Investigation of Cruzain Inhibition by Thiosemicarbazone Compounds

Chagas disease, the result of infection by the protozoan parasite Trypanosoma cruzi, is transmitted by blood-feeding insect vectors that are members of the Reduviidae family called kissing bugs. The vectors are found throughout Latin America, and predominantly in Texas, New Mexico, and Arizona in the United States. Chagas is responsible for about 12,000 deaths per year and it is estimated that 6-8 million people worldwide are infected. There is currently no approved treatment for eliminating the parasite during its chronic phase after it has migrated into tissue. Thus, there is an unmet need for treatment agents. One validated target is cruzipain, a potent cysteine protease that is necessary for the parasite’s survival throughout its life cycle including immune evasion, tissue infection, processing and degradation of proteins, and reproduction. This study used cruzain, which is a recombinant form of cruzipain, to test inhibition of enzyme activity with a focused library of thiosemicarbazone compounds. Potential inhibitors were evaluated using a fluorogenic enzyme assay to assess the cleavage of 7-amino-4-methylcoumarin (AMC) from the cruzain substrate Z-Phe-Arg-AMC. Several compounds in this series had IC50 values less than 100 nM with the substituted benzophenone thiosemicarbazone KGP434 being the most effective (IC50 =17 nM). Kinetics studies were performed to determine the mode of inhibition. Progress curves and reversibility studies indicated that KGP434 is an active site directed, time-dependent, and slowly reversible inhibitor of cruzain. Docking studies showed the positioning of the aryl groups in the hydrophobic S2 and S3 pockets places the thiosemicarbazone moiety in close proximity to cysteine-25 which provides evidence for covalent inhibition.