As the AIDS epidemic enters its third decade, the emergence of HIV variants that are drug resistant becomes the next therapeutic challenge. The six FDA-approved HIV-1 protease inhibitors are currently the most potent of the anti-viral agents used as drugs to treat infected patients. All of these inhibitors were the results of structure based drug design. Yet variants of HIV protease have evolved which are resistant to each one of these drugs alone, and some variants are now multi-drug resistant. The challenge for the community is to develop HIV-1 protease inhibitors that are either less vulnerable to drug resistance, or more active against current protease-resistant HIV-1 isolates. This project assembles a multi-disciplinary team with the goal of developing the technology and knowledge necessary to design and synthesize inhibitors that will form an effective therapy toward drug-resistant proteases. The disciplines represented span the fields of informatics and database mining, molecular virology, crystallography, thermodynamics, molecular dynamics, computational ligand design (both docking and inverse design) and high throughput organic synthesis and screening. In this highly integrated approach, the ensemble of HIV-1 protease variants becomes the therapeutic target, rather than one wild-type protease clone, and therefore the ensemble must be characterized. The structures and plasticity of these HIV-1 protease variants are at the center of the project. These data are used both to rationalize clinically observed sequence variations and to provide basis of ensemble-based inhibitor design. The successful outcome of this program project will be a better understanding of mechanisms of drug resistance and the role of structural and evolutionary constraints, a set of strategies and computational approaches to use for highly mutagenic targets, and a series of potential lead compounds with broad activity against HIV protease.