The main protease (Mpro) of SARS-CoV-2, the pathogen responsible for the COVID-19 pandemic, is a key antiviral drug target. While most SARS-CoV-2 Mpro inhibitors have a gamma-lactam glutamine surrogate at the P1 position, we recently discovered several Mpro inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II/XII, which are also active against human cathepsin L, a host-protease that is important for viral entry. To determine the binding mode of these calpain inhibitors and establish a structure-activity relationship, we solved X-ray crystal structures of Mpro in complex with calpain inhibitors II and XII, and three analogues of GC-376, one of the most potent Mpro inhibitors in vitro. The structure of Mpro with calpain inhibitor II confirmed the S1 pocket of Mpro can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. Interestingly, the structure of calpain inhibitor XII revealed an unexpected, inverted binding pose where the P1 prime pyridine inserts in the S1 pocket and the P1 norvaline is positioned in the S1 prime pocket. The overall conformation is semi-helical, wrapping around the catalytic core, in contrast to the extended conformation of other peptidomimetic inhibitors. Additionally, the structures of three GC-376 analogues UAWJ246, UAWJ247, and UAWJ248 provide insight to the sidechain preference of the S1 prime, S2, S3 and S4 pockets, and the superior cell-based activity of the aldehyde warhead compared with the alpha-ketoamide. Taken together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of Mpro inhibitors as SARS-CoV-2 antivirals.

中文翻译：

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SARS-CoV-2主蛋白酶的结构和抑制作用揭示了开发针对Mpro和组织蛋白酶L的双重抑制剂的策略。

SARS-CoV-2的主要蛋白酶（Mpro）是导致COVID-19大流行的病原体，是关键的抗病毒药物靶标。尽管大多数SARS-CoV-2 Mpro抑制剂在P1位置都具有γ-内酰胺谷氨酰胺替代物，但我们最近发现了几种Mpro抑制剂在P1位置具有疏水部分，包括钙蛋白酶抑制剂II / XII，它们对人组织蛋白酶L也具有活性。 ，一种对于病毒进入非常重要的宿主蛋白酶。为了确定这些钙蛋白酶抑制剂的结合模式并建立结构-活性关系，我们解决了钙蛋白酶抑制剂II和XII以及最有效的Mpro抑制剂之一GC-376的三个类似物与Mpro的X射线晶体结构体外。Mpro与钙蛋白酶抑制剂II的结构证实了Mpro的S1口袋可以容纳疏水性蛋氨酸侧链，挑战了在该位置必须有亲水残基的想法。有趣的是，钙蛋白酶抑制剂XII的结构显示出意外的反向结合姿势，其中P1主吡啶插入S1口袋中，而P1正缬氨酸位于S1主口袋中。与其他拟肽抑制剂的扩展构型相反，总体构型为半螺旋形，包裹在催化核周围。此外，三个GC-376类似物UAWJ246，UAWJ247和UAWJ248的结构可洞悉S1主链，S2，S3和S4口袋的侧链偏好性，以及醛弹头与基于α-的醛基相比具有出色的基于细胞的活性。酮酰胺。综合起来，生化，计算，结构，