On the Selectivity of Heparan Sulfate Recognition by SARS-CoV-2 Spike Glycoprotein

Abstract

SARS-CoV-2 infects human cells through its surface spike glycoprotein (SgP), which relies on host cell surface heparan sulfate (HS) proteoglycans that facilitate interaction with the ACE2 receptor. Targeting this process could lead to inhibitors of early steps in viral entry. Screening a microarray of 24 HS oligosaccharides against recombinant S1 and receptor-binding domain (RBD) proteins led to identification of only eight sequences as potent antagonists; results that were supported by detailed dual-filter computational studies. Competitive studies using the HS microarray suggested almost equivalent importance of IdoA2S-GlcNS6S and GlcNS3S structures, which were supported by affinity studies. Exhaustive virtual screening on a library of >93 000 sequences led to a novel pharmacophore with at least two 3-O-sulfated GlcN residues that can engineer unique selectivity in recognizing the RBD. This work puts forward the key structural motif in HS that should lead to potent and selective HS or HS-like agents against SARS-CoV-2.

Figure 1

SARS-CoV-2 spike glycoprotein (SgP) recognition of a library of HS sequences. (A) Key to the structure of 24 HS sequences printed on the microarray. (B) Images of fluorescence from the bound RBD (left) and S1 (right) proteins (20 μg/mL) detected using Alexa Fluor 488 conjugated to streptavidin. Highlighted rectangles correspond to HS sequences in (A) that exhibit preferential recognition of the two proteins done in six spot replicates (n = 6) for each HS sequence. Two independent experiments were minimally performed for each protein. (C) Plot showing quantitative fluorescence for each HS sequence as numbered in (A). Error bars represent ±1 SE. Negative control (NC) = printing buffer; positive control (PC) = biotinylated mannose. See Supporting Information for detailed experimental conditions.

Figure 2

Computational studies for understanding HS recognition of RBD. (A) Virtual screening of the library of HS sequences (HS01–HS24) for binding to the receptor binding domain (RBD) of spike glycoprotein (SgP). GOLD-based docking and scoring were performed in triplicate. Two parameters, GOLDScore and RMSD between the top six poses for each HS sequence, were calculated to evaluate preferential recognition of HS sequences and correlation with microarray data. (B) Correspondence between microarray intensity and GOLDScore for each HS sequence. HS17 → HS24 sequences containing one or more IdoA2S residues were modeled in either all ¹C₄ or ²S_O forms. See Table S1 for details.

Figure 3

Studies using fondaparinux (FPX, panel A) and HS hexasaccharide (Hexa, panel B) as competitors in RBD recognition of 24 HS sequences on the microarray. The competition experiments were performed at least twice (biological replicates) in the manner similar to that for RBD alone (Figure 1) except for the addition of either 0.5 → 50 μM FPX (green squares/lines) or 0.5 → 50 μM Hexa (brown circles/lines) (n = 6). (A) Plot showing quantitative fluorescence as a function concentration of FPX. The occurrences of slight increases in fluorescence between 0 and 0.5 μM FPX in some cases were determined to be insignificant (p > 0.05). (B) Plot showing quantitative fluorescence as a function of concentration of Hexa. (C) Plots of percent change in fluorescence as a function of increasing ligand concentration (FPX or Hexa) for the six promising sequences (HS15, HS16, HS21, HS22, HS23, and HS24). Error bars represent ±1 SE. See Supporting Information for experimental conditions.

Figure 4

Computational screening of a library of di-, tetra-, and hexasaccharide sequences of HS (total 95 976 topologies) against the RBD of SgP for identification of origin of selectivity at the atomistic level. (A) Flowchart of the dual-filter algorithm used in computational screening, which included GOLDScore as the first filter and RMSD (consistency of binding) as second filter. (B) Results following application of the first filter in the form of a histogram of the number of HS hexasaccharide topologies for every 10-unit change in GOLDScore. Inset shows promising high affinity topologies. (C) High-affinity, high-specificity sequences (shown as sticks) bound to the “open” RBD (blue rendering) of trimeric SgP (pink, cyan, and orange rendering). (D) Zoomed version of the 45 high selectivity HS hexasaccharides (sticks in white color by atom) binding to the RBD (blue rendering). See Table S2 for details on the structure of these sequences. (E) Pharmacophoric representation of the highly selective sequences (colored sticks) shown with interacting residues (ball and stick representation) of the RBD. The pink mesh engulfing sulfate groups is the pharmacophore. Hydrogen bonds are shown as white dotted lines.