Date of Award

2016-01-01

Degree Name

Doctor of Philosophy

Department

Chemistry

Advisor(s)

Mahesh Narayan

Abstract

Chagas disease, caused by the parasite Trypanosoma cruzi, is an endemic life-threatening disease that affects mainly the heart. It remains the leading cause of heart failure in Latin American countries. Since current treatments against this parasite are highly toxic and somewhat ineffective, novel and more efficacious types of interventions are desired. Cruzain, identified as the major cathepsin for T. cruzi, plays a major role in the parasite's life cycle; making this enzyme very attractive for potential trypanocidal drugs discovery. The recombinant cruzain is synthesized as a zymogenic pro-protein (PCZN) which possesses a pro domain and a catalytic domain. In this study we worked with the zymogen (inactive) form of the protease and, by utilizing bioinformatics tools, we identified undiscovered structure-impacting sites within the pro domain of pro-cruzain. PCZN DNA constructs containing point site mutations were successfully expressed and purified. Real-time catalytic activity assays and secondary structure analysis of mutants of PCZN revealed a structural and functional impact compared to the wild- type (WT-PCZN). These findings could be used to identify promising targets to aid in the development of future anti-chagastics agents as well as to support the development of a host of small-molecule compounds that could interfere in the maturation of the catalytically active state of the enzyme by disrupting critical aminoacid-aminoacid interactions within the zymogen. The outcome will possibly open the field of inhibitor design by advancing a novel, bottom-up approach, rather than the classical top-down approach that targets only the active-site of the enzyme.

Language

en

Provenance

Received from ProQuest

File Size

81 pages

File Format

application/pdf

Rights Holder

Marisol Serrano

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