Development and application of a sensitive, phenotypic, high-throughput image-based assay to identify compound activity against Trypanosoma cruzi amastigotes

Abstract

We have developed a high content 384-well, image-based assay to estimate the effect of compound treatment on Trypanosoma cruzi amastigotes in 3T3 fibroblasts. In the same well, the effect of compound activity on host cells can also be determined, as an initial indicator of cytotoxicity. This assay has been used to identify active compounds from an in-house library of compounds with either known biological activity or that are FDA approved, and separately, from the Medicines for Malaria Venture Malaria Box collection. Active compounds were screened against T. cruzi trypomastigotes, utilising an assay developed with the viability dye resazurin. Twelve compounds with reconfirmed solid sample activity, with IC50 values of less than 10 μM and selectivity indices to T. cruzi amastigotes over 3T3 host cells of between >22 and 319 times were identified from these libraries. As 3T3 cells are contact inhibited, with limited proliferation in the assay, selective compounds of interest were profiled in a separate assay to estimate the viability of compound treated, replicating HEK293 cells. Selective compounds that were not previously reported in the literature were further profiled by extending the incubation time against amastigote infected 3T3 cells to determine if there were residual amastigotes post-treatment, important for the consideration of the exposure time required for further biological characterisation. The assay development process and the suitability of identified compounds as hit molecules for Chagas disease research are discussed.

Biological characterization of chemically diverse compounds targeting the Plasmodium falciparum coenzyme A synthesis pathway

“…Compound activity against two members of another clinically important group of parasites, the kinetoplastids T. cruzi and T. b. brucei was determined. The sensitive high content imaging assay used to determine compound activity against T. cruzi assesses the intracellular amastigote life form of the parasite in the 3T3 host cell [35]. It is important to simultaneously assess compound activity against 3T3 cells to determine potential cytotoxicity against the host cell line, since this could result in a false positive outcome.…”

“…Testing of the compounds in our recently developed T. cruzi trypomastigote assay [35], revealed higher compound activities against the extracellular trypomastigote form (Additional file 1: Table S1). The three most active compounds Amb4317088, STK 740987 and Amb3377585 displayed sub-micromolar IC 50 values against T. cruzi trypomastigotes [0.392 μM, 0.682 μM and 0.744 μM, respectively (Additional file 1: Table S1)].…”

“…In the present study, the selectivity investigation was extended to a small panel of human cancer cell lines, namely the estrogen receptor (ER)-positive breast cancer cell line MCF7, the ER-negative MDA-MB-231, as well as the human prostate cancer line, PC-3. Additionally, the cytotoxic effects on mouse embryonic fibroblast cell line 3T3, which serves as the host cell for T. cruzi in our image-based amastigote assay, were investigated [35]. …”

“…A resazurin-based assay was utilized to determine cell viability as described earlier [22]. Additionally, the cytotoxic effects on the mouse embryonic fibroblast cell line 3T3 were assessed as part of the Trypanosoma cruzi amastigote image-based assay as outlined in [35]. …”

“…Compound activity against T. cruzi amastigotes was assessed in an image-based assay in single point concentration-response and three biological replicates, at a final highest compound concentration of 47.3 μM, as previously described [35]. …”