This section details the series of screening assays we will use to analyze new inhibitor leads synthesized by Bill Roush's synthetic chemistry group or predicted by the computer modeling of Fred Cohen's group. These screening methods include an automated assay of purified protease activity, in vitro culture of the parasite stages, and a murine model of infection. In parallel with identification of new leads, we will carry out initial pharmacokinetic analysis of the derivatized pseudopeptides we have already shown to be effective in lowering parasitemia. These studies will be aimed at optimizing dosing schedules for inhibitors, and identifying synthetically feasible modifications to enhance half-life, minimize toxicity, and allow oral bioavailability. We have shown that fluoromethyl ketone-derivatized peptides, which are irreversible, specific inhibitors of cysteine proteases, will arrest T. cruzi replication and transformation between stages of its life cycle. The events surrounding the transformation of trypomastigote to amastigote and early amastigote replication appear to be especially sensitive to inhibition of the protease. We will now follow up on these observations with a more detailed study of the function of the cysteine protease at different stages of the T. cruzi life cycle, and an analysis of the specific effects of inhibitors on parasite morphology at both the light and electron microscopic levels. As a foundation to these studies, we have produced monospecific, polyclonal antisera to the purified recombinant cruzain. This antisera has the advantage over previous antibody reagents in that it recognizes only protein epitopes. This should minimize cross-reactivity between carbohydrate moieties that may have clouded previous attempts at localization. Furthermore, we have developed a localization assay utilizing a biotinylated fluoromethyl ketone peptide which irreversibly binds only at the active site of the enzyme and, via a streptavidin fluorochrome derivative, can be used to confirm and supplement antibody localization studies. Coupled with the use of fluorochrome derivatives that can identify specific subcellular organelles and locales, a more definitive analysis of the localization of the protease at each stage, and studies of its intracellular trafficking can be carried out. Concurrently, we will study the effects of each new generation of inhibitors on parasite morphology at the light and ultrastructural level. Our previous work has suggested inhibition of the protease results in morphologic abnormalities at the trypomastigote to amastigote interface. These will now be confirmed and analyzed in more detail.
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| Brak, Katrien; Kerr, Iain D; Barrett, Kimberly T et al. (2010) Nonpeptidic tetrafluorophenoxymethyl ketone cruzain inhibitors as promising new leads for Chagas disease chemotherapy. J Med Chem 53:1763-73 |
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