WO1992020704A1

WO1992020704A1 - Peptidomimetic inhibitors of hiv gp120 binding to cd4

Info

Publication number: WO1992020704A1
Application number: PCT/US1992/004210
Authority: WO
Inventors: Michael Kahn; Apichaya Raktabutr; Mark I. Greene; Horacio U. Saragovi
Original assignee: Board Of Trustees Of The University Of Illinois
Priority date: 1991-05-21
Filing date: 1992-05-19
Publication date: 1992-11-26
Also published as: AU2143292A

Abstract

The invention provides materials and methods for synthesizing novel beta-turn mimetics, as well as the novel beta-turn mimetics themselves, and peptides containing the same. The invention specifically provides beta-turn mimetics that are inhibitors of the binding of HIV gp120 to amino acid residues 40-45 of human CD4.

Description

PEPTIDOMIMETIC INHIBITORS OF HIV GP120 BINDING TO CD4

BACKGROUND OF THE INVENTION

1. Field of the Invention

The T cell surface glycoprotein CD4 acts as the cellular receptor for human immunodeficiency virus type 1 through recognition of the virus envelope glycoprotein gp 120.1 The development of agents that can inhibit

CD4-gpl20 interaction is a critical goal in the field of therapeutic approaches to AIDS treatment.

Fisher et al., Nature 221; 76-78 (1988) teaches that recombinant soluble CD4 is an inhibitor of both virus replication and syncytium formation.

Harris et al., Eur. J. Biochem. JJ58: 291-300 (1990), characterizes the 368 amino acid protein secreted from Chinese Hamster Ovary cells as recombinant CD4. Layne et al., Nature 346: 277-279 (1990), discloses a mechanism for soluble CD4 inhibition of HIV infection. Since the weight of agent that must be administered varies inversely for any given affinity with the molecular size of the agent, it is especially desirable to develop inhibitory agents smaller than intact recombinant CD4 protein.

Berger et al., Proc. Natl. Acad. Sci. USA £5_: 2357-2361 (1988), teaches that a truncated 180 amino acid fragment, representing approximately the N- terminal half of the extracellular region of CD4, is capable of binding gpl20. Jameson et al., Science 240: 1335-1339 (1988), teaches that a synthetic analog of CD4 amino acid residues 25 to 58 inhibits HIV- 1 -induced cell fusion in a concentration dependent manner, and proposes that CD4 amino acids 37-53 comprise a binding site for the AIDS virus.

Kalyanaraman et al., J. Immunol. 145.: 4072-4078 (1990), teaches that benzyl or acetyl derivatized peptides corresponding to CD4 amino acids 81-92 inhibited HIV infection in vitro, whereas no peptide corresponding to any other CD4 region, including amino acids 23-56, inhibited HIV infection. Shapira-Nahor et al., Cell. Immunol. 128: 101-117 (1990), discloses that peptides corresponding to CD4 amino acids 74-95 or 81-95 inhibit HIV infection in vitro, whereas no other peptide tested produce in vitro inhibition. Rausch et al., Ann. N.Y. Acad. Sci. 6J6; 125-148 (1990), teaches that only peptides corresponding to CD4 amino acids 81-92 or 81-101 are effective antiviral agents. Wang et al., Nature 3_48 411-418 (1990) and Ryu et al., Nature MS.:

419-426 (1990) teach that amino acids within the CDR2-like region of CD4 are involved in gpl20 binding, that the region occupies a very prominent surface exposed site in the CD4 structure, and that the hairpin loop at G1N⁴⁰- Phe ^S is highly accessible.

Although synthetic peptides hold some promise for the treatment of AIDS, they have many disadvantages as well. For example, these peptides are subject to degradation by intrinsic peptidases, reducing their in vivo half -life and thus perhaps affecting their effective therapeutic dose. Moreover, such peptides may have problems with bioavailability and antigenicity. A potential means of overcoming these problems is the use of peptide mimetics. The invention further relates to peptide mimetics, which are chemical structures which serve as appropriate substitutes for peptides in interactions with receptors and enzymes. More particularly, the invention relates to the use of peptide mimetics to inhibit viral protein-receptor interactions necessary for viral infection.

2. Summary of the Related Art

Generally, peptide mimetics can be defined as structures which serve as appropriate substitutes for peptides in interactions with receptors and enzymes. The development of rational approaches for discovering peptide mimetics is a major goal of medicinal chemistry. Such development has been attempted both by empirical screening approaches and by specific synthetic design.

Specific design of peptide mimetics has utilized both peptide backbone modifications and chemical mimics of peptide secondary structure.

Spatola, Chemistry and Biochemistry of Amino Acids. Peptides and Proteins.

Vol. VII (Weinstein, Ed.) Marcel Dekker, New York (1983), p. 267, exhaustively reviews isosteric amide bond mimics which have been introduced into biologically active peptides. The beta-turn has been implicated as an important site for molecular recognition in many biologically active peptides. Consequently, peptides containing conformationally constrained mimetics of beta-turns are particularly desirable. Such peptides have been produced using either external or internal beta-turn mimetics. External beta-turn mimetics were the first to be produced. Friedinger et al., Science 210: 656-658 (1980), discloses a conformationally constrained nonpeptide beta-turn mimetic monocyclic lactam that can readily be substituted into peptide sequences via its amino and carboxy termini, and that when substituted for Gly⁶-Leu⁷ in luteinizing hormone releasing hormone (LHRH), produces a potent agonist of LHRH activity. Monocyclic lactams have generally been useful as external beta-turn mimetics for studying receptor-peptide interactions. However, the mimetic skeleton in these molecules is external to the beta-turn, which gives rise to numerous limitations. Chief among these is bulkiness, which requires the use of dipeptide mimetics, rather than mimetics of all four residues in an actual beta-turn. Substantial flexibility retained in these beta-turn mimetics makes it unsafe to assume that expected conformations are present, absent considerable conf ormational analysis. For example, Vallee et al., Int. J. Pept. Prot. Res. 3_3_: 181-190 (1989), discloses that a monocyclic lactam beta-turn mimetic did not contain an expected type IP beta-turn in its crystal structure. Another limitation of the monocyclic lactam beta-turn mimetics arises from the difficulty of producing molecules that effectively mimic the side chains of the natural peptide. These difficulties arise from steric hindrance by the mimetic skeleton, which results in a more effective mimic of the peptide backbone than of the side chains. Considering the great importance of side chains in receptor binding, these difficulties strongly limit the versatility of monocyclic lactams.

Although the use of bicyclic lactams reduces problems of flexibility somewhat, conf ormational analysis of peptides containing these mimetics may still be desirable. Moreover, the side chain hindrance in these molecules may be even worse than that in the monocyclic lactams. Finally, both monocyclic and bicyclic lactams mimic only type II and type IF beta-turns, whereas type I and type III beta-turns are more prevalent in proteins and presumably in peptides.

The limitations presented by external beta-turn mimetics may be minimized by using mimetics in which the mimetic skeleton approximately replaces the space that was occupied by the peptide backbone in the natural beta-turn. Such molecules are known as internal beta-turn mimetics. Internal beta-turn mimetics may not generally reproduce the geometry of the peptide backbone of the particular beta-turn as accurately as external beta- turn mimetics. However, the internal position of the constraint allows replacement of larger sections of peptide, thus making tetrapeptide mimetics possible. The lack of bulk also diminishes the likelihood of steric hindrance of the side chains by the mimetic skeleton.

Internal beta-turn mimetics having biological activity are known in the art. For example, Krstenasky et al., Biochem. Biophys. Commun. 109: 1368-1374 (1982), discloses a leucine enkephalin analog in which an internal beta-turn mimetic replaced the residues Gly²-GIy^s-Phe⁴-Leu^s, and which acted as an analgesic with one-third the potency of morphine. Other internal beta-turn mimetics have been described.

Kahn et al., Tetrahedron Lett. 27: 4841-4844 (1986), discloses an internal beta-turn mimetic, based upon an indolizidinone skeleton, and designed to mimic the lysine and arginine side-chain disposition of the immunosuppressing tripeptide Lys-Pro-Arg.

Kahn et al., Heterocycles 25: 29-31 (1987), discloses an internal beta- turn mimetic, based upon an indolizidinone skeleton, and designed to correctly position the aspartyl and arginyl side chains of a beta-turn in the proposed bioactive region of erabutoxin.

Kahn et al., Tetrahedron Lett. 28: 1623-1626 (1987), discloses a type I beta-turn mimetic which can be incorporated into a peptide via its amino and carboxyl termini, and which is designed to mimic an idealized type I beta- turn. See also Kahn et al., J. Am. Chem. Soc. HO: 1638-1639 (1988); Kahn et al., J. Mol. Recogn. V. 75-79 (1988).

Similarly, Kemp et al., Tetrahedron Lett. 29: 5057-5060 (1988), discloses a type II beta-turn mimetic which can be incorporated into a peptide via its amino and carboxyl termini. Arrhemius et al., Proc. Am. Peptide Symp., Rivier and Marshall, Eds.,

Escom, Leiden (1990), discloses substitution of an amide-amide backbone hydrogen bond with a covalent hydrogen bond mimic to produce an alpha- helix mimetic.

Thus, there have been numerous successes in obtaining mimetics which can force or stabilize peptide secondary structure. However, inhibition of the binding of a virus receptor by a peptide mimetic is not known in the art. In view of the devastating effect of HIV on infected individuals, there is a great need for therapeutic agents capable of inhibiting HIV infection. Peptide mimetics capable of inhibiting the binding of gpl20 to the CD4 molecule would provide a potentially life saving therapeutic. For recent reviews of the related art, see Hruby et al., Biochem. J. 268: 249- 262 (1990); Ball et al., J. Mol. Recogn. 3_: 55-64 (1990); Morgan et al., Ann. Rep. Med. Chem.24: 243-252 (1989); and Fauchere, Adv. Drug Res.ϋ: 29-69 (1986).

BRIEF SUMMARY OF THE INVENTION

The invention provides peptidomimetics that are capable of inhibiting CD4 binding to gpl20. CD4 is a glycoprotein found on the surface of T lymphocytes; during HIV infection in humans, CD4 is the receptor for the gpl20 envelope glycoprotein of HIV. Extensive mutagenesis and peptide mapping experiments have mapped a critical binding region of CD4 to a single stretch of amino acids. Recent X-ray analysis has shown that within this stretch, residues 40-45 exhibit a beta-turn conformation. The invention provides a novel approach to synthesize conformationally restricted peptidomimetics of chain reversals in peptides and proteins. Thus the invention provides a method for the design and synthesis of a mimetic of residues Gln⁴⁰-Thr⁴⁵, which form a turn between the C and C beta-strands of CD4. The method further provides a mimetic that inhibits binding of soluble gpl20 to cells expressing CD4 at micromolar concentrations, and will provide a foundation for the development of low molecular weight gpl20 binding inhibitors as therapeutic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Basic structure of mimetic ring system for residues 40-

45 of CD4.

FIG. 2 Full CD4 loop region mimetic structure; R₃ = CH₂OH or CH₂(CH)(CH₃)₂.

FIG. 3 Scheme for synthesizing peptidomimetic 1 of Figure 2.

FIG. 4 Data showing inhibition of gpl20 binding by soluble

CD4 or by peptidomimetics of the invention.

DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The invention provides a peptide mimetic (or peptidomimetic) capable of inhibiting CD4 binding to gpl20. Infection of human mononuclear cells by human immunodeficiency virus (HIV) requires the binding of the viral envelope glycoprotein gp 120 to CD4, a cell surface glycoprotein that is found principally on the helper class of T-lymphocytes. Thus, a substance that could mimic CD4 in its ability to bind gpl20 could potentially provide a valuable therapeutic. Amino acids within the CDR2-like region of CD4 have been shown to be involved in gp 120- binding. This region occupies a very prominent surface exposed site in the CD4 structure. In particular, the hairpin loop at Gln ⁰-Phe⁴³ is highly accessible. The base structure of the mimetic ring system having inhibitory effect on gpl20 binding is shown in Figure 1, where X represents -NH- or CH₂; ΕL_j^ and R₂ are residues of amino acids; and R_s is -CH₂OH or lower alkyl having 1 to 6 carbon atoms, preferably isobutyl. The full CD4 loop region mimetic structure (Mimetic 1) is shown in Figure 2, where Bn represents benzyl. Mimetics according to the invention inhibit gp 120 binding in a concentration dependent manner, with an IC_εo in the micromolar range or better, as shown in Figure 4. The invention provides a novel method to synthesize conformationally restricted peptidomimetics of chain reversals in peptides and proteins. The method of the invention provides predictable variation of side chain orientation, backbone conformations and distances. The method of the invention allows the design and synthesis of a peptidomimetic of residues Gln ⁰-Thr⁴⁵ in the C'C CDR2-like loop of CD4, which incorporates a conformationally restricted type II beta-turn mimetic. At micromolar concentration, the peptidomimetic abrogates binding of soluble gpl20 cells expressing CD4 on their surface.

An example of the method is shown in Figure 3. A third aspect of the invention provides pharmaceutical formulations suitable for use in the treatment of AIDS. Such pharmaceutical formulations comprise the hpl20-binding inhibitory peptide mimetics of the invention in a physiologically acceptable carrier or diluent.

The following examples are provided to further illustrate the invention and are not limiting in nature. EXAMPLE I Synthesis of an Inhibitory Peptide Mimetic This scheme is illustrated in Figure 3. Azetidinone 2 was prepared in

6 steps (22% yield) from dimethyl-aspartate in an analogous fashion to procedures described by Saltzmann et al., J. Amer. Chem. Soc. 102:6161 (1980), and Williams et al., J. Amer.Chem. Soc. ill: 1073 (1989). Mixed anhydride coupling of 2 to O-benzyl serine benzyl ester and subsequent hydrogenolytic cleavage of the benzyl ester provided acid 4 in 88% yield. Coupling of the Z protected phenylalanine hydrazine 5, reductive closure and saponif ication afforded the 10-membered ring beta-turn mimetic 6 in 53% yield. Dipeptide

7 was coupled to 6 using an aqueous carbodiimide protocol. Hydrogenolytic deprotection and purification by reverse phase HPLC provided homogeneous material which was characterized by 400 MHz NMR and PDMS.

EXAMPLE 2 Assessment of Inhibition of gp!20 Binding

For measuring bindings gpl20 was incubated with the mimetics or with soluble CD4 at 22°C in binding buffer (Ca²⁺, Mg²⁺ free HBSS, 0.5% BSA, 0.05% sodium azide, pH 7.4). Approximately 300,000 cells (from a lOxlO⁷ cell/ml stock) were added to tubes at 4°C in binding buffer, with a final volume of 100 microliters. Samples were incubated at 4°C for 40 min. washed in binding buffer and analyzed in FACS immediately. Data was acquired, gating on live cell population (always greater than 90%), and was consistent whether mimetics, gpl20 or other agents were added or not. Results are shown in Figure 4. Inhibition by Mimetic 1 was concentration dependent, with an IC₅₀ of 0.8 micromolar.

Claims

WHAT IS CLAIMED IS:

1. An inhibitor of the binding CD4 to gpl20 comprising a cyclic structure which is a beta-turn mimetic of amino acid residues 40-45 of CD4.

2. A compound according to claim 1 which is

wherein R_x and R₂ are residues of amino acids; R_s is -CH₂OH or lower alkyl having 1 to 6 carbon atoms; and X represents CH₂ or NH.

3. A compound according to claim 2 which is the compound of Figure 2, wherein R₃ is -CH₂OH or isobutyl and Bn is benzyl.

4. A pharmaceutical composition comprising a CD4 inhibitory amount of a compound of claim 1 in a pharmaceutical carrier.

5. A pharmaceutical composition comprising a CD4 inhibitory amount of compound of claim 2 in a pharmaceutical carrier.

6. A pharmaceutical composition comprising a CD4 inhibitory amount of a compound of claim 3 in a pharmaceutical carrier.