研究揭示肽模拟抑制剂促进、ebselen 破坏 SARS-CoV-2 M pro 二聚化的不同机制
SARS-CoV-2 main protease inhibition through dimerization promotion by peptidomimetic inhibitors and disruption by ebselen
研究以 13C 标记结合原生质谱和氢氘交换质谱（HDX-MS），考察 SARS-CoV-2 主蛋白酶 M pro 的单体-二聚体平衡及抑制剂结合引起的构象变化。结果显示两类抑制剂机制相反：PF-07321332、PF-00835231、GC376、boceprevir 等肽模拟抑制剂显著把平衡推向二聚体，抑制亚基交换并刚性化二聚界面；ebselen 则妨碍二聚体形成、增加界面柔性。串联质谱还发现 ebselen 在 C300 的新共价结合位点，分子动力学模拟与突变分析表明该修饰通过变构改变二聚界面氢键网络，直接参与 ebselen 破坏二聚体与抑制酶活。
来源：eLife · doi.org
The SARS-CoV-2 main protease (M pro ) is a key antiviral target that equilibrates between an active dimer and inactive monomer, making the dimerization interface a promising therapeutic vulnerability. This study investigates M pro monomer-dimer equilibrium and conformational changes induced by inhibitor binding using 13 C labeling with native mass spectrometry and hydrogen/deuterium exchange mass spectrometry (HDX-MS). We evaluated how peptidomimetic inhibitors (PF-07321332, PF-00835231, GC376, boceprevir) and non-peptidomimetic inhibitors (carmofur, ebselen, MR6-31-2, AT7519, pelitinib) influence M pro dimerization and subunit exchange. Key findings revealed divergent mechanisms: peptidomimetic inhibitors significantly shifted the equilibrium towards the dimeric state, suppressing subunit exchange dynamics and rigidifying the dimer interface; in contrast, ebselen impaired dimer formation and increased interface flexibility. Notably, tandem mass spectrometry identified a novel covalent binding site for ebselen at C300. Molecular dynamics simulations and mutational analyses demonstrated that this C300 modification allosterically alters the hydrogen bond network of the dimer interface, directly contributing to ebselen-mediated dimer disruption and enzymatic inhibition. Overall, this study clarifies distinct inhibitory modes between peptidomimetics and ebselen, highlighting the therapeutic potential of targeting allosteric sites at the dimer interface for designing next-generation M pro inhibitors.