Pentosan polysulfate inhibits attachment and infection by SARS-CoV-2 in vitro: insights into structural requirements for binding. (preprint)

Two years since the outbreak of the novel coronavirus SARS-CoV-2 pandemic, there remain few clinically effective drugs to complement vaccines. One is the anticoagulant, heparin, which in 2004 was found able to inhibit invasion of SARS CoV (CoV-1) and which has been employed during the current pandemic to prevent thromboembolic complications and moderate potentially damaging inflammation. Heparin has also been shown experimentally to inhibit SARS-CoV-2 attachment and infection in susceptible cells. At high therapeutic doses however, heparin increases the risk of bleeding and prolonged use can cause heparin-induced thrombocytopenia, a serious side-effect. One alternative, with structural similarities to heparin is the plant-derived, semi-synthetic polysaccharide, pentosan polysulfate (PPS). PPS is an established drug for the oral treatment of interstitial cystitis, is well-tolerated and exhibits weaker anticoagulant effects than heparin. In an established Vero cell model, PPS and its fractions of varying molecular weights, inhibited invasion by SARS-CoV-2. Intact PPS and its size-defined fractions were characterized by molecular weight distribution and chemical structure using NMR spectroscopy and LC-MS, then employed to explore the structural basis of interactions with SARS-CoV-2 spike protein receptor-binding domain (S1 RBD) and the inhibition of Vero cell invasion. PPS was as effective as unfractionated heparin, but more effective at inhibiting cell infection than low molecular weight heparin (on a weight/volume basis). Isothermal titration calorimetry and viral plaque-forming assays demonstrated size-dependent binding to S1 RBD and inhibition of Vero cell invasion, suggesting the potential application of PPS as a novel inhibitor of SARS-CoV-2 infection.

Pixatimod (PG545), a clinical-stage heparan sulfate mimetic, is a potent inhibitor of the SARS-CoV-2 virus (preprint)

A major global effort is currently ongoing to search for therapeutics and vaccines to treat or prevent infection by the SARS-CoV-2 virus. Repurposing existing entities is one attractive approach. The heparan sulfate mimetic pixatimod is a clinical-stage synthetic sulfated compound that is a potent inhibitor of the glycosidase heparanase, and has known anti-cancer, anti-inflammatory and also antiviral properties. Here we show that pixatimod binds directly to the SARS-CoV-2 spike protein S1 receptor binding domain (RBD) and alters its conformation. Notably, this site overlaps with the known ACE2 binding site in the S1 RBD. We find that pixatimod inhibits binding of recombinant S1 RBD to Vero cells which express the ACE2 receptor. Moreover, in assays with three different isolates of live SARS-CoV-2 virus we show that pixatimod effectively inhibits viral infection of Vero cells. Importantly, its potency is well within its safe therapeutic dose range. These data provide evidence that pixatimod is a potent antiviral agent against SARS-CoV-2. Together with its other known activities this provides a strong rationale for its clinical investigation as a new multimodal therapeutic for the current COVID-19 pandemic.

The 2019 coronavirus (SARS-CoV-2) surface protein (Spike) S1 Receptor Binding Domain undergoes conformational change upon heparin binding. (preprint)

Many pathogens take advantage of the dependence of the host on the interaction of hundreds of extracellular proteins with the glycosaminoglycans heparan sulphate to regulate homeostasis and use heparan sulphate as a means to adhere and gain access to cells. Moreover, mucosal epithelia such as that of the respiratory tract are protected by a layer of mucin polysaccharides, which are usually sulphated. Consequently, the polydisperse, natural products of heparan sulphate and the allied polysaccharide, heparin have been found to be involved and prevent infection by a range of viruses including S-associated coronavirus strain HSR1. Here we use surface plasmon resonance and circular dichroism to measure the interaction between the SARS-CoV-2 Spike S1 protein receptor binding domain (SARS-CoV-2 S1 RBD) and heparin. The data demonstrate an interaction between the recombinant surface receptor binding domain and the polysaccharide. This has implications for the rapid development of a first-line therapeutic by repurposing heparin and for next-generation, tailor-made, GAG-based antivirals.