Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China.
Phytomedicine. 2023 Jun;114:154796. doi: 10.1016/j.phymed.2023.154796. Epub 2023 Mar 29.
The 3C-like proteases (3CLs) are cysteine-rich homodimeric proteins and can be covalently modified by numerous natural and synthetic compounds, which in turn, block the proteolytic activity or the formation of enzymatically active dimeric forms. Although herbal medicines have been widely used to treat COVID-19, identification of the key herbal constituents that can covalently modify the 3CLs in β-coronaviruses (CoVs) remains a big challenge.
To construct a comprehensive approach for efficient discovering the covalent SARS-CoV-2 3CL inhibitors from herbal medicines. To decipher the key anti-SARS-CoV-2 3CL constituents in Ginkgo biloba extract 50 (GBE50) and to study their anti-SARS-CoV-2 3CL mechanisms.
SARS-CoV-2 3CL inhibition assay including time-dependent inhibition assays and inactivation kinetic analyses were conducted using a fluorescence-based biochemical assay. The constituents in GBE50 were analyzed by UHPLC-Q-Exactive Orbitrap HRMS. The peptides modified by herbal constituents were characterized by using nanoLC-MS/MS.
Following testing the anti-SARS-CoV-2 3CL effects of 104 herbal medicines, it was found that Ginkgo biloba extract 50 (GBE50) potently inhibited SARS-CoV-2 3CL in dose- and time-dependent manners. A total of 38 constituents were identified from GBE50 by UHPLC-Q-Exactive Orbitrap HRMS, while 26 peptides modified by 18 constituents were identified by chemoproteomic profiling. The anti-SARS-CoV-2 3CL effects of 18 identified covalent inhibitors were then validated by performing time-dependent inhibition assays. The results clearly demonstrated that most tested constituents showed time-dependent inhibition on SARS-CoV-2 3CL, while gallocatechin and sciadopitysin displayed the most potent anti-SARS-CoV-2 3CL effects.
Collectively, GBE50 and some constituents in this herbal product could strongly inhibit SARS-CoV-2 3CL in dose- and time-dependent manner. Gallocatechin and sciadopitysin were identified as potent SARS-CoV-2 3CL inhibitors, which offers promising lead compounds for the development of novel anti-SARS-CoV-2 drugs.
3C 样蛋白酶（3CLs）是富含半胱氨酸的同源二聚体蛋白，可被多种天然和合成化合物共价修饰，进而阻断蛋白水解活性或形成酶活性二聚体形式。虽然草药已被广泛用于治疗 COVID-19，但鉴定可共价修饰β-冠状病毒（CoVs）中 3CL 的关键草药成分仍是一项巨大的挑战。
构建一种从草药中高效发现共价 SARS-CoV-2 3CL 抑制剂的综合方法。解析银杏叶提取物 50（GBE50）中抗 SARS-CoV-2 3CL 的关键成分，并研究其抗 SARS-CoV-2 3CL 机制。
采用基于荧光的生化测定法进行 SARS-CoV-2 3CL 抑制测定，包括时程抑制测定和失活动力学分析。使用 UHPLC-Q-Exactive Orbitrap HRMS 分析 GBE50 中的成分。通过纳升 LC-MS/MS 鉴定草药成分修饰的肽。
在测试了 104 种草药的抗 SARS-CoV-2 3CL 作用后，发现银杏叶提取物 50（GBE50）可有效剂量和时间依赖性地抑制 SARS-CoV-2 3CL。通过 UHPLC-Q-Exactive Orbitrap HRMS 从 GBE50 中鉴定出 38 种成分，通过化学蛋白质组学分析鉴定出 18 种成分修饰的 26 种肽。然后通过进行时程抑制测定验证了 18 种鉴定的共价抑制剂的抗 SARS-CoV-2 3CL 作用。结果清楚地表明，大多数测试的成分对 SARS-CoV-2 3CL 表现出时程抑制作用，而没食子儿茶素和穗花杉双黄酮表现出最强的抗 SARS-CoV-2 3CL 作用。
总之，GBE50 和该草药产品中的一些成分可剂量和时间依赖性地强烈抑制 SARS-CoV-2 3CL。没食子儿茶素和穗花杉双黄酮被鉴定为有效的 SARS-CoV-2 3CL 抑制剂，为开发新型抗 SARS-CoV-2 药物提供了有前途的先导化合物。
