Institute of Molecular Virology, Ulm University Medical Center, 89081, Ulm, Germany.
Sci Rep. 2024 Sep 5;14(1):20697. doi: 10.1038/s41598-024-71305-6.
Human coronaviruses (hCoVs) infect millions of people every year. Among these, MERS, SARS-CoV-1, and SARS-CoV-2 caused significant morbidity and mortality and their emergence highlights the risk of possible future coronavirus outbreaks. Therefore, broadly-active anti-coronavirus drugs are needed. Pharmacological inhibition of the hCoV protease Nsp5 (3CLpro) is clinically beneficial as shown by the wide and effective use of Paxlovid (nirmatrelvir, ritonavir). However, further treatment options are required due to the risk of drug resistance. To facilitate the assessment of coronavirus protease function and its pharmacological inhibition, we developed an assay allowing rapid and reliable quantification of Nsp5 activity under biosafety level 1 conditions. It is based on an ACE2-Gal4 transcription factor fusion protein separated by a Nsp5 recognition site. Cleavage by Nsp5 releases the Gal4 transcription factor, which then induces the expression of Gaussia luciferase. Our assay is compatible with Nsp5 proteases from all hCoVs and allows simultaneous measurement of inhibitory and cytotoxic effects of the tested compounds. Proof-of-concept measurements confirmed that nirmatrelvir, GC376 and lopinavir inhibit SARS-CoV-2 Nsp5 function. Furthermore, the assay accurately predicted the impact of Nsp5 mutations on catalytic activity and inhibitor sensitivity. Overall, the reporter assay is suitable for evaluating viral protease activity.
人类冠状病毒（hCoVs）每年感染数百万人。其中，MERS、SARS-CoV-1 和 SARS-CoV-2 导致了重大的发病率和死亡率，它们的出现突显了未来可能爆发冠状病毒的风险。因此，需要广泛有效的抗冠状病毒药物。冠状病毒蛋白酶 Nsp5（3CLpro）的药理学抑制在临床上是有益的，如广泛有效的使用 Paxlovid（nirmatrelvir，ritonavir）所证明的那样。然而，由于存在药物耐药的风险，还需要进一步的治疗选择。为了便于评估冠状病毒蛋白酶的功能及其药理学抑制作用，我们开发了一种在生物安全水平 1 条件下允许快速可靠地定量 Nsp5 活性的测定法。它基于 ACE2-Gal4 转录因子融合蛋白，通过 Nsp5 识别位点隔开。Nsp5 的切割释放 Gal4 转录因子，然后诱导 Gaussia 荧光素酶的表达。我们的测定法与所有 hCoVs 的 Nsp5 蛋白酶兼容，并允许同时测量测试化合物的抑制和细胞毒性作用。概念验证测量证实，nirmatrelvir、GC376 和洛匹那韦抑制 SARS-CoV-2 Nsp5 功能。此外，该测定法准确预测了 Nsp5 突变对催化活性和抑制剂敏感性的影响。总体而言，报告基因测定法适用于评估病毒蛋白酶活性。
