Note: Descriptions are shown in the official language in which they were submitted.
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
COMPOSITIONS AND METHODS FOR TREATING HIV INFECTION
WITH CUPREDOXIN ANI) CYTOCHROME C
RELATED APPLICATIONS
This application claims priority under 35 U.S.C. 119 and 120 to co-filed
U.S.
Patent Application Serial No. , entitled "Compositions and Methods for
Treating
Malaria with Cupredoxins and Cytochrome", filed May 18, 2006, U.S. Provisional
Patent
Application Serial No. 60/780,868, filed March 10, 2006, U.S. Provisional
Patent Application
Serial No. 60/682,833, filed May 20, 2005, and U.S. Patent Application No.
11/244,105, filed
October 6, 2005, which claims priority to U.S. Provisional Patent Application
Serial No.
60/616,782, filed October 7, 2004, and U.S. Provisional Patent Application
Serial No.
60/680,500, filed May 13, 2005, and is a continuation-in-part of U.S. Patent
Application
Serial Number 10/720,603, filed November 11, 2003, which claims priority to
U.S.
Provisional Patent Application Serial No. 60/414,550, filed August 15, 2003,
and which is a
continuation-in-part of U.S. Patent Application Serial Number 10/047,710,
filed January 15,
2002, which claims priority to U.S. Provisional Patent Application Serial
Number
60/269,133, filed February 15, 2001. The entire content of these prior
applications is fully
incorporated herein by reference.
STATEMENT OF GOVERNMENTAL INTEREST
The subject matter of this application has been supported by research grants
from the
National Institutes of Health (NIH), Bethesda, Maryland, U.S.A., (Grant
Numbers AI 16790-
21, ES 04050-16, AI 45541, CA09432 and N01-CM97567). The government may have
certain rights in this invention.
FIELD OF THE INVENTION
The present invention relates to cupredoxin, specifically Pseudomonas
aeruginosa
azurin, and/or Pseudomonas aeruginosa cytochrome e551 and their use in
inhibiting of viral
infection, and in particular infection of mammalian,cells by the Human
Immunodeficiency
Virus (HIV). The invention also relates to variants and derivatives of
cupredoxin and
cytochrome c that retain the ability to inhibit viral infection, and in
particular infection by the
Human Immunodeficiency Virus (HIV). The invention also relates to research
methods for
studying viral and bacterial infection in mammalian cells.
1
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
BACKGROUND,
While human immunodeficiency virus (HIV) infection, which results in AIDS, is
a
relatively new infection in the human population, it has quickly risen to the
foremost health
problem in the world. HIV/AIDS is now the leading cause of death in sub-
Saharan Africa,
and is the fourth biggest killer worldwide. At the end of 2001, it was
estimated that 40
million people were living with HIV infection world wide. The Centers for
Disease Control
(CDC) estimates that nearly 800,000 people are living with AIDS in the US, and
40,000 new
cases diagnosed each year. While better treatment methods are now known to
prolong the
life of patients with HIV infection, there is still no cure.
Modem anti-HIV drugs target several different stages of the HIV life cycle,
and
several of the enzymes that HIV requires to replicate and survive. Some of the
commonly
used anti-HIV drugs include nucleoside reverse transcriptase inhibitors such
as AZT, ddl,
ddC, d4T, 3TC, and abacavir; nucleotide reverse transcriptase inhibitors such
as tenofovir;
non-nucleoside reverse transcriptase inhibitors such as nevirapine, efavirenz
and delavirdine;
protease inhibitors such as saquinavir, ritonavir, indinavir, nelfinavir,
amprinavir, lopinavir
and atazanavir; and fusion inhibitors such as enfuvirtide. However, in many
HIV infected
patients, none of these antiviral drugs, alone or in combination, is effective
to prevent the
progression of chronic infection or treat acute AIDS. The high mutation rate
of the HIV virus
and associated emergence of HIV strains resistant to drugs is one large factor
that results in
the inability to effectively treat HIV infection. New and better treatments
are required for
HIV infection.
SUMMARY OF THE INVENTION
The present invention relates to compositions and methods of use of
cupredoxins and
/or cytochrome c551 and their use to inhibit viral infections in mammalian
cells, and in
particular HIV infection. Specifically, the present invention relates to
compositions
comprising the peptides Pseudomonas aeruginosa azurin and/or cytochrome c551,
and/or
variants and/or derivatives thereof, and methods to use these compositions and
peptides to
inhibit the growth of HIV-1 infection in mammalian cells and patients. The
invention also
relates to variants and derivatives of cupredoxin and cytochrome c551 that
retain the ability to
inhibit the growth of viral infection, and in particular infection by the HIV.
2
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
One aspect of the invention is an isolated peptide that is a variant,
derivative or
structural equivalent of a cupredoxin or cytochrome cssl and that can inhibit
the growth of
HIV-1 infection in mammalian cells. In some embodiments, the cupredoxin is
azurin,
pseudoazurin, plastocyanin, rusticyanin, Laz or auracyanin, and specifically
azurin or Laz. In
other embodiments, the cupredoxin is from Pseudornonas aeruginosa,
Alcaligenesfaecalis,
Achrornobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp.,
Neisseria
meningitidis, Neisseria gonorrhea, Pseudornonas fluorescens, Pseudomonas
chlororaphis,
Xylella fastidiosa or Vibrio parahaernolyticus, and specifically Pseudornonas
aeruginosa or
Neisseria gonorrhea. In some embodimeints, the peptide is part of one of SEQ
ID NOS: 1-
17. In some embodiments, the peptide has at least about 90% amino acid
sequence identity to
a sequence in SEQ ID NOS: 1-17.
In some embodiments, the isolated peptide may be a truncation of cupredoxin or
cytochrome c551. The peptide may be more than about 10 residues and not more
than about
100 residues. In some embodiments, the isolated peptide comprises a region of
azurin that is
residues 36-128, residues 36-88 or residues 88-113. In other embodiments, the
isolated
peptide consists of a region of azurin that is residues 36-128, residues 36-88
or residues 88-
113. In other embodiments, the peptide comprises equivalent residues of a
cupredoxin as a
region of azurin that is residues 36-128, residues 36-88 and residues 88-113.
Another aspect of the invention is a composition which comprises at least one
cupredoxin, cytochrome c551, or isolated peptide that is a variant, derivative
or structural
equivalent of a cupredoxin or cytochrome essl and that can inhibit the growth
of HIV-1
infection in mammalian cells, in a pharmaceutical composition. In some
embodiments, the
pharmaceutical composition is formulated for intravenous administration. In
other
embodiments, the cupredoxin is from Pseudomonas aeruginosa, Alcaligenes
faecalis,
Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp.,
Neisseria
meningitidis, Neisseria gonorrhea, Pseudomonasfluorescens, Pseudomonas
chlororaphis,
Xylellafastidiosa or Vibrio parahaemolyticus, and specifically Pseudomonas
aeruginosa or
Neisseria gonorrhea. In some embodiments, the cupredoxin or cytochrome c551 is
selected
SEQ ID NOS: 1-17.
Another aspect of the invention is a method to treat a patient infected with a
virus or
bacteria, comprising administering to the patient a therapeutically effective
amount of a
composition which comprises at least one cupredoxin, cytochrome c55 1, or
isolated peptide
3
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
that is a variant, derivative or structural equivalent of a cupredoxin or
cytochrome c551 and
that can inhibit the growth of HIV- 1 infection in mammalian cells, in a
phaimaceutical
composition. The patient may be infected with HIV-1, Herpes simplex virus
(HSV), Ebola
virus, cytomeglovirus (CMV), parainfluenza viruses types A, B and C, hepatitis
virus A, B,
C, and G, the delta hepatitis virus (HDV), mumps virus, measles virus,
respiratory syncytial
virus, bunyvirus, arena virus, Dhori virus, poliovirus, rubella virus, dengue
virus; SIV or
Mycobacterium tuberculosis, and specifically HIV-1. In some embodiments, the
patient is
human. In some embodiments, the composition is administered by intravenous
injection,
intramuscular injection, subcutaneous injection, inhalation, topical
administration,
transdermal patch, suppository or oral, and specifically by intravenous
injection. The
composition may be administered within 0 minutes to 1 week of the
administration of another
anti-HIV drug., specifically at about the same time as another anti viral,
anti-bacterial and/or
anti-HIV drug.
Another aspect of the invention is a composition comprising at least two of an
isolated
cupredoxin, a cytochrome c551, and variant, derivative or structural
equivalent of a
cupredoxin or a cytochrome c551. In some embodiments, the composition is in a
pharmaceutical composition.
Another aspect of the invention is a kit comprising the composition comprising
at
least one cupredoxin, cytochrome c551, or isolated peptide that is a variant,
derivative or
structural equivalent of a cupredoxin or cytochrome c551 and that can inhibit
the growth of
HIV-1 infection in mammalian cells in a pharmaceutical composition in a vial.
In some
embodiments, the kit is designed for intravenous administration.
Another aspect of the invention is a method to study viral and bacterial
infection in
mammalian cells, comprising the steps of contacting the cells with a
cupredoxin or
cytochrome c551, or variant, derivative or structural equivalent thereof;
contacting the cells
with a bacteria or virus; and measuring the growth of infection of the virus
or bacteria. In
some embodiments, the step of contacting the cells with a cupredoxin or
cytochrome c551, or
variant, derivative or structural equivalent thereof occurs before the step of
contacting the
cells with a bacteria or virus. In other embodiments, the step of contacting
the cells with a
cupredoxin or cytochrome c551, or variant, derivative or structural equivalent
thereof occurs
after the step of contacting the cells with a bacteria or virus. In some
embodiments, the cells
are human cells. In other embodiments, the cells are lymphoma cells or
peripheral blood
4
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
mononuclear cells. In other embodiments, the virus or bacteria is HIV-1,
Herpes simplex
virus (HSV), Ebola virus, cytomeglovirus (CMV), parainfluenza viruses types A,
B and C,
hepatitis virus A, B, C, and G, the delta hepatitis virus (HDV), mumps virus,
measles virus,
respiratory syncytial virus, bunyvirus, arena virus, Dhori virus, poliovirus,
rubella virus,
dengue virus, SIV and Mycobacterium tuberculosis, specifically HIV- 1.
Another aspect of the invention is an expression vector, which encodes peptide
that is
a variant, derivative or structural equivalent of a cupredoxin or cytochrome
c551 and that can
inhibit the growth of HIV-1 infection in mammalian cells.
These and other aspects, advantages, and features of the invention will become
apparent from the following figures and detailed description of the specific
embodiments.
BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO: 1. Amino acid sequence of azurin from Pseudomonas aeruginosa.
SEQ ID NO: 2. Amino acid sequence of cytochrome c551 from Pseudomonas
aeruginosa.
SEQ ID NO: 3. Amino acid sequence of plastocyanin from Phormidium laminosum.
SEQ ID NO: 4. Amino acid sequence of rusticyanin from Thiobacillus
ferrooxidans.
SEQ ID NO: 5. Amino acid sequence of pseudoazurin from Achromobacter
cycloclastes.
SEQ ID NO: 6. Amino acid sequence of azurin from Alcaligenes faecalis.
SEQ ID NO: 7. Amino acid sequence of azurin from Achromobacter xylosoxidans
ssp.denitrificans I.
SEQ ID NO: 8. Amino acid sequence of azurin from Bordetella bronchiseptica.
SEQ ID NO: 9. Amino acid sequence of azurin from Methylomonas sp. J.
SEQ ID NO: 10. Amino acid sequence of azurin from Neisseria meningitidis Z249
1.
SEQ ID NO: 11. Amino acid sequence of azurin from Pseudomonas fluorescen.
SEQ ID NO: 12. Amino acid sequence of azurin from Pseudomonas chlororaphis.
SEQ ID NO: 13. Amino acid sequence of azurin'from Xylella fastidiosa 9a5c.
SEQ ID NO: 14. Amino acid sequence of stellacyanin from Cucumis sativus
SEQ ID NO: 15. Amino acid sequence of auracyanin A from Chloroflexus
aurantiacus
5
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
SEQ ID NO: 16. Amino acid sequence of auracyanin B from Chloroflexus
aurantiacus
SEQ ID NO: 17. Amino acid sequence of cucumber basic protein from Cucumis
sativus.
SEQ ID NO: 18 is the amino acid sequence of the H.8 region from Laz from
Neisseria
gonorrhoeae F62.
SEQ ID NO: 19 is the amino acid sequence of Laz from Neisseria gonorrhoeae
F62.
SEQ ID NO: 20 is the forward primer to PCR amplify the Laz-encoding gene (laz)
of
Neisseria gonorrhoeae.
SEQ ID NO: 21 is the reverse primer to PCR amplify the Laz-encoding gene (laz)
of
Neisseria gonorrhoeae.
SEQ ID NO: 22 is the forward primer to PCR amplify a 3.1 kb fragment of pUC 18-
laz.
SEQ ID NO: 23 is the reverse primer to PCR amplify a 3.1 kb fragment of puc18-
laz.
SEQ ID NO: 24 is the forward primer to PCR amplify a 0.4 kb fragment of pUCl9-
paz.
SEQ ID NO: 25 is the reverse primer to PCR amplify a 0.4 kb fragment of pUCl9-
paz.
SEQ ID NO: 26 is the forward primer for pGST-azu 36-128.
SEQ ID NO: 27 is the reverse primer for pGST-azu 36-128.
SEQ ID NO: 28 is the forward primer for pGST-azu 36-89.
SEQ ID NO: 29 is the reverse primer for pGST-azu 36-89.
SEQ ID NO: 30 is the forward primer for pGST-azu 88-113.
SEQ ID NO: 31 is the reverse primer for pGST-azu 88-113.
SEQ ID NO: 32 is an oligonucleotide for site directed mutagenesis for the
preparation
of pGST-azu 88-113.
SEQ ID NO: 33 is an oligonucleotide for site directed mutagenesis for the
preparation
of pGST-azu 88-113.
6
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Figure 1 depicts the inhibition of HIV-1 viral growth by azurin, H.8-
azurin
(H.8-Az) and Laz. These three proteins were incubated at different
concentrations with
PBMC followed by addition of the three subtypes of HIV-1. After 2 h
incubation, the virus
was removed but the proteins added back as described in Example 3. Suppression
of virus
growth was determined by ELISA assays of p24.
Figure 2. Figure 2 depicts surface plasmon resonance binding curves depicting
the
binding patterns of cupredoxins with CD4 and HIV-1 gp120. (A) SPR titration
curves
showing novel and specific binding of azurin, and GST-Azu 36-128 (shown as an
inset) with
immobilized CD4 on carboxymethyldextran coated gold sensor chips (CD4-CM5).
HIV-1
gp120, HIV-1 gag, and HIV-1 nef served as the positive and negative controls
respectively.
Relative binding affinities were determined via fitting the data to Req =
Rma,,/(l+(Kd/C)) with
the curve fits connecting the data points above. The CD4 binding Kd values
are: 36.9 2.0
nM (azurin), 0.34 0.04 nM (GST-Azu 36-128), and 48.1 3.1 nM (HIV-1 gp120).
(B) The
binding titrations when immobilized azurin (Az-CM5) is in contact with HIV
proteins. Due
to large nonspecific binding to the bare Au-CM5 chip, CM5 was added as an
eluent to the
running buffer (1 mg/ml CM5 to HBS-EP buffer). Curve fits gave Kd's of 25.1
3.1 nM
(CD4), and 8.9 0.8 nM (HIV-1 gp120). (C) SPR curves for the binding of ICAMs
(ICAM-
1, ICAM-2, ICAM-3 and NCAM, inset) with immobilized azurin were determined
under
similar conditions as for experiments in part (B). The selective recognition
of azurin with
ICAM-3, but not with ICAM-1 or ICAM-2, is notable and the binding strength was
19.5
5.4 nM. The Kd for NCAM binding with azurin, as shown in the inset, was 20 +
5.0 nM.
(D) SPR binding competition studies with CD4 immobilized on CM5 sensor chips.
Azurin +
HIV-1 gp120 solutions were added at different azurin concentrations (0-4500
nM, [HIV-1
gp 120] is 242 nM) to the sensor surface and the data were plotted as a ratio
of resonances, %
total response [Req (azurin+HIV-1 gp120)/(Req/(HIV-1 gpl20))]. GST-Azu 36-128
was
titrated with HIV-1 gp120 to immobilized CD4 and analyzed in a similar manner.
Competition data suggests 1:1 stoichiometry of binding between azurin and GST-
Azu 36-128
with immobilized CD4.
Figure 3. Figure 3 depicts surface plasmon resonance binding titrations
depicting the
interactions of azurin, and GST-Azurin fusions with DC-SIGN. (A) Concentration
dependent
binding of azurin, ICAM-3, and GST-Azu 36-89 with DC-SIGN were determined via
7
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
injection of various concentrations of the proteins (0 - 100 nM) over a DC-
SIGN modified
CM5 sensor surface and the extent of binding was evaluated as a function of
the equilibrium
resonance response value measured in resonance units (RU). (B) The binding
titration curve
of GST-Azu 88-113 with DC-SIGN using the same sensor chip and protocol as
described for
azurin in part A. The positive interaction of GST-Azu 88-113 with DC-SIGN
suggests its
potential role as the recognition sequence for azurin. The binding affinities
(Kd) for azurin,
ICAM-3 and GST-Azu 88-113 were determined by fitting the data to Req =
Rmax/(1+(Kd/C)) and the curve fits connect the data points in these plots. The
extrapolated
Kd values are 0.83 0.05 nM (azurin), 0.93 0.39 nM (ICAM-3), and 5.98
1.13 nM (GST-
Azu 88-113).
DETAILED DESCRIPTION OF THE.INVENTION
Definitions
As used herein, the term "cell" includes both the singular or the plural of
the term,
unless specifically described as a "single cell."
As used herein, the terms "polypeptide," "peptide," and "protein" are used
interchangeably to refer to a polymer of amino acid residues. The terms apply
to amino acid
polymers in which one or more amino acid residue is an artificial chemical
analogue of a
corresponding naturally occurring amino acid. The terms also apply to
naturally occurring
amino acid polymers. The terms "polypeptide," "peptide," and "protein" are
also inclusive of
modifications including, but not limited to, glycosylation, lipid attachment,
sulfation, gamma-
carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.
It will be
appreciated that polypeptides are not always entirely linear. For instance,
polypeptides may
be branched as a result of ubiquitination and they may be circular (with or
without
branching), generally as a result of post-translation events, including
natural processing event
and events brought about by human manipulation which do not occur naturally.
Circular,
branched and branched circular polypeptides may be synthesized by non-
translation natural
process and by entirely synthetic methods as well.
As used herein, the term "pathological condition" includes anatomic and
physiological deviations from the normal that constitute an impairment of the
normal state of
the living animal or one of its parts, that interrupts or modifies the
performance of the bodily
functions, and is a response to various factors (as malnutrition, industrial
hazards, or climate),
8
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
to specific infective agents (as worms, parasitic protozoa, bacteria, or
viruses), to inherent
defects of the organism (as genetic anomalies), or to combinations of these
factors.
As used herein, the term "condition" includes anatomic and physiological
deviations
from the normal that constitute an impairment of the normal state of the
living animal or one
of its parts, that interrupts or modifies the performance of the bodily
functions.
As used herein, the term "suffering from" includes presently exhibiting the
symptoms
of a pathological condition, having a pathological condition even without
observable
symptoms, in recovery from a pathological condition, or recovered from a
pathological
condition.
A used herein, the term "treatment" includes preventing, lowering, stopping,
or
reversing the progression or severity of the condition or symptoms associated
with a
condition being treated. As such, the term "treatment" includes medical,
therapeutic, and/or
prophylactic administration, as appropriate.
As used herein, the term " inhibit the growth of HIV infection" means any
means by
which HIV infection is decreased, or prevented from increasing in the human
body. These
means can include, but are not limited to, inhibition of replication of the
HIV genome,
inhibition of synthesis and/or assembly of the HIV coat proteins, and
inhibition of HIV entry
into uninfected cells. This definition includes any the method of action of
any of the
currently known HIV therapies. One method to determine if the growth of HIV
infection is
inhibited in found in Example 3.
A "therapeutically effective amount" is an amount effective to prevent or slow
the
development of, or to partially or totally alleviate the existing symptoms in
a particular
condition for which the subject being treated. Determination of a
therapeutically effective
amount is well within the capability of those skilled in the art.
The term "substantially pure", when used to modify the term a polypeptide or
other
compound, as used herein, refers to a polypeptide or compound, for example, a
polypeptide
isolated from the growth medium, in a form substantially free of, or
unadulterated by, active
inhibitory agents. The term "substantially pure" refers to a compound in an
amount of at
least about 75%, by dry weight, of isolated fraction, or "75% substantially
pure." More
specifically, the term "substantially pure" refers to a compound of at least
about 85%, by dry
weight, active compound, or "85% substantially pure." Most specifically, the
term
"substantially pure" refers to a compound of at least about 95%, by dry
weight, active
9
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
compound, or "95% substantially pure." The substantially pure cupredoxin or
cytochrome
c551 or a variant or derivative thereof can be used in combination with one or
more other
substantially pure compounds, or another isolated cupredoxin or cytochrome c55
1.
The phrases "isolated," "purified", or "biologically pure" refer to material
which is
substantially or essentially free from components which normally accompany the
material as
it is found in its native state. Thus, isolated peptides in accordance with
the invention
preferably do not contain materials normally associated with the peptides in
their in situ
environment. An "isolated" region refers to a region that does not include the
whole
sequence of the polypeptide from which the region was derived. An "isolated"
nucleic acid,
protein, or respective fragment thereof has been substantially removed from
its in vivo
environment so that it may be manipulated by the skilled artisan, such as but
not limited to
nucleotide sequencing, restriction digestion, site-directed mutagenesis, and
subcloning into
expression vectors for a nucleic acid fragment as well as obtaining the
protein or protein
fragment in substantially pure quantities.
The term "variant" as used herein with respect to a peptide, refers to amino
acid
sequence variants which may have amino acids replaced, deleted, or inserted as
compared to
the wild-type polypeptide. Variants may be truncations of the wild-type
peptide. Thus, a
variant peptide may be made by manipulation of genes encoding the polypeptide.
A variant
may be made by altering the basic composition or characteristics of the
polypeptide, but not
at least some of its fundamental activities. For example, a"variant" of azurin
can be a
mutated azurin that retains its ability to inhibit the growth of HIV infection
in mammalian
cells. In some cases, a variant peptide is synthesized with non-natural amino
acids, such as E-
(3,5-dinitrobenzoyl)-Lys residues. (Ghadiri & Fernholz, J. Am. Chem. Soc.,
112:9633-9635
(1990)) In some embodiments, the variant has not more than 20 amino acids
replaced,
deleted or inserted compared to wild-type peptide. In some embodiments, the
variant has not
more than 15 amino acids replaced, deleted or inserted compared to wild-type
peptide. In
some embodiments, the variant has not more than 10 amino acids replaced,
deleted or
inserted compared to wild-type peptide. In some embodiments, the variant has
not more than
6 amino acids replaced, deleted or inserted compared to wild-type peptide. In
some
embodiments, the variant has not more than 5 amino acids replaced, deleted or
inserted
compared to wild-type peptide. In some embodiments, the variant has not more
than 3 amino
acids replaced, deleted or inserted compared to wild-type peptide.
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
The term "amino acid," as used herein, means an amino acid moiety that
comprises
any naturally-occurring or non-naturally occurring or synthetic amino acid
residue, i.e., any
moiety comprising at least one carboxyl and at least one amino residue
directly linked by one,
two three or more carbon atoms, typically one (a) carbon atom.
The term "derivative" as used herein witli respect to a peptide refers to a
peptide that
is derived from the subject peptide. A derivation includes chemical
modifications of the
peptide such that the peptide still retains some of its fundamental
activities. For example, a
"derivative" of azurin can, for example, be a chemically modified azurin that
retains its
ability to inhibit the growth of HIV infection in mammalian cells. Chemical
modifications of
interest include, but are not limited to, amidation, acetylation, sulfation,
polyethylene glycol
(PEG) modification, phosphorylation or glycosylation of the peptide. In
addition, a
derivative peptide maybe a fusion of a polypeptide or fragment thereof to a
chemical
compound, such as but not limited to, another peptide, drug molecule or other
therapeutic or
pharmaceutical agent or a detectable probe.
The term "percent (%) amino acid sequence identity" is defined as the
percentage of
amino acid residues in a polypeptide that are identical with amino acid
residues in a candidate
sequence when the two sequences are aligned. To determine % amino acid
identity,
sequences are aligned and if necessary, gaps are introduced to achieve the
maximum %
sequence identity; conservative substitutions are not considered as part of
the sequence
identity. Amino acid sequence alignment procedures to determine percent
identity are well
known to those of skill in the art. Often publicly available computer software
such as
BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align peptide
sequences. In a specific embodiment, Blastp (available from the National
Center for
Biotechnology Information, Bethesda MD) is used using the default parameters
of long
complexity filter, expect 10, word size 3, existence 11 and extension 1.
When amino acid sequences are aligned, the % amino acid sequence identity of a
given amino acid sequence A to, with, or against a given amino acid sequence B
(which can
alternatively be phrased as a given amino acid sequence A that has or
comprises a certain %
amino acid sequence identity to, with, or against a given amino acid sequence
B) can be
calculated as:
% amino acid sequence identity = X/Y* 100
where
11
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
X is the number of amino acid residues scored as identical matches by the
sequence alignment program's or algorithm's alignment of A and B and
Y is the total number of amino acid residues in B.
If the length of amino acid sequence A is not equal to the length of amino
acid
sequence B, the % amino acid sequence identity of A to B will not equal the %
amino acid
sequence identity of B to A. When comparing longer sequences to shorter
sequences, the
shorter sequence will be the "B" sequence. For example, when comparing
truncated peptides
to the corresponding wild-type polypeptide, the truncated peptide will be the
"B" sequence.
General
The present invention provides compositions comprising cupredoxin and/or
cytochrome c551 and methods to inhibit viral and bacterial infection in
mammalian cells and
in mammalian patients. The present invention specifically relates to
compositions
comprising cupredoxin and/or cytochrome c551, and their use in inhibiting the
growth of
Human Immunodeficiency Virus (HIV) infection. The invention also relates to
variants,
derivatives and structural equivalents of cupredoxin and cytochrome c551 that
retain the
ability to inhibit the growth of viral infection, and in particular infection
by the HIV, and
compositions comprising the same. Most particularly, the invention provides
compositions
comprising Pseudomonas aeruginosa azurin and cytochrome c551, variants,
derivatives and
structural equivalents of azurin and cytochrome c551, and their use to treat
patients with viral
or bacterial infection, and specifically HIV-1 infection, or prevent infection
in those at risk
thereof. Finally, the invention provides methods to study infection of
mammalian cells by
viruses or bacteria by contacting the cells with cupredoxin or cytochrome
c551, or variant,
derivative or structural equivalent thereof before or after the cells are
infected, and measuring
the growth of infection.
Previously, it was know that two redox proteins elaborated by Pseudomonas
aerugisnosa, the cupredoxin azurin and cytochrome essi (cyt cssi), both
selectively enter J774
cells and show significant cytotoxic activity towards these cancerous cells
but not normal
cells. (Zaborina et al., Microbiology 146: 2521-2530 (2000).) Azurin can also
selectively
enter human melanoma UISO-Mel-2 or human breast cancer MCF-7 cells. (Yamada et
al.,
PNAS 99:14098-14103 (2002); Punj et al., Oncogene 23:2367-2378 (2004).) Azurin
from P.
aeruginosa preferentially enters J774 murine reticulum cell sarcoma cells,
forms a complex
12
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
with and stabilizes the tumor suppressor protein p53, enhances the
intracellular concentration
of p53, and induces apoptosis. (Yamada et al., Infection and Immunity 70:7054-
7062
(2002).)
Surprisingly, it is now known that azurin can induce about a 90% suppression
of
growth of HIV-1 in peripheral blood mononuclear cell (PBMC) cultures. See,
Example 3.
Azurin is now known to inhibit the growth of three strains of HIV-1, Bal (the
most
predominant clade B circulating in the US and Western Europe), a clade B
African isolate
RW/92/008/RE1, and a clade C Indian isolate IN/2167 D15. See, Example 3.
Additionally, a
cupredoxin-like protein from Neisseria, Laz, is nowalso known to inhibit the
growth of these
three HIV-1 strains, as well as a fusion of the H.8 region of the Laz protein
with P.
aeruginosa azurin. See, Example 3. Finally, it is now known that M44KM64E
mutant of
azurin and cytochrome c551 from P. aeruginosa can inhibit HIV infection in HIV-
infected
human blood lymphocytes. See, Example 1.
It is now known that azurin from Pseudomonas aeruginosa has a structural
similarly
with ICAM-1 (found in HIV-1 particles and known to enhance HIV-1 infectivity),
ICAM-2,
and CD4 (the primary cell surface receptor for HIV-1). See, Example 2. Surface
plasmon
resonance experiments have now revealed that in vitro azurin shows significant
binding to the
cell surface receptor CD4, and surprisingly a higher affinity for CD4 than the
HIV-1 surface
ligand gp120. See, Example 4. Further, GST-Azu 36-128, a glutathione S-
transferase
(GST) fusion to azurin amino acids 36-128, showed stronger binding to CD4 than
azurin
itself, while GST-Azu 88-113 showed no binding to CD4, suggesting that only a
part of the
azurin protein is responsible for binding to CD4. See, Example 4. Gp120 showed
a
somewhat stronger binding to azurin than CD4, indicating that azurin binds
both gp120 and
CD4. Further, ICAM-3 and NCAM showed strong binding the azurin in vitro in
surface
plasmon resonance assays. See, Example 5. Finally, azurin is now known to bind
another
receptor of HIV-1, dendritic cell-specific adhesion receptor (DC-SIGN). See,
Example 7.
Other proteins implicated in HIV-1 entry into host cells, such as gp4l, did
not show any
binding to azurin. See, Example 4.
Through competition experiments, it is now known that azurin can interfere
with
gp120 binding with its cognate receptor CD4 in vitro. Specifically, GST-Azu 36-
128 showed
significant ability to compete with gp 120 for CD4 binding, and GST-Azu 88-113
showed
little ability to compete. See, Example 6. Laz, an azurin like protein from
Neisseria, is now
13
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
know to inhibit the growth of HIV-1 infection in PBMC culture by 73% if added
before HIV-
1 infection, and very little if added after HIV-1 infection. See, Example 8.
While not
limiting the operation of the invention to any one mechanism, it now appears
that azurin may
inhibit the growth of HIV-1 infection in peripheral blood mononuclear cells by
interfering
with the interaction between the HIV and cell surface receptors such as ICAMs,
CD4 and
DC-SIGN, to prevent HIV from entering the cell and infecting it.
Accordingly, due to the high degree of structural similarity between
cupredoxins, that
other cupredoxins will as well inhibit the growth of HIV-1 infection in
mammalian blood
cells. It follows that in addition to HIV infection, cupredoxins also will
suppress other
infections of viruses that bind to cell surface receptors similar in structure
to the ICAMs, DC-
SIGN and CD4. For example, DC-SIGN is also a binding receptor of other viral
and
bacterial pathogens, including hepatitis C virus (HCV), Ebola virus,
cytomeglovirus (CMV)
and Mycobacterium tuberculosis. (Wang et al., Chin. Med. J. 117:1395-1400
(2004))
Compositions of the Invention
The invention provides for peptides that are variants, derivatives or
structural
equivalents of cupredoxin or Pseudomonas aeNuginosa cytochrome essl. In some
embodiments, the peptide is substantially pure. In other embodiments, the
peptide is in a
composition that comprises or consists essentially of the peptide. In other
embodiments, the
peptide is isolated. In some embodiments, the peptide is less that a full
length cupredoxin or
cytochrome c551, and retains some of the functional characteristics of the
cupredoxin or
cytochrome e551. In some embodiments, the peptide retains the ability to
inhibit the growth
of viral or bacterial infection, and more specifically HIV-1 infection in
peripheral blood
mononuclear cells. In a specific embodiment, the cytochrome c551 is SEQ ID NO:
2. In
another specific embodiment, the peptide does not raise an immune response in
a mammal,
and more specifically a human. The invention also provides compositions
comprising at least
one peptide that is a cupredoxin, Pseudoinonas aeruginosa cytochrome c551, or
variant,
derivative or structural equivalent of a cupredoxin or cytochrome c551. The
invention also
provides compositions comprising at least one peptide that is a cupredoxin,
Pseudomonas
aeruginosa cytochrome c551, or variant, derivative or structural equivalent of
a cupredoxin or
cytochrome c551 in a pharmaceutical composition.
14
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
Because of the high structural homology between the cupredoxins, it is
contemplated
that other cupredoxins will have the same anti-HIV activity as Pseudonaonas
aeruginosa
azurin with regards to inhibition of growth of HIV-1 infection. In some
embodiments, the
cupredoxin is, but is not limited to, azurin, pseudoazurin, plastocyanin,
rusticyanin or
auracyanin. In particularly specific embodiments, the azurin is derived from
Pseudomonas
aeruginosa, Alcaligenesfaecalis, Achromobacter xylosoxidans ssp.denitrificans
I, Bordetella
bronchiseptica, Methylomonas sp., Neisseria meningitidis Z2491, Neisseria
gonorrhea,
Pseudomonas f uorescens, Pseudomonas chlororaphis, Xylellafastidiosa 9a5 or
Vibrio
parahaemolyticus. In a very specific embodiment, the azurin is from
Pseudomonas
aeruginosa. In other specific embodiments, the cupredoxin comprises an amino
acid
sequence that is SEQ ID NO: 1, 3-17. In other specific embodiments, the
cupredoxin is the
Laz protein from Neisseria meningitidis or Neisseria gonorrhea.
The invention provides for amino acid sequence variants of a cupredoxin or
cytochrome c551 which have amino acids replaced, deleted, or inserted as
compared to the
wild-type polypeptide. Variants of the invention may be truncations of the
wild-type
polypeptide. In some embodiments, the composition comprises a peptide that
consists of a
region of a cupredoxin or cytochrome c551 that is less that the full length
wild-type
polypeptide. In some embodiments, the composition comprises a peptide that
consists of
more than about 10 residues, more than about 15 residues or more than about 20
residues of
a truncated cupredoxin or cytochrome c551. In some embodiments, the
composition
comprises a peptide that consists of not more than about 100 residues, not
more than about 50
residues, not more than about 40 residues or not more than about 30 residues
of a truncated
cupredoxin or cytochrome c551. In some embodiments, composition comprises a
peptide to
which a cupredoxin or cytochrome c551, and more specifically to SEQ ID NOS: 1-
17 has at
least about 90% amino acid sequence identity, at least about 95% amino acid
sequence
identity or at least about 99% amino acid sequence identity.
In specific embodiments, the variant of cupredoxin comprises P. aeruginosa
azurin
residues 36-128, azurin residues 36-88, or azurin residues 88-113. In other
embodiments, the
variant of cupredoxin consists of P. aeruginosa azurin residues 36-128, azurin
residues 36-
88, or azurin residues 88-113. In other specific embodiments, the variant
consists of the
equivalent residues of a cupredoxin other that azurin. It is also contemplated
that other
cupredoxin variants can be designed that have a similar activity to azurin
residues 36-128,
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
azurin residues 36-88, or azurin residues 88-113. To do this, the subject
cupredoxin amino
acid sequence will be aligned to the Pseudomonas aeruginosa azurin sequence
using BLAST,
BLAST2, ALIGN2 or Megalign (DNASTAR), the relevant residues located on the P.
aeruginosa azurin amino acid sequence, and the equivalent residues found on
the subject
cupredoxin sequence, and the equivalent truncated peptide thus designed.
The variants also include peptides made with synthetic amino acids not
naturally
occurring. For example, non-naturally occurring amino acids may be integrated
into the
variant peptide to extend or optimize the half-life of the composition in the
bloodstream.
Such variants include, but are not limited to, D,L-peptides (diastereomer),
(Futaki et al., J.
Biol. Chem. 276(8):5836-40 (2001); Papo et al., Cancer Res. 64(16):5779-86
(2004); Miller
et al, Biochem. Pharmacol. 36(1):169-76, (1987)); peptides containing unusual
amino acids
(Lee et al., J. Pept. Res. 63(2):69-84 (2004)), and olefin-containing non-
natural amino acid
followed by hydrocarbon stapling (Schafineister et al., J. Am. Chem. Soc.
122:5891-5892
(2000); Walenski et al., Science 305:1466-1470 (2004)) and peptides conprising
E-(3,5-
dinitrobenzoyl)-Lys residues.
In other embodiments, the peptide of the invention is a derivative of a
cupredoxin or
cytochrome c551. The derivatives of cupredoxin/or cytochrome c551 are chemical
modifications of the peptide such that the peptide still retains some of its
fundamental
activities. For example, a "derivative" of azurin can be a chemically modified
azurin that
retains its ability to inhibit the growth of HIV infection in mammalian cells.
Chemical
modifications of interest include, but are not limited to, amidation,
acetylation, sulfation,
polyethylene glycol (PEG) modification, phosphorylation and glycosylation of
the peptide.
In addition, a derivative peptide maybe a fusion of a cupredoxin or cytochrome
c551, or
variant, derivative or structural equivalent thereof to a chemical compound,
such as but not
limited to, another peptide, drug molecule or other therapeutic or
pharmaceutical agent or a
detectable probe. Derivatives of interest include chemical modifications by
which the half-
life in the bloodstream of the peptides and compositions of the invention can
be extended or
optimized, such as by several methods well known to those in the art,
including but not
limited to, circularized peptides (Monk et al., BioDrugs 19(4):261-78, (2005);
DeFreest et al.,
J. Pept. Res. 63(5):409-19 (2004)), N- and C- terminal modifications (Labrie
et al., Clin.
Invest. Med. 13(5):275-8, (1990)), and olefin-containing non-natural amino
acid followed by
16
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
hydrocarbon stapling (Schafineister et al., J. Am. Chem. Soc. 122:5891-5892
(2000);
Walenski et al., Science 305:1466-1470 (2004)).
It is contemplated that the peptides of the composition of invention may be a
variant,
derivative and/or structural equivalent of a cupredoxin or cytochrome c551.
For example, the
peptides may be a truncation of azurin that has been PEGylated, thus making it
both a variant
and a derivative. In one embodiment, the peptides of the invention are
synthesized with a,a-
disubstituted non-natural amino acids containing olefin-bearing tethers,
followed by an all-
hydrocarbon "staple" by ruthenium catalyzed olefin metathesis. (Scharmeister
et al., J. Ain.
Chem. Soc. 122:5891-5892 (2000); Walensky et al., Science 305:1466-1470
(2004))
Additionally, peptides that are structural equivalents of azurin may be fused
to other peptides,
thus making a peptide that is both a structural equivalent and a derivative.
These examples
are merely to illustrate and not to limit the invention. Variants, derivatives
or structural
equivalents of cupredoxin or cytochrome c551 may or may not bind copper.
In another embodiment, the peptide is a structural equivalent of a cupredoxin
or
cytochrome c551. Examples of studies that determine significant structural
homology between
cupredoxins and other proteins include Toth et al. (Develapmental Cell 1:82-92
(2001)).
Specifically, significant structural homology between a cupredoxin and the
structural
equivalent is determined by using the VAST algorithm (Gibrat et al., Curr Opin
Struct Biol
6:377-385 (1996); Madej et al., Proteins 23:356-3690 (1995)). In specific
einbodiments,
the VAST p value from a structural comparison of a cupredoxin to the
structural equivalent is
less than about 10-3, less than about 10"5, or less than about 10-7. In other
embodiments,
significant structural homology between a cupredoxin and the structural
equivalent is
determined by using the DALI algorithm (Holm & Sander, J. Mol. Biol. 233:123-
138
(1993)). In specific embodiments, the DALI Z score for a pairwise structural
comparison is
at least about 3.5, at least about 7.0, or at least about 10Ø
In some embodiments, the cupredoxin or cytochrome c551, or variant, derivative
or
structural equivalent thereof has some of the functional characteristics of
the P. aeruginosa
azurin or cytochrome c551. In a specific embodiment, the cupredoxin or
cytochrome c551
inhibits the growth of viral or bacterial infection, and specifically HIV
infection in
mammalian cells, more specifically in peripheral blood mononuclear cells
infected with HIV.
The invention also provides for the variants, derivatives and structural
equivalents of
cupredoxin and cytochrome c551 that retain the ability to inhibit the growth
of viral or
17
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
bacterial infection, and specifically HIV infection in mammalian cells. The
growth of HIV-1
infection in the cells may be determined by measuring the change in the
production of HIV-1
p24 antigen in the cell culture supernatant by a commercial p24 enzyme
immunoassay
(PerkinElmer Life Sciences, Inc., Wellseley, Mass.). Inhibition of a growth of
infection is
any decrease or lessening of the rate of increase of that infection that is
statistically
signification as compared to control treatments.
Because it is now known that cupredoxins and cytochrome C551 can limit the
growth
of viral or bacterial infection, and specifically infection in HIV-infected
mammalian cells, it
is now possible to design variants and derivatives of cupredoxins that retain
this anti-viral or
anti-bacterial, and specifically anti-HIV activity. Such variants and
derivatives can be made
by, for example, creating a "library" of various variants and derivatives of
cupredoxins and
cytochrome C551, and then testing each for anti-viral or anti-bacterial, and
specifically anti-
HIV activity using one of many methods known in the art, such the exemplary
method in
Example 3. It is contemplated that the resulting variants and derivatives of
cupredoxins with
anti-viral or anti-bacterial, and specifically anti-HIV activity can be used
in the methods of
the invention, in place of or in addition to cupredoxins or cytochrome c551 =
In some specific embodiments, the cupredoxin or cytochrome, or variant,
derivative
or structural equivalent thereof binds to CD4 with a binding constant that is
statistically
different a non-binding control protein. A peptide can be tested for this
activity by using
surface plasmon resonance analysis as described in Example 4. Other methods to
determine
whether one protein binds to another are well known in the art and may be used
as well.
In some specific embodiments, the cupredoxin or cytochrome, or variant,
derivative
or structural equivalent thereof binds to gp120 with a binding constant that
is statistically
different a non-binding control protein. A peptide can be tested for this
activity by using
surface plasmon resonance analysis as described in Example 4. Other methods to
determine
whether one protein binds to another are well known in the art and may be used
as well.
In some specific embodiments, the cupredoxin or cytochrome, or variant,
derivative
or structural equivalent thereof binds to ICAM3 with a binding constant that
is statistically
different a non-binding control protein. A peptide can be tested for this
activity by using
surface plasmon resonance analysis as described in Examples 4 and 5. Other
methods to
determine whether one protein binds to another are well known in the art and
may be used as
well.
18
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
In some specific embodiments, the cupredoxin or cytochrome, or variant,
derivative
or structural equivalent thereof binds to DC-SIGN with a binding constant that
is statistically
different a non-binding control protein. A peptide can be tested for this
activity by using
surface plasmon resonance analysis as described in Examples 4 and 5. Other
methods to
determine whether one protein binds to another are well known in the art and
may be used as
well.
In some specific embodiments, the peptide of the invention induces apoptosis
in a
mammalian cancer cell, more specifically a J774 cell. The ability of a
cupredoxin or other
polypeptide to induce apoptosis may be observed by mitosensor ApoAlert
confocal
microscopy using a MITOSENSORTM APOLERTTM Mitochondrial Membrane Sensor kit
(Clontech Laboratories, Inc., Palo Alto, California, U.S.A.), by measuring
caspase-8,
caspase-9 and caspase-3 activity using the method described in Zou et al. J.
Biol. Chem.
274: 11549-11556 (1999)), and by detecting apoptosis-induced nuclear DNA
fragmentation
using, for example, the APOLERTTM DNA fragmentation kit (Clontech
Laboratories, Inc.,
Palo Alto, California, U.S.A.).
In another specific embodiment, the peptide of the invention induces cellular
growth
arrest in a mammalian cancer cell, more specifically a J774 cell. Cellular
growth arrest can
be determined by measuring the extent of inhibition of cell cycle progression,
such as by the
method found in Yamada et al. (PNAS 101:4770-4775 (2004)). In another specific
embodiment, the cupredoxin or cytochrome c551, or variant, derivative or
structural equivalent
thereof inhibits cell cycle progression in a mammalian cancer cell, more
specifically a J774
cell.
Cupredoxins
These small blue copper proteins (cupredoxins) are electron transfer proteins
(10-20
kDa) that participate in bacterial electron transfer chains or are of unknown
function. The
copper ion is solely bound by the protein matrix. A special distorted trigonal
planar
arrangement to two histidine and one cysteine ligands around the copper gives
rise to very
peculiar electronic properties of the metal site and an intense blue color. A
number of
cupredoxins have been crystallographically characterized at medium to high
resolution.
The cupredoxins in general have a low sequence homology but high structural
homology. (Gough & Clothia, Structure 12:917-925 (2004); De Rienzo et al.,
Protein
Science 9:1439-1454 (2000)). For example, the amino acid sequence of azurin is
31%
19
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
identical to that of auracyanin B, 16.3% to that of rusticyanin, 20.3 % to
that of plastocyanin,
and 17.3% to that of pseudoazurin. See, Table 1. However, the structural
similarity of these
proteins is more pronounced. The VAST p value for the comparison of the
structure of
azurin to auracyanin B is 10-7'4, azurin to rusticyanin is 10-5, azurin to
plastocyanin is 10-5'6,
and azurin to psuedoazurin is 10-4'1.
All of the cupredoxins possess an eight-stranded Greek key beta-barrel or beta-
sandwich fold and have a highly conserved site architecture. (De Rienzo et
al., Protein
Science 9:1439-1454 (2000)) A prominent hydrophobic patch, due to the presence
of many
long chain aliphatic residues such as methionines and leucines, is present
around the copper
site in azurins, amicyanins, cyanobacterial plastocyanins, cucumber basic
protein and to a
lesser extent, pseudoazurin and eukaryotic plastocyanins. Id. Hydrophobic
patches are also
found to a lesser extent in stellacyanin and rusticyanin copper sites, but
have different
features. Id.
Table 1. Sequence and structure alignment of azurin (1JZG) from P. aeruginosa
to other
proteins using VAST algorithm.
PDB Alignment % aa P-value 2 Score3 RMSD 4 Description
lengthl identity
1AOZ A 2 82 18.3 10 e-7 12.2 1.9 Ascorbate oxidase
1 QHQ_A 113 31 lOe-7.4 12.1 1.9 AuracyaninB
1V54 B 1 79 20.3 lOe-6.0 11.2 2.1 Cytocrome c oxidase
1 GY2 A 92 16.3 lOe-5.0 11.1 1.8 Rusticyanin
3MSP A 74 8.1 10e-6.7 10.9 2.5 Motile Major Sperm
Protein5
1IUZ 74 20.3 10e-5.6 10.3 2.3 Plastocyanin
1KGY E 90 5.6 lOe-4.6 10.1 3.4 Ephrinb2
1PMY 75 17.3 lOe-4.1 9.8 2.3 Pseudoazurin
'Aligned Length: The number of equivalent pairs of C-alpha atoms superimposed
between the two structures, i.e. how many residues have been used to calculate
the 3D
superposition.
aP-VAL: The VAST p value is a measure of the significance of the comparison,
expressed as a probability. For example, if the p value is 0.001, then the
odds are 1000 to 1
against seeing a match of this quality by pure chance. The p value from VAST
is adjusted for
the effects of multiple comparisons using the assumption that there are 500
independent and
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
unrelated types of domains in the MMDB database. The p value shown thus
corresponds to
the p value for the pairwise comparison of each domain pair, divided by 500.
3Score: The VAST structure-similarity score. This number is related to the
number of
secondary structure elements superimposed and the quality of that
superposition. Higher
VAST scores correlate with higher similarity.
4RMSD: The root mean square superposition residual in Angstroms. This number
is
calculated after optimal superposition of two structures, as the square root
of the mean square
distances between equivalent C-alpha atoms. Note that the RMSD value scales
with the
extent of the structural alignments and that this size must be taken into
consideration when
using RMSD as a descriptor of overall structural similarity.
5 C. elegans major sperm protein proved to be an ephrin antagonist in oocyte
maturation (Kuwabara, 2003 "The multifaceted C. elegans major sperm protein:
an ephrin
signalling antagonist in oocyte maturation" Genes and Developnzent, 17:155-
161.
' Azurin
The azurins are copper containing proteins of 128 amino acid residues which
belong
to the family of cupredoxins involved in electron transfer in certain
bacteria. The azurins
include those from P. aeruginosa (PA) (SEQ ID NO: 1), A. xylosoxidans, and A.
denitrificans. (Murphy et al., J. Mol. Biol. 315:859-871 (2002)) The amino
acid sequence
identity between the azurins varies between 60-90%, these proteins showed a
strong
structural homology. All azurins have a characteristic (3-sandwich with Greek
key motif and
the single copper atom is always placed at the same region of the protein. In
addition, azurins
possess an essentially neutral hydrophobic patch surrounding the copper site.
Id.
Plastocyanins
The plastocyanins are soluble proteins of cyanobacteria, algae and plants that
contain
one molecule of copper per molecule and are blue in their oxidized form. They
occur in the
chloroplast, where they function as electron carriers. Since the determination
of the structure
of poplar plastocyanin in 1978, the structure of algal (Scenedesinus,
Enterornorpha,
Chlanaydornonas) and plant (French bean) plastocyanins has been determined
either by
crystallographic or NMR methods, and the poplar structure has been refined to
1.33 A
resolution. SEQ ID NO: 3 shows the amino acid sequence of plastocyanin from
Phornaidiufn
laminosum, a thermophilic cyanobacterium.
21
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
Despite the sequence divergence among plastocyanins of algae and vascular
plants
(e.g., 62% sequence identity between the Chlamydomonas and poplar proteins),
the three-
dimensional structures are conserved (e.g., 0.76 A rms deviation in the C
alpha positions
between the Chlamydomonas and Poplar proteins). Structural features include a
distorted
tetrahedral copper binding site at one end of an eight-stranded antiparallel
beta-barrel, a
pronounced negative patch, and a flat hydrophobic surface. The copper site is
optimized for
its electron transfer function, and the negative and hydrophobic patches are
proposed to be
involved in recognition of physiological reaction partners. Chemical
modification, cross-
linking, and site-directed mutagenesis experiments have confirined the
importance of the
negative and hydrophobic patches in binding interactions with cytochrome f,
and validated
the model of two functionally significant electron transfer paths involving
plastocyanin. One
putative electron transfer path is relatively short (approximately 4 A) and
involves the
solvent-exposed copper ligand His-87 in the hydrophobic patch, while the other
is more
lengthy (approximately 12-15 A) and involves the nearly conserved residue Tyr-
83 in the
negative patch, Redinbo et al., J. Bioenerg. Biomembr. 26:49-66 (1994).
Rusticyanins
Rusticyanins are blue-copper containing single-chain polypeptides obtained
from a
Thiobacillus (now called Acidithiobacillus). The X-ray crystal structure of
the oxidized form
of the extremely stable and highly oxidizing cupredoxin rusticyanin from
Thiobacillus
ferrooxidans (SEQ ID NO: 4) has been determined by multiwavelength anomalous
diffraction and refined to 1.9A resolution. The rusticyanins are composed of a
core beta-
sandwich fold composed of a six- and a seven-stranded b-sheet. Like other
cupredoxins, the
copper ion is coordinated by a cluster of four conserved residues (His 85,
Cys138, His143,
Met148) arranged in a distorted tetrahedron. Walter, R.L. et al., J.llfol.
Biol. 263:730-51
(1996).
Pseudoazurins
The pseudoazurins are a family of blue-copper containing single-chain
polypeptide.
The amino acid sequence of pseudoazurin obtained from AchNomobacter
cycloclastes is
shown in SEQ ID NO: 5. The X-ray structure analysis of pseudoazurin shows that
it has a
similar structure to the azurins although there is low sequence homology
between these
22
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
proteins. Two main differences exist between the overall structure of the
pseudoazurins and
azurins. There is a carboxy terminus extension in the pseudoazurins, relative
to the azurins,
consisting of two alpha-helices. In the mid-peptide region azurins contain an
extended loop,
shortened in the pseudoazurins, which forms a flap containing a short a-helix.
The only
major differences at the copper atom site are the conformation of the MET side-
chain and the
Met-S copper bond length, which is significantly shorter in pseudoazurin than
in azurin.
Phytocyanins
The proteins identifiable as phytocyanins include, but are not limited to,
cucumber
basic protein, stellacyanin, mavicyanin, umecyanin, a cucumber peeling
cupredoxin, a
putative blue copper protein in pea pods, and a blue copper protein from
Arabidopsis
thaliana. In all except cucumber basic protein and the pea-pod protein, the
axial methionine
ligand normally found at blue copper sites is replaced by glutamine.
Auracyanin
Three small blue copper proteins designated auracyanin A, auracyanin B-1, and
auracyanin B-2 have been isolated from the thermophilic green gliding
photosynthetic
bacterium Chloroflexus aurantiacus. The two B forms are glycoproteins and have
almost
identical properties to each other, but are distinct from the A form. The
sodium dodecyl
sulfate-polyacrylamide gel electrophoresis demonstrates apparent monomer
molecular
masses as 14 (A), 18 (B-2), and 22 (B-1) kDa.
The amino acid sequence of auracyanin A has been determined and showed
auracyanin A to be a polypeptide of 139 residues. (Van Dreissche et al;.
Protein Science
8:947-957 (1999).) His58, Cys123, His128, and Met132 are spaced in a way to be
expected
if they are the evolutionary conserved metal ligands as in the known small
copper proteins
plastocyanin and azurin. Secondary structure prediction also indicates that
auracyanin has a
general beta-barrel structure similar to that of azurin from Pseudomonas
aeruginosa and
plastocyanin from poplar leaves. However, auracyaniri appears to have sequence
characteristics of both small copper protein sequence classes. The overall
similarity with a
consensus sequence of azurin is roughly the same as that with a consensus
sequence of
plastocyanin, namely 30.5%. The N-terminal sequence region 1-18 of auracyanin
is
remarkably rich in glycine and hydroxy amino acids. Id. See exemplary amino
acid
23
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
sequence SEQ ID NO: 15 for chain A of auracyanin from Chloroflexus aurantiacus
(NCBI
Protein Data Bank Accession No. AAM12874).
The auracyanin B molecule has a standard cupredoxin fold. The crystal
structure of
auracyanin B from Chloroflexus aurantiacus has been studied. (Bond et al., J.
Mol. Biol.
306:47-67 (2001).) With the exception of an additional N-terminal strand, the
molecule is
very similar to that of the bacterial cupredoxin, azurin. As in other
cupredoxins, one of the
Cu ligands lies on strand 4 of the polypeptide, and the other three lie along
a large loop
between strands 7 and 8. The Cu site geometry is discussed with reference to
the amino acid
spacing between the latter three ligands. The crystallographically
characterized Cu-binding
domain of auracyanin B is probably tethered to the periplasmic side of the
cytoplasmic
membrane by an N-terminal tail that exhibits significant sequence identity
with known tethers
in several other membrane-associated electron-transfer proteins. The amino
acid sequences
of the B forms are presented in McManus et al. (JBiol Cheni. 267:6531-6540
(1992).). See
exemplary amino acid sequence SEQ ID NO: 16 for chain B of auracyanin from
ChloNoflexus
aurantiacus (NCBI Protein Data Bank Accession No. 1QHQA).
Stellacyanin
Stellacyanins are a subclass of phytocyanins, a ubiquitous family of plant
cupredoxins. An exemplary sequence of a stellacyanin is included herein as SEQ
ID NO: 14.
The crystal structure of umecyanin, a stellacyanin from horseradish root (Koch
et al., J. Am.
Chern. Soc. 127:158-166 (2005)) and cucumber stellacyanin (Hart el al.,
Protein Science
5:2175-2183 (1996)) is also known. The protein has an overall fold similar to
the other
phytocyanins. The ephrin B2 protein ectodomain tertiary structure bears a
significant
similarity to stellacyanin. (Toth et al., Developnzental Cell 1:83-92 (2001).)
An exemplary
amino acid sequence of a stellacyanin is found in the National Center for
Biotechnology
Information Protein Data Bank as Accession No. 1JER, SEQ ID NO: 14.
Cucumber basic protein
An exemplary amino acid sequence from a cucumber basic protein is included
herein
as SEQ ID NO: 17. The crystal structure of the cucumber basic protein (CBP), a
type 1 blue
copper protein, has been refined at 1.8 A resolution. The molecule resembles
other blue
copper proteins in having a Greek key beta-barrel structure, except that the
barrel is open on
24
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
one side and is better described as a "beta-sandwich" or "beta-taco". (Guss et
al., J. Mol.
Biol. 262:686-705 (1996).) The ephrinB2 protein ectodomian tertiary structure
bears a high
similarity (rms deviation 1.5A for the 50 a carbons) to the cucumber basic
protein. (Toth et
al., Developmental Cell 1:83-92 (2001).)
The Cu atom has the normal blue copper NNSS' co-ordination with bond lengths
Cu-
N(His39) = 1.93 A, Cu-S(Cys79) = 2.16 A, Cu-N(His84) = 1.95 A, Cu-S(Met89) =
2.61 A.
A disulphide link, (Cys52)-S-S-(Cys85), appears to play an important role in
stabilizing the
molecular structure. The polypeptide fold is typical of a sub-family of blue
copper proteins
(phytocyanins) as well as a non-metalloprotein, ragweed allergen Ra3, with
which CBP has a
high degree of sequence identity. The proteins currently identifiable as
phytocyanins are
CBP, stellacyanin, mavicyanin, umecyanin, a cucumber peeling cupredoxin, a
putative blue
copper protein in pea pods, and a blue copper protein from Arabidopsis
thaliana. In all except
CBP and the pea-pod protein, the axial methionine ligand normally found at
blue copper sites
is replaced by glutamine. An exemplary sequence for cucumber basic protein is
found in
NCBI Protein Data Bank Accession No. 2CBP, SEQ ID NO: 17.
Methods of Use
The invention provides a method to treat a patient infected with viral or
bacterial
infection, and specifically HIV-1 infection, comprising administering to the
patient at least
one polypeptide that is a cupredoxin or cytochrome c551, or variant,
derivative or structural
equivalent thereof, as described above. It is also contemplated that the same
method will be
effective to treat patients with other viral or bacterial infections, such as,
but not limited to,
Herpes simplex virus (HSV), Ebola virus, cytomeglovirus (CMV), parainfluenza
viruses
types A. B and C, etc., hepatitis A, B, C, G, the delta hepatitis virus (HDV),
mumps viruses,
measles viruses, respiratory syncytial viruses, bunyviruses, arena viruses,
the orthomyxo-like
insect virus called Dhori, polioviruses, rubella virus, dengue virus; SIV and
Mycobacterium
tuberculosis.
It has also been learned that cupredoxins and cytochrome c are also effective
against
malarial infections, as disclosed in a co-filed application. Further, co-
infections with HIV
and malaria are very common in many areas of the world, and in particular sub-
Saharan
Africa. In some embodiments, the patient suffering from infection by HIV is
also suffering
from infection by a malaria parasite. In some embodiments, the method of
treatment of the
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
invention also comprises administering anti-malarial drugs. In some
embodiments, the anti-
malarial drugs are co-administered. ,
The methods of the invention include contacting mammalian cells with a
composition
comprising cupredoxin or cytochrome c551, or variant, derivative or structural
equivalent
thereof. In some embodiments, the mammalian cells are infected with a virus or
bacteria,
such as HIV-1. In other embodiments, the mammalian cells will be exposed to a
virus or
bacteria, such as HIV-1.
The composition comprising cupredoxin or cytochrome c551, or variant,
derivative or
structural equivalent thereof can be administered to the patient by many
routes and in many
regimens that will be well known to those in the art. In specific embodiments,
the
cupredoxin or cytochrome c551, or variant, derivative or structural equivalent
thereof is
administered intravenously, intramuscularly or subcutaneously.
In one embodiment, the methods may comprise co-administering to a patient one
unit
dose of a composition comprising a cupredoxin or cytochrome c551, or a
variant, derivative or
structural equivalent of cupredoxin or cytochrome c551, and one unit dose of a
composition
comprising another anti-viral or anti-bacterial, and specifically anti-HIV
drug and/or an anti-
malarial drug, in either order, administered at about the same time, or within
about a given
time following the administration of the other, for example, about one minute
to about 6o
minutes following the administration of the other drug, or about 1 hour to
about 12 hours
following the administration of the other drug.
In addition, the present invention includes methods useful for studying viral
and
bacterial infection. In some embodiments, the method comprises contacting
mammalian cells
infected with a virus or bacteria with a cupredoxin or cytochrome c551, or
variant, derivative
or structural equivalent thereof, and measuring the growth of infection. In
other
embodiments, the method comprises contacting mammalian cells with a cupredoxin
or
cytochrome c551, or variant, derivative or structural equivalent thereof,
contactirig the
mammalian cells with a virus or bacteria, and measuring the growth of
infection in the cells.
In some embodiments, the bacteria or virus is HIV, Herpes simplex virus (HSV),
Ebola virus,
cytomeglovirus (CMV), parainfluenza viruses types A, B and C, etc., hepatitis
A, B, C, G, the
delta hepatitis virus (HDV), mumps viruses, measles viruses, respiratory
syncytial viruses,
bunyviruses, arena viruses, the orthomyxo-like insect virus called Dhori,
polioviruses, rubella
virus, dengue virus; SIV and Mycobacteriuna tuberculosis. In a specific
embodiment, the
26
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
virus is HIV-1, and particularly strains Bal, 2167, or RW/92/008/RE1. In other
embodiments, the mammalian cells are human. In other embodiments, the cells
are blood
lymphocytes or peripheral blood mononuclear cells. In various embodiments, the
cells are
contacted in the animal (in vivo), or outside the animal (in vitro). In some
embodiments, the
cells are contacted in vitro and them introduced into an animal.
Pharmaceutical Compositions Comprising Cupredoxin Or Cytochrome C551, Or
Variant, Derivative Or Structural Equivalent Thereof
Pharmaceutical compositions comprising cupredoxin or cytochrome c551, or
variant,
derivative or structural equivalents thereof, can be manufactured in any
conventional manner,
e.g. by conventional mixing, dissolving, granulating, dragee-making,
emulsifying,
encapsulating, entrapping, or lyophilizing processes. The substantially pure
cupredoxin
and/or cytochrome c551, and variants, derivatives and structural equivalents
thereof can be
readily combined with a pharmaceutically acceptable carrier well-known in the
art. Such
carriers enable the preparation to be formulated as a tablet, pill, dragee,
capsule, liquid, gel,
syrup, slurry, suspension, and the like. Suitable carriers or excipients can
also include, for
example, fillers and cellulose preparations. Other excipients can include, for
example,
flavoring agents, coloring agents, detackifiers, thickeners, and other
acceptable additives,
adjuvants, or binders. In some embodiments, the pharmaceutical preparation is
substantially
free of preservatives. In other embodiments, the pharmaceutical preparation
may contain at
least one preservative. General methodology on pharmaceutical dosage forms is
found in
Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems
(Lippencott
Williams & Wilkins, Baltimore MD (1999)).
The composition comprising a cupredoxin or cytochrome c551, or variant,
derivative
or structural equivalent thereof used in the invention may be administered in
a variety of
ways, including by injection (e.g., intradermal, subcutaneous, intramuscular,
intraperitoneal
and the like), by inhalation, by topical adniinistration, by suppository, by
using a transdermal
patch or by mouth. General information on drug delivery systems can be found
in Ansel et
al., Id.. In some embodiments, the composition comprising a cupredoxin or
cytochrome c551,
or variant, derivative or structural equivalent thereof can be formulated and
used directly as
injectibles, for subcutaneous and intravenous injection, among others. The
injectable
formulation, in particular, can advantageously be used to treat patients that
are at risk of,
27
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
likely to have or have a viral or bacterial infection, and specifically an HIV-
infection. The
composition comprising a cupredoxin or cytochrome c551, or variant, derivative
or structural
equivalent thereof can also be taken orally after mixing with protective
agents such as
polypropylene glycols or similar coating agents.
When administration is by injection, the cupredoxin or cytochrome c551, or
variant,
derivative or structural equivalent thereof may be formulated in aqueous
solutions,
specifically in physiologically compatible buffers such as Hanks solution,
Ringer's solution,
or physiological saline buffer. The solution may contain formulatory agents
such as
suspending, stabilizing and/or dispersing agents. Alternatively, the
cupredoxin or
cytochrome c551, or variant, derivative or structural equivalent thereof may
be in powder form
for constitution with a suitable vehicle, e.g., sterile pyrogen-free water,
before use. In some
embodiments, the pharmaceutical composition does not comprise an adjuvant or
any other
substance added to enhance the immune response stimulated by the peptide. In
some
embodiments, the pharmaceutical composition comprises a substance that
inhibits an immune
response to the peptide.
When administration is by intravenous fluids, the intravenous fluids for use
administering the cupredoxin or cytochrome c551, or variant, derivative or
structural
equivalent thereof may be composed of crystalloids or colloids. Crystalloids
as used herein
are aqueous solutions of mineral salts or other water-soluble molecules.
Colloids as used
herein contain larger insoluble molecules, such as gelatin. Intravenous fluids
may be sterile.
Crystalloid fluids that may be used for intravenous administration include but
are not
limited to, normal saline (a solution of sodium chloride at 0.9%
concentration), Ringer's
lactate or Ringer's solution, and a solution of 5% dextrose in water sometimes
called D5W, as
described in Table 2.
Table 2. Composition of Common Crystalloid Solutions
Solution Other Name [Na+], [CI"] [Glucose]
D5W 5% Dextrose 0 0 252
2/3 & 1/3 3.3% Dextrose 51 51 168
/ 0.3% saline
Half-normal 0.45% NaCl 77 77 0
saline
28
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
Normal saline 0.9% NaC1 154 154 0
Ringer's Ringer's 130 109 0
lactate* solution
*Ringer's lactate also has 28 mmol/L lactate, 4 mmol/L K+ and 3 mmol/L Ca2+.
When administration is by inhalation, the cupredoxin or cytochrome c551, or
variant,
derivative or structural equivalent thereof may be delivered in the form of an
aerosol spray
from pressurized packs or a nebulizer with the use of a suitable propellant,
e.g.,
dichlorodifluoromethane, trichlorofluoromethane, carbon dioxide or other
suitable gas. In the
case of a pressurized aerosol, the dosage unit may be determined by providing
a valve to
deliver a metered amount. Capsules and cartridges of, e.g., gelatin, for use
in an inhaler or
insufflator may be forrnulated containing a powder mix of the proteins and a
suitable powder
base such as lactose or starch.
When administration is by topical administration, the cupredoxin or cytochrome
cssi,
or variant, derivative or structural equivalent thereof may be formulated as
solutions, gels,
ointments, creams, jellies, suspensions, and the like, as are well known in
the art. In some
embodiments, administration is by means of a transdermal patch. When
administration is by
suppository (e.g., rectal or vaginal), cupredoxin and/or cytochrome c and
variants and
derivatives thereof compositions may also be formulated in compositions
containing
conventional suppository bases.
When administration is oral, a cupredoxin or cytochrome c551, or variant,
derivative or
structural equivalent thereof can be readily formulated by combining the
cupredoxin or
cytochrome cssl, or variant, derivative or structural equivalent thereof with
pharmaceutically
acceptable carriers well known in the art. A solid carrier, such as mannitol,
lactose,
magnesium stearate, and the like may be employed; such carriers enable the
cupredoxin
and/or cytochrome c and variants and derivatives thereof to be formulated as
tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like,
for oral ingestion
by a subject to be treated. For oral solid formulations such as, for example,
powders,
capsules and tablets, suitable excipients include fillers such as sugars,
cellulose preparation,
granulating agents, and binding agents.
Other convenient carriers, as well-known in the art, also include multivalent
carriers,
such as bacterial capsular polysaccharide, a dextran or a genetically
engineered vector. In
29
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
addition, sustained-release formulations that include a cupredoxin or
cytochrome C551, or
variant, derivative or structural equivalent thereof allow for the release of
cupredoxin or
cytochrome c551, or variant, derivative or structural equivalent thereof over
extended periods
of time, such that without the sustained release formulation, the cupredoxin
or cytochrome
c551, or variant, derivative or structural equivalent thereof would be cleared
from a subject's
system, and/or degraded by, for example, proteases and simple hydrolysis
before eliciting or
enhancing a therapeutic effect.
The half-life in the bloodstream of the compositions of the invention can be
extended
or optimized by several methods well known to those in the art, including but
not limited to,
circularized peptides (Monk et al., BioDrugs 19(4):261-78, (2005); DeFreest et
al., J. Pept.
Res. 63(5):409-19 (2004)), D,L-peptides (diastereomer), (Futaki et al., J.
Biol. Chem. Feb
23;276(8):5836-40 (2001); Papo et al., Cancer Res. 64(16):5779-86 (2004);
Miller et al.,
Biochem. Pharmacol. 36(l):169-76, (1987)); peptides containing unusual amino
acids (Lee et
al., J. Pept. Res. 63(2):69-84 (2004)), N- and C- terminal modifications
(Labrie et al., Clin.
Invest. Med. 13(5):275-8, (1990)), and hydrocarbon stapling (Schafineister et
al., J. Am.
Chem. Soc. 122:5891-5892 (2000); Walenski et al., Science 305:1466-1470
(2004)). Of
particular interest are d-isomerization (substitution) and modification of
peptide stability via
D-substitution or L- amino acid substitution.
In various embodiments, the pharmaceutical composition includes carriers and
excipients (including but not limited to buffers, carbohydrates, mannitol,
proteins,
polypeptides or amino acids such as glycine, antioxidants, bacteriostats,
chelating agents,
suspending agents, thickening agents and/or preservatives), water, oils,
saline solutions,
aqueous dextrose and glycerol solutions, other pharmaceutically acceptable
auxiliary
substances as required to approximate physiological conditions, such as
buffering agents,
tonicity adjusting agents, wetting agents and the lilce. It will be recognized
that, while any
suitable carrier known to those of ordinary skill in the art may be employed
to administer the
compositions of this invention, the type of carrier will vary depending on the
mode of
administration. Compounds may also be encapsulated within liposomes using well-
known
technology. Biodegradable microspheres may also be employed as carriers for
the
pharmaceutical compositions of this invention. Suitable biodegradable
microspheres are
disclosed, for example, in U.S. Patent Nos. 4,897,268; 5,075,109; 5,928,647;
5,811,128;
5,820,883; 5,853,763; 5,814,344 and 5,942,252.
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
The pharmaceutical compositions may be sterilized by conventional, well-known
sterilization techniques, or may be sterile filtered. The resulting aqueous
solutions may be
packaged for use as is, or lyophilized, the lyophilized preparation being
combined with a
sterile solution prior to administration.
Administration Of Cupredoxin Or Cytochrome Cssl, Or Variant, Derivative Or
Structural Equivalent Thereof
The cupredoxin or cytochrome e551, or variant, derivative or structural
equivalent
thereof can be administered formulated as pharmaceutical compositions and
administered by
any suitable route, for example, by oral, buccal, inhalation, sublingual,
rectal, vaginal,
transurethral, nasal, topical, percutaneous, i.e., transdermal or parenteral
(including
intravenous, intramuscular, subcutaneous and intracoronary) administration.
The
pharmaceutical formulations thereof can be administered in any amount
effective to achieve
its intended purpose. More specifically, the composition is administered in a
therapeutically
effective amount. In specific embodiments, the therapeutically effective
amount is generally
from about 0.01-20 mg/day/kg of body weight.
The compounds comprising cupredoxin or cytochrome cssl, or variant, derivative
or
structural equivalent thereof are useful for the treatment and/or prophylaxis
of HIV infection
or other viral or bacterial infections, alone or in combination with other
active agents. The
appropriate dosage will, of course, vary depending upon, for example, the
compound of
cupredoxin or cytochrome c551, or variant, derivative or structural equivalent
thereof
employed, the host, the mode of administration and the nature and severity of
the conditions
being treated. However, in general, satisfactory results in humans are
indicated to be
obtained at daily dosages from about 0.01-20 mg/kg of body weight. An
indicated daily
dosage in humans is in the range from about 0.7 mg to about 1400 mg of a
compound of
cupredoxin or cytochrome e551, or variant, derivative or structural equivalent
thereof
conveniently administered, for example, in daily doses, weekly doses, monthly
doses, and/or
continuous dosing. Daily doses can be in discrete dosages from 1 to 12 times
per day.
Alternatively, doses can be administered every other day, every third day,
every fourth day,
every fifth day, every sixth day, every week, and similarly in day increments
up to 31 days.
Alternatively, dosing can be continuous using patches, i.v. administration and
the like.
31
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
The method of introducing cupredoxin or cytochrome cssl, or variant,
derivative or
structural equivalent thereof to patients is, in some embodiments, the same as
currently used
to introduce anti-HIV drugs, such as the protease-inhibitor-containing
cocktails. Such
methods are well-known in the art. In a specific embodiment, the cupredoxin or
cytochrome
c551, or variant, derivative or structural equivalent thereof are part of an
cocktail or co-dosing
containing or with other anti-HIV therapeutics. Other anti-HIV drugs include,
but are not
limited to, reverse transcriptase inhibitors: AZT (zidovudine [Retrovir]), ddC
(zalcitabine
[Hivid], dideoxyinosine), d4T (stavudine [Zerit]), and 3TC (lamivudine
[Epivir]),
nonnucleoside reverse transcriptase inhibitors (NNRTIS): delavirdine
(Rescriptor) and
nevirapine (Viramune), protease inhibitors: ritonavir.(Norvir), a lopinavir
and ritonavir
combination (Kaletra), saquinavir (Invirase), indinavir sulphate (Crixivan),
amprenavir
(Agenerase), and nelfinavir (Viracept). Presently, a combination of several
drugs called
highly active antiretroviral therapy (HAART) is used to treat people with HIV.
The method of introducing cupredoxin or cytochrome, or variant, derivative or
structural equivalent thereof to patients is, in some embodiments, through the
co-
administration of cupredoxin or cytochrome, or variant, derivative or
structural equivalent
thereof with drugs used for malaria therapy. Such methods are well-known in
the art. In a
specific embodiment, the cupredoxin and/or cytochrome c are part of an
cocktail or co-dosing
containing or with malaria therapeutics. Malaria therapeutics of interest
include, but are not
limited to, proguanil, chlorproguanil, trimethoprim, chloroquine, mefloquine,
lumefantrine,
atovaquone, pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halofantrine,
quinine,
quinidine, amodiaquine, amopyroquine, sulphonamides, artemisinin, arteflene,
artemether,
artesunate, primaquine, pyronaridine, proguanil, chloroquine, mefloquine,
pyrimethamine-
sulfadoxine, pyriinethamine-dapsone, halofantrine, quinine, proguanil,
chloroquine,
mefloquine, 1,16-hexadecamethylenebis(N-methylpyrrolidinium)dibromide, and
combinations thereof.
The exact formulation, route of administration, and dosage is determined by
the
attending physician in view of the patient's condition. Dosage amount and
interval can be
adjusted individually to provide plasma levels of the active cupredoxin or
cytochrome c551, or
variant, derivative or structural equivalent thereof which are sufficient to
maintain therapeutic
effect. Generally, the desired cupredoxin or cytochrome c551, or variant,
derivative or
structural equivalent thereof is administered in an admixture with a
pharmaceutical carrier
32
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
selected with regard to the intended route of administration and standard
pharmaceutical
practice.
In one aspect, the cupredoxin or cytochrome c551, or variant, derivative or
structural
equivalent thereof is delivered as DNA such that the polypeptide is generated
in situ. In one
embodiment, the DNA is "naked," as described, for example, in Ulmer et al.,
(Science
259:1745-1749 (1993)) and reviewed by Cohen (Science 259:1691-1692 (1993)).
The uptake
of naked DNA may be increased by coating the DNA onto a carrier, e.g.,
biodegradable
beads, which are then efficiently transported into the cells. In such methods,
the DNA may
be present within any of a variety of delivery systems known to those of
ordinary skill in the
art, including nucleic acid expression systems, bacterial and viral expression
systems.
Techniques for incorporating DNA into such expression systems are well known
to those of
ordinary skill in the art. See, e.g., W090/11092, W093/24640, WO 93/17706, and
U.S. Pat.
No. 5,736,524.
Vectors, used to shuttle genetic material from organism to organism, can be
divided
into two general classes: Cloning vectors are replicating plasmid or phage
with regions that
are essential for propagation in an appropriate host cell and into which
foreign DNA can be
inserted; the foreign DNA is replicated and propagated as if it were a
component of the
vector. An expression vector (such as a plasmid, yeast, or animal virus
genome) is used to
introduce foreign genetic material into a host cell or tissue in order to
transcribe and translate
the foreign DNA, such as the DNA of a cupredoxin. In expression vectors, the
introduced
DNA is operably-linked to elements such as promoters that signal to the host
cell to highly
transcribe the inserted DNA. Some promoters are exceptionally useful, such as
inducible
promoters that control gene transcription in response to specific factors.
Operably-linking a
cupredoxin and/or cytochrome c and variants and derivatives thereof
polynucleotide to an
inducible promoter can control the expression of the cupredoxin and/or
cytochrome c and
variants and derivatives thereof in response to specific factors. Examples of
classic inducible
promoters include those that are responsive to a-interferon, heat shock, heavy
metal ions, and
steroids such as glucocorticoids (Kaufman, Methods Enzymol. 185:487-511
(1990)) and
tetracycline. Other desirable inducible promoters include those that are not
endogenous to
the cells in which the construct is being introduced, but, are responsive in
those cells when
the induction agent is exogenously supplied. In general, useful expression
vectors are often
33
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
plasmids. However, other forms of expression vectors, such as viral vectors
(e.g., replication
defective retroviruses, adenoviruses and adeno-associated viruses) are
contemplated.
Vector choice is dictated by the organism or cells being used and the desired
fate of
the vector. In general, vectors comprise signal sequences, origins of
replication, marker
genes, polylinker sites, enhancer elements, promoters, and transcription
termination
sequences.
Kits Comprising Cupredoxin Or Cytochrome C551, Or Variant, Derivative Or
Structural Equivalent Thereof
In one aspect, the invention provides kits containing one or more of the
following in a
package or container: (1) a biologically active composition comprising at
least one
cupredoxin or cytochrome c551, or variant, derivative or structural equivalent
thereof; (2) an
anti-viral or anti-bacterial drug, specifically an anti-HIV drug, including,
but not limited to,
reverse transcriptase inhibitors: AZT (zidovudine [Retrovir]), ddC
(zalcitabine [Hivid],
dideoxyinosine), d4T (stavudine [Zerit]), and 3TC (lamivudine [Epivir]),
nonnucleoside
reverse transcriptase inhibitors (NNRTIS): delavirdine (Rescriptor) and
nevirapine
(Viramune), protease inhibitors: ritonavir (Norvir), a lopinavir and ritonavir
combination
(Kaletra), saquinavir (Invirase), indinavir sulphate (Crixivan), amprenavir
(Agenerase), and
nelfinavir (Viracept); (3) a pharmaceutically acceptable excipient; (4) a
vehicle for
administration, such as a syringe; (5) instructions for administration.
Embodiments in which
two or more of components (1) - (5) are found in the same packaging or
container are also
contemplated.
In some embodiments, the kit also comprises an anti-malarial therapeutic. Anti-
malarial therapeutics of interest include, but are not limited to, proguanil,
chlorproguanil,
trimethoprim, chloroquine, mefloquine, lumefantrine, atovaquone, pyrimethamine-
sulfadoxine, pyrimethamine-dapsone, halofantrine, quinine, quinidine,
amodiaquine,
amopyroquine, sulphonamides, artemisinin, arteflene, artemether, artesunate,
primaquine,
pyronaridine, proguanil, chloroquine, mefloquine, pyrimethamine-sulfadoxine,
pyrimethamine-dapsone, halofantrine, quinine, proguanil, chloroquine,
mefloquine, 1,16-
hexadecamethylenebis(N-methylpyrrolidinium)dibromide.
When a kit is supplied, the different components of the composition may be
packaged
in separate containers and admixed immediately before use. Such packaging of
the
34
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
components separately may permit long-term storage without losing the active
components'
functions.
The reagents included in the kits can be supplied in containers of any sort
such that
the life of the different components are preserved and are not adsorbed or
altered by the
materials of the container. For example, sealed glass ampules may contain
lyophilized
cupredoxin and/or cytochrome c and variants and derivatives thereof , or
buffers that have
been packaged under a neutral, non-reacting gas, such as nitrogen. Ampules may
consist of
any suitable material, such as glass, organic polymers, such as polycarbonate,
polystyrene,
etc., ceramic, metal or any other material typically employed to hold similar
reagents. Other
examples of suitable containers include simple bottles that may be fabricated
from similar
substances as ampules, and envelopes, that may comprise foil-lined interiors,
such as
aluminum or an alloy. Other containers include test tubes, vials, flasks,
bottles, syringes, or
the like. Containers may have a sterile access port, such as a bottle having a
stopper that can
be pierced by a hypodermic injection needle. Other containers may have two
compartments
that are separated by a readily removable membrane that upon removal permits
the
components to be mixed. Removable membranes may be glass, plastic, rubber,
etc.
Kits may also be supplied with instructional materials. Instructions may be
printed on
paper or other substrate, and/or may be supplied as an electronic-readable
medium, such as a
floppy disc, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, flash memory
device etc.
Detailed instructions may not be physically associated with the kit; instead,
a user may be
directed to an internet web site specified by the manufacturer or distributor
of the kit, or
supplied as electronic mail.
Modification Of Cupredoxin And/Or Cytochrome C And Variants And Derivatives
Thereof
Cupredoxin or cytochrome c551, or variant, derivative or structural
equivalents thereof
may be chemically modified or genetically altered to produce variants and
derivatives as
explained above. Such variants and derivatives may be synthesized by standard
techniques.
In addition to naturally-occurring allelic variants of cupredoxin and
cytochrome c551,
changes can be introduced by mutation into cupredoxin or cytochrome c551
coding sequence
that incur alterations in the amino acid sequences of the encoded cupredoxin
or cytochrome
c551 that do not significantly alter the ability of cupredoxin or cytochrome c
to inhibit the
growth of a viral or bacterial infection, and specifically an HIV infection in
mammalian cells.
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
A "non-essential" amino acid residue is a residue that can be altered from the
wild-type
sequences of the cupredoxin without altering biological activity, whereas an
"essential"
amino acid residue is required for such biological activity. For example,
amino acid residues
that are conserved among the cupredoxins are predicted to be particularly non-
amenable to
alteration, and thus "essential."
Amino acids for which conservative substitutions that do not change the
activity of
the polypeptide can be made are well known in the art. Useful conservative
substitutions are
shown in Table 3, "Preferred substitutions." Conservative substitutions
whereby an amino
acid of one class is replaced with another amino acid of the same type fall
within the scope of
the invention so long as the substitution does not materially alter the
biological activity of the
compound.
Table 3. Preferred substitutions
Original residue Exemplary substitutions Preferred
substitutions
Ala (A) Val, Leu, Ile Val
Arg (R) Lys, Gln, Asn Lys
Asn (N) Gln, His, Lys, Arg Gln
Asp (D) Glu Glu
Cys (C) Ser Ser
Gln (Q) Asn Asn
Glu (E) Asp Asp
Gly (G) Pro, Ala Ala
His (H) Asn, Gln, Lys, Arg Arg
Ile (I) Leu, Val, Met, Ala, Phe, Leu
Norleucine
Leu (L) Norleucine, Ile, Val, Met, Ala, Ile
Phe
Lys (K) Arg, Gln, Asn Arg
Met (M) Leu, Phe, Ile Leu
Phe (F) Leu, Val, Ile, Ala, Tyr Leu
Pro (P) Ala Ala
Ser (S) Thr Thr
Thr (T) Ser Ser
Trp (W) Tyr, Phe Tyr
Tyr (Y) Trp, Phe, Thr, Ser Phe
Val (V) Ile, Leu, Met, Phe, Ala, Leu
Norleucine
36
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
Non-conservative substitutions that affect (1) the structure of the
polypeptide
backbone, such as a(3-sheet or a-helical conformation, (2) the charge, (3)
hydrophobicity, or
(4) the bulk of the side chain of the target site can modify the cytotoxic
factor function.
Residues are divided into groups based on common side-chain properties as
denoted in Table
4. Non-conservative substitutions entail exchanging a member of one of these
classes for
another class. Substitutions may be introduced into conservative substitution
sites or more
specifically into non-conserved sites.
Table 4. Amino acid classes
Class Amino acids
hydrophobic Norleucine, Met, Ala, Val, Leu, Ile
neutral hydrophilic Cys, Ser, Thr
acidic Asp, Glu
basic Asn, Gln, His, Lys, Arg
disrupt chain conformation Gly, Pro
aromatic Trp, Tyr, Phe
The variant polypeptides can be made using methods known in the art such as
oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and
PCR
mutagenesis. Site-directed mutagenesis (Carter, Biochem J. 237:1-7 (1986);
Zoller and
Smith, Methods Enzymol. 154:329-350 (1987)), cassette mutagenesis, restriction
selection
mutagenesis (Wells et al., Gene 34:315-323 (1985)) or other known techniques
can be
performed on the cloned DNA to produce the cupredoxin or cytochrome c551
variant DNA.
Known mutations of cupredoxins and cytochrome c551 can also be used to create
variant cupredoxin and cytochrome c551 to be used in the methods of the
invention. For
example, the Cl 12D and M44KM64E mutants of azurin are known to have cytotoxic
and
growth arresting activity that is different from the native azurin, and such
altered activity can
be useful in the treatment methods of the present invention. One embodiment of
the methods
of the invention utilize cupredoxin and/or cytochrome c551 and variants and
derivatives
thereof retaining the ability inhibit the growth of an viral or bacterial
irifection, and
specifically HIV infection in mammalian cells. In another embodiment, the
methods of the
37
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
present invention utilize cupredoxin variants such as the M44KM64E mutant,
having the
ability to cause cellular growth arrest.
A more complete understanding of the present invention can be obtained by
reference
to the following specific Examples. The Examples are described solely for
purposes of
illustration and are not intended to limit the scope of the invention. Changes
in form and
substitution of equivalents are contemplated as circumstances may suggest or
render
expedient. Although specific terms have been employed herein, such terms are
intended in a
descriptive sense and not for purposes of limitations. Modifications and
variations of the
invention as hereinbefore set forth can be made without departing from the
spirit and scope
thereof, and, therefore, only such limitations should be imposed as are
indicated by the
appended embodiments.
EXAMPLES
Example 1: In Vivo Inhibition Of HIV Infection Of Lymphocytes By Azurin Mutant
And Cytochrome Cs5i=
The M44KM64E mutant of azurin was mixed with cytochrome c551 on a 1:1 basis (1
M azurin : 1 M cytochrome c551). HIV-infected human blood lymphocytes were
incubated
with the mixed azurin/cytochrome c551 proteins at concentrations of 0, 500 to
1000 g/ml
protein for 7 days. The HIV p241evels were then measured in the infected
lymphocytes. p24
levels are known to be colinear with HIV virus levels in infected blood.
Measuring the
change in p24 concentrations in blood will indicate the change of HIV virus
titer in the blood.
Controls with non-infected human blood lymphocytes were also run in a parallel
manner.
After the 7 day incubation, the HIV p241evels in the infected lymphocytes were
reduced by
25% to 90% as compared to the control infected lymphocytes with 0 g/ml azurin
and
cytochrome c551. In the non-infected control cells, after 7 days of incubation
with the protein
mixture, neither cell death nor cytotoxicity was found.
Example 2. Azurin exhibits structural similarity with ICAM-1 from HIV-1
Previous studies (Gough & Chothia, Structure 12:917-925 (2004); Stevens et
al., J.
Mol. Recognit. 18:150-157 (2005)) have shown that cupredoxins show structural
similarity to
the variable domains of the immunoglobulin superfamily members. The DALI
algorithm
(Holm & Park, Bioinformatics 16:566-567 (2000)) was used to search the 3D
databases for
38
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
structural homologs of azurin (1JZG) from P. aeruginosa. See, Table 5. Azurin
exhibits
structural similarity to not only the fab fragment of the monoclonal antibody
in complexation
with PfMSP1-19, but also to ICAM-1 (Table 5) involved in cerebral malaria
(Smith et al.,
Proc. Natl. Acad. Sci. USA 97:1766-1771 (2000); Franke-Fayard et al., Proc.
Natl. Acad. Sci.
USA 102:11468-11473 (2005))
ICAM-1 is not only implicated as a receptor on the endothelial cells in the
microvasculture of the brain and other tissues for sequestering P. falciparum -
infected
erythrocytes, but is also found in HIV-1 particles during their passage
through the host cells
and is known to enhance HIV-1 infectivity by enhancing cytosolic delivery of
the viral
materials. (Fortin et al., J. Virol. 71:3588-3596 (1997); Tardif & Tremblay,
J. Virol.
77:12299-12309 (2003)) ICAM-1 is known also to be subverted as receptors for
major
groups of rhinoviruses and coxsackie viruses. (Bella & Rossmann, J. Struct.
Biol. 128:69-74
(1999))
Azurin demonstrates structural similarity to CD4 (Table 5), the primary host
cell
surface receptor for HIV-1. (Maddon et al., Ce1147:333-348 (1986)) This
example shows
that cupredoxins including azurin demonstrate structural similarities in
having two anti-
parallel 0 sheets packed face to face and linked by a disulfide bridge to the
variable domains
of the immunoglobulin superfamily members as well as extracellular domains of
the
intercellular adhesion molecules (ICAM) and their ligands.
39
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
Table 5. Structural similarity of P. aeruginosa azurin with various
pathogenesis-related
proteins
Azurin (ljzg)
PDB Annotation Reference DALI z RMSD Alignment
score(1) to length
Azurin~2)
1VCA B1 Human Vascular Cell 17 3.5 3.2 80
Adhesion
1 CISH CD4 (D 1 D2 Fragment) 18 3.4 2.8 73
Type I Crystal Form
1ZXQ1 Crystal Structure of 19 3.3 2.9 80
ICAM-2
lIAM1 Structure of the Two 20 3.3 3.1 74
Amino-Terminal Adhesion
Molecule-1, ICAM-1
IOBl Al Crystal Structure of a Fab 21 2.9 3.7 84
Complex with Plasmodium
falciparum MSP1-19
Structural alignment of azurin were made using DALI (Holm & Park,
Bioinformatics 16:566-
567 (2000)). Structure pairs with DALI z scores <2 are considered dissimilar.
RMSD-Root-mean-square deviation of backbone residues in angstroms between the
aligned
parts of the pair of structure.
Example 3. Effect of Azurin, H.8-Azurin and Laz on HIV-1 Entry and Viral
Growth.
The effect of various concentrations of azurin, H.8-azurin and Laz on the
growth of
three subtypes of HIV-1 in peripheral blood mononuclear cells (PBMCs), Bal,
RW/92/008/RE 1 clade A and IN/2157 D15 clade C
Cloning and Expression of the paz and laz Genes. The laz gene from Neisseria
gonorrhoeae was cloned based on its known sequence (SEQ ID NO: 19). The P.
aeruginosa
azurin gene (SEQ ID NO: 1), termed paz , and the sequence of the H.8 epitope
of laz from N.
gonnerrhoeae (SEQ ID NO: 18), were used to clone in frame the H.8 epitope gene
in the 5'-
end of paz to produce H.8-paz or in the 3'-end of paz to generate paz-H.8.
See, Table 6 for
bacterial strains and genetic constructs used in this and related Examples.
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
Table 6. Bacterial strains and genetic constructs
Cells/strains/ Relevant characteristics* Reference
plasmids
P. aeruginosa Prototroph, FP- (sex factor minus) Holloway, et al., Microbiol.
PAO1 Rev. 43:73-102 (1979)
E. coli JM109 Cloning and azurin expression strain Yanisch-Perron, et al.,
Gene 33:103-119 (1985)
E. coli BL21 GST expression strain Novagen
(DE3)
N. gonorrhoeae Prototroph used for DNA isolation American Type Culture
F62 Collection
pUC18 General cloning vector, Apr Yanisch-Perron, et al., id.
pUC19 General cloning vector, Apr Yanisch-Perron, et al., id.
pUC18-laz A 1 kb PCR fragment from genomic Herein
DNA of N. gonorrhoeae F62 cloned into
pUC 18
pUC19 paz A 0.55 kb PCR fragment from P. Yamada, et al., Proc. Natl.
aeruginosa PAO1 cloned into Hindlll Acad. Sci. USA 99:14098-
and Pstl digested pUC19, Apr 14103 (2002); Yamada, et
al., Proc. Natl. Acad. Sci.
USA 101:4770-4775 (2004)
pUC18-H.8-paz Fusion plasmid encoding H.8 from N. Herein
gonorrhoeae and azurin from P.
aeruginosa PAO 1, Ap'
pGEX-5X-3 GST gene fusion vectors, Apr Amersham
pET29a E. coli expression vector, Km' Novagen
pET29a-gst pET29a derivative containing the gst Herein
gene, Km'
pGEX-5X-3- pGEX-5X-3 derivative containing H.8- Herein
H.8 encoding region, Ap'
pET29a-gst-H.8 pET29a derivative containing gst-H.8 Herein
gene, Km'
*Ap, ampicillin; Km, kanamycin; GST, Glutathione S-transferase.
41
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
The cloning and hyperexpression of the azurin gene has been described.
(Yamada, et
al., Proc. Natl. Acad. Sci. USA 99:14098-14103 (2002); Punj, et al., Oncogene
23:2367-2378
(2004)) The Laz-encoding gene (laz) of Neisseria gonorrhoeae was amplified by
PCR with
genomic DNA of N. gonorrhoeae strain F62 as template DNA. The forward and
reverse
primers used were 5'-CCGGAATTCCGGCAGGGATGTTGTAAATATCCG-3' (SEQ ID
NO: 20) and 5'-GGGGTACCGCCGTGGCAGGCATACAGCATTTCAATCGG-3' (SEQ ID
NO: 21) where the additionally introduced restriction sites of EcoRI and KpnI
sites are
underlined respectively. The amplified DNA fragment of 1.0 kb, digested with
EcoRl and
KpnI, was inserted into the corresponding sites of pUC 18 vector (Yanisch-
Perron, et al.,
Gene 33:103-119 (1985)) so that the laz gene was placed downstream of the lac
promoter to
yield an expression plasmid pUC18-lcrz (Table 6).
The plasmids expressing fusion H.8 of N. gonorrhoeae Laz and azurin of P.
aeruginosa (Paz) were constructed by PCR with pUC 19-paz and pUC 1 8-laz as
templates.
For H.8-Paz fusion, a 3.1 kb fragment was amplified with pUC 18-laz as a
template and
primers, 5'-(phosphorylated)GGCAGCAGGGGCTTCGGCAGCATCTGC-3' (SEQ ID NO:
22) and 5'-CTGCAGGTCGACTCTAGAGGATCCCG-3' (SEQ ID NO: 23) where a SaII site
is underlined. A PCR amplified a 0.4 kb fragment was obtained from pUC 19paz
as a
template and primers, 5'-(phosphorylated)GCCGAGTGCTCGGTGGACATCCAGG-3' (SEQ
ID NO: 24) and 5'-TACTCGAGTCACTTCAGGGTCAGGGTG-3' (SEQ ID NO: 25) where
a XhoI site is underlined. A SaII digested PCR fragment from pUC 18-laz and
XhoI digested
PCR fragment from pUC 19-paz were cloned to yield an expression plasmid pUC 18-
H.8 paz
(Table 6).
E. coli JM109 was used as a host strain for expression of azurin and its
derivative
genes. Recombinant E. coli strains were cultivated in 2 X YT medium containing
100 g/ml
ampicillin, 0.1 mM IPTG and 0.5 mM CuSd4 for 16 h at 37 C to produce the
azurin proteins.
When E. coli strains harboring these plasmids were grown in presence of IPTG,
cells
lysed and the proteins purified as described for azurin (Yamada, et al., Proc.
Natl. Acad. Sci.
USA 99:14098-14103 (2002); Punj, et al., Oncogene 23:2367-2378 (2004);Yamada,
et al.,
Cell. Microbiol. 7:1418-1431 (2005)), the various azurin derivatives migrated
on SDS-PAGE
as single components, although the H.8 containing proteins (about 17 kDa)
showed
anomalous migrations, as noted before. (Cannon, Clin. Microbiol. Rev. 2:S1-S4
(1989);
Fisette, et al., J. Biol. Chem. 278:46252-46260 (2003))
42
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
HIV-1 Suppression Assay. Azurin, H.8-azurin and Laz were filter sterilized
through
a 0.45 M filter. Peripheral blood mononuclear cells (PBMC) were treated with
polybrene (5
g/ml) for 1 h and seeded at 250,000 cells/well in a microtiter plate. The
plate was spun at
800 rpm for 5 min to collect the cells. The supernatant was taken off and
media with protein
(at concentrations of 0.3, 0.6, 1.2, 6.0 and 30 M) was added (100 l). The
cells were then
incubated for 1 h. AZT (25 M) was used as a control. The proteins were left
on cells and
100 l of virus (Bal, 2167, or RW/92/008/RE1) was added and incubated for 2 h.
The plate
was spun again at 800 rpm for 5 minutes and protein and virus was removed.
Protein and
media were added back for a total volume of 100 l and incubated for 5 days.
At the end of
the 5 day period, the culture supernatant was tested for HIV/p24 by ELISA.
The results in Figure 1 show that azurin at a concentration of 6.0 M shows
about
90% suppression of the growth of HIV-1 Bal, the most predominant clade B
circulating in the
US and Western Europe, a clade B African isolate RW/92/008/RE1 and a clade C
Indian
isolate IN/2167 D15. However, H.8-azurin (azurin with the H.8 epitope in the N-
terminal)
had high inhibitory activity against all the three subtypes at concentrations
as low as 0.3 M,
particularly for the African and the Indian subtypes (Fig. 1).
The Neisserial protein Laz, which also harbors the H.8 epitope in the N-
terminal part
of the Neisserial azurin homolog (Gotschlich & Seiff FEMS Microbiol. Lett.
43:253-255
(1987); Kawula et al., Mol. Microbiol. 1:179-185 (1987)), had similar
inhibitory activity for
the three subtypes, particularly for the African and the Indian subtypes (Fig.
1),
demonstrating a role of the H.8 epitope in promoting enhanced anti-HIV-1
activity by azurin.
No effect on host cell (PBMC) death by MTT assay (Yamada et al., Proc. Natl.
Acad. Sci.
USA 99:14098-14103 (2002); Punj et al., Oncogene 23:2367-2378 (2004)) was
discernible
for all concentrations of these three proteins, suggesting that inhibition of
HIV-1 growth was
not due to death of the host cells.
Example 4. Azurin Binding with gp120 and CD4 as Studied by Surface Plasmon
Resonance.
Surface Plasmon Resonance experiments were conducted to determine the extent
of
azurin binding not only to CD4 but also to HIV-1 surface proteins such as gp
120 or gp41
known to be involved in HIV- 1 entry and other proteins such as Nef or Gag
that are involved
in intracellular virus multiplication.
43
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
Plasmid Construction for Fusion GST Proteins. Plasmids expressing fusion
glutathione S-transferase (GST)-truncated wt-azurin (azu) derivatives were
constructed by a
polymerase chain reaction using proofreading DNA polymerase. For pGST-azu 36-
128, an
amplified PCR fragment was introduced into the BamHl and EcoRl sites of the
commercial
GST expression vector pGEX-5X (Amersham Biosciences, Piscataway, NJ). The
fragment
was amplified with pUC19-azu as a template and primers, 5'-CGGGATCC CCG GCA
ACC
TGC CGA AGA ACG TCA TGG GC-3'(SEQ ID NO: 26) and 5'-CGGAATTC GCA TCA
CTT CAG GGT CAG GG-3' (SEQ ID NO: 27), where the additionally introduced
BarnHl
and EcoRl sites are underlined respectively. Carboxyl-terminus truncation of
azu gene was
cumulatively performed by introducing a stop codon using QuickChange site-
direct
mutagenesis kit (Stratagene, La Jolla, CA).
For pGST-azu 36-89, a stop codon were introduced into G1y90. The plasmid
carrying
pGST-azu 36-128 was used as template DNA. Three sets of oligonuclotides for
site-direct
mutagenesis are shown as follows. For pGST-azu 36-89: 5'-CCA AGC TGA TCG GCT
CGT GAG AGAAGG ACT CGG TGA CC-3' (SEQ ID NO: 28), and 5'-GGT CAC CGA
GTC CTT CTC TCA CGA GCC GAT CAG CTT GG-3 (SEQ ID NO: 29).
For pGST-azu 88-113, carboxyl terminus truncation of azu gene was cumulatively
performed by introducing stop codon using QuickChange site directed
mutagenesis kit
(Stratagene, La Jolla, CA). For pGST-azu 88-113, a stop codon was introduced
into Phe114.
The plasmid carrying pGST-azu 88-128 was used as the template. For pGST-azu 88-
128 an
amplified PCR fragment was introduced into the BamHl and EcoRl sites of the
commercial
GST expression vector pGEX-5X (Amersham Biosciences). The fragment was
amplified
with pUC19-azu as the template and primers, 5'-CGGGGATCC CCG GCT CGG GCG AGA
AGG AC-3' (SEQ ID NO: 30) and 5'-CGGGAATTC TCC ACT TCA GGG TCA GGG TG-
3' (SEQ ID NO: 31) where the additionally introduced BanaHl and EcoRl sites
are
underlined respectively.
One set of oligonucleotides for site directed mutagenesis are shown as follows
for the
preparation of pGST-azu 88-113: 5'-GTT CTT CTG CAC CTA GCC GGG CCA CTC CG-
3' (SEQ ID NO: 32) and 5'-CGG AGT GGC CCG GCT AGG TGC AGA AGA AC-3' (SEQ
ID NO: 33). pGST-azu 88-113 was used to transform E. coli XL-1 Blue strains.
Plasmid
extraction was performed using a commercial kit (Qiagen, Venlo, The
Netherlands) and PCR
sequencing were performed to assess plasmid insertion and transfection.
44
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
E. coli BL21 (DE3) was used as a host strain for expression of the gst and its
fusions
derivatives. E. coli strain XL1-Blue transforrned with pGST-azu plasmids was
grown in LB
media with ampicillin for three hours at 37 C upon which IPTG induction (0.4
mM) was
performed an subsequent incubation for 2-4 h at 37 C to maximize the
expression levels.
Cells were isolated by centrifugation, resuspended in 25 mL of 1X PBS buffer.
Subsequent
cell lysis involved two sequential treatments of the cell suspension via
sonication (20 min on
ice) and heat-cold shock in acetone-dry ice bath (using the appropriate
protease inhibitors).
Supernatants of the cell lysis mixture were isolated and passed through a
freshly packed and
PBS equilibrated 1 mL glutathione-sepharose 4B (Amersham Biosciences) column.
After
column washing and subsequent elution of GST-azu product using 10 mM
glutathione in 20
mM Tris-HCl pH 8. GST-Azu 88-113 purity was tested via electrophoresis using a
10%
SDS-PAGE Tris-Gly gel stained with Coomassie Brilliant Blue R reagent. Protein
concentration was determined using the Bradford Method.
Surface Plasmon Resonance (SPR) Studies. In vitro protein-protein interactions
were evaluated using a Biacore X spectrometer from Biacore AB International
(Uppsala,
Sweden). All experiments were conducted at 25 C in HBS-EP running buffer (0.01
M
HEPES, pH 7.4, 0.15 M NaCI, 3 mM EDTA, 0.005% v/v Surfactant P20)) using Au-
CM5
sensor chips purchased from Biacore. Protein stock solutions were prepared in
PBS after
desalting on G-75 column and lyophilizaiton in order to preconcentrate and
exchange the
buffer.
Protein immobilizations on CM5 chips were conducted according to the amine
coupling procedure. Due to differences in protein crosslinking efficiencies,
proteins were
immobilized under various conditions after NHS/EDC preactivation of the CM5
surface: 50
l injections of azurin (510 M), or 35 l injections of CD4 (25 M, 2x), or
HIV-1 gp120
(10 M). Subsequent treatment of CM5 surface with ethanolamine (1M, pH 8.8)
removed
uncrosslinked proteins prior to binding studies. Binding studies were
performed by injecting
protein eluents (50 l) over the protein-CM5 surface at flow rates of 30
l/min with a 120 sec
time delay at the end of the injections. Protein eluents included CD4 (Protein
Sciences Corp.,
Meriden, CT), HIV-1 gp120 (Immunodiagnostics Inc., Woburn, MA), HIV-1 gp41
(Bioclone
Inc., San Diego, CA), HIV-1 gag and HIV-1-nef (Chemicon International,
Temecula, CA)
and GST-azurin fusion proteins (GST, GST-Azu 36-128, GST-Azu 36-89, and GST-
Azu 88-
113, expressed in inventor's laboratory). Sensor chip surfaces were
regenerated between
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
protein injections using 100 mM NaOH (10 l injection pulse). All binding
studies were run
against a negative flow channel containing bare Au-CM5 to correct for
nonspecific binding
effects. For the binding experiments wherein CD4 and HIV-1 gp120 served as the
eluents
(not immobilized), 1 mg/mL of carboxymethyldextran (CarboMer Inc., San Diego
CA) was
added to the running buffer in order to reduce nonspecific protein binding to
the bare Au-
CM5 flow channel surface.
To generate binding constant data, titration experiments were designed via
injection
of increasing concentrations of protein eluents (0.05-2000 nM) and the data
collected. The
SPR data could be fit to a Langmuir equilibrium binding model [Req = Rmax/(1 +
Kd/C]
form which binding constants (Kd) were determined. Similar to the binding
constant studies
described above, competition studies with CD4-CM5 were performed using similar
protocols
but with injections of HIV-1 gp 120 + the competitor proteins (azurin, GST-Azu
36-128 and
GST-Azu 88-113).
With CD4 immobilized in the sensor chip, both azurin and gp120 showed
significant
binding to CD4 (Fig. 2A). .Azurin demonstrated a higher affinity for binding
CD4 (Kd = 36.9
nM) than the HIV-1 ligand gpl20 (Kd = 48.1 nM). While a GST-azurin fusion such
as GST-
Azu 88-113 showed no binding (Fig. 2A), another GST-azurin fusion protein, GST-
Azu 36-
128 showed even stronger binding than azurin itself with a Kd value of 0.34 nM
(Fig. 4A
inset), suggesting that parts of azurin might retain a stronger binding
affinity than the full
length protein. When azurin was immobilized on the sensor chip, gp120 showed
somewhat
stronger binding to azurin than CD4 (Fig. 2B), clearly demonstrating that
azurin binds both to
gp120 and CD4 with a high affinity. Interestingly, gp41, also involved in HIV-
1 entry into
the host cell, did not show any binding to azurin (Fig. 2B). Similar lack of
binding was
demonstrated for Gag and Nef.
Example 5. Azurin Binding with ICAMs and CD5 as Studied by Surface Plasmon
Resonance.
There is a structural similarity between azurin and ICAMs (Table 5) that are
known to
be involved as receptors HIV-1 infections. (Liao et al.,. AIDS Res. Hum.
Retroviruses
16:355-366 (2000); Hioe et al., J. Virol. 75:1077-1082 (2001)) ICAM-3 has been
implicated
in stimulating HIV-1 transcription and viral production, thereby contributing
additionally to
intracellular viral growth. (Barat et al., J. Virol. 78, 6692-6697 (2004))
46
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
Protein-protein interactions as measured by SPR between azurin and ICAMs such
as
ICAM-1, ICAM-2, ICAM-3 and NCAM were therefore studied. With immobilized
azurin on
the CM5 chip, ICAM-3 (Fig. 2C, Kd = 19.5 + 5.4 nM) and NCAM (Fig. 2C, inset),
but not
ICAM-1 and ICAM-2, showed strong binding. While not limiting the operation of
the
invention to any one mechanism, part of azurin suppression of HIV-1 growth
might also be
mediated through its interaction with ICAM-3 or NCAM.
Example 6. Azurin Compeition with gp120 for CD4 as Studied by Surface Plasmon
Resonance.
Due to the higher affinity of binding of azurin to CD4, as compared to gp 120
(Fig.
2A), a competition experiment was performed to see if azurin can interfere in
gp 120 binding
with its cognate receptor CD4. As the concentration of the competitor protein
(azurin, GST-
Azu 36-128 or GST-Azu 88-113) was increased in presence of a fixed
concentration of gp120
adsorbed to the immobilized CD4 chip, both azurin and GST-Azu 36-128
demonstrated
significant decrease in the total protein binding of gp120 from the CD4-CM5
chip (Fig. 2D).
Such apparent displacement of gp120 from the chip was not observed in case of
GST-Azu
88-113 (Fig. 2D). GST-Azu 88-113 is known not to bind CD4 (Fig. 2A). While not
limiting
the operation of the invention to any one mechanism, this indicates that
azurin or GST-Azu
36-128 fusion protein may successfully inhibit the complex formation between
gp120 and
CD4.
Example 7. Azurin and ICAM-3 Binding with DC-SIGN as Studied by Surface
Plasmon Resonance.
The strong binding of azurin with gp120, CD4 and ICAM-3 (Fig. 2) mimics the
binding of another very important HIV-1 binding protein present on the surface
of dendritic
cells (DC) known as DC-SIGN (DC-specific intercellular adhesion molecule 3-
grabbing
nonintegrin) and a related protein called DC-SIGN/R. DC-SIGN is expressed
abundantly on
DC while DC-SIGN/R is expressed primarily on sinusoidal and endothelial cells.
DC-SIGN
plays a major role in HIV-1 immunopathogenesis by allowing DC, which are
professional
antigen presenting cells, to capture and present pathogens including HIV-1 to
resting T cells
through their interactions with ICAM-3 on the T cell surface. (Geijtenbeek et
al., Cell 100,
575-585 (2000); Soilleux, Clin. Sci. 104, 437-446 (2003); Geijtenbeek et al.,
Placenta 22,
47
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
S19-S23 (2001)). DC-SIGN has also been shown to bind avidly to HIV-1 envelope
protein
gp 120, thereby capturing HIV- 1 and transporting it to CD4+ T cells, where
HIV- 1 can
replicate freely. (Snyder et al., J. Virol. 79:4589-4598 (2005))
In SPR experiments with immobilized DC-SIGN on the sensor chip, both azurin
(Kd=0.83+0.05 nM) and ICAM-3 (Kd=0.93 0.39 nM) bound strongly to DC-SIGN
(Fig.
3A). While the GST-fusion derivative GST-Azu 36-89 showed very little binding
(Fig. 3A),
another GST-fusion derivative GST-Azu 88-113 exhibited relatively strong
binding
(Ka=5.98 1.13 nM), demonstrating the involvement of the C-terminal part of
azurin in DC-
SIGN binding (Fig. 3B). GST-Azu 88-113, however, does not bind with CD4 (Fig.
2A),
suggesting that different parts of azurin have different binding
specificities.
While not limiting the operation of the invention to any one mechanism, such
binding
with DC-SIGN demonstrates azurin's potential ability to interfere in the
binding of HIV-1
with DCs. Thus DC-SIGN, a critical molecule on DC surface responsible for
transmitting
HIV-1 from the mucosal cells to the lymphoid T cells, may well find a strong
competitor in
azurin or Laz that can also avidly bind gp 120, CD4 and ICAM-3.
Example 8. Azurin/Laz Acts in the Entry Stage of HIV-1 Infection.
To determine if azurin acts at the entry or post entry step of HIV-1
infection, the
effect of Laz on the Indian isolate IN/2167 of HIV-1 was investigated. In one
experiment,
activated PBMC (25,000 cells/well) were incubated with 6.0 M Laz and HIV-1
for 2 h. The
mixture was centrifuged to remove Laz and HIV-1, fresh medium without Laz was
added
back, and the culture was grown for 5 days. HIV-1 growth was monitored by
measurement
of p24 in the culture supernatant. Under this condition, Laz (6.0 M)
suppressed the HIV-1
growth by 43%. With higher concentration of Laz (30 pM), the extent of
suppression was
76%. In a parallel experiment, when the Laz (6 or 30 M) was added to the PBMC
after the
HIV-1 infection and its removal, very little suppression of viral growth was
observed. As a
positive control, when Laz (6.0 M) was present both during infection and
after removal of
the virus with fresh medium during 5 days of the culture, the extent of
inhibition was about
93%. While not limiting the operation of the invention to any one mechanism.
such data
clearly indicate that azurin or Laz exerts its effect primarily at the entry
stage of infection.
48
CA 02608398 2007-11-13
WO 2006/127514 PCT/US2006/019565
Example 9: Treatment of HIV-infected patients with azurin
Twenty four patients with AIDS presents with low to non-detectable HIV viral
loads
(RNA PCR) in the plasma as measured by PCR techniques, and increased CD4+
counts.
Next, CD4+RO+ cells are enriched by magnetic separation and FACS sorting, and
assayed to
determine infectivity with respect to naive and uninfected cell co-culture
experiments. This
analysis of CD4+RO+ memory cells shows the presence of infective HIV.
Azurin is therefore administered to twenty of the patients at a dose of 4
mg/m2 by
intravenous infusion once every two weeks for a period of 3 months until CD4+
cells,
including memory cells, are at low levels. Four patients receive placebo
infusions. During
administration of azurin and for a period of approximately 1-2 months
thereafter, or until
CD4+ cells recover, the patients are maintained with antibiotics and
antifungal therapy. Stem
cell or precursor cell replacement is provided through a bone marrow
transplant and cytokine
therapy, both of which are performed according to conventional techniques.
During and following therapy, the patients are followed at frequent intervals
and
monitored for CD34 cell level, reestablishment of CD4+ cells and quantitation
of CD4+RO+
cells. Additionally, the patients' plasma is assayed for viral load by cell co-
culture
experiments. On reducing virus load in active and memory CD4+ T cells to low
or non-
detectable concentrations, the patients are weaned from azurin. After 3
months, the patients
are weaned from antibiotic and antifungal therapy. Following this, the
patients are followed
at 6 month intervals and assayed for viral content. The results demonstrate
the effectiveness
of azurin therapy for patients with HIV infection.
49
