Rational Design of Macrocyclic Noncovalent Inhibitors of SARS-CoV‑2 M^pro from a DNA-Encoded Chemical Library Screening Hit That Demonstrate Potent Inhibition against Pan-Coronavirus Homologues and Nirmatrelvir-Resistant Variants

The recent global COVID-19 pandemic has highlighted treatments for coronavirus infection as an unmet medical need. The main protease (Mpro) has been an important target for the development of SARS-CoV-2 direct-acting antivirals. Nirmatrelvir as a covalent Mpro inhibitor was the first such approved therapy. Although Mpro inhibitors of various chemical classes have been reported, they are generally less active against nirmatrelvir-resistant variants and have limited pan-coronavirus potential, presenting a significant human health risk upon future outbreaks. We here present a novel approach and utilized DNA-encoded chemical library screening to identify the noncovalent Mpro inhibitor 5, which demonstrated a distinct binding mode to nirmatrelvir. A macrocyclization strategy designed to lock the active conformation resulted in lactone 12 with significantly improved antiviral activity. Further optimization led to the potent lactam 26, which demonstrated exceptional potency against nirmatrelvir-resistant variants as well as against a panel of viral main proteases from other coronaviruses.近期暴发的全球COVID-19疫情凸显了冠状病毒感染治疗手段存在未被满足的医疗需求。主蛋白酶（main protease, Mpro）是开发SARS-CoV-2直接作用抗病毒药物的重要靶点。奈玛特韦（Nirmatrelvir）作为共价结合型Mpro抑制剂，是首款获批的此类治疗药物。尽管已有多种化学类别的Mpro抑制剂被报道，但它们通常对奈玛特韦耐药变异株的活性较弱，且泛冠状病毒潜力有限，在未来疫情暴发时仍会对人类健康构成重大威胁。本研究提出了一种全新策略，并利用DNA编码化合物库筛选（DNA-encoded chemical library screening）鉴定出非共价结合型Mpro抑制剂5，该化合物与奈玛特韦展现出截然不同的结合模式。本研究设计大环化策略以锁定活性构象，得到了内酯（lactone）12，其抗病毒活性显著提升。经过进一步优化，得到了活性强劲的内酰胺（lactam）26，该化合物不仅对奈玛特韦耐药变异株展现出优异的活性，同时对一组源自其他冠状病毒的病毒主蛋白酶也表现出极强的抑制效力。