Note: Descriptions are shown in the official language in which they were submitted.
WO 94121660 PCT/US94/02609
21~83~
TITLE OF THE INVENTION
CLONED HUMAN ALPHAlC ADREN~RGIC 2ECEPTOR
.. :
CROSS REFERENCES TO RELATED APPLICATIONS
This is a continll~tion-in-part of U.S. Application serial
number 08/032,849, filed on March 15, 1993, pending.
BACKGROUND OF THE ~VENTION
i. Field of the Invention:
This invention relates to a method for defining the potency
and selectivity of compounds for use as hllm~n alphalC adrenergic
receptor antagonists using cloned hllm~n alpha 1 receptors. The
invention also relates to the cloned receptors themselves, to compounds
15 identified according to the method of this invention, and to methods of
use of such compounds, alone or in combination with other agents.
Particularly preferred are combinations of compounds identified
according to this invention and testosterone 5-alpha reductase inhibitors,
to alleviate pathologic conditions, particularly benign prostatic
20 hyperplasia (also known as benign prostatic hypertrophy, BPH).
ii. Background:
Human adrenergic receptors are integral membrane
proteins which have been classified into two broad classes, the alpha and
25 the beta adrenergic receptors. Both types mediate the action of the
peripheral sympathetic nervous system upon binding of catechol~mines,
norepinephrine and epinephrine.
Norepinephrine is produced by adrenergic nerve endings,
while epinephrine is produced by the adrenal medulla. The binding
30 affinity of adrenergic receptors for these compounds forms one basis of
the classification: alpha receptors bind norepinephrine more strongly
than epinephrine and much more strongly than the synthetic compound
isoproterenol. The binding affinity of these hormones is reversed for
the beta receptors. In many tissues, the functional responses, such as
WO 94/21660 2 ~ ~ ~ 3 4 5 PCTIUS94tO2609
smooth muscle contract`ion, induced by alpha receptor activation are
opposed to responses induced by beta receptor binding.
Subsequently, the functional distinction between alpha and
beta receptors was further highlighted and refined by the
pharmacological characterization of these receptors from various
~nim~l and tissue sources. As a result, alpha and beta adrenergic
receptors were further subdivided into a1, a2, ~1, and ~2 subtypes.
Functional differences between oc1 and a2 receptors have been
recognized, and compounds which exhibit selective binding between
these two subtypes have been developed. Thus, in WO 92/0073, the
selective ability of the R(+) enantiomer of terazosin to selectively bind
to adrenergic receptors of the alpha 1 subtype was reported. The al/a2
selectivity of this compound was disclosed as being significant because
agonist stim~ tion of the a2 receptors was said to inhibit secretion of
epinephrine and norepinephrine, while antagonism of the a2 receptor
was said to increase secretion of these ho~nones. Thus, the use of non-
selective alpha-adrenergic blockers, such as phenoxyben7~mine and
phentol~mine, is limited by their a2 adrenergic receptor mediated
induction of increased plasma catecholamine concentration and the
attendant physiological sequelae (increased heart rate and smooth muscle
contraction).
For a general background on the a-adrenergic receptors,
the reader's attention is directed to Robert R. Ruffolo, Jr., ~c-
Adrenoreceptors: Molecular Biolo~y~ Biochemistry and Pharmacolo~y.
(Progress in Basic and Clinical Ph~rm~colo~y series, Karger, 1991),
wherein the basis of ocl/a2 subclassification, the molecular biology,
signal transduction (G-protein interaction and location of the significant
site for this and ligand binding activity away from the 3'-terminus of
alpha adrenergic receptors), agonist structure-activity relationships,
receptor functions, and therapeutic applications for compounds
exhibiting a-adrenergic receptor affinity was explored.
The cloning, sequencing and expression of alpha receptor
subtypes from ~nim~l tissues has led to the subclassification of the al
receptors into a1A, (Lomasney, et al., J. Biol. Chem.. ~:6365-6369
WO 94121660 2 1 5 8 3 ~ ~ PCT/US94/02609
(1991), rat a1A; Bruno et al., BBRC. 179:1485-1490 (1991), hllm~n
a1A)~ alg (Cotecchia, et al., PNAS. 85;7159-7163 (1988), hamster
alg; Libert, et al., Science. (1989), dog a1g; Ramarao, et al., J. Biol.
Chem.. 267:21936-21945 (1992), hllm~n alB)~ and most recently, in a
5 study using bovine brain, a new alC subtype was proposed (Schwinn, et
al.. J. Biol. Chem.. 265:8183-8189, 1990; Hirasawa et al., BBRC
195:902-909 (1993), desc~ibed the cloning, functional expression and
tissue distribution of a hllm~n alC adrenergic receptor; Hoehe et al.,
Human Mol. Genetics 1(5):349 (8/92) noted the existence of a two-allele
o Pstl restriction fragment polymorphism in the a1C adrenergic receptor
gene; another study suggests that there may even be an alpha-lD
receptor subtype, see Perez et al., Mol. Pharm.., 40:876-883, 1992).
Each al receptor subtype exhibits its own pharmacologic and tissue
specificities. Schwinn and coworkers noted that the cloned bovine alC
15 receptor exhibited ph~rm~cological properties proposed for the alA
subtype. Nonetheless, based on its non-expression in tissues where the
alA subtype is expressed, and its sensitivity to chloroethylclonidine, the
receptor was given a new designation.
The differences in the a-adrenergic receptor subtypes have
20 relevance in pathophysiologic conditions. Benign prostatic
hypertrophy, BPH, is an illness typically affecting men over fifty years
of age, increasing in severity with increasing age. The symptoms of the
condition include, but are not limted to, increased difficulty in urination
and sexual dysfunction. These sylllpLollls are induced by enlargement,
25 or hypertrophy, of the prostate gland. As the prostate increases in size,
it impinges on free-flow of fluids through the male urethra.
Concommitantly, the increased noradrenergic innervation of the
enlarged prostate leads to an increased adrenergic tone of the bladder
neck and urethra, further restricting the flow of urine through the
30 urethra.
The mech~ni~m of prostatic hypertrophy is well
understood. The male hormone, 5a-dihydrotestosterone has been
identified as the principal culprit. The contiml~l production of 5a-
dihydrotestosterone by the male testes induces incremental growth of
WO 94/21660 ~ 15 8 3 ~ 5 PCT/US94/02609
the prostate gland throughout the life of the male. Beyond the age of
about fifty years, in many-men, this enlarged gland begins to obstruct
the urethra with the pathologic s~lllptolns noted above.
The elucidation of the mech~ni~m sllmm~rized above has
5 resulted in the recent development of effective agents to control, and in
many cases reverse, the pernicious advance of BPH. In the forefront of
these agents is Merck & Co., Inc.s' product PROSCAR(~) (finasteride).
The effect of this compound is to inhibit the enzyme testosterone 5-
alpha reductase, which converts testosterone into Sa-dihydrotesterone,
resulting in a reduced rate of prostatic enlargement, and often reduction
in prostatic mass.
The development of such agents as PROSCAR(~ bodes well
for the long-teFm control of BPH. However, as may be appreciated
from the lengthy development of the syndrome, its reversal also is not
15 immediate. In the interim, those males ~urrt;lillg with BPH continue to
suffer, and may in fact lose hope that the agents are working sufficiently
rapidly.
In response to this problem, one solution is to identify
ph~rm~reutically active compounds which complement slower-acting
20 therapeutics by providing acute relief. Agents which induce relaxation
of the urethral smooth muscle, by binding to alpha-1 adrenergic
receptors, thus reducing ~e increased adrenergic tone due to the
disease, would be good c~n~l1d~tes for this activity. Thus, one such
agent is alfuzosin, which is reported in EP 0 204597 to induce urination
25 in cases of prostatic hypertrophy. Likewise, in WO 92/0073, the
selective ability of the R(+) enantiomer of terazosin to bind to
adrenergic receptors of the al subtype was reported. In addition, in
WO 92/161213, hereby incorporated by reference, combinations of 5-
alpha-reductase inhibitory compounds and alphal-adrenergic receptor
blockers (terazosin, doxazosin, prazosin, bunazosin, indoramin,
3 alfuzosin) were disclosed. However, no information as to the alA,
a1B, or a1C subtype specificity of these compounds was provided as
these refinements were not yet available. The instant invention changes
this situation by providing a cloned hllm~n a1C adrenergic receptor and
WO 94/21660 PCT/US94/02609
2i583~5
a method for idellliryillg compounds which bind the hllm~n oc1c
receptor. ~ `
Typically, identification of active compounds is through use
of ~nim~l tissues known to be enriched in adrenergic receptors. Thus,
rat tissues have been used to screen for potential adrenergic receptor
antagonists. However, bec~llce of species variability, compounds which
appear active in ~nim~l tissue may not be active or sufficiently selective
in hllm~n~. This results in substantial wastage of time and effort,
particularly where high volume compound screening programs are
employed. There is also the danger that compounds, which might be
highly effective in hllm~nc, would be missed because of their absence of
appreciable affinity for the heterologous ~nim~l receptors. In this
regard, it has been noted that even single amino acid changes between
the sequence of biologically active proteins in one species may give rise
to substantial ph~rm~cological differences. Thus, Fong et al., (J. Biol.
Chem.~ 267:25668-25671, 1992) showed that there are 22 divergent
amino acid residues between the sequence of the hllm~n neurokinin-l
receptor and the homologous rat receptor. They further showed, in
studies with mllt~nt receptors, that substitution of only two amino acid
residues was both necessary and sufficient to reproduce the rat
receptor's antagonist binding ar~ iLy in the hllm~n receptor. Oksenberg
et al., (Nature. 360:161-163, 1992) showed that a single amino-acid
difference confers major pharmacological variation between the hllm~n
and the rodent 5-hydroxytrypt~mine receptors. Likewise, Kuhse et al.,
(Neuron. 5:867-~73, 1990) showed that a single amino-acid exçh~n~e
alters the ph~rm~cology of the neonatal rat glycine receptor subunit.
This difficulty and unpredictability has resulted in a need for a
compound screen which will identify compounds that will be active in
hllm~n.c.
The in~t~nt inventors have solved these problems by
cloning a novel hllm~n adrenergic receptor of the a1C subtype. Their
efforts have led to the development of a novel screening assay which
enables them to identify compounds which specifically interact with the
hllm~n o~lC adrenergic receptor. Marshall et al (Br. J. Pharm.,
WO 94/21660 PCTNS94/02609
21583~ ~
107:327 (1992)) speculated that compounds which specifically interact
with the alC adrenergic receptor may be responsible for contraction of
the hllm~n prostate. The instant invention provides a method for
identifying compounds which bind ~e hllm~n alC receptor. In
addition, if the compounds are further tested for binding to other 1.
hllm~n alpha 1 receptor subtypes, as well as counterscreened against
other types of receptors, the specificity of the compounds for the hllm~n
alC adrenergic receptor may be defined.
Compounds identified according to this invention may be
used to reduce the acute symptoms of BPH. New agents identified in
this m~nnt~.r, or already known agents showing activity in this assay,
may now be employed in a novel way to help BPH ~ufrerers contend
with the acute symptoms of the syndrome. Thus, this invention is useful
to identify compounds which may be used alone or in conjunction with a
lS more long-term anti-BPH therapeutics, such as PROSCAR(~. Other
uses for ~e invention include identi~lcation of compounds which induce
highly tissue-specific, localized alC adrenergic receptor blockade.
Effects of ~is blockade include reduction of intra-ocular pressure,
control of cardiac arrhythmias, and possibly a host of alpha-lC receptor
mediated central nervous system events. In addition, the cloned alC
receptor can be used for screening of tissue specific expression of ~lC
adrenergic receptors. Effects such as these, induced by or available to
analysis with the alC adrenergic receptor also form part of this
invention.
2s
SUMMARY OF THE INVENTION
The hllm~n adrenergic receptor of the alphalC subtype is
cloned and used in an in vitro assay to screen for compounds that bind
to the receptor, including compounds which specifically inhibit the
activity of the receptor. The invention includes the assay, the cloned
receptor used in the assay (cDNA), an isolated hllm~n alphalC
adrenergic receptor, cells expressing the cloned receptor, and
compounds identified through the use of this novel, cloned receptor,
which selectively bind to the hllm~n alphalC adrenergic receptor,
WO 94/21660 2 ~ ~ 8 3 ~ 5 PCT/US94/02609
including specific antagonists of the receptor. One embodiment of this
invention is a method of treating benign prostatic hypeplasia (BPH)
employing compounds having an afrll~ily for the hllm~n alpha lC
receptor that is at least 12 fold greater than for either the hllm~n alpha
s lA or the hllm~n alpha lB receptors.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1: Sequence of cDNA obtained by PCR of hllm~n heart mRNA,
SEQ. ID:4:.
Fig. 2: Comparison of the open reading frame obtained from hllm~n
heart, SEQ ID:5:, and the bovine alpha-lC adrenergic receptor
sequence, SEQ. ID:6:.
Fig. 3: Sequence of cDNA obtained by screenin~ a hllm~n hippocampus
cDNA library using the heart mRNA derived sequence from figure 1,
SEQ. ID:7:
20 Fig. 4: Sequence of 3' coding region of hllm~n alpha-lC gene, obtained
by PCR amplification of a h~m~n genomic DNA library with
oligonucleotides, SEQ. ID:10:.
Fig. 5: Sequence of the ligated portions of hllm~n alpha-lC DNA
25 shown in figures 3 and 4, SEQ. ID:ll:.
Fig. 6: The amino acid sequence of the hllm~n alpha-lC adrenergic
receptor, SEQ ID:12:.
30 Fig. 7: The ~li nment of the nucleotide and amino acid sequence of the
hllm~n alpha-lC adrenergic receptor, showing the 5'-untranslated
region, SEQ. ID:ll: and SEQ. ID:12:.
WO 94121660 21~ 8 3 ~ ~; PCT/US94/02609
Fig. 8: Expression of the hllm~n alpha-lC adrenergic receptor in COS
cells: Binding data using membranes from cells transfected with the
expression vector alone and ~e expression vector Cont~inin.~; the hl!m~n
alpha-lC adrenergic receptor coding sequences.
Fig. 9: Binding curves of compounds using membranes from COS cells
transfected with the hllm~n alpha-lC adrenergic receptor Cont~inin~
expression vector.
o Fig. 10: Nucleotide sequence of the hllm~n alphalA receptor, SEQ.
ID:13:
Fig. 11: Amino acid sequence of the hllm~n alphalA adrenergic
receptor, SEQ. ID:14:
Fig. 12: Partial sequence of ~e hllm~n alphalB adrenergic receptor,
SEQ. ID:17:
Fig. 13: Partial sequence of the hllm~n alphalB adrenergic receptor,
SEQ. ID-20-
-
Pig. 14: Partial sequence of the hllm~n alphalB adrenergic receptor,
SEQ. ID:23:
Fig. 15: Composite hllm~n/rat alphalB adrenoreceptror, SEQ. ID:24:
Fig. 16: Amino acid sequence of ~e composite hl-m~n/rat alphalB
adrenergic receptor, SEQ. ID:25:
Fig. 17: Binding curves of compounds using membranes from COS
cells transfected wi~ the hllm~n alphalA, lB, and lC adrenergic
receptor expression vectors.
WO 94/21660 PCT/IJS94/02609
~1~834~
Pig. 18: Sequence of truncated hllm~n alphalC adrenergic receptor,
SEQ. ID:26:.
Fig. 19: Nucleotide sequence of the hllm~n a1C adrenergic receptor
5 having a Pstl site, SEQ.ID:27:.
Fig. 20: Amino acid sequence of the hllm~n a1C adrenergic receptor
encoded by the Pstl site encoding allele, SEQ.ID:28:.
Fig. 21: Alignment of the nucleotide and amino acid sequences of
figures 19 and 20, SEQ.ID:27: and SEQ.ID:28:.
FIG. 22: Nucleotide sequence of the hllm~n a1A adrenergic receptor,
Seq.ID:29:.
Fig. 23: Amino acid sequence of the hllm~n a1A adrenergic receptor,
SEQ.ID:30:.
Fig. 24: Alignment of the nucleotide and amino acid sequences of
20 figures 22 and 23, SEQ.ID:29: and SEQ.ID:30:.
DETAILED DESCRIPTION OF THE INVENTION
The hllm~n alpha adrenergic receptor of the 1-C subtype
was i(lentified, cloned and expressed by the instant inventors. A partial
25 coding region for this receptor was generated by reverse transcriptase-
polymerase chain reaction technology, RT-PCR. Accordingly,
degenerate oligonucleotides encoding amino acids conserved in the fifth
and sixth transmembrane domains of all three al receptor subtypes (A,
B, C) were used to prime RT-PCR reactions using hllm~n heart mRNA
30 as template. The predicted sized products were cloned and sequenced.
Tr~n~l~tion of the amplified cDNA yielded an open reading frame
encoding a protein 95~o homologous to the bovine alC receptor (Fig.2,
SEQ. ID:~: and SEQ. ID:6:). This partial sequence was used to obtain a
larger cDNA clone from a hllm~n hippocampus library (Fig. 3, SEQ.
WO 94/21660 PCT/US94102609
~S~3~ ~
-- 10 --
ID:6:). The rem~inin~ coding region was ~btained by PCR
amplification of hllm~n genomic DNA using primers based on the
cDNA sequence and the last six amino acids of bovine alC receptor
(Fig. 4, SEQ.ID:10:). The complete receptor was then assembled using
5 the partial sequences shown in Fig. 3, SEQ.ID:6: and Fig. 4, SEQ.
ID:10:, to generate the sequence shown in Fig. 5, SEQ. ID:11:. The
translation of this sequence is shown in Fig. 6, SEQ. ID:12:, and the
~lignment of the nucleotide and amino acid sequences, and the 5'-
untranslated sequences, is shown in Fig. 7, SEQ. ID: 11: and SEQ.
ID-12-
- The 3'-terminal six amino acids of the hllm~n a1C
adrenergic receptor were confirmed by screening a hllm~n genomic
library with the radiolabeled 3'-terminal 512 nucleotides of the SEQ.
ID:10: clone previously obtained. A complete hllm~n exon 2 was
15 generated in this manner and sequenced. The nucleotide sequence of
this gene is provided in figure 19, SEQ. ID:27: and the amino acid
sequence is provided in figure 20, SEQ. ID:28:. We discovered that this
clone was identical to the original 3'-terminal portion of the gene,
except that:
2 0 1 ) There are five silent nucleotide changes between the new clone and
the previously obtained clone (the last f;ve codons, including the stop
codon, each have a silent change in the third nucleotide); and
2) At nucleotide position 1636 (amino acid 347), there is a cytosine to
thymine base change resul~ng in the formation of a Pstl site at that
25 location and a concommit~n~ single amino acid change of Arg to Cys.
Thus, we have confirmed and localized the site of the two-allele Pstl
restriction fragment polymoIphism (RFLP) noted by Hoehe et al.,
~Human Mol. Genetics. 1 (5):349 (8/92)]. Through ph~ cological
studies using clones of both alleles, we have confirmed that the Arg to
0 Cys change appear to be ph~rm~rologically indistinguishable (see Table
II, Example 11, below).
The cloned hl-m~n alC receptor, when expressed in
m~mm~ n cell lines (see Fig. 8), is used to discover ligands that bind
to ~e receptor and alter its function. In addition, the cloned al C
WO 94/21660 PCT/US94/02609
~1 2158~5
receptor enables quanititation of mRNA levels in hllm~n tissues,
including the aorta and prostate, by RNase protection assays. For these
purposes, a complete coding sequence of the receptor is provided.
However, as long as the ligand binding and signal transduction segments
5 of the receptor (G-protein interaction) are intact, truncation at the 3'
end of the sequence does not affect the functioning of the receptor.
Thus, in addition to the sequence provided in SEQ. ID: 11:, a sequence,
truncated at the 3' end, SEQ. ID:26: is disclosed, which consists entirely
of human alphalC sequence.
The specificty of binding of compounds showing affinity
for the alC receptor is shown by comparing affinity to membranes
obtained from COS cells tranfected with the cloned alC receptor and
membranes from tissues known to express other types of alpha or beta
adrenergic receptors. In addition, the cloned hllm~n alA and a hybrid
hllm~n/rat alB (with only the cytoplasmic, carboxy termin~l region
being rat sequence) could be used for this purpose, along with the
hllm~n alC receptor expressed in COS cells . Expression of the cloned
hllm~n alA, alB, and alC receptors and comparison of their binding
properties with known selective antagonists provides a rational way for
20 selection of compounds and discovery of new compounds with
predictable ph~nn~cological activities.
Once the hllm~n receptor is cloned and expressed in a cell
such as COS cells or CHO cells, the receptor is free of other hllm~n
proteins. The membranes from cells expressing different hllm~n alpha
25 adrenergic receptor subtypes are then isolated according to methods
well known in the art for membrane associated receptor binding assays.
For example, the method of Schwinn, et al., (J. Biol. Chem.. 265:8183-
8189, 1990) may be used. A compound of interest is used to compete
with the binding of a known, qll~ntifi~ble alpha receptor ligand. Thus,
30 radiolabled prazosin, niguldipine, 5-methyl urapidil, terazosin,
dozazosin, phenoxyben7~mine, WB4101, benox~thi~n, HEAT (2-[~-(4-
hydroxy-3-iodophenyl)ethylaminomethyl]tetralone, or phentol~mine
may be used for this purpose (see, for example, Robert R. Ruffolo, Jr.,
a-Adrenoreceptors: Molecular Biolo~v. Biochemistry and
WO 94/21660 PCT/US94/02609
2~83~ ~
-- 12 --
Pharmacolo~y. (Progress in Basic and Clinical Pharmacolo~y series,
Karger, 1991), page 29). Because of the ease of 125Iodine detection,
125I-HEAT may be preferred for ~is purpose. By increasing the
amount of unlabeled, test compound, the labeled compound is competed
5 off the receptor. From these experiments, Ic50 values for each test
compound and receptor subtype is dele,ll,illed.
Thus, according to this invention, a method is provided for
identifying compounds specific for the hllm~n alphalC receptor
comprising the following steps:
a. Cloning the hllm~n alphalC adrenerglc receptor;
b. Splicing the the cloned alphalC adrenergic receptor into an
expression vector to produce a construct such that the alphalC receptor
is operably linked to transcription and translation signals sufficient to
15 induce expression of said receptor upon introduction of said construct
into a prokaryotic or eukaryotic cell;
c. Introducing said construct into a prokaryotic or eukaryotic cell which
does not express a hllm~n alphalC adrenergic receptor in the absence of
said introduced construct;
20 d. Incubating cells or membranes isolated from cells produced in step c.
with a qll~ntifi~ble compound known to bind to hllm~n alpha adrenergic
receptors, and subsequently ~lclin~ test compounds at a range of
concentrations so as to compete the qll~ntifi~ble compound from the
receptor, such that an ICso for the test compound is obtained as the
25 concentration of test compound at which 50% of the qll~ntifi~ble
compound becomes displaced from the receptor;
e. Incubating cells or membranes of cells which naturally express or
have an introduced, cloned hllm~n alpha adrenregic receptor of a
subtype other than the hllm~n alphalC receptor under identical
conditions to the incubation conducted in step d, and obt~inin~ the IC50
of the test compound for the non-alphalC receptor; and
f. Comparing ~e IC50 for ~e test compound for the alphalC receptor
and for the alpha adrenergic receptor of a subtype other than the
WO 94/Z1660 PCT/US94/02609
2158345
alphalC to identify compounds having a lower IC50 for the alphalC
receptor.
In addition to providing a sequence for the hllm~n al C
adrenergic receptor, the instant inventors have also discovered a
different sequence than that reported by Bruno et al., rBBRC 179:1485-
1490 (1991)] for the human alA adrenergic receptor. The new
sequence is more homologous to the rat alA adrenergic receptor
sequence. Disclosed and claimed herein is the sequence for this new
hllm~n alA adrenergic receptor (see Fx~mple 12 and figures 22, 23,
and 24, SEQ. ID:29: and SEQ. ID:30:). While no difference in ligand
binding has thus far been observed based on the dirre-~l-t amino
terminal amino acid sequences between these two receptors, such
differences cannot be ruled out except by screening compounds against
both clones. Since a new hllm~n sequence is provided herein,
compounds illenti~ed according to the method of this invention using
the earlier reported hllm~n alA adrenergic receptor sequence can now
be collfi-,ned against this clone.
As a result of the cloning, sequencing, expression, and
screening efforts described above and further exemplified below,
numerous compounds have been tested for their ability to specifically
bind to the cloned hllm~n alC receptor with high affinity. Compounds
specific for the hllm~n alpha lC adrenergic receptor, that is compounds
having an ~îril.ily for the hllm~n alpha lC receptor that is at least 12
fold greater than for either the hllm~n alpha lA or the hllm~n alpha lB
receptors, are identified by this method. Thus, the compounds S(+)
niguldipine, (S(+)-1,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-
pyridinedicarboxylic acid 3-(4,4-diphenyl-1-piperidinyl)-propyl methyl
ester hydrochloride), and 5-methyl urapidil (5-methyl-6[[3-[4-(2-
methoxyphenyl)-l-piperazinyl]propyl]amino]-1,3-dimethyluracil) have
been discovered to selectively bind to the hllm~n alC adrenergic
receptor. These compounds may be ~lminictered in dosages effective to
antagonize the alphalC receptor where such treatment is needed, as in
BPH.
WO 94/21660 PCT/US94/02609
~5~3~
- 14 -
Compounds identified according to the method of this
invention as being selective h~-rn~n al C adrenergic receptor antagonists
may fur~er be defined by counterscreening. This is accomplished
according to methods known in the art.using other receptors responsible
5 for mediating diverse biological functions Compounds which are both
selective amongst the various h~ n alphal adrenergic receptor
subtypes and which have low affinity for other receptors, such as the
alpha2 adrenergic receptors, the ~-adrenergic receptors, the muscarinic
receptors, the serotonin receptors, and others are particularly
preferred. The absence of these non-specific activities may be
confirmed by using cloned and expressed receptors in an analogous
fashion to the method disclosed herein for identifying compounds which
have high affinity for the various hll~m~n alphal adrenergic receptors.
Furthermore, functional biological tests are used to confirm the effects
15 of identified compounds as alphalC adrenergic receptor antagonists.
Compounds identified according to this patent disclosure
may be used alone at a~r~liate dosages defined by routine testing in
order to obtain optimal inhibition of the human alC adrenergic
receptor while minimi7.in~ any potential toxicity. In addition, co-
20 ?~lmini~tration or sequential ~lmini~tration of other agents whichalleviate the effects of BPH is desirable. Thus, in one embodiment, this
includes ~lmini~tration of compounds identified according to this
disclosure and a hllm~n testosterone 5-a reductase inhibitor. Many such
compounds are now well known in the art and include such compounds
25 as PROSCAR~, (also lmown as finasteride, a 4-Aza-steroid; see US
Patents 4,377,584 and 4,760,071, for example, hereby incorporated by
reference). In addition to PROSCAR~, which is principally active in
prostatic tissue due to its selectiv*y for hllm~n 5-a reductase isozyme 2,
combinations of compounds which are specifically active in inhibiting
30 isozyme 1 (found particularly in skin) and compounds which act at both
of these isozymes, are useful in combination with compounds identified
according to this invention.
In the treatment of hyperandrogenic disease conditions, e.g.
benign prostatic hyperplasia (BPH) and/or the prevention and treatment
WO 94121660 21 5 8 34 ~ PCT/US94/02609
- 15 -
of prostatic cancer, and the treatment of prostatitis, it would be
desirable to have one drug entity which is active against both isozymes
to significantly inhibit dihydrotesterone production. It would also be
desirable to have one drug entity that is active as a dual inhibitor of both
5 isozymes for the treatment of conditions of the skin and scalp, e.g. acne
vulgaris, seborrhea, female hirsutism, and androgenic alopecia.
Additionally, such a dual inhibitor of 5a-reductase 1 and 2 could be
used in combination with a 5a-reductase 1 inhibitor or wi~ a 5a-
reductase 2 inhibitor, e.g. finasteride (PROSCAR~), for combination
therapy in the treatment of hyperandrogenic conditions, in combination
with compounds identified according to this inventionas being selective
hllm~n alphalC adrenergic receptor antagonists. The dual 5a-reductase
isozyme inhibitor could also be used in combination with a potassium
channel opener, e.g. minoxidil, for the treatment of male pattern
15 baldness, and such combinations in combination with selective hllm~n
alphalC adrenergic receptor antagonists also form part of the in~t~nt
invention . Compounds that are active as dual 5a-reductase 1 and 2
inhibitors have been described in W093/23420, EP 0572166; WO
93/23050; W093/23038,; W093/23048; WO93/23041; W093/23040;
20 W093/23039; W093/23376; WO93/23419, EP 0572165; WO93/230~1,
each of which is hereby incorporated by reference.
The present invention also has the objective of providing
suitable topical, oral, systemic and parenteral ph~rm~ceutical
form~ tions for use in the novel methods of treatment of the present
invention. The compositions cont~ining compounds identified
according to this invention as the active ingredient for use in the
specific antagonism of hllm~n alphalC adrenergic receptors can be
~-lmini~tered in a wide variety of therapeutic dosage forms in
conventional vehicles for systemic ~lmini~tration. For example, the
compounds can be ~-lmini~tered in such oral dosage forms as tablets,
capsules (each including timed release and sustained release
form~ tions), pills, powders, granules, elixirs, tinctures, solutions,
suspensions, syrups and emulsions, or by injection. Likewise, they
may also be ~lmini~tered in intravenous (both bolus and infusion),
WO 94/21660 PCT/US94/02609
2~83~ ~
- 16 -
. i .
intraperitoneal, subcutariéous, topical with or without occlusion, or
intramuscular form, all using forms well known to those of ordinary
skill in the pharmaceutical arts. An effective but non-toxic amount
of the compound desired can be employed as an alphalC antagonistic
agent.
The daily dosage of the products may be varied over a
wide range from 0.01 to 1,000 mg per adult hllm~n/per day. For
oral ~lminictration~ the compositions are preferably provided in the
form of scored or unscored tablets cont~inin~ 0.01, 0.05, 0.1, 0.5,
1.0, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active
ingredient for the sy~ tolllatic adjustment of the dosage to the
patient to be treated. An effective amount of ~e drug is ordinarily
supplied at a dosage level of from about 0.0002 mg./l~g to about 50
mg.~g. of body weight per day. The range is more par~icularly
15 from about 0.001 mg./kg to 7 mg.~g. of body weight per day. The
dosages of the alphalC adrenergic receptor and testosterone 5-alpha
reductase inhibitors are adjusted when combined to achieve desired
effects. As those skilled in the art will appreciate, less 5-alpha
reductase inhibitor may be required when the acute symptoms of
20 BPH are alleviated by treatment with alphalC adrenergic receptor
inhibitors has been initi~ted. On the other hand, dosages of these
various agents may be independently optimized and combined to
achieve a synergistic result wherein the pathology is reduced more
than it would be if either agent were used alone.
Advantageously, compounds of the present invention
may be ~timini~tered in a single daily dose, or the total daily dosage
may be ~lmini~tered in divided doses of two, three or four times
daily. Furthermore, compounds for the present invention can be
2~1mini~tered in intranasal form via topical use of suitable intranasal
30 vehicles, or via transdermal routes, using those forms of transdermal
skin patches well known to those of ordinary skill in that art. To be
~(lmini~tered in the form of a transdermal delivery system, the
dosage ~tlmini~tration will, of course, be continuous rather than
intermittent throughout the dosage regimen.
-
WO 94/21660 21~ 8 34 5 PCT/US94/02609
-- 17 --
For the treatment of acne vulgaris, androgenic alopecia
including male pattern baldness, seborrhea, female hirsutism, benign
prostatic hyperplasia, prostatitis and the prevention and/or treatment
of prostatic cancer, compounds exhibiting at least 12 fold selectivity
5 for inhibition of the alphalC adrenergic receptor can be combined
with a therapeutically effective amount of a Sa-reductase 2 inhibitor,
such as finasteride, in addition to a Sa-reductase 1 inhibitor, such as
4,7~-dimethyl-4-aza-Sa-cholestan-3-one, in a single oral, systemic,
or parenteral pharmaceutical dosage form~ tion. Alternatively, a
o combined therapy can be employed wherein the alphalC adrenergic
receptor antagonist and the 5a-reductase 1 or 2 inhibitor are
~lmini~tered in separate oral, systemic, or parenteral dosage
form~ tions. Also, for the skin and scalp related disorders of acne
vulgaris, androgenic alopecia including male pattern baldness,
seborrhea, and female hirsutism, the compounds of the instant
invention and dual inhibitors of Sa-reductase 1 and 2 could be
formnl~ted for topical ~(lmini~tration. For example, niguldipine or
S-methyl urapidil and finasteride can be ~lmini~tered in a single oral
or topical dosage formlll~tion, or each active agent can be
20 ~lmini~tered in a separate dosage form~ tion, e.g., in separate oral
dosage form~ ions, or an oral dosage formlll~tion of finasteride in
combination with a topical dosage formlll~tion of a compound
exhibiting dual inhibiton of both isozymes of 5a-reductase. See,
e.g., U.S. Patent No.'s 4,377,584 and 4,760,û71 which describe
2 dosages and form~ ions for 5a-reductase inhibitors.
Furthermore, since ~lmini~tration of 5a-reductase
inhibitors have been found to be useful in combination with a
therapeutically effective amount of a potassium ch~nnel opener, such
as minoxidil, crom~k~lin, pinacidil, a compound selected from the
0 classes of S-triazine, thiane-1-oxide, benzopyran, and pyridinopyran
derivatives or a ph~rm~ceutically acceptable salt thereof, compounds
of this invention may also be used in combination therapy for the
treatment of androgenic alopecia including male pattern baldness.
The active agents can be ~lmini~tered in a single topical dosage
WO 94/21660 PCT/US94/02609
21583~5~i ~
- 18 -
form~ tion, or each active agent can be ~lmini~tered in a separate
dosage formulation, e.g., in separate topical dosage formulations, or
an oral dosage formulation of a compound of formula I in
combination with a topical dosage fonn~ tion of, e.g., minoxidil.
See, e.g., U.S. Patent No.'s 4,596,812, 4,139,619 and WO 92/02225,
published 20 February 1992, for dosages and forrmll~tions of
calcium channel openers. ~
For combination tre~trnent with more than one active
agent, where the active agents are in separate dosage forml-l~tions,
~e active agents can be ~lmini~tered concurrently, or they each can
be ~lmini~tered at separately staggered times.
The dosage regimen lltili7in~ the compounds of the
present invention is selected in accordance with a variety of factors
including type, species, age, weight, sex and medical condition of the
15 patient; the severity of the condition to be treated; the route of
~lmini~tration; the renal and hepatic function of the patient; and the
particular compound thereof employed. A physician or veterinarian
of ordinary skill can readily determine and prescribe the effective
amount of the drug required to prevent, counter or arrest the
20 progress of the condition. Optimal precision in achieving
concentration of drug within the range that yields efficacy without
toxicity requires a regimen based on the kinetics of ~e drug's
availability to target sites. This involves a consideration of the
distribution, equilibrium, and elimin~tion of a drug.
In the methods of the present invention, the compounds
herein described in detail can form the active ingredient, and are
typically ~c~mini~tered in ~imixlllre with suitable ph~ eutical
diluents, excipients or carriers (collectively referred to herein as
"carrier" materials) suitably selected with respect to the intended
30 form of ~lrnini~ctration~ that is, oral tablets, capsules, elixirs, syrups
and the like, and consistent with conventional pharmaceutical
practices.
For instance, for oral ~(lminictration in the form of a
tablet or capsule, the active drug component can be combined with
WO 94/21660 2 15 ~ 3 ~ 5 PCT/US94/02609
- 19 -
an oral, non-toxic pharmaceutically acceptable inert carrier such as
ethanol, glycerol, water and the like. Moreover, when desired or
necessary, suitable binders, lubricants, disintegrating agents and
coloring agents can also be incorporated into the mixture. Suitable
5 binders include, without limit~tion, starch, gelatin, natural sugars
such as glucose or beta-lactose, corn sweeteners, natural and
synthetic gums such as acacia, tr~g~c~nth or sodium ~l~in~te,
carboxymethylcellulose, polyethylene glycol, waxes and the like.
Lubricants used in these dosage forms include, without limit~tion,
sodium oleate, sodium stearate, m~gnesium stearate, sodium
benzoate, sodium acetate, sodium chloride and the like.
Disintegrators include, without limit~tion, starch, methyl cellulose,
agar, bentonite, x~nth~n gum and ~e like.
The liquid forms in suitably flavored suspending or
dispersing agents such as the synthetic and natural gums, for example,
tragacanth, acacia, methyl-cellulose and the like. Other dispersing
agents which may be employed include glycerin and the like. For
parenteral ~lmini~tration~ sterile suspensions and solutions are desired.
Isotonic preparations which generally contain suitable preservatives are
employed when intravenous ~lmini~tration is desired.
Topical preparations cont~ining the active drug component
can be admixed with a variety of carrier materials well known in the
art, such as, e.g., alcohols, aloe vera gel, ~ ntoin, glycerine, vitamin A
and E oils, mineral oil, PPG2 myristyl propionate, and the like, to
2 form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin
gels, skin lotions, and shampoos in cream or gel formnl~tions. See,
e.g., EP 0 285 382.
The compounds of the present invention can also be
~clmini~tered in the form of liposome delivery systems, such as small
nnil~mellar vesicles, large llnil~mellar vesicles and multilamellar
vesicles. Liposomes can be formed from a variety of phospholipids,
such as cholesterol, stearylamine or phosphatidylcholines.
Compounds of the present invention may also be
delivered by the use of monoclonal antibodies as individual carriers
WO 94/21660 PCT/US94/02609
2~583~5 ~
- 20 -
to which ~e compound molecules are coupled. The compounds of
the present invention may alsa b~ coupled wi~ soluble polymers as
targetable drug carriers, Such polymers can include polyvinyl-
pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-
5 amidephenol, polyhydroxy-ethylaspartamidephenol, or polyethyl-
eneoxidepolylysine substituted with palmitoyl residues.
Furthermore, the compounds of the present invention may be
coupled to a class of biodegradable polymers useful in achieving
controlled release of a drug, for example, polylactic acid,
polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters,
polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked
or amphipathic block copolymers of hydrogels.
The following examples are provided to further define the
15 invention without, however, limitin~ ~e invention to the particulars of
these examples.
~,XAMP~ F 1
20 PCR amplification. cloning and sequencing of phalX:
Based on the amino acid homologies of human alA, rat alB and bovine
alC receptors, degenerate oligonucleotides were designed to amplify
cDNAs encoding all ~ree receptor subtypes. These oligonucleotides
are-
WL' (SEQ. ID:1) l l l lCTAGAT TRTTNARRTA NCCNAGCC 28
30 MYC (SEQ. ID:2) TlTACTAGTA TCSTNGTNAT GTAYTG 16WC' (SEQ. ID:3) lTl lCTAGAG AARAANGGNA RCCARC 26
WO 94t21660 2 1 5 ~ 3 ~ 5 PCT/US94/02609
.
-- 21 --
Oligonucleotides MYC and WL' were used as primers in a reverse
transcription PCR amplification of hllm~n heart mRNA (Clontech)
using the RNA PCR kit from Perkin Elmer Cetus. Briefly, 0.5 ug of
mRNA was reverse transcribed in a volume of 20 ul using either
s random oligonucleotide primers (reaction 1) or oligo dT primer
(reaction 2). Reactions 1 and 2 were pooled and served as template for
PCR amplification as follows:
PCR Reactions:
Primary reaction (50 ul)
5 ul lOX buffer from Perkin Elmer Cetus GeneAmp Kit
8 ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP
3ul first strand cDNA
1 ul 25 pMoles oligo MYC
1 ul 25 pMoles oligo WL'
0.25 ul 1.25 units Amplitaq DNA polymersase
31.75 ul water
Reaction conditions; 40 cycles of 94C 1'; 45C 2'; 72C 2'
Secondary reaction (100 ul)
9.5 ul lOX buffer from Perkin Elmer Cetus GeneAmp Kit
16ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP
5ul first strand cDNA
2 ul 50 pMoles oligo MYC
2 ul 50 pMoles oligo WC'
0.5 ul 2.5 units Amplitaq DNA polymersase
65 ul water
Reaction conditions; 40 cycles of 94C 1'; 45C 2; 72C 2'
Prep scale tertiary reaction 3 X 200 ul:
WO 94/21660 PCT/US94/02609
3~ ~
19.5 ul lOX buffer
32 ul l.25 mM each stock of dATP, dCTP, dGTP, a~d dl~P
5 ul secondary PCR reaction
4 ul 100 pMoles oligo MYC
4 ul 100 pMoles oligo WC'
1 ul 5 units Amplitaq DNA polymerase
134.5 ul water -
Reaction conditions; 30 cycles of 94C 1'; 50C 2'; 72C 2'
The PCR product was purified by Qiagen spin columns and
digested with restriction endonucleases SpeI and XbaI. The fragment
was then ligated into SpeVXbaI cut pGEM9Zf(-). The ligation mix was
used to transform E. coli XL-1 blue. Plasmid DNA was isolated from
white transform~nts and sequenced by the dideoxy chain termination
method. The base sequence obtained is shown in Fig. 1, SEQ. ID:4:.
l~,XAMP~
Isolation of partial alphalC cDNA Clone:
A cDNA library prepared from rnRNA isolated from hllm~n
hippocampus (Stratagene) was screened by plaque hybridization using
phalX as a probe. Hybridization conditions were as follows:
SXSSC ( lXSSC is O.l5M sodiurn chloride, 0.015M sodium
citrate,
50% Form~mide
5X Denhardt's Solution ( 1% Ficoll, 1%
polyvinylpyrrolidone, 1% bovine serum albumin)
0.15 mg/ml salmon sperm DNA
hybridize overnight at 42 C.
WO 94121660 PCT/US94/02609
* 2158345
- 23 -
Filters were washed 3 times in 2XSSC, 0.1% SDS at room
temperature for 5', then 1 time in lXSSC, 0.1% SDS at 50C for 30'.
Positive clones were identified by autoradiography. Phagemid DNA
was rescued from ~e positive plaques and sequenced by the dideoxy
chain termin~tion method. The base sequence obtained is shown in Fig.
3, SEQ. ID:7:.
FXAMP~,F 3
PCR amplification~ clonin~ and sequencing of 3'CG of alphalC:
The 3' end of the coding region of hllm~n alphalC adrenergic
receptor was amplified from hllm~n genomic DNA using two
oligonucleotides:
S3C (SEQ ID:8:)
S' 'l~l'l'GAATTCT GAl~CAAGC CCTCTG 3'
and
3'C (SEQ ID:9:)
5' TTTGAATTCT TANACYTCYT CNCCRTTYTC 3'
as follows:
10 ul lOX buffer from Perkin Elmer Cetus GeneAmp Kit
16 ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP
6 ul 1 ug hl-m~n genomic DNA (Promega)
2 ul 50 pMoles oligo S3C
2 ul 50 pMoles oligo 3'C
0.5 ul 2.5 units Amplitaq DNA polymersase
63.5 ul water
WO 94/21660 PCT/US94/02609
2 ~
- 24 -
. . ,;,:
Reaction conditions; 40 cycles of 94C 1'; 50C 2';
72C 2'
The PCR product was purified by Qiagen spin columns and
digested with restriction endonuclease EcoRI. The fragment was then
ligated into EcoRI cut pGEM3Zf(-). The ligation mix was used to
transform E. coli XL-l blue. Plasmid DNA was isolated from white
transform~nt~ and sequenced by the dideoxy chain termin~tion method.
The base sequence is shown in Fig. 4, SEQ. ID:10:.
F,X AMP~ ,F, 4
Assembly of complete coding region of hllm~n alphalc adrenergic
1 5 receptor:
The complete coding region of hllm~n alphalc adrenergic receptor
was assembled by ligating the cDNA clone (see Fx~mple 2, figure 3,
SEQ ID:7:) and 3'CG (see Fx~mple 3, figure 4, SEQ ID:10: ) at their
20 common PvuII site ( 1552-1557 of figure 3, SEQ ID:7: and 59-64 of
figure 4, SEQ ID:10:). The complete nucleotide sequence is shown in
figure 5, SEQ ID: l l :. The amino acid sequence is shown in figure 6,
SEQ. ID:12:. Figure 7 shows the structure of the cDNA, including the
5'-untranslated sequences. The very 3' twenty seven nucleotides (6
25 amino acids) shown is the sequence of the PCR primer used to generate
the sequence. However, the function of the receptor, both for ligand
binding and signal transduction depends on sequences far removed from
the carboxy terminll~ of the receptor. A completely hl-m~n sequence is
shown in figure 18, SEQ. ID:26: which is truncated at the 3' terimuns.
F,XAMP~,F, ~
Expression of the cloned alphalC adrenergic receptor:
WO 94/21660 PCT/US94/02609
~ 2158345
- 25 -
The complete sequence (SEQ ID:11:) of the human alphalC
adrenergic receptor was subcloned into the eukaryotic expression vector
pcDNAI-neo (Invitrogen). The resulting plasmid was transfected into
COS-7 cells by electroporation. Cells were harvested after 72 hours
5 and the membranes cont~ining the expressed receptor protein were
prepared as described in Schwinn, et al.. J. Biol. Chem.. 265:8183-
8189, 1990. Membranes (5-25 ug, see figure 8) prepared from the
COS-7 cells transfected with the vector cont~ining the alphalC receptor
gene specifically bound the alpha 1 antagonist [125 Il-HEAT;
membranes prepared from the COS-7 cells transfected with the vector
alone did not bind the alpha 1 antagonist [125 I]-HEAT (figure 8),
proving the expression of the alphalC adrenergic receptor. Binding
reactions (total volume = 200 ul) cont~inP~l 50 mM Tris-HCl pH. 7.4, 5
mM EDTA, 150 mM NaCl, 100 pM [125 I]-HEAT, and membranes
15 prepared from COS-7 cells transfected with expression plasmids.
Reactions were incubated at room temperature for one hour with
~h~king. Reactions were filtered onto Wh~tm~n GF/C glass fiber filters
with a Brandel cell harvester. Filters were washed three times with ice
cold buffer and bound radioactivity was detelmi"ed. Non specific
20 binding was determinP~l in the presence of 10 uM prazosin.
W O 94/21660 2 ~ 5 8 ~ 4 ~ PCTnUS94/02609
- 2 6
~XAMP~ ~ 6
Screening assay: Alpha 1 C Adrenergic Receptor Binding
Membranes prepared from the transfected COS-7 cells may also be
used to identify compounds that bind to the hllm~n alphalC adrenergic
receptor. These competition binding reactions (total volume = 200 ul)
contain 50 mM Tris-HCl pH. 7.4, 5 mM EDTA, 150 mM NaCl, 100 pM
[125 Il-HEAT, membranes prepared from COS-7 cells transfected with the
alphalC expression plasmid and increasing amounts of unlabeled ligand.
Reactions are incubated at room temperature for one hour with sh~kin~.
Reactions were filtered onto Wll~tm~n GF/C glass fiber filters with a
Brandel cell harvester. Filters were washed three times with ice cold buffer
and bound radioactivity was determined. Binding data were analyzed and
ICSOs determined by an iterative curve fitting program. Results are shown
in Figure 9.
hX ~MP~ F 7
Expression of hllm~n alphalA adrenergic receptor:
The complete coding region for the hllm~n alphalA adrenergic
receptor (Bruno, et al., BBRC., 179:1485-1490, (1991); see figure 10,
25 SEQ. ID:13: and figure 11, SEQ. ID:14: herein) was subcloned into the
eukaryotic expression vector pcDNAI-neo (Invitrogen). The resulting
pl~cmi(l was transfected into COS-7 cells by electroporation. Cells were
harvested after 72 hours and the membranes cont~ining the expressed
receptor protein were prepared as described in Schwinn, et al.~ J. Biol.
30 Chem.~ 265:8183-8189, 1990. Membranes prepared from the COS-7
cel!s transfected with the vector cont~ining the alphalA receptor gene
specifically bound the alpha 1 antagonist [125 Il-HEAT; membranes
prepared from the COS-7 cells transfected with the vector alone did not
bind the alpha 1 antagonist [125 I]-HEAT.
WO 94121660 PCT/US94/02609
21~i83~
- 27 -
Bindir.g reactions (total volume = 200 ul) contained 50 mM Tris-
HCl pH. 7.4, 5 mM EDTA, 150 mM NaCl, 100 pM [125 I]-HEAT, and
membranes prepared from COS-7 cells transfected wi~ expression
plasmids. Reactions are incubated at room temperature for one hour
5 with ~h~kin~. Reactions were filtered onto Wh~tm~n GF/C glass fiber
filters with a Brandel cell harvester. Filters were washed three times
with ice cold buffer and bound radioactivity was determined. Non-
specific binding was determined in the presence of 10 uM prazosin.
~,XAMPT,F, 8
Expression of hllm~n alphalB adrenergic receptor:
1. PCR amplification of partial cDNA for human alphalB adrenergic
receptor:
Amplification of SXB clones
5XB, SEQ. ID:15: 5' TCT AGA CCA TGA AYC CNG AYC TGG 3'
AlB, SEQ. ID:16: 5' 'l l-l GAA TTC ACA TWC CGA CYA CAA
TGC CC 3'
Oligonucleotides SXB and AlB were used as primers in a reverse
transcription PCR amplification of hl-m~n heart mRNA (Clontech)
using the Invitrogen Copy Kit. Briefly, 1.0 ug of mRNA was reverse
transcribed in a volume of 20 ul using oligonuleotide WC' as primer.
Primary reaction (50 ul)
5 ul lOX buffer from Perkin Elmer Cetus
GeneAmp Kit
WO 94/21660 PCT/US94tO2609
~ ~8~45
- 28 -
8 ul 1.25 mM each stock of dATP,dCTP,dGTP, and
dTTP
2.5 ul first strand cDNA
1 ul 25 pMoles oligo SXB
1 ul 25 pMoles oligo AlB
0.25 ul 1.25 units Amplitaq DNA polymersase
32.75 ul water
Reaction conditions; 40 cycles of 94C 1'; 58C 2'; 72C 2'
The PCR product was directly ligated into pCR vector (Invitrogen)
and used to transform E. coli INVaF' (Invitrogen). Plasmid DNA was
isolated from white transform~nt~ and sequenced by the dideoxy chain
termin~tion method. The base sequence is shown in Fig. 12, SEQ.
ID:17:
2. Amplification of EFK clones
EFK, SEQ. ID:18: 5' GAAGGCGCGCTTGAACTC 3'
SB1, SEQ. ID:19: 5' AGAGAACCACCAAGAACC 3'
Oligonucleotides EFK and SB 1 were used as primers in a reverse
transcription PCR amplification of hllm~n aorta mRNA (Clontech)
using the Invitrogen Copy Kit. Bnefly, 1.0 ug of mRNA was reverse
transcribed in a volume of 20 ul using oligo dT as primer.
Primary reaction (50 ul)
5 ul 10X buffer from Perkin Elmer Cetus
GeneAmp Kit
8 ul 1.25 mM each stock of dATP,dCTP,dGTP, and
dTTP
2.0 ul first strand cDNA
WO 94/21660 PCT/US94/02609
21S834~i
~; :
- 29 -
1 ul 25 pMoles oligo EF~
1 ul 25 pMoles oligo 5Bl
0.25 ul 1.25 units Amplitaq DNA polymersase
33.25 ul water
Reaction conditions; 40 cycles of 94C 1'; 58C 2'; 72C 2'
The PCR product was directly ligated into pCR vector (Invitrogen)
and used to transform E. coli INVaF' (Invitrogen). Plasmid DNA was
O isolated from white transforrn~nt,~ and sequenced by the dideoxy chain
termination method. The base sequence is shown in Fig. 13, SEQ.
ID:20:.
3. Assembly of partial cDNA for hllm~n alphalB adrenergic receptor
A partial cDNA clone encoding the hllm~n alphalB adrenergic
receptor was assembled by joining the SXB sequence (SEQ. ID: 17:) and
20 the EFK sequence (SEQ. ID:20:) at their common BamHI site.
4. Amplification of the 3' end of rat alphalB adrener~ic receptor
S4B, SEQ. ID:21: 5' TTT GAA l~C ATG TTC AAG GTG GTG TTC
3'
3'B2, SEQ. ID:22: 5' TTT GAA TTC TAA AASTGN CCN GGN SCC
AGN GGC AT 3'
0 Oligonucleotides S4B and 3'B2 were used as primers in a reverse
transcription PCR amplification of rat heart mRNA using the Invitrogen
Copy Kit. Briefly, 0.6 ug of mRNA was reverse transcribed in a
volume of 20 ul using oligo dT as primer.
WO 94/21660 PCT/US94/02609
2~S~3~5
A 3 0
Primary reaction (50 ul)
5 ul lOX buffer from Perkin Elmer Cetus
GeneAmp Kit
8 ul 1.25 mM each stock of dATP,dCTP,dGTP, and
dTTP
2.0 ul first strand cDNA
1 ul 25 pMoles oligo EFE~
1 ul 25 pMoles oligo SBl
0.25 ul 1.25 units Amplitaq DNA polymersase
33.25 ul water
Reaction conditions; 40 cycles of 94C 1'; 58C 2'; 72C 2'
The PCR product was directly ligated into pCR vector (Invitrogen)
and used to transform E. coli INVaF' (Invitrogen). Plasmid DNA was
isolated from white transform~nt~ and sequenced by the dideoxy chain
termin~tion method. The base sequence is shown in Fig. 14, SEQ.
ID:23:.
5. Assembly and expression of a functional h~lm~n/rat hybrid alphalB
adrenergic receptor
The partial hllm~n alphalB adrenergic receptor cDNA was joined
25 to the 3' end of the rat alphalB adrenergic receptor cDNA at their
common BssHII restriction endonuclease site. This composite sequence
is shown in figure 15, SEQ. ID:24:, and the amino acid sequence is
shown in Fig. 16, SEQ. ID:25:
The complete coding region for the hllm~n/rat alphalB
30 adrenergic receptor was subcloned into the eukaryotic expression vector
pcDNAI-neo (Invitrogen). The resulting plasmid was transfected into
COS-7 cells by electroporation. Cells were harvested after 72 hours
and the membranes cont~ining the expressed receptor protein were
prepared as described in Schwinn, et al.~ J. Biol. Chem., 265:8183-
WO 94/21660 PCT/US94/02609
21583~5
-- 31 --
8189, 1990. Membranes prepared from the COS-7 cells transfected
with the vector collt~ the alphalB receptor gene specifically bour.d
the alpha 1 antagonist [125 I]-HEAT; membranes prepared from the
COS-7 cells transfected with the vector alone did not bind the alpha 1
antagonist [125 I]-HEAT. Binding reactions (total volume = 200 ul)
cont~ine-l 50 mM Tris-HCl pH. 7.4, 5 mM EDTA, 150 mM NaCl, 100
pM [125 I]-HEAT, and membranes prepared from COS-7 cells
transfected with expression plasmids. Reactions are incubated at room
temperature for one hour with ~hakin~. Reactions were filtered onto
o Whatman GF/C glass fiber filters with a Brandel cell harvester. Filters
were washed three times with ice cold buffer and bound radioactivity
was determined. Non specific binding was determined in the presence
of 10 uM prazosin.
~,XAMP~, 9
Selective Binding assays
Membranes prepared from COS-7 cells transfected with the hllman
alpha 1 receptor subtype expression vectors may also be used to identify
compounds that selectively bind to the hllman alphalC adrenergic receptor.
These competition binding reactions (total volume = 200 ul) contain 50
mM Tris-HCl pH. 7.4, 5 mM EDTA, 150 mM NaCl, 100 pM [125 I]-
HEAT, membranes prepared from COS-7 cells transfected with the
respective alpha 1 subtype expression plasmid and increasing amounts of
unlabeled ligand. Reactions are incubated at room temperature for one
hour with ~hakin~. Reactions were filtered onto Whatman GF/C glass fiber
filters with a Brandel cell harvester. Filters were washed three times with
ice cold buffer and bound radioactivity was determined. Binding data were
analyzed and IC50s determined by an iterative curve fitting program.
Table I shows the results from such an analysis.
W O 94/21660 PCTnUS94102609
215g34~ ~
IC~o (nM)
Compound a1A oc1B alC
prazosin 0.6 ~ 2.4
terazosin 4 5 19
doxazosin 5 2 9
phenoxyben7~minP 6.1 4.3 4.0
WB4101 1 16
benox~thi~n 2.5 68 1.5
phentol~mine 36 650 14
5-methyl urapidil 42 270 3.5
S(+) niguldipine 130 670 1.4
F,XAMP-,F, 10
IDENTIFICATION AND CLONING OF A NEW ALLELE FOR THE
HUMAN ALPHAl-C ADRENERGIC RECEPTOR
Probes:
3'CG: A 525 bps fragment, specific to complete exon.2 of
hllm~n alphalc AR, was PCR amplified from hllm~n genomic DNA
3 0 using a sense primer based on the isolated cDNA clone and an antisense
primer based on the last six amino acids of bovine alphalc cDNA. This
PCR product was subcloned and confirmed by sequencing (see Example
3, SEQ.ID:10:).
~WO 94/21660 2 ~ 5 8 ~ ~ ~ PCT/US94/02609
(~enomic ~,ihr~ry ~creenin~:
Human W138 Fibroblast genomic library synthesized in
the Lambda Fix II vector (2 x 106 recombinants; Stratagene,La Jolla,
CA) was screened with (3'CG). This probe was labelled with 32P-dCTP
5 (Amersham) by random-primed labelling kit (}3oehringer
~nnheim,Tndi~n~polis,~). A Total 800,000 plaques were screened,
using duplicate Hybond-N nylone filters (Amersham,UK). Prior to
hybridization, filters were denatured (1.5M NaCl+0.5M NaOH),
neutralized (1.SM NaCl+ lM Tris.Cl,pH 8.0) and washed (0.2M Tris.Cl
pH 7.5 + 2 SSC), 5' for each. DNA was cross-linked with UV cross-
linker (Stratagene,La Jolla,CA). The filters were, then, hybridized in
50% formamide, 5 x SSC(lxSSC= 0.15M NaCl, 0.015M Na citrate,
pH7.0), 0.02% polyvinylpyrophosphate, 0.2% Ficoll, 0.2% bovine
serurn albumin, 150,ug of sheared & boiled Salmon sperm DNA, 106
cpm of 32P-labelled probe at 42C for 40hrs. Filters were washed in
0.1x SSC +1% SDS solution at 60C for 20'. Two more rounds of
screening for 20 "positive" plaques/clones with 3'CG probe confirmed
two clones for the alphalC adrenergic receptor, which were named
48.1C and 53.1C. Clone 53.1C was subjected to further
20 aIlalysislinvestigation.
h-clonnin~ of l~xon.~:
53.1C lambda DNA was amplified by plate lysis method
and purified with Qiagen midi-lambda kit (Qiagen,Chatsworth,CA). A
25 2.6Kb band excised with EcoRI restriction enzyme was identified by
Southern analysis using 3'CG probe. This fr~nent was then subcloned
into pGEM3Zf(+) vector.
DNA se~llencin~:
Nucleotide sequence analysis of DNA in both direction was
performed by Sanger chain termination method.
W O 94/21660 21~ 8 3 4 5 PCTrUS94/02609
- 3 4
Resl-lt ~nd Oisc-l~sion:
Sequencing analysis of this genomic clone confirmed that
clone 53.1c contains sequences for complete exon.2 flanked by an intron
at 5'-end. It also revels that there is a nucleotide change from cytosine
(C) to thymine(T) at nucleotide position 1636, amino acid position 347.
This change creates a PstI site and changes the codon for arginine (Arg)
to cystine (Cys). This data differs from the known/published cDNA
sequen_e of the gene. Southern analysis of hllm~n genomic DNA
confirms the PstI site in the gene/exon.2.
F,XAMPT,F 11
COMPARATIVE PHARMACOLOGY OF ALPHAl-C ~T .T FT FS
We have cloned two gerles for the hllm~n alpha-lc
receptor. The coding regions differ by a single nucleotide. The genes
15 encode either Cys or Arg at amino acid 347 near the C terminllc of the
receptor. The nucleotide difference lies within a PstI restriction
enzyme recognition site thus creating a Restriction Fragment Length
Polymorphism (RFLP). The frequency of allele 1 (LRR) is 0.34; allele
2 (LCR) is 0.66 in 83 unrelated individuals (Hoehe et al "A two-allele
20 PstI RFLP for the alpha-lC adrenergic receptor gene" Human
Molecular Genetics 1: 349, 1992; Allele I is defined by a 2.1 kb PstI
fragment; allele 2 yields two bands of 1.6 and 0.5 kb). Since the amino
acid dirre~ ce occurs within the intracellular tail of the receptor we
would not expect any ph~ cological dirre,~;nces between the
25 expressed receptors. To investigate the ph~ cological profiles of the
two allelic forms of the hl-m~n alpha-lc adrenergic receptor we ligated
the genomic exon 2 fragment of allele 2 to a cDNA clone of allele 1 at a
common PvuII restriction site. The two allelic forms were transientl
expressed in COS-7 cells using pcDNAI/NEO (Invitrogen) expression
vector. Competitive inhibition studies performed in the presence of
125I-HEAT with various antagonists showed no significant difference in
their ph~ cological profiles (Table II):
~WO 94/21660 ~ 1 5 8 3 4 ~ PCT/US94/02609
Table II
COMPARATIVE PHARMACOLOGY OF ALPHAl-C ~T T FT FS
I~ (nM)
LRR LCR
phentol~mine 15 17
niguldipine 0.8 1.8
prazosin 1.0 0.9
5-me~yl urapidi 3.1 4.3
WB4101 0.9 1.0
~,X~MP~,F, 1 ~
CLON~G OF A NOVEL ALPHAl-A ADRENERGIC RECEPTOR
A cosmid library cont~inin~ FG293 cell line genomic DNA in
the double-cos vector sCos-l was screened as follows: The published
hllm~n ala receptor cDNA clone (Bruno et al., BBRC. 179:1485-1490
(1991), and see Fig. 10, SEQ.ID:13:) was cloned into ~e vector
20 pcDNA1 neo to generate the clone pEXala. Filters cont~inin~
approxim~tely 200,000 clones were screened by colony hybridization
([Sambrook, Molecular Cloning, Cold Spring Harbor Laboratory Press,
New York, 1989 ]) using a mixed exon 1 probe generated by PCR
corresponding to ala (TMDl-3), alb (TMDl-S) and oc1c (TMDl-5):
25 25 cycles of 95 C 1'; 52 C 30 sec; 72 C 1.5' using 10 ng of pEX
alb, pEX alc or pEX ala and 10 pmoles each of primers 5' MET (5'
GAATCCCGACCTGGAC ), SEQ.ID:31:, and 3' BAM
(5'GGATCCTCAGGGTC ), SEQ.ID:32:, for alb, 5' 597 (5'
CCATGGTGTTTCTCTCGGG), SEQ.ID:33: and 3' 1219 (5'
30 GACGCGGCAGTACATGAC ), SEQ.ID:34: for alc or 5' 76 (5'
GTCATGATGGCTGGGTACTTG ), SEQ.ID:35:, for ala in a 12 ,ul
reaction cont~inin~ 1.5 ,uM each unlabelled dNTP and 50 ,uCi 3000
Ci/mmol a-[32P] dCTP. The filters were incubated with 1 x 106
cpm/ml of probe in SX SSC, 35% Formamide, 0.02% SDS, 0.1 %
-
W O 94/21660 E~CTnUS94/02609
215~34~
-- 36 --
lauroyl sarcosine, 2% blocking buffer (Bohrenger ~nnheim), at 42 C
for 18 hours. The ~llters were washed with 2 liters of 0.5X SSC, 0.1%
SDS, 55 C and exposed to Kodak XAR-5 film. Twelve primary
positives were picked from master plates and re-screened using the a1a-
specific probe. Cosmid DNA was prepared from second round positiveclones, digested with endonucleases Eco RI or Hind m and subjected to
Southern blot analysis: Fragments were resolved by electrophoresis,
and transferred to a nitrocellulose membrane (Bohrenger ~nnheim)
with 20X SSC (lX SSC = 0.15M Sodium chloride, 0.015M Sodium
citrate, pH 7.0) according to the method of Southern ([Southern, 1975
#14]) . The membrane was hybridized, washed and analyzed as
described above. Alpha-la, a1b, and a1C receptor clones were
identified by co~ alison of restriction patterns with genomic southern
blots performed with a1a, a1b, or a1C specific probes. A Cosmid
cont~inin~ a1a receptor exon 1 DNA was subjected to restriction
digestion by endonuclease Pst I and subjected to southern blot analysis as
above using the a1a -specific probe. Two fM~m~nt~ of 2.3 and 1.6 kb
were detected and subcloned into the Pst I site of PGEM 3ZF . The
presence of the correct S' terminal sequences in the 2.3 kb fr~rne~t was
20 confirrned by sequencing across the junction between inverted repeat
and non-repeat sequences. The 5' end of the ala receptor gene was
ligated to the cDNA clone at ~eir comrnon PstI site, see figures 22-24,
SEQ.ID:29:, and SEQ.ID:30:.
2s F,XAMPT,F 13
EXEMPLARY COUN~RSCREENS
1. A~s~y Title: Doparnine D2,D4 in vitro screen
Objective of the Ass~
The objective of this assay is to elimin~te agents which
specifically affect binding of [3H] spiperone to cells expressing hllm~n
dopamine receptors D2, D3 or D4.
WO 94/21660 PCT/US94tO2609
21~i83g5
Metho~:
Modified from VanTol et al (1991); Nature (Vol 350) Pg
610-613.
Frozen pellets cont~ining specific dopamine receptor
subtypes stably expressed in clonal cell lines are lysed in 2 ml lysing
buffer (lOmM Tris-HCl/~mM Mg, pH 7.4). Pellets obtained after
centrifuging these membranes (15' at 24,450 rpm) are resuspended in
50mM Tris-HCl pH 7.4 co-lt~i-lill~ EDTA, MgCl[2], KCl, NaCl, CaCl[2]
and ascorbate to give a 1 Mg/mL suspension. The assay is initiated by
~lcling 50-75 ~g membranes in a total volume of 500 ,ul cont~ining 0.2
nM [3H]-spiperone. Non-specific binding is defined using 10 ,uM
apomorphine. The assay is tennin~ted after a 2 hour incubation at
room temperature by rapid filtration over GF/B filters presoaked in
0.3% PEI, using 50mM Tris-HCl pH 7.4.
. Ass~,y Title: Serotonin SHTla
Ohjective of the Assav
The objective of this assay is to elimin~te agents which
specifically affect binding to cloned hllm~n 5HTla receptor
Method:
Modified from Schelegel and Peroutka Biochemical
Pharmacology 35: 1943-1949 (1986).
l~mm~ n cells expressing cloned hllm~n SHTla receptors
are lysed in ice-cold 5 mM Tris-HCl, 2 mM EDTA (pH 7.4) and
homogenized with a polytron homogenizer. The homogenate is
centrifuged at 1000Xg for 30', and then the supernatant is centrifuged
again at 38,000Xg for 30'. The binding assay contains 0.25 nM [3H]8-
OH-DPAT in 50 mM Tris-HCl, 4 mM CaCl2 and lmg/ml ascorbate.
Non-specific binding is defined using 10 ,uM propranolol. The assay is
termin~ted after a 1 hour incubation at room temperature by rapid
filtration over GF/Cfilters
W O 94/21660 PCTrUS94/02609
2~5~3~
- 38 -
F,XAMPT,F, 14
EXEMPLARY FUNCTIONAL ASSAYS
.:
In order to confirm the specificity of compounds for the
hllm~n alphalC adrenergic receptor and to define the biological activity
of the compounds, the following functional tests may be performed:
. I N V I T R O FU~T , D O G ~D H~nML~N P R O S T A T E
A N D D O G U R E T H R A
Taconic Farms Sprague-Dawley male rats, weighing 250-
400 grams are sacrificed by cervical dislocation under anesthesia
(methohexital; 50 mg~g, i.p.). An incision is made into the lower
abdomen to remove the ventral lobes of the prostate. Each prostate
removed from a mongrel dog is cut into 6-8 pieces lon~itlltlin~lly along
the Ul`t~ d opening and stored in ice-cold oxygenated Krebs solution
overnight before use if necessary. Dog urethra proximal to prostate is
cut into approximately 5 mrn rings, the rings are then cut open for
contractile measurement of circular muscles. Human prostate chips
from transurethral ~ulgely of benign prostate hyperplasia are also
stored overnight in ice-cold Krebs solution if needed.
The tissue is placed in a Petri dish co-~t~il,il,g oxygenated
Krebs solution [NaCl, 118 mM; KCl, 4.7 mM; CaC12, 2.5 mM;
KH2P04, 1.2 mM; MgS04, 1.2 mM; NaHC03, 2.0 mM; dextrose, 11
mM] warmed to 37C. Excess lipid material and connective tissue are
carefully removed. Tissue segments are attached to glass tissue holders
with 4-0 surgical silk and placed in a 5 ml jacketed tissue bath
cont~ining Krebs buffer at 37C, bubbled with 5% C02/95% 2- The
tissues are connected to a St~th~m-Gould force transducer; 1 gram (rat,
hllm~n) or 1.5 gram (dog) of tension is applied and the tissues are
allowed to equilibrate for one hour. Contractions are recorded on a
Hewlett-Packard 7700 series strip chart recorder.
After a single pr.~ g dose of 3 ,uM (for rat), 10 ~M (for
dog) and 20 ~M (for hlm~n) of phenylephrine, a cllm~ tive
WO 94/21660 PCT/US94/02609
2~8345
-- 39 --
concentration response curve to an agonist is generated; the tissues are
washed every 10 minutes for one hour. Vehicle or antagonist is added
to the bath and allowed to incubate for one hour, then another
cllmlll~tive concentration response curve to the agonist is generated.
EC50 values are calculated for each group using GraphPad
Inplot software. pA2 (-log Kb) values were obtained from Schild plot
when three or more concentrations were tested. When less than three
concentrations of antagonist are tested, Kb values are calculated
according to the following formula Kb = rBl,
x-l
where x is the ratio of EC50 of agonist in the presence and absence of
antagonist and [B] is the antagonist concentration.
~. M~A~URli'Mli',NT ()F INTRA-UR}iTHR~, PRF,~URF ~N
AN~THF'~TI7~ ) l)OG~
PURPOSE: Benign prostatic hyperplasia causes decreased urine flow
rate that may be produced by both passive physical obstruction of the
prostatic uretlll~ from increased prostate mass as well as active
20 obstruction due to prostatic contraction. Alpha adrenergic receptor
antagonists such as prazosin and terazosin prevent active prostatic
contraction, thus improve urine flow rate and provide symptomatic
relief in man. However, these are non-selective alpha-1 receptor
antagonists which also have pronounced vascular effects. Because we
25 have identified the alpha-lC receptor subtype as the predominent
subtype in the hllm~n prostate, it is now possible to specifically target
this receptor to inhibit prostatic contraction without concomitant
changes in the vasculature. The following model is used to measure
adrenergically mediated changes in intra-urethral pressure and arterial
3 0 pressure in anesthetized dogs in order to evaluate the efficacy and
potency of selective alpha adrenergic receptor antagonists. The goals
are to: 1) identify the alpha-1 receptor subtypes responsible for
prostatic/urethral contraction and vascular responses, and 2) use this
model to evaluate novel selective alpha adrenergic antagonists. Novel
WO 94/21660 PCT/US94/02609
215~3~
- 40 -
and standard alpha adrenergic antagonists may be evaluated in this
m~nner.
METHODS: Male mongre~ dogs (7-12 kg) are used in this study.
5 The dogs are anesthetized with pentobarbital sodium (35 mg/kg, i.v.
plus 4 mg~g/hr iv infusion). An endotracheal tube is inserted and the
~nim~l ventilated with room air using a Harvard instruments positive
displacement large ~nim~l ventilator. Catheters (PE 240 or 260) are
placed in the aorta via the femoral artery and vena cava via the femoral
veins (2 catheters, one in each vein) for the measurement of arterial
pressure and the ~tlmini~kation of drugs, respectively. A supra-pubic
incision ~1/2 inch lateral to the penis is made to expose the urethers,
bladder and urethra. The urethers are ligated and c~nn~ ted so that
urine flows freely into beakers. The dome of the bladder is retracted to
facilitate dissection of ~e proximal and distal urethra. Umbilical tape is
passed beneath the urethra at the bladder neck and another piece of
umbilical tape is placed under the distal urethra approxim~tely 1-2 cm
distal to the prostate. The bladder is incised and a Millar micro-tip
pressure transducer is advanced into the urethra. The bladder incision
20 iS sutured with 2-0 or 3-0 silk (purse-string suture) to hold the
transducer. The tip of the transducer is placed in ~e prostatic urethra
and the position of the Millar catheter is verified by gently squeezing
the prostate and noting the large change in urethral pressure.
Phenylephrine, an alpha-1 adrenergic agonist, is
~clmini~tered (0.1-100 ug/kg, iv; 0.05 ml/l~g volume) in order to
construct dose response curves for changes in intra-urethral and arterial
pressure. Following ~tlminictration of increasing doses of an alpha
adrenergic antagonist (or vehicle), the effects of phenylephrine on
arterial pressure and intra-urethral pressure are re-evaluated. Four or
30 five phenylephrine dose-response curves are generated in each ~nim~l
(one control, three or four doses of antagonist or vehicle). The relative
antagonist potency on phenylephrine induced changes in arterial and
intra-urethral pressure are determined by Schild analysis. The family
of averaged curves are fit simultaneously (using ALLFIT software
WO 94/21660 PCT/US94/02609
~' 215~5
- 41 -
package) with a four paramenter logistic equation constraining the
slope, .,~i.,i...l..~ response, and ma~hl-ulll response to be constant among
curves. The dose ratios for the antagonist doses (rightward shift in the
dose-response curves from control) are calculated as the ratio of the
EDso's for the respective curves. These dose-ratios are then used to
construct a Schild plot and the Kb (expressed as ug/l~g, iv) determined.
The Kb (dose of antagonist c~ ing a 2-fold rightward shift of the
phenylephrine dose-response curve) is used to compare the relative
potency of the antagonists on inhibiting phenylephrine responses for
intra-ulctl,l~l and arterial pressure. The relative selectivity is
calculated as the ratio of arterial pressure and intra-urethral pressure
Kb's. Effects of the alpha-l antagonists on baseline arterial pressure are
also monitored. Comparison of the relative antagonist potency on
changes in arterial pressure and intra-urethral pressure provide insight
15 as to whether the alpha receptor subtype responsible for increasing
intra-ur~ l pressure is also present in the systemic vasculature.
According to this method, one is able to confirm the selectivity of
alphalC adrenergic receptor antagonists that prevent the increase in
intra-urethral pressure to phenylephrine without any activity at the
vasculature
At the end of the experiment, the dogs are killed via an
overdose of intravenously ~lmini~tered pentobarbital or saturated KCl.
WO 94/21660 PCT/US94/02609
21~83~S ~
-- 42 --
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Bayne, Marvin L
Clineschmidt, Bradley V
Strader, Catherine D
(ii) TITLE OF INVENTION: CLONED HUMAN ALPHAlC ADRENERGIC RECEPTOR
(iii) NUMBER OF SEQUENCES: 35
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Merck & Co , Inc.
(B) STREET: 126 Lincoln Avenue
(C) CITY: Rahway
(D) STATE: New Jersey
(E) COUNTRY: United States of America
(F) ZIP: 07065
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) CONPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/032,849
(B) FILING DATE: 15-MAR-1993
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Bencen, Gerard H
(B) REGISTRATION NUMBER: 35,746
(C) Kh~KEN~:/DOCKET NUMBER: 18943IA
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (908)594-3901
(B) TELEFAX: (908)594-4720
(C) TELEX: 138825
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
3 0 ( c ) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
WO 94121660 PCTIUS94/02609
21583~
- 43 -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
TTTTCTAGAT TRTTNARRTA NCCNAGCC 28
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
TTTACTAGTA TCSTNGTNAT GTAYTG 26
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 ba3e pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) nY~snr:llCAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
25 TTTTCTAGAG AARAANGGNA RCCARC 26
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 235 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
wo 94/21660 2 ~ 5 8 3 4 ~ - PCT/US94/02609
- 44 -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GCCGCGTCTA C~l w rGGCC AAGAGGGAGA,G~.~GCCT CAAGTCTGGC CTCAAGACCG 60
ACAAGTCGGA CTCGGAGCAA GTGACGCTCC GCATCCATCG GAAAAACGCC CCGGCAGGAG 120 .
GCAGCGGGAT GGCCAGCGCC AAGACCAAGA CGCACTTCTC AGTGAGGCTC CTCAAGTTCT 180
CCCGGGAGAA GAAAGCGGCC AAAACGCTGG GCATCGTGGT ~G~~ C GTCCT 235
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 78 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Arg Val Tyr Val Val Ala Lys Arg Glu Ser Arg Gly Leu Lys Ser Gly
1 5 10 15
Leu Lys Thr Asp Lys Ser Asp Ser Glu Gln Val Thr Leu Arq Ile His
Arg Lys Asn Ala Pro Ala Gly GIy Ser Gly Met AIa Ser Ala Lys Thr
~0 45
Lys Thr His Phe Ser Val Arg Leu Leu Lys Phe Ser Arg Glu Lys Lys
Ala Ala Lys Thr Leu Gly Ile Val Val Gly Cys Phe Val Leu
65 70 75
25 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HY~l'H~:l LCAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
WO 94/21660 PCT/US94/02609
21~8345
- 45 -
(xi) SEQUENCE DESCRIPTION: SEQ ID N3:6:
- Leu Val Met Tyr Cys Arq Val Tyr Val Val Ala Lys Arg Glu Ser Arg 1 5 10 15
Gly Leu Lys Ser Gly Leu Lys Thr Asp Lys Ser Asp Ser Glu Gln Val
20 25 30
Thr Leu Arg Ile His Arg Lys Asn Ala Gln Val Gly Gly Ser Gly Val
35 40 45
Thr Ser Ala Lys Asn Lys Thr His Phe Ser Val Arg Leu Leu Lys Phe
50 55 60
Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu Gly Ile Val Val Gly Cys
65 70 75 80
Phe Val Leu Cys Trp Leu Pro Phe Phe Leu Val Met Pro
~2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1601 base pairs
(B) TYPE: nucleic acid
( c ) STRANDEDNESS: both
(D) TQPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(iii) HYHO'l'~:'l'lCAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GAATTCCCTC CTAGAAGCTG GAGAGAGCAG GA~C~llCG~ TGGGGCAGCT CAAAATGTAG 60
GTAACTGC wG GCCAGGAGCA rrGcrrArA-T GCCATCGGTC ~l~C~ ~ AGCGTCGACG 120
GCTGATCTTT TGGTTTGAGG GAGAGACTGG CGCTGGAGTT TTGAATTCCG M TCATGTGC 180
25 AG M TCGTGA AT~llCC~ AGC~A~ACG M TAAGACAG CGCGGM AAG CAGATTCTCG 240
TAATTCTGGA ATTGCATGTT GCAAGGAGTC TCCTGGATCT TCGCACCCAG CTTCGGGTAC 300
GGGAGGGAGT ~G W~l~CC~ GCTAGGCCAG CCCGCAGGTG GAGAG w TCC CCGGCAGCCC 360
CGCGCGCCCC TGGCCATGTC TTT MTGCCC 'l~CC~ A l~l~G~ TGAGGGTTCC 420
3 o CAG~rG~ CAGGGTTGTC TCCCACCCGC ~CGC~l~ TCACCCCCAG CCAAACCCAC 480
CTw CAGGGC TCCCTCCAGA AGAGACCTTT TGATTCCCGG ~l~GC~l ~C~C~l~ 540
CGCCAGCCCG GGA wGTGGCC CTGGACAGCC GGACCTCGCC ~G~GC TGGGACCATG 600
~ ~l CGGGAAATGC TTCCGACAGC TCCAACTGCA CCCAACCGCC GGCACCGGTG 660
AACATTTCCA AGGCCATTCT GCTCGGGGTG ATCTTGGGGG GCCTCATTCT lll~GG w l~ 720
W O 94/21660 PCTnUS94/02609
21~83~
- 4 6
CTGGGTAACA TCCTAGTGAT LL1L1~LL-1A GCCTGTCACC GACACCTGCA CTCAGTCACG 780
CACTACTACA TCGTCAACCT GGCGGTGGCC GACCTCCTGC TCACCTCCAC GGTGCTGCCC 840
~11L1CCGCCA TCTTCGAGGT CCTAGGCTAC TG~LLLll~ GCAGGGTCTT CTGCAACATC 900
TGGGCGGCAG TGGATGTGCT GTGCTGCACC GCGTCCATCA TGGGCCTCTG CATCATCTCC 960
ATCGACCGCT ACATCGGCGT GAGCTACCCG CTGCGCTACC CAACCATCGT CACCCAGAGG 1020
AGGG~1~1CA TGGL1~1GL1 CTGCGTCTGG GCACTCTCCC TGGTCATATC CATTGGACCC 1080
L1~11~G~L1 GGAGGCAGCC ~GCLLLL~AG GACGAGACCA TCTGCCAGAT CAACGAGGAG 1140
CCGGGCTACG TGL'1'L11~'1'L GGL1~ L~GGC TCCTTCTACC T~C~l~lGGC CATCATCCTG 1 2 0 0
GTCATGTACT GCCL~LL1L~1~A CL~1~G~1~G~CC AAGAGGGAGA GCCGGGGCCT CAAGTCTGGC 1 2 6 0
CTCAAGACCG ACAAGTCGGA CTCGGAGCAA GTGACGCTCC GCATCCATCG GAAAAACGCC 13 2 0
CCGGCAGGAG ~A~Cr.~ T GGCCAGCGCC AAGACCAAGA CGCACTTCTC AGTGAGGCTC 1380
CTCAAGTTCT CCCGGGAGAA GAAAGCGGCC AAAACGCTGG GCATCGTGGT CGGL1~L11C 1440
L~ CL~1~L1GL1 GGL1~LL111 TTTCTTAGTC ATGCCCATTG ~L1L111~11 CCCTGATTTC 1500
AA~LLL1~1~ AAACAGTTTT TAAAATAGTA 1111~L-L1CG GATATCTAAA CAGCTGCATC 1 5 6 0
AACCCCATCA TATACCCATG CTCCAGCCAA GAGGGAATTC C 16 0
(2) INFORMATION FOR SEQ ID NO 8
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 26 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY 1 inear
(ii) MOLECULE TYPE CDNA
(iii) ~Y~1~11CAL: NO
(iV) ANTI-SENSE NO
(Xi) SEQUENCE DESCRIPTION SEQ ID NO:8:
TTTGAATTCT GATTTCAAGC CCTCTG 2 6
( 2 ) INFORMATION FOR SEQ ID NO g:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 30 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE CDNA
(iii) HYPOTHETICAL NO
~WO 94/21660 2 1 5 8 3 4 5 PCT/US94/02609
- 47 -
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
TTTGAATTCT TANACYTCYT CNCCRTTYTC 30
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 512 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(iii) HY~ln~ CAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
CTGATTTCAA GCCCTCTGAA ACAGTTTTTA AAATAGTATT TTGGCTCGGA TATCTAAACA 60
GCTGCATCAA CCCCATCATA TACCCATGCT CCAGCCAAGA GTTCAAAAAG ~'l"l"l'~'AGA 120
ATGTCTTGAG AATCCAGTGT CTCCGCAGAA AGCAGTCTTC CAAACATGCC ~l~G~l~CA 180
CCCTGCACCC GCCCAGCCAG GCCGTGGAAG GGCAACACAA GGACATGGTG CGCATCCCCG 240
TGGGATCAAG AGAGACCTTC TACAGGATCT CCAAGACGGA TGG~lsl~l GAATGGAAAT 300
llllcl~l"l~ CAT~C~l GGATCTGCCA GGATTACAGT GTCCAAAGAC CAATCCTCCT 360
GTACCACAGC CCGGGTGAGA AGTAAAAGCT TTTTGCAGGT ~lG~i~ GTAGGGCCCT 420
CAACCCCCAG CCTTGACAAG AACCATCAAG TTCCAACCAT TAAGGTCCAC ACCATCTCCC 480
TCAGTGAGAA CGGCGAAGAG GTTTAAGAAT TC 512
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2004 base pairs
(B) TYPE: nucleic acid
(C~ STRANDEDNESS: both
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
WO 94/21660 2 1 5 ~ 3 ~ 5 PCT/US94/02609
- 48 -
GAATTCCCTC CTAGAAGCTG GAGAGAGCAG GAGCCTTCGG TGGGGCAGCT CAAAATGTAG 60
GTAACTGCGG GCCAGGAGCA GCGCCCAGAT GCCATCGGTC LL~l~CLl..G AGCGTCGACG 120
GCTGATCTTT TGGTTTGAGG GAGAGACTGG CGCTGGAGTT TTGAATTCCG AATCATGTGC 180
AGAATCGTGA A1~ LCCCC AGCCAGGACG AATAAGACAG CGCGGAAAAG CAGATTCTCG 240
TAATTCTGGA ATTGCATGTT GCAAGGAGTC TCCTGGATCT TCGCACCCAG ~l.CGGLlAC 300
GGGAGGGAGT CCGGGTCCCG GCTAGGCCAG CCCGCAGGTG GAGAGGGTCC CCGGCAGCCC 360
rrrrrrrrrr TGGCCATGTC TTTAATGCCC I~CLLL.l~A ~l~.~GLL.l~ TGAGGGTTCC 420
CA wLL.GGL CA~..~l~ TCCCACCCGC GCGCLCLL-lC TCACCCCCAG CCAAACCCAC 480
CTGGCAGGGC TCCCTCCAGA AGAGACCTTT TGATTCCCGG L.~LCGLLL. LLLLCLl~CG 540
CGCCAGCCCG GGAGGTGGCC CTGGACAGCC GGACCTCGCC CG~LLLLG~ TGGGACCATG 600
Ll~lll.~ CGGGAAATGC TTCCGACAGC TCCAACTGCA CCCAACCGCC GGCACCGGTG 660
AACATTTCCA AGGCCATTCT LLlC w G~l~ ATCTTGGGGG GCCTCATTCT .-.LGG W lG 720
CTGGGTAACA TCCTAGTGAT CCTCTCCGTA GCCTGTCACC GACACCTGCA CTCAGTCACG 780
CACTACTACA TCGTCAACCT GGCGGTGGCC GACL.CL.GL TCACCTCCAC w lGLlLCCC 840
-LlCCGCCA TCTTCGAGGT CCTAGGCTAC T~CL~lCG GCAGGGTCTT CTGCAACATC 900
TGrGrGGrAr. TGGATGTGCT GTGCTGCACC GCGTCCATCA TGGGC~l--~ CATCATCTCC 960
ATCGACCGCT ACATCGGCGT GAGCTACCCG CTGCGCTACC CAACCATCGT CACCCAGAGG 1020
AGGLL.~lLA TGGCTCTGCT CTGCGTCTGG GCACTCTCCC TGGTCATATC CATTGGACCC 1080
CTCTTCGGCT GGAGGCAGCC GGCCCCCGAG GACGAGACCA TCTGCCAGAT CAACGAGGAG 1140
CCGGGCTACG ~l~L~ L GGLlLl~GC TCCTTCTACC I~CLlL-GGL CATCATCCTG 1200
GTCATGTACT GCCGCGTCTA CL1~L1~L-L'C AAGAGGGAGA GCCGGGGCCT CAAGTCTGGC 1260
CTCAAGACCG ACAAGTCGGA CTCGGAGCAA GTGACGCTCC GCATCCATCG GAAAAACGCC 1320
CCGGCAGGAG GCAGCGGGAT GGCCAGCGCC AAGACCAAGA CGCACTTCTC AGTGAGGCTC 1380
CTCAAGTTCT CCCGGGAGAA GAAAGCGGCC AAAACGCTGG GCATCGTGGT CGGCTGCTTC 1440
L~l~LL.LlGL. GGLl~CL-1. TTTCTTAGTC ATGCCCATTG ~ CCCTGATTTC 1500
AAGCCCTCTG AAACAGTTTT TAAAATAGTA ...GG~lLG GATATCTAAA CAGCTGCATC 1560
3 o AACCCCATCA TATACCCATG CTCCAGCCAA GAGTTCAAAA AGGCCTTTCA GAATGTCTTG 1620
AGAATCCAGT ~.~lLC~AG AAAGCAGTCT TCCAAACATG CCCTGGGCTA CACCCTGCAC 1680
CCGCCCAGCC AGGCCGTGGA AGGGCAACAC AAGGACATGG TGCGCATCCC CGTGGGATCA 1740
AGAGAGACCT TCTACAGGAT CTCCAAGACG GATGGCGTTT GTGAATGGAA A-~ L1 1800
TCCATGCCCC GTGGATCTGC CAGGATTACA GTGTCCAAAG ACCAATCCTC CTGTACCACA 1860
~WO 94/21660 PCTIUS94/02609
2158345
- 49 -
G~G~l~A GAAGTAAAAG -llll~CAG ~ 'rG~l GTGTAGGGCC CTCAACCCCC 1920
AGCCTTGACA AGAACCATCA AGTTCCAACC ATTAAGGTCC ACACCATCTC CCTCAGTGAG 1980
AACGGCGAAG AGGTTTAAGA ATTC 2004
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 466 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv~ ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Met Val Phe Leu Ser Gly Asn Ala Ser Asp Ser Ser Asn Cys Thr Gln
1 5 10 15
Pro Pro Ala Pro Val Asn Ile Ser Lys Ala Ile Leu Leu Gly Val Ile
Leu Gly Gly Leu Ile Leu Phe Gly Val Leu Gly Asn Ile Leu Val Ile
~0 ~5
Leu Ser Val Ala Cys His Arg His Leu His Ser Val Thr His Tyr Tyr
Ile Val Asn Leu Ala Val Ala Asp Leu Leu Leu Thr Ser Thr Val Leu
Pro Phe Ser Ala Ile Phe Glu Val Leu Gly Tyr Trp Ala Phe Gly Arg
Val Phe Cys Asn Ile Trp Ala Ala Val Asp Val Leu Cys Cys Thr Ala
100 105 110
Ser Ile Met Gly Leu Cys Ile Ile Ser Ile Asp Arg Tyr Ile Gly Val
115 120 125
Ser Tyr Pro Leu Arg Tyr Pro Thr Ile Val Thr Gln Arg Arg Gly Leu
1~0 1~5 140
Met Ala Leu Leu Cys Val Trp Ala Leu Ser Leu Val Ile Ser Ile Gly
145 150 155 160
Pro Leu Phe Gly Trp Arg Gln Pro Ala Pro Glu Asp Glu Thr Ile Cys
165 170 175
Gln Ile Asn Glu Glu Pro Gly Tyr Val Leu Phe Ser Ala Leu Gly Ser
180 185 190
WO 94/21660 PCTIUS94/02609
~158345
-- 50 --
Phe Tyr Leu Pro Leu Ai~,a Ile Ile Leu Val Met Tyr Cys .brg Va] Tyr
195 200 205
Val Val Ala Lys Arg Glu Ser Arg Gly Leu Lys Ser Gly Leu Lys Thr
210 215 220
Asp Lys Ser Asp Ser Glu Gln Val Thr Leu Arg Ile His Arg Lys Asn
225 230 235 240
Ala Pro Ala Gly Gly Ser Gly Met Ala Ser Ala Lys Thr Lys Thr His
245 250 255
Phe Ser Val Arg Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala Ala Lys
260 265 270
Thr Leu Gly Ile Val Val Gly Cys Phe Val Leu Cys Trp Leu Pro Phe
0 275 . 280 285
Phe Leu Val Met Pro Ile Gly Ser Phe Phe Pro Asp Phe Lys Pro Ser
290 295 300
Glu Thr Val Phe Lys Ile Val Phe TrP Leu Gly Tyr Leu Asn Ser Cys
305 310 315 320
Ile Asn Pro Ile Ile Tyr Pro Cys Ser Ser Gln Glu Phe Lys Lys Ala
325 330 335
Phe Gln Asn Val Leu Arg Ile Gln Cys Leu Arg Arg Lys Gln Ser Ser
340 345 350
Lys His Ala Leu Gly Tyr Thr Leu His Pro Pro Ser Gln Ala Val Glu
355 360 365
Gly Gln His Lys Asp Met Val Arg Ile Pro Val Gly Ser Arg Glu Thr
370 375 380
Phe Tyr Arg Ile Ser Lys Thr Asp Gly Val Cys Glu Trp Lys Phe Phe
385 390 3g5 400
Ser Ser Met Pro Arg Gly Ser Ala Arg Ile Thr Val Ser Lys Asp Gln
405 ~10 415
Ser Ser Cys Thr Thr Ala Arg Val Arg Ser Lys Ser Phe Leu Gln Val
420 425 430
Cys Cys Cys Val Gly Pro Ser Thr Pro Ser Leu Asp Lys Asn His Gln
435 440 445
Val Pro Thr Ile Lys Val His Thr Ile Ser Leu Ser Glu Asn Gly Glu
450 455 460
Glu Val
465
(2) INFORMATION FOR SEQ ID NO:13:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1621 base pairs
(B) TYPE: nucieic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
_WO 94/21660 PCTtUS94tO2609
21S83~5
(ii) MOLECULE TYPE cDNA
- (iii) HYPOTHETICAL NO
(iv) ANTI-SENSE NO
(xi) SEQ~ENCE DESCRIPTION SEQ ID NO 13
CCCGTGCAGG GGCCCTACGG ACACCACCAG GGCTACGACC CAGAGCAGGG CCAGGATGGC 60
GGCCGCCTTG CGCTCGGTCA TGATGGCTGG GTACTTGAGT GAGTG OE GCA CGCCCACGTA 120
CCOE TCCACG GAGATGGTGC AGAG OE TGAG GATGGAGGCC GTGCA OEACA OEACGTCCAC 180
~G~ OE ~l. GGGGGACTGG TGGTGAGCGC GCAGG OE GTG OE CGT WGCG I~llC~l~ OE 240
AGCCTTCATC CTTATGGCCG TGGCAGGTAA OE lG~ll~lC ATCCTCTCAG l~GCLl~AA 300
CCGCCACCTG CAGACCGTCA CCAACTATTT CATCGTGAAC ~lG~C~l~ CCGACCTGCT 360
GCTGAGCGCC ACCGTACTGC OE ll~lC~ CACCATGGAG ~ll~lG~ OE l T.lGGGC~ll 420
TGGCCGC OE~ TTCTGCGACG TATGGGCCGC CGTGGACGTG ~l~lG~ A CGGCCTCCAT 480
CCTCAGCCTC TGCACCATCT CCGTGGACCG GTACGTGGGC GTGCGCCACT CACTCAAGTA 540
CCCAGCCATC ATGACCGAGC GCAAGGCGGC CGCCATCCTG ~ l W ~lC~lAGC 600
CCTGGTGGTG TCCGTAGGGC ~l~cl~GG CTGGAAGGAG COE ~l~C CTGACGAGCG 660
~'l"l~'l'~C~l ATCACCGAGG AG OE ~lA C OE l~l~llC I~l OE~l~l OE l~ll~lA 720
2 0 CCTGCCCATG GCGGTCATCG TGGTCATGTA CTGCC OE GTG TA~l W lCG CGCGCAGCAC 780
CACGCGCAGC CTCGAGGCAG GCGTCAAGCG CGAGCGAGGC AAGGCCTCCG A~l~ 840
GCGCATCCAC `T~l~ CGGCCACGGG CGCCGACGGG OE GCACGGCA TGCGCAGCGC 900
CAAGGGCCAC A OE ll.C~A G~lC OE l~l~ ~lGC~l~ CTCAAGTTCT CCCGTGAGAA 960
GAAAGCGGCC AAGACTCTGG CCATCGTCGT GG~l~l~ll~ ~1 OE l~l~l G~llCC~lll 1020
2 5~ 1 1 . 1 1 1 ~ 1 . CTGCCGCTCG G~l OE ll~ll CCCGCAGCTG AAGCCATCGG AGG~l~ll 1080
CAAGGTCATC TTCTGGCTCG GCTACTTCAA CAGCTGCGTG AACCC OE TCA TCTACCCCTG 1140
TTCCAGCC OE GAGTTCAAGC GC~C~ll.~l CC~l~l~lG CGCTGCCAGT ~C~lC~l~G 1200
CC OE CGCC OE ~'l'~'l'~l GGC~l~l~lA CGGCCACCAC T~ OE ~l CCACCA OE GG 1260
3 o CCTGCGCCAG GACTGCGCCC CGAGTTCGGG CGACGCGCCC CCCGGAGCGC CGCTGGCCCT 1320
CACCGCGCTC CCCGACCCCG ACCCCGAACC CCCAG OEACG CCCGAGATGC AGGCTCCGGT 1380
CGCCAGCCGT CGAAGCCACC CAGC OE CTTC CGCGAGTGGA GOE l~lGGG ~ OE ~llCCG~ 1440
AGACCCACGA CCCAGCTGCG CGCCAAAGTC GCCAGCCTGT CGCACAAGAT CGCCGCCGGG 1500
GGCGCGCAGC GCGCAGAGGC AGCGTGCGCC CAGCGCTCAG AGGTGGAGGC TGTGTCCCTA 1560
WO 94/21660 PCT/US94/02609 ~
2~ ~3~
-- 52 --
GGCGTCCCAC ACGAGGTGGC CGAGGGCGCC ACCTGCCAGG CCTACGAATT GGCCGACTAC 1620
A , 1621
2) INFORMATION FOR SEQ ID NO:14-
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 501 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) ~Y~u~ CAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Met Ala Ala Ala Leu Arg Ser Val Met Met Ala Gly Tyr Leu Ser Glu
1 5 10 15
Trp Arg Thr Pro Thr Tyr Arg Ser Thr Glu Met Val Gln Arg Leu Arg
20 25 30
Met Glu Ala Val Gln His Ser Thr Ser Thr Ala Ala Val Gly Gly Leu
35 40 45
Val Val Ser Ala Gln Gly Val Gly Val Gly Val Phe Leu Ala Ala Phe
50 55 60
Ile Leu Met Ala Val Ala Gly Asn Leu Leu Val Ile Leu Ser Val Ala
65 70 75 80
Cys Asn Arg His Leu Gln Thr Val Thr Asn Tyr Phe Ile Val Asn Leu
85 90 95
Ala Val Ala Asp Leu Leu Leu Ser Ala Thr Val Leu Pro Phe Ser Ala
loo 105 110
Thr Met Glu Val Leu Gly Phe Trp Ala Phe Gly Arg Ala Phe Cys Asp
115 120 125
Val Trp Ala Ala Val Asp Val Leu Cys Cys Thr Ala Ser Ile Leu Ser
130 1~5 140
Leu Cys Thr Ile Ser Val Asp Arg Tyr Val Gly Val Arg His Ser Leu
145 150 155 160
Lys Tyr Pro Ala Ile Met Thr Glu Arg Lys Ala Ala Ala Ile Leu Ala
165 170 175
Leu Leu Trp Val Val Ala Leu Val Val Ser Val Gly Pro Leu Leu Gly
180 185 190
Trp Lys Glu Pro Val Pro Pro Asp Glu Arg Phe Cys Gly Ile Thr Glu
WO 94121660 PCT/US94/02609
2158~5
195 200 205
Glu Ala Gly Tyr Ala Val Phe Ser Ser Val Cy8 Ser Phe Tyr Leu Pr-
210 215 220
Met Ala Val Ile Val Val Met Tyr Cys Arg Val Tyr Val Val Ala Arq
225 230 235 240
Ser Thr Thr Arg Ser Leu Glu Ala Gly Val Lys Arg Glu Arg Gly Lys
245 250 255
Ala Ser Glu Val Val Leu Arg Ile His Cys Arg Gly Ala Ala Thr Gly
260 265 270
Ala Asp Gly Ala His Gly Met Arg Ser Ala Lys Gly His Thr Phe Arg
275 280 285
0 Ser Ser Leu Ser Val Arg Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala
290 295 300
Ala Lys Thr Leu Ala Ile Val Val Gly Val Phe Val Leu Cys Trp Phe
~05 310 315 320
Pro Phe Phe Phe Val Leu Pro Leu Gly Ser Leu Phe Pro Gln Leu Lys
325 330 335
Pro Ser GlU Gly Val Phe Lys Val Ile Phe Trp Leu Gly Tyr Phe Asn
340 345 350
Ser Cys Val Asn Pro Leu Ile Tyr Pro Cys Ser Ser Arg Glu Phe Lys
355 360 365
Arg Ala Phe Leu Arg Leu Leu Arg Cys Gln Cys Arg Arg Arg Arg Arg
370 375 380
Arg Arg Pro Leu Trp Arg Val Tyr Gly Hi~ His Trp Arg Ala Ser Thr
385 390 395 400
Ser Gly Leu Arg Gln Asp Cys Ala Pro Ser Ser Gly Asp Ala Pro Pro
405 410 415
Gly Ala Pro Leu Ala Leu Thr Ala Leu Pro Asp Pro Asp Pro Glu Pro
420 425 430
Pro Gly Thr Pro Glu Met Gln Ala Pro Val Ala Ser An~ Arg Ser His
435 440 445
Pro Ala Pro Ser Ala Ser Gly Gly Cys Trp Gly Arg Ser Gly Asp Pro
450 455 460
Arg Pro Ser Cys Ala Pro Lys Ser Pro Ala Cys Arg Thr Arg Ser Pro
465 470 475 480
3 o Pro Gly Ala Arg Ser Ala Gln Arg Gln Arg Ala Pro Ser Ala Gln Arg
485 490 495
Trp Arg Leu Cys Pro
500
(2) INFORMATION FOR SEQ ID NO:15:
( i ) SEQUENC E CHARACTERI ST I CS:
WO 94/21660 PCT/US94/02609
2~583~ ~
- 54 -
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic aci.d
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) ~Y~u~i~n~ CAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
TCTAGACCAT GAAYCCNGAY CTGG 24
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ii~) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
20 TTTGAATTCA CATWCCGACY ACAATGCCC 29
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 921 base pairs
(B) TYPE: nucleic acid
(C) STF~NDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(iii) ~Y~ul~llCAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
TCTAGACCAT GAATCCCGAC CTGGACACCG GCCACAACAC ATCAGCACCT GCCCACTGGG 60
GAGAGTTGAA AAATGCCAAC TTCACTGGCC CCAACCAGAC CTCGAGCAAC TCCACACTGC 120
CCCAGCTGGA CATCACCAGG GCCATCTCTG TGGGCCTGGT GcTGGGcGcr TTCATCCTCT 180
TTGCCATCGT GGGCAACATC CTAGTCATCT TGTCTGTGGC CTGCAACCGG CACCTGCGGA 240
~wo 94nl660 2 1 5 ~ 3 ~ ~ PCT/US94/02609
- 55 -
CGCCCACCAA CTACTTCATT GTCAACCTGG CCATGGCCGA C~lG~l~.lG AGCTTCACCG 300
s TC~L~CCCL1 CTCAGCGGCC CTAGAGGTGC TCGGCTACTG G~.~c.~GGG CGGATCTTCT 360
GTGACATCTG GGCAGCCGTG GAT~l~.~l GCTGCACAGC GTCCATTCTG A~l~-~C~ 420
CCATCTCCAT CGATCGCTAC ATCGGGGTGC GCTACTCTCT GCAGTATCCC ACGCTGGTCA 480
CCCGGAGGAA GGCCATCTTG GCC~lG~-CA ~-~.c.GG~l CTTGTCCACC GTCATCTCCA 540
TC~G~.cl C~.,~ AAGGAGCCGG CACCCAACGA TGACAAGGAG TGCGGGGTCA 600
CCGAAGAACC CTTCTATGCC ~----.lCCT CTCTGGGCTC CTTCTACATC C~lclG~CGG 660
TCATTCTAGT CATGTACTGC CGTGTCTATA TAGTGGCCAA GAGAACCACC AAGAACCTAG 720
AGGCAGGAGT CATGAAGGAG ATGTCCAACT CCAAGGAGCT GACCCTGAGG ATCCATTCCA 780
AGAACTTTCA CGAGGACACC CTTAGCAGTA CCAAGGCCAA GGGCCACAAC CCCAGGAGTT 840
CCATAGCTGT CAAACTTTTT AAGTTCTCCA GGGAAAAGAA AGCAGCTAAG ACGTTGGGCA 900
.. ~L~l~G~ TATGTGAATT C 921
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(Dl TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GAAGGCGCGC TTGAACTC 18
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(ivl ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
2 ~ ~ 83~ 5 PCT/US94/02609
- 56 ~
AGAGAACCAC CAAGAACC 18
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 389 base pairs
(B) TYPE: nucleic acid
(c) STRANDEDNESS: both
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(iii) HY~u~rH~ CAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
AAGAGAACCA CCAAGAACCT AGAGGCAGGA GTCATGAAGG AGATGTCCAA CTCCAAGGAG 60
CTGACCCTGA GGATCCATTC CAAGAACTTT CACGAGwACA CCCTTAGCAG TACCAAGwCC 120
15 AAGGGCCACA ACCCCAGGAG TTCCATAGCT GTCAAACTTT TTAAGTTCTC CAGGGAAAAG 180
AAAGCAGCTA AGACGTTGGG CATTGTGGTC GGTATGTTCA ~l~-l~l~rG GCTACCCTTC 240
TTCATCGCTC TAC~ullGG ~rC~ll~l-c TCCACCCTGA AGCCCCCCGA ~CC~r~ll. 300
AA W rG~l~l Tul~ ~ CTACTTCAAC A~l~C~luA ACCCCATCAT CTACCCATGC 360
TCCAGCAAGG AGTTCAAGCG CG~lll~l 389
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: ~ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) ~Y~OlHkllCAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
30 TTTGAATTCA TGTTCAAGGT GGTGTTC 27
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
W O 94/21660 8 ~ 5 PCT~US94/02609
~ 5 7
(ii) MOLECULE TYPE: cDNA
(iii~ ~YPus~ CAL: NO
(iv) ANTI-SENSE: NO
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
TTTGAATTCT AAAASTGNCC NGGNSCCAGN GGCAT 35
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 582 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(iii) ~YPOl~ CAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
GAATTCATGA TTCAAGGTGG T~.l~l~G~. GGGCTACTTC AACAGCTGCC TCAATCCCAT 60
CATCTACCCG TGCTCCAGCA AGGAGTTCAA ~C~CG~llC ATGCGTATCC I-lG~ CA 120
~l~CC~G~l GGCCGCCGCC GrcGrrrrcG IC~C~ .A ~GCGC~lGCG CTTACACCTA 180
CC~C~l~ ACCCGCGGCG ~lC~TGGA GAGATCACAG TCGCGGAAGG A~l~l~l~GA 240
TGACAGCGGC AGCTGCATGA GCGGCCAGAA GAGGACCCTG CC~.C w C~ CGCCCAGCCC 300
GGGCTACCTG GGTCGAGGAA CGCAGCCACC CGTGGAGCTG TGCGC~--CC CCGAGTGGAA 360
ACCCGGGGCG CTGCTCAGCT TGCCAGAGCC .~ G~CG~ ~ G~ .C TCGACTCTGG 420
GCCACTCTTC ACCTTCAAGC TCCTGGGCGA TCCTGAGAGC CCGGGAACCG AAGCGACAGC 480
CAGCAACGGG GGCTGCGACA CCACGACCGA CCTGGCCAAC GGGCAGCCCG GCTTCAAGAG 540
CAACATGCCC ~l~G~C~ GCCACTTTTA AAAGCCGAAT TC 582
(2) INFORMATION FOR SEQ ID No:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1567 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
~ (D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
WO 94/21660 PCT/US94/02609
3~5
-- 58 --
(iv) ANTI-SENSE NO
(xi) SEQUENCE DESCRIPTION SEQ ID-~0 24
TCTAGACCAT GAATCCCGAC CTGGACACCG GCCACAACAC ATCAGCACCT GCCCACTGGG 60
GAGAGTTGAA AAATGCCAAC TTCACTGGCC CCAACCAGAC CTCGAGCAAC TCCACACTGC 120
CCCAGCTGGA CATCACCAGG GCCATCTCTG TGGGCCTGGT GCTGGGCGCC TTCATCCTCT 180
TTGCCATCGT GGGCAACATC CTAGTCATCT I~ ~l~lGGC CTGCAACCGG CACCTGCGGA 240
c~rr~A~A~ CTACTTCATT GTCAACCTGG CCATGGCCGA ~lG~l~ ~ AGCTTCACCG 300
TCCTGCCCTT CTCAGCGGCC CTAGAGGTGC TCGGCTACTG GGTGCTGGGG CGGATCTTCT 360
GTGACATCTG GGCAGCCGTG GAI~l~l~l GCTGCACAGC GTCCATTCTG AGCCTGTGCG 420
CCATCTCCAT CGATCGCTAC AT~G w ~ ~ GCTACTCTCT GCAGTATCCC A ~lG~l~A 480
CCCGGAGGAA GGCCATCTTG GCCCTGCTCA ~l~'l'~'l'~ CTTGTCCACC GTCATCTCCA 540
TCGGGCCTCT C~'l"l~'l'~ AAGGAGCCGG CACCCAACGA TGACAAGGAG TGCGGGGTCA 600
CCGAAGAACC CTTCTATGCC ~l~ll~lC~l ~ ~l~ CTTCTACATC ~l~lG~GG 660
TCATTCTAGT CATGTACTGC CGTGTCTATA TAGTGGCCAA GAGAACCACC AAGAACCTAG 720
AGGCAGGAGT CATGAAGGAG ATGTCCAACT CCAAGGAGCT GACCCTGAGG ATCCATTCCA 780
AGAACTTTCA CGAGGACACC CTTAGCAGTA CCAAGGCCAA GGGCCACAAC CCCAGGAGTT 840
2 0 CCATAGCTGT CAAACTTTTT AAGTTCTCCA GGGAAAAGAA AGCAGCTAAG ACGTTGGGCA 900
l.~.G~lC w TATGTTCATC l"l~l~"lG~C TACCCTTCTT CATCGCTCTA ~G~ll~G~'l' 960
C~ l~ CACCCTGAAG CCCCCCGACG ~ ~llCAA W'l'~1~11~ T~G~l~ W~'l' 1020
ACTTCAACAG CTGCCTCAAC CCCATCATCT ACCCATGCTC CAGCAAGGAG TTCAAGCGCG 1080
CCTTCATGCG TATCCTTGGG TGCCAGTGCC GCGGTGGCCG ~ C~GC W~ 1140
25 GTCTAGGCGC ~lG~G~l AC ACCTACCGGC CGTGGACCCG CGGCGGCTCG CTGGAGAGAT 1200
CACAGTCGCG GAAGGACTCT CTGGATGACA GCGGCAGCTG CATGAGCGGC CAGAAGAGGA 1260
CCCTGCCCTC GGG~lCGCCC AGCCCGGGCT AC~lG w lCG AGGAACGCAG CCACCCGTGG 1320
AGCTGTGCGC CTTCCCCGAG TGGAAACCCG GGGCGCTGCT CAGCTTGCCA GAGCCTCCTG 1380
3 o GCCGCCGCGG CCGTCTCGAC TCTGGGCCAC TCTTCACCTT CAAGCTCCTG wCGATCCTG 1440
AGAGCCCG w AACCGAAGCG ACAGCCAGCA ACGGGGGCTG CGACACCACG ACCGACCTGG 1500
CCAACGGGCA GCCCGGCTTC AAGAGCAACA TGCCCCTGGG CCCGGGCCAC TTTTAAAAGC 1560
CGAATTC 1567
(2) INFORMATION FOR SEQ ID NO 25
~WO 94/21660 PCT/US94/02609
2~583~
- 59 -
ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 515 amino acids
~ lB) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HY~OIHrllCAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID No 25
Met Asn Pro Asp Leu Asp Thr Gly His Asn Thr Ser Ala Pro Ala His
1 5 10 15
Trp Gly Glu Leu Lys Asn Ala Asn Phe Thr Gly Pro Asn Gln Thr Ser
Ser Asn Ser Thr Leu Pro Gln Leu Asp Ile Thr Arg Ala Ile Ser Val
Gly Leu Val Leu Gly Ala Phe Ile Leu Phe Ala Ile Val Gly Asn Ile
Leu Val Ile Leu Ser Val Ala Cys Asn Arg His Leu Arq Thr Pro Thr
Asn Tyr Phe Ile Val Asn Leu Ala Met Ala Asp Leu Leu Leu Ser Phe
85 90 95
Thr Val Leu Pro Phe Ser Ala Ala Leu Glu Val Leu Gly Tyr Trp Val
100 105 110
Leu Gly Arg Ile Phe Cys Asp Ile Trp Ala Ala Val Asp Val Leu Cys
115 120 125
Cys Thr Ala Ser Ile Leu Ser Leu Cys Ala Ile Ser Ile Asp Arg Tyr
130 135 140
Ile Gly Val Arg Tyr Ser Leu Gln Tyr Pro Thr Leu Val Thr Arq Arg
145 150 155 160
Lys Ala Ile Leu Ala Leu Leu Ser Val Trp Val Leu Ser Thr Val Ile
165 170 175
Ser Ile Gly Pro Leu Leu Gly Trp Lys Glu Pro Ala Pro Asn Asp Asp
180 185 190
Lys Glu Cys Gly Val Thr Glu Glu Pro Phe Tyr Ala Leu Phe Ser Ser
195 200 205
Leu Gly Ser Phe Tyr Ile Pro Leu Ala Val Ile Leu Val Met Tyr Cys
210 215 220
Arg Val Tyr Ile Val Ala Lys Arg Thr Thr Lys Asn Leu Glu Ala Gly
WO 94/21660 PCTIUS94/02609
2~8~4~
- 60 -
225 230 235 240
Val Met Lys Glu Met Ser Asn Ser Lys ~lu Leu Thr Leu Arg Ile His
245 250 255
Ser Lys Asn Phe His Glu Asp Thr Leu Ser Ser Thr Lys Ala Lys Gly
260 265 270
S His Asn Pro Arg Ser Ser Ile Ala Val Lys Leu Phe Lys Phe Ser Arg 275 280 ~.- 285
Glu Lys Lys Ala Ala Lys Thr Leu Gly Ile Val Val Gly Met Phe Ile
290 295 300
Leu Cys Trp Leu Pro Phe Phe Ile Ala Leu Pro Leu Gly Ser Leu Phe
305 310 315 320
0 Ser Thr Leu Lys Pro Pro Asp Ala Val Phe Lys Val Val Phe Trp Leu
325 330 335
Gly Tyr Phe Asn Ser Cys Leu Asn Pro Ile Ile Tyr Pro Cys Ser Ser
340 345 350
Lys Glu Phe Lys Arg Ala Phe Met Arg Ile Leu Gly Cys Gln Cys Arg
355 360 365
Gly Gly Arg Arg Arg Arg Arg Arg Arg Arg Leu Gly Ala Cys Ala Tyr
370 375 380
Thr Tyr Arg Pro Trp Thr Arg Gly Gly Ser Leu Glu Arg Ser Gln Ser
385 390 395 400
Arg Lys Asp Ser Leu Asp Asp Ser Gly Ser Cys Met Ser Gly Cln Lys
405 410 415
2 0 Arg Thr Leu Pro Ser Ala Ser Pro Ser Pro Gly Tyr Leu Gly Arg Gly
420 425 430
Thr Gln Pro Pro Val Glu Leu Cys Ala Phe Pro Glu Trp Lys Pro Gly
435 440 445
Ala Leu Leu Ser Leu Pro Glu Pro Pro Gly Arg Arg Gly Arg Leu Asp
450 455 460
2 5 Ser Gly Pro Leu Phe Thr Phe Lys Leu Leu Gly Asp Pro Glu Ser Pro
465 470 475 480
Gly Thr Glu Ala Thr Ala Ser Asn Gly Gly Cys Asp Thr Thr Thr Asp
485 490 495
Leu Ala Asn Gly Gln Pro Gly Phe Lys Ser Asn Met Pro Leu Gly Pro
500 505 510
3 oGly His Phe
515
(2) INFORMATION FOR SEQ ID NO:26:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1987 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
~WO 94nl660 PCT/US94tO2609
2~834~
-- 61 --
(D) TOPOLOGY both
(ii) MOLECULE TYPE cDNA
(iii) HYPOTHETICAL NO
(iv) ANTI-SENSE NO
(xi) SEQUENCE DESCRIPTION SEQ ID NO 26
GAATTCCCTC CTAGAAGCTG G~r~A~Ar~rAG GAGCCTTCGG TGGGGCAGCT CAAAATGTAG 60
GTAACTGCGG GCCAGGAGCA GcGccrArAT GCCATCGGTC CCTGCCTTTG AGCGTCGACG 120
GCTGATCTTT TGGTTTGAGG GAGAGACTGG CGCTGGAGTT TTGAATTCCG AATCATGTGC 180
AGAATCGTGA ATCTTCCCCC AGCCAGGACG AATAAGACAG CGCGGAAAAG CAGATTCTCG 240
TAATTCTGGA ATTGCATGTT GCAAGGAGTC TCCTGGATCT TCGCACCCAG ~llCGG~ AC 300
GGGAGGGAGT CCG~l~ GCTAGGCCAG CCCGCAGGTG GAGAGGGTCC CCGGCAGCCC 360
CGCGCGCCCC TGGCCATGTC TTTAATGCCC TGCCCCTTCA TGTGGCCTTC TGAGGGTTCC 420
l 5 CAGGGCTGGC CAG~ C TCCCACCCGC GCG~l. TCACCCCCAG CCAAACCCAC 480
CTGGCAGGGC TCCCTCCAGA AGAGACCTTT TGATTCCCGG ~lCCCGCG~l ~CGC~l~C~ 540
CGCCAGCCCG GGAGGTGGCC CTGGACAGCC GGACCTCGCC ~(Gr~ l;l;r TGGGACCATG 600
~l~l,l~l~l CGGGAAATGC TTCCGACAGC TCCAACTGCA CCCAACCGCC GGCACCGGTG 660
AACATTTCCA AGGCCATTCT GCTCGGGGTG AT~llGGG~ GCCTCATTCT TTTCGGGGTG 720
CTGGGTAACA TCCTAGTGAT C~l~lCC~lA GCCTGTCACC GACACCTGCA CTCAGTCACG 780
CACTACTACA TCGTCAACCG CTAGTGGCGG TGGCCGACCT CCTGCTCACC TCCACGGTGC 840
TG~~ ~ CGCCATCTTC GAGGTCCTAG GCTACTGGGC CTTCGGCAGG ~l~ll~lGCA 900
ACATCTGGGC GGCAGTGGAT ~lG~l~lG~l GrArrr~rGTc CATCATGGGC CTCTGCATCA 960
25 TCTCCATCGA CCGCTACATC GGCGTGAGCT ACCCGCTGCG CTACCCAACC ATCGTCACCC 1020
AGAGGAGGGG TCTCATGGCT CTGCTCTGCG TCTGGGCACT CTCCCTGGTC ATATCCATTG 1080
GA~l~ll CGGCTGGAGG CAGC~ CC CCGAGGACGA GACCATCTGC CAGATCAACG 1140
AGGAGCCGGG CTACGTGCTC .l~l~G~lC l~G~l~l~l CTACCTGCCT CTGGCCATCA 1200
TCCTGGTCAT GTACTGCCGC GTCTACGTGG TGGCCAAGAG GGAGAGCCGG GGCCTCAAGT 1260
~lG~C~l~AA GACCGACAAG TCGGACTCGG AGCAAGTGAC GCTCCGCATC CATCGGAAAA 1320
A~CCCCG~ AGGAGGCAGC GGGATGGCCA GCGCCAAGAC CAAGACGCAC TTCTCAGTGA 1380
GG~ CAA GTTCTCCCGG GAGAAGAAAG CGGCCAAAAC GCTGGGCATC ~l~lC~G~l 1440
G~~ CCT CTGCTGGCTG C~~TAGTCATGCC CATTGGGTCT l~ll~lG 1500
WO 94/21660 PCT/US94/02609
~1~83~5
- 62 -
ATTTCAAGCC CTCTGAAACA GTTTTTAAAA TAGTATTTTG GCTCGGATAT CTAAACAGCT 1560
GCATCAACCC CATCATATAC CCATGCTCCA GCCAAGAGTT CAAAAAGGCC TTTCAGAATG 1620
TCTTGAGAAT CCAGTGTCTC CGCAGAAAGC AGTCGCTAGT TCCAAACATG CC~-~GIi-lA 1680
CACCCTGCAC CCGCCCAGCC AGGCCGTGGA AGGGCAACAC AAGGACATGG TGCGCATCCC 1740
CGTGGGATCA AGAGAGACCT TCTACAGGAT CTCCAAGACG GATGGCGTTT GTGAATGGAA 1800
A1llll~ TCCATGCCCC GTGGATCTGC CAGGATTACA GTGTCCAAAG ACCAATCCTC 1860
CTGTACCACA GCC~Ii~l~A GAAGTAAAAG CTTTTTGCAG GTCTGCTGCT GTGTAGGGCC 1920
CTCAACCCCC AGCCTTGACA AGAACCATCA AGTTCCAACC ATTAAGGTCC ACACCATCTC 1980
CCTCAGT 1987
(2) INFORMATION FOR SEQ ID N3:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1997 base pairs
~B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID No:27:
AA-lCC~ TAGAAGCTGG AGAGAGCAGG AGC~-~ GGGGCAGCTC AAAATGTAGG 60
TAACTGCGGG CCAGGAGCAG CGCCCAGATG CCATCGGTCC ~lG~LllGA GCGTCGACGG 120
CTGATCTTTT GGTTTGAGGG AGAGACTGGC GCTGGAGTTT TGAATTCCGA ATCATGTGCA 180
GAATCGTGAA ~ CC~A Gc~Ac~ArGA ATAAGACAGC GCGGAAAAGC AGATTCTC~T 240
M TTCTGGAA TTGCATGTTG CAAGGAGTCT CCTGGATCTT CGCACCCAGC TTCGGGTACG 300
GGAGGGAGTC CGGGTCCCGG CTAGGCCAGC CCGCAGGTGG AGAGGGTCCC CGGCAGCCCC 360
GC~GC~- GGCCATGTCT TTAATGCCCT GCCCCTTCAT ~lGGC~ll~l GAGG~ll~C~ 420
AG~G~lGGCC AGGGTTGTCT CCCACCCGCG CGCG~C~l-l CACCCCCAGC CAAACCCACC 480
TGGCAGGGCT CCCTCCAGAA GAGACCTTTT GATTCCCGGC TCCCGCGCTC CCGCCTCCGC 540
GCCAGCCCGG GAGGTGGCCC TGGACAGCCG GAC~l~GCC~ iCCC(I.GI_l GGGACCATGG 600
Tlj~1"1"1~rL~1~ GGGAAATGCT TCCGACAGCT CCAACTGCAC CCAACCGCCG GCACCGGTGA 660
ACATTTCCAA GGCCATTCTG CTCGGGGTGA '~ li~G CCTCATTCTT l~GGG~lGC 720
TGGGTAACAT CCTAGTGATC ~ CC~ AG CCTGTCACCG ACACCTGCAC TCAGTCACGC 780
~WO 94121660 PCT/US94/02609
2~8~
- 63 -
ACTACTACAT CGTCAACCTG ~l~CCG ACCTCCTGCT CACCTCCACG GTGCTGCCCT 840
T~ GCCAT CTTCGAGGTC CTAGGCTACT GGGC~l CGG CAGGGTCTTC TGCAACATCT 900
GGGCGGCAGT GGATGTGCTG TGCTGCACCG CGTCCATCAT G~ C ATCATCTCCA 960
TCGACCGCTA CATCGGC~liG AGCTACCCGC TGCGCTACCC AACCATCGTC ACCCAGAGGA 1020
5 GGGGTCTCAT GG~l~rG~lC TGC~l~lGG~ CA~l~lCC~l GGTCATATCC ATTGGACCCC 1080
TCTTCGGCTG GAGGCAGCCG GCCCCC~A~G ACGAGACCAT CTGCCAGATC AACGAGGAGC 1140
CGGGCTACGT ~l~ll~l~ GCTCTGGGCT CCTTCTACCT ~-'~l~l~GCC ATCATCCTGG 1200
TCATGTACTG C~G~ lAC ~lG~l~GCCA AGAGGGAGAG ~r~GGGccTc AAGTCTGGCC 1260
TC M GACCGA CAAGTCGGAC TCGGAGCAAG TGACGCTCCG CATCCATCGG AAAAACGCCC 1320
CGGCAGGAGG CAGCGGGATG GCCAGCGCCA AGACCAAGAC GCACTTCTCA GTGAGGCTCC 1380
TCAAGTTCTC CCGGGAGAAG AAAGCGGCCA AAACGCTGGG CATC~l~l~ G~lG-llCG 1440
TC~l~lG~l~ G~r~l,,, TTCTTAGTCA TGCCCATTGG ~~ CCTGATTTCA 1500
AGC~ lGA AACAGTTTTT AAAATAGTAT TTTGGCTCGG ATATCTAAAC AGCTGCATCA 1560
ACCCCATCAT ATACCCATGC TCCAGCCAAG AGTTCAAAAA GGCCTTTCAG AATGTCTTGA 1620
GAATCCAGTG TCTCTGCAGA AAGCAGTCTT CCAAACATGC ~l~G~lAC ACCCTGCACC 1680
CGCCCAGCCA G~ AA GGGCAACACA AGGACATGGT GCGCATCCCC GTGGGATCAA 1740
GAGAGACCTT CTACAGGATC TCCAAGACGG AT~lll~ TGAATGGAAA llllr~l~ll 1800
CCATGCCCCG TGGATCTGCC AGGATTACAG TGTCCAAAGA CCAATCCTCC TGTACCACAG 1860
CC~G~lGAG AAGTAAAAGC TTTTTGCAGG T.l~ lG TGTAGGGCCC TCAACCCCCA 1920
GCCTTGACAA GAACCATCAA GTTCCAACCA TTAAGGTCCA CACCATCTCC CTCAGTGAGA 1980
ACGGGGAGGA AGTCTAG 1997
t2) INFORMATION FOR SEQ ID NO 28
2 5 ~ i ) SEQUENCE CHARACTERISTICS
(A) LENGTH 466 amino acids
(B) TYPE amino acid
(C) STRANu~N~SS single
(D) TOPOLOGY linear
(ii) MOLEC~LE TYPE protein
(iii) HYPOTHETICAL NO
(v) FRAGMENT TYPE N-terminal
(xi) SEQ~ENCE DESCRIPTION SEQ ID NO 28
Met Val Phe Leu Ser Gly Asn Ala Ser Asp Ser Ser Asn Cys Thr Gln
1 5 10 15
W O 94/21660 PCTrUS94/02609
21~3~5
- 6 4
Pro Pro Ala Pro Val Asn Ile Ser Lys Ala Ile Leu Leu Gly Val Ile
Leu Gly Gly Leu Ile Leu Phe Gly Val l~eu Gly Asn Ile Leu Val Ile
' 45
Leu Ser Val Ala Cys His Arg His Leu His Ser Val Thr His Tyr Tyr
50 55 60
Ile Val Asn Leu Ala Val Ala Asp Leu Leu Leu Thr Ser Thr Val Leu
65 70 75 80
Pro Phe Ser Ala Ile Phe Glu Val Leu Gly Tyr Trp Ala Phe Gly Arg
85 90 95
Val Phe Cys Asn Ile Trp Ala Ala Val Asp Val Leu Cys Cys Thr Ala
100 105 110
Ser Ile Met Gly Leu Cys Ile Ile Ser Ile Asp Arq Tyr Ile Gly Val
115 120 125
Ser Tyr Pro Leu Arg Tyr Pro Thr Ile Val Thr Gln Arg Arg Gly Leu
130 135 140
Met Ala Leu Leu Cys Val Trp Ala Leu Ser Leu Val Ile Ser Ile Gly
145 150 155 160
Pro Leu Phe Gly Trp Arg Gln Pro Ala Pro Glu Asp Glu Thr Ile Cys
165 170 175
Gln Ile Asn Glu G1U Pro Gly Tyr Val Leu Phe Ser Ala Leu Gly Ser
180 185 190
Phe Tyr Leu Pro Leu Ala Ile Ile Leu Val Met Tyr Cys Arg Val Tyr
195 200 205
Val Val Ala Lys Arg Glu Ser Arq Gly Leu Lys Ser Gly Leu Lys Thr
210 215 220
Asp Lys Ser Asp Ser Glu Gln Val Thr Leu Arg Ile His Arg Lys Asn
225 230 235 2~0
Ala Pro Ala Gly Gly Ser Gly Met Ala Ser Ala Lys Thr Lys Thr His
245 250 255
Phe Ser Val Arg Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala Ala Lys
260 265 270
Thr Leu Gly Ile Val Val Gly Cys Phe Val Leu Cys Trp Leu Pro Phe
275 280 285
Phe Leu Val Met Pro Ile Gly Ser Phe Phe Pro Asp Phe Lys Pro Ser
290 295 300
Glu Thr Val Phe Lys Ile Val Phe Trp Leu Gly Tyr Leu Asn Ser Cys
305 310 315 320
Ile Asn Pro Ile Ile Tyr Pro Cys Ser Ser Gln Glu Phe Lys Lys Ala
325 330 335
Phe Gln Asn Val Leu Arg Ile Gln Cys Leu Cys Arg Lys Gln Ser Ser
W0 94/21660 PCTtUS94tO2609
2~8345
-- 65 --
340 345 350
Lys His Ala Leu Gly Tyr Thr Leu His Pro Pro Ser Gln Ala Val Glu
~ 355 360 365
Gly Gln His Lys Asp Met Val Arg Ile Pro Val Gly Ser Arq Glu Thr
370 375 380
Phe Tyr Arg Ile Ser Lys Thr Asp Gly Val Cys Glu Trp Lys Phe Phe
385 390 395 400
Ser Ser Met Pro Arg Gly Ser Ala Arq Ile Thr Val Ser Lys Asp Gln
405 410 415
Ser Ser Cys Thr Thr Ala Arg Val Arg Ser Lys Ser Phe Leu Gln Val
420 425 430
Cys Cys Cys Val Gly Pro Ser Thr Pro Ser Leu Asp Lys Asn His Gln
435 440 445
Val Pro Thr Ile Lys Val His Thr Ile Ser Leu Ser Glu Asn Gly Glu
450 455 460
Glu Val
465
(2) INFORMATION FOR SEQ ID NO:29:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1776 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(iii) ~Y~OTn~ CAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
2 5 CTCCCTGCCG G~r~~ r~l~ GCC~GCCAC CGACGGCCGG CGTTGAGATG 60
ACTTTCCGCG ATCTCCTGAG CGTCAGTTTC GAGGGACCCC GCCCGGACAG CAGCGCAGGG 120
GGCTCCAGCG CGG~GCGG CGGG~C ~C~GC~ ~ C(:u~ ~ GGAGGGCCCG 180
~1~ ~l~C~ ~ '~G~ GG~G~ ~C~l~l~ CGCAGGCAGC 240
GGCGAGGACA ACCGGAGCTC fGe~'~x:~ G CCGGGGAGCG ~G CGGCGACGTG 300
AATGGCACGG CGGCCGTCGG GGGACTGGTG GTGAGCGCGC A~ lGGG ~lGGG~lC 360
ll~l~AG CCTTCATCCT TATG~C~lG GCAGGTAACC l~8l~lCAT CCTCTCAGTG 420
GCCTGCAACC GCCACCTGCA GACCGTCACC AACTATTTCA TCGTGAACCT ~CC~l~CC 480
GA~ TGAGCGCCAC CGTACTGCCC TTCTCGGCCA CCATGGAGGT T~8G~-llC 540
WO 94/21660 PCT/US94/02609
2~ ~3~ ~
- 66 -
TGG~C~IlL~ ~CC~GC~l~ CTGCGACGTA TGGGCCGCCG TGGACGTGCT GTGCTGCACG 600
GCCTCCATCC TCAGCCTCTG CACCATCTCC GTGGACCGGT ACGTGGGCGT GCGCCACTCA 660
CTCAAGTACC CAGCCATCAT GACCGAGCGC AA~C w C~ CCATCCTGGC ~ s~G 720
GTCGTAGCCC TG~l~s~lC CGTAGGGCCC CTGCTGGGCT GGAAGGAGCC CGTGCCCCCT 780
GACGAGCGCT TCTGCGGTAT CACCGAGGAG GCGGGCTACG ~l~l~ll~lC CTCCGTGTGC 840
T~ll~lAcc TGCCCATGGC GGTCATCGTG GTCATGTACT GCCGCGTGTA CGTGGTCGCG 900
CGCAGCACCA CGCGCAGCCT CGAGGCGGGC GTCAAGCGCG AGCGAGGCAA GGCCTCCGAG 960
~l~lG~s~ GCATCCACTG Trrrr~rrrr GCCACGGGCG CCGACGGGGC GCACGGCATG 1020
CGCAGCGCCA AGGGCCACAC CTTCCGCAGC TCGCTCTCCG I~CGC~lGCT CAAGTTCTCC 1080
CGTGAGAAGA AAGrrrrrAA GACTCTGGCC ATC~l~l~ ~l~l~ll~l G~ l~G 1140
ll~lll~l T~lll~lC~l GCCGCTCGGC TC~ll~llCC CGCAGCTGAA GCCATCGGAG 1200
G~C~lcllCA AGGTCATCTT CTGGCTCGGC TACTTCAACA GCTGCGTGAA CCCGCTCATC 1260
TACCCCTGTT CCAGCCGCGA GTTCAAGCGC GC~ ~l~C ~l.lC~l~CG CTGCCAGTGC 1320
CGTCGTCGCC GGCGCCGCCG ~C~ lGG CGTGTCTACG GrrA-rrArTG GCGGGCCTCC 1380
ACCAGCGGCC TGCGCCAGGA CTGCGCCCCG AGTTCGGGCG A(~GcG(~ rr CGGAGCGCCG 1440
CTGGCCCTCA CCGCGCTCCC CGACCCCGAC CCCGAACCCC CAGGCACGCC CGAGATGCAG 1500
GCTCCGGTCG CCAGCCGTCG AAAGCCACCC AGCGCCTTCC GCGAGTGGAG G~~ G~G 1560
CCGTTCCGGA GACCCACGAC CCAGCTGCGC GCCAAAGTCT CCAGCCTGTC GCACAAGATC 1620
~G~ w GCGCGCAGCG CGCAGAGGCA G~lG~CCC AGCGCTCAGA GGTGGAGGCT 1680
~l~l~lAG GCGTCCCACA CGAGGTGGCC GAGGGCGCCA CCTGCCAGGC CTACGAATTG 1740
GCCGACTACA GCAACCTACG GGAGACCGAT ATTTAA 1776
~ 2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 572 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID No:30:
~WO 94121660 PCT/US94/02609
21~8345
-- 67 --
Met Thr Phe Arg Asp Leu Leu Ser Val Ser Phe Glu Gly Pro Arg Pro
5 10 15
Asp Ser Ser Ala Gly Gly Ser Ser Ala Gly Gly Gly Gly Gly Gly Ala
20 25 30
Gly Gly Ala Ala Pro Ser Glu Gly Pro Ala Val Gly Gly Val Pro Gly
35 40 45
Gly Ala Gly Gly Gly Gly Gly Val Val Gly Ala Gly Ser Gly Glu Asp
50 55 60
Asn Arg Ser Ser Ala Gly Glu Pro Gly Ser Ala Gly Ala Gly Gly Asp
65 70 75 80
Val Asn Gly Thr Ala Ala Val Gly Gly Leu Val Val Ser Ala Gln Gly
85 90 95
Val Gly Val Gly Val Phe Leu Ala Ala Phe Ile Leu Met Ala Val Ala
100 105 110
Gly Asn Leu Leu Val Ile Leu Ser Val Ala Cys Asn Arg His Leu Gln
115 120 125
Thr Val Thr Asn Tyr Phe Ile Val Asn Leu Ala Val Ala Asp Leu Leu
130 135 140
Leu Ser Ala Thr Val Leu Pro Phe Ser Ala Thr Met Glu Val Leu Gly
145 150 155 160
Phe Trp Ala Phe Gly Arg Ala Phe Cys Asp Val Trp Ala Ala Val Asp
165 170 175
Val Leu Cys Cys Thr Ala Ser Ile Leu Ser Leu Cys Thr Ile Ser Val
180 185 190
Asp Arg Tyr Val Gly Val Arg His Ser Leu Lys Tyr Pro Ala Ile Met
195 200 205
Thr Glu Arg Lys Ala Ala Ala Ile Leu Ala Leu Leu Trp Val Val Ala
210 215 220
Leu Val Val Ser Val Gly Pro Leu Leu Gly Trp Lys Glu Pro Val Pro
225 230 235 240
Pro Asp Glu Arg Phe Cys Gly Ile Thr Glu Glu Ala Gly Tyr Ala Val
245 250 255
Phe Ser Ser Val Cys Ser Phe Tyr Leu Pro Met Ala Val Ile Val Val
260 265 270
Met Tyr Cys Arg Val Tyr Val Val Ala Arg Ser Thr Thr Arg Ser Leu
275 280 285
Glu Ala Gly Val Lys Arg Glu Arg Gly Lys Ala Ser Glu Val Val Leu
290 295 300
Arg Ile His Cys Arg Gly Ala Ala Thr Gly Ala Asp Gly Ala His Gly
305 310 315 320
Met Arg Ser Ala Lys Gly His Thr Phe Arg Ser Ser Leu Ser Val Arg
325 330 335
wo 94~21660 2 ~. S 8 3 ~ ~ PCT/US94/02609
- 68 -
Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala. Ala Lys Thr Leu Ala Ile
340 345 350
Val Val Gly Val Phe Val Leu Cys Tr,p Phe Pro Phe Phe Phe Val Leu
355 360:. 365
Pro Leu Gly Ser Leu Phe Pro Gln Leu Lys Pro Ser Glu Gly Val Phe
370 375 380
Lys Val Ile Phe Trp Leu Gly Tyr Phe Asn Ser Cys Val Asn Pro Leu
385 390 395 400
Ile Tyr Pro Cys Ser Ser Arg Glu Phe Lys Arg Ala Phe Leu Arg Leu
405 410 415
Leu Arg Cys Gln Cys Arg Arg Arg Arg Arg Arg Arg Pro Leu Trp Arg
0 420 425 430
Val Tyr Gly His His Trp Arg Ala Ser Thr Ser Gly Leu Arg Gln Asp
435 440 445
Cys Ala Pro Ser Ser Gly Asp Ala Pro Pro Gly Ala Pro Leu Ala Leu
450 455 460
Thr Ala Leu Pro Asp Pro Asp Pro Glu Pro Pro Gly Thr Pro Glu Met
465 470 475 480
Gln Ala Pro Val Ala Ser Arg Ars Lys Pro Pro Ser Ala Phe ArSI Glu
485 490 495
Trp Arg Leu Leu Gly Pro Phe Arg Arq Pro Thr Thr Gln Leu Arg Ala
500 505 510
Lys Val Ser Ser Leu Ser His Lys Ile Arg Ala Gly Gly Ala Gln Arg
515 520 525
Ala Glu Ala Ala Cys Ala Gln Arg Ser Glu Val Glu Ala Val Ser Leu
530 535 540
Gly Val Pro His Glu Val AIa Glu Gly Ala Thr Cys Gln Ala Tyr Glu
545 550 555 560
Leu Ala Asp Tyr Ser Asn Leu Arg Glu Thr Asp Ile
565 570
(2) INFORMATION FOR SEQ ID NO:31:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: ~oth
( D ) TOPOLOGY: l inear
(ii) MOLECULE TYPE: cDN~
(iii) hY~o~ cAL: NO
(iv) ANTI-SENSE: NO
WO 94/21660 PCT/US94/02609
2~583~
- 69 -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
GAATCCCGAC CTGGAC 16
(2) INFORMATION FOR SEQ ID No:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID No:32:
GGATCCTCAG GGTC 14
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TQPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID No 33
CCATGGTGTT I~ CG~ 19
25 (2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
WO 94/21660 PCT/US94/02609
3~
-- 70 --
GACGCGGCAG TACATGAC 18
(2) INFORMATION FOR SEQ ID No 35
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO 35
GTCATGATGG CTGGGTACTT G 21
