Development of dual inhibitors targeting the viral main protease and the host cathepsin L as SARS-CoV-2 antivirals

项目概要 严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2)，也称为新型冠状病毒 2019 （nCoV-19）于 2019 年 12 月左右开始在人类中传播，目前已作为全球性病毒广泛传播 大流行。目前尚无针对 SARS-CoV-2 的疫苗或抗病毒药物。在这笔赠款中，我们建议开发双重 针对病毒主要蛋白酶和组织蛋白酶 L 的抑制剂作为 SARS-CoV-2 抗病毒药物。使用基于 FRET 的 通过酶测定，我们最近鉴定了几种抑制剂，包括 boceprevir、GC-376 和钙蛋白酶抑制剂 II XII，在酶测定中具有单位数至亚微摩尔 IC50 值的有效活性。 值得注意的是，所有四种化合物均以 EC50 值抑制细胞培养物中传染性 SARS-CoV-2 的复制 范围为 0.5 至 3.4 µM。总体而言，所确定的化合物为进一步研究提供了有希望的起点 SARS-CoV-2 疗法的开发。我们发现钙蛋白酶抑制剂 II 作为一种有效的抑制剂 SARS-CoV-2 具有创新性，因为它表明通过以下方式开发 SARS-CoV-2 抗病毒药物可能是可行的： 同时靶向病毒 Mpro 和宿主组织蛋白酶 L，这两者对于病毒复制至关重要。 与最近报道的 Mpro 抑制剂相比，我们研究中确定的命中代表了最有效和最有效的抑制剂。 具有新颖作用机制的选择性候选药物，因此值得进一步开发。 鉴于我们令人鼓舞的初步数据，我们建议优化双抑制剂作为 SARS-CoV-2 抗病毒药物。 该提案的目标是开发双重抑制剂作为有效的 SARS-CoV-2 抗病毒药物， 选择性、良好的药代动力学特性以及针对密切接触者的广谱抗病毒活性 相关冠状病毒，如 SARS 和中东呼吸综合征 (MERS) 冠状病毒。 我们这笔赠款的目标是通过高通量筛选和 对我们最近发现的双抑制剂进行基于结构的先导优化。通过针对 SARS-CoV-2 Mpro， 拟议研究的预期结果是广泛作用的冠状病毒抗病毒药物，并已得到证实 作用机制、高选择性指数和良好的体外药代动力学特性 用于相关动物模型的体内抗病毒功效测试。总体而言，这笔赠款是基于强有力的初步 数据和我们在开发针对半胱氨酸蛋白酶的抗病毒药物方面的专业知识。

Project Summary The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also called novel coronavirus 2019 (nCoV-19), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. There is no vaccine or antiviral available for SARS-CoV-2. In this grant, we propose to develop dual inhibitors targeting viral main protease and cathepsin L as SARS-CoV-2 antivirals. Using the FRET-based enzymatic assay, we recently identified several inhibitors including boceprevir, GC-376, and calpain inhibitors II and XII, that have potent activity with single-digit to submicromolar IC50 values in the enzymatic assay. Significantly, all four compounds inhibit infectious SARS-CoV-2 replication in cell culture with EC50 values ranging from 0.5 to 3.4 µM. Overall, the compounds identified provide promising starting points for the further development of SARS-CoV-2 therapeutics. Our discovery of calpain inhibitor II as a potent inhibitor against SARS-CoV-2 is innovative as it suggests it might be feasible to develop SARS-CoV-2 antivirals by simultaneously targeting both viral Mpro and host cathepsin L, both of which are essential for viral replication. Compared to recently reported Mpro inhibitors, the hits identified from our study represent the most potent and selective drug candidates with a novel mechanism of action, therefore warranting further development. Given our encouraging preliminary data, we propose to optimize dual inhibitors as SARS-CoV-2 antivirals. The objective of this proposal is to develop dual inhibitors as potent SARS-CoV-2 antivirals with high potency, selectivity, favorable pharmacokinetic properties, as well as broad-spectrum antiviral activity against closely related coronaviruses such as SARS and Middle East respiratory syndrome (MERS) coronaviruses. Our goals of this grant are to identify additional dual inhibitors through both high-throughput screening and structure-based lead optimization of our recently identified dual inhibitors. By targeting the SARS-CoV-2 Mpro, the expected outcomes of the proposed research are broad-acting coronavirus antivirals with a confirmed mechanism of action, a high selectivity index, and favorable in vitro pharmacokinetic properties that are ready for in vivo antiviral efficacy testing in relevant animal models. Overall, this grant is based on strong preliminary data and our expertise in developing antivirals targeting cysteine proteases.