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

项目摘要 严重急性呼吸综合征冠状病毒2(SARS-CoV-2)，又称新型冠状病毒2019 (NCoV-19)，大约在2019年12月开始在人类之间传播，现在作为一种全球 大流行。目前还没有针对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 针对病毒主要蛋白水解酶和组织蛋白酶的抑制剂L作为SARS-CoV-2的抗病毒药物。使用基于FRET的 酶分析，我们最近发现了几种抑制剂，包括博西普利韦，GC-376和钙蛋白酶抑制剂II 和XII，在酶试验中具有较强的活性，IC50值在个位数到亚微摩尔之间。 值得注意的是，所有四个化合物都以EC50值抑制传染性SARS-CoV-2在细胞培养中的复制 总体而言，从0.5到3.4微米的范围内，所鉴定的化合物为进一步 SARS-CoV-2治疗药物的研究进展我们发现的钙蛋白酶抑制剂II是一种有效的抗肿瘤药物 SARS-CoV-2具有创新性，因为它表明通过以下方法开发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L，这两者都是病毒复制所必需的。 与最近报道的MPRO抑制剂相比，我们研究发现的HITS代表了最有效和 具有新的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，我们建议将双重抑制剂优化为SARS-CoV-2抗病毒药物。 这项建议的目的是开发双重抑制剂作为高效的SARS-CoV-2抗病毒药物， 选择性、良好的药代动力学特性以及广谱抗病毒活性 SARS和中东呼吸综合征(MERS)冠状病毒等相关冠状病毒。 我们这笔赠款的目标是通过高通量筛选和 我们最近确定的双重抑制剂的基于结构的先导优化。通过以SARS-CoV-2 MPRO为目标， 拟议研究的预期结果是广泛作用的冠状病毒抗病毒药物，其确认的 作用机理、高选择性指数和良好的体外药代动力学特性 用于在相关动物模型中进行体内抗病毒效果测试。总体而言，这笔赠款是基于强大的初步 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.