Chagas disease is caused by Trypanosoma cruzi infection and remains a relevant cause of chronic heart failure in Latin America. The pharmacological arsenal for Chagas disease is limited, and the available anti-T. cruzi drugs are not effective when administered during the chronic phase. Cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) have the potential to accelerate the process of drug discovery for Chagas disease, through predictive preclinical assays in target human cells. Here, we aimed to establish a novel high-content screening- (HCS-) based method using hiPSC-CMs to simultaneously evaluate anti-T. cruzi activity and cardiotoxicity of chemical compounds. To provide proof-of-concept data, the reference drug benznidazole and three compounds with known anti-T. cruzi activity (a betulinic acid derivative named BA5 and two thiazolidinone compounds named GT5A and GT5B) were evaluated in the assay. hiPSC-CMs were infected with T. cruzi and incubated for 48 h with serial dilutions of the compounds for determination of EC50 and CC50 values. Automated multiparametric analyses were performed using an automated high-content imaging system. Sublethal toxicity measurements were evaluated through morphological measurements related to the integrity of the cytoskeleton by phalloidin staining, nuclear score by Hoechst 33342 staining, mitochondria score following MitoTracker staining, and quantification of NT-pro-BNP, a peptide released upon mechanical myocardial stress. The compounds showed EC₅₀ values for anti-T. cruzi activity similar to those previously described for other cell types, and GT5B showed a pronounced trypanocidal activity in hiPSC-CMs. Sublethal changes in cytoskeletal and nucleus scores correlated with NT-pro-BNP levels in the culture supernatant. Mitochondrial score changes were associated with increased cytotoxicity. The assay was feasible and allowed rapid assessment of anti-T. cruzi action of the compounds, in addition to cardiotoxicity parameters. The utilization of hiPSC-CMs in the drug development workflow for Chagas disease may help in the identification of novel compounds.

中文翻译：

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一种基于高内涵筛选的新型抗克氏锥虫药物发现方法，使用人诱导多能干细胞衍生的心肌细胞

南美锥虫病是由克氏锥虫感染引起的，并且仍然是拉丁美洲慢性心力衰竭的相关原因。南美锥虫病的药理学武器库是有限的，并且可用的抗克氏锥虫药物在慢性期给药时无效。源自人类诱导多能干细胞 (hiPSC-CM) 的心肌细胞有可能通过在靶标人类细胞中进行预测性临床前分析来加速南美锥虫病的药物发现过程。在这里，我们旨在建立一种新的基于高内涵筛选（HCS）的方法，使用 hiPSC-CM 同时评估抗克氏锥虫化合物的活性和心脏毒性。为了提供概念验证数据，在分析中评估了参考药物苯并硝唑和三种具有已知抗克氏锥虫活性的化合物（一种名为 BA5 的桦木酸衍生物和两种名为 GT5A 和 GT5B 的噻唑烷酮化合物）。hiPSC-CM 感染了克氏锥虫并与化合物的系列稀释液一起温育 48 小时，以确定 EC50 和 CC50 值。使用自动化高内涵成像系统进行自动化多参数分析。通过与鬼笔环肽染色的细胞骨架完整性相关的形态学测量、Hoechst 33342 染色的核评分、MitoTracker 染色后的线粒体评分以及 NT-pro-BNP（一种在机械心肌应激时释放的肽）的量化，对亚致死毒性测量进行了评估。这些化合物显示出抗克氏锥虫的EC ₅₀值GT5B 的活性类似于先前描述的其他细胞类型，并且 GT5B 在 hiPSC-CM 中显示出明显的杀锥虫活性。细胞骨架和细胞核评分的亚致死变化与培养上清液中的 NT-pro-BNP 水平相关。线粒体评分变化与细胞毒性增加有关。除了心脏毒性参数外，该测定是可行的并且允许快速评估化合物的抗克氏锥虫作用。在南美锥虫病药物开发工作流程中使用 hiPSC-CM 可能有助于识别新化合物。