U.S. patent application number 15/019558 was filed with the patent office on 2016-06-02 for compositions and methods for the treatment of viral infections.
The applicant listed for this patent is Dana-Farber Cancer Institute, Inc.. Invention is credited to Gregory H. Bird, Loren D. Walensky.
Application Number | 20160152667 15/019558 |
Document ID | / |
Family ID | 41016644 |
Filed Date | 2016-06-02 |
United States Patent
Application |
20160152667 |
Kind Code |
A1 |
Walensky; Loren D. ; et
al. |
June 2, 2016 |
COMPOSITIONS AND METHODS FOR THE TREATMENT OF VIRAL INFECTIONS
Abstract
The invention provides compositions, kits and methods utilizing
polypeptides having a viral alpha-helix heptad repeat domain in a
stabilized .alpha.-helical structure (herein also referred to as
SAH). The compositions are useful for treating and/or preventing
viral infections. The invention is based, at least in part, on the
result provided herein demonstrating that viral hydrocarbon stapled
alpha helical peptides display excellent proteolytic, acid, and
thermal stability, restore the native alpha-helical structure of
the peptide, are highly effective in interfering with the viral
fusogenic process, and possess superior pharmacokinetic properties
compared to the corresponding unmodified peptides.
Inventors: |
Walensky; Loren D.; (Newton,
MA) ; Bird; Gregory H.; (Pelham, NH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dana-Farber Cancer Institute, Inc. |
Boston |
MA |
US |
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|
Family ID: |
41016644 |
Appl. No.: |
15/019558 |
Filed: |
February 9, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12864375 |
Jul 23, 2010 |
9290545 |
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15019558 |
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PCT/US2009/000438 |
Jan 23, 2009 |
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12864375 |
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61062007 |
Jan 23, 2008 |
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Current U.S.
Class: |
424/139.1 ;
424/188.1; 435/375; 530/322; 530/387.9 |
Current CPC
Class: |
C12N 2760/16022
20130101; A61P 31/14 20180101; C12N 2760/18022 20130101; C12N
2760/18634 20130101; C12N 2740/16222 20130101; C12N 2740/15034
20130101; C07K 16/1063 20130101; C12N 2740/16122 20130101; A61P
31/18 20180101; C12N 2740/15022 20130101; C07K 2317/76 20130101;
C12N 2740/16234 20130101; C12N 2760/18034 20130101; C12N 2760/16034
20130101; A61K 39/21 20130101; C12N 2760/18671 20130101; C12N
2760/14071 20130101; A61K 39/155 20130101; A61K 38/162 20130101;
A61P 31/12 20180101; C12N 2740/16271 20130101; C12N 2760/18522
20130101; C12N 2740/15071 20130101; A61P 31/16 20180101; C12N
2760/14022 20130101; C12N 2760/14034 20130101; C12N 2760/18571
20130101; C12N 2760/18622 20130101; C12N 2740/16134 20130101; C12N
2760/18071 20130101; C12N 2760/16071 20130101; A61K 39/145
20130101; C07K 14/005 20130101; C12N 2760/18534 20130101; C12N
2740/16171 20130101 |
International
Class: |
C07K 14/005 20060101
C07K014/005; A61K 39/145 20060101 A61K039/145; A61K 39/155 20060101
A61K039/155; A61K 39/21 20060101 A61K039/21; C07K 16/10 20060101
C07K016/10 |
Claims
1. A modified polypeptide comprising a stabilized alpha helix of
HIV gp41 heptad repeat domain.
2.-61. (canceled)
62. A method for inhibiting transmission of HIV to a cell
comprising contacting the virus in the presence of the cell with an
effective dose of a modified polypeptide comprising a stabilized
alpha helix of HIV gp41 heptad repeat domain, so that the infection
of the cell by the virus is inhibited.
63.-70. (canceled)
71. A method for treating or delaying the onset of AIDS in an HIV
infected individual, comprising administering to the individual an
effective dose of a pharmaceutical composition comprising a
modified polypeptide comprising a stabilized alpha helix of HIV
gp41 heptad repeat domain.
72.-85. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/864,375, filed on Jul. 23, 2010, which is a continuation of
International Application No. PCT/US2009/000438 (WO2009/108261)
filed on Jan. 23, 2009, which claims priority to U.S. Provisional
Patent Application Ser. No. 61/062,007 filed on Jan. 23, 2008, each
of which is incorporated herein by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web, and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jun. 24, 2011, is named 69208US7.txt and is 79,397 bytes in
size.
BACKGROUND
[0003] The molecular process of viral fusion, in which viral coat
proteins recognize and bind to surface receptors of the host cell,
is a critical target in the prevention and treatment of viral
infections. Upon recognition of the viral glycoprotein by host
cellular receptors, viral fusion proteins undergo conformational
changes that are essential to viral fusion and infection. A series
of hydrophobic amino acids, located at the N- and C-termini
organize to form a complex that pierces the host cell membrane.
Adjacent viral glycoproteins containing two amphipathic heptad
repeat domains fold back on each other to form a trimer of
hairpins, consisting of a bundle of six .alpha.-helices. This
six-helix bundle motif is highly conserved among many viral
families, including Filovirus (ebola), (Malashkevich, V. N., et
al., PNAS, 1999. 96(6): p. 2662-2667; Weissenhorn, W., et al.,
Molecular Cell, 1998. 2(5): p. 605-616), Orthomyxovirus (influenza)
(Wilson, I. A., J. J. Skehel, and D. C. Wiley, Nature, 1981.
289(5796): p. 366-3'7; Bullough, P. A., et al., Nature, 1994.
371(6492): p. 37-43), Coronavirus (SARS) (Xu, Y. H., et al. Journal
of Biological Chemistry, 2004. 279(47): p. 49414-49419),
Paramyxovirus (HRSV) (Zhao, X., et al., PNAS, 2000. 97(26): p.
14172-14177) and Retrovirus (HIV) (Chan, D. C., et al., Cell, 1997.
89(2): p. 263-27; Weissenhorn, W., et al., Nature, 1997. 387(6631):
p. 426-430).
[0004] HIV envelope proteins gp120 and gp41 non-covalently
associate with each other to form a trimer of dimers. On the host
cell, gp120 specifically interacts with CD4, CXCR4, and CCR5, which
are the glycoproteins involved in host-cell recognition. gp41, the
viral membrane spanning glycoprotein, is responsible for fusing the
viral and cellular membranes, resulting in viral particle uptake by
the host cell. Once gp120 binds to CD4, gp41 undergoes a
conformational change, transforming from its native state into a
fusogenic six-helix bundle. The regions of gp41 involved in this
change are 43 (C43) residues of the C-terminal heptad repeat (CHR
or HR-2), near the transmembrane domain, and 51 (N51) residues of
the N-terminal heptad repeat (NHR or HR-1), found just proximal to
the fusion peptide domain. Peptides N51 and C43 orient to form
helical antiparallel heterodimers, which associate to form a higher
order trimeric complex that is thermo- and proteolytically
stable.
[0005] Peptides which interfere with this viral fusogenic process
can be used for the prevention and treatment of viral infections.
For example, peptides corresponding to residues 553-590 of the gp41
N-terminal heptad repeat domain (HR-1) and residues 630-659 and
648-673 of the C-terminal heptad repeat domain (HR-2) of HIV have
been shown to inhibit the replication of a variety of HIV strains.
Studies have determined that these peptides inhibit cell-cell
fusion by interacting with the HIV envelope glycoproteins.
[0006] T20 or enfuvirtide, is the first fusion inhibitor peptide
developed based on the CHR region of gp41 for the treatment of HIV.
Enfuvirtide is active at nanomolar concentrations against many
strains and subtypes of HIV, including the common lab strains and
primary isolates of HIV-1 and HIV-2 (Wild, C. T., et al., PNAS,
1994. 91(21): p. 9770-9774).
[0007] However, enfuvirtide has remained a tertiary treatment
option due to a variety of factors which include cost, no oral
bioavailability (subcutaneous injections limit accessibility and
compliance) and poor in vivo stability (Kilby, J. M., et al.,
Nuclic Aids Research and Human Retroviruses, 2002. 18(10): p.
685-693), and loss of bioactive secondary structure. Thus, although
peptide-based inhibition of viral fusion processes is
mechanistically feasible and clinically effective, the biophysical
and biochemical properties of amphipathic fusion peptides present
numerous challenges which hinder their use.
SUMMARY OF THE INVENTION
[0008] The present invention is directed to compositions, kits and
methods utilizing polypeptides with stabilized .alpha.-helical
structures (herein also referred to as SAH). The compositions are
useful for treating and/or preventing viral infections. The
invention is based, at least in part, on the result provided herein
demonstrating that viral hydrocarbon stapled alpha helical peptides
display excellent proteolytic, acid, and thermal stability, restore
the native alpha-helical structure of the peptide, are highly
effective in interfering with the viral fusogenic process, and
possess superior pharmacokinetic properties compared to the
corresponding unmodified peptides.
[0009] In a first aspect, the invention is directed to a modified
polypeptide having a stabilized viral alpha helix heptad repeat
domain. Preferably the alpha helix heptad repeat domain is
stabilized with at least one hydrocarbon staple, but could include
two, three or more hydrocarbon staples. Suitable hydrocarbon
staples (e.g., tethers) are described herein. Suitable viral alpha
helix heptad repeat domains are derived from any virus with an
alpha helix domain or analog thereof that is directly or indirectly
involved in cell attachment and/or fusion. Suitable stabilized
alpha helical heptad repeat domains can be derived from numerous
viruses, including respiratory syncytial virus, parainfluenza
virus, influenza virus, coronavirus, ebolavirus and HIV. The
modified polypeptides of the invention can include a stabilized HIV
gp41 heptad repeat domain (e.g., heptad repeat domain 1 or 2, or
portions thereof).
[0010] Any of the modified polypeptides of the invention can be
included in compositions and kits.
[0011] In another aspect, the invention is directed to a method for
inhibiting the transmission of HIV to a cell. In the method, the
HIV virus is contacted with an effective dose of a modified
polypeptide so that the HIV virus is inhibited from infecting the
cell. Preferably, the modified polypeptide has an HIV gp41 heptad
repeat domain (e.g., heptad repeat domain 1 or 2, or portions
thereof) that is stabilized with a hydrocarbon staple.
[0012] The invention may also include a method for treating or
delaying the onset of AIDS in an HIV infected individual. A
pharmaceutical composition having a modified polypeptide with a
stabilized HIV gp41 heptad repeat domain (e.g., heptad repeat
domain 1 or 2, or portions thereof) is administered to an
individual infected with HIV, thus treating or delaying the onset
of AIDS. Preferably the HIV gp41 heptad repeat domain is stabilized
with a hydrocarbon staple.
[0013] In still another aspect, the invention is directed to a
method for increasing the number of CD4+ cells in an individual
infected with HIV. The method involves administering to the
individual infected with HIV an effective dose of a pharmaceutical
composition having a modified polypeptide with a stabilized HIV
gp41 heptad repeat domain (e.g., heptad repeat domain 1 or 2, or
portions thereof). The administration of the composition results in
an increase in the number of CD4+ cells in the individual.
Preferably the HIV gp41 heptad repeat domain is stabilized with a
hydrocarbon staple.
[0014] In yet another aspect, the invention is directed to a method
for inhibiting syncytia formation between an HIV infected cell and
an uninfected cell. The method involves contacting the infected
cell with an effective dose of a modified polypeptide having a
stabilized HIV gp41 heptad repeat domain (e.g., heptad repeat
domain 1 or 2, or portions thereof), thereby inhibiting syncytia
formation between the cells. Preferably the HIV gp41 heptad repeat
domain is stabilized with a hydrocarbon staple.
[0015] In still another aspect, the invention is directed to a
method for inactivating HIV. The method involves contacting the
virus with an effective dose of a modified polypeptide having a
stabilized HIV gp41 heptad repeat domain (e.g., heptad repeat
domain 1 or 2, or portions thereof) so that the HIV is rendered
inactive (e.g., non-infectious). Preferably the HIV gp41 heptad
repeat domain is stabilized with a hydrocarbon staple.
[0016] In still another aspect, the invention is directed to a
method for preventing an HIV infection in an individual. The method
involves administering to an individual an effective dose of a
pharmaceutical composition having modified polypeptide with a
stabilized HIV gp41 heptad repeat domain (e.g., heptad repeat
domain 1 or 2, or portions thereof). Administration of the
stabilized HIV gp41 heptad repeat domain interferes with the
ability of the HIV to infect the individual. Preferably the HIV
gp41 heptad repeat domain is stabilized with a hydrocarbon
staple.
[0017] The modified polypeptides can be used to inhibit the
transmission of RSV to a cell. The virus is contacted with an
effective dose of a modified polypeptide having a stabilized RSV
viral alpha helix heptad repeat domain analog thereby inhibiting
transmission of the virus to a cell. Preferably the heptad repeat
domain analog is stabilized with the hydrocarbon staple.
[0018] The modified polypeptides can also be used to inhibit the
transmission of a parainfluenza virus to a cell. The virus is
contacted with an effective dose of a modified polypeptide having a
stabilized parinfluenza viral alpha helix heptad repeat domain
analog, thereby inhibiting transmission of the virus to a cell.
Preferably the heptad repeat domain analog is stabilized with the
hydrocarbon staple.
[0019] In another aspect, the modified polypeptides can also be
used to inhibit the transmission of an influenza virus to a cell.
The virus is contacted with an effective dose of a modified
polypeptide having a stabilized influenza viral alpha helix heptad
repeat domain analog, thereby inhibiting transmission of the virus
to a cell. Preferably the heptad repeat domain analog is stabilized
with the hydrocarbon staple.
[0020] In still another aspect, the invention is directed to a
method for inhibiting the transmission of a coronavirus to a cell.
The method includes contacting the coronavirus with an effective
dose of a modified polypeptide having a stabilized coronavirus
alpha helix heptad repeat domain analog, thereby inhibiting
transmission of the virus to a cell. Preferably the heptad repeat
domain analog is stabilized with the hydrocarbon staple.
[0021] In yet still another aspect, the invention is directed to a
method for inhibiting the transmission of an ebolavirus to a cell.
The method includes contacting the ebolavirus with an effective
dose of a modified polypeptide having a stabilized ebolavirus alpha
helix heptad repeat domain analog, thereby inhibiting transmission
of the virus to the cell. Preferably the heptad repeat domain
analog is stabilized with a hydrocarbon staple.
[0022] In an aspect of the invention, the invention provides
modified peptides of the inventions as a pharmaceutical
composition. In some embodiments, the pharmaceutical composition is
for enteral administration, preferably oral administration.
[0023] In yet another aspect, the alpha helix heptad repeat domains
and analogs thereof are used to generate an antibody response to
the polypeptides by administering the polypeptides to a subject.
Furthermore, the antibodies generated directly or indirectly (e.g.,
humanized antibodies) by the administration of the polypeptides may
then be used to prevent or treat a viral infection (e.g., HIV, RSV,
parainfluenza, influenza, coronavirus, ebolavirus).
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 illustrates the domains of the gp41 glycoprotein.
[0025] FIG. 2A and FIG. 2B illustrate the amino acid sequence for
FIG. 2A) HX-strain of gp160 (SEQ ID NO: 49) and FIG. 2B) YU2-strain
of gp160 (SEQ ID NO: 50), with HR-1 domain bolded and underlined
and HR-2 domain bolded and italicized.
[0026] FIG. 3 illustrates the amino acid sequences for HIV-1 gp41
HR-1 and HR-2 domains and homologous regions in other viruses (SEQ
ID NOS: 14, 7, 9, 11, 51-54, 8, 10, 12, and 55-57, respectively, in
order of appearance).
[0027] FIG. 4A illustrates the HIV six-helix bundle and key
interhelix interactions of the helicies N36 and C34. One of the N36
and two C34 helicies are faded for clarity. The helical wheel
further illustrates key contacts among the helicies based upon the
a, b, c, d, e, f, g, nomenclature.
[0028] FIG. 4B illustrates the fusogenic bundle formed by HR-analog
domains from RSV, influenza, SARS and Ebola. The six-helix
fusogenic bundle is highly conserved across many species.
[0029] FIG. 5A provides examples of amino acid sequence templates
from within the HIV-1 HR-2 domain polypeptides with sequential
N-terminal truncations. Shown are N-terminal truncations of the
polypeptides of SEQ ID NOS: 1, 13, 58, and 59, respectively.
[0030] FIG. 5B provides examples of amino acid sequence templates
from within the HIV-1 HR-2 domain polypeptides with sequential
C-terminal truncations. Shown are C-terminal truncations of the
polypeptides of SEQ ID NOS: 1, 13, 58, and 59, respectively.
[0031] FIG. 6 provides examples of sequence templates from within
the HIV-1 HR2 domain depicting staggered N- and C-terminal
truncations. Shown are staggered N- and C-terminal truncations of
the polypeptides of SEQ ID NOS: 1, 13, 58, and 59,
respectively.
[0032] FIG. 7 illustrates a synthetic design of a truncated
SAH-gp41 compound, SAH-gp41.sub.(626-645)(A) (SEQ ID NO: 61), which
is based on gp41.sub.(626-645) (SEQ ID NO: 60). X=S5 amino acid,
B=norleucine.
[0033] FIG. 8 provides examples of sequence templates from within
the HR2 domains of SIV (SEQ ID NO: 62) and the HX (SEQ ID NOS: 1
and 13) and YU2 (SEQ ID NOS: 58 and 59) strains of HIV-1 depicting
the generation of chimeras (SEQ ID NO: 62).
[0034] FIG. 9A-FIG. 9D illustrate the heptad repeat domain motif as
applied to HIV gp41 (626-663) (SEQ ID NO: 66) and associated
preferred amino acid residues.
[0035] FIG. 9A presents the heptad repeat domain. FIG. 9B presents
HIV gp41 (626-663). FIG. 9C presents the heptad position a, d. FIG.
9D presents a sequence associated with the heptad repeat domain.
Examples of sequence template from within the HIV-1 HR2 domain
depicting the specific amino acid residues necessary to preserve
the HR1 interaction are provided (SEQ ID NOS: 44 and 45). Thus, the
positions indicated with a dash may be amenable to
substitution/mutation without disruption of activity.
[0036] FIGS. 10A-FIG. 10D illustrate the possible combinations of
helix-stabilizing crosslinks formed at positions FIG. 10A) i, and
i+4 across one turn in the helix using two S5 amino acids; FIG.
10B) i, and i+7, across two turns of the helix using one S8 and one
R5 amino acid or one R8 and one S5 amino acid; FIG. 10C) a double
crosslink employing two i, i+4, two i, i+7, or one i, i+4 and one
i, i+7 crosslink; and FIG. 10D) a triple crosslink employing any
combination of i, i+4, i+7, or other crosslinks (e.g. i, i+3).
[0037] FIG. 11 illustrates SAH-gp41 singly stapled peptides (SEQ ID
NOS: 67-71, 1, and 72-79, in order of appearance). (e.g., N-term:
Ac, FITC-.beta.Ala, Biotin-.beta.Ala; C-term: CONH.sub.2, COOH).
X=S5 amino acid, B=norleucine
[0038] FIG. 12 includes sequences of doubly and triply stapled SAH
gp41 peptides (SEQ ID NOS 80-87, 1 and 88-98, in order of
appearance). (e.g., N-term: Ac, FITC-.beta.Ala, Biotin-.beta.Ala;
C-term: CONH.sub.2, COOH). X=S5 amino acid, B=norleucine.
[0039] FIG. 13 illustrates unstapled, singly stapled and doubly
stapled gp41 HR-2 peptides (SEQ ID NOS 99-100 and 82-85, in order
of appearance) and illustrates a strategy for locating the staples
in the helix. Staples are positioned so as to preserve and/or
optimize inter-helix interaction surfaces. X=S5 amino acid,
B=norleucine
[0040] FIGS. 14A-FIG. 14F illustrate that singly and doubly stapled
SAH-gp41 compounds exhibit greater helical stability as compared to
the unmodified gp41 peptides at pH 7 and pH2. Percent helicity for
each compound is indicated in parenthesis; FIG. 14A)
SAH-gp41.sub.(626-662) singly- and doubly-stapled peptides at pH 7,
FIG. 14B) SAH-gp41.sub.(638-673) singly-stapled peptides at pH 7,
FIG. 14C) SAH-gp41.sub.(638-673) doubly-stapled peptides at pH 7,
FIG. 14D) SAH-gp41.sub.(626-662) singly- and doubly-stapled
peptides at pH 2, FIG. 14E) SAH-gp41.sub.(638-673) singly- and
doubly-stapled peptides at pH 2, FIG. 14F) Table comparing
calculated percent helicities of SAH-gp41 compounds at pH 7 and pH
2.
[0041] FIGS. 15A-FIG. 15C illustrate that singly and doubly stapled
SAH-gp-41 compounds exhibit greater thermal stability compared to
the unmodified gp41 peptides at pH 7 FIG. 15A)select singly- and
doubly-stapled SAH-gp41.sub.(626-662) compounds; FIG. 15B)
singly-stapled SAH-gp41.sub.(638-673) compounds; and FIG. 15C)
doubly-stapled SAH-gp41.sub.(638-673).
[0042] FIGS. 16A-FIG. 16F illustrate that SAH-gp41 compounds
exhibit greater protease resistance to chymotrypsin at pH 7 and
pepsin at pH 2 compared to the unmodified gp41 peptides; FIG. 16A)
SAH-gp41.sub.(626-662), chymotrypsin pH 7, FIG. 16B)
SAH-gp41.sub.(638-673) chymotrypsin pH 7, FIG. 16C) Table of
half-lives of SAH-gp41 compounds in the presence of chymotrypsin,
pH 7, FIG. 16D) SAH-gp41.sub.(626-662), pepsin pH 2, FIG. 16E)
SAH-gp41.sub.(638-673) pepsin, pH 2, FIG. 16F) Table of half-lives
of SAH-gp41 compounds in the presence of pepsin, pH 2.
[0043] FIG. 17 shows a fluorescence polarization binding analysis
of HIV fusion inhibitor peptides to the gp41 five-helix bundle
illustrating enhanced binding of SAH-gp41 to the five-helix bundle
compared to the unmodified peptides.
[0044] FIG. 18 shows improved inhibition of syncytia formation by a
truncated SAH-gp41 compound (A) compared to enfuvirtide (T20:
gp41.sub.(638-673)), highlighting the potential to retain, and even
enhance, anti viral activity with shorter, stapled peptides.
[0045] FIG. 19 demonstrates the anti-viral activity of select
SAH-gp41 compounds against HIV strains HXBc2, ADA, and HXBc2P 3.2,
and YU2. AMLV serves as a negative control.
[0046] FIGS. 20A-FIG. 20B demonstrate that FIG. 20A) SAH-gp41
compounds overcome HIV-1 HR1 resistance mutations that block the
binding of unmodified gp41-based fusion peptides. FIG. 20B)
Tabulated values indicate fraction of HR2 peptide input bound to
the indicated FITC-HR1 peptide; and B) Select SAH-gp41 compounds
are notably superior to the corresponding unmodified peptides in
blocking the infectivity of a resistant HIV-1 strain, YU2.
[0047] FIG. 21 shows that a doubly-stapled gp41 peptide has
markedly enhanced pharmacologic properties in vivo (stability and
bioavailability) compared to the corresponding unmodified
peptide.
DETAILED DESCRIPTION
[0048] The present invention is directed to compositions, kits and
methods utilizing polypeptides with stabilized alpha helical
structures. The compositions are useful for treating and/or
preventing viral infections. The invention is based, at least in
part, on the results provided herein demonstrating that viral
hydrocarbon stapled alpha helical peptides have excellent
structural, proteolytic, acid, and thermal stability, are highly
effective in interfering with virus/cell fusion, and have superior
pharmacologic properties in vivo compared to their unmodified
counterparts.
[0049] The alpha helix heptad repeat domain is stabilized with at
least one hydrocarbon staple, but could include two, three or more
hydrocarbon staples. The inclusion of multiple hydrocarbon staples
is particularly suited for alpha helical peptides that are 20 or
more amino acids in length. In fact the inclusion of two more
hydrocarbon staples, as shown herein, provides for exceptional
structural, acid and thermal stability of the modified
polypeptides, yielding bioactive peptides with strikingly enhanced
pharmacologic properties in vivo.
DEFINITIONS
[0050] As used herein, the term "hydrocarbon stapling", refers to a
process for stably cross-linking a polypeptide having at least two
modified amino acids that helps to conformationally bestow the
native secondary structure of that polypeptide. Hydrocarbon
stapling allows a polypeptide, predisposed to have an alpha-helical
secondary structure, to maintain its native alpha-helical
conformation. This secondary structure increases resistance of the
polypeptide to proteolytic cleavage and heat, and also may increase
hydrophobicity. Accordingly, the hydrocarbon stapled (cross-linked)
polypeptides described herein have improved biological activity
relative to a corresponding non-hydrocarbon stapled (uncrosslinked)
polypeptide. For example the cross-linked polypeptide can include
an alpha-helical domain of an HIV polypeptide (e.g., HR-1/HR-2
domain), which can interfere with HIV attachment, fusion with, and
infection of a cell. In some instances, the cross-linked
polypeptide can be used to inhibit virus entry into a cell. The
cross-linked polypeptides described herein can be used
therapeutically, e.g., to treat HIV infection and/or AIDS.
[0051] The hydrocarbon stapled polypeptides include one or more
tethers (linkages) between two non-natural amino acids, which
tether significantly enhances the alpha helical secondary structure
of the polypeptide. Generally, the tether extends across the length
of one or two helical turns (i.e., about 3.4 or about 7 amino
acids). Accordingly, amino acids positioned at i and i+3; i and
i+4; or i and i+7 are ideal candidates for chemical modification
and cross-linking. Thus, for example, where a peptide has the
sequence . . . X1, X2, X3, X4, X5, X6, X7, X8, X9 . . . ,
cross-links between X1 and X4, or between X1 and X5, or between X1
and X8 are useful as are cross-links between X2 and X5, or between
X2 and X6, or between X2 and X9, etc. The use of multiple
cross-links (e.g., 2, 3, 4 or more) is also contemplated. The use
of multiple cross-links is very effective at stabilizing and
optimizing the peptide, especially with increasing peptide length,
as is the case for some gp41 fusion peptides. Thus, the invention
encompasses the incorporation of more than one crosslink within the
polypeptide sequence to either further stabilize the sequence or
facilitate the structural stabilization, proteolytic resistance,
acid stability, thermal stability, and biological activity
enhancement of longer polypeptide stretches.
[0052] The term "stable" or "stabilized", as used herein with
reference to a polypeptide, refers to polypeptides which have been
hydrocarbon-stapled to maintain their natural alpha-helical
structure and/or improve protease resistance and/or improve acid
stability and/or improve thermal stability.
[0053] As used herein, "HIV" is meant to include HIV-1 and HIV-2
and SIV. "HIV-1" means the human immunodeficiency virus type-1.
HIV-1 includes but is not limited to extracellular virus particles
and the forms of HIV-1 associated with HIV-1 infected cells.
"HIV-2" means the human immunodeficiency virus type-2. HIV-2
includes but is not limited to extracellular virus particles and
the forms of HIV-2 associated with HIV-2 infected cells. The term
"SIV" refers to simian immunodeficiency virus which is an HIV-like
virus that infects monkeys, chimpanzees, and other nonhuman
primates. SIV includes but is not limited to extracellular virus
particles and the forms of SIV associated with SIV infected
cells.
[0054] As used herein a "heptad repeat domain" and "HR domain"
refers to a polypeptide that forms an alpha-helix when properly
folded. The terms, "heptad repeat domain" and "HR domain" include
"HR-like" and "HR-analog" polypeptides. Numerous viral proteins
involved in cell attachment and fusion contain HR, HR-like and
HR-analog domains including, HIV, parainfluenza, coronavirus, and
others. Generally, HR domains are derived from gp41 of HIV, while
HR-analog domains are derived from the envelope glycoproteins of
non-HIV viruses. Many HR and HR-analog domain polypeptides are
known in the art and described herein. In one embodiment, the HR
domain has an amino acid sequence which is 40%, 50%, 60%, 70%, 80%,
or more identical to FIG. 5, FIG. 6 or SEQ ID NO:1-14. It should be
noted that HR and HR-like domains may have low homology but will
share a common alpha helical structure, with more conservation on
the interaction surfaces than non-interacting surfaces (see FIGS. 4
and 9).
[0055] In one embodiment, the HR modified polypeptide includes a
heptad repeat domain having the formula: a b c d e f g, wherein a
and d are hydrophobic amino acid residues and b, c, e, f and g are
any amino acid. Preferably, the formula is repeated in tandem two
or more times.
[0056] For example, in a further embodiment the heptad repeat
domain of the modified polypeptide has the formula: W(a), b, c,
W(d), e, f, g, I(a), b, c, Y(d), e, f, g, I(a), b, c, L(d), e, f,
g, S(a), b, c, Q(d), e, f, g, N(a), b, c, E(d), e, f, g, L(a), or
conservative amino acid substitutions thereof and wherein the b, c,
e, f and g can be any amino acid (e.g., residues 3-38 of SEQ ID NO:
44).
[0057] In a further, embodiment the heptad repeat domain of the
modified polypeptide has the formula: T(g), W(a), b, c, W(d), D(e),
R(f), g, I(a), b, c, Y(d), e, f, g, I(a), b, c, L(d), I(e), f, g,
a, Q(b), c, d, Q(e), E(f), K(g), a, E(b), c, d, L(e), f, E(g),
L(a), or conservative amino acid substitutions thereof and wherein
non-designated amino acids can be any amino acid (e.g., residues
2-38 of SEQ ID NO: 45).
[0058] The HR regions are known to comprise a plurality of 7 amino
acid residue stretches or "heptads" (the 7 amino acids in each
heptad designated "a" through "g"), wherein the amino acids in the
"a" position and "d" position are generally hydrophobic. Generally
the HR region will include one or more leucine zipper-like motifs
(also referred to as "leucine zipper-like repeats") comprising an 8
amino acid sequence initiating with, and ending with, an isoleucine
or leucine. Heptads and leucine zipper like-motifs contribute to
formation of a coiled coil structure of gp41, and of a coiled coil
structure of peptides derived from the HR regions. Generally,
coiled coils are known to be comprised of two or more helices that
wrap around each other in forming oligomers, with the hallmark of
coiled coils being a heptad repeat of amino acids with a
predominance of hydrophobic residues at the first ("a") and fourth
("d") positions, charged residues frequently at the fifth ("e") and
seventh ("g") positions, and with the amino acids in the "a"
position and "d" position being primary determinants that influence
the oligomeric state and strand orientation (see, e.g., Akey et
al., 2001, Biochemistry, 40:6352-60).
[0059] The effect on stability and oligomerization state of a model
coiled coil, by substituting various amino acids at various
positions including the "a" and "d" positions, have been reported
previously, wherein formation of a trimeric structure was
particularly dependent on the substitution at the "d" position
(see, e.g., Tripet et al., J. Mol. Biol. 300:377-402 (2000);
Wagschal et al., J. Mol. Biol. 285:785-803 (2000); and Dwyer et
al., PNAS USA. 104; 12772-12777 (2007).
[0060] It will be apparent to one skilled in the art that any
peptide derived from the native sequence of the HR1 domain or HR2
domain of HIV gp41 which has antiviral activity (as can be
determined using methods standard in the art without undue
experimentation), and which contains all or a fraction of the
region can be used as a native sequence into which one or more
amino acid substitutions, preferably conservative, in the domain
may be introduced to produce a synthetic peptide provided with the
present invention. For purposes of illustration, such HR2 peptides
derived from the native sequence, and from which a synthetic
peptide may be produced, may include, but are not limited to, those
illustrated in FIGS. 5 and 6.
[0061] It is apparent to those of ordinary skill in the art that
some HR and HR-analog domain residues are less prone to
substitution while others are more accepting of changes. For
example, it is preferable not to mutate or to only conservatively
mutate the amino acids at positions a and d of the heptad repeat
(See FIG. 9). In one embodiment, the heptad repeat domain has the
formula a, b, c, d, e, f, g, wherein a and d are hydrophobic amino
acids. In a further embodiment, the heptad repeat domain has two or
more repeats of the formula a, b, c, d, e, f, g. For example, in
one embodiment the HR domain will have the amino acid sequences
illustrated in FIG. 9 or conservative substitutions thereof. Thus,
the HR and HR-like domains have significant variability in amino
acid sequence but will maintain an alpha helical structure and
antiviral activity.
[0062] In one embodiment, the modified polypeptide includes a
heptad repeat domain having the formula: a b c d e f g, wherein a
and d are hydrophobic amino acid residues and b, c, e, f and g are
any amino acid. Preferably, the formula is repeated in tandem two
or more times.
[0063] For example, in a further embodiment the heptad repeat
domain of the modified polypeptide has the formula: W(a), b, c,
W(d), e, f, g, I(a), b, c, Y(d), e, f, g, I(a), b, c, L(d), e, f,
g, S(a), b, c, Q(d), e, f, g, N(a), b, c, E(d), e, f, g, L(a), or
conservative amino acid substitutions thereof and wherein the b, c,
e, f and g can be any amino acid (e.g., residues 3-38 of SEQ ID NO:
44).
[0064] In a further, embodiment the heptad repeat domain of the
modified polypeptide has the formula: T(g), W(a), b, c, W(d), D(e),
R(f), g, I(a), b, c, Y(d), e, f, g, I(a), b, c, L(d), I(e), f, g,
a, Q(b), c, d, Q(e), E(f), K(g), a, E(b), c, d, L(e), f, E(g),
L(a), or conservative amino acid substitutions thereof and wherein
non-designated amino acids can be any amino acid (e.g., residues
2-38 of SEQ ID NO: 45).
[0065] The HR, HR-like and HR-analog domains are readily
identifiable by those possessing ordinary skill in the art by
sequence based homology, structural homology and/or functional
homology. Such methods are well known in the art and include
bioinformatics programs based on pairwise residue correlations
(e.g., on the world wide web at:
ch.embnet.org/software/COILS_form.html), which have the ability to
recognize coiled coils from protein sequences and model their
structures (See Lupas, A., et al. Science 1991. 252(5009); p.
1162-1164). Additional methods for identifying HR, HR-like and
HR-analog domains are described in U.S. Pat. No. 6,824,783; U.S.
Pat. No. 7,273,614; U.S. Pat. No. 5,464,933; and U.S. Pat. No.
7,122,190, all of which are herein incorporated by reference in
their entirety.
[0066] In one embodiment, the modified polypeptide of the invention
is 70% or more similar at the interacting face to the amino acid
sequence of SEQ ID NO:1-14, FIG. 5 or FIG. 6. The "interacting
face" of the alpha helix includes those amino acid residues which
interact with other amino acid residues. For example, in the HIV
gp41 HR-2 domain the interacting face includes the "a" and "d"
position amino acids (See FIGS. 4A and 9), while the interacting
face of the HIV gp41 HR-1 domain includes amino acids at positions
e, g that interact with HR-2 and a, d that engage in HR1-HR1
interactions (See FIG. 4A). Methods for identifying heptad repeats
and the interacting face residues are well known in the art and
described herein.
[0067] An "HR-1 domain of HIV" or "heptad repeat one domain of HIV"
is an N-terminal portion of the gp41 protein of HIV (the
transmembrane subunit of HIV envelope) that forms an alpha-helix
when properly folded. The HR-1 domain of HIV gp41 can include
between 5 and 55 amino acid residues and is based on the sequence
of the native HR-1 domain of HIV gp41, or a combination or chimera
thereof. The HR-1 domain of HIV can include the N36 domain which
encompasses amino acid residues 546-581 HIV-1 Env (See FIG. 2 and
Bewley et al., J. Biol. Chem. 277:14238-14245 (2002)). HR-1 domain
polypeptides are known in the art and described herein. In one
embodiment, the HR-1 domain has an amino acid sequence which is 30%
or more identical to SEQ ID NO:2 or 14.
[0068] An "HR-2 domain of HIV" or a heptad repeat two domain of HIV
is a C-terminal portion of the gp41 protein of HIV (the
transmembrane subunit of HIV envelope) that forms an alpha-helix
when properly folded. The HR-2 domain of HIV can include the C34
domain which encompasses amino acid residues 628-661 of HIV-1 Env
(See FIG. 2). HR-2 domain polypeptides are known in the art and
described herein. In one embodiment, the HR-2 domain has an amino
acid sequence which is 40% or more identical to SEQ ID NO:1 or
13.
[0069] As used herein, the term "chimera" or "chimeric", with
reference to the polypeptides of the invention refers to a
polypeptide having at least two different HR domains or having a
single HR domain region that is combined in a manner not found in
nature (FIG. 8). For example, the chimera polypeptide may have a
first portion of an HIV-1 gp41 HR-2 domain and a second portion
from a SIV gp41 HR-2 domain. These chimeric polypeptides are
encoded by nucleotide sequences which can be been fused or ligated
together resulting in a coding sequence which does not occur
naturally. The chimera includes any functional derivative,
fragments, variants, analogues, or chemical derivatives which may
be substantially similar to the wild-type HR polypeptides (HIV-1
gp41 HR-2) and which possess similar activity (i.e., most
preferably, 90%, more preferably, 70%, preferably 40%, or at least
10% of the wild-type HR activity, e.g., inhibiting fusion, viral
infectivity).
[0070] The terms "treat," and "treating," as used herein, shall
mean decrease, suppress, attenuate, diminish, arrest, or stabilize
the development or progression of a disease or decrease the
occurrence of pathological cells (e.g., infected cells) in an
animal who is infected with the viral disorder. The treatment may
be complete, e.g., the total absence of HIV in a subject. The
treatment may also be partial, such that the occurrence of infected
cells in a subject is less than that which would have occurred
without the present invention. Treatment results in the
stabilization, reduction or elimination of the infected cells, an
increase in the survival of the patient or decrease of at least one
sign or symptoms of the disease.
[0071] The terms "prevent," "preventing," and "prevention," as used
herein, shall refer to a decrease in the occurrence of a disease,
or decrease in the risk of acquiring a disease, or a decrease in
the presentation of at least one sign or associated symptom of the
disease in a subject. The prevention may be complete, e.g., the
total absence of disease or pathological cells in a subject. The
prevention may also be partial, such that the occurrence of the
disease or pathological cells in a subject is less than that which
would have occurred without the present invention.
[0072] The term "inhibits" as used herein with reference to a viral
infection refers to a decrease in viral transmission, decrease in
virus binding to a cellular target or decrease in disease. For
example, the polypeptides of the present invention are used to
inhibit viral transmission, syncytia formation, and disease
associated with the virus (e.g. AIDS). A compound of the invention
can be screened by many assays, known in the art and described
herein, to determine whether the compound inhibits the virus (e.g.,
infectivity, transmission, etc.). For example, a compound of the
invention can be assayed for its ability to inhibit viral
infectivity by contacting a cell culture that is incubated with the
virus with a test compound. The compound is found to inhibit viral
infectivity when viral infectivity is 90%, 80%, 75%, 70%, 60%, 50%,
40%, 30%, 20%, 10%, 5% or less in the presence of the test compound
as compared to a suitable control (population of cells not
subjected to inhibitor).
[0073] The term "inhibit transmission", as used herein, refers to
the agent's ability to inhibit viral infection of cells, via, for
example, cell-cell fusion or free virus infection. Such infection
may involve membrane fusion, as occurs in the case of enveloped
viruses, or some other fusion event involving a viral structure and
a cellular structure.
[0074] The term "inhibiting syncytia formation", as used herein,
refers to an agent's ability to inhibit or reduce the level of
membrane fusion events between two or more moieties relative to the
level of membrane fusion which occurs between said moieties in the
absence of the agent. The moieties may be, for example, cell
membranes or viral structures, such as viral envelopes.
[0075] The terms "effective amount," or "effective dose" refers to
that amount of an agent to produce the intended pharmacological,
therapeutic or preventive result. The pharmacologically effective
amount results in the amelioration of one or more symptoms of a
viral disorder, or prevents the advancement of a viral disease, or
causes the regression of the disease or decreases viral
transmission. For example, a therapeutically effective amount
preferably refers to the amount of a therapeutic agent that
decreases the rate of transmission, decreases HIV viral load, or
decreases the number of infected cells, by at least 5%, preferably
at least 10%, at least 15%, at least 20%, at least 25%, at least
30%, at least 35%, at least 40%, at least 45%, at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, or more. A
therapeutically effective amount, with reference to HIV, also
refers to the amount of a therapeutic agent that increases CD4+
cell counts, increases time to progression to AIDS, or increases
survival time by at least 5%, preferably at least 10%, at least
15%, at least 20%, at least 25%, at least 30%, at least 35%, at
least 40%, at least 45%, at least 50%, at least 55%, at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, or more.
[0076] The term "amino acid" refers to a molecule containing both
an amino group and a carboxyl group. Suitable amino acids include,
without limitation, both the D- and L-isomers of the 20 common
naturally occurring amino acids found in peptides (e.g., A, R, N,
C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V (as known by the
one letter abbreviations)) as well as the naturally occurring and
non-naturally occurring amino acids prepared by organic synthesis
or other metabolic routes.
[0077] A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of a polypeptide (e.g., an
HR-1 or HR-2 domain) without abolishing or substantially altering
its activity/secondary structure (alpha-helical structure). An
"essential" amino acid residue is a residue that, when altered from
the wild-type sequence of the polypeptide, results in abolishing or
substantially abolishing the polypeptide activity and/or secondary
structure. Substantially abolishing is understood as reducing the
activity of the peptide to less than about 30%, less than about
20%, less than about 10%, less than about 5% of the wild-type
peptide in an appropriate assay (e.g., a syncytia formation assay,
a viral fusion assay). The essential and non-essential amino acid
residues of the HR and HR-like domains can readily be determined by
methods well known in the art and are described herein. In one
embodiment, an essential amino acid residue is in the "a" or "d"
position of a heptad repeat domain, while non-essential amino acids
may occur in a "b", "c", "e", "f" or "g" position (FIG. 9). The
term "essential" amino acid residue as used herein, includes
conservative substitutions of the essential amino acid. Generally,
the "essential" amino acid residues are found at the interacting
face of the alpha helix. For example, in the HIV gp41 HR-2 domain
the interacting face includes the "a" and "d" position amino acids.
(See FIGS. 4A and 9). In another embodiment, a modified polypeptide
comprises a gp41 HR-1 domain having a Leu-556, Leu-565, Val-570,
Gly-572, and Arg-579 (Lu, M., et al., J. Vir, 2001. 75(22); p.
11146-11156).
[0078] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. For example, families of amino acid residues
having similar side chains have been defined in the art. These
families include amino acids with basic side chains (e.g., lysine,
arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine, glutamine, serine, threonine, tyrosine, cysteine),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan), beta-branched side
chains (e.g., threonine, valine, isoleucine) and aromatic side
chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Other conserved amino acid substitutions can also occur across
amino acid side chain families, such as when substituting an
asparagine for aspartic acid in order to modify the charge of a
peptide. Thus, a predicted nonessential amino acid residue in a HR
domain polypeptide, for example, is preferably replaced with
another amino acid residue from the same side chain family or
homologues across families (e.g. asparagine for aspartic acid,
glutamine for glutamic acid).
[0079] As used herein, the terms "identity" or "percent identity",
refers to the subunit sequence similarity between two polymeric
molecules, e.g., two polynucleotides or two polypeptides. When a
subunit position in both of the two molecules is occupied by the
same monomeric subunit, e.g., if a position in each of two peptides
is occupied by serine, then they are identical at that position.
The identity between two sequences is a direct function of the
number of matching or identical positions, e.g., if half (e.g., 5
positions in a polymer 10 subunits in length), of the positions in
two peptide or compound sequences are identical, then the two
sequences are 50% identical; if 90% of the positions, e.g., 9 of 10
are matched, the two sequences share 90% sequence identity. The
identity between two sequences is a direct function of the number
of matching or identical positions. Thus, if a portion of the
reference sequence is deleted in a particular peptide, that deleted
section is not counted for purposes of calculating sequence
identity. Identity is often measured using sequence analysis
software e.g., BLASTN or BLASTP (available at the world wide web
site ("www") of the National Center for Biotechnology Information
(".ncbi") of the National Institutes of Health (".nih") of the U.S.
government (".gov"), in the "Blast" directory ("/BLAST/"). The
default parameters for comparing two sequences (e.g., "Blast"-ing
two sequences against each other), by BLASTN (for nucleotide
sequences) are reward for match=1, penalty for mismatch=-2, open
gap=5, extension gap=2. When using BLASTP for protein sequences,
the default parameters are reward for match=0, penalty for
mismatch=0, open gap=11, and extension gap=1. Additional, computer
programs for determining identity are known in the art.
[0080] "Similarity" or "percent similarity" in the context of two
or more polypeptide sequences, refer to two or more sequences or
subsequences that are the same or have a specified percentage of
amino acid residues, or conservative substitutions thereof, that
are the same when compared and aligned for maximum correspondence,
as measured using one of the following sequence comparison
algorithms, or by visual inspection. By way of example, a first
polypeptide can be considered similar to an HIV-1 HR-1 domain when
the amino acid sequence of the first polypeptide is at least 20%,
50%, 60%, 70%, 75%, 80%, 90%, or even 95% or more identical, or
conservatively substituted, to a region of the HIV-1 HR-1 domain
when compared to any sequence of an equal number of amino acids as
the number contained in the first polypeptide as aligned by a
computer similarity program known in the art and described herein.
Preferably, the polypeptide region of the first protein and the
second protein includes one or more conserved amino acid
residues.
[0081] As used herein, an "antibody" includes any reactive fragment
or fragments of antibodies such as Fab molecules, Fab proteins,
single chain polypeptides, or the multi-functional antibodies
having binding affinity for the antigen. The term includes chimeric
antibodies, altered antibodies, univalent antibodies, bi-specific
antibodies, monoclonal antibodies, polyclonal antibodies, human
antibodies, and humanized antibodies. Methods for preparing
antibodies are well known in the art.
[0082] The symbol [0083] "" when used as part of a molecular
structure refers to a single bond or a trans or cis double
bond.
[0084] The term "amino acid side chain" refers to a moiety attached
to the .alpha.-carbon in an amino acid. For example, the amino acid
side chain for alanine is methyl, the amino acid side chain for
phenylalanine is phenylmethyl, the amino acid side chain for
cysteine is thiomethyl, the amino acid side chain for aspartate is
carboxymethyl, the amino acid side chain for tyrosine is
4-hydroxyphenylmethyl, etc. Other non-naturally occurring amino
acid side chains are also included, for example, those that occur
in nature (e.g., an amino acid metabolite) or those that are made
synthetically (e.g., an alpha di-substituted amino acid).
[0085] The term polypeptide encompasses two or more naturally
occurring or synthetic amino acids linked by a covalent bond (e.g.,
an amide bond). Polypeptides as described herein include full
length proteins (e.g., fully processed proteins) as well as shorter
amino acids sequences (e.g., fragments of naturally occurring
proteins or synthetic polypeptide fragments).
[0086] The term "halo" refers to any radical of fluorine, chlorine,
bromine or iodine. The term "alkyl" refers to a hydrocarbon chain
that may be a straight chain or branched chain, containing the
indicated number of carbon atoms. For example, C.sub.1-C.sub.10
indicates that the group may have from 1 to 10 (inclusive) carbon
atoms in it. In the absence of any numerical designation, "alkyl"
is a chain (straight or branched) having 1 to 20 (inclusive) carbon
atoms in it. The term "alkylene" refers to a divalent alkyl (i.e.,
--R--).
[0087] The term "alkenyl" refers to a hydrocarbon chain that may be
a straight chain or branched chain having one or more carbon-carbon
double bonds. The alkenyl moiety contains the indicated number of
carbon atoms. For example, C.sub.2-C.sub.10 indicates that the
group may have from 2 to 10 (inclusive) carbon atoms in it. The
term "lower alkenyl" refers to a C.sub.2-C.sub.8 alkenyl chain. In
the absence of any numerical designation, "alkenyl" is a chain
(straight or branched) having 2 to 20 (inclusive) carbon atoms in
it.
[0088] The term "alkynyl" refers to a hydrocarbon chain that may be
a straight chain or branched chain having one or more carbon-carbon
triple bonds. The alkynyl moiety contains the indicated number of
carbon atoms. For example, C.sub.2-C.sub.10 indicates that the
group may have from 2 to 10 (inclusive) carbon atoms in it. The
term "lower alkynyl" refers to a C.sub.2-C.sub.8 alkynyl chain. In
the absence of any numerical designation, "alkynyl" is a chain
(straight or branched) having 2 to 20 (inclusive) carbon atoms in
it.
[0089] The term "aryl" refers to a 6-carbon monocyclic or 10-carbon
bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of
each ring may be substituted by a substituent. Examples of aryl
groups include phenyl, naphthyl and the like. The term "arylalkyl"
or the term "aralkyl" refers to alkyl substituted with an aryl. The
term "arylalkoxy" refers to an alkoxy substituted with aryl.
[0090] The term "cycloalkyl" as employed herein includes saturated
and partially unsaturated cyclic hydrocarbon groups having 3 to 12
carbons, preferably 3 to 8 carbons, and more preferably 3 to 6
carbons, wherein the cycloalkyl group additionally may be
optionally substituted. Preferred cycloalkyl groups include,
without limitation, cyclopropyl, cyclobutyl, cyclopentyl,
cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and
cyclooctyl.
[0091] The term "heteroaryl" refers to an aromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be
substituted by a substituent. Examples of heteroaryl groups include
pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl,
thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the
like. The term "heteroarylalkyl" or the term "heteroaralkyl" refers
to an alkyl substituted with a heteroaryl. The term
"heteroarylalkoxy" refers to an alkoxy substituted with
heteroaryl.
[0092] The term "heterocyclyl" refers to a nonaromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2 or 3 atoms of each ring may be
substituted by a substituent. Examples of heterocyclyl groups
include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl,
tetrahydrofuranyl, and the like.
[0093] The term "substituents" refers to a group "substituted" on
an alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl group at
any atom of that group. Suitable substituents include, without
limitation, halo, hydroxy, mercapto, oxo, nitro, haloalkyl, alkyl,
alkaryl, aryl, aralkyl, alkoxy, thioalkoxy, aryloxy, amino,
alkoxycarbonyl, amido, carboxy, alkanesulfonyl, alkylcarbonyl, and
cyano groups.
[0094] Ranges provided herein are understood to be shorthand for
all of the values within the range. For example, a range of 1 to 50
is understood to include any number, combination of numbers, or
sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, or 50.
[0095] Unless specifically stated or obvious from context, as used
herein, the term "or" is understood to be inclusive.
[0096] Unless specifically stated or obvious from context, as used
herein, the terms "a", "an", and "the" are understood to be
singular or plural.
[0097] Unless specifically stated or obvious from context, as used
herein, the term "about" is understood as within a range of normal
tolerance in the art, for example within 2 standard deviations of
the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%,
5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated
value.
[0098] The recitation of a listing of chemical groups in any
definition of a variable herein includes definitions of that
variable as any single group or combination of listed groups. The
recitation of an embodiment for a variable or aspect herein
includes that embodiment as any single embodiment or in combination
with any other embodiments or portions thereof.
[0099] Any compositions or methods provided herein can be combined
with one or more of any of the other compositions and methods
provided herein.
Polypeptides
[0100] Described herein are modified peptides which exhibit
antiviral activity. It is believed that the modified peptides
exhibit antiviral activity via their ability to inhibit virus-cell
fusion by interfering with viral coat proteins. The modified
peptides of the invention may include a stabilized alpha helix
heptad repeat domain derived from a virus. Preferably, the alpha
helix heptad repeat domain is stabilized with hydrocarbon staples.
Suitable viral alpha helix heptad repeat domains can be derived
from any virus with an alpha helical domain (e.g., RSV, influenza,
parainfluenza, conronavirus, ebolavirus, HIV) that is directly or
indirectly involved in cell attachment or entry.
[0101] While not limited to any theory of operation, the following
model is proposed to explain the potent anti-viral activity of the
modified polypeptides described herein. When synthesized as
stabilized peptides, the modified polypeptides of the invention are
potent inhibitors of viral infection and fusion, likely by virtue
of their ability to form complexes with viral glycoproteins and
interfere with the fusogenic process; e.g., during the structural
transition of the viral protein from the native structure to the
fusogenic state. While not being bound by theory, it is believed
the modified peptides gain access to their respective binding sites
on the viral glycoprotein, and exert a disruptive influence which
inhibits fusion of the virus with the cell. The modified
polypeptides are particularly useful as a result of their increased
stability and efficacy.
[0102] In a first aspect, the invention is directed to a modified
polypeptide having a stabilized viral alpha helix heptad repeat
domain (e.g., HR-1, HR-2, HR-like or HR-analogs) or active fragment
thereof. The modified polypeptide may also comprise a chimera of an
HR domain. Suitable viral alpha helix heptad repeat domains can be
derived from any virus with an alpha helix domain that is directly
or indirectly involved in cell attachment or entry.
[0103] In another aspect, the invention is directed to a modified
polypeptide having a stabilized HIV gp41 heptad repeat domain
(e.g., heptad repeat domain 1 or 2 of HIV-1 or HIV-2). The amino
acid sequences of heptad repeat-1 and heptad repeat-2 domains are
well known in the art and include those represented by SEQ ID NO:2
and SEQ ID NO:1, respectively. In one embodiment, the heptad repeat
domain 1 is 30% or more identical to an amino acid sequence of SEQ
ID NO:2, SEQ ID NO:3 or SEQ ID NO:14 and forms an alpha helix.
Alternatively, the heptad repeat one domain of the modified
polypeptide may differ by more than 30% as long as the residues of
the interacting face are identical to those of SEQ ID NO:1 or 2 or
are conservative substitutions thereof. Methods for identifying the
interacting face residues of the heptad repeat are well known in
the art and described herein.
In another embodiment, the heptad repeat domain 2 is 30% or more
identical to an amino acid sequence of FIG. 4, FIG. 6 or SEQ ID
NO:1 and forms an alpha-helix. Alternatively, the heptad repeat two
domain of the modified polypeptide may differ by more than 30% as
long as the residues of the interacting face are identical to those
of SEQ ID NO:1 or 2 or have conservative substitutions thereof.
Methods for identifying the interacting face residues of the heptad
repeat are well known in the art and described herein.
[0104] In one embodiment, the modified polypeptide includes a
heptad repeat domain having the formula: a b c d e f g, wherein a
and d are hydrophobic amino acid residues and b, c, e, f and g are
any amino acid. Preferably, the formula is repeated in tandem two
or more times.
[0105] For example, in a further embodiment the heptad repeat
domain of the modified polypeptide has the formula: W(a), b, c,
W(d), e, f, g, I(a), b, c, Y(d), e, f, g, I(a), b, c, L(d), e, f,
g, S(a), b, c, Q(d), e, f, g, N(a), b, c, E(d), e, f, g, L(a), or
conservative amino acid substitutions thereof and wherein the b, c,
e, f and g can be any amino acid (e.g., residues 3-38 of SEQ ID NO:
44).
[0106] In a further, embodiment the heptad repeat domain of the
modified polypeptide has the formula: T(g), W(a), b, c, W(d), D(e),
R(f), g, I(a), b, c, Y(d), e, f, g, I(a), b, c, L(d), I(e), f, g,
a, Q(b), c, d, Q(e), E(f), K(g), a, E(b), c, d, L(e), f, E(g),
L(a), or conservative amino acid substitutions thereof and wherein
non-designated amino acids can be any amino acid (e.g., residues
2-38 of SEQ ID NO: 45).
[0107] In another embodiment, the modified polypeptide of the
invention is has the same amino acid residues, or conservative
substitutions thereof, of the interacting face of the amino acid
sequence of SEQ ID NO:1-14, FIG. 5 or FIG. 6. The "interacting
face" of the alpha helix are those amino acid residues which
interact with other amino acid residues in a coiled coil structure.
For example, in the HIV gp41 HR-2 domain the interacting face
includes the "a" and "d" position amino acids. (See FIGS. 4A and
9), while the interacting face of the HIV gp41 HR-1 domain includes
amino acids at positions e, g that interact with HR-2 and a, d that
engage in HR1-HR1 interactions (See FIG. 4A). Methods for
identifying heptad repeats and the interacting face residues are
well known in the art and described herein.
[0108] Preferably the alpha helix heptad repeat domain is
stabilized with a hydrocarbon staple (e.g., FIG. 10). Hydrocarbon
staples suitable for use with any of the modified polypeptides are
described herein and in U.S. Publication No. 2005/0250680, which is
incorporated by reference in its entirety. Hydrocarbon stapling
allows a polypeptide, predisposed to have an alpha-helical
secondary structure, to maintain its native alpha-helical
conformation and increase its stability and efficacy. In one
embodiment, the modified polypeptide has at least 10%, 20%, 30%,
35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% or more alpha helicity in
an aqueous solution as determined by circular dichroism. Assays for
determining circular dichroism are known in the art and described
herein.
[0109] The hydrocarbon stapled polypeptides include a tether
(linkage) between two amino acids, which tether significantly
enhances the alpha helical secondary structure of the polypeptide.
Generally, the tether extends across the length of one or two
helical turns (i.e., about 3.4 or about 7 amino acids).
Accordingly, amino acids positioned at i and i+3; i and i+4; or i
and i+7 are ideal candidates for chemical modification and
cross-linking. Thus, any of the amino acid residues of the modified
polypeptides of the invention may be tethered (e.g., cross-linked)
in conformity with the above. Suitable tethers are described herein
and in U.S. Patent Publication No. 2005/0250680.
[0110] In a further embodiment, the hydrocarbon staple(s) is
positioned so as to link a first amino acid (i) and a second amino
acid (i+3) which is 3 amino acids downstream of the first amino
acid. In another embodiment, the hydrocarbon staple links a first
amino acid (i) and a second amino acid (i+4) which is 4 amino acids
downstream of the first amino acid. In yet another embodiment, the
hydrocarbon staple links a first amino acid (i) and a second amino
acid (i+7) which is 7 amino acids downstream of the first amino
acid.
[0111] In yet a further embodiment, the modified polypeptides
include a heptad repeat domain with the sequence:
TABLE-US-00001 (SEQ ID NO: 15) BTWXEWDXEINNYTSLIHSL, (SEQ ID NO:
16) BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 17)
BTWBXWDRXINNYTSL, (SEQ ID NO: 18)
BTWBEWDREINNYTSLIHSLIEXSQNXQEKNEQELLE, (SEQ ID NO: 19)
BTWBXWDRXINNYTSLIHSLIEESQNQQEKNEQELLE, (SEQ ID NO: 20)
BTWBXWDRXINNYTSLIHSLIEXSQNXQEKNEQELLE, (SEQ ID NO: 21)
BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 22)
BTWBXWDRXINNYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 23)
BTWBEWDXEINXYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 24)
BTWBEWDREINXYTSXIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 25)
BTWBEWDREINNYTSXIHSXIEESQNQQXKNEXELLE, (SEQ ID NO: 26)
BTWBXWDRXINNYTSXIHSXIEESQNQQXKNEXELLE, (SEQ ID NO: 27)
YTSXIHSXIEESQNQQEKNEQELLELDKWASLWNWF, (SEQ ID NO: 28)
YTSLIXSLIXESQNQQEKNEQELLELDKWASLWNWF, (SEQ ID NO: 29)
YTSLIHSLIEXSQNXQEKNEQELLELDKWASLWNWF, (SEQ ID NO: 30)
YTSLIHSLIEESQNQQXKNEXELLELDKWASLWNWF, (SEQ ID NO: 31)
YTSLIHSLIEESQNQQEXNEQXLLELDKWASLWNWF, (SEQ ID NO: 32)
YTSLIHSLIEESQNQQEKNEQXLLEXDKWASLWNWF, (SEQ ID NO: 33)
YTSLIHSLIEESQNQQEKNEQELLELDXWASXWNWF, (SEQ ID NO: 34)
YTSLIHSLIEESQNQQEKNEQELLELDKWXSLWXWF, (SEQ ID NO: 35)
YTSLIHSLIEXSQNXQEKNEQXLLEXDKWASLWNWF, (SEQ ID NO: 36)
YTSXIHSXIEESQNQQEKNEQELLELDKWXSLWXWF, (SEQ ID NO: 37)
YTSLIHSLIEESQNQQXKNEXELLELDKWXSLWXWF, (SEQ ID NO: 38)
YTSXIHSXIEESQNQQXKNEXELLELDKWASLWNWF, (SEQ ID NO: 39)
YTSXIHSXIEESQNQQEKNEQELLELDXWASXWNWF, (SEQ ID NO: 40)
YTSLIHSLIEXSQNXQEKNEQELLELDXWASXWNWF, (SEQ ID NO: 41)
YTSXIHSXIEESQNQQXKNEXELLELDXWASXWNWF, (SEQ ID NO: 42)
BTWBXWDRXINNYTSLIHSLIEESQNQXEKNXQELLE, or (SEQ ID NO: 43)
BTWBXWDRXINNYTSLIHSLIEESQNXQEKXEQELLE;
wherein X is any amino acid and further identifies the amino acid
residues which are linked by a hydrocarbon staple, and B is
methionine or norleucine. The modified polypeptides will generally
have the structure of Formula (I), (II) or (III), as described
herein.
[0112] The invention is also, inter alia, directed to modified
polypeptides from other viruses with alpha helical domains that are
either directly or indirectly involved in the attachment and/or
fusion of a virus to a cell. For example, in one aspect the
invention is directed to a modified polypeptide having a stabilized
viral alpha helix (e.g., heptad repeat domain) that is derived from
respiratory syncytial virus. The alpha helix may include any alpha
helical domain derived from RSV that is involved in viral
infectivity. Suitable RSV alpha helix domains include those which
are 30% or more identical to
YTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST (SEQ ID NO:4);
FYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL (SEQ ID NO:5);
SGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKSYINNQ LLPI-(SEQ ID
NO: 11) or
[0113] PIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIM (SEQ
ID NO: 12); and form an alpha-helix.
[0114] Alternatively, the heptad repeat analog domain of the
modified polypeptide may differ by more than 30% as long as the
residues of the interacting face are identical to those of SEQ ID
NOs: 4, 5, 11 and 12 or are conservative substitutions thereof.
Methods for identifying the interacting face residues of the heptad
repeat analogs are well known in the art and described herein.
[0115] In yet another aspect, the invention is directed to a
modified polypeptide having a stabilized viral alpha helix heptad
repeat domain that is derived from a parainfluenza virus. Suitable
parainfluenza virus heptad repeat domains include those which are
30% or more identical to ALGVATSAQITAAVALVEAKQARSDIEKLKEAIR (SEQ ID
NO:6) and form an alpha-helix. Alternatively, the heptad repeat
domain of the modified parainfluenza polypeptide may differ by more
than 30% as long as the residues of the interacting face are
identical to those of SEQ ID NO: 6 or are conservative
substitutions thereof. Methods for identifying the interacting face
residues of the heptad repeat are well known in the art and
described herein.
[0116] In another aspect, the invention is directed to a modified
polypeptide having a stabilized viral alpha helix heptad repeat
domain derived from a paramyxovirus, orthomyxovirus coronavirus,
and a filovirus.
[0117] Coronavirus alpha helix heptad repeat domains are known in
the art and include those which have an amino acid sequence which
are 30% or more identical to
NVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQ
LSSNFGAISSVLNDILSRLDKVEAE (SEQ ID NO:7) or
TSPDVDFGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKY (SEQ ID NO:8) and
form an alpha-helix. Alternatively, the heptad repeat domain of the
modified coronavirus polypeptide may differ by more than 30% as
long as the residues of the interacting face are identical to those
of SEQ ID NOs: 7 and 8 or are conservative substitutions thereof.
Methods for identifying the interacting face residues of the heptad
repeat are well known in the art and described herein.
[0118] Similarly, filovirus alpha helix heptad repeat domains are
known in the art and include those that are 30% or more identical
to DGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLL (SEQ ID NO:9) or
DWTKNITDKIDQIIHDFVDKTLPD (SEQ ID NO:10) and form an alpha-helix.
Alternatively, the heptad repeat domain of the modified filovirus
polypeptide may differ by more than 30% as long as the residues of
the interacting face are identical to those of SEQ ID NO: 10 or are
conservative substitutions thereof. Methods for identifying the
interacting face residues of the heptad repeat are well known in
the art and described herein.
[0119] Influenza heptad repeat domains are also known in the art.
For example, a heptad repeat domain in Influenza A Virus (strain
A/Aichi/2/68) occurs at residues 379-436, 387-453, and 380-456.
Similarly, residues 383-471 were shown by Carr and Kim to be an
extended coiled coil when under acidic pH (Carr and Kim, 1993, Cell
73: 823-832).
[0120] The modified polypeptides of the invention will generally
include the structure of Formula (I), (II) or (III) provided
below.
[0121] Any of the modified polypeptides described herein can be
present in a composition (e.g., pharmaceutical composition) or kit.
In some embodiments of the invention, the composition or kit
comprises two or more modified polypeptides. For example, the
composition may include two or more modified polypeptides having a
stabilized HIV gp41 heptad repeat domain.
[0122] For clarity of discussion, the invention will be further
described primarily for HR-1 and HR-2 modified polypeptides of HIV.
However, the principles may be analogously applied to other
viruses, both enveloped and nonenveloped, and to other non-viral
organisms. As used herein the term "heptad repeat" includes HR-2
and HR-1 peptides.
HR-2 and HR-2-Peptides
[0123] The modified polypeptides of the invention include the HR-2
peptides (SEQ ID NO:1 and 13) which corresponds to amino acid
residues 638 to 673 and 626 and 662 respectively of gp160 from the
HIV-1 (SEQ ID NO:13), and has the 36 and 37 amino acid sequences,
respectively, of (reading from amino to carboxy terminus):
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO:1) and
MTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE (SEQ ID NO:13).
[0124] Other useful HR-2 polypeptides for use with the current
invention are described in U.S. Pat. No. 7,273,614, which is
incorporated herein by reference in its entirety.
[0125] In addition to the use of full-length HR-2 (SEQ ID NO:1 and
13) 36 and 37-mers and the corresponding sequences and variants
thereof found in the diversity of HIV-1 strains and mutants, the
peptides of the invention may include truncations of the HR-2 (SEQ
ID NO: 1 and 13) peptide, gp41 polypeptide sequences that flank the
HR-2 domain (ie immediately upstream or downstream sequences), or
chimeras which exhibit antifusogenic activity and antiviral
activity. Truncations of HR-2 (SEQ ID NO:1 and 13) peptides may
comprise peptides of between 3 and 36 amino acid residues, as shown
in FIGS. 5 and 6. Peptide sequences in this figure are listed from
amino (left) to carboxy (right) terminus.
[0126] The modified peptides of the invention also include
HR-2-like peptides. "HR-2-like" or "heptad repeat-like", as used
herein, refers to full-length and truncated and chimeric HR-2
polypeptides which contain one or more amino acid substitutions,
insertions and/or deletions as well as peptide sequences identified
or recognized by homology searching. Representative HR-2 like
polypeptides include those illustrated in FIG. 5 or FIG. 6. The
modified HR-2-like peptides of the invention may exhibit
antifusogenic or antiviral activity. In one embodiment, the heptad
repeat domain 2 is 30% or more identical to an amino acid sequence
of FIG. 5, FIG. 6, SEQ ID NO:1 or SEQ ID NO:13 and form an
alpha-helix. Alternatively, the heptad repeat domain 2 of the
modified polypeptide may differ by more than 30% as long as the
residues of the interacting face are identical to those of FIG. 5,
FIG. 6, SEQ ID NO:1 or SEQ ID NO:13 or are conservative
substitutions thereof. Methods for identifying the interacting face
residues of the heptad repeat are well known in the art and
described herein.
[0127] HIV-1 and HIV-2 enveloped proteins are structurally
distinct, but there exists a striking amino acid conservation
within the HR-2 regions of HIV-1 and HIV-2. The amino acid
conservation is of a periodic nature, suggesting some conservation
of structure and/or function. Therefore, one possible class of
amino acid substitutions would include those amino acid changes
which are predicted to stabilize the structure of the HR-2 peptides
of the invention. Utilizing the HR-2 and HR-2 analog sequences
described herein, the skilled artisan can readily compile HR-2
consensus sequences and ascertain from these, conserved amino acid
residues which would represent preferred amino acid
substitutions.
[0128] The amino acid substitutions may be of a conserved or
non-conserved nature. Conserved amino acid substitutions consist of
replacing one or more amino acids of the HR-2 (SEQ ID NO:1 or 13)
peptide sequence with amino acids of similar charge, size, and/or
hydrophobicity characteristics, such as, for example, a glutamic
acid (E) to aspartic acid (D), aspartic acid (D) to asparagine (N),
and glutamic acid (E) to glutamine (Q) amino acid substitution.
Non-conserved substitutions consist of replacing one or more amino
acids of the HR-2 peptide sequence with amino acids possessing
dissimilar charge, size, and/or hydrophobicity characteristics,
such as, for example, a glutamic acid (E) to valine (V)
substitution.
[0129] Amino acid insertions may consist of single amino acid
residues or stretches of residues. The insertions may be made at
the carboxy or amino terminal end of the full length or truncated
HR-2 peptides, as well as at a position internal to the peptide.
Such insertions will generally range from 2 to 15 amino acids in
length. It is contemplated that insertions made at either the
carboxy or amino terminus of the peptide of interest may be of a
broader size range, with about 2 to about 50 amino acids being
preferred. One or more such insertions may be introduced into the
full-length (SEQ ID NO:1 or 13) or truncated HR-2 polypeptides as
long as such insertions result in modified peptides that exhibit
antifusogenic or antiviral activity.
[0130] Preferred amino or carboxy terminal insertions are peptides
ranging from about 2 to about 50 amino acid residues in length,
corresponding to gp41 protein regions either amino to or carboxy to
the actual HR-2 gp41 amino acid sequence, respectively. Thus, a
preferred amino terminal or carboxy terminal amino acid insertion
would contain gp41 amino acid sequences found immediately amino to
or carboxy to the HR-2 region of the gp41 protein.
[0131] Deletions of full-length (SEQ ID NO:1 or 13) or truncated
HR-2 polypeptides are also within the scope of the invention. Such
deletions consist of the removal of one or more amino acids from
the HR-2 or HR-2-like peptide sequence, with the lower limit length
of the resulting peptide sequence being 4 to 6 amino acids. Such
deletions may involve a single contiguous or greater than one
discrete portion of the peptide sequences. One or more such
deletions may be introduced into full-length (SEQ ID NO: 1 or 13)
or truncated HR-2 polypeptides, as long as such deletions result in
peptides which may still exhibit antifusogenic or antiviral
activity.
HR-1 and HR-1-Peptides
[0132] Further, the modified peptides of the invention include
peptides having amino acid sequences corresponding to HR-1 analogs.
HR-1 includes 38- and 51-amino acid peptides which exhibits potent
antiviral activity, and corresponds to residues 553 to 590 and
542-592, respectively, of HIV-1 transmembrane (TM) gp41 protein, as
shown below:
TABLE-US-00002 (SEQ ID NO: 2)
NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLQDQ or (SEQ ID NO: 14)
RQLLSGIVQQQ NNLLRAIEAQQHLLQLTVWGIKQLQARI LAVERYLQDQQL.
[0133] In addition to the full-length HR-1 38-mer, the modified
peptides of the invention include truncations of the HR-1 peptide
which exhibit antifusogenic activity or antiviral activity.
Truncations of HR-1 peptides can be made in a similar manner as
those exemplified for the HR-2 peptides in FIG. 5 and FIG. 6.
[0134] The modified peptides of the invention also include
HR-1-like peptides. "HR-1-like" or "heptad-repeat like", as used
herein, refers to full-length and truncated HR-1 polypeptides which
contain one or more amino acid substitutions, insertions and/or
deletions and exhibiting antifusogenic or antiviral activity. In
one embodiment, the heptad repeat domain 1 is 30% or more identical
to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID
NO:14 and form an alpha-helix. Alternatively, the heptad repeat
domain 1 of the modified polypeptide may differ by more than 30% as
long as the residues of the interacting face are identical to those
of SEQ ID NOs 2, 3 or 14 or are conservative substitutions thereof.
Methods for identifying the interacting face residues of the heptad
repeat are well known in the art and described herein.
[0135] HIV-1 and HIV-2 enveloped proteins are structurally
distinct, but there exists a striking amino acid conservation
within the HR-1-corresponding regions of HIV-1 and HIV-2. The amino
acid conservation is of a periodic nature, suggesting some
conservation of structure and/or function. Therefore, one possible
class of amino acid substitutions would include those amino acid
changes which are predicted to stabilize the structure of the HR-1
peptides of the invention. Utilizing the HR-1 and HR-1 analog
sequences described herein, the skilled artisan can readily compile
HR-1 consensus sequences and ascertain from these, conserved amino
acid residues which would represent preferred amino acid
substitutions.
[0136] The amino acid substitutions may be of a conserved or
non-conserved nature. Conserved amino acid substitutions consist of
replacing one or more amino acids of the HR-1 peptide sequence with
amino acids of similar charge, size, and/or hydrophobicity
characteristics, such as, for example, a glutamic acid (E) to
aspartic acid (D), aspartic acid (D) to asparagine (N), and
glutamic acid (E) to glutamine (Q) amino acid substitution.
Non-conserved substitutions consist of replacing one or more amino
acids of the HR-1 peptide sequence with amino acids possessing
dissimilar charge, size, and/or hydrophobicity characteristics,
such as, for example, a glutamic acid (E) to valine (V)
substitution.
[0137] Amino acid insertions may consist of single amino acid
residues or stretches of residues. The insertions may be made at
the carboxy or amino terminal end of the full-length or truncated
HR-1 peptides, as well as at a position internal to the peptide.
Such insertions will generally range from 2 to 15 amino acids in
length. It is contemplated that insertions made at either the
carboxy or amino terminus of the peptide of interest may be of a
broader size range, with about 2 to about 50 amino acids being
preferred. One or more such insertions may be introduced into
full-length or truncated HR-1 polypeptides, as long as such
insertions result in modified peptides which may still exhibit
antifusogenic or antiviral activity.
[0138] Preferred amino or carboxy terminal insertions are peptides
ranging from about 2 to about 50 amino acid residues in length,
corresponding to gp41 protein regions either amino to or carboxy to
the actual HR-1 gp41 amino acid sequence, respectively. Thus, a
preferred amino terminal or carboxy terminal amino acid insertion
would contain gp41 amino acid sequences found immediately amino to
or carboxy to the HR-1 region of the gp41 protein.
[0139] Deletions of full-length or truncated HR-1 polypeptides are
also within the scope of the invention. Such deletions consist of
the removal of one or more amino acids from the HR-1 or HR-1-like
peptide sequence, with the lower limit length of the resulting
peptide sequence being 4 to 6 amino acids. Such deletions may
involve a single contiguous or greater than one discrete portion of
the peptide sequences. One or more such deletions may be introduced
into full-length or truncated HR-1 polypeptides, as long as such
deletions result in peptides which may still exhibit antifusogenic
or antiviral activity
HR-1 and HR-2 Analogs
[0140] Peptides corresponding to analogs of the full-length and
truncated HR-1 and HR-2 polypeptides, described, above, may be
found in other viruses. The term "HR-1 and HR-2-analogs", as used
herein, refers to a peptide which is recognized or identified as
having a heptad repeat-analog domain in a non-HIV virus. Methods
for identifying heptad repeat-analog polypeptides are known in the
art, for example, bioinformatics programs based on pairwise residue
correlations (e.g., on the world wide web at:
ch.embnet.org/software/COILS_form.html), which have the ability to
recognize coiled coils from protein sequences and model their
structures (See Lupas, A., et al. Science 1991. 252(5009); p.
1162-1164, which is herein incorporated by reference). Further,
such modified peptides exhibit antifusogenic or antiviral
activity.
[0141] Such HR-2 and HR-1 analogs may, for example, correspond to
peptide sequences present in transmembrane proteins of other
enveloped viruses. Such peptides may exhibit antifusogenic activity
or antiviral activity.
[0142] HR-2 analogs are peptides whose amino acid sequences are
comprised of the amino acid sequences of peptide regions of, for
example, other viruses that correspond to the gp41 peptide region
from which HR-2 (SEQ ID NO: 1) was derived. Such viruses may
include, but are not limited to, other HIV-1 isolates, HIV-2
isolates, SIV isolates, influenza, parainfluenza virus,
coronavirus, RSV, etc.
[0143] HR-1 analogs are peptides whose amino acid sequences are
comprised of the amino acid sequences of peptide regions of, for
example, other viruses that correspond to the gp41 peptide region
from which HR-1 (SEQ ID NO: 2) was derived. Such viruses may
include, but are not limited to, other HIV-1 isolates HIV-2
isolates, SIV isolates, parainfluenza virus, coronavirus, RSV,
etc.
[0144] HR-1 and HR-2 analogs or other heptad repeat polypeptides
include peptides whose amino acid sequences are comprised of the
amino acid sequences of peptide regions of, for example, other
viruses that correspond to the gp41 peptide region from which HR-1
(SEQ ID NO: 2 or SEQ ID NO:3) and HR-2 (SEQ ID NO:1) were derived.
These polypeptides include:
[0145] RSV heptad repeat domains which are 30% or more identical to
(SEQ ID NO:4), (SEQ ID NO:5), (SEQ ID NO: 11), or (SEQ ID NO: 12)
and form an alpha-helix.
[0146] Parainfluenza virus heptad repeat domains which are 30% or
more identical to (SEQ ID NO:6) and form an alpha-helix.
[0147] Coronavirus alpha helix heptad repeat domains which are 30%
or more identical to (SEQ ID NO:7) or (SEQ ID NO:8) and form an
alpha-helix.
[0148] Filovirus alpha helix heptad repeat domains which are 30% or
more identical to (SEQ ID NO:9) or (SEQ ID NO:10) and form an
alpha-helix.
[0149] The modified polypeptides of the invention also contemplate
the use of influenza virus heptad repeat domains.
[0150] Alternatively, the heptad repeat domains of the modified
polypeptides may differ by more than 30% as long as the residues of
the interacting face are identical to those of the interacting face
of the reference sequence or are conservative substitutions
thereof. Methods for identifying the interacting face residues of
the heptad repeat are well known in the art and described
herein.
[0151] Heptad repeats or heptad repeat--analogs are recognized or
identified, for example, by utilizing computer-assisted search
strategies known in the art. For example, bioinformatics programs
based on pairwise residue correlations (e.g., on the world wide web
at: ch.embnet.org/software/COILS_form.html), which have the ability
to recognize coiled coils from protein sequences and model their
structures (See Lupas, A., et al. Science 1991. 252(5009); p.
1162-1164, and U.S. Pat. No. 7,273,614 both of which are herein
incorporated by reference in its entirety. The search strategy can
identify additional peptide regions which are predicted to have
structural and/or amino acid sequence features similar to those of
HR-1 and/or HR-2.
Stabilization of Heptad Repeat Polypeptides
[0152] The modified polypeptides of the present invention have
stabilized (e.g., cross-linked) alpha helical domains. Preferable
the polypeptides are hydrocarbon-stapled. Hydrocarbon stapling is
described in U.S. Patent Publication No. 2005/0250680, which is
herein incorporated by reference in its entirety.
[0153] The peptide .alpha.-helix participates in critically
important protein interactions by presenting specific amino acid
residues in an ordered and precise arrangement over a relatively
large contact surface area (Chittenden, T., et al., Embo Journal,
1995. 14(22): p. 5589-5596; Kussie, P. H., et al. Science, 1996.
274(5289): p. 948-953; Ellenberger, T. E., et al., Cell, 1992.
71(7): p. 1223-1237). Alpha-helical domains are frequently
stabilized by scaffold sequences in the remainder of the protein,
which facilitate the preorganization of .alpha.-helical structure.
When taken out of context, .alpha.-helical peptide motifs can
unfold, leading to loss of biological activity. Critical challenges
is developing .alpha.-helical peptides include maintaining their
natural .alpha.-helical structure and preparing peptides that can
resist proteolytic, acid and thermal degradation, and thereby
remain intact in vivo.
[0154] Hydrocarbon stapling, refers to a process for stably
cross-linking a polypeptide via at least two amino acids that helps
to conformationally bestow the native secondary structure of that
polypeptide. Hydrocarbon stapling allows a polypeptide, predisposed
to have an alpha-helical secondary structure, to maintain its
native alpha-helical conformation. This secondary structure
increases resistance of the polypeptide to proteolytic cleavage and
heat, and also may increase hydrophobicity. Accordingly, the
hydrocarbon stapled (cross-linked) polypeptides described herein
have improved biological activity relative to a corresponding
non-hydrocarbon stapled (uncrosslinked) polypeptide. For example
the cross-linked polypeptide can include an alpha-helical domain of
an HIV polypeptide (e.g., HR-1/HR-2 domain), which can interfere
with HIV attachment, fusion with, and infection of a cell. In some
instances, the cross-linked polypeptide can be used to inhibit
virus entry into a cell. The cross-linked polypeptides described
herein can be used therapeutically, e.g., to treat HIV.
[0155] The hydrocarbon stapled polypeptides include a tether
(linkage) between two amino acids, which tether significantly
enhances the alpha helical secondary structure of the polypeptide.
Generally, the tether extends across the length of one or two
helical turns (i.e., about 3.4 or about 7 amino acids).
Accordingly, amino acids positioned at i and i+3; i and i+4; or i
and i+7 are ideal candidates for chemical modification and
cross-linking. Thus, for example, where a peptide has the sequence
. . . X1, X2, X3, X4, X5, X6, X7, X8, X9 . . . , cross-links
between X1 and X4, or between X1 and X5, or between X1 and X8 are
useful as are cross-links between X2 and X5, or between X2 and X6,
or between X2 and X9, etc. The use of multiple cross-links (e.g.,
2, 3, 4 or more) has also been achieved, compounding the benefits
of individual stapled adducts (e.g. improved helicity and activity;
improved helicity and thermal stability; improved helicity and acid
stability). Thus, the invention encompasses the incorporation of
more than one crosslink within the polypeptide sequence to either
further stabilize the sequence or facilitate the structural
stabilization, proteolytic resistance, thermal stability, acid
stability, and biological activity enhancement of longer
polypeptide stretches.
[0156] In one embodiment, the modified polypeptides of the
invention have the formula (I),
##STR00001##
wherein; each R.sub.1 and R.sub.2 are independently H or a C.sub.1
to C.sub.10 alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl,
heteroarylalkyl, or heterocyclylalkyl; R.sub.3 is alkyl, alkenyl,
alkynyl; [R.sub.4--K--R.sub.4].sub.n; each of which is substituted
with 0-6 R.sub.5; R.sub.4 is alkyl, alkenyl, or alkynyl; R.sub.5 is
halo, alkyl, OR.sub.6, N(R.sub.6).sub.2, SR.sub.6, SOR.sub.6,
SO.sub.2R.sub.6, CO.sub.2R.sub.6, R.sub.6, a fluorescent moiety, or
a radioisotope; K is O, S, SO, SO.sub.2, CO, CO.sub.2, CONR.sub.6,
or
##STR00002##
R.sub.6 is H, alkyl, or a therapeutic agent; n is an integer from
1-4; x is an integer from 2-10; each y is independently an integer
from 0-100; z is an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7,
8, 9, 10); and each Xaa is independently an amino acid. The
modified polypeptides may include an amino acid sequence which
forms an alpha-helix and is 30% or more identical to an amino acid
sequence of SEQ ID NO:1-14, FIG. 5, FIG. 6,
TABLE-US-00003 (SEQ ID NO: 15) BTWXEWDXEINNYTSLIHSL, (SEQ ID NO:
16) BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 17)
BTWBXWDRXINNYTSL, (SEQ ID NO: 18)
BTWBEWDREINNYTSLIHSLIEXSQNXQEKNEQELLE, (SEQ ID NO: 19)
BTWBXWDRXINNYTSLIHSLIEESQNQQEKNEQELLE, (SEQ ID NO: 20)
BTWBXWDRXINNYTSLIHSLIEXSQNXQEKNEQELLE, (SEQ ID NO: 21)
BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 22)
BTWBXWDRXINNYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 23)
BTWBEWDXEINXYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 24)
BTWBEWDREINXYTSXIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 25)
BTWBEWDREINNYTSXIHSXIEESQNQQXKNEXELLE, (SEQ ID NO: 26)
BTWBXWDRXINNYTSXIHSXIEESQNQQXKNEXELLE, (SEQ ID NO: 27)
YTSXIHSXIEESQNQQEKNEQELLELDKWASLWNWF, (SEQ ID NO: 28)
YTSLIXSLIXESQNQQEKNEQELLELDKWASLWNWF, (SEQ ID NO: 29)
YTSLIHSLIEXSQNXQEKNEQELLELDKWASLWNWF, (SEQ ID NO: 30)
YTSLIHSLIEESQNQQXKNEXELLELDKWASLWNWF, (SEQ ID NO: 31)
YTSLIHSLIEESQNQQEXNEQXLLELDKWASLWNWF, (SEQ ID NO: 32)
YTSLIHSLIEESQNQQEKNEQXLLEXDKWASLWNWF, (SEQ ID NO: 33)
YTSLIHSLIEESQNQQEKNEQELLELDXWASXWNWF, (SEQ ID NO: 34)
YTSLIHSLIEESQNQQEKNEQELLELDKWXSLWXWF, (SEQ ID NO: 35)
YTSLIHSLIEXSQNXQEKNEQXLLEXDKWASLWNWF, (SEQ ID NO: 36)
YTSXIHSXIEESQNQQEKNEQELLELDKWXSLWXWF, (SEQ ID NO: 37)
YTSLIHSLIEESQNQQXKNEXELLELDKWXSLWXWF, (SEQ ID NO: 38)
YTSXIHSXIEESQNQQXKNEXELLELDKWASLWNWF, (SEQ ID NO: 39)
YTSXIHSXIEESQNQQEKNEQELLELDXWASXWNWF, (SEQ ID NO: 40)
YTSLIHSLIEXSQNXQEKNEQELLELDXWASXWNWF, (SEQ ID NO: 41)
YTSXIHSXIEESQNQQXKNEXELLELDXWASXWNWF, (SEQ ID NO: 42)
BTWBXWDRXINNYTSLIHSLIEESQNQXEKNXQELLE, or (SEQ ID NO: 43)
BTWBXWDRXINNYTSLIHSLIEESQNXQEKXEQELLE;
wherein X is any amino acid and further identifies the amino acid
residues which are linked by a hydrocarbon staple, and B is
methionine or norleucine.
[0157] The tether can include an alkyl, alkenyl, or alkynyl moiety
(e.g., C.sub.5, C.sub.8 or C.sub.11 alkyl or a C.sub.5, C.sub.8 or
C.sub.11 alkenyl, or C.sub.5, C.sub.8 or C.sub.11 alkynyl). The
tethered amino acid can be alpha disubstituted (e.g.,
C.sub.1-C.sub.3 or methyl).
[0158] In some instances, x is 2, 3, or 6.
[0159] In some instances, each y is independently an integer
between 3 and 15.
[0160] In some instances each y is independently an integer between
1 and 15.
[0161] In some instances, R.sub.1 and R.sub.2 are each
independently H or C.sub.1-C.sub.6 alkyl.
[0162] In some instances, R.sub.1 and R.sub.2 are each
independently C.sub.1-C.sub.3 alkyl.
[0163] In some instances, at least one of R.sub.1 and R.sub.2 are
methyl. For example R.sub.1 and R.sub.2 are both methyl.
[0164] In some instances R.sub.3 is alkyl (e.g., C.sub.8 alkyl) and
x is 3.
[0165] In some instances, R.sub.3 is C.sub.11 alkyl and x is 6.
[0166] In some instances, R.sub.3 is alkenyl (e.g., C.sub.8
alkenyl) and x is 3.
[0167] In some instances x is 6 and R.sub.3 is C.sub.11
alkenyl.
[0168] In some instances, R.sub.3 is a straight chain alkyl,
alkenyl, or alkynyl.
[0169] In some instances R.sub.3 is
--CH.sub.2--CH.sub.2--CH.sub.2--CH.dbd.CH--CH.sub.2--CH.sub.2--CH.sub.2---
.
[0170] In certain embodiments the two alpha, alpha disubstituted
stereocenters are both in the R configuration or S configuration
(e.g., i, i+4 cross-link), or one stereocenter is R and the other
is S (e.g., i, i+7 cross-link). Thus, where formula I is depicted
as
##STR00003##
the C' and C'' disubstituted stereocenters can both be in the R
configuration or they can both be in the S configuration, for
example when X is 3. When x is 6, the C' disubstituted stereocenter
is in the R configuration and the C'' disubstituted stereocenter is
in the S configuration. The R.sub.3 double bond may be in the E or
Z stereochemical configuration.
[0171] In some instances R.sub.3 is [R.sub.4--K--R.sub.4].sub.n;
and R4 is a straight chain alkyl, alkenyl, or alkynyl.
[0172] In some embodiments the modified polypeptide comprises at
least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 45, 50, or more contiguous amino acids of a heptad
repeat or heptad repeat like domain, e.g., a HIV-1 HR-1 or HR-2
domain. Each [Xaa]y is a peptide that can independently comprise at
least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25
or more contiguous amino acids of a heptad repeat or heptad repeat
like domain, e.g., a HIV-1 HR-1 or HR-2 domain, e.g., a polypeptide
depicted in any of FIGS. 5 and 6. [Xaa].sub.x is a peptide that can
comprise 3 or 6 contiguous amino acids of acids of a heptad repeat
or heptad repeat like domain, e.g., a HIV-1 HR-1 domain or HR-2,
e.g., a polypeptide having the amino acid sequence of SEQ ID
NO:1-14 or FIG. 5 or 6.
[0173] The modified polypeptide can comprise 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50
contiguous amino acids of acids of a heptad repeat or heptad repeat
like domain, e.g., a HIV-1 HR-1 domain or HR-2, e.g., a polypeptide
having the amino acid sequence of SEQ ID NO:1-14 or FIG. 5 or 6,
wherein two amino acids that are separated by two, three, or six
amino acids are replaced by amino acid substitutes that are linked
via R.sub.3. Thus, at least two amino acids can be replaced by
tethered amino acids or tethered amino acid substitutes. Thus,
where formula (I) is depicted as
##STR00004##
[Xaa].sub.y' and [Xaa].sub.y'' can each comprise contiguous
polypeptide sequences from the same or different heptad repeat or
heptad repeat like domains.
[0174] The invention features cross-linked polypeptides comprising
10 (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,
35, 40, 45, 50 or more) contiguous amino acids of a heptad repeat
or heptad repeat like domain, e.g., a HIV-1 HR-1 domain or HR-2,
e.g., a polypeptide having the amino acid sequence of SEQ ID
NO:1-14 or FIG. 5 or 6, wherein the alpha carbons of two amino
acids that are separated by two, three, or six amino acids are
linked via R.sub.3, one of the two alpha carbons is substituted by
R.sub.1 and the other is substituted by R.sub.2 and each is linked
via peptide bonds to additional amino acids.
[0175] In another embodiment, the modified polypeptides of the
invention have the formula (II),
##STR00005##
wherein each R.sub.1 and R.sub.2 are independently H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkylalkyl; heteroarylalkyl; or
heterocyclylalkyl; each n is independently an integer from 1-15; x
is 2, 3, or 6 each y is independently an integer from 0-100; z is
an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); each
Xaa is independently an amino acid.
[0176] The modified polypeptide forms an alpha-helix and can have
an amino acid sequence which is 30% or more identical to an amino
acid sequence of SEQ ID NO:1-14, FIG. 5, FIG. 6,
the modified polypeptides include a heptad repeat domain with the
sequence:
TABLE-US-00004 (SEQ ID NO: 15) BTWXEWDXEINNYTSLIHSL, (SEQ ID NO:
16) BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 17)
BTWBXWDRXINNYTSL, (SEQ ID NO: 18)
BTWBEWDREINNYTSLIHSLIEXSQNXQEKNEQELLE, (SEQ ID NO: 19)
BTWBXWDRXINNYTSLIHSLIEESQNQQEKNEQELLE, (SEQ ID NO: 20)
BTWBXWDRXINNYTSLIHSLIEXSQNXQEKNEQELLE, (SEQ ID NO: 21)
BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 22)
BTWBXWDRXINNYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 23)
BTWBEWDXEINXYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 24)
BTWBEWDREINXYTSXIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 25)
BTWBEWDREINNYTSXIHSXIEESQNQQXKNEXELLE, (SEQ ID NO: 26)
BTWBXWDRXINNYTSXIHSXIEESQNQQXKNEXELLE, (SEQ ID NO: 27)
YTSXIHSXIEESQNQQEKNEQELLELDKWASLWNWF, (SEQ ID NO: 28)
YTSLIXSLIXESQNQQEKNEQELLELDKWASLWNWF, (SEQ ID NO: 29)
YTSLIHSLIEXSQNXQEKNEQELLELDKWASLWNWF, (SEQ ID NO: 30)
YTSLIHSLIEESQNQQXKNEXELLELDKWASLWNWF, (SEQ ID NO: 31)
YTSLIHSLIEESQNQQEXNEQXLLELDKWASLWNWF, (SEQ ID NO: 32)
YTSLIHSLIEESQNQQEKNEQXLLEXDKWASLWNWF, (SEQ ID NO: 33)
YTSLIHSLIEESQNQQEKNEQELLELDXWASXWNWF, (SEQ ID NO: 34)
YTSLIHSLIEESQNQQEKNEQELLELDKWXSLWXWF, (SEQ ID NO: 35)
YTSLIHSLIEXSQNXQEKNEQXLLEXDKWASLWNWF, (SEQ ID NO: 36)
YTSXIHSXIEESQNQQEKNEQELLELDKWXSLWXWF, (SEQ ID NO: 37)
YTSLIHSLIEESQNQQXKNEXELLELDKWXSLWXWF, (SEQ ID NO: 38)
YTSXIHSXIEESQNQQXKNEXELLELDKWASLWNWF, (SEQ ID NO: 39)
YTSXIHSXIEESQNQQEKNEQELLELDXWASXWNWF, (SEQ ID NO: 40)
YTSLIHSLIEXSQNXQEKNEQELLELDXWASXWNWF, (SEQ ID NO: 41)
YTSXIHSXIEESQNQQXKNEXELLELDXWASXWNWF, (SEQ ID NO: 42)
BTWBXWDRXINNYTSLIHSLIEESQNQXEKNXQELLE, or (SEQ ID NO: 43)
BTWBXWDRXINNYTSLIHSLIEESQNXQEKXEQELLE;
wherein X is any amino acid and further identifies the amino acid
residues which are linked by a hydrocarbon staple, and B is
methionine or norleucine.
[0177] In still another embodiment, the modified polypeptides of
the invention have the formula (III),
##STR00006##
wherein; each R.sub.1 and R.sub.2 are independently H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or
heterocyclylalkyl; R.sub.3 is alkyl, alkenyl, alkynyl;
[R.sub.4--K--R.sub.4].sub.n or a naturally occurring amino acid
side chain; each of which is substituted with 0-6 R.sub.5; R.sub.4
is alkyl, alkenyl, or alkynyl; R.sub.5 is halo, alkyl, OR.sub.6,
N(R.sub.6).sub.2, SR.sub.6, SOR.sub.6, SO.sub.2R.sub.6,
CO.sub.2R.sub.6, R.sub.6, a fluorescent moiety, or a radioisotope;
K is O, S, SO, SO.sub.2, CO, CO.sub.2, CONK.sub.6, or
##STR00007##
R.sub.6 is H, alkyl, or a therapeutic agent; R.sub.7 is alkyl,
alkenyl, alkynyl; [R.sub.4--K--R.sub.4].sub.n or an naturally
occurring amino acid side chain; each of which is substituted with
0-6 R.sub.5; n is an integer from 1-4; x is an integer from 2-10;
each y is independently an integer from 0-100; z is an integer from
1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); and each Xaa is
independently an amino acid;
[0178] The polypeptide forms and alpha-helix and includes an amino
acid sequence which is about 30% or more identical to an amino acid
sequence of SEQ ID NO:1-14, FIG. 5, FIG. 6 or
the modified polypeptides include a heptad repeat domain with the
sequence:
TABLE-US-00005 (SEQ ID NO: 15) BTWXEWDXEINNYTSLIHSL, (SEQ ID NO:
16) BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 17)
BTWBXWDRXINNYTSL, (SEQ ID NO: 18)
BTWBEWDREINNYTSLIHSLIEXSQNXQEKNEQELLE, (SEQ ID NO: 19)
BTWBXWDRXINNYTSLIHSLIEESQNQQEKNEQELLE, (SEQ ID NO: 20)
BTWBXWDRXINNYTSLIHSLIEXSQNXQEKNEQELLE, (SEQ ID NO: 21)
BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 22)
BTWBXWDRXINNYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 23)
BTWBEWDXEINXYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 24)
BTWBEWDREINXYTSXIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 25)
BTWBEWDREINNYTSXIHSXIEESQNQQXKNEXELLE, (SEQ ID NO: 26)
BTWBXWDRXINNYTSXIHSXIEESQNQQXKNEXELLE, (SEQ ID NO: 27)
YTSXIHSXIEESQNQQEKNEQELLELDKWASLWNWF, (SEQ ID NO: 28)
YTSLIXSLIXESQNQQEKNEQELLELDKWASLWNWF, (SEQ ID NO: 29)
YTSLIHSLIEXSQNXQEKNEQELLELDKWASLWNWF, (SEQ ID NO: 30)
YTSLIHSLIEESQNQQXKNEXELLELDKWASLWNWF, (SEQ ID NO: 31)
YTSLIHSLIEESQNQQEXNEQXLLELDKWASLWNWF, (SEQ ID NO: 32)
YTSLIHSLIEESQNQQEKNEQXLLEXDKWASLWNWF, (SEQ ID NO: 33)
YTSLIHSLIEESQNQQEKNEQELLELDXWASXWNWF, (SEQ ID NO: 34)
YTSLIHSLIEESQNQQEKNEQELLELDKWXSLWXWF, (SEQ ID NO: 35)
YTSLIHSLIEXSQNXQEKNEQXLLEXDKWASLWNWF, (SEQ ID NO: 36)
YTSXIHSXIEESQNQQEKNEQELLELDKWXSLWXWF, (SEQ ID NO: 37)
YTSLIHSLIEESQNQQXKNEXELLELDKWXSLWXWF, (SEQ ID NO: 38)
YTSXIHSXIEESQNQQXKNEXELLELDKWASLWNWF, (SEQ ID NO: 39)
YTSXIHSXIEESQNQQEKNEQELLELDXWASXWNWF, (SEQ ID NO: 40)
YTSLIHSLIEXSQNXQEKNEQELLELDXWASXWNWF, (SEQ ID NO: 41)
YTSXIHSXIEESQNQQXKNEXELLELDXWASXWNWF, (SEQ ID NO: 42)
BTWBXWDRXINNYTSLIHSLIEESQNQXEKNXQELLE, or (SEQ ID NO: 43)
BTWBXWDRXINNYTSLIHSLIEESQNXQEKXEQELLE;
wherein X is any amino acid and further identifies the amino acid
residues which are linked by a hydrocarbon staple, and B is
methionine or norleucine.
[0179] While hydrocarbon tethers have been described, other tethers
are also envisioned. For example, the tether can include one or
more of an ether, thioether, ester, amine, or amide moiety. In some
cases, a naturally occurring amino acid side chain can be
incorporated into the tether. For example, a tether can be coupled
with a functional group such as the hydroxyl in serine, the thiol
in cysteine, the primary amine in lysine, the acid in aspartate or
glutamate, or the amide in asparagine or glutamine. Accordingly, it
is possible to create a tether using naturally occurring amino
acids rather than using a tether that is made by coupling two
non-naturally occurring amino acids. It is also possible to use a
single non-naturally occurring amino acid together with a naturally
occurring amino acid.
[0180] It is further envisioned that the length of the tether can
be varied. For instance, a shorter length of tether can be used
where it is desirable to provide a relatively high degree of
constraint on the secondary alpha-helical structure, whereas, in
some instances, it is desirable to provide less constraint on the
secondary alpha-helical structure, and thus a longer tether may be
desired.
[0181] Additionally, while examples of tethers spanning from amino
acids i to i+3, i to i+4; and i to i+7 have been described in order
to provide a tether that is primarily on a single face of the alpha
helix, the tethers can be synthesized to span any combinations of
numbers of amino acids.
[0182] As can be appreciated by the skilled artisan, methods of
synthesizing the compounds of the described herein will be evident
to those of ordinary skill in the art. Additionally, the various
synthetic steps may be performed in an alternate sequence or order
to give the desired compounds. Synthetic chemistry transformations
and protecting group methodologies (protection and deprotection)
useful in synthesizing the compounds described herein are known in
the art and include, for example, those such as described in R.
Larock, Comprehensive Organic Transformations, VCH Publishers
(1989); T. W. Greene and P. G. M. Wuts, Protective Groups in
Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser
and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis,
John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of
Reagents for Organic Synthesis, John Wiley and Sons (1995), and
subsequent editions thereof.
Synthesis of Peptides
[0183] The peptides of this invention can be made by chemical
synthesis methods, which are well known to the ordinarily skilled
artisan and described herein. See, for example, Fields et al.,
Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H.
Freeman & Co., New York, N.Y., 1992, p. 77. Hence, peptides can
be synthesized using the automated Merrifield techniques of solid
phase synthesis with the alpha-NH.sub.2 protected by either t-Boc
or F-moc chemistry using side chain protected amino acids on, for
example, an Applied Biosystems Peptide Synthesizer Model 430A or
431 or the AAPPTEC multichannel synthesizer APEX 396.
[0184] One manner of making of the peptides described herein is
using solid phase peptide synthesis (SPPS). The C-terminal amino
acid is attached to a cross-linked polystyrene resin via an acid
labile bond with a linker molecule. This resin is insoluble in the
solvents used for synthesis, making it relatively simple and fast
to wash away excess reagents and by-products. The N-terminus is
protected with the Fmoc group, which is stable in acid, but
removable by base. Any side chain functional groups are protected
with base stable, acid labile groups.
[0185] Longer peptides could be made by conjoining individual
synthetic peptides using native chemical ligation. Alternatively,
the longer synthetic peptides can be synthesized by well known
recombinant DNA techniques. Such techniques are provided in
well-known standard manuals with detailed protocols. To construct a
gene encoding a peptide of this invention, the amino acid sequence
is reverse translated to obtain a nucleic acid sequence encoding
the amino acid sequence, preferably with codons that are optimum
for the organism in which the gene is to be expressed. Next, a
synthetic gene is made, typically by synthesizing oligonucleotides
which encode the peptide and any regulatory elements, if necessary.
The synthetic gene is inserted in a suitable cloning vector and
transfected into a host cell. Furthermore, the host cell is
engineered so as to be able to incorporate the non-natural amino
acids for the hydrocarbon staple. The peptide is then expressed
under suitable conditions appropriate for the selected expression
system and host. See Liu et al. Proc. Nat. Acad. Sci (USA),
94:10092-10097 (1997). The peptide is purified and characterized by
standard methods.
[0186] The peptides can be made in a high-throughput, combinatorial
fashion, e.g., using a high-throughput polychannel combinatorial
synthesizer available from Advanced Chemtech.
Assaying Anti-Viral Activity
[0187] Described herein, are methods for evaluating the ability of
a compound, such as the peptides of the invention, to inhibit
membrane fusion and/or exhibit anti-viral activity both in vitro
and in vivo. Specifically, such assays are described below and in
Examples 4 and 5. Additional assays for evaluating anti-vial
activity are well known to those with ordinary skill in the
art.
[0188] The antiviral activity exhibited by the peptides of the
invention may be measured, for example, by easily performed in
vitro assays, such as those described herein and known by those of
ordinary skill in the art, which can test the peptides' ability to
inhibit syncytia formation, or their ability to inhibit infection
by cell-free virus (Madani, N., et al., Journal of Virology, 2007.
81(2): p. 532-538; Si, Z. H., M. Cayabyab, and J. Sodroski, Journal
of Virology, 2001. 75(9): p. 4208-4218; Si, Z. H., et al., PNAS
USA, 2004. 101(14): p. 5036-5041).
[0189] Using these assays, such parameters as the relative
antiviral activity of the peptides exhibit against a given strain
of virus and/or the strain specific inhibitory activity of the
peptide can be determined.
[0190] Assays to test a peptide's antiviral capabilities are
contemplated with the present invention. Taking HIV as an example,
a reverse transcriptase (RT) assay may be utilized to test the
peptides' ability to inhibit infection of CD-4.sup.+ cells by
cell-free HIV. Such an assay may comprise culturing an appropriate
concentration (i.e., Tissue Culture Infectious Dose 50) of virus
and CD-4+ cells in the presence of the peptide to be tested.
Culture conditions well known to those in the art are used. A range
of peptide concentrations may be used, in addition to a control
culture wherein no peptide has been added. After incubation for an
appropriate period (e.g., 7 days) of culturing, a cell-free
supernatant is prepared, using standard procedures, and tested for
the present of RT activity as a measure of successful infection.
The RT activity may be tested using standard techniques such as
those described by, for example, Goff et al. (Goff, S. et al.,
1981, J. Virol. 38:239-248) and/or Willey et al. (Willey, R. et
al., 1988, J. Virol. 62:139-147). These references are incorporated
herein by reference in their entirety.
[0191] Standard methods which are well-known to those of skill in
the art may be utilized for assaying non-retroviral activity. See,
for example, Pringle et al. (Pringle, C. R. et al., 1985, J.
Medical Virology 17:377-386) for a discussion of respiratory
syncytial virus and parainfluenza virus activity assay techniques.
Further, see, for example, "Zinsser Microbiology", 1988, Joklik, W.
K. et al., eds., Appleton & Lange, Norwalk, Conn., 19th ed.,
for a general review of such techniques. These references are
incorporated by reference herein in their entirety.
[0192] It is known that HIV positive patients who respond to
initial treatment with enfuvirtide, may ultimately develop a viral
rebound that typically occurs within a maximum of 80 weeks.
Resistance to enfuvirtide derives from mutations within the HR-1
region of gp41, although some genetic changes are found in the HR-2
domain (Xu, L., et al., Antimicrobial Agents and Chemotherapy,
2005. 49(3): p. 1113-1119; Perez-Alvarez, L., et al. Journal of
Medical Virology, 2006. 78(2): p. 141-147). These mutations, such
as I37V, V38A/E/M, Q39R, Q40H, N42T/Q/H, N43D/Q, are only found in
enfuvirtide-experienced patients (Poveda, E., et al., Journal of
Medical Virology, 2004. 74(1): p. 21-28; Melby, T., et al., Aids
Research and Human Retroviruses, 2006. 22(5): p. 375-385; Sista, P.
R., et al., Aids, 2004. 18(13): p. 1787-1794; Wei, X. P., et al.,
Antimicrobial Agents and Chemotherapy, 2002. 46(6): p.
1896-1905).
[0193] Modified polypeptides of the invention can be developed
which are able to inhibit these enfuvirtide resistant HIV strains.
One suitable method for assessing the ability of the modified
polypeptides to treat these enfuvirtide resistant HIV strains is a
five-helix bundle assay as described in Root, M. J., M. S. Kay, and
P. S. Kim, Science, 2001. 291(5505): p. 884-888.
[0194] Briefly, the five-helix bundle assay would include
polypeptides that incorporate resistance mutations. FITC-labeled
SAH-gp41 compounds can then be screened against these mutant
five-helix bundle proteins to determine if any native SAH-gp41
compounds retain activity despite HR domain mutations. The FITC
labeled mutants SAH-gp41 (mSAH-gp41) compounds can be screened for
binding affinity to mutant five-helix bundle proteins and for
suppression of HIV infectivity using primary resistance
strains.
[0195] In another aspect, the modified polypeptides of the
invention can be used to monitor the evolution of resistance in HIV
isolates. To explore the evolution of potential resistance to
SAH-gp41 compounds, HIV strains can be incubated in the presence of
increasing concentrations of lead SAH-gp41 compounds in a cell
culture. Resistant strains can be genotyped to monitor the
evolution of resistance. (See Dwyer et al. Proc. Natl. Acad. Sci.,
104:12772 (2007)). Because resistance to one modified polypeptide
of the invention may not affect susceptibility to other variants,
(Ray, N., et al., Journal of Virology, 2007. 81(7): p. 3240-3250)
it is contemplated that treatment may include a combination of
different SAH-gp41 polypeptides that are able to treat resistant
strains of HIV.
[0196] In vivo assays may also be utilized to test, for example,
the antiviral activity of the peptides of the invention. To test
for anti-HIV activity, for example, the in vivo model described in
Barnett et al. (Barnett, S. W. et al., 1994, Science 266:642-646)
may be used.
[0197] Additionally, anti-RSV activity can be assayed in vitro
using the RSV plaque assay and in vivo via well known mouse models
(Kong et al., Virology J. 2(1):3 (2005). For example, RSV can be
administered intranasally to mice of various inbred strains. Virus
replicates in lungs of all strains, but the highest titers are
obtained in P/N, C57L/N and DBA/2N mice. Infection of BALB/c mice
produces an asymptomatic bronchiolitis characterized by lymphocytic
infiltrates and pulmonary virus titers of 104 to 10.sup.5 pfu/g of
lung tissue (Taylor, G. et al., 1984, Infect. Immun. 43:649-655).
Cotton rat models of RSV are also well known. Virus replicates to
high titer in the nose and lungs of the cotton rat but produces few
if any signs of inflammation. Additional assays for evaluating the
effectiveness of the modified viral polypeptides are well known to
those of ordinary skill in the art.
Pharmaceutical Compositions and Routes of Administration
[0198] As used herein, the compounds of this invention (e.g., the
modified polypeptides described herein), are defined to include
pharmaceutically acceptable derivatives or prodrugs thereof. A
"pharmaceutically acceptable derivative or prodrug" means any
pharmaceutically acceptable salt, ester, salt of an ester, or other
derivative of a compound of this invention which, upon
administration to a recipient, is capable of providing (directly or
indirectly) a compound of this invention. Particularly favored
derivatives and prodrugs are those that increase the
bioavailability of the compounds of this invention when such
compounds are administered to a mammal (e.g., by allowing an orally
administered compound to be more readily absorbed into the blood)
or which enhance delivery of the parent compound to a biological
compartment (e.g., the brain or lymphatic system) relative to the
parent species. Preferred prodrugs include derivatives where a
group which enhances aqueous solubility or active transport through
the gut membrane is appended to the structure of formulae described
herein.
[0199] The compounds of this invention may be modified by appending
appropriate functionalities to enhance selective biological
properties. Such modifications are known in the art and include
those which increase biological penetration into a given biological
compartment (e.g., blood, lymphatic system, central nervous
system), increase oral availability, increase solubility to allow
administration by injection, alter metabolism and alter rate of
excretion. Pharmaceutically acceptable salts of the compounds of
this invention include those derived from pharmaceutically
acceptable inorganic and organic acids and bases. Examples of
suitable acid salts include acetate, adipate, benzoate,
benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate,
formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate,
hydrochloride, hydrobromide, hydroiodide, lactate, maleate,
malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate,
nitrate, palmoate, phosphate, picrate, pivalate, propionate,
salicylate, succinate, sulfate, tartrate, tosylate and undecanoate.
Salts derived from appropriate bases include alkali metal (e.g.,
sodium), alkaline earth metal (e.g., magnesium), ammonium and
N-(alkyl).sub.4+ salts. This invention also envisions the
quaternization of any basic nitrogen-containing groups of the
compounds disclosed herein. Water or oil-soluble or dispersible
products may be obtained by such quaternization.
[0200] The compounds of the invention can, for example, be
administered by injection, intravenously, intraarterially,
subdermally, intraperitoneally, intramuscularly, or subcutaneously;
or orally, buccally, nasally, transmucosally, intravaginally,
cervically, topically, in an ophthalmic preparation, or by
inhalation, with a dosage ranging from about 0.001 to about 100
mg/kg of body weight, or according to the requirements of the
particular drug and more preferably from 0.5-10 mg/kg of body
weight. The methods herein contemplate administration of an
effective amount of compound or compound composition to achieve the
desired or stated effect. Typically, the pharmaceutical
compositions of this invention will be administered from about 1 to
about 6 times per day or alternatively, as a continuous infusion,
or for example as an intravaginal foam or formulated for a cervical
ring if used singly or in combination with a contraceptive. Such
administration can be used as a chronic or acute therapy. The
amount of active ingredient that may be combined with the carrier
materials to produce a single dosage form will vary depending upon
the host treated and the particular mode of administration. A
typical preparation will contain from about 1% to about 95% active
compound (w/w). Alternatively, such preparations contain from about
20% to about 80% active compound.
[0201] Lower or higher doses than those recited above may be
required. Specific dosage and treatment regimens for any particular
patient will depend upon a variety of factors, including the
activity of the specific compound employed, the age, body weight,
general health status, sex, diet, time of administration, rate of
excretion, drug combination, the severity and course of the
disease, condition or symptoms, the patient's disposition to the
disease, condition or symptoms, and the judgment of the treating
physician.
[0202] Upon improvement of a patient's condition or prevention of
infection, a maintenance dose of a compound, composition or
combination of this invention may be administered, if necessary.
Subsequently, the dosage or frequency of administration, or both,
may be reduced, as a function of the symptoms, to a level at which
the improved condition is retained. Patients may, however, require
intermittent treatment on a long-term basis upon any recurrence of
disease symptoms (e.g. increase in HIV viral load).
[0203] Pharmaceutical compositions of this invention comprise a
compounds of the invention or a pharmaceutically acceptable salt
thereof; an additional agent including for example, morphine or
codeine; and any pharmaceutically acceptable carrier, adjuvant or
vehicle. Alternate compositions of this invention comprise a
compound of the invention or a pharmaceutically acceptable salt
thereof; and a pharmaceutically acceptable carrier, adjuvant or
vehicle. The compositions delineated herein include the compounds
of the invention delineated herein, as well as additional
therapeutic agents if present, in amounts effective for achieving a
modulation of disease or disease symptoms, including HIV mediated
disorders or symptoms thereof.
[0204] The term "pharmaceutically acceptable carrier or adjuvant"
refers to a carrier or adjuvant that may be administered to a
patient, together with a compound of this invention, and which does
not destroy the pharmacological activity thereof and is nontoxic
when administered in doses sufficient to deliver a therapeutic
amount of the compound.
[0205] Pharmaceutically acceptable carriers, adjuvants and vehicles
that may be used in the pharmaceutical compositions of this
invention include, but are not limited to, ion exchangers, alumina,
aluminum stearate, lecithin, self-emulsifying drug delivery systems
(SEDDS) such as d-.alpha..-tocopherol polyethyleneglycol 1000
succinate, surfactants used in pharmaceutical dosage forms such as
Tween.RTM. or other similar polymeric delivery matrices, serum
proteins, such as human serum albumin, buffer substances such as
phosphates, glycine, sorbic acid, potassium sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts
or electrolytes, such as protamine sulfate, disodium hydrogen
phosphate, potassium hydrogen phosphate, sodium chloride, zinc
salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, cellulose-based substances, polyethylene glycol,
sodium carboxymethylcellulose, polyacrylates, waxes,
polyethylene-polyoxypropyle-ne-block polymers, polyethylene glycol
and wool fat. Cyclodextrins such as alpha-, beta-, and
gamma-cyclodextrin, may also be advantageously used to enhance
delivery of compounds of the formulae described herein.
[0206] The pharmaceutical compositions of this invention may be
administered enterally for example by oral administration,
parenterally, by inhalation spray, topically, rectally, nasally,
buccally, vaginally or via an implanted reservoir, preferably by
oral or vaginal administration or administration by injection. The
pharmaceutical compositions of this invention may contain any
conventional non-toxic pharmaceutically-acceptable carriers,
adjuvants or vehicles. In some cases, the pH of the formulation may
be adjusted with pharmaceutically acceptable acids, bases, or
buffers to enhance the stability of the formulated compound or its
delivery form. The term parenteral as used herein includes
subcutaneous, intracutaneous, intravenous, intramuscular,
intraarticular, intraarterial, intrasynovial, intrastemal,
intrathecal, intralesional, and intracranial injection or infusion
techniques.
[0207] Examples of dosage forms include, but are not limited to:
tablets; caplets; capsules, such as soft elastic gelatin capsules;
cachets; troches; lozenges; dispersions; suppositories; ointments;
cataplasms (poultices); pastes; powders; dressings; creams;
plasters; solutions; patches; aerosols (e.g., nasal sprays or
inhalers); gels; liquid dosage forms suitable for oral or mucosal
administration to a patient, including suspensions (e.g., aqueous
or non-aqueous liquid suspensions, oil-in-water emulsions, or a
water-in-oil liquid emulsions), solutions, and elixirs; liquid
dosage forms suitable for parenteral administration to a patient;
and sterile solids (e.g., crystalline or amorphous solids) that can
be reconstituted to provide liquid dosage forms suitable for
parenteral administration to a patient.
[0208] The pharmaceutical compositions may be in the form of a
sterile injectable preparation, for example, as a sterile
injectable aqueous or oleaginous suspension. This suspension may be
formulated according to techniques known in the art using suitable
dispersing or wetting agents (such as, for example, Tween.RTM. 80)
and suspending agents. The sterile injectable preparation may also
be a sterile injectable solution or suspension in a non-toxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are mannitol, water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose, any bland fixed oil may be
employed including synthetic mono- or diglycerides. Fatty acids,
such as oleic acid and its glyceride derivatives are useful in the
preparation of injectables, as are natural
pharmaceutically-acceptable oils, such as olive oil or castor oil,
especially in their polyoxyethylated versions. These oil solutions
or suspensions may also contain a long-chain alcohol diluent or
dispersant, or carboxymethyl cellulose or similar dispersing agents
which are commonly used in the formulation of pharmaceutically
acceptable dosage forms such as emulsions and or suspensions. Other
commonly used surfactants such as Tweens or Spans and/or other
similar emulsifying agents or bioavailability enhancers which are
commonly used in the manufacture of pharmaceutically acceptable
solid, liquid, or other dosage forms may also be used for the
purposes of formulation.
[0209] The pharmaceutical compositions of this invention may be
orally administered in any orally acceptable dosage form including,
but not limited to, capsules, tablets, emulsions and aqueous
suspensions, dispersions and solutions. In the case of tablets for
oral use, carriers which are commonly used include lactose and corn
starch. Lubricating agents, such as magnesium stearate, are also
typically added. For oral administration in a capsule form, useful
diluents include lactose and dried corn starch. When aqueous
suspensions and/or emulsions are administered orally, the active
ingredient may be suspended or dissolved in an oily phase is
combined with emulsifying and/or suspending agents. If desired,
certain sweetening and/or flavoring and/or coloring agents may be
added.
[0210] The pharmaceutical compositions of this invention may also
be administered in the form of suppositories for rectal
administration. These compositions can be prepared by mixing a
compound of this invention with a suitable non-irritating excipient
which is solid at room temperature but liquid at the rectal
temperature and therefore will melt in the rectum to release the
active components. Such materials include, but are not limited to,
cocoa butter, beeswax and polyethylene glycols.
[0211] The pharmaceutical compositions of the invention may be
administered topically or intravaginally. The pharmaceutical
composition will be formulated with a suitable ointment containing
the active components suspended or dissolved in a carrier. Carriers
for topical administration of the compounds of this invention
include, but are not limited to, mineral oil, liquid petroleum,
white petroleum, propylene glycol, polyoxyethylene polyoxypropylene
compound, emulsifying wax and water. Alternatively, the
pharmaceutical composition can be formulated with a suitable lotion
or cream containing the active compound suspended or dissolved in a
carrier. In still another embodiment, the pharmaceutical
composition is formulated as a vaginal ring. Suitable carriers
include, but are not limited to, mineral oil, sorbitan
monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,
2-octyldodecanol, benzyl alcohol and water. The pharmaceutical
compositions of this invention may also be topically applied to the
lower intestinal tract by rectal suppository formulation or in a
suitable enema formulation. Topically-transdermal patches and
iontophoretic administration are also included in this invention.
In one embodiment, the compound of the invention is administered
vaginally as a prophylactic treatment for a sexually transmitted
disease, e.g., HIV.
[0212] The pharmaceutical compositions of this invention may be
administered by nasal aerosol or inhalation. Such compositions are
prepared according to techniques well-known in the art of
pharmaceutical formulation and may be prepared as solutions in
saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to enhance bioavailability, fluorocarbons,
and/or other solubilizing or dispersing agents known in the
art.
[0213] When the compositions of this invention comprise a
combination of a compound of the formulae described herein and one
or more additional therapeutic or prophylactic agents, both the
compound and the additional agent should be present at dosage
levels of between about 1 to 100%, and more preferably between
about 5 to 95% of the dosage normally administered in a monotherapy
regimen. The additional agents may be administered separately, as
part of a multiple dose regimen, from the compounds of this
invention. Alternatively, those agents may be part of a single
dosage form, mixed together with the compounds of this invention in
a single composition.
[0214] With respect to HIV, peptides of the invention may be used
as therapeutics in the treatment of HIV infection and/or AIDS. In
addition, the peptides may be used as prophylactic measures in
previously uninfected individuals after acute exposure to an HIV
virus (e.g. post-exposure prophylaxis). Examples of such
prophylactic use of the peptides may include, but are not limited
to, prevention of virus transmission from mother to infant and
other settings where the likelihood of HIV transmission exists,
such as, for example, sexual transmission or accidents in health
care settings wherein workers are exposed to HIV-containing blood
products.
[0215] Effective dosages of the peptides of the invention to be
administered may be determined through procedures well known to
those in the art which address such parameters as biological
half-life, bioavailability, and toxicity.
[0216] A therapeutically effective dose refers to that amount of
the compound sufficient to result in amelioration of symptoms or a
prolongation of survival in a patient. Toxicity and therapeutic
efficacy of such compounds can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD.sub.50/ED.sub.50. Compounds which exhibit
large therapeutic indices are preferred. The data obtained from
these cell culture assays and animal studies can be used in
formulating a range of dosage for use in humans. The dosage of such
compounds lies preferably within a range of circulating
concentrations that include the ED50 with little or no toxicity.
The dosage may vary within this range depending upon the dosage
form employed and the route of administration utilized. For any
compound used in the method of the invention, the therapeutically
effective dose can be estimated initially from cell culture assays.
A dose may be formulated in animal models to achieve a circulating
plasma concentration range that includes the IC.sub.50 (e.g., the
concentration of the test compound which achieves a half-maximal
inhibition of the fusogenic event, such as a half-maximal
inhibition of viral infection relative to the amount of the event
in the absence of the test compound) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography (HPLC) or mass spectrometry
(MS).
Prophylactic Vaccine
[0217] The peptides of the invention may, further, serve the role
of a prophylactic vaccine, wherein the host raises antibodies
against the peptides of the invention, which then serve to
neutralize a virus (e.g., HIV, RSV, influenza, parainfluenza,
coronavirus, ebolavirus) by, for example, inhibiting further
infection. Administration of the peptides of the invention as a
prophylactic vaccine, therefore, would comprise administering to a
host a concentration of peptides effective in raising an immune
response which is sufficient to neutralize the virus, by, for
example, inhibiting virus ability to infect cells. The exact
concentration will depend upon the specific peptide to be
administered, but may be determined by using standard techniques
for assaying the development of an immune response which are well
known to those of ordinary skill in the art. The peptides to be
used as vaccines are usually administered intramuscularly.
[0218] The peptides may be formulated with a suitable adjuvant in
order to enhance the immunological response. Such adjuvants may
include, but are not limited to mineral gels such as aluminum
hydroxide; surface active substances such as lysolecithin, pluronic
polyols, polyanions; other peptides; oil emulsions; and potentially
useful human adjuvants such as BCG and Corynebacterium parvum. Many
methods may be used to introduce the vaccine formulations described
here. These methods include but are not limited to oral,
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, and intranasal routes.
[0219] Alternatively, an effective concentration of polyclonal or
monoclonal antibodies raised against the peptides of the invention
may be administered to a host so that no uninfected cells become
infected by the virus. The exact concentration of such antibodies
will vary according to each specific antibody preparation, but may
be determined using standard techniques well known to those of
ordinary skill in the art. Administration of the antibodies may be
accomplished using a variety of techniques, including, but not
limited to those described in this section.
[0220] In one aspect, the invention is directed to a method of
generating an antibody to a modified polypeptide. The method
includes administering a modified polypeptide(s) of the invention
to a subject so as to generate an antibody to the modified
polypeptide.
[0221] In yet another aspect, the invention is directed to an
antibody that specifically binds a modified polypeptide, wherein
the modified polypeptide has an amino acid sequence of any of the
sequences of FIGS. 5, 6, or
the modified polypeptides include a heptad repeat domain with the
sequence:
TABLE-US-00006 (SEQ ID NO: 15) BTWXEWDXEINNYTSLIHSL, (SEQ ID NO:
16) BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 17)
BTWBXWDRXINNYTSL, (SEQ ID NO: 18)
BTWBEWDREINNYTSLIHSLIEXSQNXQEKNEQELLE, (SEQ ID NO: 19)
BTWBXWDRXINNYTSLIHSLIEESQNQQEKNEQELLE, (SEQ ID NO: 20)
BTWBXWDRXINNYTSLIHSLIEXSQNXQEKNEQELLE, (SEQ ID NO: 21)
BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 22)
BTWBXWDRXINNYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 23)
BTWBEWDXEINXYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 24)
BTWBEWDREINXYTSXIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 25)
BTWBEWDREINNYTSXIHSXIEESQNQQXKNEXELLE, (SEQ ID NO: 26)
BTWBXWDRXINNYTSXIHSXIEESQNQQXKNEXELLE, (SEQ ID NO: 27)
YTSXIHSXIEESQNQQEKNEQELLELDKWASLWNWF, (SEQ ID NO: 28)
YTSLIXSLIXESQNQQEKNEQELLELDKWASLWNWF, (SEQ ID NO: 29)
YTSLIHSLIEXSQNXQEKNEQELLELDKWASLWNWF, (SEQ ID NO: 30)
YTSLIHSLIEESQNQQXKNEXELLELDKWASLWNWF, (SEQ ID NO: 31)
YTSLIHSLIEESQNQQEXNEQXLLELDKWASLWNWF, (SEQ ID NO: 32)
YTSLIHSLIEESQNQQEKNEQXLLEXDKWASLWNWF, (SEQ ID NO: 33)
YTSLIHSLIEESQNQQEKNEQELLELDXWASXWNWF, (SEQ ID NO: 34)
YTSLIHSLIEESQNQQEKNEQELLELDKWXSLWXWF, (SEQ ID NO: 35)
YTSLIHSLIEXSQNXQEKNEQXLLEXDKWASLWNWF, (SEQ ID NO: 36)
YTSXIHSXIEESQNQQEKNEQELLELDKWXSLWXWF, (SEQ ID NO: 37)
YTSLIHSLIEESQNQQXKNEXELLELDKWXSLWXWF, (SEQ ID NO: 38)
YTSXIHSXIEESQNQQXKNEXELLELDKWASLWNWF, (SEQ ID NO: 39)
YTSXIHSXIEESQNQQEKNEQELLELDXWASXWNWF, (SEQ ID NO: 40)
YTSLIHSLIEXSQNXQEKNEQELLELDXWASXWNWF, (SEQ ID NO: 41)
YTSXIHSXIEESQNQQXKNEXELLELDXWASXWNWF, (SEQ ID NO: 42)
BTWBXWDRXINNYTSLIHSLIEESQNQXEKNXQELLE, or (SEQ ID NO: 43)
BTWBXWDRXINNYTSLIHSLIEESQNXQEKXEQELLE;
wherein X is any amino acid and further identifies the amino acid
residues which are linked by a hydrocarbon staple, and B is
methionine or norleucine.
Uses of the Modified Polypeptides
[0222] The antifusogenic capability of the modified peptides of the
invention may additionally be utilized to inhibit or
treat/ameliorate symptoms caused by processes involving membrane
fusion events. Such events may include, for example, virus
transmission via cell-cell fusion and virus-cell fusion. The
peptides of the invention may be used to inhibit free viral, such
as retroviral, e.g., HIV, transmission to uninfected cells wherein
such viral infection involves membrane fusion events or involves
fusion of a viral structure with a cell membrane.
[0223] In one aspect, the invention is directed to a method for
inhibiting transmission of HIV to a cell. The method includes
contacting the HIV virus with an effective dose of a modified
polypeptide so that the HIV virus is inhibited from infecting the
cell. Preferably, the modified polypeptide has a HIV gp41 heptad
repeat domain (e.g., heptad repeat domain 1 or 2, or combinations
thereof) that is stabilized with a hydrocarbon staple. Suitable
modified polypeptides include those directed to the heptad repeat
domain 1, wherein the polypeptide is 30% or more identical to the
amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:14 and
forms an alpha-helix. Other suitable modified polypeptides include
those directed to the heptad repeat domain 2, wherein the
polypeptide is 30% or more identical to the amino acid sequence of
FIG. 5, FIG. 6, SEQ ID NO:1 or 14 and forms an alpha-helix.
[0224] In yet another aspect, the invention is directed to a method
for treating or delaying the onset of AIDS in an HIV infected
individual. The method entails administering to an individual
infected with HIV an effective dose of a pharmaceutical composition
having a modified polypeptide with a stabilized HIV gp41 heptad
repeat domain, thus treating or delaying the onset of AIDS.
Preferably the HIV gp41 heptad repeat domain is stabilized with a
hydrocarbon staple(s). Suitable polypeptides include those directed
to the heptad repeat domain 1, wherein the polypeptide is 30% or
more identical to an amino acid sequence of SEQ ID NO:2, SEQ ID
NO:3 or SEQ ID NO:14 and forms an alpha-helix. Other suitable
polypeptides include those directed to the heptad repeat domain 2,
wherein the polypeptide is 30% or more identical to an amino acid
sequence of FIG. 5, FIG. 6, SEQ ID NO:1 or 14 and forms an
alpha-helix.
[0225] In still another aspect, the invention is directed to a
method for increasing the number of CD4+ cells in an individual
infected with HIV. The method involves administering to the
individual infected with HIV an effective dose of a pharmaceutical
composition having a modified polypeptide with a stabilized HIV
gp41 heptad repeat domain. The administration of the composition
results in an increase in the number of CD4+ cells in the
individual. Preferably the HIV gp41 heptad repeat domain is
stabilized with a hydrocarbon staple(s). Suitable polypeptides
include those directed to the heptad repeat domain 1, wherein the
polypeptide is 30% or more identical to an amino acid sequence of
SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:14 and forms an alpha-helix.
Other suitable polypeptides include those directed to the heptad
repeat domain 2, wherein the polypeptide is 30% or more identical
to an amino acid sequence selected of FIG. 5, FIG. 6, or SEQ ID
NO:1 and forms an alpha-helix.
[0226] In yet another aspect, the invention is directed to a method
for inhibiting syncytia formation between an HIV infected cell and
an uninfected cell. The method involves contacting the infected
cell with an effective dose of a composition having a modified
polypeptide with a stabilized HIV gp41 heptad repeat domain,
thereby inhibiting syncytia formation between the cells. Preferably
the HIV gp41 heptad repeat domain is stabilized with a hydrocarbon
staple. Suitable polypeptides include those that are 30% or more
identical to an amino acid sequence of FIG. 5, FIG. 6, SEQ ID NO:2,
SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:13, or SEQ ID NO:14 and forms
an alpha-helix.
[0227] In still another aspect, the invention is directed to a
method for inactivating HIV. The method involves contacting the
virus with an effective dose of a modified polypeptide having a
stabilized HIV gp41 heptad repeat domain so that the HIV is
rendered inactive (e.g., non-infectious). Preferably the HIV gp41
heptad repeat domain is stabilized with a hydrocarbon staple(s).
Suitable polypeptides include those that are 30% or more identical
to an amino acid sequence of FIG. 5, FIG. 6, SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:1, SEQ ID NO:13 or SEQ ID NO:14 and forms an
alpha-helix.
[0228] In still another aspect, the invention is directed to a
method for preventing an HIV infection in an individual. The method
involves administering to an individual an effective dose of a
pharmaceutical composition having modified polypeptide with a
stabilized HIV gp41 heptad repeat domain, wherein the stabilized
HIV gp41 heptad repeat domain interferes with the ability of the
HIV to infect the individual. Suitable polypeptides include those
directed to the heptad repeat domain 1, wherein the polypeptide is
30% or more identical to an amino acid sequence of SEQ ID NO:2, SEQ
ID NO:3 or SEQ ID NO:14 and forms an alpha-helix. Other suitable
polypeptides include those directed to heptad repeat domain 2,
wherein the polypeptide is 30% or more identical to an amino acid
sequence of FIG. 5, FIG. 6, SEQ ID NO:1 or 13 and forms an
alpha-helix.
[0229] In another aspect, the invention is directed to a method for
inhibiting the transmission of RSV to a cell. The method includes
contacting the virus with an effective dose of a modified
polypeptide having a stabilized RSV viral alpha helix heptad
repeat-analog domain, thereby inhibiting transmission of the virus
to a cell. Preferably the heptad repeat domain is stabilized with a
hydrocarbon staple(s) Suitable modified polypeptides include those
which are 30% or more identical to SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:11 and SEQ ID NO:12 and forms an alpha-helix.
[0230] In yet another aspect, the invention is directed to a method
for inhibiting the transmission of influenza virus to a cell. The
method includes contacting the virus with an effective dose of a
modified polypeptide having a stabilized influenza viral alpha
helix heptad repeat-analog domain, thereby inhibiting transmission
of the virus to a cell. Preferably the heptad repeat domain is
stabilized with a hydrocarbon staple(s). Suitable polypeptides are
known in the art.
[0231] In yet another aspect, the invention is directed to a method
for inhibiting the transmission of a parainfluenza virus to a cell.
The method includes contacting the virus with an effective dose of
a modified polypeptide having a stabilized parinfluenza viral alpha
helix heptad repeat-analog domain, thereby inhibiting transmission
of the virus to a cell. Preferably the heptad repeat domain is
stabilized with a hydrocarbon staple(s). Suitable polypeptides
include those which are 30% or more identical to (SEQ ID NO:6) and
forms an alpha-helix.
[0232] In still another aspect, the invention is directed to a
method for inhibiting the transmission of a coronavirus to a cell.
The method includes contacting the coronavirus with an effective
dose of a modified polypeptide having a stabilized coronavirus
alpha helix heptad repeat-analog domain, thereby inhibiting
transmission of the virus to a cell. Preferably the heptad repeat
domain is stabilized with a hydrocarbon staple(s). Suitable
polypeptides include those which are 30% or more identical to (SEQ
ID NO:7) or (SEQ ID NO:8) and forms an alpha-helix.
[0233] In yet still another aspect, the invention is directed to a
method for inhibiting the transmission of an ebola virus to a cell.
The method includes contacting the ebolavirus with an effective
dose of a modified polypeptide having a stabilized ebolavirus alpha
helix heptad repeat-analog domain, thereby inhibiting transmission
of the virus to a cell. Preferably the heptad repeat domain is
stabilized with a hydrocarbon staple(s). Suitable polypeptides
include those having an amino acid sequence which is 30% identical
to (SEQ ID NO:9) or (SEQ ID NO:10) and forms an alpha-helix.
[0234] Preferably, any of the above modified polypeptides used in
the methods of the invention have the structure of Formula (I),
(II) or (III) as described herein.
Kits
[0235] The present invention also encompasses a finished packaged
and labeled pharmaceutical product. This article of manufacture
includes the appropriate unit dosage form in an appropriate vessel
or container such as a glass vial or other container that is
hermetically sealed. The pharmaceutical product may contain, for
example, a compound of the invention in a unit dosage form in a
first container, and in a second container, sterile water for
injection. Alternatively, the unit dosage form may be a solid
suitable for oral, transdermal, intranasal, intravaginal, cervical
ring, or topical delivery.
[0236] In a specific embodiment, the unit dosage form is suitable
for intravenous, intramuscular, intranasal, oral, intravaginal,
cervical, topical or subcutaneous delivery. Thus, the invention
encompasses solutions, solids, foams, gels, preferably sterile,
suitable for each delivery route.
[0237] As with any pharmaceutical product, the packaging material
and container are designed to protect the stability of the product
during storage and shipment. Further, the products of the invention
include instructions for use or other informational material that
advise the physician, technician, or patient on how to
appropriately prevent or treat the disease or disorder in question.
In other words, the article of manufacture includes instruction
means indicating or suggesting a dosing regimen including, but not
limited to, actual doses, monitoring procedures (e.g. detection and
quantitation of infection), and other monitoring information.
[0238] Specifically, the invention provides an article of
manufacture comprising packaging material, such as a box, bottle,
tube, vial, container, sprayer, insufflator, intravenous (i.v.)
bag, envelope and the like; and at least one unit dosage form of a
pharmaceutical agent contained within said packaging material,
wherein said pharmaceutical agent comprises a compound of the
invention, and wherein said packaging material includes instruction
means which indicate that said compound can be used to prevent,
manage, treat, and/or ameliorate one or more symptoms associated
with a viral disease by administering specific doses and using
specific dosing regimens as described herein.
[0239] The following examples are provided merely as illustrative
of various aspects of the invention and shall not be construed to
limit the invention in any way.
EXAMPLES
Example 1
Synthesis of Hydrocarbon Stapled Alpha Helical Polypeptides
[0240] A combined strategy of structural analysis and chemical
synthesis is applied to construct the modified polypeptides.
Asymmetric syntheses of .alpha.,.alpha.-disubstituted amino acids
is first performed as previously reported (Schafmeister, C. E., J.
Po, and G. L. Verdine, Journal of the American Chemical Society,
2000. 122(24): p. 5891-5892; Walensky, L. D., et al., Science,
2004. 305(5689): p. 1466-1470). The modified polypeptide compounds
are generated by replacing at least two naturally occurring amino
acids with the .alpha.,.alpha.-disubstituted non-natural amino
acids at discrete locations flanking either 2, 3 or 6 amino acids,
namely the "i, i+3," "i, i+4" or "i, i+7" positions,
respectively.
[0241] Locations for the non-natural amino acids and subsequent
hydrocarbon staple(s) are carefully chosen so as not to interfere
with N36 interactions (Chan, D. C., et al., Cell, 1997. 89(2): p.
263-273). Residues in positions a and d interact directly with N36,
whereas, residues e and g may contact the N36 core as a result of
the pitch of the six-helix bundle. Residues b, f, and c localize to
the opposite face of the .alpha.-helix and are thus ideally located
for placement of the hydrocarbon staple(s).
[0242] The modified polypeptides can be generated using solid phase
Fmoc chemistry and ruthenium-catalyzed olefin metathesis, followed
by peptide deprotection and cleavage, purification by reverse-phase
high performance liquid chromatography, and chemical
characterization using LC/MS mass spectrometry and amino acid
analysis.
[0243] Alternatively an established fragment-based approach can be
pursued (Wray, B. L. Nature Reviews Drug Discovery, 2003. 2(7): p.
587-593; MYUNG-CHOL KANG, B. B., et al., Methods and compositions
for peptide synthesis, U.S.P.a.T. Office, Editor. Jan. 18, 2000
USA). In this strategy, the peptide is divided into 3 fragments,
such that an N-terminal, central, and C-terminal portion are
synthesized independently. These polypeptide fragments should be
generated using solid phase Fmoc chemistry and ruthenium-catalyzed
olefin metathesis on super-acid cleavable resins, which will yield
fully protected peptides having an Fmoc at the N-terminus, and
either a C-terminal amide (for the C-terminal fragment) or a free
carboxylate (for the central and N-terminal fragments). These fully
protected fragments are purified by reverse-phase high performance
liquid chromatography, followed by sequential deprotection,
coupling, and purification, to yield the full length, fully
protected polypeptides. Global deprotection, followed by
reverse-phase high performance liquid chromatography will yield the
final products, which can be characterized using LC/MS mass
spectrometry and amino acid analysis.
Example 2
Determining the Secondary Structure and Proteolytic Stability of
the Modified Polypeptides
[0244] The .alpha.-helicity of stapled modified polypeptides can be
compared to their unmodified counterparts by circular dichroism. CD
spectra can be obtained on a Jasco J-710 or Aviv spectropolarimeter
at 20.degree. C. using the following standard measurement
parameters: wavelength, 190-260 nm; step resolution, 0.5 nm; speed,
20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm;
path length, 0.1 cm. The .alpha.-helical content of each peptide is
calculated by dividing the mean residue ellipticity
[.theta.]222.sub.obs by the reported [.theta.]222.sub.obs for a
model helical peptide (Forood, B., E. J. Feliciano, and K. P.
Nambiar, PNAS, 1993. 90(3): p. 838-842; J. Martin Scholtz,
Biopolymers, 1991. 31(13): p. 1463-1470; Lawless, M. K., et al.,
Biochemistry, 1996. 35(42): p. 13697-13708) or using, for example,
the Aviv machine using CDNN software developed by Brohm in order to
deduce five different secondary structure fractions (helix,
parallel and antiparallel beta-sheet, beta-turn and random coil).
Protein Engineering, 1992. 5(3); p. 191-195
[0245] To assess whether helix stabilization confers enhanced
protease resistance and serum stability, the modified polypeptides
can be subjected to trypsin/chymotrypsin degradation assays and in
vitro and in vivo serum stability assays, and compared to their
unmodified counterparts as previously described (Walensky, L. D.,
et al., Science, 2004. 305(5689): p. 1466-1470). Recovery of intact
compound is determined, for example, by flash freezing the in vitro
or serum specimens in liquid nitrogen, lyophilization, and
extraction in 50:50 acetonitrile/water containing 0.1%
trifluoroacetic acid, followed by LC/MS based detection and
quantitation.
Example 3
Optimization of the Biophysical and Biochemical Properties of the
Modified Polypeptides by Evaluating Diversified Modified Peptide
Libraries Synthesized in High-Throughput Fashion
[0246] High-throughput technologies can be used to optimize the
modified polypeptides activities for cellular and in vivo studies.
For example, an Apex 396 multichannel synthesizer (AAPPTEC;
Louisville, Ky.) can be used to produce polypeptide libraries for
biological evaluation. The polypeptide compounds can be diversified
by extension, truncation, or amino acid substitution across natural
and select non-natural amino acids, and differential staple
localization can be made to maximize their biophysical and
biochemical properties. The libraries are generated using
high-throughput solid phase Fmoc chemistry and ruthenium-catalyzed
olefin metathesis and peptide deprotection and cleavage. Peptide
purification is achieved by reverse phase C18 HPLC, and products
characterized by LC/MS mass spectrometry and amino acid
analysis.
Example 4
Evaluating the Modified Polypeptides Ability to Target and Inhibit
HIV Fusion
[0247] The binding activity and functional effects of the HIV
modified polypeptides can be assessed in fluorescence polarization,
syncytial fusion, and HIV infectivity assays. Equilibrium binding
constants can be determined by fluorescence polarization assays
(FPA) using fluorescein isothiocyanate (FITC)-labeled modified
polypeptides and titrated recombinant five-helix bundle protein.
FPA experiments can be performed using a BMG Labtech FLUOstar
optima microplate reader, and dissociation constants determined by
regression analysis using GraphPad software (Prism). The
recombinant 5-helix bundle protein, first developed by Root et al.,
contains five of the six helices that comprise the core of the gp41
trimer-of-hairpins, which are connected by short peptide linkers
(Root, M. J., M. S. Kay, and P. S. Kim, Science, 2001. 291(5505):
p. 884-888). Because the 5-helix bundle lacks the third C-peptide
helix and under experimental conditions is soluble, stable, and
helical, incorporation of the sixth C-peptide in the form of
FITC-modified polypeptide would provide a direct measure of binding
activity. In this manner, modified polypeptides, differing in
peptide sequence, staple location, and staple number, can be
screened for maximal in vitro binding activity. Binding activity
can also be determined indirectly by competition assays in which
the 5-helix bundle is combined with a FITC-labeled unmodified HIV
fusion inhibitor peptide and then unlabeled stapled gp41 peptides
are added at increasing concentrations followed by measurement of
fluorescence polarization and then calculation of Ki by nonlinear
regression analysis, as indicated above.
[0248] Alternatively, an alternative binding assay can be employed
based upon the "gp41-5" construct of Frey et al. Gp41-5 binds with
high affinity to added peptides that contain all or part of the
missing CHR. For example, using gp41-5 and fluorescein-labeled C38
(residues 117-154), Frey et al. successfully generated an FPA
binding curve that revealed a K.sub.d of 3.6 nM (Frey, G., et al.,
PNAS, 2006. 103(38): p. 13938-13943).
[0249] Functional assays can also be used to evaluate the modified
polypeptides activity. In culture, multinucleated giant cells or
"syncytia" form as a result of direct cell-cell fusion between
HIV-1-infected and uninfected CD4-positive cells. In the syncytia
formation assay, an indicator cell line expressing the CD4
receptor, and a fusogenic cell line that lacks the CD4 receptor but
contains HIV-1 proteins on the surface, fuse to generate 70-100
multinucleated giant cells in culture within 48 h. Syncytia are
then counted using an inverted microscope. The ability of
stabilized alpha helix of gp41 (SAH-gp41) compounds to inhibit
syncytia formation in a dose-responsive fashion is used as a
functional measure of fusion inhibition, for which IC.sub.50s can
be determined and compared with peptides T20 and T649 (Brenner, T.
J., et al. The Lancet, 1991. 337(8748): p. 1001-1005; Madani, N.,
et al., Journal of Virology, 2007. 81(2): p. 532-538).
[0250] Also the anti-viral properties of the modified polypeptides
can be quantified based upon their capacity to directly block HIV
infection of CD4-positive and CCR5-expressing canine thymus cells.
Recombinant HIV-1 viruses (eg. HXBc2, YU2, and additional strains
available through the NIH AIDS Research and Reference Reagent
Program) expressing firefly luciferase and containing the indicated
envelope glycoproteins can be used to infect Cf2Th-CD4-CCRS/CXCR4
cells in the presence of serially diluted HIV modified
polypeptides. After 48 hours, the cells are lysed and luciferase
activity is quantified (Si, Z. H., M. Cayabyab, and J. Sodroski,
Journal of Virology, 2001. 75(9): p. 4208-4218 Si, Z. H., et al.,
PNAS, 2004. 101(14): p. 5036-5041). The identical experiment is
performed with the amphotropic murine leukemia virus (AMLV), to
monitor for any nonspecific effects of the modified polypeptides.
Similar control assays may be performed with non-HIV modified
polypeptides of the invention and are known in the art.
Example 5
Evaluate the Ability of SAH-Gp41 Compounds to Overcome Resistance
to Enfuvirtide
[0251] Heavily antiretroviral-treated HIV-positive patients who
respond to initial treatment with enfuvirtide, may ultimately
develop a viral rebound that typically occurs within a maximum of
80 weeks. Resistance to enfuvirtide derives from mutations within
the HR-1 region of gp41, although some genetic changes are found in
the HR-2 domain (Xu, L., et al., Antimicrobial Agents and
Chemotherapy, 2005. 49(3): p. 1113-1119; Perez-Alvarez, L., et al.
Journal of Medical Virology, 2006. 78(2): p. 141-147). These
mutations, such as I37V, V38A/E/M, Q39R, Q40H, N42T/Q/H, N43D/Q,
are only found in enfuvirtide-experienced patients (Poveda, E., et
al., Journal of Medical Virology, 2004. 74(1): p. 21-28; Melby, T.,
et al., Aids Research and Human Retroviruses, 2006. 22(5): p.
375-385; Sista, P. R., et al., Aids, 2004. 18(13): p. 1787-1794;
Wei, X. P., et al., Antimicrobial Agents and Chemotherapy, 2002.
46(6): p. 1896-1905).
[0252] Structural analysis and molecular modeling can be used to
evaluate the impact of these mutations on the binding interface of
the HR-1 domain with enfuvirtide. Five-helix bundle proteins
incorporating resistance mutations can then be generated for
binding analysis as described in Example 4. FITC-labeled SAH-gp41
compounds can then be screened against these mutant five-helix
bundle proteins to determine if any native SAH-gp41 compounds
retain activity despite HR domain mutations. Alternatively, HR1
peptides that contain resistance mutations are synthesized and can
be directly incubated with SAH-gp41 compounds, and then run on
native gels to detect and quantitate the formation of
heteroduplexes, which represent HR1-SAH-gp41 complex, detectable by
fluorescence scanning of the gel (FIG. 20A). SAH-gp41 compounds
should contain T649 sequences known to contact two gp41 residues
(Leu-568 and Trp-571) that are critical for fusion activity. By
incorporating this sequence functionality, the SAH-gp41 compounds
may overcome enfuvirtide-resistant virus and are less likely to
elicit a resistant virus, in contrast to analogs, like T20, that
lack such residues at the N-terminal region of the HR-2 domain
(Cao, J., et al., Journal of Virology, 1993. 67(5): p. 2747-2755;
Chan, D. C., C. T. PNAS 1998. 95(26): p. 15613-15617; Rimsky, L.
T., D. C. Shugars, and T. J. Matthews, J. Virol., 1998. 72(2): p.
986-993). Follow-up HIV infectivity studies would evaluate the
functional activity of such SAH-gp41 compounds against the
corresponding primary resistant isolates.
[0253] To monitor for restoration of SAH-gp41 activity, FITC
labeled mutants SAH-gp41 (mSAH-gp41) compounds can be screened for
binding affinity to mutant five-helix bundle proteins and for
suppression of HIV infectivity using primary resistance
strains.
[0254] To explore the evolution of potential resistance to SAH-gp41
compounds, HIV strains can be evolved in the presence of increasing
concentrations of lead SAH-gp41 compounds. Resistant strains can be
genotyped for comparative mutational analysis between these mutants
and enfuvirtide-resistant mutants (Van Laethem, K., et al., Journal
of Virological Methods, 2005. 123(1): p. 25-34). Because resistance
to one type of entry inhibitor may not affect susceptibility to
other variants, (Ray, N., et al., Journal of Virology, 2007. 81(7):
p. 3240-3250) combined SAH-gp41 and mSAH-gp41 polypeptide
compositions can be formulated.
[0255] Alternative, a phage display strategy can be employed. Lai
et al. successfully used phage display to restore
heterodimerization of a coiled-coil pair of .alpha.-helices after
destabilizing mutations were introduced (Lai, J. R., et al.,
Journal of the American Chemical Society, 2004. 126(34); p.
10514-10515). Whereas complimentary electrostatic pairing
preferences among helical residues that flank the core are readily
apparent, less is known about the packing preferences of non-polar
residues located at core positions (Lumb, K. J. and P. S. Kim,
Science, 1995. 268(5209): p. 436-439) Using phage display, one can
screen all possible amino acid combinations at up to 7 variable
locations of the HR-2 domain for binding affinity to a mutant HR-1
domain, using the corresponding five-helix bundle. In addition,
phage display screening of fully randomized HR-2 domains against
combinations of known mutations in HR-1 domains could be undertaken
in order to determine the SAH-gp41 sequence capable of forming the
most stable complex with the 5-helix bundle (Xu, L., et al.,
Antimicrobial Agents and Chemotherapy, 2005. 49(3): p. 1113-1119;
Perez-Alvarez, L., et al., Journal of Medical Virology, 2006.
78(2): p. 141-147). After three cycles of "panning", phage DNA
sequencing would reveal those peptide sequences having the highest
binding affinities for the mutant 5-helix bundle. The corresponding
SAH-gp41 derivatives would then be synthesized and evaluated in
binding and activity studies as described above.
Example 6
Analyze the In Vivo Stability, Pharmacokinetics, and
Biodistribution of SAH-gp41 Compounds
[0256] A rigorous assessment of the in vivo pharmacology of
SAH-gp41 compounds can be used to determine and optimize the
therapeutic potential of the modified polypeptides. For in vivo
serum half-life studies, 5-50 mg/kg of FITC-labeled or unlabeled
SAH-gp41 polypeptides can be injected or delivered orally into
control mice and blood specimens withdrawn for example at 0, 0.25,
0.5, 1, 2, 4, 8, 12, and 24 hours post-injection to measure levels
of intact compound by HPLC as previously described (Walensky, L.
D., et al., Science, 2004. 305(5689): p. 1466-14701) or by
reverse-phase LC/MS, followed by mathematical determination of
pharmacokinetic parameters using formulas and software known in the
art. LC/MS-based characterization of metabolites can also be
performed. 111In-DOTA-derivatized compounds can be synthesized and
injected into control mice for measurement of tissue uptake,
excretion, and biodistribution of the modified polypeptide
compounds over time by radioisotope scintillation counting.
SPECT/NMR imaging of control mice injected with
111In-DOTA-derivatized modified compounds will provide high
resolution images of biodistribution in real time as previously
performed by the Walensky lab in collaboration with Ralph
Weissleder, MD of Massachusetts General Hospital (Hird V, V. M., et
al. Br J Cancer, 1991. 64(5): p. 911-4). Chemical modifications,
including lipidation, polysialylation, or antibody-conjugation,
could be performed should optimization of pharmacokinetics and
tissue targeting of modified compounds.
Example 7
Native Gp41 C-Terminal Heptad Peptides are Predominantly Random
Coils in Solution
[0257] gp41 HR-2-derived peptides based upon the sequences of T20
(residues 638-673) and a T649 variant, T649v (residues 626-662,
rather than T649 is 628-663) were prepared and the circular
dichroism (CD) spectra determined at physiologic pH. The native
peptides display only modest minima at 222 nm and 208 nm,
suggesting predominantly random coil structure in solution. Indeed,
the calculated .alpha.-helical content (Forood, B., E. J.
Feliciano, and K. P. Nambiar, PNAS, 1993. 90(3): p. 838-84; J.
Martin Scholtz, Biopolymers, 1991. 31(13): p. 1463-1470; Lawless,
M. K., et al., Biochemistry, 1996. 35(42): p. 13697-13708) was only
.about.25% for T20 and 14% for T649v. Thus, synthetic gp41-derived
HR-2 peptides are predominantly disordered in solution, reflecting
a significant loss of bioactive structure.
Example 8
Truncated C-Terminal Heptad Peptides Display Enhanced
.alpha.-Helicity Upon Incorporation of an all-Hydrocarbon
Staple
[0258] In order to improve the biochemical properties of HIV
gp41-HR-2 peptides the T649v peptide was truncated to yield a
20-mer consisting of residues 626-645 (FIG. 7). The truncated
SAH-gp41 compound, SAH-gp41(626-645)(A), was successfully
synthesized in high yield. Analysis of comparative CD spectra
revealed marked enhancement of .alpha.-helical content for
SAH-gp41(626-645)(A) compared to its unstapled counterpart (48% vs.
20%). Evaluation of the compounds in an HIV syncytial formation
assay revealed markedly enhanced inhibitory activity of
SAH-gp41(626-645)(A) compared to its unstapled derivative. Thus, in
spite of eliminating more than 40% of the residues of T649v, the
hydrocarbon staple successfully transformed a 20-mer gp41
truncation with little .alpha.-helicity and only modest
anti-syncytial activity, into an .alpha.-helical compound with
significant structural stabilization and potent anti-syncytial
activity (IC90, .about.100 nM) (FIG. 18).
[0259] The activity of SAH-gp41(626-645)(A) peptide was compared to
the clinically approved T20 peptide in an HIV infectivity assay
using the HXBc2 strain. The SAH-gp41(626-645)(A) displayed
significant anti-HXBc2 activity, particularly given the markedly
shortened construct.
Example 9
SAH-Gp41 Compounds Demonstrate Marked .alpha.-Helical
Stabilization, Proteolytic Stability, Thermal Stability, 5-Helix
Bundle Binding Affinity
[0260] To optimize the activity of tSAH-gp41 peptides, an
alternative strategy based upon inserting one or more hydrocarbon
staples into the full-length gp41-HR-2 constructs was pursued (FIG.
11, 12). Unmodified enfuvirtide and T649v were predominantly
unstructured in pH 7 aqueous solution at 21.degree. C., exhibiting
less than 20% .alpha.-helicity (FIG. 14A,B). All stapled
derivatives displayed comparatively increased .alpha.-helical
content, with up to 4.7-fold structural stabilization (FIG. 14A-C).
The insertion of either one or two hydrocarbon staples consistently
transformed the circular dichroism spectra from a random coil
pattern with a predominant single minimum at 204 nm to an
.alpha.-helical contour with double minima at 208 and 222 nm. For
select peptide templates, single C-terminal stapling conferred a
greater degree of .alpha.-helical stabilization than single
N-terminal stapling. Select doubly stapled SAH-gp41 compounds
exhibited an intermediate enhancement in .alpha.-helical structure,
balancing the effects of the N- and C-terminal singly stapled
peptides. Enhancement of peptide .alpha.-helicity was likewise
observed at pH2, and in most cases, SAH-gp41 compounds were even
more helical at pH2 than at pH7 (FIG. 14D-F).
[0261] To assess the resistance of SAH-gp41 peptides to thermal
unfolding, we performed circular dichroism studies across a
1-91.degree. C. temperature range. We observed that select single
and double stapling of HIV-1 fusion inhibitor peptides conferred
.alpha.-helical stabilization that was remarkably heat-resistant,
sustaining an up to 2.3-fold enhancement in .alpha.-helicity even
at 91.degree. C. (FIG. 15).
[0262] A major limitation of peptides as therapeutics is their
susceptibility to rapid proteolytic degradation. Biologically
active peptides such as enfuvirtide that are lengthy, unfolded, and
replete with protease sites are particularly vulnerable. One of the
potential benefits of a covalent crosslinking strategy to enforce
peptide .alpha.-helicity is shielding of the vulnerable amide bonds
from proteolysis. Because proteases require that peptides adopt an
extended conformation to hydrolyze amide bonds, the structural
constraint afforded by the hydrocarbon staple can render
crosslinked peptides protease-resistant. To determine if
hydrocarbon stapling, and especially double stapling, could protect
the 36 to 37-mer HIV-1 fusion peptides from proteolysis, we
subjected enfuvirtide, T649v, and SAH-gp41 peptides to direct
protease exposure in vitro. To especially challenge the stapled
peptides, we selected chymotrypsin, which can cleave gp41 HR2
peptides at numerous consensus cleavage sites, including 9-11
locations for SAH-gp41(638-673) and 7 locations for
SAH-gp41(626-662).
[0263] In the presence of 0.5 ng/.mu.L chymotrypsin, enfuvirtide
and T649v (25 .mu.M) exhibited rapid degradation, with half-lives
of 12 and 14 minutes, respectively (FIG. 16A-C). In comparison,
singly stapled SAH-gp41 compounds displayed longer half-lives that
ranged from 21 to 200 minutes. The majority of doubly stapled
compounds markedly surpassed their singly stapled counterparts,
with select doubly stapled peptides achieving half-lives of up to
1275 minutes. In most cases, double stapling had a stronger
influence on proteolytic stability than overall peptide
.alpha.-helicity, as select doubly stapled peptides had lower
.alpha.-helicity than select singly stapled peptides, but still
exhibited superior protease resistance. Almost all stapled peptides
had the identical number of chymotrypsin cleavage sites as the
corresponding unmodified peptides, emphasizing that the observed
protease resistance derived from peptide stapling itself, rather
than elimination of cleavage sites.
[0264] Peptides have poor oral bioavailability in part due to rapid
acid hydrolysis in the proximal digestive tract. The compelling
protease resistance of doubly stapled SAH-gp41 compounds at neutral
pH prompted us to explore their stability under acidic conditions.
In each case, acidification of the peptide solutions significantly
enhanced their .alpha.-helical content as measured by CD (FIG.
16D-F). Upon exposure to pepsin at 0.5 ng/.mu.L, enfuvirtide and
T649v (25 .mu.M) exhibited rapid degradation, with half-lives of 4
and 11 minutes, respectively. Select doubly stapled SAH-gp41
compounds displayed half-lives ranging from approximately
80-800-fold greater than the unmodified peptides, and consistently
surpassed their singly stapled counterparts. Remarkably, select
doubly-stapled SAH-gp41 peptides remained 80% intact after exposure
to pepsin at pH 2 for more than 12 hours. As observed for
chymotrypsin resistance, double stapling itself, rather than
overall peptide .alpha.-helicity or number of cleavage sites,
correlated with the superior resistance to pepsin hydrolysis. These
studies highlight the capacity of double stapling to generate HIV-1
fusion inhibitor peptides with unprecedented resistance to
proteolytic hydrolysis at both neutral and acidic pH.
[0265] The compounds of the invention were also measured for their
affinity to gp41 in a five-helix binding assay as described herein.
As shown in FIG. 17 the modified compounds bound substantially
better than the unmodified control polypeptides.
Example 10
SAH-Gp41 Compounds Demonstrate Anti-Syncytial Formation Activity
and Anti-HIV Viral Fusion Activity
[0266] The compounds of the invention were assayed for inhibition
of syncytial formation using methods well known to those skilled in
the art. The results of the assay are shown in FIG. 18. Equal
amounts of either T20/gp41.sub.(638-673) or SAH-gp41.sub.(626-645)A
were added to the media. As shown, the modified compounds inhibited
syncytial formation more so than unmodified control
polypeptides.
[0267] In order to determine the functional impact of
hydrocarbon-stapling on gp41-based fusion inhibitor activity,
SAH-gp41 compounds were tested and compared to their unmodified
counterparts in a luciferase-based HIV infectivity assay (Si, Z.
H., M. Cayabyab, and J. Sodroski, Journal of Virology, 2001. 75(9):
p. 4208-4218; Si, Z. H., et al., PNAS, 2004. 101(14): p.
5036-5041). Recombinant HIV-1 bearing the envelope glycoproteins
from three distinct HIV-1 strains, HXBc2, ADA, and HXBc2P 3.2, and
a negative control virus bearing the amphotropic murine leukemia
virus (A-MLV) envelope glycoproteins, were evaluated. Compared to
enfuvirtide, select SAH-gp41(638-673) peptides exhibited a 3- to
15-fold enhancement of inhibitory activity across all three HIV-1
strains (FIG. 19). T649v, an HR2 peptide that encompasses a
37-amino acid fragment terminating 11 residues upstream of
enfuvirtide's C-terminus, displayed 26-, 40-, and 16-fold greater
inhibitory activity than enfuvirtide against viruses with the
HXBc2, ADA, and HXBc2P 3.2 envelope glycoproteins, respectively.
Given the marked potency of T649v against these viral strains, we
found that the corresponding SAH-gp41 peptides showed essentially
comparable activity in infectivity assays. In order to probe for
differential anti-viral potencies among T649v-based stapled
peptides, we screened the compounds against viruses with envelope
glycoproteins derived from the more resistant primary R5 isolate,
YU2. Compared to T649v, select SAH-gp41(626-662) peptides
demonstrated enhanced anti-YU2 activity (FIG. 19, 20B). The ability
of SAH-gp41 peptides to overcome HIV-1 HR1 resistance mutations,
was further underscored by the superior binding activity of select
SAH-gp41 peptides to mutant HR1 peptides, as compared to unmodified
gp41-based fusion peptides, when assayed by fluorescence scan of
electopheresed mixtures of HR1 and HR2/SAH-gp41 peptides (FIG.
20A).
[0268] These functional data reveal that insertion of one or more
hydrocarbon staples can yield SAH-gp41 peptides with potent and
broad anti-HIV-1 activity. The importance of striking a balance
between .alpha.-helical stabilization, proteolytic stability, and
anti-viral activity is underscored by the doubly stapled
SAH-gp41(626-662)(A, F) peptide, which combines intermediate
.alpha.-helical stabilization, the striking anti-proteolysis
feature of double stapling, and potent anti-viral activity, to
yield a pharmacologically optimized HIV-1 fusion inhibitor
peptide.
Example 11
A Doubly Stapled SAH-Gp41 Peptide Demonstrates Striking Enhancement
of In Vivo Stability and Bioavailabilty Compared to the
Corresponding Unmodified Peptide
[0269] Male C57/BL6 mice were administered intravenously or by oral
gavage 10 mg/kg of either SAH-gp41.sub.(626-662)(A,F) or the
corresponding unmodified peptide. Blood samples withdrawn at 30
minutes by retro-orbital bleed were subjected to quantitation using
LC/MS-based blood tests. The level of SAH-gp41.sub.(626-662)(A,F)
measured in the blood was more than 6-fold greater than the
measured level of the corresponding unmodified peptide. Strikingly,
30 minutes after oral administration, intact
SAH-gp41.sub.(626-662)(A,F) was detected in the blood at measurable
levels, whereas the unmodified peptide was undetectable (FIG. 21).
These data emphasize that hydrocarbon stapling confers unique
pharmacologic properties to gp41-based fusion peptide sequences,
enhancing their in vivo stability and even conferring measurable
oral bioavailability. This single dose experiment demonstrates that
the SAH-gp41 peptides could be dosed at a level to provide serum
levels of the compound comparable to the level of an unmodified
peptide (e.g., enfuvirtide) suggesting that a therapeutically
effective dose could be administered orally.
[0270] All patents, patent applications, GenBank numbers, and
published references cited herein are hereby incorporated by
reference in their entirety as if they were incorporated
individually. While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
100136PRTHuman immunodeficiency virus-1 1Tyr Thr Ser Leu Ile His
Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys Asn Glu
Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30 Trp Asn
Trp Phe 35 238PRTHuman immunodeficiency virus-1 2Asn Asn Leu Leu
Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu 1 5 10 15 Thr Val
Trp Gly Ile Lys Gln Leu Gln Ala Arg Ile Leu Ala Val Glu 20 25 30
Arg Tyr Leu Gln Asp Gln 35 320PRTHuman immunodeficiency
virus-1MOD_RES(1)..(1)Met or Nle 3Xaa Thr Trp Xaa Glu Trp Asp Arg
Glu Ile Asn Asn Tyr Thr Ser Leu 1 5 10 15 Ile His Ser Leu 20
448PRTRespiratory syncytial virus 4Tyr Thr Ser Val Ile Thr Ile Glu
Leu Ser Asn Ile Lys Glu Asn Lys 1 5 10 15 Cys Asn Gly Thr Asp Ala
Lys Val Lys Leu Ile Lys Gln Glu Leu Asp 20 25 30 Lys Tyr Lys Asn
Ala Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr 35 40 45
537PRTRespiratory syncytial virus 5Phe Tyr Asp Pro Leu Val Phe Pro
Ser Asp Glu Phe Asp Ala Ser Ile 1 5 10 15 Ser Gln Val Asn Glu Lys
Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys 20 25 30 Ser Asp Glu Leu
Leu 35 634PRTParainfluenza virus 6Ala Leu Gly Val Ala Thr Ser Ala
Gln Ile Thr Ala Ala Val Ala Leu 1 5 10 15 Val Glu Ala Lys Gln Ala
Arg Ser Asp Ile Glu Lys Leu Lys Glu Ala 20 25 30 Ile Arg
777PRTCoronavirus 7Asn Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn
Gln Phe Asn Lys 1 5 10 15 Ala Ile Ser Gln Ile Gln Glu Ser Leu Thr
Thr Thr Ser Thr Ala Leu 20 25 30 Gly Lys Leu Gln Asp Val Val Asn
Gln Asn Ala Gln Ala Leu Asn Thr 35 40 45 Leu Val Lys Gln Leu Ser
Ser Asn Phe Gly Ala Ile Ser Ser Val Leu 50 55 60 Asn Asp Ile Leu
Ser Arg Leu Asp Lys Val Glu Ala Glu 65 70 75 847PRTCoronavirus 8Thr
Ser Pro Asp Val Asp Phe Gly Asp Ile Ser Gly Ile Asn Ala Ser 1 5 10
15 Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys
20 25 30 Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys
Tyr 35 40 45 943PRTFilovirus 9Asp Gly Leu Ile Cys Gly Leu Arg Gln
Leu Ala Asn Glu Thr Thr Gln 1 5 10 15 Ala Leu Gln Leu Phe Leu Arg
Ala Thr Thr Glu Leu Arg Thr Phe Ser 20 25 30 Ile Leu Asn Arg Lys
Ala Ile Asp Phe Leu Leu 35 40 1024PRTFilovirus 10Asp Trp Thr Lys
Asn Ile Thr Asp Lys Ile Asp Gln Ile Ile His Asp 1 5 10 15 Phe Val
Asp Lys Thr Leu Pro Asp 20 1157PRTRespiratory syncytial virus 11Ser
Gly Ile Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn 1 5 10
15 Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu
20 25 30 Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val Leu Asp Leu
Lys Ser 35 40 45 Tyr Ile Asn Asn Gln Leu Leu Pro Ile 50 55
1254PRTRespiratory syncytial virus 12Pro Ile Ile Asn Tyr Tyr Asp
Pro Leu Val Phe Pro Ser Asp Glu Phe 1 5 10 15 Asp Ala Ser Ile Ser
Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala 20 25 30 Phe Ile Arg
Arg Ser Asp Glu Leu Leu His Asn Val Asn Thr Gly Lys 35 40 45 Ser
Thr Thr Asn Ile Met 50 1337PRTHuman immunodeficiency virus-1 13Met
Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu 1 5 10
15 Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu
20 25 30 Gln Glu Leu Leu Glu 35 1451PRTHuman immunodeficiency
virus-1 14Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu
Leu Arg 1 5 10 15 Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr
Val Trp Gly Ile 20 25 30 Lys Gln Leu Gln Ala Arg Ile Leu Ala Val
Glu Arg Tyr Leu Gln Asp 35 40 45 Gln Gln Leu 50 1520PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 15Xaa
Thr Trp Xaa Glu Trp Asp Xaa Glu Ile Asn Asn Tyr Thr Ser Leu 1 5 10
15 Ile His Ser Leu 20 1637PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 16Xaa Thr Trp Xaa Glu Trp
Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu 1 5 10 15 Ile His Ser Leu
Ile Glu Glu Ser Gln Asn Gln Gln Xaa Lys Asn Glu 20 25 30 Xaa Glu
Leu Leu Glu 35 1716PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 17Xaa Thr Trp Xaa Xaa Trp Asp Arg Xaa
Ile Asn Asn Tyr Thr Ser Leu 1 5 10 15 1837PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
18Xaa Thr Trp Xaa Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu 1
5 10 15 Ile His Ser Leu Ile Glu Xaa Ser Gln Asn Xaa Gln Glu Lys Asn
Glu 20 25 30 Gln Glu Leu Leu Glu 35 1937PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
19Xaa Thr Trp Xaa Xaa Trp Asp Arg Xaa Ile Asn Asn Tyr Thr Ser Leu 1
5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn
Glu 20 25 30 Gln Glu Leu Leu Glu 35 2037PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
20Xaa Thr Trp Xaa Xaa Trp Asp Arg Xaa Ile Asn Asn Tyr Thr Ser Leu 1
5 10 15 Ile His Ser Leu Ile Glu Xaa Ser Gln Asn Xaa Gln Glu Lys Asn
Glu 20 25 30 Gln Glu Leu Leu Glu 35 2137PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
21Xaa Thr Trp Xaa Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu 1
5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Xaa Lys Asn
Glu 20 25 30 Xaa Glu Leu Leu Glu 35 2237PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
22Xaa Thr Trp Xaa Xaa Trp Asp Arg Xaa Ile Asn Asn Tyr Thr Ser Leu 1
5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Xaa Lys Asn
Glu 20 25 30 Xaa Glu Leu Leu Glu 35 2337PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
23Xaa Thr Trp Xaa Glu Trp Asp Xaa Glu Ile Asn Xaa Tyr Thr Ser Leu 1
5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Xaa Lys Asn
Glu 20 25 30 Xaa Glu Leu Leu Glu 35 2437PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
24Xaa Thr Trp Xaa Glu Trp Asp Arg Glu Ile Asn Xaa Tyr Thr Ser Xaa 1
5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Xaa Lys Asn
Glu 20 25 30 Xaa Glu Leu Leu Glu 35 2537PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
25Xaa Thr Trp Xaa Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Xaa 1
5 10 15 Ile His Ser Xaa Ile Glu Glu Ser Gln Asn Gln Gln Xaa Lys Asn
Glu 20 25 30 Xaa Glu Leu Leu Glu 35 2637PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
26Xaa Thr Trp Xaa Xaa Trp Asp Arg Xaa Ile Asn Asn Tyr Thr Ser Xaa 1
5 10 15 Ile His Ser Xaa Ile Glu Glu Ser Gln Asn Gln Gln Xaa Lys Asn
Glu 20 25 30 Xaa Glu Leu Leu Glu 35 2736PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
27Tyr Thr Ser Xaa Ile His Ser Xaa Ile Glu Glu Ser Gln Asn Gln Gln 1
5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser
Leu 20 25 30 Trp Asn Trp Phe 35 2836PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
28Tyr Thr Ser Leu Ile Xaa Ser Leu Ile Xaa Glu Ser Gln Asn Gln Gln 1
5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser
Leu 20 25 30 Trp Asn Trp Phe 35 2936PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
29Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Xaa Ser Gln Asn Xaa Gln 1
5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser
Leu 20 25 30 Trp Asn Trp Phe 35 3036PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
30Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1
5 10 15 Xaa Lys Asn Glu Xaa Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser
Leu 20 25 30 Trp Asn Trp Phe 35 3136PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
31Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1
5 10 15 Glu Xaa Asn Glu Gln Xaa Leu Leu Glu Leu Asp Lys Trp Ala Ser
Leu 20 25 30 Trp Asn Trp Phe 35 3236PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
32Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1
5 10 15 Glu Lys Asn Glu Gln Xaa Leu Leu Glu Xaa Asp Lys Trp Ala Ser
Leu 20 25 30 Trp Asn Trp Phe 35 3336PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
33Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1
5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Xaa Trp Ala Ser
Xaa 20 25 30 Trp Asn Trp Phe 35 3436PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
34Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1
5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Xaa Ser
Leu 20 25 30 Trp Xaa Trp Phe 35 3536PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
35Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Xaa Ser Gln Asn Xaa Gln 1
5 10 15 Glu Lys Asn Glu Gln Xaa Leu Leu Glu Xaa Asp Lys Trp Ala Ser
Leu 20 25 30 Trp Asn Trp Phe 35 3636PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
36Tyr Thr Ser Xaa Ile His Ser Xaa Ile Glu Glu Ser Gln Asn Gln Gln 1
5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Xaa Ser
Leu 20 25 30 Trp Xaa Trp Phe 35 3736PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
37Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1
5 10 15 Xaa Lys Asn Glu Xaa Glu Leu Leu Glu Leu Asp Lys Trp Xaa Ser
Leu 20 25 30 Trp Xaa Trp Phe 35 3836PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
38Tyr Thr Ser Xaa Ile His Ser Xaa Ile Glu Glu Ser Gln Asn Gln Gln 1
5 10 15 Xaa Lys Asn Glu Xaa Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser
Leu 20 25 30 Trp Asn Trp Phe 35 3936PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
39Tyr Thr Ser Xaa Ile His Ser Xaa Ile Glu Glu Ser Gln Asn Gln Gln 1
5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Xaa Trp Ala Ser
Xaa 20 25 30 Trp Asn Trp Phe 35 4036PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
40Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Xaa Ser Gln Asn Xaa Gln 1
5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Xaa Trp Ala Ser
Xaa 20 25 30 Trp Asn Trp Phe 35 4136PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
41Tyr Thr Ser Xaa Ile His Ser Xaa Ile Glu Glu Ser Gln Asn Gln Gln 1
5 10 15 Xaa Lys Asn Glu Xaa Glu Leu Leu Glu Leu Asp Xaa Trp Ala Ser
Xaa 20 25 30 Trp Asn Trp Phe 35 4237PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
42Xaa Thr Trp Xaa Xaa Trp Asp Arg Xaa Ile Asn Asn Tyr Thr Ser Leu 1
5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Xaa Glu Lys Asn
Xaa 20 25 30 Gln Glu Leu Leu Glu 35 4337PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
43Xaa Thr Trp Xaa Xaa Trp Asp Arg Xaa Ile Asn Asn Tyr Thr Ser Leu 1
5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln Asn Xaa Gln Glu Lys Xaa
Glu 20 25 30 Gln Glu Leu Leu Glu 35 4438PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
44Xaa Xaa Trp Xaa Xaa Trp Xaa Xaa Xaa Ile Xaa Xaa Tyr Xaa Xaa Xaa 1
5 10 15 Ile Xaa Xaa Leu Xaa Xaa Xaa Ser Xaa Xaa Gln Xaa Xaa Xaa Asn
Xaa 20 25 30 Xaa Glu Xaa Xaa Xaa Leu 35 4538PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
45Xaa Thr Trp Xaa Xaa Trp Asp Arg Xaa Ile Xaa Xaa Tyr Xaa Xaa Xaa 1
5 10 15 Ile Xaa Xaa Leu Ile Xaa Xaa Xaa Gln Xaa Xaa Gln Glu Lys Xaa
Glu 20 25 30 Xaa Xaa Leu Xaa Glu Leu 35 4639PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
46Trp Gln Glu Trp Glu Gln Lys Ile Thr Ala Leu Leu Glu Gln Ala Gln 1
5 10 15 Ile Gln Gln Glu Lys Asn Glu Tyr Glu Leu Gln Lys Leu Asp Lys
Trp 20 25 30 Ala Ser Leu Trp Glu Trp Phe 35 4722PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 47Xaa
Thr Trp Xaa Xaa Glu Trp Asp Xaa Arg Glu Ile Asn Asn Tyr Thr 1 5 10
15 Ser Leu Ile His Ser Leu 20 4839PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 48Xaa Thr Trp Xaa Glu
Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu 1 5 10 15 Ile His Ser
Leu Ile Glu Glu Ser Gln Asn Gln Gln Xaa Glu Lys Asn 20 25 30 Glu
Xaa Gln Glu Leu Leu Glu 35 49856PRTHuman immunodeficiency virus-1
49Met Arg Val Lys Glu Lys Tyr Gln His Leu Trp Arg Trp Gly Trp Arg 1
5 10 15 Trp Gly Thr Met Leu Leu Gly Met Leu Met Ile Cys Ser Ala Thr
Glu 20 25
30 Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala
35 40 45 Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp
Thr Glu 50 55 60 Val His Asn Val Trp Ala Thr His Ala Cys Val Pro
Thr Asp Pro Asn 65 70 75 80 Pro Gln Glu Val Val Leu Val Asn Val Thr
Glu Asn Phe Asn Met Trp 85 90 95 Lys Asn Asp Met Val Glu Gln Met
His Glu Asp Ile Ile Ser Leu Trp 100 105 110 Asp Gln Ser Leu Lys Pro
Cys Val Lys Leu Thr Pro Leu Cys Val Ser 115 120 125 Leu Lys Cys Thr
Asp Leu Lys Asn Asp Thr Asn Thr Asn Ser Ser Ser 130 135 140 Gly Arg
Met Ile Met Glu Lys Gly Glu Ile Lys Asn Cys Ser Phe Asn 145 150 155
160 Ile Ser Thr Ser Ile Arg Gly Lys Val Gln Lys Glu Tyr Ala Phe Phe
165 170 175 Tyr Lys Leu Asp Ile Ile Pro Ile Asp Asn Asp Thr Thr Ser
Tyr Lys 180 185 190 Leu Thr Ser Cys Asn Thr Ser Val Ile Thr Gln Ala
Cys Pro Lys Val 195 200 205 Ser Phe Glu Pro Ile Pro Ile His Tyr Cys
Ala Pro Ala Gly Phe Ala 210 215 220 Ile Leu Lys Cys Asn Asn Lys Thr
Phe Asn Gly Thr Gly Pro Cys Thr 225 230 235 240 Asn Val Ser Thr Val
Gln Cys Thr His Gly Ile Arg Pro Val Val Ser 245 250 255 Thr Gln Leu
Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val Val Ile 260 265 270 Arg
Ser Val Asn Phe Thr Asp Asn Ala Lys Thr Ile Ile Val Gln Leu 275 280
285 Asn Thr Ser Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg
290 295 300 Lys Arg Ile Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val
Thr Ile 305 310 315 320 Gly Lys Ile Gly Asn Met Arg Gln Ala His Cys
Asn Ile Ser Arg Ala 325 330 335 Lys Trp Asn Asn Thr Leu Lys Gln Ile
Ala Ser Lys Leu Arg Glu Gln 340 345 350 Phe Gly Asn Asn Lys Thr Ile
Ile Phe Lys Gln Ser Ser Gly Gly Asp 355 360 365 Pro Glu Ile Val Thr
His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr 370 375 380 Cys Asn Ser
Thr Gln Leu Phe Asn Ser Thr Trp Phe Asn Ser Thr Trp 385 390 395 400
Ser Thr Glu Gly Ser Asn Asn Thr Glu Gly Ser Asp Thr Ile Thr Leu 405
410 415 Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Lys Val Gly
Lys 420 425 430 Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln Ile Arg Cys
Ser Ser Asn 435 440 445 Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly
Asn Ser Asn Asn Glu 450 455 460 Ser Glu Ile Phe Arg Pro Gly Gly Gly
Asp Met Arg Asp Asn Trp Arg 465 470 475 480 Ser Glu Leu Tyr Lys Tyr
Lys Val Val Lys Ile Glu Pro Leu Gly Val 485 490 495 Ala Pro Thr Lys
Ala Lys Arg Arg Val Val Gln Arg Glu Lys Arg Ala 500 505 510 Val Gly
Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser 515 520 525
Thr Met Gly Ala Ala Ser Met Thr Leu Thr Val Gln Ala Arg Gln Leu 530
535 540 Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile
Glu 545 550 555 560 Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly
Ile Lys Gln Leu 565 570 575 Gln Ala Arg Ile Leu Ala Val Glu Arg Tyr
Leu Lys Asp Gln Gln Leu 580 585 590 Leu Gly Ile Trp Gly Cys Ser Gly
Lys Leu Ile Cys Thr Thr Ala Val 595 600 605 Pro Trp Asn Ala Ser Trp
Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn 610 615 620 His Thr Thr Trp
Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser 625 630 635 640 Leu
Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn 645 650
655 Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp
660 665 670 Phe Asn Ile Thr Asn Trp Leu Trp Tyr Ile Lys Leu Phe Ile
Met Ile 675 680 685 Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe Ala
Val Leu Ser Ile 690 695 700 Val Asn Arg Val Arg Gln Gly Tyr Ser Pro
Leu Ser Phe Gln Thr His 705 710 715 720 Leu Pro Thr Pro Arg Gly Pro
Asp Arg Pro Glu Gly Ile Glu Glu Glu 725 730 735 Gly Gly Glu Arg Asp
Arg Asp Arg Ser Ile Arg Leu Val Asn Gly Ser 740 745 750 Leu Ala Leu
Ile Trp Asp Asp Leu Arg Ser Leu Cys Leu Phe Ser Tyr 755 760 765 His
Arg Leu Arg Asp Leu Leu Leu Ile Val Thr Arg Ile Val Glu Leu 770 775
780 Leu Gly Arg Arg Gly Trp Glu Ala Leu Lys Tyr Trp Trp Asn Leu Leu
785 790 795 800 Gln Tyr Trp Ser Gln Glu Leu Lys Asn Ser Ala Val Ser
Leu Leu Asn 805 810 815 Ala Thr Ala Ile Ala Val Ala Glu Gly Thr Asp
Arg Val Ile Glu Val 820 825 830 Val Gln Gly Ala Cys Arg Ala Ile Arg
His Ile Pro Arg Arg Ile Arg 835 840 845 Gln Gly Leu Glu Arg Ile Leu
Leu 850 855 50843PRTHuman immunodeficiency virus-1 50Met Arg Ala
Thr Glu Ile Arg Lys Asn Tyr Gln His Leu Trp Lys Gly 1 5 10 15 Gly
Thr Leu Leu Leu Gly Met Leu Met Ile Cys Ser Ala Ala Glu Gln 20 25
30 Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr
35 40 45 Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr
Glu Val 50 55 60 His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr
Asp Pro Asn Pro 65 70 75 80 Gln Glu Val Lys Leu Glu Asn Val Thr Glu
Asn Phe Asn Met Trp Lys 85 90 95 Asn Asn Met Val Glu Gln Met His
Glu Asp Ile Ile Ser Leu Trp Asp 100 105 110 Gln Ser Leu Lys Pro Cys
Val Lys Leu Thr Pro Leu Cys Val Thr Leu 115 120 125 Asn Cys Thr Asp
Leu Arg Asn Ala Thr Asn Thr Thr Ser Ser Ser Trp 130 135 140 Glu Thr
Met Glu Lys Gly Glu Ile Lys Asn Cys Ser Phe Asn Ile Thr 145 150 155
160 Thr Ser Ile Arg Asp Lys Val Gln Lys Glu Tyr Ala Leu Phe Tyr Asn
165 170 175 Leu Asp Val Val Pro Ile Asp Asn Ala Ser Tyr Arg Leu Ile
Ser Cys 180 185 190 Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Val
Ser Phe Glu Pro 195 200 205 Ile Pro Ile His Tyr Cys Ala Pro Ala Gly
Phe Ala Ile Leu Lys Cys 210 215 220 Asn Asp Lys Lys Phe Asn Gly Thr
Gly Pro Cys Thr Asn Val Ser Thr 225 230 235 240 Val Gln Cys Thr His
Gly Ile Arg Pro Val Val Ser Thr Gln Leu Leu 245 250 255 Leu Asn Gly
Ser Leu Ala Glu Glu Glu Ile Val Ile Arg Ser Glu Asn 260 265 270 Phe
Thr Asn Asn Ala Lys Thr Ile Ile Val Gln Leu Asn Glu Ser Val 275 280
285 Val Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile Asn
290 295 300 Ile Gly Pro Gly Arg Ala Leu Tyr Thr Thr Gly Glu Ile Ile
Gly Asp 305 310 315 320 Ile Arg Gln Ala His Cys Asn Leu Ser Lys Thr
Gln Trp Glu Asn Thr 325 330 335 Leu Glu Gln Ile Ala Ile Lys Leu Lys
Glu Gln Phe Gly Asn Asn Lys 340 345 350 Thr Ile Ile Phe Asn Pro Ser
Ser Gly Gly Asp Pro Glu Ile Val Thr 355 360 365 His Ser Phe Asn Cys
Gly Gly Glu Phe Phe Tyr Cys Asn Ser Thr Gln 370 375 380 Leu Phe Thr
Trp Asn Asp Thr Arg Lys Leu Asn Asn Thr Gly Arg Asn 385 390 395 400
Ile Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Glu 405
410 415 Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Arg Gly Gln Ile Arg
Cys 420 425 430 Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly
Gly Lys Asp 435 440 445 Thr Asn Gly Thr Glu Ile Phe Arg Pro Gly Gly
Gly Asp Met Arg Asp 450 455 460 Asn Trp Arg Ser Glu Leu Tyr Lys Tyr
Lys Val Val Lys Ile Glu Pro 465 470 475 480 Leu Gly Val Ala Pro Thr
Lys Ala Lys Arg Arg Val Val Gln Arg Glu 485 490 495 Lys Arg Ala Val
Gly Leu Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala 500 505 510 Ala Gly
Ser Thr Met Gly Ala Ala Ser Ile Thr Leu Thr Val Gln Ala 515 520 525
Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg 530
535 540 Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly
Ile 545 550 555 560 Lys Gln Leu Gln Ala Arg Val Leu Ala Val Glu Arg
Tyr Leu Arg Asp 565 570 575 Gln Gln Leu Leu Gly Ile Trp Gly Cys Ser
Gly Lys Leu Ile Cys Thr 580 585 590 Thr Thr Val Pro Trp Asn Thr Ser
Trp Ser Asn Lys Ser Leu Asn Glu 595 600 605 Ile Trp Asp Asn Met Thr
Trp Met Lys Trp Glu Arg Glu Ile Asp Asn 610 615 620 Tyr Thr His Ile
Ile Tyr Ser Leu Ile Glu Gln Ser Gln Asn Gln Gln 625 630 635 640 Glu
Lys Asn Glu Gln Glu Leu Leu Ala Leu Asp Lys Trp Ala Ser Leu 645 650
655 Trp Asn Trp Phe Asp Ile Thr Lys Trp Leu Trp Tyr Ile Lys Ile Phe
660 665 670 Ile Met Ile Val Gly Gly Leu Ile Gly Leu Arg Ile Val Phe
Val Val 675 680 685 Leu Ser Ile Val Asn Arg Val Arg Gln Gly Tyr Ser
Pro Leu Ser Phe 690 695 700 Gln Thr His Leu Pro Ala Gln Arg Gly Pro
Asp Arg Pro Asp Gly Ile 705 710 715 720 Glu Glu Glu Gly Gly Glu Arg
Asp Arg Asp Arg Ser Gly Pro Leu Val 725 730 735 Asp Gly Phe Leu Ala
Ile Ile Trp Val Asp Leu Arg Ser Leu Cys Leu 740 745 750 Phe Ser Tyr
His Arg Leu Arg Asp Leu Leu Leu Ile Val Thr Arg Ile 755 760 765 Val
Glu Leu Leu Gly Arg Arg Gly Trp Gly Val Leu Lys Tyr Trp Trp 770 775
780 Asn Leu Leu Gln Tyr Trp Ile Gln Glu Leu Lys Asn Ser Ala Val Ser
785 790 795 800 Leu Leu Asn Ala Thr Ala Ile Ala Val Ala Glu Gly Thr
Asp Arg Val 805 810 815 Ile Glu Ile Leu Gln Arg Ala Phe Arg Ala Val
Leu His Ile Pro Val 820 825 830 Arg Ile Arg Gln Gly Leu Glu Arg Ala
Leu Leu 835 840 5165PRTHuman parainfluenza virus 51Ala Leu Gly Val
Ala Thr Ser Ala Gln Ile Thr Ala Ala Val Ala Leu 1 5 10 15 Val Glu
Ala Lys Gln Ala Arg Ser Asp Ile Glu Lys Leu Lys Glu Ala 20 25 30
Ile Arg Asp Thr Asn Lys Ala Val Gln Ser Val Gln Ser Ser Ile Gly 35
40 45 Asn Leu Ile Val Ala Ile Lys Ser Val Gln Asp Tyr Val Asn Lys
Glu 50 55 60 Ile 65 5250PRTHuman T-lymphotropic virus 1 52Met Ser
Leu Ala Ser Gly Lys Ser Leu Leu His Glu Val Asp Lys Asp 1 5 10 15
Ile Ser Gln Leu Thr Gln Ala Ile Val Lys Asn His Lys Asn Leu Leu 20
25 30 Lys Ile Ala Gln Tyr Ala Ala Gln Asn Arg Arg Gly Leu Asp Leu
Leu 35 40 45 Phe Trp 50 5342PRTMarburg virus 53Asn Asn Leu Val Cys
Arg Leu Arg Arg Leu Ala Asn Gln Thr Ala Lys 1 5 10 15 Ser Leu Glu
Leu Leu Leu Arg Val Thr Thr Glu Glu Arg Thr Phe Ser 20 25 30 Leu
Ile Asn Arg His Ala Ile Asp Phe Leu 35 40 5443PRTHuman
immunodeficiency virus-1 54Trp Asn Asn Met Thr Trp Met Glu Trp Glu
Lys Glu Ile Asp Asn Tyr 1 5 10 15 Thr Ser Ile Ile Tyr Thr Leu Leu
Glu Thr Ser Gln Asn Gln Gln Glu 20 25 30 Lys Asn Glu Gln Glu Leu
Leu Glu Leu Asp Lys 35 40 5556PRTHuman parainfluenza virus 55Tyr
Thr Pro Asn Asp Ile Thr Leu Asn Asn Ser Val Ala Leu Asp Pro 1 5 10
15 Ile Asp Ile Ser Ile Glu Leu Asn Lys Ala Lys Ser Asp Leu Glu Glu
20 25 30 Ser Lys Glu Trp Ile Arg Arg Ser Asn Gln Lys Leu Asp Ser
Ile Gly 35 40 45 Asn Trp His Gln Ser Ser Thr Thr 50 55 5630PRTHuman
T-lymphotropic virus 1 56Cys Cys Phe Leu Asn Ile Thr Asn Ser His
Val Ser Ile Leu Gln Glu 1 5 10 15 Arg Pro Pro Leu Glu Asn Arg Val
Leu Thr Gly Trp Gly Leu 20 25 30 5720PRTMarburg virus 57Ile Glu Asp
Leu Ser Arg Asn Ile Ser Glu Gln Ile Asp Gln Ile Lys 1 5 10 15 Lys
Asp Glu Gln 20 5836PRTHuman immunodeficiency virus-1 58Tyr Thr His
Ile Ile Tyr Ser Leu Ile Glu Gln Ser Gln Asn Gln Gln 1 5 10 15 Glu
Lys Asn Glu Gln Glu Leu Leu Ala Leu Asp Lys Trp Ala Ser Leu 20 25
30 Trp Asn Trp Phe 35 5936PRTHuman immunodeficiency virus-1 59Met
Thr Met Lys Trp Glu Arg Glu Ile Asp Asn Tyr Thr His Ile Ile 1 5 10
15 Tyr Ser Leu Ile Glu Gln Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln
20 25 30 Glu Leu Leu Ala 35 6020PRTHuman immunodeficiency
virus-1MOD_RES(1)..(1)Nle 60Xaa Thr Trp Xaa Glu Trp Asp Arg Glu Ile
Asn Asn Tyr Thr Ser Leu 1 5 10 15 Ile His Ser Leu 20
6120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 61Xaa Thr Trp Xaa Glu Trp Asp Xaa Glu Ile Asn Asn
Tyr Thr Ser Leu 1 5 10 15 Ile His Ser Leu 20 6227PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
62Trp Gln Glu Trp Glu Arg Val Asp Phe Leu Glu Glu Asn Ile Thr Ala 1
5 10 15 Leu Leu Glu Glu Ala Gln Ile Gln Gln Glu Lys 20 25
636PRTSimian immunodeficiency virus 63Gln Gln Glu Lys Asn Glu 1 5
648PRTHuman immunodeficiency virus-1 64Leu Asp Lys Trp Ala Ser Leu
Trp 1 5 655PRTSimian immunodeficiency virus 65Trp Gln Glu Trp Glu 1
5 6638PRTHuman immunodeficiency virus-1 66Met Thr Trp Met Glu Trp
Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu 1 5 10 15 Ile His Ser Leu
Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu 20 25 30 Gln Glu
Leu Leu Glu Leu 35 6748PRTHuman immunodeficiency
virus-1MOD_RES(1)..(1)Nle 67Xaa Thr Trp Xaa Glu Trp Asp Arg Glu Ile
Asn Asn Tyr Thr Ser Leu 1 5 10 15 Ile His Ser Leu Ile Glu Glu Ser
Gln Asn Gln Gln Glu Lys Asn Glu 20 25
30 Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe
35 40 45 6837PRTHuman immunodeficiency virus-1MOD_RES(1)..(1)Nle
68Xaa Thr Trp Xaa Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu 1
5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn
Glu 20 25 30 Gln Glu Leu Leu Glu 35 6937PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
69Xaa Thr Trp Xaa Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu 1
5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Xaa Glu Lys Asn
Xaa 20 25 30 Gln Glu Leu Leu Glu 35 7037PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
70Xaa Thr Trp Xaa Glu Trp Asp Xaa Glu Ile Asn Asn Tyr Thr Ser Leu 1
5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn
Glu 20 25 30 Gln Glu Leu Leu Glu 35 7137PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
71Xaa Thr Trp Xaa Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu 1
5 10 15 Ile His Ser Leu Ile Xaa Glu Ser Gln Xaa Gln Gln Glu Lys Asn
Glu 20 25 30 Gln Glu Leu Leu Glu 35 7236PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
72Tyr Thr Xaa Leu Ile His Xaa Leu Ile Glu Glu Ser Gln Asn Gln Gln 1
5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser
Leu 20 25 30 Trp Asn Trp Phe 35 7336PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
73Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Xaa 1
5 10 15 Glu Lys Asn Xaa Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser
Leu 20 25 30 Trp Asn Trp Phe 35 7436PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
74Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1
5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Xaa Lys Trp Ala Xaa
Leu 20 25 30 Trp Asn Trp Phe 35 7536PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
75Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1
5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Xaa Ala Ser
Leu 20 25 30 Xaa Asn Trp Phe 35 7636PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
76Tyr Thr Ser Leu Xaa His Ser Leu Xaa Glu Glu Ser Gln Asn Gln Gln 1
5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser
Leu 20 25 30 Trp Asn Trp Phe 35 7736PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
77Tyr Thr Ser Leu Ile His Ser Leu Ile Xaa Glu Ser Gln Xaa Gln Gln 1
5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser
Leu 20 25 30 Trp Asn Trp Phe 35 7836PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
78Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1
5 10 15 Xaa Lys Asn Glu Xaa Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser
Leu 20 25 30 Trp Asn Trp Phe 35 7936PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
79Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1
5 10 15 Glu Lys Asn Glu Xaa Glu Leu Leu Xaa Leu Asp Lys Trp Ala Ser
Leu 20 25 30 Trp Asn Trp Phe 35 8048PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
80Xaa Thr Trp Xaa Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu 1
5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn
Glu 20 25 30 Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp
Asn Trp Phe 35 40 45 8137PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 81Xaa Thr Trp Xaa Glu Trp
Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu 1 5 10 15 Ile His Ser Leu
Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu 20 25 30 Gln Glu
Leu Leu Glu 35 8237PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 82Xaa Thr Trp Xaa Glu Trp Asp Xaa
Glu Ile Asn Asn Tyr Thr Ser Leu 1 5 10 15 Ile His Ser Leu Ile Glu
Glu Ser Gln Asn Gln Xaa Glu Lys Asn Xaa 20 25 30 Gln Glu Leu Leu
Glu 35 8337PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 83Xaa Thr Trp Xaa Glu Trp Xaa Arg Glu Ile Xaa
Asn Tyr Thr Ser Leu 1 5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln
Asn Gln Xaa Glu Lys Asn Xaa 20 25 30 Gln Glu Leu Leu Glu 35
8437PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 84Xaa Thr Trp Xaa Glu Trp Asp Arg Glu Ile Xaa
Asn Tyr Thr Xaa Leu 1 5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln
Asn Gln Xaa Glu Lys Asn Xaa 20 25 30 Gln Glu Leu Leu Glu 35
8537PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 85Xaa Thr Trp Xaa Glu Trp Asp Arg Glu Ile Asn
Asn Tyr Thr Xaa Leu 1 5 10 15 Ile His Xaa Leu Ile Glu Glu Ser Gln
Asn Gln Xaa Glu Lys Asn Xaa 20 25 30 Gln Glu Leu Leu Glu 35
8637PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 86Xaa Thr Trp Xaa Glu Trp Asp Xaa Glu Ile Asn
Asn Tyr Thr Ser Leu 1 5 10 15 Ile His Ser Leu Ile Xaa Glu Ser Gln
Xaa Gln Gln Glu Lys Asn Glu 20 25 30 Gln Glu Leu Leu Glu 35
8737PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 87Xaa Thr Trp Xaa Glu Trp Asp Xaa Glu Ile Asn
Asn Tyr Thr Xaa Leu 1 5 10 15 Ile His Xaa Leu Ile Glu Glu Ser Gln
Asn Gln Xaa Glu Lys Asn Xaa 20 25 30 Gln Glu Leu Leu Glu 35
8836PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 88Tyr Thr Xaa Leu Ile His Xaa Leu Ile Glu Glu
Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu
Leu Asp Lys Xaa Ala Ser Leu 20 25 30 Xaa Asn Trp Phe 35
8936PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 89Tyr Thr Xaa Leu Ile His Xaa Leu Ile Glu Glu
Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu
Leu Xaa Lys Trp Ala Xaa Leu 20 25 30 Trp Asn Trp Phe 35
9036PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 90Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu
Ser Gln Asn Gln Xaa 1 5 10 15 Glu Lys Asn Xaa Gln Glu Leu Leu Glu
Leu Asp Lys Xaa Ala Ser Leu 20 25 30 Xaa Asn Trp Phe 35
9136PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 91Tyr Thr Xaa Leu Ile His Xaa Leu Ile Glu Glu
Ser Gln Asn Gln Xaa 1 5 10 15 Glu Lys Asn Xaa Gln Glu Leu Leu Glu
Leu Asp Lys Trp Ala Ser Leu 20 25 30 Trp Asn Trp Phe 35
9236PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 92Tyr Thr Ser Leu Ile His Ser Leu Ile Xaa Glu
Ser Gln Xaa Gln Gln 1 5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu
Leu Xaa Lys Trp Ala Xaa Leu 20 25 30 Trp Asn Trp Phe 35
9336PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 93Tyr Thr Ser Leu Ile His Ser Leu Ile Xaa Glu
Ser Gln Xaa Gln Gln 1 5 10 15 Glu Lys Asn Glu Xaa Glu Leu Leu Xaa
Leu Asp Lys Trp Ala Ser Leu 20 25 30 Trp Asn Trp Phe 35
9436PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 94Tyr Thr Xaa Leu Ile His Xaa Leu Ile Glu Glu
Ser Gln Asn Gln Xaa 1 5 10 15 Glu Lys Asn Xaa Gln Glu Leu Leu Glu
Leu Xaa Lys Trp Ala Xaa Leu 20 25 30 Trp Asn Trp Phe 35
9537PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 95Xaa Thr Trp Xaa Xaa Trp Asp Arg Xaa Ile Asn
Asn Tyr Thr Ser Leu 1 5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln
Asn Gln Gln Glu Lys Asn Glu 20 25 30 Gln Glu Leu Leu Glu 35
9637PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 96Xaa Thr Trp Xaa Glu Trp Asp Arg Glu Ile Asn
Asn Tyr Thr Ser Leu 1 5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln
Asn Xaa Gln Glu Lys Xaa Glu 20 25 30 Gln Glu Leu Leu Glu 35
9737PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 97Xaa Thr Trp Xaa Xaa Trp Asp Arg Xaa Ile Asn
Asn Tyr Thr Ser Leu 1 5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln
Asn Gln Xaa Glu Lys Asn Xaa 20 25 30 Gln Glu Leu Leu Glu 35
9837PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 98Xaa Thr Trp Xaa Xaa Trp Asp Arg Xaa Ile Asn
Asn Tyr Thr Ser Leu 1 5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln
Asn Xaa Gln Glu Lys Xaa Glu 20 25 30 Gln Glu Leu Leu Glu 35
9937PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 99Xaa Thr Trp Xaa Glu Trp Asp Arg Glu Ile Asn
Asn Tyr Thr Ser Leu 1 5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln
Asn Gln Gln Glu Lys Asn Glu 20 25 30 Gln Glu Leu Leu Glu 35
10037PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 100Xaa Thr Trp Xaa Glu Trp Asp Arg Glu Ile
Asn Asn Tyr Thr Ser Leu 1 5 10 15 Ile His Ser Leu Ile Glu Glu Ser
Gln Asn Gln Xaa Glu Lys Asn Xaa 20 25 30 Gln Glu Leu Leu Glu 35
* * * * *