PRO-2000 exhibits SARS-CoV-2 antiviral activity by interfering with spike-heparin binding

Here, we report on the anti-SARS-CoV-2 activity of PRO-2000, a sulfonated polyanionic compound. In Vero cells infected with the Wuhan, alpha, beta, delta or omicron variant, PRO-2000 displayed EC₅₀ values of 1.1 μM, 2.4 μM, 1.3 μM, 2.1 μM and 0.11 μM, respectively, and an average selectivity index (i.e. ratio of cytotoxic versus Antiviral concentration) of 172. Its anti-SARS-CoV-2 activity was confirmed by virus yield assays in Vero cells, Caco2 cells and A549 cells overexpressing ACE2 and TMPRSS2 (A549-AT). Using pseudoviruses bearing the SARS-CoV-2 spike (S), PRO-2000 was shown to block the S-mediated pseudovirus entry in Vero cells and A549-AT cells, with EC₅₀ values of 0.091 μM and 1.6 μM, respectively. This entry process is initiated by interaction of the S glycoprotein with angiotensin-converting Enzyme 2 (ACE2) and heparan sulfate proteoglycans. Surface Plasmon Resonance (SPR) studies showed that PRO-2000 binds to the receptor-binding domain (RBD) of S with a K_D of 1.6 nM. Similar K_D values (range: 1.2 nM-2.1 nM) were obtained with the RBDs of the alpha, beta, delta and omicron variants. In an SPR neutralization assay, PRO-2000 had no effect on the interaction between the RBD and ACE2. Instead, PRO-2000 was proven to inhibit binding of the RBD to a heparin-coated sensor chip, yielding an IC₅₀ of 1.1 nM. To conclude, PRO-2000 has the potential to inhibit a broad range of SARS-CoV-2 variants by blocking the heparin-binding site on the S protein.

Antiviral; Covid-19; Heparan sulfate; Polyanionic; Spike.