STRUCTURE OF HIV-1 PROTEASE WITH KNI-272, A TIGHT-BINDING TRANSITION-STATE ANALOG CONTAINING ALLOPHENYLNORSTATINE

Background: HIV-1 protease (HIV PR), an aspartic protease, cleaves Phe -Pro bonds in the Gag and Gag-Pol viral polyproteins. Substrate-based peptide mimics constitute a major class of inhibitors of HIV PR presen tly being developed for AIDS treatment. One such compound, KNI-272, wh ich incorporates allophenylnorstatine (Apns)-thioproline (Thp) in plac e of Phe-Pro, has potent antiviral activity and is undergoing clinical trials. The structure of the enzyme-inhibitor complex should lead to an understanding of the structural basis for its tight binding propert ies and provide a framework for interpreting the emerging resistance t o this drug. Results: The three-dimensional crystal structure of KNI-2 72 bound to HIV PR has been determined to 2.0 Angstrom resolution and used to analyze structure-activity data and drug resistance for the Ar g8-->Gln and Ile84-->Val mutations in HIV PR. The conformationally con strained Apns-Thp linkage is favorably recognized in its low energy tr ans conformation, which results in a symmetric mode of binding to the active-site aspartic acids and also explains the unusual preference of HIV PR for the S, or syn, hydroxyl group of the Apns residue. The inh ibitor recognizes the enzyme via hydrogen bonds to three bridging wate r molecules, including one that is coordinated directly to the catalyt ic Asp125 residue. Conclusions: The structure of the HIV PR/KNI-272 co mplex illustrates the importance of limiting the conformational degree s of freedom and of using protein-bound water molecules for building p otent inhibitors. The binding mode of HIV PR inhibitors can be predict ed from the stereochemical relationship between adjacent hydroxyl-bear ing and side chain bearing carbon atoms of the P1 substituent. Our str ucture also provides a framework for designing analogs targeted to dru g-resistant mutant enzymes.