Recombinant expression systems will be established for papain family cysteine proteases that have been implicated in parasitic diseases. In particular, cysteine proteases of protozoan and helminth parasites including Trypanosoma cruzi, Trypanosoma. brucei, Leishmania donovani and Plasmodium falciparum will be expressed in E. coli, P. pastoris, and/or mammalian cell expression systems. The extended substrate specificity of these enzymes will be determined using combinatorial methods that can identify the preferred peptide substrates for the binding pockets both N-terminal and C-terminal to the scissile peptide bond. Optimal substrates will be identified for the proteases and used to assist in high throughput screening efforts of the program project to identify inhibitors. Furthermore, the specificity profiling information will be combined with data from related cysteine proteases to identify key amino acids that line the binding pocket and serve as putative determinants of substrate specificity. Site directed substitutions will be made and the variant enzymes profiled to test the role of these amino acids in substrate recognition and enzyme turnover. Using cruzain as a strategic template for the other cysteine proteases, peptide-based acyloxymethyl ketone inhibitors and single chain antibody inhibitors will be made that are highly specific and map the extended substrate binding pockets. These inhibitors will be used to probe the active site of the enzyme in solution using NMR spectroscopy. Cruzain will be uniformly labeled and/or labeled at specific amino acids with 13C/15N. NMR will be used to determine if conformational flexibility exists in the active site and binding pockets of cruzain when free in solution and upon binding different inhibitors. The active site histidine will also be monitored in NMR experiments that measure the coupling constants of the imadazole ring to follow the protonation state and tautomeric state of the histidine. Correlating these changes with inhibitor binding will assist in the concerted efforts of the program project to develop cysteine protease inhibitors as new anti-parasitic drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Program Projects (P01)
Project #
5P01AI035707-13
Application #
7449702
Study Section
Special Emphasis Panel (ZAI1)
Project Start
Project End
Budget Start
2007-06-01
Budget End
2008-05-31
Support Year
13
Fiscal Year
2007
Total Cost
$145,299
Indirect Cost
Name
University of California San Francisco
Department
Type
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
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
Huang, Niu; Jacobson, Matthew P (2010) Binding-site assessment by virtual fragment screening. PLoS One 5:e10109
Chen, Yen Ting; Brinen, Linda S; Kerr, Iain D et al. (2010) In vitro and in vivo studies of the trypanocidal properties of WRR-483 against Trypanosoma cruzi. PLoS Negl Trop Dis 4:
Chen, Chiung-Kuang; Leung, Siegfried S F; Guilbert, Christophe et al. (2010) Structural characterization of CYP51 from Trypanosoma cruzi and Trypanosoma brucei bound to the antifungal drugs posaconazole and fluconazole. PLoS Negl Trop Dis 4:e651
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

Showing the most recent 10 out of 126 publications