The rational design of drugs that can inhibit the action of viral proteases depends on obtaining accurate structures of these enzymes. The crystal structure of chemically synthesized HIV-1 protease has been determined at 2.8 angstrom resolution (R factor of 0.184) with the use of a model based on the Rous sarcoma virus protease structure. In this enzymatically active protein, the cysteines were replaced by alpha-amino-n-butyric acid, a nongenetically coded amino acid. This structure, in which all 99 amino acids were located, differs in several important details from that reported previously by others. The interface between the identical subunits forming the active protease dimer is composed of four well-ordered beta strands from both the amino and carboxyl termini and residues 86 to 94 have a helical conformation. The observed arrangement of the dimer interface suggests possible designs for dimerization inhibitors.

The structure of a complex between a peptide inhibitor with the sequence N-acetyl-Thr-Ile-Nle-psi[CH2-NH]-Nle-Gln-Arg.amide (Nle, norleucine) with chemically synthesized HIV-1 (human immunodeficiency virus 1) protease was determined at 2.3 A resolution (R factor of 0.176). Despite the symmetric nature of the unliganded enzyme, the asymmetric inhibitor lies in a single orientation and makes extensive interactions at the interface between the two subunits of the homodimeric protein. Compared with the unliganded enzyme, the protein molecule underwent substantial changes, particularly in an extended region corresponding to the "flaps" (residues 35 to 57 in each chain), where backbone movements as large as 7 A are observed.

The crystal structure of a complex between chemically synthesized human immunodeficiency virus type 1 (HIV-1) protease and an octapeptide inhibitor has been refined to an R factor of 0.138 at 2.5-A resolution. The substrate-based inhibitor, H-Val-Ser-Gln-Asn-Leu psi [CH(OH)CH2]Val-Ile-Val-OH (U-85548e) contains a hydroxyethylene isostere replacement at the scissile bond that is believed to mimic the tetrahedral transition state of the proteolytic reaction. This potent inhibitor has Ki less than 1 nM and was developed as an active-site titrant of the HIV-1 protease. The inhibitor binds in an extended conformation and is involved in beta-sheet interactions with the active-site floor and flaps of the enzyme, which form the substrate/inhibitor cavity. The inhibitor diastereomer has the S configuration at the chiral carbon atom of the hydroxyethylene insert, and the hydroxyl group is within H-bonding distance of the two active-site carboxyl groups in the enzyme dimer. The two subunits of the enzyme are related by a pseudodyad, which superposes them at a 178 degrees rotation. The main difference between the subunits is in the beta turns of the flaps, which have different conformations in the two monomers. The inhibitor has a clear preferred orientation in the active site and the alternative conformation, if any, is a minor one (occupancy of less than 30%). A new model of the enzymatic mechanism is proposed in which the proteolytic reaction is viewed as a one-step process during which the nucleophile (water molecule) and electrophile (an acidic proton) attack the scissile bond in a concerted manner.

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...