemias.
Both the novel nucleic acid encoding the ENDOX protein, and the ENDOX protein
of
the invention, or fragments thereof, may also be useful in diagnostic
applications, wherein the
presence or amount of the nucleic acid or the protein are to be assessed. A
further use could
be as an anti-bacterial molecule (i. e., some peptides have been found to
possess anti-bacterial
properties). These materials are further useful in the generation of
antibodies which
immunospecifically-bind to the novel substances of the invention for use in
therapeutic or
diagnostic methods.
EXAMPLES
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The following examples illustrate by way of non-limiting example various
aspects of the
invention.
A family of new human genes, related to the gene encoding human acyl-CoA
binding
protein (ACBP) / Diazepam- binding Inhibitor (DBI), was identified by an
analysis of
expressed sequences and genomic DNA sequences. See example 6. The human family
consists
of 7 novel and 3 known ENDO genes that all contain a highly conserved domain
of 20 amino
acids. ACBP/DBI is processed to produce a biologically active 18 amino acid
peptide (ODN)
that influences organismal energy metabolism. It is expected that biological
processing of the
other family members would lead to other metabolism-regulating peptides.
Synthetic peptides derived from the conserved domain of each human family
member,
were used in studies of metabolism and to generate polyclonal antibodies in
rabbits. The
synthetic peptides from the conserved domain of each human and a rat family
member were
injected into various mouse strains. The injected peptides elicited distinct
changes in
organismal energy metabolism that effect adipose stores, muscle mass, insulin
secretion,
glucose utilization and serum lipid levels (triglycerides and cholesterol).
See example 1.
Consensus sequences can be derived from the full-length proteins and
individual
peptides having specific metabolic effects. See example 2 and 3. These human
peptides,
peptides from non-human species, mutant peptides derived by rational or
combinatorial
changes based upon the consensus sequences, antibodies and small molecule
drugs that
interact with the full length proteins, peptides, processing enzymes and/or
receptors could have
important therapeutic value in the treatment of metabolic disorders. See
example 1 and 4 .
These disorders include anorexia, cancer-associated cachexia, obesity, Type I
and Type II
diabetes and the various dyslipidemias.
The invention is also useful as a marker for various cancer types. See example
S
Example 1 Peptide-Elicited Metabolic Effects in AKR Mice
A study was performed to determine the effect of daily ip doses (14 days) of
the
metabolic regulating peptides (MRPs) derived from the human and rat
endozepines on
metabolic parameters in AKR (obesity and diabetes prone) and C57B1/6 (control)
mice. This
example summarizes the effects of the peptides on AKR mice. Serum cholesterol,
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triglycerides, insulin and glucose were monitored along with body weight, body
weight
change, relative organ weights of liver, reproductive caudal abdominal fat
pads, pancreatic and
mesenteric tissue weights, quadriceps muscle weight, food and water intake.
Histopathologic
analysis was performed on selected tissue samples.
S
Peptides were injected into both AKR and C57B1/6 mice. The AKR mice are
heavier
and have higher serum glucose levels (particularly in response to
manipulation) than control
C57B1/6 mice. As such, the AKR mice serve as a polygenic model for the forms
of obesity
and type II diabetes prevalent in the human population. Table 29 summarizes
the metabolic
effects elicited by each MRP in the AKR mice. MRPs and a peptide with a
sequence
randomized from the amino acids found in ODN were given by intraperitoneal
injection at the
indicated dose in 1 % DMSO. Control animals were either not manipulated,
injected with 1
DMSO or injected with phosphate-buffered saline (PBS). The data in the Table
29 is
recorded separately for males and females where indicated and combined for
both sexes
otherwise. The change from baseline (pre-injection) on serum glucose is
reported in mg/dl.
The insulin is in microU/ml, the "relative" measures of mesenteric (males and
females) fat,
uterine fat (females) and quadriceps muscle are recorded as a percent of body
weight. The
other findings represent consistent findings for both males and females.
Statistically
significant findings (p<0.05) are shaded red (or dark gray) for increases
(increased glucose for
MRPI~g, MRP2zo, MRP3lg, MRP4,8, MRPSZO, MRP72o, and MRPlOzo; and increased
relative
muscle for MRP 1 2o and MRP 1 OZO) and blue-green (or light gray) for
decreases (decreased
glucose for MRP 1 Zo, MRP42o and MRP62o; decreased ending insulin for MRP 1
zo, MRP 1, g,
MRP2zo, MRP3~8, MRP4,g, MRP8zo, MRP102o and MRP1,$ (Rat); decreased relative
fat ratio
for MRP 1 Zo, MRPSZO, MRP 1 OZO and MRP 1 ~ g (Rat); and decreased relative
muscle for MRP22o
and MRP7zo).
A separate test group was established for each peptide examined, a vehicle
control and
a non- manipulated group. Each test group consisted of five male and five
female AKR mice
( 10 per peptide). The mice were acclimated for 2 days following shipment. The
mice were
weighed and divided into groups. Food weight and water volume per cage was
measured and
recorded.
The mice acclimated another 5 days. On day (-2) pre-injection glucose
measurements
were made utilizing a glucometer (Johnson & Johnson; One touch Sure Step).
Blood samples
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were obtained by, first, heating the mice under a heat lamp for approximately
5 minutes before
obtaining a drop of blood via the tail vein. Dosing of the various peptides
began on day (0).
Peptides were prepared identically and dosed at the same concentration
(O.OSmg/ml in PBS
with 1%DMSO, with a dose volume of lOml/kg) except as noted in Table 29. Stock
peptide
solutions were prepared by dissolving 2.5 mg of peptide in 0.5 ml of DMSO with
1-2 minutes
of sonication, if required, followed by the addition of O.SmI of sterile PBS
to each vial.
Dosing solutions were prepared weekly by the addition of O.SmI of the peptide
stock solution
to 24.5 mls of sterile PBS. All dose solutions were kept refrigerated
throughout the entire
study.
The mice were weighed and given daily ip injections of the various peptides or
vehicle
for 14 consecutive days. Every seven days food weight and water volume was
measured and
recorded per cage. Blood glucose measurements (via glucometer) were also taken
and
recorded every 7 days. Blood glucose measurements were taken between 8:00 and
10:00 am
and the animals were dosed between 9:00 and 11:00 am daily.
On day 14, one hour prior to necropsy and blood collection, the mice were
injected
with the final dose of the various peptides or vehicle. Blood (heparinized
plasma) was
obtained via cardiac or orbital puncture. Plasma chemistries, glucose and
insulin, as well as a
final glucometer reading were taken at the same time to calibrate/corroborate
the glucometer
readings. Animals were dissected and placed in formalin for histological
sample preparation.
Mesenteric fat and pancreas, liver, quadriceps muscle, and caudal abdominal
fat pads were
removed and weighed for each animal. This study illustrates that the ENDOX
peptides can be
used to modulate various metabolic functions and treat metabolic disorders.
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Table 29
Pepti Dose O Glucose Ending Relative Relative Other
Fat


de (mg/kg) (M /F ) Insulin (Mesenteric/Muscle Findings


Name (vehicle (M /F ) Uterine) (M /F
)


MRPlzo0. 5 r . ,


DMSO ~a


MRP1180.5 1.76f0.08 0.72f0.02toholesterol


(ODN) DMSO 1.44f0.21 0.6510.02
ttriglycerid


MRP2zp0.5 1.7210.15 icholesterol


DMSO i triglyceride
25
1
24f0


. iCPK iLiver
.


MRP3zo0.5 -3.6 (-3~)3.7010.32 1.95f0.10 0.7310.03lLiver


PBS +32.6 4.2810.98 1.74f0.27 0.6310.02


MRP3180.5 ~ 2.0110.04 0.6610.03


DMSO 1.5310.22 0.6310.02
v


MRP4zo0.1 3.3310.33 2.00f0.16 0.76f0.09


DMSO ~ 2.9010.17 1.56f0.22 0.61f0.05


MRP4ia0.5 1.73f0.09 0.68f0.09toholesterol


DMSO 1.16f0.18 0.63f0.02lLiver
rcPx


MRPSzo0.5 2.4610.32 0.7710.07


DMSO 2.4010.93 0.6910.03


MRP6zo0.5 2.9510.35 2.0410.12 0.7910.07


DMSO 2.70f0.80 1.9210.18 0.7910.05


MRP7zp0.5 2.3510.44 1.7010.08 ~ icholesterol


ttriglyceride
DMSO 1.8810.51 1.4710.36 ~CPK iLiver


q


MRP8zo0.5 +30.6 1.6610.25 0.7410.06icholesterol


DMSO (+26~) 1.99f0.45 0.62f0.02vtriglyceride


MRP9zo0.5 +28.6 4.08f1.72 1.74f0.09 0.70f0.03tcholesterol


DMSO (+22$) 2.6010.44 1.2410.14 0.6710.04


MRPlOz0.5 ~ ~ itCPK
~.
~ ~


p DMSO
u~


MRPlie0.5 +7.8 (+6$) 0.7910.06


DMSO +17.2 0.7110.04
x .


0.5 +99.9 1.4210.52 1.7010.08 0.6810.03


Rando DMSO (+40~) 0.6210.12 1.7010.09 0.6710.03


PBS +8.8 (+6~)3.9210.35 1.8910.15 0.7010.04


Vehic +25.1 3.5810.22 2.5010.32 0.7810.05


DMSO +40.2 2.7210.62 1.7710.08 0.7310.03


Vehic (+30~) 0.79f0.20 1.3110.15 0.6310.04


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Example 2 Clustal W Alignments
Table 30 shows the Clustal W alignment of all 10 human endozepines. The family
consists of
two classes of polypeptides - 7 short polypeptides of about 90 amino acids and
3 longer
polypeptides containing regions of homology to the other family members at
their N termini.
There is no homology between the longer forms at their C-termini. This
alignment and the
phylogenetic distances suggest that the family may have evolved by gene
duplications, fusions
and independent evolution.
Table 30
Multiple Alignment:
Endo1_MRP-6 1 ------------------------------------TASTPC K~SSSCAALI~Q G-- 22
Endo8_MRP-1 ACBP- 1 -----------------------MWGDLWLLPPASANPGGTE~ E~RH - 35
Endol_MRP-9 1 _________________________________________SQA ~ KDfTfH E -- 17
Endo3_MRP-3 1 -----MAKPISTKNTKISRHGWHAAVITAAREAEAENHLWEEKKKK RCAG~I".KHF - 53
Endo4_MRP-4 1 -------------------------------MLLLFVCLFF(L~K ~ (~- E~ RK A-- 27
Endo2_MRP-5 1 ---------------------------------------MS~t~Q °MVTED
K - 19
EndoS_MRP-7 1 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
_ _ _ _ _ _ _ MA;Q~ E IiK P - - 19
Endo9_MRP-2 PECI_ 1 --------------------------------------MRASQKb NSMNQ kL K--
20
EndolO_MRP-10 ELP 1 MASSFLPAGAITGDSGGELSSGDDSGEVEFPHSPEIEETS~C;GAEL AHIQG IQ--
58
Endo6_MRP-8 1 -MFQFHAGSWESWCCCCLIPADRPWDRGQHWOLEMADTRS~VHETR A VKV'QS PKNG 59
Enda1_ MRP-6 23 - t~
- 77


Endo8_ MRP-1 36 - 90
ACBP -


Endo7_ MRP-9 18 - -72
-


Endo3_ MRP-3 54 - MKA~
- 110


Endo4_ MRP-4 28 - TS
- 82


Endo2_ MRP-5 20 - -
- M
~
74


EndoS_ MRP-7 20 - TS
. V
- 75


Endo9_ MRP-2 21 - RON
PEC1 - 75


Endo10 MRP-1 59 - QE
D_ELP - 113


. Endo6- _ 60 F I
_ S ~
MRP-8 116


Endo1_ MRP-6 78 AATK E-_________.______________________________________

89


Endo8_ MRP-1 91 N KK ~'$G----------------------------------------------

ACBP_ 104


Endo7 MRP-9 73 N RNK tR______________________________________________

D 86


_ MRP-3 111 K KKFARRETGIVASHAFVLN--------------------------------

Endo3_ 138


Endo4_ MRP-4 83 S AK IEY~G______________________________________________

96


Endo2_ MRP-5 75 S A IEYG______________________________________________

88


EndoS_ MRP-7 76 S AK -------------------------------------------------

S PIE 86


Endo9_ MRP-2 76 VDLSS
PECI SPSLESSSQVEPGTDRKSTGFETLVVTSEDGITKIMFNRPKKKNAINTEMYH

135


Endo10 MRP-10 114 AVKK
ELP DPGY11NPQIPEKKG-KEANTGFGGPVISSLYHEETIR---EEDKNIFDYCREN

169


Endo6 _ 117 EEKKK
MRP-B IETMPTEKVEELLRVIGPFYEIVEDKKSGRS5D1TSVRLEKISKCLEDLGN

176


-
lO



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WO 01/25436 PCT/US00/41077
Endo1_MRP-6 ~ __._________...._..____.__._._._______.___..__._._._..____
Endo8_MRP-1 ACBP ~* __._______......__._________.________________________.____
Endol_MRP-9 -~ _._____.____.__.__.____.____._____..______..___.._.__.______
.~.,.
Endo3_MRP-3 *** ...____.__......____________._____..___________._____.____
Endo4 MRP-4 ~ _.____..____......____.._.....___...._____...__....__._.__
Enda2-MRP-5 ~* _._____.___..._.__._________._____..___________._____.____
EndoS_MRP-7 ~ __..._.____.______._________..___...__...___.___..._._.___
Endo9_MRP-2 PECI_ 136 E IMRALKAASKDD51 I TVLTGNGDYYSSGNDLTNFTD I
PPGGVEEKAKNNAVLLREFVGC 195
Endo10_MRP-10 ELP 170 N I DH I TKA I KSKNVDVNVKDEE GRALLHWACDRGHKE LVTVLLQHRAD
I NCQDNEGQTAL 229
Endo6 MRP-8 177 VLTSTPNAKTVNGKAESSDSGAESEEEEAQEEVKGAEHSDNDKKMMKKSADHKNLEVIVT
236
Endo1_MRP-6 ~ _.__._..._..._._____________...._.__..____...__._._____.__
EndoB_MRP-1 ACBP_ ~ ___._.____.......______._.__________.__.___._________.____
Endo7_MRP-9 ~ ___...___...______..._____________..___.._........._.__...
Endo3_MRP-3 H* ___....._........_._.____._...______.__.___.______________
Endo4_MRP-4 *** _._...._._..._____..______________..___.__..___._..__._.__
Endo2_MRP-5 ~ .__..______._..___..___.__._.._.__...__.__.______.._______
Endo5_MRP-7 ~ _._.______________._________._______.__.__.._____.___.____
Endo9_MRP-2 PECI 196
FIDFPKPLIAVVNGPAVGISVTLLGLFDAVYASDRATFHTPFSHLGQSPEGCSSYTFPK1 255
Endo10_MRP-10 ELP 230 HYASACEFLDIVELLLQSGADPTLRDQDGCLPEEVTGCKTVSLVLQRHTTGKA----
--- 282
Endo6 MRP-8 237 NGYDKDGFVaDIQNDIHASSSLNGRSTEEVKPIDENLGQTGKSAVCIHQGINDDHVEDVT
296
Endo1_MRP-6 ~ ___________.._________.._...________....._.._._______._.._
Endo8_MRP-1 ACBP_ ~ ___...____.__.____..._..__._.__.____.._..___________._____
Endo7_MRP-9 x** .._________.__._.__________.._______.__..______._.__.._.__
Endo3_MRP-3 ~ ___.__________...__._________..__....__._____.._..________
Endo4_MRP-4 ~ .._.__.___.______......__.________.._______..._______..._.__ ..x
Endo2_MRP-5 ~ .._________....________._.________...__..___._.._.__._____
Endo5_MRP-T ~ .._...._____._.___..._.._...______...______...________..._
Endo9_MRP-2 PECI 256
MSPAKATEMLIFGKKLTAGEACAQGLVTEVFPDSTFQKEVWTRLKAFAKLPPNALRISKE 315
Endo10_MRP-10ELP ~**
_.______.__...._.__.___.____.__.____________._______._____
Endo6 MRP-8 297 GIQHLTSDSDSE-VYCDSMEQFGQEESLDSFTSNNGPFQYYLGGHSSQPMENSGFREDIQ
355
Endo1_MRP-6 ~* __...________..__.________.__________._.________________._
Endo8_MRP-1 ACBP_ ~ ___....__.____......._.______.._..._.._.______.___._______
Endo7_MRP-9 ~* _......._..___....._________._______.._.__.___.___.__.____
Endo3_MRP-3 ~ ____..________..__.__________._...._.._______..__.._______
Endo4_MRP-4 ~ __________.__._________.__._._______.._______.._________._
Endo2_MRP-5 ~ ___..._._________...._.___________._..____________..______
EndoS_MRP-1 ~ _________..__..________.__._________.._______..___._______
Endo9_MRP-2 PEC1 316 VIRKREREKLHAVNAEECNVLQGRWLSDECTNAVVNFLSRKSKL--------------
-- 359
Endo10_MRP-10ELP ~ ___.__..........______._..._________.._______..__._____.._
Endo6 MRP-8 356 VPPGNGNIGNMQVVAVEGKGEVKHGGEDGRNNSGAPHREKRGGETDEFSNVRRGRGHRMQ
415
Endo1_MRP-6 ~ ________._._._.____._..__...__.__..__._.__._________._____
EndoB_MRP-1 ACBP_ ~ ________._.________._.___________.___._.___.__________._._
Endo7_MRP-9 ~ ._._______.__...___.__.__._.__.______._.___.________._____
Endo3_MRP-3 ~ ________.__________._.________..___________._______..__...
Endo4_MRP-4 ~ ___________......__._____._.._._________.__._______.._____
Endo2_MRP-5 ~*~~ .._________..._______....__._______.___.._______.__..___._
EndoS_~MRP-7 ~* _.___.___._____.__......._____..___._______.________.__.____
..~
Endo9_MRP-2 PECI ~ _._________..________..._._____.___.__________._.__.._____
Endo10_MRP-10ELP ~* _________._.__..._.___.___._.______________________.._____
Enda6 MRP-8 416 HLSEGTKGRaVGSGGDGERWGSDRGSRGSLNE01ALVLMRLQEDMQNVLQRLQKLETLTA
475
Endo1_MRP-6 ~ __________._________.________..__.__.____....._______._
EndoB_MRP-1ACBP ~__________.._..___.______......_________.__________..__
Endo7_MRP-9 ~ _______._..________.._______..._________._.___.___.....
Endo3_MRP-3 ~ _______....__..__.__........__.___._..._.____._.__.____
Endo4_MRP-4 *~ __________.______....______._.______..____.___.______._
Endo2_MRP-5 ~ __..._____.____.__....__.____.__._.__________________..
Endo5_MRP-7 ~ __...________.______.._.._._____..._.._._____..________
Endo9_MRP-2 PECI ~ __...._.._.________...._..______..__.__________________
Endo10_MRP-10ELP m _____________________.___.________________..__.____....
Endo6 MRP-8 476 AKSSTSTLQTAPQPTSSQRPSWWPFEMSPGVLTFAIIWPFIAQWLVYLYYQRRRR 530
Example 3 Consensus Sequences for the Metabolism - Regulating Peptides
All of the MRPs and several subsets of peptides, that have similar metabolic
effects in AKR
mice were identified in Table 29. These can be aligned to identify consensus
sequences which
114


CA 02386199 2002-03-28
WO 01/25436 PCT/US00/41077
are identified in Table 31. The general and preferred consensus sequences are
deduced by
inspection. The most conserved amino acid at each position in the 20 amino
acid motif is in
bold print. If that amino acid is invariant in a particular metabolic subset
then it is also
underlined. If the amino acid at a particular position is highly variable it
is represented by an
"X." When a small number of amino acids are found at a particular position
then they are
enclosed within [brackets]. The "general" consensus allows for the most
variability in a
peptide sequence that could have similar metabolic effects. The "preferred"
consensus
sequence is limited to the amino acids found in the inspected peptide subset.
Particular
subsets of peptides were identified that are associated with cholesterol-
lowering properties, fat
mass reducing, insulin-lowering, glucose lowering, glucose-raising and muscle
mass building
properties
Table 31
Consensus Sequences for the Metabolism - Regulating Peptides
Global Alignment:
25
EndoB-MRP11 ~ R FT 20
T


Endo3-MRP31 R N Ft ' F 20
KT


Endo2-MRPS1 '~(~ N _ EL 20
t GSE ~


EndoS-MRP71 I I ~,' F 20
YL


Endo4-MRP41 I I ~RC ~ 20


Endol-MRP91 H L' R L 20
T


EndolO-MRP10 1 _ PSFF FE 20
NG
T


Endo9-MRP21 E PC P4C IN 20
F


Endo6-MRP81 E PGK~t~SR P 20
F I
~ R


Endo1-MRP61 Q CDIPG RAFT 20
PAS



Global
Consensus
Sequence:


QAT[V/I/E]G[D/N/P][I/L/C][N/K][I/L
/M/T][E/S/P][K/R]PGMLD[L/F]KG[K/R]
(SEQ
ID



N0:37)
Cholesterol - Lowering Peptides:
EndoS-MRP7 1 I I ~D I =(~ YL F 20
Endol-MRP9 1 HD L T R 20
Endo9-MRP2 1 P CPK F I N 20
Endv6-MRP8 1 P K[~SR FU4~P I F~ 20
Cholesterol-Lowering Consensus Sequence:
General SAT[E/V/I]G[D/P][C/I/L][N/K]X[E/X]XP[G/R][M/X[[L/X]_D[L/X]XG[K/R] (SEQ
ID N0:38)
PreferredQATEG[DP]C[KRN][AITVFLM]X[KR]PG[AITVFLM][WAITVFLM]D[PAITVFLM]IX[KR]
(SEQ
ID N0:39)
115


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WO 01/25436 PCT/US00/41077
Adipose - Lowering Peptides:
EndoB-MRP1 1 D R~G 20
Endo2-MRP5 1 [~1, I CSE L 20
Endo10-MRP10 1 C PK~S FF E 20
Adipose-Lowering Consensus Sequence:
General QAX[VI]GNIN[T/I]EXPXML[DE]FXGK (SEQ ID N0:40~
Preferred Q[AITVFLM]X[AITVFLM]G[DEN]XNXEXXX [AITVFLM][DE]XXGK
(SEQ ID N0:41
Insulin - Lowering Peptides:
EndoB-MRP1 1 D I E L 20
Endo3-MRP3 1 R N I K E V ~ 20
Endo4-MRP4 1 I D I I AC L ~L 20
Endo10-MRP10 1 NC Pl~ S F F ~ E 20
Endo9-MRP2 1 E PC MPK VF ELI N 20
Endo6-MRP8 1 E PCKLS F PI R 20
Insulin-Lowering Consensus Sequence:
QATVG[D/N/P][I/C]N[T/I/M/L]X[R/K]PG[M/X]XD[F/L]XG[K/R] (SEQ ID N0:42)
Glucose - Lowering Peptides:
EndoB-MRP1 1 ER' FT 20
Endo4-MRP4 1 I C LK~ 20
Endo1-MRP6 1 Q CD PG PAS ,;RArFt 20
Glucose-Lowering Consensus Sequence:
QATVGD[I/C]NIXXP[G/P][M/A][L/S]DXX[G/A][K/R] (SEQ ID N0:43)
Glucose - Raising Peptides:
Endo2-MRPS -I~ ~~~-~
1 CSE 20


EndoS-MRP71 YL 20
I
!>
D


Endo3-MRP31 KTR 20
R


Endo9-MRP21 ~P~ ~F 20
E _ LI
PC N
1 ~
!


Endo10-MRP101 P,~ FF 20
4C S E
T



Glucose-Raising sensus
Con Sequence:


QAXXG[N/D/P][I/C]N[T/I/M][E/P]X[P/X][G/X][M/X]XD[F/L][K/X]GK (SEQ ID N0:44)
45
Muscle Mass Raising Peptides:
'~ EndoB-MRP1 1 ~D I ~ER~GML~2~
EndolO-MRP10 1 NC PK SFF~E 20
116


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Muscle-Mass Raising Consensus Sequence:
General QXXVGXXNTX(R/K)PXXXDFXGK
Preferred Q[AITVFLM]XVG[DEN]XNTX[RK]PXX[AITVFLM]DFXGK (SEQ
ID N0:45)
Example 4 Effects on Mesenteric Adipocyte Size
Figure 1 consists of 6 photomicrographs of mesenteric adipose fixed from male
AKR mice
after treatment with the agent indicated beneath each panel. In the top panels
(A-C) large
adipocytes predominate whereas in the bottom panels (D-F) smaller adipocytes
predominate.
This correlates with the decrease in the mass of that particular adipose
deposit in response to
the specific treatment. There are subsets of MRPs that affect the mass of
specific adipose
deposits and subsets of MRPs that have influence on other metabolic
parameters.
Example 5 Gene Expression, Cloning & Antibody Production Summary
Table 32 lists the members of the endozepine family (EndoX) and the
corresponding bio-
active Metabolism-Regulating Peptide (MRP-#) synthesized from each endozepine.
In
addition, the tissues) where the gene is most highly expressed is indicated
from the result of
real-time quantitative RTQ-PCR (TaqMan) using total RNA as well as by
traditional PCR
using ds cDNA as a template. PCR products corresponding to the coding regions
of the
various endozepines have been amplified for cloning purposes as noted. Anti-
peptide
antibodies have been generated in rabbits for each human synthetic 20 amino
acid peptide.
Anti-peptide titers that have been determined (ELISA) are indicated.
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Table 32
Gene
Fxpression
Dist<ib~rtlon


Polycl~al
Antisera


Bio-Alive Peptide traditional production
Seq RT-


Bio-Active raqAAan
- Principal
Tissues


Peptide



20 - mars Normal ~ issue Ptrysical w9~ rter
Tissues Product
For


TissuesDistritxrtionaoning
or aortal


~I~~ebrain
GAs


F~doU~lial Heart
cells


Liver


ParxreasYes Antibody
Availade


Skeletal
Muscle


Small
Intestine-


MRP5QA~~Z~K n on Pdipose


Yes AntibodyAvailade


Fetal
Brain


Fetal
Liver


Er>do3 MRP3 Adipose


Skeletal Brain
Mode


Liver


Pars
Yes 2,000


Skeletal
Mode


Small
Intestine


Yes A~ntibodyAvailade


6cpression


Antibody
Availade


Endo6 MF~-8 S~Ivl~sdeMultipleBran


Liver Yes 13,000


M~sde


Endo7 MRP-9~ AdiposeOver ip.


Fetal Yes 12000
Brain


Fetal
Liver


~g _1 mrrF.r~c~o~x Heart BreastBrain
CA -


(Aa3P SkeletalCdon Liver
I DBI Mode CA
I


a~(.~ Liver Mdanorrra Yes 14.000


Endotf~lial
cells


-_ ~_2__~fi~xi?GVrvLiritc Bran
_


(PEa) user


(D2,D3 SkeletalYes 3,600
FAA Muscle


CoA
Isomerase) -


-10 x>SSFF~~x Fetal
Brain


(ELP) Foal 1
Liver
~


- . - ___ Y __
-- ._


18 - mars ,


Er>do8 MRP-isQA -


(ACeP (a~rq
I oel
I


-


l~t MRP-Rn.
CDN


_ _ ~~ I
. _ g~T.p~3TI~D~


Example 6 Phylogenetic Relationships Among the Novel and Known Endozepines
S Table 33 indicates the relatedness of the various full-length endozepines as
well as the MRPs
encoded within each endozepine. The distances are computed by the program
"Phylip" that
calculates neighbor joining distances, a method for describing the relative
relatedness of the
input sequences.
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Table 33
Full Protein
Neighbor-joining method
Negative branch lengths allowed
+Hndo8 MAP
+--6
! ! +----Hndo7 MRP
! +_____1
! +----------------Hndo3 HRP
! -
! +__________________________________gndo9_HAP
--8---5
, +_______________________-___________________gndol0 MR
I +------------------Hndol MRP
+______p
I 1 +_____________________________________gndo6 HRP
! +---Hndo4_HRP
+--3
+------4 +------HndoS_HRP
!
+----Hndo2 HRP
Metabolism - Regulating Peptide Sequence Only
Neighbor-joining method
Negative branch lengths allowed
+--------Hndo2-HRP5
+---4
+---5 +--HndoS-HRP7
! !
+--6 +Hndo4-HRP4
! !
! +----------8ndo7-HRD9
! +------------HndolO-MRP
! !
__g__________________3 +_____gndo9-HAP2
! ! +__________1
+------2 +------------------8ndo6-HAPB
! !
+___________________________gndol-HRP6
!
! +Hndo8-HAP1
+--7
+-------Hndo3-HAP3
Example 7 Identification of Related Peptides In Other Proteins
Tables 34 and 35 illustrate how the specific consensus motifs can be used to
identify
polypeptides in other animal species that may be involved in the regulation of
the same
metabolic parameter. The GenBank database was searched using two general
consensus
sequences, the first associated with muscle mass building (Table 34) and the
second associated
with fat mass reduction (Table 35). The consensus sequences identified in each
peptide are
shown in bold font. Many polypeptides from multiple species were identified
with 0, 1 or 2
mismatches from the consensus sequence. The highest similarity is seen in
ACBPs from other
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CA 02386199 2002-03-28
WO 01/25436 PCT/US00/41077
species. This is also seen in Figure 1, which illustrates that orthologous
sequences can be
found for 6 of the 10 members of the human endozepine family.
S TABLE 34
Muscle-Raising Motif
Pattern Submitted: 'qxxvgxxntx[kr)pxxxdfxgk'
Database Analyzed: Non-Redundant Composite
Matching Sequences To Show: Top 50
Mismatches Allowed: 3
Allow Insertions/Deletions: no
1$ Allow matches to database ambiguities: no
Database contains 553,883 sequences.
Reporting all 29 matching sequences (did not trigger cutoff of 50 matches).
There are 4 equivalence classes of equally good matches.
SWISSPROT-ACC:P07107 ACYL-COA-BINDING PROTEIN (ACBP) (DIAZEPAM BINDING
INHIBITOR) (DBI) (ENDOZEPINE) (EP) - Bos taurus (Bovine), 86 aa.
2$ # Mismatches Match Position Match Context
0 33-52 IYSHYKQATVGDINTERPGMLDFKGKAKWDAW
SWISSPROT-ACC:P07108 ACYL-COA-BINDING PROTEIN (ACBP) (DIAZEPAM BINDING
INHIBITOR) (DBI) (ENDOZEPINE) (EP) - Homo sapiens (Human), 86 aa.
## Mismatches Match Position Match Context
0 33-52 IYGHYKQATVGDINTERPGMLDFTGKAKWDAW
SPTREMBL-ACC:Q9VLS4 CG8498 PROTEIN - Drosophila melanogaster (Fruit fly),
90 aa..
# Mismatches Match Position Match Context
0 35-54 LYSLYKQATVGDCNTDKPGFLDFKGKAKWEAW
SPTREMBL-ACC:Q9PRL8 ACYL-COENZYME A BINDING PROTEIN, ACBP - Gallus gallus
(Chicken), 86 aa.
# Mismatches Match Position Match Context
0 33-52 VYSHYKQATVGDVNTDRPGMLDFKGKAKWDAW
REMTREMBL-ACC:CAA44618 ACYL-COA-BINDING PROTEIN /DIAZEPAM-BINDING INHIBITOR
- synthetic construct, 87aa
# Mismatches Match Position Match Context
0 34-53 IYSHYKQATVGDINTERPGMLDFKGKAKWDAW
a:"T~TTTT endozepine [validated] - human, 87 aa.
# Mismatches Match Position Match Context
0 34-53 IYGHYKQATVGDINTERPGMLDFTGRAKWDAW
pir-id:S63593 acyl-coenzyme A-binding protein - turtle, 86 aa.
SS # Mismatches Match Position Match Context
0 33-52 IYSHFKQATVGDINTERPGFLDFKGKAKWDAW
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CA 02386199 2002-03-28
WO 01/25436 PCT/US00/41077
pir-id:S63594 acyl-coenzyme A-binding protein - mallard, 86 aa.
# Mismatches Match Position Match Context
S 0 33-52 VYSHYKQATVGDVNTDRPGMLDFRGKAKWDAW
P31786 ACYL-COA-BINDING PROTEIN (ACBP) (DIAZEPAM BINDING INHIBITOR) (DBI)
(ENDOZEPINE) (EP) - Mus musculus (Mouse), 86 aa.
# Mismatches Match Position Match Context
1O 1 33-52 IYSHFKQATVGDVNTDRPGLLDLRGKAKWDSW
SWISSPROT-ACC:P12026 ACYL-COA-BINDING PROTEIN (ACBP) (DIAZEPAM BINDING
INHIBITOR) (DBI) (ENDOZEPINE) (EP) [CONTAINS: DBI(32-86)] '- Sus scrofa
(Pig), 86 aa.
1S ## Mismatches Match Position Match Context
1 33-52 IYSHYKQATVGDINTERPGILDLRGRAKWDAW
Table 35
Identification of Related Peptides In Other Proteins
Adipose-Lowering Motif
Pattern Submitte~'..' ' qax [vi ] gnin [ti] expxml [de] fxgk'
Database Analyzed: Nor.-nPdundant Composite
Matching Sequences To Show: Top 50
Mismatches Allowed: 3
Allow Insertions/Deletions: no
Allow matches to database ambiguities: no
Database contains 553,883 sequences.
Reporting all 14 matching sequences (did not trigger cutoff of 50
matches).
3' There are 3 equivalence classes of equa).ly good matches.
4O
SWISSPROT-ACC:P07107 ACYL-COA-BINDING PROTEIN (ACBP) (DIAZEPAM BINDING
INHIBITOR) (DBI) (ENDOZEPINE) (EP) - Bos taurus (Bovine), 86 aa.
# Mismatches Match Position Match Context
1 33-52 IYSHYKQATVGDINTERPGMLDFRGRAKWDAW
SWISSPROT-ACC:P07108 ACYL-COA-BINDING PROTEIN (ACBP) (DIAZEPAM BINDING
4S INHIBITOR) (DBI) (ENDOZEPINE) (EP) - Homo sapi~°ns (Human), 86 aa.
# Mismatches Match Position Match Context
1 33-52 IYGHYKQATVGDINTERPGMLDFTGRAKWDAW
SO REMTREMBL-ACC:CAA44618 ACYL-COA-BINDING PROTEIN /DIAZEPAM-BINDING INHIBITOR
- synthetic construct,87aaaa.
# Mismatches Match Position Match Context
1 3 4-53 IYSHYKQATVGDINTERPGMLDFRGRAKWDAW
SS pir-id:NZHU endozepine [validated] - human, 87 aa.
# Mismatches Match Position Match Context
1 34-53 IYGHYKQATVGDINTERPGMLDFTGKAKWDAW
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CA 02386199 2002-03-28
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SWISSPROT-ACC:P45882 ACYL-COA-BINDING PROTEIN (ACBP) (DIAZEPAM BINDING
INHIBITOR) (DBI) (ENDOZEPINE) (EP) - Anas platyrhynchos (Domestic duck),
103 aa.
# Mismatches Match Position Match Context
S 2 50-69 LYGFYKQATVGDINIECPGMLDLRGKAKWEAW
pir-id:S63593 acyl-coenzyme A-binding protein - turtle, 86 aa.
# Mismatches Match Position Match Context
1O 2 33-52 IYSHFKQATVGDINTERPGFLDFRGRAKWDAW
Example 8 Orthologous Gene Products in Multiple Species
1 S Table 36 illustrates the high degree of conservation of the MRP motifs
across diverse species.
The human MRP sequences were used to search GenBank for related sequences.
Eleven other
sequences with a high degree of similarity were identified. Using a larger
peptide centered on
the 20 amino acid MRP domain will identify additional polypeptides. The 11
entries from
other species are represented by their accession numbers. The indicated set of
21 peptides
20 from species ranging from frogs to humans was examined for their
relationship to the set of 10
human peptides by the PHYLIP, PILEUP and Clustal W algorithms. As can be seen
in
Table 36, there are non-human peptides that are more like some human sequences
than other
human sequences. This suggests that the peptide-containing genes arose and
followed
divergent paths in energy metabolism early in evolution.
2S
122


CA 02386199 2002-03-28
WO 01/25436 PCT/US00/41077
Table 36
Orthologous Gene Products in Multiple Species
PHYLIP
+-HndoB-MRP1
+-18
! t------Hndo3-MRP3
!
! +--------Hndo2-MRPS
i +___g
! -10 +--BndoS-MRP7
! !
! +-1 tHndo4-MRP4
! !
! +-16 -U09205
! ! !
! ! +---------Bndo7-MRP9
! !
! ! +U04823
! ! +____7
! ! ! +X75596
! ! !
+--9 +------- --HndolO-MRP
i i i i i
! ! ! ! ! +------AF006493
! ! ! ! ! +___2
19-17 ! +------------6 ! +AF153613
! ! ! ! +-- ---4
! ! ! ! ! ! +----Hndo9-MRP2
! ! +-11 ! ! +--3
! ! ! ! +____5 ! +--M15888
! ! ! ! ! +__________________1
! ! ! ! ! +Hndo6-MRP8
! ! ! ! !
! ! +-14 ! +__ __________-____________gndol-MRP6
i i i i i
! ! ! ! +AL096874
! ! ! !
! +-15 ! +X61431
! ! +-12
! ! +M20268
! !
! +AB019792
!
+M15886
Table 37 also illustrates the high degree of conservation of the MRP motifs
across diverse
species.
123


CA 02386199 2002-03-28
WO 01/25436 PCT/US00/41077
Table 37
PILEUP CLUSTAL W
u09205 M15886 1 20


endo4mrp4 Endo8-MRP1 1 20


endo2mrp5 Endo3-MRP3 1 20


endo5mrp7 Endo2-MRP5 1 20


u04823 EndoS-MRP7 1 20


x75596 Endo4-MRP4 1 20


a1096874 U09205 1 20


m15886 U04823 1 20


endo8mrp1 X75596 1 20


x61431 AL09687d 1 20


m20268 X61431 1 20


ab019792 M20268 1 20


endo3mrp3 AB019792 1 20


endolmrp9 Endo7-MRP9 1 20


m15888 Endo10-MRP10 1 20


endo6mrp8 AFD06493 1 20


af006493 AF153613 1 20


af153613 Endo9-MRP2 1 20


endo9mrp2 M15888 1 20


endo10mrp10 Endo6-MRP8 1 20


endol mrp6 Endo1-MRP6 1 20


Example 9 PCR Products Of Endozepine Coding Sequences
The coding sequence of each human endozepine was cloned into numerous
expression vectors
in order to examine the complex ways in which these secreted proteins and
their biologically
active MRPs affect energy metabolism. Some of the cDNAs have been cloned in
bacteria in
order to prepare recombinant proteins. The complete set of proteins were
expressed in
bacteria, yeast and mammalian cells to study their individual biochemical
activities, protein-
protein interactions and how they modulate energy metabolism in isolated cells
or whole
animals. Physical DNA molecules for this purpose were generated by PCR from
the various
tissues, noted above in Table 29, and can be seen on this ethidium bromide-
stained agarose gel
(Figure 2). The individual PCR products for Endos 1-4, and Endos 6-10 are of
the expected
size as deduced from the sequences (including 42 by of flanking cloning
sequence here).
Other Embodiments
It is to be understood that while the invention has been described in
conjunction with
the detailed description thereof, the foregoing description is intended to
illustrate and not limit
the scope of the invention, which is defined by the scope of the appended
claims. Other
aspects, advantages, and modifications are within the scope of the following
claims.
124

Representative Drawing
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Administrative Status

Title Date
Forecasted Issue Date Unavailable
(86) PCT Filing Date 2000-10-05
(87) PCT Publication Date 2001-04-12
(85) National Entry 2002-03-28
Examination Requested 2005-08-24
Dead Application 2007-10-05

Abandonment History

Abandonment Date Reason Reinstatement Date
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2006-10-05 FAILURE TO PAY APPLICATION MAINTENANCE FEE

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Application Fee $300.00 2002-03-28
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Request for Examination $800.00 2005-08-24
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Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
CURAGEN CORPORATION
Past Owners on Record
EISEN, ANDREW
MAJUMDER, KUMUD
PRAYAGA, SUDHIRDAS K.
SHIMKETS, RICHARD A.
SPADERNA, STEVEN K.
VERNET, CORINE
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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