title: Evaluation of Parameters Impacting Drug Susceptibility in Intracellular Trypanosoma cruzi Assay Protocols.
abstract: In order to understand the key parameters influencing drug susceptibility, different Trypanosoma cruzi assay protocols were evaluated using a comparative assay design. The assays compared in this study were an image-based intracellular T. cruzi assay quantified through an image-mining algorithm and an intracellular assay utilizing a β-galactosidase-expressing T. cruzi strain. Thirty-one reference compounds known to exhibit activities against intracellular T. cruzi were used as benchmarks. Initial comparison using EC50values from two assays showed a very poor correlation, with an R2value of 0.005. Nitroheterocyclics and CYP51 inhibitors were inactive in an image-based assay, but were highly active in a colorimetric assay. In order to identify the differentiating factor, we synchronized the compound-parasite incubation times or the sequential cell and compound seeding schemes between assays, but the correlation remained low. A high correlation ( R2= 0.86) was observed only after both compound incubation time and cell seeding were synchronized between assays. Further analysis of EC50and maximum inhibition values showed that nitroheterocyclics and CYP51 inhibitors exhibit relatively large deviations in activity between experimental protocols routinely used for in vitro intracellular T. cruzi assays. These findings suggest that the factors mentioned are critical when designing an intracellular T. cruzi assay.
authors.affiliation: 1 Leishmania Research Laboratory, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, Republic of Korea.|2 Interdisciplinary Programs of Functional Genomics, Yonsei University, Seoul, Republic of Korea.
authors.forename: Gyongseon
authors.initials: G
authors.lastname: Yang
authors.affiliation: 1 Leishmania Research Laboratory, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, Republic of Korea.
authors.forename: Nakyung
authors.initials: N
authors.lastname: Lee
authors.affiliation: 3 Drugs for Neglected Diseases initiative (DND i), Geneva, Switzerland.
authors.forename: Jean-Robert
authors.initials: JR
authors.lastname: Ioset
authors.affiliation: 1 Leishmania Research Laboratory, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, Republic of Korea.
authors.forename: Joo Hwan
authors.initials: JH
authors.lastname: No
mesh_terms: D058888:14-alpha Demethylase Inhibitors / Q000494:pharmacology; D002460:Cell Line / Q000187:drug effects; D014355:Chagas Disease / Q000188:drug therapy* / Q000469:parasitology; D000072505:Cytochrome P450 Family 51 / Q000037:antagonists & inhibitors*; D003593:Cytoplasm / Q000187:drug effects; D004353:Drug Evaluation, Preclinical; D057166:High-Throughput Screening Assays; D006801:Humans; D014344:Trypanocidal Agents / Q000494:pharmacology; D014349:Trypanosoma cruzi / Q000187:drug effects* / Q000472:pathogenicity
chemical_list: D058888:14-alpha Demethylase Inhibitors; D014344:Trypanocidal Agents; D000072505:Cytochrome P450 Family 51
keywords: Trypanosoma cruzi; cell-based assay; high-content assay; susceptibility