SARS-CoV-2 virus mutations might increase its virulence, and thus the severity and duration of the ongoing pandemic. Global drug discovery campaigns have successfully developed several vaccines to reduce the number of infections by the virus. However, finding a small molecule pharmaceutical that is effective in inhibiting SARS-CoV-2 remains a challenge. Natural products are the origin of many currently used pharmaceuticals and, for this reason, a library of in-house fungal extracts were screened to assess their potential to inhibit the main viral protease M^pro in vitro. The extract of Penicillium citrinum, TDPEF34, showed potential inhibition and was further analysed to identify potential M^pro inhibitors. Following bio-guided isolation, a series of benzodiazepine alkaloids cyclopenins with good-to-moderate activity against SARS-CoV-2 M^pro were identified. The mode of enzyme inhibition of these compounds was predicted by docking and molecular dynamic simulation. Compounds 1 (isolated as two conformers of S- and R-isomers), 2, and 4 were found to have promising in vitro inhibitory activity towards M^pro, with an IC₅₀ values range of 0.36–0.89 µM comparable to the positive control GC376. The in silico investigation revealed compounds to achieve stable binding with the enzyme active site through multiple H-bonding and hydrophobic interactions. Additionally, the isolated compounds showed very good drug-likeness and ADMET properties. Our findings could be utilized in further in vitro and in vivo investigations to produce anti-SARS-CoV-2 drug candidates. These findings also provide critical structural information that could be used in the future for designing potent M^pro inhibitors.

中文翻译：

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生物引导下从柑橘青霉 TDPEF34 中分离作为潜在 SARS-CoV-2 Mpro 抑制剂的环青素类似物


SARS-CoV-2 病毒突变可能会增加其毒力，从而增加当前大流行的严重程度和持续时间。全球药物发现活动已成功开发出多种疫苗来减少病毒感染的数量。然而，寻找有效抑制 SARS-CoV-2 的小分子药物仍然是一个挑战。天然产物是许多目前使用的药物的来源，因此，我们筛选了内部真菌提取物库，以评估它们在体外抑制主要病毒蛋白酶 M ^pro的潜力。柑橘青霉提取物 TDPEF34 显示出潜在的抑制作用，并经过进一步分析以确定潜在的 M ^pro抑制剂。经过生物引导分离后，鉴定出一系列对 SARS-CoV-2 M ^pro具有良好至中等活性的苯二氮卓类生物碱环青素。通过对接和分子动力学模拟预测了这些化合物的酶抑制模式。发现化合物1 （分离为 S 和 R 异构体的两种构象异构体）、 2和4对 M ^pro具有良好的体外抑制活性，与阳性对照 GC376 相比，IC ₅₀值范围为 0.36–0.89 µM 。计算机研究揭示了化合物通过多重氢键和疏水相互作用实现与酶活性位点的稳定结合。此外，分离的化合物显示出非常好的药物相似性和 ADMET 特性。我们的研究结果可用于进一步的体外和体内研究，以生产抗 SARS-CoV-2 候选药物。 这些发现还提供了关键的结构信息，可在未来用于设计有效的 M ^pro抑制剂。