The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an important role in viral replication and transcription and received great attention as a vital target for drug/peptide development. Therapeutic agents such as small-molecule drugs or peptides that interact with the Cys–His present in the catalytic site of Mpro are an efficient way to inhibit the protease. Although several emergency-approved vaccines showed good efficacy and drastically dropped the infection rate, evolving variants are still infecting and killing millions of people globally. While a small-molecule drug (Paxlovid) received emergency approval, small-molecule drugs have low target specificity and higher toxicity. Besides small-molecule drugs, peptide therapeutics are thus gaining increasing popularity as they are easy to synthesize and highly selective and have limited side effects. In this study, we investigated the therapeutic value of 67 peptides targeting Mpro using molecular docking. Subsequently, molecular dynamics (MD) simulations were implemented on eight protein–peptide complexes to obtain molecular-level information on the interaction between these peptides and the Mpro active site, which revealed that temporin L, indolicidin, and lymphocytic choriomeningitis virus (LCMV) GP1 are the best candidates in terms of stability, interaction, and structural compactness. These peptides were synthesized using the solid-phase peptide synthesis protocol, purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and authenticated by mass spectrometry (MS). The in vitro fluorometric Mpro activity assay was used to validate the computational results, where temporin L and indolicidin were observed to be very active against SARS-CoV-2 Mpro with IC₅₀ values of 38.80 and 87.23 μM, respectively. A liquid chromatography–MS (LC–MS) assay was developed, and the IC₅₀ value of temporin L was measured at 23.8 μM. The solution-state nuclear magnetic resonance (NMR) structure of temporin L was determined in the absence of sodium dodecyl sulfate (SDS) micelles and was compared to previous temporin structures. This combined investigation provides critical insights and assists us to further develop peptide inhibitors of SARS-CoV-2 Mpro through structural guided investigation.

中文翻译：

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通过计算筛选和体外蛋白酶测定研究抑制 SARS-CoV-2 主要蛋白酶的抗病毒肽

严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的主要蛋白酶（Mpro）在病毒复制和转录中发挥着重要作用，作为药物/肽开发的重要靶点而受到高度关注。与 Mpro 催化位点中存在的 Cys-His 相互作用的小分子药物或肽等治疗剂是抑制蛋白酶的有效方法。尽管几种紧急​​批准的疫苗显示出良好的功效并大幅降低了感染率，但不断演变的变种仍在全球范围内感染和杀死数百万人。虽然小分子药物（Paxlovid）获得紧急批准，但小分子药物的靶点特异性较低，毒性较高。除小分子药物外，肽类药物因其易于合成、选择性高且副作用有限而越来越受欢迎。在这项研究中，我们利用分子对接研究了 67 种靶向 M​​pro 的肽的治疗价值。随后，对八种蛋白质-肽复合物进行了分子动力学 (MD) 模拟，以获得这些肽与 Mpro 活性位点之间相互作用的分子水平信息，结果揭示了 temporin L、indolicidin 和淋巴细胞性脉络膜脑膜炎病毒 (LCMV) GP1就稳定性、相互作用和结构紧凑性而言，它们是最佳候选者。这些肽是使用固相肽合成方案合成的，通过反相高效液相色谱 (RP-HPLC) 纯化，并通过质谱 (MS) 验证。体外荧光 Mpro 活性测定用于验证计算结果，其中观察到 temporin L 和 indolicidin 对 SARS-CoV-2 Mpro 非常活跃，IC ₅₀值分别为 38.80 和 87.23 μM。开发了液相色谱-质谱 (LC-MS) 检测方法，测得 temporin L 的 IC ₅₀值为 23.8 μM。在不存在十二烷基硫酸钠 (SDS) 胶束的情况下测定了 temporin L 的溶液态核磁共振 (NMR) 结构，并与之前的 temporin 结构进行了比较。这项综合研究提供了重要的见解，并帮助我们通过结构引导研究进一步开发 SARS-CoV-2 Mpro 的肽抑制剂。