The utility of native MS for understanding the mechanism of action of repurposed therapeutics in COVID-19: heparin as a disruptor of the SARS-CoV-2 interaction with its host cell receptor

Abstract

The emergence and rapid proliferation of the novel coronavirus (SARS-CoV-2) resulted in a global pandemic, with over six million cases and nearly four hundred thousand deaths reported worldwide by the end of May 2020. A rush to find the cures 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 non-covalent 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 of 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-pI 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.

Unfractionated heparin inhibits live wild type SARS‐CoV‐2 cell infectivity at therapeutically relevant concentrations

“…Regarding the underlying mechanisms, we provided additional evidence here for the action of heparin to destabilise the RBD protein structure (Figure 3) and notably the first evidence for the ability of heparin to directly inhibit binding of the RBD to its protein receptor human ACE2 (Figure 4). Both these pieces of data are supported by recent preliminary observations using native MS (Yang, Du, & Kaltashov, 2020). Thus, there is a clear mechanistic basis supporting the notion of targeting these interactions with heparin and related compounds.…”

A comprehensive guide to the pharmacologic regulation of angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 entry receptor

“…In a recent study, it has been shown that heparin forms 1:1 complexes with the receptor-binding domain of the S1 protein and disrupts its binding to ACE2 (Y. Yang, Du, & Kaltashov, 2020 ). In the context of SARS-CoV-2, a growing body of evidence suggests that SARS-CoV-2 can bind the glycosaminoglycans HS and unfractionated heparin (UFH), dependent on their level of sulphation (W. Hao et al, 2020 ; Li et al, 2020 , Mycroft-West et al, 2020 ; Tree et al, 2020 ).…”

“…However, another recent study indicates that corticosteroid use is associated with increased mortality and delayed SARS–CoV-2 coronavirus RNA clearance in 409 COVID-19 patients ( J. Liu et al, 2020 ). While some of the meta-analyses conclude that the use of corticosteroids is associated with a higher rate of ARDS in COVID-19 patients ( Z. Yang et al, 2020 ; J. J. Y. Zhang, Lee, Ang, Leo, & Young, 2020 ), a recent randomized study of several thousands of hospitalized COVID-19 patients reports that dexamethasone reduces the mortality among those receiving invasive mechanical ventilation or oxygen ( Horby et al, 2020 ). In another study of 396 COVID-19 patients, the use of steroids significantly decreased in-hospital mortality ( Fernandez Cruz et al, 2020 ), an observation that has been corroborated by several additional recent publications ( Bani-Sadr et al, 2020 ; Chopra et al, 2020 ; Majmundar et al, 2020 ).…”

Platelet Activating Immune Complexes Identified in COVID-19 Associated Coagulopathy

“…21 This is further supported by inhibition with therapeutic heparin, since heparin binds the SARS-CoV-2 RBD to cause a conformational change with altered binding specificity, potentially disrupting the ICs and inhibiting platelet activation. 22 Regardless, the data presented here clearly outline the characteristics of these ICs and differentiate them from other severe coagulation disorders, including HIT and TTP.…”