人冠状病毒OC43对主蛋白酶抑制剂FB2001的耐药性

The resistance characteristics and mechanisms of HCoV-OC43 to protease inhibitor FB2001

摘要:

【背景】人冠状病毒OC43 (human coronavirus OC43, HCoV-OC43)属β冠状病毒，其感染会对免疫低下的老人和幼儿的健康带来严重威胁。FB2001作为已进入临床试验的抗冠状病毒主蛋白酶3CLpro抑制剂，前期研究表明其对HCoV-OC43也具有良好的抗病毒活性，但HCoV-OC43对FB2001的耐药特征有待阐明。【目的】研究HCoV-OC43 3CLpro对FB2001产生的耐药突变位点及耐药机制，发现冠状病毒对3CLpro抑制剂耐药的共性特征，对广谱抗冠状病毒3CLpro小分子药物的开发和优化改造具有重要意义。【方法】通过在体外进行药物压力下连续传代实验获得具有明显耐药表型的HCoV-OC43耐药适应株，二代测序(next generation sequencing, NGS)发现病毒3CLpro上存在多个突变位点，然后构建HCoV-OC43 3CLpro Flip GFP活细胞酶活系统验证耐药突变特征，最后通过Alphafold 3和DiffDock人工智能平台获得突变蛋白-小分子的结合模式，分析耐药机制并通过生长竞争试验研究耐药适应株的生长适应性变化。【结果】耐药适应株3CLpro出现D65G、L165V、I300T、D65G+L165V氨基酸突变，这些突变均会导致3CLpro在HEK-293T细胞中对FB2001的敏感性降低，其中L165V造成的耐药特征最显著；3CLpro^L165V-FB2001复合物结构预测显示，突变后第165位的Val侧链残基改变3CLpro活性中心S1位局部构象，导致蛋白酶与FB2001的P1位亲和力下降；生长竞争试验结果表明，耐药适应株在与HCoV-OC43野生株的生长竞争中不具有生长竞争优势。【结论】本研究提示冠状病毒主蛋白酶对P1位为γ-内酰胺环的拟肽抑制剂具有耐药共性机制。

Abstract:

[Background] Human coronavirus OC43 (HCoV-OC43) belongs to betacoronavirus, and its infection poses a serious threat to the health of immunocompromised elderly and young children. As a reported 3CLpro inhibitor that has entered phase II/III clinical trial against coronaviruses, FB2001 has strong inhibitory activity against HCoV-OC43, while the resistance characteristics of HCoV-OC43 to FB2001 remain to be clarified. [Objective] To study the mutation sites and resistance mechanism of HCoV-OC43 3CLpro to FB2001 and discover the common characteristics of coronavirus resistance to 3CLpro inhibitors, which is of great significance for the development and optimization of broad-spectrum small molecule drugs targeting coronavirus 3CLpro. [Methods] A HCoV-OC43 strain with obvious resistance was obtained by continuous passage under drug pressure in vitro. Next generation sequencing revealed multiple mutation sites on 3CLpro. Then, a HCoV-OC43 3CLpro Flip GFP system was constructed to verify the characteristics of resistance mutations. Finally, the mutant protein-small molecule binding mode was predicted by Alphafold 3 and DiffDock to reveal the drug resistance mechanism, and a growth competition experiment was carried out to investigate the adaptive growth changes of the resistant strain. [Results] The amino acid mutations of D65G, L165V, I300T, and D65G+L165V were present in 3CLpro of the resistant strain. All the mutations reduced the sensitivity of 3CLpro to FB2001 in HEK-293T cells, among which L165V caused the most significant resistance. Structural prediction of the 3CLpro^L165V-FB2001 complex showed that the Val side chain residue at position 165 changed the local conformation of the 3CLpro active center at position S1 after mutation, resulting in a decrease in affinity between protease and FB2001 at position P1. The results of the growth competition experiment showed that the resistant strain did not have a growth competitive advantage over the wild-type strain HCoV-OC43. [Conclusion] This study suggests that the main protease of coronaviruses has a common mechanism of resistance to peptide-like inhibitors with γ-lactam ring at P1.