Evidence for multiple binding modes in the initial contact between SARS-CoV-2 spike S1 protein and cell surface glycans.

Infection of host cells by SARS-CoV-2 begins with recognition by the virus S (spike) protein of cell surface heparan sulfate (HS), tethering the virus to the extracellular matrix environment, and causing the subunit S1-RBD to undergo a conformational change into the 'open' conformation. These two events promote the binding of S1-RBD to the angiotensin converting enzyme 2 (ACE2) receptor, a preliminary step toward viral-cell membrane fusion. Combining ligand-based NMR spectroscopy with molecular dynamics, oligosaccharide analogues were used to explore the interactions between S1-RBD of SARS CoV-2 and HS, revealing several low-specificity binding modes and previously unidentified potential sites for the binding of extended HS polysaccharide chains. The evidence for multiple binding modes also suggest that highly specific inhibitors will not be optimal against protein S but, rather, diverse HS-based structures, characterized by high affinity and including multi-valent compounds, may be required.

Synthetic Heparan Sulfate Mimetic Pixatimod (PG545) Potently Inhibits SARS-CoV-2 by Disrupting the Spike–ACE2 Interaction

Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a coreceptor with the ACE2 protein for the S1 spike protein on SARS-CoV-2 virus, providing a tractable new therapeutic target. Clinically used heparins demonstrate an inhibitory activity but have an anticoagulant activity and are supply-limited, necessitating alternative solutions. Here, we show that synthetic HS mimetic pixatimod (PG545), a cancer drug candidate, binds and destabilizes the SARS-CoV-2 spike protein receptor binding domain and directly inhibits its binding to ACE2, consistent with molecular modeling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of SARS-CoV-2 virus show that pixatimod potently inhibits the infection of monkey Vero E6 cells and physiologically relevant human bronchial epithelial cells at safe therapeutic concentrations. Pixatimod also retained broad potency against variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, in a K18-hACE2 mouse model, pixatimod significantly reduced SARS-CoV-2 viral titers in the upper respiratory tract and virus-induced weight loss. This demonstration of potent anti-SARS-CoV-2 activity tolerant to emerging mutations establishes proof-of-concept for targeting the HS–Spike protein–ACE2 axis with synthetic HS mimetics and provides a strong rationale for clinical investigation of pixatimod as a potential multimodal therapeutic for COVID-19. Heparan sulfate (HS) has emerged as a SARS-CoV-2 coreceptor. Pixatimod (PG545), an HS mimetic, inhibits infectivity of multiple variants offering a novel therapeutic approach against COVID-19.

Structural Insights into Pixatimod (PG545) Inhibition of Heparanase, a Key Enzyme in Cancer and Viral Infections.

Pixatimod (PG545), a heparan sulfate (HS) mimetic and anticancer agent currently in clinical trials, is a potent inhibitor of heparanase. Heparanase is an endo-ß-glucuronidase that degrades HS in the extracellular matrix and basement membranes and is implicated in numerous pathological processes such as cancer and viral infections, including SARS-CoV-2. To understand how PG545 interacts with heparanase, we firstly carried out a conformational analysis through a combination of NMR experiments and molecular modelling which showed that the reducing end ß-D-glucose residue of PG545 adopts a distorted conformation. This was followed by docking and molecular dynamics simulations to study the interactions of PG545 with heparanase, revealing that PG545 is able to block the active site by binding in different conformations, with the cholestanol side-chain making important hydrophobic interactions. While PG545 blocks its natural substrate HS from binding to the active site, small synthetic heparanase substrates are only partially excluded, and thus pentasaccharide or larger substrates are preferred for assaying this class of inhibitor. This study provides new insights for the design of next-generation heparanase inhibitors and substrates.

From Cancer to COVID-19: A Perspective on Targeting Heparan Sulfate-Protein Interactions.

Heparan sulfate (HS) is a complex, polyanionic polysaccharide ubiquitously expressed on cell surfaces and in the extracellular matrix. HS interacts with numerous proteins to mediate a vast array of biological and pathological processes. Inhibition of HS-protein interactions is thus an attractive approach for new therapeutic development for cancer and infectious diseases, including COVID-19; however, synthesis of well-defined native HS oligosaccharides remains challenging. This has aroused significant interest in the development of HS mimetics which are more synthetically tractable and have fewer side effects, such as undesired anticoagulant activity. This account provides a perspective on the design and synthesis of different classes of HS mimetics with useful properties, and the development of various assays and molecular modelling tools to progress our understanding of their interactions with HS-binding proteins.

Evidence of a putative glycosaminoglycan binding site on the glycosylated SARS-CoV-2 spike protein N-terminal domain.

SARS-CoV-2 has rapidly spread throughout the world's population since its initial discovery in 2019. The virus infects cells via a glycosylated spike protein located on its surface. The protein primarily binds to the angiotensin-converting enzyme-2 (ACE2) receptor, using glycosaminoglycans (GAGs) as co-receptors. Here, we performed bioinformatics and molecular dynamics simulations of the spike protein to investigate the existence of additional GAG binding sites on the receptor-binding domain (RBD), separate from previously reported heparin-binding sites. A putative GAG binding site in the N-terminal domain (NTD) of the protein was identified, encompassing residues 245-246. We hypothesized that GAGs of a sufficient length might bridge the gap between this site and the PRRARS furin cleavage site, including the mutation S247R. Docking studies using GlycoTorch Vina and subsequent MD simulations of the spike trimer in the presence of dodecasaccharides of the GAGs heparin and heparan sulfate supported this possibility. The heparan sulfate chain bridged the gap, binding the furin cleavage site and S247R. In contrast, the heparin chain bound the furin cleavage site and surrounding glycosylation structures, but not S247R. These findings identify a site in the spike protein that favors heparan sulfate binding that may be particularly pertinent for a better understanding of the recent UK and South African strains. This will also assist in future targeted therapy programs that could include repurposing clinical heparan sulfate mimetics.

Heparin Inhibits Cellular Invasion by SARS-CoV-2: Structural Dependence of the Interaction of the Spike S1 Receptor-Binding Domain with Heparin.

The dependence of development and homeostasis in animals on the interaction of hundreds of extracellular regulatory proteins with the peri- and extracellular glycosaminoglycan heparan sulfate (HS) is exploited by many microbial pathogens as a means of adherence and invasion. Heparin, a widely used anticoagulant drug, is structurally similar to HS and is a common experimental proxy. Exogenous heparin prevents infection by a range of viruses, including S-associated coronavirus isolate HSR1. Here, we show that heparin inhibits severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) invasion of Vero cells by up to 80% at doses achievable through prophylaxis and, particularly relevant, within the range deliverable by nebulisation. Surface plasmon resonance and circular dichroism spectroscopy demonstrate that heparin and enoxaparin, a low-molecular-weight heparin which is a clinical anticoagulant, bind and induce a conformational change in the spike (S1) protein receptor-binding domain (S1 RBD) of SARS-CoV-2. A library of heparin derivatives and size-defined fragments were used to probe the structural basis of this interaction. Binding to the RBD is more strongly dependent on the presence of 2-O or 6-O sulfate groups than on N-sulfation and a hexasaccharide is the minimum size required for secondary structural changes to be induced in the RBD. It is likely that inhibition of viral infection arises from an overlap between the binding sites of heparin/HS on S1 RBD and that of the angiotensin-converting enzyme 2. The results suggest a route for the rapid development of a first-line therapeutic by repurposing heparin and its derivatives as antiviral agents against SARS-CoV-2 and other members of the Coronaviridae.