Trypanosoma cruzi Malic Enzyme Is the Target for Sulfonamide Hits from the GSK Chagas Box.

Chagas disease, an infectious condition caused by Trypanosoma cruzi, lacks treatment with drugs with desired efficacy and safety profiles. To address this unmet medical need, a set of trypanocidal compounds were identified through a large multicenter phenotypic-screening initiative and assembled in the GSK Chagas Box. In the present work, we report the screening of the Chagas Box against T. cruzi malic enzymes (MEs) and the identification of three potent inhibitors of its cytosolic isoform (TcMEc). One of these compounds, TCMDC-143108 (1), came out as a nanomolar inhibitor of TcMEc, and 14 new derivatives were synthesized and tested for target inhibition and efficacy against the parasite. Moreover, we determined the crystallographic structures of TcMEc in complex with TCMDC-143108 (1) and six derivatives, revealing the allosteric inhibition site and the determinants of specificity. Our findings connect phenotypic hits from the Chagas Box to a relevant metabolic target in the parasite, providing data to foster new structure-activity guided hit optimization initiatives.

In Vitro Image-Based Assay for Trypanosoma cruzi Intracellular Forms.

The advances in development and popularization of automated fluorescence microscopes and pipetting robots allowed scientists to establish high-throughput compound screening using image-based assays for Trypanosoma cruzi intracellular forms, which are associated to chronic Chagas disease. An intracellular T. cruzi image-based assay is a valuable tool to early stage drug discovery for Chagas disease, because it allows scientists to assess a compound's efficacy and safety in the same experiment. During the last 10 years, several improvements have been incorporated into intracellular T. cruzi assay protocols to make them more predictable in what happens with parasites within an infected organism. In the present chapter, a protocol will be presented for an intracellular T. cruzi assay, but at a low-throughput scale, more compatible with facilities in many academic laboratories.