Core: This section details the series of screening assays used to analyze new inhibitor leads synthesized by Bill Roush's synthetic chemistry group, predicted by the computer modeling of the computation group, or provided by our other synthetic chemistry collaborators. These latter screening methods include automated fluorescence-based assays of protease activity, in vitro culture of the parasite stages, and murine models of infection for T. cruzi, T. brucei, and Leishmania species. In parallel with identification I lof new leads, we can carry out initial pharmacokinetic and rodent toxicity analyses of the most promising IIleads. These studies are aimed at optimizing dosing schedules for inhibitors, and identifying synthetically I feasible modifications to enhance half-life, minimize toxicity, and maximize oral bioavailability. Leadsthat pass the first level of animal screening will be reevaluated in a model of chronic Chagas' disease or, in the case of Leishmania, models of L. donovani, L. major and L. mexicana infection. Target validation includes confirmation of signature ultrastructural defects shown to be due to protease inhibition, RNAi or targeted gene disruption, and competition studies with radiolabeled or biotinylated active site 'tags'. Further preclinical testing of leads is coordinated in collaboration with Chuck Litterst at NIAID.In addition to Core support of inhibitor screening, we are continuing several studies to analyzebiological function for protease targets and evaluate which target is most exploitable in a parasite like Leishmania, which has several cysteine protease gene families. Gene knockout is feasible in Leishmania, and this will be applied to selected cathepsin L and cathepsin B gene clusters. These results will be correlated with those of our collaborator, Jeremy Mottram, who is carrying out similar studies in L. mexicana. In parallel, we will be carrying out 'chemical knockout' of selected proteases in Trypanosoma brucei and L. donovani. In experiments similar to that which identified the function of falcipain 1 in malaria merozoites, highly selective inhibitors of the target proteases will be identified in our compound library, and a combination of radiolabeled or fluorescent-labeled active site tags will be used to confirm targeting of a specific protease species. In the case of T. brucei, RNAi knockdown has proven to be a useful technique for dissecting the function of cathepsin B and cathepsin L-like proteases. This technology will be combined again with chemical knockout to identify rhodesain or T. brucei cathepsin B as the most promising target for further inhibitor development.
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