Analysis of Thiosemicarbazone Warhead Inhibitors of Cruzain as Potential Therapeutic Agents of Chagas' Disease

American Trypanosomiasis or Chagas’ disease is a tropical disease caused by Trypanosoma cruzi, a parasite commonly transmitted by an insect vector or by a blood transfusion. The parasite requires cruzain, a cysteine protease, throughout its life cycle for multiple functions including evasion of the body's immune response and cell invasion. This study aims to find novel inhibitors for cruzain, a validated target for Chagas’, to understand the inhibitor’s mechanism of action as well as to create potential therapeutic treatments. Current drug treatments, including nifurtimox and benznidazole, are not very effective during the chronic stage and have serious side effects. Consequently, there is an urgent need for new treatment agents. This study investigates thiosemicarbazone compounds, synthesized by the Pinney group in a collaborative project between the Trawick and Pinney laboratories at Baylor University. Compounds containing the thiosemicarbazone warhead were previously shown to be inhibitors of cruzain. Fluorescence plate enzyme assays that measured the cleavage of the fluorescent product, 7-amino-methylcoumarin (AMC), from the cruzain substrate Z-FR-AMC allowed us to measure the enzymatic activity and inhibition. Advanced kinetics, including progress curves, reversibility studies, and IC50 assays based on differing incubation times provided methods to evaluate the effectiveness and the mode of action of these compounds as inhibitors. Data from these analyses suggest that the inhibitors form transient covalent bonds with the enzyme, and are non-classically competitive and time-dependent. Further studies are necessary to determine the precise mechanism of inhibition for these compounds and ultimately to evaluate whether they can be pursued as promising avenues for the treatment of Chagas’ disease.