Small Molecule Inhibitors of Cruzain for Treatment of Chagas' Disease

Chagas’ disease, a neglected tropical disease, affects millions worldwide. This zoonosis occurs as a result of parasitic invasion. The etiological organism, Trypanosoma cruzi (T. cruzi) enters mammalian host cells by triaatomine insects. There are two phases of the disease with different presented symptoms. Current treatment proves largely ineffective in the chronic phase and highly toxic, however no other treatment exists. Due to the treatments’ negative side effects, there is an urgent unmet need for alternative treatments. A potential therapy lies in targeting cruzain, the recombinant form of cruzipain, because this cysteine protease is involved in nutrition, cellular remodeling, immune evasion, and parasitic replication throughout the life cycle of T. cruzi. Treatment of an infected mouse with the cruzain inhibitor K777 resulted in the eradication of the T. cruzi infection. This validates cruzain as a potential target for developing a curative treatment. A series of thiosemicarbazone derivatives synthesized by the Pinney laboratories as part of a collaborative project between the Trawick and Pinney laboratories at Baylor University were evaluated as inhibitors of cruzain. A plasmid for cruzain production by recombinant DNA was provided by the James McKerrow laboratory at UCSF. This study intended to evaluate these compounds as potential inhibitors of cruzain by ascertaining IC50 values against the enzyme. Moreover, advanced kinetic investigations of the most effective inhibitors were conducted so their mechanisms could be understood. Cruzain activity was monitored using a fluorogenic enzyme assay, which measured the concentration of 7-amino-4-methylcoumarin (AMC) product released from Z-FR-AMC, a synthetic substrate. A number of thiosemicarbazone compounds proved to be effective inhibitors of cruzain in the nanomolar range (29.5-312 nM). Progress curves and pre-incubation studies show these compounds as time-dependent inhibitors. Several cruzain inhibitors evaluated in these studies should advance as potential therapeutic agents for Chagas’ disease.