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.
| Lee, Gregory M; Balouch, Eaman; Goetz, David H et al. (2012) Mapping inhibitor binding modes on an active cysteine protease via nuclear magnetic resonance spectroscopy. Biochemistry 51:10087-98 |
| Doyle, Patricia S; Zhou, Yuan M; Hsieh, Ivy et al. (2011) The Trypanosoma cruzi protease cruzain mediates immune evasion. PLoS Pathog 7:e1002139 |
| Boyom, Fabrice Fekam; Fokou, Patrick Valere Tsouh; Yamthe, Lauve Rachel Tchokouaha et al. (2011) Potent antiplasmodial extracts from Cameroonian Annonaceae. J Ethnopharmacol 134:717-24 |
| Swenerton, Ryan K; Zhang, Shuyi; Sajid, Mohammed et al. (2011) The oligopeptidase B of Leishmania regulates parasite enolase and immune evasion. J Biol Chem 286:429-40 |
| Robertson, Stephanie A; Renslo, Adam R (2011) Drug discovery for neglected tropical diseases at the Sandler Center. Future Med Chem 3:1279-88 |
| 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 |
| Guiguemde, W Armand; Shelat, Anang A; Bouck, David et al. (2010) Chemical genetics of Plasmodium falciparum. Nature 465:311-5 |
| Na, Byoung-Kuk; Bae, Young-An; Zo, Young-Gun et al. (2010) Biochemical properties of a novel cysteine protease of Plasmodium vivax, vivapain-4. PLoS Negl Trop Dis 4:e849 |
| Sun, Lingzhi; Shah, Falgun; Helal, Mohamed A et al. (2010) Design, synthesis, and development of novel guaianolide-endoperoxides as potential antimalarial agents. J Med Chem 53:7864-8 |
| Loser, Reik; Gut, Jiri; Rosenthal, Philip J et al. (2010) Antimalarial activity of azadipeptide nitriles. Bioorg Med Chem Lett 20:252-5 |
Showing the most recent 10 out of 126 publications
