The
cysteine protease cruzipain is essential for the viability, infectivity, and virulence of Trypanosoma cruzi, the causative agent of
Chagas disease. Thus, inhibitors of
cruzipain are considered promising
anti-T. cruzi chemotherapeutic agents. Reversible
cruzipain inhibitors containing a
nitrile "warhead" were prepared and demonstrated 50% inhibitory concentrations (IC50s) as potent as 1 nM in baculovirus-generated
cruzipain enzyme assays. In epimastigote and intracellular amastigote in vitro assays, the most potent compounds demonstrated
antiparasitic behavior in the 5 to 10 μM IC50 range; however, trypomastigote production from the amastigote form was ∼90 to 95% inhibited at 2 μM. Two key compounds, Cz007 and Cz008, with IC50s of 1.1 and 1.8 nM, respectively, against the recombinant
enzyme were tested in a murine model of acute T. cruzi
infection, with oral dosing in chow for 28 days at doses from 3 to 50 mg/kg of
body weight. At 3 mg/kg of Cz007 and 3 mg/kg of Cz008, the blood
parasitemia areas under the concentration-time curves were 16% and 25% of the untreated group, respectively. At sacrifice, 24 days after immunosuppression with
cyclophosphamide, parasite presence in blood, heart, and esophagus was evaluated. Based on negative quantitative PCR results in all three tissues, cure rates in surviving animals were 90% for Cz007 at 3 mg/kg, 78% for Cz008 at 3 mg/kg, and 71% for
benznidazole, the control compound, at 50 mg/kg.