Department of Chemistry, University of Massachusetts-Amherst, 240 Thatcher Way, Amherst, Massachusetts 01003, United States.
Anal Chem. 2020 Aug 18;92(16):10930-10934. doi: 10.1021/acs.analchem.0c02449. Epub 2020 Jul 27.
The emergence and rapid proliferation of the novel coronavirus (SARS-CoV-2) resulted in a global pandemic, with over 6,000,000 cases and nearly 400,000 deaths reported worldwide by the end of May 2020. A rush to find a cure prompted re-evaluation of a range of existing therapeutics vis-à-vis their potential role in treating COVID-19, placing a premium on analytical tools capable of supporting such efforts. Native mass spectrometry (MS) has long been a tool of choice in supporting the mechanistic studies of drug/therapeutic target interactions, but its applications remain limited in the cases that involve systems with a high level of structural heterogeneity. Both SARS-CoV-2 spike protein (S-protein), a critical element of the viral entry to the host cell, and ACE2, its docking site on the host cell surface, are extensively glycosylated, making them challenging targets for native MS. However, supplementing native MS with a gas-phase ion manipulation technique (limited charge reduction) allows meaningful information to be obtained on the noncovalent complexes formed by ACE2 and the receptor-binding domain (RBD) of the S-protein. Using this technique in combination with molecular modeling also allows the role of heparin in destabilizing the ACE2/RBD association to be studied, providing critical information for understanding the molecular mechanism of its interference with the virus docking to the host cell receptor. Both short (pentasaccharide) and relatively long (eicosasaccharide) heparin oligomers form 1:1 complexes with RBD, indicating the presence of a single binding site. This association alters the protein conformation (to maximize the contiguous patch of the positive charge on the RBD surface), resulting in a notable decrease in its ability to associate with ACE2. The destabilizing effect of heparin is more pronounced in the case of the longer chains due to the electrostatic repulsion between the low-p ACE2 and the heparin segments not accommodated on the RBD surface. In addition to providing important mechanistic information on attenuation of the ACE2/RBD association by heparin, the study demonstrates the yet untapped potential of native MS coupled to gas-phase ion chemistry as a means of facilitating rational repurposing of the existing medicines for treating COVID-19.
新型冠状病毒（SARS-CoV-2）的出现和迅速传播导致了一场全球大流行，截至 2020 年 5 月底，全球报告的病例已超过 600 万例，死亡近 40 万例。为了寻找治疗方法，人们匆忙评估了一系列现有的治疗药物，以期确定它们在治疗 COVID-19 方面的潜在作用，这就需要能够支持此类研究的分析工具。天然质谱（MS）长期以来一直是支持药物/治疗靶标相互作用机制研究的首选工具，但在涉及结构异质性水平较高的系统的情况下，其应用仍然有限。新型冠状病毒的刺突蛋白（S 蛋白）和 ACE2 都是病毒进入宿主细胞的关键元素，而且它们都高度糖基化，这使得它们成为天然 MS 的挑战性靶标。然而，通过将气相离子操纵技术（有限电荷还原）与天然 MS 结合使用，可以获得有关 ACE2 和 S 蛋白受体结合域（RBD）形成的非共价复合物的有意义信息。使用该技术与分子建模相结合，还可以研究肝素在破坏 ACE2/RBD 结合方面的作用，为理解其干扰病毒与宿主细胞受体结合的分子机制提供关键信息。短（五糖）和相对长（二十糖）肝素寡糖都与 RBD 形成 1:1 复合物，表明存在一个单一的结合位点。这种结合改变了蛋白质构象（以最大限度地增加 RBD 表面正电荷的连续斑块），导致其与 ACE2 结合的能力显著下降。由于低 p ACE2 与未在 RBD 表面上容纳的肝素片段之间的静电排斥，较长链的肝素的去稳定作用更为明显。除了提供肝素对 ACE2/RBD 结合的抑制作用的重要机制信息外，该研究还证明了天然 MS 与气相离子化学相结合作为一种促进现有药物合理重新用于治疗 COVID-19 的手段，具有尚未开发的潜力。
