X-ray crystallographic structure of a complex between a synthetic protease of human immunodeficiency virus 1 and a substrate-based hydroxyethylamine inhibitor.

Abstract

The structure of a crystal complex of the chemically synthesized protease of human immunodeficiency virus 1 with a heptapeptide-derived inhibitor bound in the active site has been determined. The sequence of the inhibitor JG-365 is Ac-Ser-Leu-Asn-Phe-#[CH(OH)CH2N]-Pro-Ile-ValOMe; the K; is 0.24 nM. The hydroxyethylamine moiety, in place of the normal scissile bond of the substrate, is believed to mimic a tetrahedral reaction intermediate. The structure of the complex has been rermed to an R factor of 0.146 at 2.4A resolution by using restrained least squares with rms deviations in bond lengths of 0.02 A and bond angles of 4'. The bound inhibitor diastereomer has the S configuration at the hydroxyethylamine chiral carbon, and the hydroxyl group is positioned between the active site aspartate carboxyl groups within hydrogen bonding distance. Comparison of this structure with a reduced peptide bond inhibitor-protease complex indicates that these contacts confer the exceptional binding strength of JG-365.Reverse transcriptase, integrase, and protease are the three virally encoded enzymes necessary for replication of human immunodeficiency virus 1 (HIV-1), so each is a potential target for drug design. Rational design of drugs directed against AIDS would be greatly facilitated by knowledge of the three-dimensional structures of the target molecules, yet the protease is the only one of these enzymes for which the structure of the native form (1-3) or of an inhibitor complex (4) is known. The protease is a member of the wellcharacterized family of aspartic proteases, which also includes mammalian enzymes such as renin, pepsin, and chymosin. Whereas cell-encoded aspartic proteases are monomers with distinct amino and carboxyl domains, the retroviral proteases are dimers of identical subunits that are analogous to these domains (5). The function of the HIV-1 protease is to cleave the translated viral gag-pol polyprotein into discrete components. Without protease activity, the viral particle remains noninfective (6, 7) and this property makes the protease an attractive candidate for therapeutic drug design against AIDS (8)(9)(10)(11)(12)(13)(14).Inhibitors -of aspartic proteases have been developed as potential pharmaceutical agents for modulating the biological processes catalyzed by this class of enzymes (15,16 Herein we report the structure of a synthetic HIV-1 protease complexed with an HEA inhibitor, JG-365, and compare it with a complex structure in which the reduced peptide bond inhibitor MVT-101 was 3000 times less potent (4). The results substantiate a previously proposed mechanism of action for this class of enzymes (23) protease (where Aba is L-a-amino-n-butyric acid) was chemically synthesized as previously described (24,25). The sequence used was that of the SF2 isolate with cysteines replaced by L-a-amino-n-butyric acid (2). The inhibitor JG-365 was synthesized as described (22) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby...

Crystal structure of a complex of HIV‐1 protease with a dihydroxyethylene‐containing inhibitor: Comparisons with molecular modeling

“…Of course, in the orthorhombic space group to which these crystals belong, molecules 1 and 2 are distinct crystallographically and make different lattice contacts. As judged by the definition above, these two molecules are similar to those previously observed in the same space group for MVT-101 , JG-365 (Swain et al, 1990), and U85548e (Jaskolski et al, 1991). Although this result might indicate strict correlation between crystal packing forces and orientation of the flaps, recent results from this and other laboratories have indicated that this is not the case, because in some other isomorphous structures the opposite arrangement of the two molecules was also observed (J.K.M.…”

“…The distance between OPl(203) and OP2(204) is 2.6 A and the torsion around (H0)C-C(0H) is 50" (Kinemage 1). The two asymmetric centers (C * 203,204) at the scissile bond both have the R configuration, unlike some previously reported inhibitors with one OH substituent at the scissile bond, such as hydroxyethylamine (Swain et al, 1990) and hydroxyethylene (Jaskolski et al, 1991), which have the S diastereomers preferentially bound by the enzyme. However, the R configuration reported here is equivalent to S in the inhibitors mentioned above, with the difference being the result of the nomenclature only.…”

Structural engineering of the HIV‐1 protease molecule with a β‐turn mimic of fixed geometry

“…The synthetic enzyme has been prepared in crystalline form and was used to solve the three-dimensional structure of both the native molecule and the enzyme complexed with several dif- ferent substrate-derived inhibitors Swain et al, 1990;Jaskolski et al, 1991). The chemical synthesis approach brings the entire world of organic chemistry into the realm of proteins.…”

“…The range of chemical alterations that can be made to a native protein is limited only by the imagination and by any peculiarities of the particular chemical methodology being employed. In addition to the synthetic accessibility of the HIV-1 protease, knowledge of its three-dimensional structure Wlodawer et al, 1989;Erickson et al, 1990;Swain et al, 1990;Bone et al, 1991;Jaskolski et al, 1991) is invaluable in designing experiments to probe the interrelationship of enzyme structure with catalytic function.…”

“…However, in the numerous protease-inhibitor complexes that have been solved, the geometry of this turn varies and is not necessarily identical in both subunits. For example, in the complex with JG-365, the geometries are type I' and 11' (Swain et al, 1990); with U85548e, type I' and I1 (Jaskolski et al, 1991); and with MVT-101, type I' and IV (Milier et al, 1989). In all cases, the residues of these turns possess high thermal factors, indicating high mobility (Jaskolski et al, 1991).…”