Inhibitors of Trypanothione Reductase

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DESCRIPTION (provided by applicant): This proposal focuses on the design, synthesis, and evaluation of potential inhibitors of trypanothione reductase (TR). TR is an NADPH-dependent flavoenzyme that catalyzes the reduction of the antioxidant trypanothione (N1, N8-bis (glutathionyl) spermidine) from its disulfide to its dithiol form. The enzyme is found in the trypanosomatid parasites, Trypanosoma and Leishmania, causative agents of a host of diseases of both humans and domestic animals. Examples include African sleeping sickness (Trypanosoma brucei), Chagas' disease (T. cruzi), and numerous leishmanial infections, such as oriental sore (Leishmania tropica) and kalazar (L. donovani). The health and economic effects of these diseases are enormous; however, truly effective treatments for them are lacking. Since its discovery in the mid-1980s, TR has emerged as a prime target for the development of antiparasitic drugs. Much attention has been focused on the identification of reversible TR inhibitors. While this study will add to that body of knowledge with a number of proposed competitive TR inhibitors, its primary focus is to identify mechanism-based irreversible inhibitors, a class of TR inhibitor that has received scant attention in the literature. The approach taken in the design of the proposed irreversible inhibitors is to prepare analogues of trypanothione that incorporate electrophilic moieties in place of the substrate's disulfide group. It is expected that the electrophilic inhibitors will form a covalent bond with a nucleophilic cysteine residue in the enzyme active site, which is known to be a key residue in the catalytic mechanism of TR. The experimental methods will be those of organic chemistry, in particular, nuclear magnetic resonance (NMR) spectroscopy and electro spray ionization (ESI) mass spectrometry will play a large role in the preparation and analysis of the proposed compounds. The inhibitory activity of potential inhibitors will be evaluated using both kinetic and mass spectrometric approaches. In particular, analysis of inhibited protein via HPLC/ESI ion trap MS/MS will be important in verifying the covalent nature of the proposed irreversible inhibitors. Carleton College undergraduate chemistry majors will carry out the research. The principal investigator has had significant experience in crafting successful undergraduate research experiences.