Polysulfates Block SARS-CoV-2 Uptake through Electrostatic Interactions*

Abstract

Here we report that negatively charged polysulfates can bind to the spike protein of SARS-CoV-2 via electrostatic interactions. Using a plaque reduction assay, we compare inhibition of SARS-CoV-2 by heparin, pentosan sulfate, linear polyglycerol sulfate (LPGS) and hyperbranched polyglycerol sulfate (HPGS). Highly sulfated LPGS is the optimal inhibitor, with an IC₅₀ of 67 μg mL^-1 (approx. 1.6 μm). This synthetic polysulfate exhibits more than 60-fold higher virus inhibitory activity than heparin (IC₅₀ : 4084 μg mL^-1 ), along with much lower anticoagulant activity. Furthermore, in molecular dynamics simulations, we verified that LPGS can bind more strongly to the spike protein than heparin, and that LPGS can interact even more with the spike protein of the new N501Y and E484K variants. Our study demonstrates that the entry of SARS-CoV-2 into host cells can be blocked via electrostatic interactions, therefore LPGS can serve as a blueprint for the design of novel viral inhibitors of SARS-CoV-2.

Keywords: SARS-CoV-2; electrostatic interactions; inhibition; polysulfates; virus binding.

Figure 1

Left: binding of SARS‐CoV‐2 to surface‐exposed heparan sulfate facilitates virus entry; right: competitive binding to soluble synthetic polyglycerol sulfates shields the viral surface and therefore finally reduces infectivity.

Figure 2

a) Crystal structure of the SARS‐CoV‐2 spike protein RBD (PDB ID: 6M0J) with a few important cationic residues that interact with polyanionic ligands. b) The electrostatic potential map of RBD. c) Schematic illustrations of polyglycerol sulfates in linear and hyperbranched architectures, and of the natural polysulfates, respectively. The negatively charged groups are marked red.

Figure 3

a) Plaque reduction ratios for the samples at different inhibitor doses. Values are expressed as mean ±SD, n=4. Mw shown here refers to the LPG and HPGS precursors. b) CLSM image for the virus binding to Vero E6 cells in presence of LPGS. Scale bar: 10 μm. c) Analysis of virus binding to Vero E6 cells from CLSM images for the number of virions per cells. More detailed images are shown in Figure S4, Supporting Information. d) Immunofluorescent images revealing the infected cells in the post‐infection inhibition assay. The cell nuclei are stained blue, while the infected cells are stained green by antibodies against the nucleocapsid protein (N) of SARS‐CoV‐2. Scale bar: 50 μm. More images are shown in Figure S6, Supporting Information. e) Ratios of infected cells in each group. “LPGS” in (b)–(e) refers to LPG₂₀S_0.94. Values are expressed as mean ±SD, n=4. **p<0.01 from Student t‐test.

Figure 4

a) Affinity measurements of RBD of wild‐type SARS‐CoV‐2 with LPGS, HPGS, heparin and ACE2 using MST. Each data point represents mean values with N≥4 experiments, and the error bars show the standard deviation. Data points were fitted according to the mass‐action law function to obtain K _d values (see Table 1). The differences in the slopes of the dose‐response curves depend on changes of the hydration shell areas and effective charges, but do not affect the determinations of K _d‐values from the inflection points of the curves. b) Crystal structure of RBD bound with ACE2 (PDB ID: 6MOJ). ACE2 is shown in secondary structure representation (red), while RBD is shown in surface representation (green). The amino acid residues of RBD (R346, A348, A352, N354, R355, K356, R357, S359, Y396, K444, N450, R466, I468) found in MD simulations to form contacts with the polysulfates are highlighted in VDW representation (blue), denoting the putative HS‐binding site. More detailed images are shown in Figure S7, Supporting Information. c) Mass spectra of 4.0 μL RBD solution mixed with 0, 0.4, 0.8, and 1.2 μL heparin (light traces) or LPGS (dark trace). The charge states are marked with a single dot for the RBD monomer and with a double dot for the RBD dimer, while the calculated m/z for the 10–13+ charge states of the 34 kDa RBD are marked with orange lines.

Figure 5

Simulation setup for studying interactions of the RBD of wild‐type SARS‐CoV‐2 with a single a) LPGS undecamer and b) heparin pentamer. The protein is shown in secondary structure representation (tan), whereas polymers are shown in ball and stick representation with each atom type colored differently (hydrogen in white, carbon in cyan, oxygen in red, and sulfur in yellow). Water molecules and ions are omitted for clarity. To the right, snapshots after 500 ns of MD simulations are shown for a) RBD–LPGS and b) RBD–heparin complexes. c) The number of contacts LPGS and heparin forms with each residue of wild‐type RBD averaged over the last 100 ns simulation time. d) End‐to‐end distance distributions for LPGS and heparin free in aqueous solutions, which reveal the different flexibility of the polymers. Relevant parameters of the polymers are given in the inset; see text for details. e) Comparison of interaction energies for the different polymers and RBD variants. The electrostatic (Elec.) and van der Waals (VDW) contributions to the total interaction energy for each protein–polymer complex are given. f) The number of contacts LPGS forms with each residue of the different RBD mutants. To the left, snapshots after 500 ns of MD simulations are provided, representing the complex formation of LPGS with each RBD mutant.