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 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 I 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.

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.…”

Use of Enoxaparin to Counteract COVID-19 Infection and Reduce Thromboembolic Venous Complications: A Review of the Current Evidence

“…These findings are implied in the process of repurposing heparin a first line therapeutic agent as an antiviral agent and tailor made GAG based antiviral agent. Yang et al (2020) also showed by native mass spectrometry that both short (pentasaccharide) and relatively long (eicosasaccharide) heparin oligomers form 1:1 complexes with S1 protein receptor binding domain, supporting the existence of a single binding site. This association induces a conformational change with an important reduction of the ability to associate with ACE2.…”

“… Yang et al. (2020) also showed by native mass spectrometry that both short (pentasaccharide) and relatively long (eicosasaccharide) heparin oligomers form 1:1 complexes with S1 protein receptor binding domain, supporting the existence of a single binding site.…”

Proteomic Approaches to Study SARS-CoV-2 Biology and COVID-19 Pathology

“… 75 While SARS-CoV-2 S protein and ACE2 are challenging to study by nMS given their extensive glycosylation, nMS with limited charge reduction 76 provided meaningful information about the complex between ACE2 and the receptor-binding domain (RBD) of the S protein as well as the role of heparin in destabilizing the ACE2/RBD association. 77 Yang et al showed that both short (pentasaccharide) and long (eicosasaccharide) heparin oligomers form a stoichiometric complex with the RBD, indicating a single binding site which alters the protein conformation and subsequently results in a decrease in its ability to associate with ACE2. 77 This study suggests that nMS might be a powerful method for studying the interaction between drugs and their therapeutic targets.…”