Title:  Compounds which inhibit HIV replication

United States Patent:  6,573,078

Issued:  June 3, 2003

Inventors:  Wild; Carl T. (Durham, NC); Matthews; Thomas J. (Durham, NC); Bolognesi; Dani P. (Durham, NC)

Assignee:  Duke University (Durham, NC)

Appl. No.:  464003

Filed:  June 2, 1995

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.

SUMMARY OF THE INVENTION

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:

NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ (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").

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:

NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ (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).

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:

NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ (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.

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.

DETAILED DESCRIPTION OF THE INVENTION

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_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.

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).

The biological activity of the eptide 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 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.

As noted above, 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:

NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ (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: I)

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.

Peptides of the invention may be provided as multimers, particularly as dimers and tetramers. When provided in such form the multimer may 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.

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.

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.

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_m of the heterodimer will be observed (this T_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_m 's) will be observed.

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 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.

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., dimer, 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.

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:

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).

Claim 1 of 26 Claims

What is claimed is:

1. An isolated peptide having the DP-107 amino acid sequence listed in SEQ ID NO:1.
 

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.