Well-Defined Heparin Mimetics Can Inhibit Binding of the Trimeric Spike of SARS-CoV-2 in a Length-Dependent Manner

Abstract

The emergence of new SARS-CoV-2 variants and the dangers of long-covid necessitate the development of broad-acting therapeutics that can reduce viral burden. SARS-CoV-2 employs heparan sulfate (HS) as an initial cellular attachment factor, and therefore, there is interest in developing heparin as a therapeutic for SARS-CoV-2. Its use is, however, complicated by structural heterogeneity and the risk of causing bleeding and thrombocytopenia. Here, we describe the preparation of well-defined heparin mimetics by a controlled head-to-tail assembly of HS oligosaccharides having an alkyne or azide moiety by copper-catalyzed azide-alkyne cycloaddition (CuAAC). Alkyne- and azide-containing sulfated oligosaccharides were prepared from a common precursor by modifying an anomeric linker with 4-pentynoic acid and by enzymatic extension with an N -acetyl-glucosamine having an azide moiety at C-6 (GlcNAc6N 3 ), respectively, followed by CuAAC. The process of enzymatic extension with GlcNAc6N 3 followed by CuAAC with the desired alkyne-containing oligosaccharides could be repeated to give compounds composed of 20 and 27 monosaccharides, respectively. The heparin mimetics could inhibit the binding of the SARS-CoV-2 spike or RBD to immobilized heparin or to Vero E6 cells. The inhibitory potency increased with increasing chain length, and a compound composed of four sulfated hexasaccharides linked by triazoles had a similar potency as unfractionated heparin. Sequence analysis and HS microarray binding studies with a wide range of RBDs of variants of concern indicate that they have maintained HS-binding capabilities and selectivities. The heparin mimetics exhibit no or reduced binding to antithrombin-III and platelet factor 4, respectively, which are associated with side effects.

Block Synthesis and Step-Growth Polymerization of C-6-Sulfonatomethyl-Containing Sulfated Malto-Oligosaccharides and Their Biological Profiling

“…It has been shown that heparin and heparinoid derivatives can prevent the binding of SARS-CoV-2 to human cells and inhibit the entry of different SARS-CoV-2 strains into cells [ 46 , 47 , 48 ]. Therefore, we hypothesized that our heparin-analogue oligosaccharides may be effective against SARS-associated coronaviruses.…”

The accomplices: Heparan sulfates and N-glycans foster SARS-CoV-2 spike:ACE2 receptor binding and virus priming

“…They are composed of a core protein with covalently attached long, variably sulfated polysaccharide chains. HSPGs are co-receptors for SARS-CoV-2 infection and bind spike via its basic domains (Clausen et al, 2020; Kim et al, 2020; Liu et al, 2021; Sun et al, 2023). The variability of sulfation patterns in HS across different individuals, tissues, and cell types may contribute to virus tropism, although this variability hinders a full comprehension of their mechanistic role (Kearns et al, 2022).…”

“…Heparin, a long, linear, highly-sulfated polyanionic oligomer, is frequently used as a model for HS in experimental studies due to their structural similarity (Hogwood et al, 2023). Computational and experimental studies have demonstrated that heparin can act as a potent antiviral agent by competing with HSPGs for binding to spike (Clausen et al, 2020; Kim et al, 2022; Kim et al, 2020; Liu et al, 2021; Mycroft-West et al, 2020; Paiardi et al, 2022; Sun et al, 2023; Tree et al, 2021) and, intriguingly, that spike forms a ternary complex by simultaneously binding ACE2 and heparin or HS (Cecon et al, 2022; Clausen et al, 2020; Kim et al, 2023). We previously characterized the anti-viral effects of heparin on SARS-CoV-2 by performing over 20µs of all-atom molecular dynamics (MD) simulation of spike in closed and open conformations, each with zero, one or three heparin chains bound.…”

Heparan Sulfate-Mimicking Glycopolymers Bind SARS-CoV-2 Spike Protein in a Length- and Sulfation Pattern-Dependent Manner

“…Binding of SARS-CoV-2 to heparin and HS is mediated by ionic interactions between the positively charged amino acid cluster in the heparin-binding domains (HBDs) of the spike (Figure A) and the negatively charged sulfate and carboxylate groups of HS and heparin. Computational and experimental studies have reported three putative HBDs in the spike sequence, including in the RBD, at the S1/S2 furin cleavage site, and at the S2′ TMPRSS2 cleavage site (Figure A). ,− As a result, the purpose of the studies presented here is to determine whether HS-mimicking glycopolymers (Figure C) could compete with HS (Figure B) to bind the spike protein in a length- and sulfation pattern-dependent manner and whether these compounds can bind to the S1/S2 junction and consequently shield it from cleavage by furin.…”

“…Computational and experimental studies have reported three putative HBDs in the spike sequence, including in the RBD, at the S1/S2 furin cleavage site, and at the S2′ TMPRSS2 cleavage site (Figure 1A). 12,37−39 As a result, the purpose of the studies presented here is to determine whether HS-mimicking glycopolymers (Figure 1C) could compete with HS (Figure 1B) to bind the spike protein in a length-and sulfation pattern-dependent manner 40 and whether these compounds can bind to the S1/ S2 junction and consequently shield it from cleavage by furin.…”