The development of broad-spectrum antivirals against human coronaviruses (HCoVs) is critical to combat the current coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, as well as future outbreaks of emerging CoVs. We have previously identified a polyethylene glycol-conjugated (PEGylated) lipopeptide, EK1C4, with potent pan-CoV fusion inhibitory activity. However, PEG linkers in peptide or protein drugs may reduce stability or induce anti-PEG antibodies in vivo. Therefore, we herein report the design and synthesis of a series of dePEGylated lipopeptide-based pan-CoV fusion inhibitors featuring the replacement of the PEG linker with amino acids in the heptad repeat 2 C-terminal fragment (HR2-CF) of HCoV-OC43. Among these lipopeptides, EKL1C showed the most potent inhibitory activity against infection by SARS-CoV-2 and its spike (S) mutants, as well as other HCoVs and some bat SARS-related coronaviruses (SARSr-CoVs) tested. The dePEGylated lipopeptide EKL1C exhibited significantly stronger resistance to proteolytic enzymes, better metabolic stability in mouse serum, higher thermostability than the PEGylated lipopeptide EK1C4, suggesting that EKL1C could be further developed as a candidate prophylactic and therapeutic for COVID-19 and other coronavirus diseases.

开发针对人类冠状病毒 (HCoV) 的广谱抗病毒药物对于对抗由严重急性呼吸系统综合症冠状病毒 2 (SARS-CoV-2) 及其变体以及当前引起的 2019 年冠状病毒病 (COVID-19) 大流行至关重要新兴冠状病毒的未来爆发。我们之前已经鉴定出一种聚乙二醇缀合（PEG 化）脂肽 EK1C4，具有强大的泛冠状病毒融合抑制活性。然而，肽或蛋白质药物中的 PEG 接头可能会降低稳定性或在体内诱导抗 PEG 抗体. 因此，我们在此报告了一系列基于去聚乙二醇化脂肽的泛冠状病毒融合抑制剂的设计和合成，其特点是用 HCoV-OC43 的七肽重复 2 C 末端片段 (HR2-CF) 中的氨基酸替换 PEG 接头。 . 在这些脂肽中，EKL1C 对 SARS-CoV-2 及其刺突 (S) 突变体以及其他 HCoV 和一些与蝙蝠 SARS 相关的冠状病毒 (SARSr-CoV) 的感染表现出最有效的抑制活性。与聚乙二醇化脂肽EK1C4相比，去聚乙二醇化脂肽EKL1C对蛋白水解酶的抵抗力明显更强，在小鼠血清中的代谢稳定性更好，热稳定性更高，这表明EKL1C可以进一步开发为COVID-19和其他冠状病毒疾病的候选预防和治疗剂。