Fragment Based Drug Design in HIV Protease

点击翻译按钮获取中文摘要

We will apply fragment based screening to identify small molecule binding sites in HIV protease (PR) leading to the design of potent inhibitors that maintain efficacy against multi-site PR mutants. Mutations giving rise to resistance in PR affect both protein-ligand interactions and protein conformation. Efficient binding to multi-site PR mutants requires that the compound be able to bind to conserved structural features, be inherently flexible, trap PR in an inactive conformation, and/or exploit induced fit conformational changes. Fragment based screening is a novel approach to drug design that employs a small library of chemically diverse small molecules (~500 compounds, MW ~150) to identify weak but specific binders. Inhibitor design entails subsequent elaboration, assay, and linkage of the 'fragments'. A very large area of chemical space is screened, and the inhibitor (drug) is derived from smaller fragments each having high ligand efficiency. With the development of high throughput X-ray crystallography, it is now feasible to screen many small molecules, i.e. fragments, for specific binding sites in protein crystals. We have developed good diffracting crystals of ligand free wild-type PR and a 6-residue mutant (6X) resistant to the broad spectrum inhibitor, TL-3. The six mutations commonly occur in clinical isolates of resistant HIV strains. Based on a screen of 68 compounds we have already identified three small molecule fragments that bind at novel sites in the PR dimer. The experimental strategy entails three Aims. (1) Complete the fragment screen against wild-type PR, and repeat the screen with cocrystals containing fragments (F1) from the initial screen. Identification of second site binders (F2) will exploit induced fit conformational changes in PR, and provide a scaffold on which to develop linked high affinity inhibitors, i.e. F1-F2. (2) Repeat the two-stage fragment screen against 6X PR. Fragments binding to the 6X mutant may differ (f1), while fragments binding in the second stage (f2) may differ due to altered conformational response of the protein. Derivative fragment libraries, i.e. FT, F2', f1', f2', identified in silico and/or synthesized in Project 3, will be screened for improved binding. The results will provide an empirical picture of the chemical affinity of PR, and how it is altered by resistance causing mutations. (3) Develop and assay a potent dual site binding inhibitor. An iterative design cycle of synthesis - assay - structure - synthesis will be employed within the context of the Program Project. Structures of linked fragment derivatives bound to HIV PR, based on the results in Aims 1 and 2, and synthesized in Project 3, will be determined. The goal is to discover high affinity inhibitors, e.g. F1'-f2', having novel modes of binding and the ability to bind tightly to multi-site, drug resistant PR mutants. Inhibitors to HIV PR developed in this fashion should be beneficial in addressing the evolution of drug resistance in HIV.