We propose to form a National Cooperative Drug Discovery Group (NCDDG) whose overall goal is to develop antiviral therapeutic agents for the treatment of acquired immune deficiency syndrome (AIDS). The focus of this NCDDG will be to discover and design inhibitors of the viral encoded protease of the human immunodeficiency virus (HIV), which is the causative agent of AIDS. The NCDDG will employ a multidisciplinary, highly integrated approach to achieve these goals and includes five projects which deal with the molecular biology, protein biochemistry, chemical synthesis, X-ray crystallography, and NMR of HIV protease and protease inhibitors. In Project 1, an HIV protease gene will be synthesized, cloned and expressed. Purification procedures and assays for the protease will be developed in Project 2. Protease inhibitors will be chemically synthesized in Project 3. Three- dimensional crystal structure determinations of HIV protease and protease-inhibitor complexes will be carried out by X-ray crystallographic methods in Project 4. NMR studies in Project 5 will provide information about the conformations of bound inhibitors, active site environment, and the structure of the protease in solution. The structural information provided by Projects 4 and 5 will be used to design novel and more potent inhibitors. In addition to these primary aims, site-directed mutagenesis studies in Project 1 will be used to probe the functional importance of various amino acids in the protease structure. The five projects are supported by six research cores. Core B will provide assistance with amino acid and nucleic acid sequence analysis, and with peptide and oligonucleotide synthesis. Core C will perform large scale soil screening for protease inhibitors (using the assays developed in Project 2). Core D will provide lysates of HIV infected cells for natural protease isolation, and also will perform antiviral assays (on compunds synthesized in Project 3). Core E will provide state-of-the-art expertise in graphical and theoretical techniques currently employed in modern drug design. Cores F and G will provide electron microscopy and fluorescence spectroscopy support, respectively, to various project components. Initially, the focus will be on HIV-1 protease. Later on, protease inhibitors of HIV- I will be tested against HIV-2 protease for cross-reactivity. If necessary, the strategies developed for the design of HIV-1 protease inhibitors will be used to design inhibitors to HIV-2 protease.
