Excellent Center for Drug Discovery, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Int J Mol Sci. 2024 Jan 24;25(3):1427. doi: 10.3390/ijms25031427.
Establishing a drug-screening platform is critical for the discovery of potential antiviral agents against SARS-CoV-2. In this study, we developed a platform based on human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to investigate SARS-CoV-2 infectivity, with the aim of evaluating potential antiviral agents for anti-SARS-CoV-2 activity and cardiotoxicity. Cultured myocytes of iPSC-CMs and immortalized human cardiomyocyte cell line (AC-16) were primarily characterized for the expression of cardiac markers and host receptors of SARS-CoV-2. An infectivity model for the wild-type SARS-CoV-2 strain was then established. Infection modeling involved inoculating cells with SARS-CoV-2 at varying multiplicities of infection (MOIs) and then quantifying infection using immunofluorescence and plaque assays. Only iPSC-CMs, not AC16 cells, expressed angiotensin-converting enzyme 2 (ACE-2), and quantitative assays confirmed the dose-dependent infection of iPSC-CMs by SARS-CoV-2, unlike the uninfectable AC16 cells lacking the expression of ACE2. Cytotoxicity was evaluated using MTT assays across a concentration range. An assessment of the plant-derived compound panduratin A (panA) showed cytotoxicity at higher doses (50% cytotoxic concentration (CC) 10.09 μM) but promising antiviral activity against SARS-CoV-2 (50% inhibition concentration (IC) 0.8-1.6 μM), suppressing infection at concentrations 10 times lower than its CC. Plaque assays also showed decreased viral production following panA treatment. Overall, by modeling cardiac-specific infectivity, this iPSC-cardiomyocyte platform enables the reliable quantitative screening of compound cytotoxicity alongside antiviral efficacy. By combining disease pathogenesis and pharmacology, this system can facilitate the evaluation of potential novel therapeutics, such as panA, for drug discovery applications.
建立药物筛选平台对于发现针对 SARS-CoV-2 的潜在抗病毒药物至关重要。在这项研究中，我们开发了一个基于人诱导多能干细胞衍生的心肌细胞（iPSC-CMs）的平台，以研究 SARS-CoV-2 的感染性，旨在评估潜在的抗病毒药物对 SARS-CoV-2 的活性和心脏毒性。原代培养的 iPSC-CMs 和永生化的人心肌细胞系（AC-16）主要用于鉴定心脏标志物和 SARS-CoV-2 的宿主受体。然后建立了野生型 SARS-CoV-2 株的感染模型。感染建模涉及用 SARS-CoV-2 在不同感染复数（MOI）下接种细胞，然后通过免疫荧光和蚀斑分析定量感染。只有 iPSC-CMs 而不是 AC16 细胞表达血管紧张素转换酶 2（ACE-2），并且定量测定证实了 SARS-CoV-2 对 iPSC-CMs 的剂量依赖性感染，而缺乏 ACE2 表达的不可感染的 AC16 细胞则不然。通过 MTT 测定评估细胞毒性。对植物衍生化合物 panduratin A（panA）的评估显示，在较高剂量（50%细胞毒性浓度（CC）10.09 μM）下具有细胞毒性，但对 SARS-CoV-2 具有有前途的抗病毒活性（50%抑制浓度（IC）0.8-1.6 μM），抑制感染浓度比其 CC 低 10 倍。蚀斑分析还表明，panA 处理后病毒产量减少。总的来说，通过对心脏特异性感染建模，该 iPSC 心肌细胞平台能够可靠地对化合物的细胞毒性和抗病毒功效进行定量筛选。通过结合疾病发病机制和药理学，该系统可以促进对潜在新型治疗药物（如 panA）的评估，用于药物发现应用。
