The long-term objective of this proposal is to utilize an will develop novel small molecules to lock conformational states in these proteins both in vitro and in cells. Key to this is a powerful site-directed screening method called disulfide-trapping, or Tethering, that allows the discovery of specific thiol-containing small molecules which are selected by natural or engineered cysteines. The site-directed character of this approach permits one to target any site on the protein surface, whether active or allosteric site. This approach was used to identify specific inhibitors for caspase-1 that bind to a cysteine in a cavity about 15[unreadable] from the active site. Structural studies show that the compounds trap a natural off-state of matured caspase-1. Preliminary data show these allosteric inhibitors are reversible and highly selective for caspase-1 over caspase-4 or -5. Mutational and kinetic analysis show there is a critical hydrogen-bonding circuit that radiates from this allosteric site to the active sites and that the protease exhibits positive cooperativity that is unique among proteases. Remarkably, the compounds can diffuse into cells and inhibit enzyme activity in cells. We hypothesize that this allosteric transition and cavity are commonly held in the caspase family. Thus, mutational and kinetic studies of the other inflammatory caspases should reveal similar results and disulfide trapping screens should identify selective inhibitors. These inhibitors will be used to trap specific allosteric transitions in caspases that can be visualized by X-ray crystallography and studied by mutational methods. This will enable identification of common features that control the allosteric circuitry in this highly regulated enzyme family. In addition, the selective inhibitors will permit investigation for the first time of the specific role of each caspase in processing pro-inflammatory proteins in cells and cell extracts. These studies will provide better validation of these enzymes as potential drug targets for inflammatory diseases and fully characterize a novel allosteric site that may be more tractable than the active site for drug discovery efforts. Ultimately, this research may result in novel starting points in drug development efforts aimed at inflammatory and autoimmune diseases. These drugs would be anticipated to have more selectivity and less side effects than existing drugs.