Department of Molecular Genetics and Microbiology, Duke University School of Medicine , Durham, North Carolina, USA.
Medical Scientist Training Program, Duke University School of Medicine , Durham, North Carolina, USA.
J Virol. 2023 Aug 31;97(8):e0059723. doi: 10.1128/jvi.00597-23. Epub 2023 Aug 14.
Multiple coronaviruses (CoVs) can cause respiratory diseases in humans. While prophylactic vaccines designed to prevent infection are available for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), incomplete vaccine efficacy, vaccine hesitancy, and the threat of other pathogenic CoVs for which vaccines do not exist have highlighted the need for effective antiviral therapies. While antiviral compounds targeting the viral polymerase and protease are already in clinical use, their sensitivity to potential resistance mutations as well as their breadth against the full range of human and preemergent CoVs remain incompletely defined. To begin to fill that gap in knowledge, we report here the development of an improved, noninfectious, cell-based fluorescent assay with high sensitivity and low background that reports on the activity of viral proteases, which are key drug targets. We demonstrate that the assay is compatible with not only the SARS-CoV-2 M protein but also orthologues from a range of human and nonhuman CoVs as well as clinically reported SARS-CoV-2 drug-resistant M variants. We then use this assay to define the breadth of activity of two clinically used protease inhibitors, nirmatrelvir and ensitrelvir. Continued use of this assay will help define the strengths and limitations of current therapies and may also facilitate the development of next-generation protease inhibitors that are broadly active against both currently circulating and preemergent CoVs. IMPORTANCE Coronaviruses (CoVs) are important human pathogens with the ability to cause global pandemics. Working in concert with vaccines, antivirals specifically limit viral disease in people who are actively infected. Antiviral compounds that target CoV proteases are already in clinical use; their efficacy against variant proteases and preemergent zoonotic CoVs, however, remains incompletely defined. Here, we report an improved, noninfectious, and highly sensitive fluorescent method of defining the sensitivity of CoV proteases to small molecule inhibitors. We use this approach to assay the activity of current antiviral therapies against clinically reported SARS-CoV-2 protease mutants and a panel of highly diverse CoV proteases. Additionally, we show this system is adaptable to other structurally nonrelated viral proteases. In the future, this assay can be used to not only better define the strengths and limitations of current therapies but also help develop new, broadly acting inhibitors that more broadly target viral families.
多种冠状病毒（CoV）可引起人类呼吸道疾病。虽然针对严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）的预防性疫苗可预防感染，但疫苗效力不完全、疫苗犹豫以及针对尚无疫苗的其他致病性 CoV 的威胁，突出表明需要有效的抗病毒疗法。虽然针对病毒聚合酶和蛋白酶的抗病毒化合物已在临床中使用，但它们对潜在耐药突变的敏感性以及对全人类和新兴 CoV 的广谱性仍不完全确定。为了开始填补这一知识空白，我们在此报告了一种改进的、非感染性的、具有高灵敏度和低背景的基于细胞的荧光测定法的开发，该测定法可报告病毒蛋白酶的活性，而病毒蛋白酶是关键的药物靶标。我们证明，该测定法不仅与 SARS-CoV-2 M 蛋白兼容，而且与来自一系列人类和非人类 CoV 的同源物以及临床上报告的 SARS-CoV-2 耐药 M 变体兼容。然后，我们使用该测定法来定义两种临床使用的蛋白酶抑制剂（nirmatrelvir 和 ensitrelvir）的广泛活性。继续使用该测定法将有助于确定当前疗法的优势和局限性，并且还可能促进开发对当前流行和新兴 CoV 均具有广泛活性的下一代蛋白酶抑制剂。
冠状病毒（CoV）是重要的人类病原体，具有引起全球大流行的能力。与疫苗一起，抗病毒药物专门限制积极感染的人群中病毒疾病的发展。针对 CoV 蛋白酶的抗病毒化合物已在临床中使用；然而，它们对变异蛋白酶和新兴的人畜共患 CoV 的疗效仍不完全确定。在这里，我们报告了一种改进的、非感染性的、高度敏感的荧光方法，用于定义小分子抑制剂对 CoV 蛋白酶的敏感性。我们使用这种方法来测定针对临床上报告的 SARS-CoV-2 蛋白酶突变体和一组高度多样化的 CoV 蛋白酶的当前抗病毒疗法的活性。此外，我们表明该系统可适应其他结构上无关联的病毒蛋白酶。在未来，该测定法不仅可以更好地定义当前疗法的优势和局限性，还可以帮助开发新的、广谱作用的抑制剂，更广泛地针对病毒家族。
