WO1991017762A1

WO1991017762A1 - Selective retroviral proteinase inhibitor

Info

Publication number: WO1991017762A1
Application number: PCT/US1991/003452
Authority: WO
Inventors: Terry D. Copeland; Ewald M. Wondrak; Jozsef Tozser; Michael M. Roberts; Stephen Oroszlan
Original assignee: The United States Of America, Represented By The Secretary, United States Department Of Commerce
Priority date: 1990-05-24
Filing date: 1991-05-17
Publication date: 1991-11-28
Also published as: AU8089191A

Abstract

An inhibitor of retroviral proteinase comprises a peptide of the formula: (Z)nX-Y(Z)m wherein X is any naturally occurring amino acid (such as tyrosine or phenylalanine) or modified amino acid; Y is an α-imino acid having a 4-, 6-, 7- or 8-membered ring structure; Z is an α-amino acid; and n and m are independently 0 to 7 when the N- or C-terminal amino acids are carrying free NH2 or carboxyl or amide groups, or when the terminal amino acids are blocked at the NH2 or carboxy end. These peptides serve as substrates for the retroviral proteinases and thereby inhibit retroviral proteinase activity without inhibiting nonretroviral proteolytic enzymes of the same family, that is, aspartic proteinases. The peptides selectively inhibit retroviral proteinases (PR) such as HIV-1 PR, HIV-2 PR and EIAV PR. Pharmaceutical compositions comprising the inhibitor and methods of treatment involving same are included.

Description

SELECTIVE RETROVIRAL PROTEINASE INHIBITOR

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a selective inhibitor of retroviral proteinase (PR) . In particular, the present invention relates to a selective inhibitor of

HIV-l PR, HIV-2 PR and EIAV PR.

Background Information During the maturation of retroviruses, the virally encoded proteinase (PR) cleaves the Gag and Gag- Pol polyproteins into smaller units to produce the struc¬ tural proteins and enzymes of the infectious virion. A striking feature of this proteolytic processing event is that it involves cleavage at the NH side of proline residues in all retroviruses. Without exception, the N terminus of all retroviral capsid (CA) proteins is pro¬ line. In many retroviruses, similar cleavages occur at more than a single site. For example, in the mammalian C-type viruses the Gag polyprotein is cleaved twice at proline residues and in the human immunodeficiency virus type-1 (HIV-l) the PR and reverse transcriptase (RT) have a proline N terminus [Copeland, et al.. Gene Anal. Techn. 5:109-115 (1988); and Veronese, et al.. Science 231:1289- 1291 (1986)]. Since cellular proteinases have not been known to efficiently hydrolyze peptide bonds involving the proline nitrogen, it was this unusual proteolytic cleavage that suggested that a "unique" virus-specified proteinase might be involved in the proteolytic processing of retroviral polyproteins [Oroszlan et al., Proc. Natl. Acad. Sci. USA 75:1404-1408 (1978)]. Oligopeptides having the naturally occurring Phe-Pro or Tyr-Pro cleavage sites were found to be good substrates for both HIV-l and HIV-2 proteinases [Copeland, et al.. Gene Anal. Techn. 5:109-115 (1988); Darke et al., Biochem. Biophys. Res. Commun. 156:297-303 (1988); and Moore et al., Biochem. Biophys. Res. Commun_ 159:420-425 (1989)]. A pressing health issue worldwide is the AIDS epidemic. The retrovirus human immuno-deficiency virus

(HIV) is the etiologic agent of AIDS. At present, there is no known cure for AIDS. There is also no vaccine or means for immunizing against HIV infection.

The HIV PR is a potential target for chemother¬ apy [Krausslich et al.. In Current Communications in Molecular Biology: Viral Proteinase as Targets for Chemotherapy. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)]. In order to design drugs effective against HIV, inhibitors must be specific. That is, they must be able to inhibit the retroviral PR and yet not interfere with other essential cellular and extracel¬ lular proteinases. Proline (2-pyrrolidinecarboxylic acid) and

4-hydroxyproline are the only imino acids naturally occur¬ ring in proteins. They have a five membered ring struc¬ ture. In the present invention, the size of the ring structure of the proline residue at the retroviral PR cleavage site is modified, providing a selective inhibitor of the retroviral proteinase, such as, for example, HIV PR.

SUMMARY OF THE INVENTION It is an object of the present invention to provide therapeutic agents for the treatment of retroviral infections, particularly HIV infection.

It is another object of the present invention to provide a means for inhibiting retroviral proteinases.

In one embodiment, the present invention relates to an inhibitor of retroviral proteinase comprising a peptide of formula I:

(Z)_nX-Y(Z)_m (I) wherein

X is any naturally occurring amino acid (such as tyrosine or phenylalanine) or modified amino acid;

Y is an α-imino acid having a 4-, 6-, 7-or 8- membered ring structure; Z is an α-amino acid; and n and m are independently 0 to 7 when the N- or

C- terminal amino acids are carrying free NH₂ or carboxyl or amide groups, or when the terminal amino acids are blocked at the NH₂ or carboxy end.

In another embodiment, the present invention relates to an anti-retroviral composition comprising an amount of the retroviral inhibitor of the present inven- tion sufficient to inhibit the retroviral proteinase, and a pharmaceutically acceptable carrier.

In a further embodiment, the present invention relates to a method of treating retroviral infection comprising administering to a patient in need of such treatment the composition of the present invention in an amount sufficient to effect the treatment.

Various other objects and advantages of the present invention will be apparent to one skilled in the art from the drawings and the description of the invention that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the structure of proline and pipecolic acid (Pip) and the amino acid sequence of a nonapeptide substrate (SP-211) and inhibitor SP-346. Notations according to Schechter et al. [Biochem. Biophys.

Res. Commun. 27:157-162 (1967)].

Figure 2 shows an SDS-PAGE analysis of the protein components of equine infectious anemia virus

(EIAV) capsids after incubation at 37°C in Tris-EDTA buffer containing 10 mM DTT, pH 7.6 for 0 hr, 6 hr without inhibitor (-) and 6 hr with inhibitor SP-346 (+) . The total protein concentration of the capsids during the incubation was 300 μg/ml and that of the inhibitor ranged in various experiments from 100 μg - 750 μg/ml. The minimum concentration of SP-346 tested gave results similar to that shown here with 750 μg/ml. M: molecular weight markers; the numbers to the left are the respective molecular weights x 10^"3. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an inhibitor of retroviral proteinases, such as HIV-l, HIV-2, and EIAV proteinases, comprising a peptide of formula I: (Z)_nX-Y(Z)_m (I) wherein

X is any naturally occurring amino acid (such as tyrosine or phenylalanine) or modified amino acid including modifications of the scissle bond between X-Y to provide transition state analogs of substrate;

Y is an α-imino acid having a 4-, 6-, 7-or 8- membered ring structure; Z is an α-amino acid; and n and m are independently 0 to 7 when the N- and/or C- terminal amino acids are carrying free

NH₂ and/or carboxyl/amide groups, or when the terminal amino acids are appropriately blocked at the NH₂ and/or carboxyl end. Y may be unsubstituted or substituted by, for example, methyl or longer aliphatic groups at carbon 3 and/or 4 of the 4 membered ring, at carbon 3, 4, 5 and/or 6 of the 6 membered ring, at carbon 3, 4, 5, 6 and/or 7 carbon of the

7 membered ring, at carbon 3, 4, 5, 6, 7 and/or 8 of the 8 membered ring or any other substitutions at the above carbons which would provide a tighter binding inhibitor including substitutions capable of forming a covalent bond with the enzyme. When n and/or m are greater than zero, the peptide can have as Z any amino acid(s) so long as the resulting peptide can function as an inhibitor of the retroviral proteinase.

The term "scissle bond" as used herein refers to the bond between proline and the amino acid immediately next to it on its N-terminus side (see in Figure 1, scissle bond is between PI and PI'). Naturally, the scissle bond is formed in the retroviral cleavage site between an amino acid, preferably tyrosine or phenylala¬ nine, and proline. However, in the inhibitory peptide of the present invention, the proline residue of the scissle bond is replaced with an α-imino acid having a 4-, 6-, 7- or 8-membered ring, such as, for example, L-pipecolic acid (2-piperidine-carboxylic acid) . Incorporation of such a proline analog, with its differing ring size, into a peptide results in a selective inhibitor of retroviral proteinases which does not affect cellular proteinases.

The inhibitor of the present invention comprises at least the two amino acids forming the "scissle bond" and may comprise up to an additional 14 amino acids. Preferably, the peptide comprises 9 amino acids, and most preferably, 4 amino acids before the scissle bond-forming amino acid pair and 3 amino acids following the scissle bond-forming amino acid pair. The present invention further relates to a pharmaceutical composition suitable for use in treating a retroviral infection comprising the above-described inhibitor in a anti-retrovirally effective amount, togeth¬ er with a pharmaceutically acceptable carrier. Suitable carriers include, but are not limited to reagents which facilitate cellular uptake of the active inhibitor.

The present invention also relates to a method of treating retroviral infection comprising administering to a patient with such an infection the therapeutic composition of the present invention, in an amount suffi¬ cient to inhibit retroviral function. Preferably, between 1 μg and 5 g of the composition is administered per kg of the patient.

The following non-limiting examples are provided to aid in the understanding of the present invention.

EXAMPLES Synthetic Peptides

Peptides were synthesized on solid supports [Merrifield, R.B. J. Am. Che . Soc. 85:2149-2154 (1963)]. Synthetic peptide SP-211 was described elsewhere [Copeland, et al.. Gene Anal. Techn. 5:109-115 (1988)]. L-Pipecolic acid and 2-(tert-butoxycarbonyloxyimino)-2- phenylacetonitrile) (BOC-ON) were purchased from Aldrich Chemical Co. (Milwaukee, WI) . L-Pipecolic acid was protected at the N terminus using BOC-ON as recommended by the manufacturer. SP-346 was synthesized as SP-211 except that the coupling time for Boc-pipecolic acid was 3 times that of Boc-Pro. Purified peptides were taken for amino acid analysis and automated Edman N-terminal amino acid sequence determination in a gas phase sequenator [Hewick et al., J. Biol. Chem. 256:7990-7997 (1981)].

The proline residue of a nonapeptide substrate (SP-211) for HIV-l and related retroviral proteinases, representing the cleavage site at the junction of the matrix (MA) and CA proteins in HIV-l Gag precursor polyprotein was replaced by L-pipecolic acid. The struc¬ tures of proline and pipecolic acid together with the amino acid sequences of the synthetic substrate (SP-211) and its analog (SP-346) are shown in Figure 1. Retroviral Proteinases and Their Assays

Recombinant HIV-l PR [Louis et al., Biochem. Biophys. Res. Commun. 159:87-94 (1989)] and EIAV PR from a virus were purified by subsequent chromatography on a hydrophobic interaction column, pepstatin A agarose, and a cation exchange column. HIV-2 PR was chemically synthe¬ sized [Copeland, et al.. Gene Anal. Techn. 5:109-115 (1988)], purified by RP-HPLC, refolded from guanidine-HCl and further purified by gel filtration. Proteinase activity was assayed with a synthetic nonapeptide sub¬ strate (SP-211) as described earlier [Copeland, et al.. Gene Anal. Techn. 5:109-115 (1988); and Louis, J.M. , et al., Biochem. Biophvs. Res. Commun. 159:87-94 (1989)]. Pepsin. Cathepsin D. Renin. Trypsin and Chymotrypsin Assays

These enzymes were purchased from Sigma and assayed according to published methods [Will et al., Clin.

Chem. 30:707-711 (1984); Agarwal et al., Anal. Biochem. 130:158-165 (1983); Galen et al., Biochem. Biophvs. Acta

523:485-493 (1978); Nelson et al., Proc. Natl. Acad. Sci.

USA 78:1670-1674 (1981); and Starkey, P.M., In Proteinases in Mammalian Cells and Tissues (A.J. Barrett, Ed.), pp. 57-89. North-Holland, New York, NY (1977)]. SP-211 cleavage by these proteinases was assayed under the conditions described in the above references using RP-HPLC to detect cleavage products. EIAV Capsid

Capsids of EIAV were prepared as previously described [Roberts et al., Biochem. Biophvs. Res. Commun. 160:486-494 (1989)]. These capsides were incubated at 37°C in 10 mM Tris HC1 1 mM EDTA buffer at pH 7.6 con¬ taining 10 mM DTT. The extent of the nucleocapsid (NC) protein cleavage was determined by SDS-PAGE as described [Roberts et al., Biochem. Biophys. Res. Commun. 160:486- 494 (1989)].

When tested with retroviral PRs the rate of cleavage of SP-346, as compared with that of SP-211, was reduced to such an insignificant level (100 fold or more) that the peptide could not be considered as a suitable substrate anymore. However, SP-346 was found to be an effective inhibitor of the SP-211 cleavage. The results are shown in Table 1 below.

TABLE 1. The effect of SP-346 on retroviral and cellular (pepsin-like) aspartic proteinases

Substrate Cleaved Inhibition nmolxs"¹ xmg"¹ by SP-346

Enzyme pH SP-211 Other IC₅₀ μM

^a Substrates other than SP-211 were: azocoll (Sigma) for pepsin, H-Phe-Ala-Ala-pN0₂-Phe-Phe-Val-Leu-4-hydroxy methyl pyridine ester (Bachem Bioscience) for cathepsin D and synthetic porcine renin substrate tetradecapeptide (Sigma) for renin. ^b nt, not tested ^c Expressed as mg of substrate hydrolyzed per second per mg of enzyme. ^d nc, not cleaved ^e ni, not inhibited at 300 μM, the highest concentration tested The IC₅₀ values were determined to be 1.4 μM for HIV-l PR, 0.6 μM for HIV-2 PR and 1.7 μM for the PR of the related lentivirus, equine infectious anemia virus (EIAV) .

It was previously found that the nonapeptide substrate SP-211, was not hydrolyzed by cellular enzymes present in lysates of E. coli cells [Louis et al., Biochem. Biophvs. Res. Commun. 159:87-94 (1989)]. To further examine the resistance of SP-211 to a number of nonretroviral proteolytic enzymes, the susceptibility of SP-211 to cellular (pepsin-like) aspartic proteinases (since the retroviral PR belongs to this family of en- zymes) was studied. As shown in Table 1, the results indicated that neither renin nor cathepsin D cleaved SP- 211. The value found for the substrate cleavage by pepsin was very small. Further as expected SP-211 was also resistant to serine proteinases such as trypsin and chymotrypsin.

The specificity of the SP-346 retroviral PR inhibitor was studied with the same set of cellular enzymes at the optimal pH in assays utilizing suitable substrates (see Table 1) . The results (presented in Table 1) clearly showed that SP-346 did not inhibit any of these nonviral proteinases with the exception of a very slight inhibitory effect against pepsin. The assayed serine proteinases were also not inhibited. Thus, the nonapeptide SP-346 containing the L-pipecolic acid residue instead of L-proline in the PI' position is a highly selective substrate based inhibitor of the proteinase of HIVs and EIAV. The synthetic peptide substrate used in these studies represents a maturation cleavage site.

Recent studies indicated that the retroviral PR in addition to its crucial role in virus maturation may also have an important function in the early phase of virus replication. This involves the in situ cleavage of the nucleocapsid (NC) protein inside the viral capsid [Roberts et al., Biochem. Biophvs. Re«- cmrnn^p. 160:486- 494 (1989)]. To test the effect of the inhibitor SP-346 on this cleavage, purified capsids of EIAV were used and the results are shown in Figure 2. As can clearly be seen, in the absence of inhibitor, incubation of the capsids in Tris-EDTA, pH 7.6 buffer containing DTT result¬ ed in cleavage of the NC protein pll to smaller peptides (band labeled p4) with p6 as the intermediate. The cleavage was inhibited by SP-346. These results demon¬ strate that the SP-346 also effectively inhibits the cleavage of a natural substrate of EIAV PR.

All publications mentioned hereinabove are hereby incorporated by reference.

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. An inhibitor of retroviral proteinase comprising a peptide of formula I:

(Z)_nX-Y(Z)_m (I) wherein

X is any naturally occurring or modified amino acid;

Y is an α-imino acid having a 4-, 6-, 7-or 8- membered ring structure;

Z is an α-amino acid; and n and m are independently 0 to 7 when the N- or

2. The inhibitor according to claim 1 wherein X is tyrosine or phenylalanine.

3. The inhibitor according to claim 1 wherein said Y is substituted by methyl or longer aliphatic groups at carbon 3 or 4 of the 4 membered ring, at carbon 3, 4, 5 or 6 of the 6 membered ring, at carbon 3, 4, 5, 6 or 7 of the 7 membered ring, and at carbon 3, 4, 5, 6, 7 or 8 of the 8 membered ring.

4. The inhibitor according to claim 1 wherein n is 4 and is 3.

5. The inhibitor according to claim 4 which is H-Val-Ser-Gln-Asn-Tyr-Pip-Ile-Val-Gln-NH₂.

6. An anti-retroviral composition comprising an amount of said inhibitor according to claim 1 suffi¬ cient to inhibit retroviral proteinase, and a pharmaceu¬ tically acceptable carrier.

7. A method of treating retroviral infection comprising administering to a patient in need of such treatment an inhibitor according to claim 1 in an amount sufficient to effect said treatment.

8. The method according to claim 7 wherein said retrovirus is human immunodeficiency virus-1, human immunodeficiency virus-2 or equine infectious anemia virus.

9. The inhibitor of claim 1 for use in treat¬ ing a retroviral infection in a patient.

10. The inhibitor of claim 9, wherein said retroviral infection is caused by human immunodeficiency virus-1, human immunodeficiency virus-2, or equine infec¬ tious anemia virus.

11. Use of the inhibitor of claim 1 in the manufacture of a medicament for treating a retroviral infection in a patient.

12. The use according to claim 11, wherein said retroviral infection is caused by human immunodeficiency virus-1, human immunodeficiency virus-2, or equine infec¬ tious anemia virus.