The 3-chymotrypsin-like cysteine protease (3CLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is considered a major target for the discovery of direct antiviral agents. We previously reported the evaluation of SARS-CoV-2 3CLpro inhibitors in a novel self-assembled monolayer desorption ionization mass spectrometry (SAMDI-MS) enzymatic assay (Gurard-Levin et al., 2020). The assay was further improved by adding the rhinovirus HRV3C protease to the same well as the SARS-CoV-2 3CLpro enzyme. High substrate specificity for each enzyme allowed the proteases to be combined in a single assay reaction without interfering with their individual activities. This novel duplex assay was used to profile a diverse set of reference protease inhibitors. The protease inhibitors were grouped into three categories based on their relative potency against 3CLpro and HRV3C including those that are: equipotent against 3CLpro and HRV3C (GC376 and calpain inhibitor II), selective for 3CLpro (PF-00835231, calpain inhibitor XII, boceprevir), and selective for HRV3C (rupintrivir). Structural analysis showed that the combination of minimal interactions, conformational flexibility, and limited bulk allows GC376 and calpain inhibitor II to potently inhibit both enzymes. In contrast, bulkier compounds interacting more tightly with pockets P2, P3, and P4 due to optimization for a specific target display a more selective inhibition profile. Consistently, the most selective viral protease inhibitors were relatively weak inhibitors of human cathepsin L. Taken together, these results can guide the design of cysteine protease inhibitors that are either virus-specific or retain a broad antiviral spectrum against coronaviruses and rhinoviruses.

严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 的 3-胰凝乳蛋白酶样半胱氨酸蛋白酶 (3CLpro) 被认为是发现直接抗病毒药物的主要靶点。我们之前报道过在新型自组装单层解吸电离质谱 (SAMDI-MS) 酶测定中对 SARS-CoV-2 3CLpro 抑制剂的评估（Gurard-Levin 等人，2020）。通过将鼻病毒 HRV3C 蛋白酶添加到与 SARS-CoV-2 3CLpro 酶相同的孔中，进一步改进了该测定。每种酶的高底物特异性使得蛋白酶可以在单个测定反应中组合，而不干扰它们各自的活性。这种新颖的双链测定用于分析一组不同的参考蛋白酶抑制剂。根据蛋白酶抑制剂针对 3CLpro 和 HRV3C 的相对效力，将蛋白酶抑制剂分为三类，包括：对 3CLpro 和 HRV3C 等效（GC376 和钙蛋白酶抑制剂 II）、对 3CLpro 选择性（PF-00835231、钙蛋白酶抑制剂 XII、boceprevir）、对 HRV3C (rupintrivir) 具有选择性。结构分析表明，最小的相互作用、构象灵活性和有限的体积相结合，使得 GC376 和钙蛋白酶抑制剂 II 能够有效抑制这两种酶。相比之下，由于针对特定靶标的优化，体积较大的化合物与口袋 P2、P3 和 P4 相互作用更紧密，显示出更具选择性的抑制特性。一致地，最具选择性的病毒蛋白酶抑制剂是相对较弱的人组织蛋白酶 L 抑制剂。总之，这些结果可以指导半胱氨酸蛋白酶抑制剂的设计，这些抑制剂要么是病毒特异性的，要么保留了针对冠状病毒和鼻病毒的广泛抗病毒谱。