U.S. patent application number 10/414192 was filed with the patent office on 2003-09-25 for compounds which inhibit hiv replication.
This patent application is currently assigned to Duke University. Invention is credited to Bolognesi, Dani P., Matthews, Thomas J., Wild, Carl T..
Application Number | 20030181382 10/414192 |
Document ID | / |
Family ID | 27129697 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030181382 |
Kind Code |
A1 |
Wild, Carl T. ; et
al. |
September 25, 2003 |
Compounds which inhibit HIV replication
Abstract
This invention relates to human immunodeficiency virus (HIV)
protein fragments which have antiviral activity, and particularly
relates to HIV peptides derived from the HIV transmembrane
glycoprotein (gp41) which inhibit HIV-induced cell-cell fusion.
This invention further relates to methods for the inhibition of
enveloped viral infection, and to methods that modulate biochemical
processes which involve coiled coil peptide interactions.
Inventors: |
Wild, Carl T.; (Durham,
NC) ; Matthews, Thomas J.; (Durham, NC) ;
Bolognesi, Dani P.; (Durham, NC) |
Correspondence
Address: |
PENNIE AND EDMONDS
1155 AVENUE OF THE AMERICAS
NEW YORK
NY
100362711
|
Assignee: |
Duke University
|
Family ID: |
27129697 |
Appl. No.: |
10/414192 |
Filed: |
April 15, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10414192 |
Apr 15, 2003 |
|
|
|
08464003 |
Jun 2, 1995 |
|
|
|
6573078 |
|
|
|
|
08464003 |
Jun 2, 1995 |
|
|
|
08374666 |
Jan 27, 1995 |
|
|
|
5656480 |
|
|
|
|
08374666 |
Jan 27, 1995 |
|
|
|
07927532 |
Aug 7, 1992 |
|
|
|
07927532 |
Aug 7, 1992 |
|
|
|
07916540 |
Jul 20, 1992 |
|
|
|
Current U.S.
Class: |
435/70.1 ;
514/3.8; 530/395 |
Current CPC
Class: |
A61P 31/12 20180101;
C07K 14/005 20130101; A61K 38/00 20130101; C12N 2740/16122
20130101; C12N 2760/18522 20130101; Y10S 530/806 20130101; A61P
31/18 20180101; C12N 2760/16022 20130101 |
Class at
Publication: |
514/12 ;
530/395 |
International
Class: |
A61K 038/16; C07K
014/16 |
Goverment Interests
[0002] The present invention was made with Government support under
grants numbers R01-AI30411 and P30-AI28662 from the National
Institute of Allergy & Infectious Diseases.. The Government has
certain rights to this invention.
Claims
What is claimed is:
1. An isolated peptide comprising a DP-107 amino acid sequence
listed in SEQ ID:1.
2. An isolated peptide ranging from about 14 to about 60 amino
acids in length, capable of forming a heterodimer with the peptide
of claim 1.
3. The peptide of claim 1 or 2 wherein the amino terminus of the
peptide is acetylated.
4. The peptide of claim 1 or 2 wherein the carboxy terminus of the
peptide is amidated.
5. An isolated multimer of the peptide of claim 1 or 2.
6. The multimer of claim 5 wherein the multimer is a tetramer.
7. The multimer of claim 5 wherein the multimer is a dimer
consisting of two peptide monomers.
8. The dimer of claim 6 wherein the monomers of the dimer are
covalently bound to one another.
9. A method for inhibiting HIV-induced cell fusion comprising
contacting an HIV-infected cell with an effective amount of a
peptide comprising a DP-107 amino acid sequence listed in SEQ ID:1
so that the cell fusion is inhibited.
10. The method of claim 9 wherein the HIV is HIV-1.
11. A method for inhibiting HIV-induced cell fusion comprising
contacting an HIV-infected cell with an effective amount of a
peptide comprising the peptide of claim 2 so that the cell fusion
is inhibited.
12. The method of claim 11 wherein the HIV is HIV-1.
13. The method of claim 9 wherein the peptide is present as a
multimer.
14. The method of claim 11 wherein the peptide is present as a
multimer.
15. The method of claim 13 or 14 wherein the multimer is a dimer
having two peptide monomers.
16. The method of claim 12 wherein the monomers are covalently
bound to one another.
17. A method for testing compounds capable of inhibiting the
ability of HIV to infect cells, comprising: (a) contacting a test
compound to a multimer of a peptide comprising a DP-107 amino acid
sequence listed in SEQ ID:1; and (b) detecting whether the test
compound disrupts the multimer, the ability of the test compound to
disrupt the multimer indicating the test compound is capable of
inhibiting HIV infection of cells.
18. The method of claim 17 wherein the HIV is HIV-1.
19. The method of claim 17 wherein the multimer is a dimer or a
tetramer.
20. The method of claim 17 wherein the contacting step is carried
out in an aqueous solution.
21. A method for testing compounds capable of inhibiting the
ability of HIV to infect cells, comprising: (a) contacting a test
compound to a multimer of the peptide of claim 2; and (b) detecting
whether the test compound disrupts the multimer, the ability of the
test compound to disrupt the multimer indicating the test compound
is capable of inhibiting HIV infection of cells.
22. The method of claim 21 wherein the HIV is HIV-1.
23. The method of claim 21 wherein the multimer is a dimer or a
tetramer.
24. The method of claim 21 wherein the contacting step is carried
out in an aqueous solution.
25. A method for inhibiting enveloped viral infection comprising
contacting an uninfected cell with an effective amount of a peptide
capable of contributing to the formation of a coiled coil peptide
structure so that an enveloped virus is inhibited from infecting
the uninfected cell.
26. The method of claim 25 wherein the enveloped virus is a
retrovirus.
27. The method of claim 26 wherein the retrovirus is HIV-2, HTLV-I,
or HTLV-II.
28. The method of claim 25 wherein the enveloped virus is an
influenza virus.
29. The method of claim 25 wherein the enveloped virus is a
respiratory syncytial virus.
29. A method for testing compounds capable of inhibiting the
ability of an enveloped virus to infect cells, comprising: (a)
contacting a test compound to a multimer of a peptide capable of
contributing to the formation of a coiled coil peptide structure;
and (b) detecting whether the test compound disrupts the multimer,
the ability of the test compound to disrupt the multimer indicating
the test compound is capable of inhibiting enveloped viral
infection of cells.
30. The method of claim 29 wherein the enveloped virus is a
retrovirus.
31. The method of claim 30 wherein the retrovirus is HIV-2, HTLV-I,
or HTLV-II.
32. The method of claim 29 wherein the enveloped virus is an
influenza virus.
33. The method of claim 29 wherein the multimer is a dimer or a
tetramer.
34. The method of claim 29 wherein the contacting step is carried
out in an aqueous solution.
Description
[0001] This application-is a continuation-in-part of co-pending
application Ser. No. 07/927,532 of C. Wild, T. Matthews and D.
Bolognesi, filed Aug. 7, 1992, the disclosure of which is to be
incorporated by reference herein in its entirety.
1. INTRODUCTION
[0003] This invention relates to human immunodeficiency virus (HIV)
protein fragments which have antiviral activity, and particularly
relates to HIV peptides derived from the HIV transmembrane
glycoprotein (gp41) which inhibit HIV-induced cell-cell fusion.
This invention further relates to methods for the inhibition of
enveloped viral infection, and to methods that modulate biochemical
processes which involve coiled coil peptide interactions.
2. BACKGROUND OF THE INVENTION
[0004] Numerous HIV protein fragments, or peptides, have been
identified in an effort to develop an effective HIV vaccine. See
generally B. Spalding, Biotechnology 10, 24 (Jan. 1992). Examples
of patent applications which are directed to antigenic epitopes of
the gp41 protein include J. Rosen et al., PCT Application WO
87/06005 and R. Duncan, EPO Application 0 371 817. To date, the
development of an anti-HIV vaccine has been difficult.
[0005] N. Qureshi et al., Aids 1990 4, 553-558, describe a segment
of the HIV transmembrane protein (designated "gp41") which inhibits
T-cell activation in vitro. This segment, designated "CS3", when
conjugated to human serum albumin and labeled with fluorescein,
binds specifically to CD4+ cell lines, and is said to have
antiviral activity. CS3 comprises amino acids 581 to 597 of the
gp41 protein.
[0006] B. Kemp et al., EPO Application 0 323 157, describes a
fragment comprised of amino acids 572 to 591 of the gp41 protein
which is said to have antiviral activity.
3. SUMMARY OF THE INVENTION
[0007] A first aspect of the present invention is a peptide
selected from the group consisting of: (a) the peptide DP-107,
which has the formula, from amino. terminus to carboxy terminus,
of:
[0008] NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ
[0009] (SEQ ID NO: 1); and (b) peptides of from 14 to 60 amino
acids in length which form a heterodimer with the peptide DP-107
(SEQ ID NO: 1) (hereinafter on occasion referred to as "active
compounds").
[0010] A second aspect of the present invention is a process for
inhibiting HIV-induced cell fusion. The process comprises
contacting to an HIV-infected cell an effective fusion-inhibiting
amount of a peptide selected from the group consisting of: (a) the
peptide DP-107, which has the formula, from amino terminus to
carboxy terminus, of:
[0011] NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ
[0012] (SEQ ID NO: 1); and (b) peptides of from about 14 to 60
amino acids in length which form a heterodimer with the peptide
DP-107 (SEQ ID NO: 1).
[0013] A third aspect of the present invention is a process for
testing-compounds for the ability to inhibit the ability of HIV to
infect cells. The process comprises (a) contacting a test compound
to a multimer of a peptide selected from the group consisting of:
(i) the peptide DP-107, which has the formula, from amino terminus
to carboxy terminus of:
[0014] NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ
[0015] (SEQ ID NO: 1); and (ii) peptides of from 14 to 60 amino
acids in length which form a heterodimer with the peptide DP-107
(SEQ ID NO: 1); and then (b) detecting whether the test compound
disrupts said multimer, the ability of the test compound to disrupt
the multimer indicating the test compound is capable of inhibiting
HIV infection of cells.
[0016] A further aspect of the invention is a method for inhibiting
enveloped viral infection comprising contacting an uninfected cell
with an effective amount of a peptide capable of contributing to
the formation of a coiled coil peptide structure so that an
enveloped virus is inhibited from infecting the uninfected
cell.
[0017] The foregoing and other objects and aspects of the present
invention are explained in detail in the drawings herein and the
specification set forth below.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 shows the sequences of various peptides studied. In
DP-107 (SEQ ID NO: 1), DP-121 (SEQ ID NO: 2), and DP-125 (SEQ ID
NO: 3) the leucine or isoleucine heptad repeat units are
underlined. DP-107, DP-121, and DP-125 are acetylated at the
NH.sub.2 terminus and amidated at the COOH terminus. DP-116 (SEQ ID
NO:4) (identical to the CS3 peptide) is amidated at the carboxy
terminus and has a free amine terminus. DP-31 (SEQ ID NO: 5) is
neither acetylated nor amidated. Amino acid residues are numbered
according to Human Retroviruses and AIDS (1991).
[0019] FIG. 2 shows the CD spectra of 10 mM solutions of DP-107(m),
bP-121(+ ), and DP-116(1) at 0.degree. C. (A) and 37.degree. C.
(B). Concentration dependence of the midpoint of the temperature
dependence (T.sub.m) of the CD signal (C). T.sub.m corresponds to
the maxima of the first derivative of the CD melt curve. The CD
spectra were obtained in 10 mM sodium phosphate, 150 mM sodium
chloride buffer at pH 7.0.
[0020] FIG. 3 shows the-test for peptide blockade of AA5 cell
infection by HIV-1.sub.LAI About 500 TCID.sub.50 HIV-LAI were added
to 2.times.10.sup.4 AA5 cells and test peptides
(final-concentrations shown) in a final volume of 100 ml. Cell
cultures were maintained in 96-well microtiter plates for 8 days by
addition of fresh medium (but no further addition of peptides)
every other day. On the eighth day post-infection, supernatant was
tested for reverse transcriptase activity as evidence of successful
infection.
[0021] FIG. 4 shows the test for direct virocidal effect of
peptides and soluble CD4 (sT4) The HIV-1.sub.LAI virus stock was
divided into two portions. Samples of one portion (shown in figure
as virus pelleted +) were treated for 2 hr at 37.degree. C. in
medium alone, with DP-107 at 40 mg/ml, or with sT4 at 10 mg/ml.
Virus was then pelleted through a 5% sucrose layer to separate
virus from non-associated inhibitor. The virus-containing pellets
were dispersed in media and serial dilutions tested for infectivity
on the AA5 cells. Serial four fold dilutions of the other portion
of virus (virus pelleted -) were tested directly for infection of
the cells with each dilution of virus incubated in the absence or
presence of DP-107 at 40 mg/ml.
[0022] FIG. 5 shows a comparison of peptides and soluble CD4 to
inhibit primary virus isolates. About 25 TCID.sub.100 of two
primary isolates and HIV-1.sub.LAI were added to PRA activated
human PBMCs (about 1.5.times.10.sup.6/ml) containing the indicated
final concentrations of test peptide or 5T4. Each treatment
condition was tested in duplicate and the cells were cultured in
RPMI1640 containing 10% FCS 5% IL2. Four days post infection an
equal number of fresh PBMCs were added to each well. This step
allows for further expansion of virus and increases the noise to
background ratio in the final RT assay. Supernatant was harvested 8
days after infection and tested for the presence of reverse
transcriptase activity.
5. DETAILED DESCRIPTION OF THE INVENTION
[0023] The-term "HIV" as used herein refers to HIV-1, and the
numbering of amino acids in HIV proteins and fragments thereof
given herein is with respect to the HIV1.sub.LAI isolate. It is to
be understood, however, that while HIV viral infection and the
effects of DP-107 on such HIV infection are being used herein as a
model systems in which the potential anti-viral properties of
peptides capable of forming coiled coils are described, such
properties of coiled coil peptides may represent generalized
mechanisms by which a broad spectrum of enveloped viral infections
may be inhibited. Enveloped viruses whose infectivity may be
inhibited using the coiled coil peptides of the invention may
include, but are not limited to, other HIV strains such as HIV-2,
as well as influenza viruses, syncytial respiratory viruses, an
herpes viruses.
[0024] The DP107 peptide sequence is based on a highly conserved
region in the transmembrane protein (TM) which was predicted by
Gallaher et al., AIDS Res. and Human Retro. 5, 431 (1989), to form
an extended amphipathic .alpha.-helix with structural analogues in
the TM proteins of several fusogenic viruses such as influenza and
other retroviruses. The function of the site is not known but may
be related to multimerization of the envelope glycoprotein. The
site has been shown to contain a "leucine zipper" repeat. See E.
Delwart et al., AIDS Res. and Human Retro. 6, 703-(1990). The use
of peptides such as DP-107 capable of forming coiled coils,
therefore, may serve to interfere, block, or in some way modulate
many biochemical processes which involve such coiled coil peptide
formations. Such biochemical process may include, but are not
limited to transcription factors (Abel and Maniatis, Nature 341:24)
and physiological processes involving membrane fusion (White, J.
M., 1992, Science 258:1917-1924).
[0025] The biological activity of the peptide DP-107 was unexpected
and its mechanism is not readily apparent. The results shown herein
suggest that it does not act directly on the cell-free virions.
Qureshi et al. (AIDS 4, 553 (1990)) have reported that an
overlapping peptide, CS3 (DP-116 herein), inhibited infection when
coupled to albumin and suggested that this occurred by attachment
to a second receptor on the cell surface required for membrane
fusion. These investigators have tentatively identified a candidate
for the receptor as a 44 kD protein. Although such a mechanism
would be consistent with the DP-107 result shown in FIG. 4
described hereinbelow, other observations argue that these two
peptides are quite dissimilar and as such might function through
different mechanisms. Most importantly, the CS3 peptide was only
active after conjugation to albumin which contrasts with the
striking anti-viral effect of the free (non-conjugated) DP-107
peptide. Also, the CS3 peptide showed no evidence for stable
secondary structure by CD even at low temperatures and high
concentrations. Our experiments indicate that structure or capacity
to assume stable secondary structure is a requirement for
biological activity. For example, the DP-107 analogue containing a
helix breaking proline substitution (DP-121) and several truncated
versions of DP-107 (not shown) that either disrupted or failed to
show stable solution structure did not exhibit anti-viral
activity.
[0026] As noted above, a first aspect of the present invention is a
peptide selected from the group consisting of:
[0027] (a) the peptide DP-107,which has the formula, from amino
terminus to carboxy terminus, of:
[0028] NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ (SEQ ID NO: 1);
and
[0029] (b) peptides of from 14 to 60 amino acids in length which
form a heterodimer with the peptide DP-107 (SEQ ID NO:I).
[0030] In general, the peptide may be of any suitable length, but
is preferably from 14 to 60 amino acids in length, and more
preferably from 16 to 38 amino acids in length. In addition, it
will be appreciated that minor variations can be made to the
peptide. For example, the peptide may be acetylated at the amino
terminus thereof and/or amidated at the carboxy terminus
thereof.
[0031] Peptides of the invention may be provided as multimers,
particularly as dimers and tetramers. When provided in such form
the multimermay be stabilized by covalently joining the monomers to
one another. For example, a cysteine residue may be added to either
(or both) ends of the monomer and monomers of the multimer
covalently joined to one another by a disulfide bond between
cysteine residues. Reactions are carried out in accordance with
known techniques. In this manner two monomers of a dimer may be
covalently joined to form a covalently stabilized dimer, and if
desired two such covalently stabilized dimers conjugated to one
another to form a tetramer. In another example, all four members of
a tetramer could be covalently joined to one another through
disulfide linkages between terminally positioned cysteine
residues.
[0032] Other techniques for stabilizing the multimeric forms of
these peptides include crosslinking the monomer components to one
another through the formation of intermolecular amide bonds. This
process involves the reaction of the amine moiety of a basic amino
acid residue i.e. lysine, with the carboxy moiety of an acidic
amino acid residue i.e. aspartic or glutamic acid.
[0033] Several techniques can used to determine the multimerization
state of a given peptide or peptide mixture (homodimer or
heterodimer). The most straightforward methods involve determining
the apparent molecular weight of the multimer complex and from this
determining the number of associated monomer components (this can
be accomplished by dividing this apparent molecular weight by the
molecular weight of the monomer). Analytical ultracentrifugation is
a particularly suitable technique for this purpose. The specifics
of this method are known to those skilled in the art. See., e.g.,
P. Graceffa et al., J. Biol. Chem. 263, 14196-14202 (1988), and can
be summarized as follows. The material of interest is placed in a
sample cell and spun very rapidly in a model E ultracentrifuge
equipped with the appropriate detection devices. Information
collected during the experiment combined with the amino acid
composition of the peptide allows for the determination of the
apparent MW of the multimer complex. Fast Protein Liquid
Chromatography (FPLC) can also be used for this purpose. This
technique is different from the above in that, as a type of
chromatography, it ultimately requires reference back to some
primary standard (determined by analytical ultracentrifugation).
Pharmica Biosystems supplies the SUPERDEX 75.TM. column, which
allows for the separation of the various multimeric forms of
self-associating peptides. These determinations are carried out
under non-denaturing (native) conditions and when referenced to the
appropriate standards can be used to identify peptide and protein
oligomerization states.
[0034] As will also be apparent to those skilled in the art, the
test for heterodimerization may be carried out using either of the
above two methods or through the use of CD combined with one or the
other of these methods. This latter technique, in brief, involves
adding known amounts of peptide to a solution containing a known
amount of either the same peptide (for homodimerization) or a
different peptide (for heterodimerization) and following the CD
signal as a function of this addition. An increase in the magnitude
of the signal as peptide is added indicates that the added material
is participating in multimer formation. Homo vs heterodimerization
is determined by carrying out this same experiment using FPLC or
ultracentrifugation, which would determine if the resulting system
is either single (hetero) or multi (homo) component. A second, and
particularly preferred, approach to this same end is to conduct a
CD melt on this same sample. If heterodimerization has occurred,
then a single transition corresponding to the T.sub.m of the
heterodimer will be observed (this T.sub.m value will probably be
different from the value for either of the mixture components). If
only homodimerization takes place then two transitions (two
T.sub.m's) will be observed.
[0035] A process for inhibiting HIV-induced cell fusion, as also
disclosed herein, comprising contacting to an HIV-infected cell an
effective fusion-inhibiting amount of a peptide as given above. The
process may be carried out in vitro in an aqueous solution, or may
be carried out in vivo in a cellular culture assay for HIV
infection (e.g., the CEM-SS cell monolayer plaque assay described
in L. Kucera et al., Aids Research and Human Retroviruses 6, 491
(1990) or in an animal subject afflicted with the HIV virus The
process may be carried out with peptides of the invention in the
form of multimers (particularly dimers) thereof as discussed above.
The process may be carried out in a human or animal subject to
prevent HIV-induced cell fusion, in which case the compounds may be
combined with a suitable pharmaceutically acceptable carrier (such
as sterile, pyrogen-free physiological saline solution, or sterile,
pyrogen free phosphate-buffered saline solution), and administered
to the subject by a suitable route (i.e., by intramuscular
injection, subcutaneous injection, or intravenous injection). The
therapeutic dosage is about 1 to 10,000 .mu.g/Kg of patient weight
per day, more particularly from about 10 to 1,000 .mu.g/Kg of
patient weight per day, and most particularly about 100 .mu.g/Kg of
patient weight per day. Thus, the present invention provides a
method of combatting HIV (and particularly HIV-induced cell fusion)
in a human or animal subject by administering an active compound as
given herein in an effective in an effective HIV (or more
particularly HIV-induced cell fusion)combatting amount. The present
invention also provides the use of an active compound as given
herein for the preparation of a medicament for combatting HIV (or
more particularly HIV-induced cell fusion) in a human or animal
subject in need of such treatment.
[0036] A still further aspect of the present invention is a
process, useful in rational drug design, for testing compounds for
the ability to inhibit the ability of HIV to infect cells. The
process comprises (a) contacting a test compound to a multimer
(e.g., diner, tetramer) of a peptide as given above, and then (b)
detecting whether the test compound disrupts said multimer, the
ability of said test compound to disrupt said multimer indicating
the test compound is capable of inhibiting HIV infection of cells.
This process is noteworthy for its ability to identify compounds
directed to a site which has not heretofore been explored in
rational drug design. The process may be conveniently carried out
in vitro in an aqueous solution containing the multimer by adding
the test compound to the aqueous solution, and then determining
whether or not the multimer structure has been disrupted.
Disruption of multimer structure may be determined in the same
manner as set forth above.
[0037] Note that amino acid sequences disclosed herein are
presented in the amino to carboxy direction, from left to right.
The amino and carboxy groups are not presented in the sequence.
Amino acids are represented herein by one letter code or three
letter code as follows:
1 Ala; A = Alanine Leu; L = Leucine Arg; R = Arginine Lys; K =
Lysine Asn; N = Asparagine Met; M = Methionine Asp; D = Aspartic
acid Phe; F = Phenylalanine Cys; C = Cysteine Pro; P = Proline Gln;
Q = Glutamine Ser; S = Serine Glu; E = Glutamic Acid Thr; T =
Threonine Gly; G-- = Glycine Trp; W = Tryptophan His; H = Histidine
Tyr; Y = Tyrosine Ile; I = Isoleucine Val; V = Valine
[0038] The foregoing abbreviations are in accordance with
established usage. See, e.g., U.S. Pat. No. 4,871,670 to Hudson et
al. at Col. 3 lines 20-43 (applicants specifically intend that the
disclosure of this and all other patent references cited herein be
incorporated herein by reference).
[0039] The present invention is explained in greater detail in the
following Examples. These Examples are for illustrative purposes
only, and are not to be taken as limiting of the invention.
6. EXAMPLE
[0040] Peptide Synthesis
[0041] Peptides DP-107 (SEQ ID NO: 1) , DP121 (SEQ ID NO: 2),
DP-125 (SEQ ID NO: 3), DP-116 (SEQ ID NO: 4), and DP-31 (SEQ ID NO:
5) were synthesized using FAST MOC.TM. chemistry on an Applied
Biosystems Model 431A peptide synthesizer. Amidated peptides were
prepared using Rink resin (Advanced Chemtech) while peptides
containing free carboxy termini were synthesized on Wang
(p-alkoxy-benzyl-alcohol) resins (Bachem). First residues were
double coupled to the appropriate resin and subsequent residues
were single coupled. Each coupling step was followed by acetic
anhydride capping. Peptides were cleaved from the resin by
treatment with TFA (10 ml), H.sub.2O (0.5 ml), thioanisole (0.5
ml), ethanedithiol (0.25 ml), and crystalline phenol (0.75 g).
Purification was carried out by reverse phase HPLC. Approximately
50 mg samples of crude peptide were chromatographed on a Waters
DELTA PAK.TM. C18 column (19 mm.times.30 cm, 15 m spherical) using
a linear gradient: H.sub.2O/acetonitrile 0.1% TFA. Lyophilized
peptides were stored desiccated and peptide solutions were made in
water at about 5 mg/ml. Peptides stored in solution were stable for
an extended period of time at 4.degree. C. and could be repeatedly
frozen and thawed with little apparent effect on biological
activity.
[0042] The amino acid sequences of the peptides synthesized are
shown in FIG. 1. DP-107 is a 38 amino acid peptide corresponding to
residues 558 to 595 of the HIV-1 TM protein. Gallaher et al. (AIDS
Res. and Human Retro. 5, 431 (1989)) and Delwart et al. (AIDS Res.
and Human Reix-o. 6, 703 (1990)) observed that the primary sequence
of this region was strongly predictive of a helical secondary
structure and also contained a "leucine zipper" repeat. The amino
terminus of the peptide was acetylated and the carboxy terminus
amidated to reduce unnatural charge effects at those positions.
DP-107 and each of the other peptides used in this study were
purified by reverse phase HPLC and in each case the purified
peptides gave a single symmetrical peak by analytical HPLC. The
identity of each peptide was confirmed by electro-spray mass
spectrometry, which yielded the following results: DP-107: 4526.71
(calculated 4526.31); DP-121: 4510.75 (calculated 4510.27); DP-116:
2057.32 (calculated 2056.44); DP-125: 4743.46 (calculated 4743.55);
DP-31: 2479.35 (calculated 2480.93).
7. EXAMPLE
[0043] Evidence for Secondary Structure in Solution
[0044] Circular dichroism spectra were measured in 10 mM sodium
phosphate, 150 mM sodium chloride, pH 7.0 buffer at approximately
10 mM concentrations using a 1 cm pathlength cell on a Jobin/Yvon
Autodichrograph Mark V CD spectrometer. Peptide concentrations were
determined from A.sub.280 using Edlehoch's method (Biochemistry 6,
1948 (1967)).
[0045] A summary of the ultraviolet CD analyses of DP-107 is shown
in FIG. 2. The results suggest a considerable amount of secondary
structure for the peptide under physiologic conditions. The double
minima at 222 and 208 nm are characteristic of alpha helices and
mean molar ellipticity values ([Q].sub.222) of -32,000 at 0.degree.
C. (FIG. 2A) and -27,000 at 37.degree. C. (FIG. 2B) indicate that
the peptide is approximately 100% and 85% folded at these
temperatures (Y. Chen et al., Biochemistry 13, 3350 (1974); N.
Greenfield & G. Fasman, Biochemistry 8, 4108 (1969)). The
stability of the observed structure is illustrated by the thermal
melt data shown in FIG. 2C. For example, at a 10 mM concentration
of DP-107, the midpoint of the melting curve (T.sub.m) was
approximately 72.degree. C. Also apparent in FIG. 2C is that the
T.sub.m for DP-107 varies as a function of peptide concentration.
This concentration dependence is characteristic of leucine
zipper-type structures and is indicative of stabilization of
secondary structural elements by self-association (E. O'Shea et
al., Science 243, 538 (1989)). Oligomerization of DP-107 in
solution to form dimers and tetramers is also suggested by
sedimentation equilibrium studies. Taken together, the results
shown in FIG. 2 tend to support the predictions (W. Gallaher et
al., supra; E. Delwart et al., supra) that the region of gp41
corresponding to DP-107 contains a leucine zipper-like (coiled
coil) motif which may play a role in envelope oligomerization. This
type of structure can be described as a homodimer formed by the
specific (and often parallel) association of two alpha helices.
This interaction is characterized by the alignment of the
hydrophobic faces of the helices. The unusually stable secondary
structure exhibited by peptides involved in these types of systems
is due to these higher order interactions. Preliminary analysis of
the solution structure of DP-107 by multi-dimensional nuclear
magnetic resonance (NMR) spectroscopy indicates a large number of
sequential NH--NH.sub.i crosspeaks in the NOESY spectra which is
consistent with the CD evidence that under physiologic conditions,
the peptide exhibits significant a-helical secondary structure.
[0046] The CD spectra of two other synthetic peptides are also
shown in FIG. 2. One of these (DP-121) is identical to DP-107 but
with the isoleucine at position 578 replaced with a proline
residue. The other peptide, DP-116, is a 17 mer and overlaps the
carboxy terminus of DP-107. This peptide was synthesized to contain
the same amino acids and blocking groups as CS3, a peptide
described by Qureshi et al. (AIDS 4, 553 (1990)) and reported to
exhibit anti-viral activity when coupled to albumin. The CD spectra
observed for these two peptides indicate that both exist in random
coil conformations at 37.degree. C. in direct contrast to the
results obtained for DP-107. This outcome was expected for the
proline substituted analogue, DP-121, in that the proline residue
would tend to both break helix formation as well as disrupt
hydrophobic interactions thought to Stabilize coiled coil
structures.
8. EXAMPLE
[0047] Reverse Transcriptase (RT) Assay
[0048] The micro RT assay was adapted from Goff et al. (J. Virol.
38, 239 (1981)) and Willey et al. (J. Virol. 62, 139 (1988)).
Supernatants from virus/cell cultures are made 1% in Triton-X100. A
10 ml sample of supernatant was added to 50 ml of RT cocktail in a
96 well U bottom microtiter plate and the samples incubated at 370
C. for 90 min. The cocktail contained 75 mM KCl, 2 mM
dithiothreitol, 5 mm Mgcl2l 5 mg/ml poly A (Pharmacia cat. No.
27-4110-01), 0.25 units/ml oligo dT (Pharmacia cat. No.
27-7858-01), 0.05% NP40, 50 mM Tris-HCl, pH 7.8, 0.5 mM
non-radioactive dTTP, and 10 mCi/ml .sup.32p-dTTP (Amersham cat.
No. PB.10167). After the incubation period, 40 ml of reaction
mixture was applied to a Schleicher and Schuell NA45 membrane (or
DE81 paper) saturated in 2.times.SSC buffer (0.3M NaCl and 0.003M
sodium citrate) held in a Schleicher and Schuell Minifold over one
sheet of GBOO3 filter paper. Each well of the minifold was washed
four times with 200 ml 2.times.SSC. The membrane was removed from
the minifold and washed 2 more times in a pyrex dish with an excess
of 2.times.SSC. Finally the membrane was drained on absorbent
paper, placed on Whatman #3 paper, covered with saran wrap, and
exposed to film overnight.
9. EXAMPLE
[0049] HIV-1 Virus Propagation
[0050] The HIV-1.sub.LAI virus was obtained from R. Gallo (see M.
Popovic et al., Science 224, 497 (1984)) and propagated in CEM
cells cultured in RPMI 1640 containing 10-% FCS. Supernatant from
the infected CEM cells was passed through a 0.2 mm filter and the
infectious titer estimated in a microinfectivity assay using the
AA5 cell line to support virus replication. For this purpose 25 ml
of serially diluted virus was added to 75 ml AA5 cells at
2.times.10.sup.5/ml in a 96 well microtiter plate. Each virus
dilution was tested in triplicate. Cells were cultured for eight
days by addition of fresh medium every other day. On day 8 post
infection supernatant samples were tested for virus replication as
evidenced by RT activity released to the supernatant in accordance
with the procedure described above. The TCID.sub.50 was calculated
according to the Reed and Muench formula in accordance with known
techniques. See L. Reed et al., Amer. J. Hygiene 27, 493 (1938).
The titer of the HIV-1.sub.LAI stock used for these studies, as
measured on the AA5 cell line, was approximately
1.times.10.sup.7TCID.sub.50/ml. The two primary isolates were
obtained from PBMCs of two infected donors, one from Brazil
(HIV-1.sub.Br3) and the other Trinidad (HIV-1.sub.QZ2775) by
co-cultivation with PHA-blasted normal donor PBMCs in RPMI 1640
containing IL2. The infectious titers of the primary virus stocks
were estimated by titration onto normal human PHA blasted PBMCs in
a 96 well microtiter plate, again using RT activity released to the
supernatant as evidence for successful infection. The infectious
titer of both of these isolates was estimated to be approximately
1.times.10.sup.3 TCID.sub.100/ml.
10. EXAMPLE
[0051] Peptide Inhibition of Infected Cell Induced Syncytia
Formation
[0052] The initial screen for antiviral activity of the peptides
shown in FIG. 1 was for blockade of syncytium formation induced by
overnight co-cultivation of uninfected Molt4 cells with chronically
infected (HIV-1.sub.IIIB) CEM cells.
[0053] Approximately 7.times.10.sup.4 Molt cells were incubated
with 1.times.10.sup.4 CEM cells chronically infected with the
HIV-1.sub.LAI virus in 96 well plates (one-half area cluster
plates;Costar. Cambridge, Mass.) in a final volume of 100 ml
culture media in accordance with known techniques (T. Matthews et
al., Proc. Natl. Acad. Sci. U. S. A. 84, 5424 (1987)). Peptide
inhibitors were added in a volume of 10 ml and the cell mixtures
were incubated for 24 hr at 37.degree. C. At that time point,
multinucleated giant cells were estimated by microscopic
examination at a 40.times.magnification which allowed visualization
of the entire well in a single field.
[0054] The results of three such experiments are shown in Table 1.
In the first of these, serial peptide concentrations between 50
Kg/ml and 1.5 mg/ml were tested for blockade of the cell fusion
process. It is shown that DP-107 afforded complete protection down
to a concentration of 6 mg/ml. The overlapping 17 mer peptide,
DP-116, which is analogous to the previously described CS3 by
Qureshi et al. (AIDS 4, 553 (1990)) exhibited no evidence of
anti-fusogenic activity even at 50 mg/ml. This observation is in
agreement with that study which only found anti-viral activity for
CS3 after conjugation to albumin. A second peptide DP-31
representing an overlapping immunodominant site (M. Oldstone et
al., J. Virol. 65, 1727 (1991); J. Wang et al., Proc. Natl. Acad.
Sci. USA 83, 1659 (1986)) also failed to show inhibitory
activity.
2TABLE 1 Test for Peptide Blockade of HIV-1 Induced Cell-Cell
Fusion Syncytia Number Peptide Peptide Concentration
(micrograms/ml) 50 25 12 6 3 1.5 0 EXP. 1 DP-31 85 80 78 87 90 75
89 DP-116 89 82 93 92 89 82 89 DP-107 0 0 0 0 46 80 89 40 20 10 5
2.5 1.25 0 EXP. 2 DP-107 0 0 0 36 83 98 93 DP-125 0 0 0 0 3 35 93
EXP. 3 DP-121 69 71 65 60 68 ND 76 DP-125 0 0 0 0 0 0 76
[0055] The inhibitory activity of DP-107 did not appear related to
cytotoxic or cytostatic effects since in other studies CEM cells
grown in the presence of DP-107 at 50 mg/ml (the highest
concentration tested) for three days with fresh peptide added each
day displayed the same viability and growth rate as control
cultures. We also found that DP-107 blocked fusion mediated by the
other prototypic isolates: HIV-1.sub.MN, RF., and .sub.SF2.
[0056] The concentration dependence of the CD spectra described
suggests that the structure of the DP-107 is stabilized by peptide
self association. In similar studies, O'Shea et al. (Science 243,
538 (1989)) reported that disulfide bridging of a peptide of the
leucine zipper domain in the GCN4 protein (a transcriptional
regulatory factor) to form covalently bonded homodimers stabilized
the coiled coil structure. Following similar reasoning we sought to
determine if the limiting effective concentration for cell fusion
blockade by DP-107 might in part be related to the concentration
dependence of peptide self-association. In order to test this
possibility we synthesized a DP-107 analogue with a cysteine
containing "tail" which after purification could be air oxidized to
yield a homodimer. The resulting peptide, DP-125, exhibited about
twice the apparent molecular weight of DP-107 in SDS-PAGE under
non-reducing conditions indeed suggesting that a covalently bonded
homodimer was generated. In the syncytial blocking assays (Table 1
experiments 2 and 3) this analogue was, in fact, more efficacious
than DP-107, requiring one-half to one-fourth the concentration for
inhibition. The increased activity exhibited by DP-125 has
reproduced in all assays performed to date and suggests that a
dimer or higher order multimer might actually represent the
biologically active form of the peptide. Also, CD measurements of
DP-125 were found to yield similar ellipticity values as the
parental DP-107. Taken together these observations indicate that
the individual peptide components of the putative multimers are
arranged in a parallel rather than anti-parallel orientation.
[0057] To gain further insights as to whether the solution
structure observed for DP-107 in the CD and NMR studies is required
for biological activity, the proline-containing analog (DP-121)
which failed to exhibit helix related signals at 37.degree. C. in
the CD experiments was tested for activity in the cell fusion
assay. The results showed no sign of inhibitory activity as
indicated in Table 1. Although this does not prove that structure
is necessary for biological activity, it is consistent with that
possibility. In a similar fashion, each of the DP-107 peptide
analogues tested to date that failed to block cell fusion have also
failed to show evidence of stable solution structure in CD studies.
Also, a peptide which formed a coiled coil structure (GCN4-p1,
provided by R. Rutkowski) exhibited no biological activity.
11. EXAMPLE
[0058] Peptide Inhibition of Infection by Cell-Free Virus
[0059] The peptides were next tested for blockade of infection by
cell-free virus. The results shown in FIG. 3 are representative of
several experiments in which the DP-107, 125, and 116 (CS3)
peptides were compared for potency in the blockade of HIV.sub.LAI,
infection of AA5 cells. Each level of peptide was incubated in
triplicate with about 500 TCID.sub.50 of virus and cells. After 7
days of culture, cell-free supernatant was tested for the presence
of RT activity as a measure of successful infection. The results
are shown in FIG. 3 and demonstrate that both the DP-107 and DP-125
reagents inhibited the de-novo infection process at about the same
effective doses as noted in the fusion assays described above.
Moreover the dose required for inhibitory effect was lower for the
disulfide bridged DP-125 analogue and no hint of anti-viral effect
was noted for the DP-116 peptide.
12. EXAMPLE
[0060] Peptide Inhibition of PBMC Infection by Primary Isolates
[0061] It is now apparent that substantial differences in
sensitivity to anti-viral agents can exist between laboratory
adapted prototypic isolates and primary field isolates passaged
only through PBMCs. This problem was first highlighted by Ho and
colleagues (E. Dear et al., Proc. Natl. Acad. Sci. 87, 6574 (1990))
in studies with soluble CD4. To test if the gp41peptides might
exhibit a similar discordance in reactivity, the DP-107, DP-125
(cysteine analogue), and sT4 (same reagent as used in the preceding
experiment) were compared for inhibition of PBMC infection by two
primary HIV-1 isolates and HIV.sub.LAI. The results of these
studies (FIG. 5) show that the peptides inhibit both the primary
and prototypic isolates tested. Only a single dose of sT4 (10
mg/ml) was included for comparative purposes and it is apparent
that this reagent is substantially more active on the cell-line
adapted HIV-1.sub.LAI virus in comparison to the two primary
isolates.
13. EXAMPLE
[0062] Synthesis of DP-107 Analogs
[0063] Analogs of DP-107 were synthesized in accordance with known
techniques as given in Example 1 above. Such analogs are set forth
in Table 2 below.
3TABLE 2 DP-107 Analogs. DP-118
QQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKYLKDQ (SEQ ID NO:5 DP-122
GIKQLQARILAVERYLKDQQ (SEQ ID NO:6 DP-123
IEAQQHLLQLTVWGIKQLQARILAVERYLKDQ (SEQ ID NO:7 DP-127
NNLLRAIEAQQHLLQLTVWGIKQLQARILAV (SEQ ID NO:8 DP-129
NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQGGC (SEQ ID NO:9 DP-130
CGGNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQGGC (SEQ ID NO:10
14. EXAMPLE
[0064] Activity of DP-107 Analogs
[0065] The activity of various DP-107 analogs as described herein
was tested by the syncytia assay described in Example 5 above.
These data are given in Table 3 below.
4TABLE 3 No. Syncytia/well at peptide concentration, ug/ml on IIIB
Peptide [CONC] 40 20 10 5 2.5 1.25 0 DP116 8.0 mg/ml 77 DP107 4.5
mg/ml 0 0 0 4 68 88 DP118 3.9 mg/ml 0 0 77 80 78 74 DP121 3.9 mg/ml
79 DP122 3.4 mg/ml 73 DP123 4.0 mg/ml 4 55 86 76 69 80 DP125 3.1
mg/ml 0 0 0 0 0 0 DP127 3.3 mg/ml 0 0 0 20 53 87 DP129 3.4 mg/ml 0
0 0 0 0 0 DP130 4.5 mg/ml 0 0 0 0 0 47 Ctrl 85 cells only
15. EXAMPLE
[0066] Biological Activity of DP-107 and Analogs Thereof
[0067] The biological activity of DP-107 and various analogs
thereof in the fusion assay described in Example 5 above and the
neutralization assay described in Example 6 above is summarized in
Table 4 below. The quantity of compound required to produce a
ninety percent reduction in number of syncytia is shown in the
column marked "Fusion"; the quantity of compound required to
produce a ninety percent reduction in infectivity is given in the
column marked "Neutraliz". Compounds active in the fusion assay at
a range between 20 and 40 4 .mu.g/ml are considered moderately
active; compounds active at a range between 10 and 20 .mu.g/ml are
considered potent; and compounds active in an amount of less than
10 .mu.g/ml are considered very potent.
5TABLE 4 BIOLOGY OF DP-107 AND ANALOGS Fusion Neutraliz
[ug/ml].sup.1 [ug/ml (ug/ml)].sup.2 DP-107 (SEQ ID No:1)
NNLLRAIEAQQGLLQLTVWGIKQLQARILAVERYL- KDQ 5 10 DP-116 (SEQ ID No:1)
LQARILAVERYLKDQQL >40 >30 DP-121 (SEQ ID No:4)
NNLLRAIEAQQGLLQLTVWGIKQLQARILAVERYLKDQ >40 >30 DP-122 (SEQ ID
No:2) GIKQLQARILAVERYLKDQQ >40 >30 DP-123 (SEQ ID No:6)
IEAQQGLLQLTVWGIKQLQARILAVERYLKDQ 40 >30 DP-125 (SEQ ID No:7)
CGGNNLLRAIEAQQGLLQLTVWGIKQLQARILAVERYLKD- Q <1 2 DP-127 (SEQ ID
No:8) NNLLRAIEAQQGLLQLTVWGIKQLQARILAV 7 23 DP-129 (SEQ ID No:9)
NNLLRAIEAQQGLLQLTVWGIKQLQARILAVERYLKDQGGC <1 <1 DP-130 (SEQ
ID No:10) CGGNNLLRAIEAQQGLLQLTVWGIKQLQARIL- AVERYLKDQGGC 2 <1
DP-136 (SEQ ID No:11) CGGNNLLRAIEAQQGLLQLTVWGIKQLQARILAV >40
>30 DP-137 (SEQ ID No:12)
LSGIVQQQNNLLRAIEI4QQHLLQLTVWGIKQLQARILAV 12 12 DP-138 (SEQ ID
No:13) CGGLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAV >40 >30
DP-139 (SEQ ID No:14) NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ >40
>30 Dp-140 (SEQ ID No:15) NNLLRAIEAQQHLLQLTVWCIKQLQARILAVERYLKDQ
17 14 .sup.190% reduction in number of syncytia (control = 90)
.sup.290% reduction in infectivity Bold letters = non-naturally
occurring amino acid residues
[0068] The foregoing examples are illustrative of the present
invention, and are not to be construed as limiting-thereof. The
invention is defined by the following claims, with equivalents of
the claims to be included therein.
Sequence CWU 1
1
* * * * *