Following translation of the SARS-CoV-2 RNA genome into two viral polypeptides, the main protease Mpro cleaves at eleven sites to release non-structural proteins required for viral replication. M^Pro is an attractive target for antiviral therapies to combat the coronavirus-2019 disease (COVID-19). Here, we have used native mass spectrometry (MS) to characterize the functional unit of Mpro. Analysis of the monomer-dimer equilibria reveals a dissociation constant of K_d = 0.14 ± 0.03 µM, revealing M^Pro has a strong preference to dimerize in solution. Developing an MS-based kinetic assay we then characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, which are effectively flash-frozen as they transition from solution to the gas phase. We screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the catalytically active dimer, slow the rate of substrate processing by ~35%. This information was readily obtained and, together with analysis of the x-ray crystal structures of these enzyme-small molecule complexes, provides a starting point for the development of more potent molecules that allosterically regulate M^Pro activity.

中文翻译：

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SARS-CoV-2主要蛋白酶的变构抑制作用-基于质谱分析的见解

SARS-CoV-2 RNA基因组翻译成两个病毒多肽后，主要蛋白酶Mpro在11个位点裂解，释放出病毒复制所需的非结构蛋白。M ^Pro是对抗冠状病毒2019疾病（COVID-19）的抗病毒治疗的有吸引力的靶标。在这里，我们使用了天然质谱（MS）来表征Mpro的功能单元。单体-二聚体平衡分析表明解离常数K _d = 0.14±0.03 µM，表明M ^Pro强烈希望在溶液中二聚。然后，通过开发基于质谱的动力学分析，我们通过跟踪酶-底物复合物的时间变化来表征底物周转率，当酶从溶液过渡到气相时，酶-底物复合物可以进行快速冷冻。我们筛选了与活动位点结合较远的小分子的调节活性的能力。这些化合物（包括一种建议破坏催化活性二聚体的化合物）会使底物处理速度降低约35％。这些信息很容易获得，并且与这些酶-小分子复合物的X射线晶体结构分析一起，为开发变构调节M ^Pro活性的更强分子提供了起点。