Madani Abdeldjalil, Alvarez Nadine, Park Steven, Murugan Madhuvika, Perlin David S
Center for Discovery and Innovation, Hackensack Meridian Health, 111 Ideation Way. Nutley, New Jersey 07110, United States.
Center for Discovery and Innovation, Hackensack Meridian Health, 111 Ideation Way. Nutley, New Jersey 07110, United States.
SLAS Discov. 2025 Mar;31:100211. doi: 10.1016/j.slasd.2025.100211. Epub 2025 Jan 15.
The COVID-19 pandemic has emphasized the necessity for rapid and adaptable drug screening platforms against live pathogenic viruses that require high levels of biosafety containment. Conventional antiviral testing is time-consuming and labor-intensive. Here, we outline the design and validation of a semi-automated drug-screening platform for SARS-CoV-2 that utilizes multiple liquid handlers, a stable A549 cell line expressing ACE2 and TMPRSS2 receptors, and a recombinant SARS-CoV-2 strain harboring the nano-luciferase gene. This platform allows for accelerated low-, mid-, and high-throughput screenings by bypassing the virus inactivation and the staining steps compared to assays utilizing fluorescent reporter viruses or immunofluorescence. First, we demonstrated that the luminescence signal obtained at 24 h post-infection is robust and can be used as a surrogate for fluorescent reporter viruses and immunofluorescence assays that require 48 h incubation post infection. We confirmed the susceptibility of the reporter virus to a panel of reference drugs and validated the luminescence signal in 96- and 384-well plates in accordance with NIH criteria for high-throughput screening. The validation assays showed reproducible results, robust Z factor of ≥0.5, and a coefficient of variation of <20% achieved in both 96 and 384-well plate formats. Lastly, we assessed the assay's performance by screening 240 compounds from the MMV Global Health Library, using the 384-well plate format and remdesivir as a control compound. The single point screening resulted in the identification of 48 hits that inhibited more than 50% of the viral growth. We selected the 15 most active compounds to evaluate their inhibitory concentration and their cytotoxicity, which resulted in the confirmation of the 3 most potent and least toxic compounds that were never reported as antivirals. These results confirm that our platform can be reliably employed for rapid drug screening against SARS-CoV-2 and can be easily adapted to other nano-luciferase reporter viruses.
新冠疫情凸显了针对需要高水平生物安全防护的活致病病毒建立快速且适应性强的药物筛选平台的必要性。传统的抗病毒检测既耗时又费力。在此，我们概述了一种用于严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的半自动药物筛选平台的设计与验证，该平台利用了多种液体处理仪、表达血管紧张素转换酶2（ACE2）和跨膜丝氨酸蛋白酶2（TMPRSS2）受体的稳定A549细胞系，以及携带纳米荧光素酶基因的重组SARS-CoV-2毒株。与使用荧光报告病毒或免疫荧光的检测方法相比，该平台通过绕过病毒灭活和染色步骤，实现了低、中、高通量筛选的加速。首先，我们证明了感染后24小时获得的发光信号很强，可作为需要感染后孵育48小时的荧光报告病毒和免疫荧光检测的替代指标。我们确认了报告病毒对一组参考药物的敏感性，并根据美国国立卫生研究院（NIH）的高通量筛选标准，在96孔板和384孔板中验证了发光信号。验证试验结果具有可重复性，稳健的Z因子≥0.5，且在96孔板和384孔板两种形式下变异系数均<20%。最后，我们使用384孔板形式，以瑞德西韦作为对照化合物，对疟疾药物研发公司（MMV）全球健康库中的240种化合物进行筛选，评估了该检测方法的性能。单点筛选鉴定出48种能抑制超过50%病毒生长的活性化合物。我们选择了15种活性最强的化合物来评估它们的抑制浓度和细胞毒性，结果确认了3种最有效且毒性最小的化合物，这些化合物从未被报道为抗病毒药物。这些结果证实，我们的平台可可靠地用于针对SARS-CoV-2的快速药物筛选，并且可以轻松适应其他纳米荧光素酶报告病毒。
