Interactions of heparin with key glycoproteins of human respiratory syncytial virus

Introduction: The unexpected surge of respiratory syncytial virus (RSV) cases following pandemic phase of COVID-19 has drawn much public attention. Drawing on the latest antiviral research, revisiting this heightened annual outbreak of respiratory disease could lead to new treatments. The ability of sulfated polysaccharides to compete for a variety of viruses binding to cell surface heparan sulfate, suggests several drugs that might have therapeutic potential for targeting RSV–glycosaminoglycan interactions. Methods: In the current study, the binding affinity and kinetics of two RSV glycoproteins (RSV-G protein and RSV-F protein) to heparin were investigated by surface plasmon resonance. Furthermore, solution competition studies using heparin oligosaccharides of different lengths indicated that the binding of RSV-G protein to heparin is size-dependent, whereas RSV-F protein did not show any chain length preference. Results and discussion: The two RSV glycoproteins have slightly different preferences for heparin sulfation patterns, but the N-sulfo group in heparin was most critical for the binding of heparin to both RSV-G protein and RSV-F protein. Finally, pentosan polysulfate and mucopolysaccharide polysulfate were evaluated for their inhibition of the RSV-G protein and RSV-F protein–heparin interaction, and both highly negative compounds showed strong inhibition.

Suramin binds and inhibits infection of SARS-CoV-2 through both spike protein-heparan sulfate and ACE2 receptor interactions.

SARS-CoV-2 receptor binding domains (RBDs) interact with both the ACE2 receptor and heparan sulfate on the surface of host cells to enhance SARS-CoV-2 infection. We show that suramin, a polysulfated synthetic drug, binds to the ACE2 receptor and heparan sulfate binding sites on the RBDs of wild-type, Delta, and Omicron variants. Specifically, heparan sulfate and suramin had enhanced preferential binding for Omicron RBD, and suramin is most potent against the live SARS-CoV-2 Omicron variant (B.1.1.529) when compared to wild type and Delta (B.1.617.2) variants in vitro. These results suggest that inhibition of live virus infection occurs through dual SARS-CoV-2 targets of S-protein binding and previously reported RNA-dependent RNA polymerase inhibition and offers the possibility for this and other polysulfated molecules to be used as potential therapeutic and prophylactic options against COVID-19.

Structural Characteristics of Heparin Binding to SARS-CoV-2 Spike Protein RBD of Omicron Sub-Lineages BA.2.12.1, BA.4 and BA.5.

The now prevalent Omicron variant and its subvariants/sub-lineages have led to a significant increase in COVID-19 cases and raised serious concerns about increased risk of infectivity, immune evasion, and reinfection. Heparan sulfate (HS), located on the surface of host cells, plays an important role as a co-receptor for virus-host cell interaction. The ability of heparin and HS to compete for binding of the SARS-CoV-2 spike (S) protein to cell surface HS illustrates the therapeutic potential of agents targeting protein-glycan interactions. In the current study, phylogenetic tree of variants and mutations in S protein receptor-binding domain (RBD) of Omicron BA.2.12.1, BA.4 and BA.5 were described. The binding affinity of Omicron S protein RBD to heparin was further investigated by surface plasmon resonance (SPR). Solution competition studies on the inhibitory activity of heparin oligosaccharides and desulfated heparins at different sites on S protein RBD-heparin interactions revealed that different sub-lineages tend to bind heparin with different chain lengths and sulfation patterns. Furthermore, blind docking experiments showed the contribution of basic amino acid residues in RBD and sulfo groups and carboxyl groups on heparin to the interaction. Finally, pentosan polysulfate and mucopolysaccharide polysulfate were evaluated for inhibition on the interaction of heparin and S protein RBD of Omicron BA.2.12.1, BA.4/BA.5, and both showed much stronger inhibition than heparin.

The diverse role of heparan sulfate and other GAGs in SARS-CoV-2 infections and therapeutics.

In December 2019, the global coronavirus disease 2019 (COVID-19) pandemic began in Wuhan, China. COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which infects host cells primarily through the angiotensin-converting enzyme 2 (ACE2) receptor. In addition to ACE2, several studies have shown the importance of heparan sulfate (HS) on the host cell surface as a co-receptor for SARS-CoV-2-binding. This insight has driven research into antiviral therapies, aimed at inhibiting the HS co-receptor-binding, e.g., by glycosaminoglycans (GAGs), a family of sulfated polysaccharides that includes HS. Several GAGs, such as heparin (a highly sulfated analog of HS), are used to treat various health indications, including COVID-19. This review is focused on current research on the involvement of HS in SARS-CoV-2 infection, implications of viral mutations, as well as the use of GAGs and other sulfated polysaccharides as antiviral agents.

The diverse role of heparan sulfate and other GAGs in SARS-CoV-2 infections and therapeutics

In December 2019, the global coronavirus disease 2019 (COVID-19) pandemic began in Wuhan, China. COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which infects host cells primarily through the angiotensin-converting enzyme 2 (ACE2) receptor. In addition to ACE2, several studies have shown the importance of heparan sulfate (HS) on the host cell surface as a co-receptor for SARS-CoV-2-binding. This insight has driven research into antiviral therapies, aimed at inhibiting the HS co-receptor-binding, e.g., by glycosaminoglycans (GAGs), a family of sulfated polysaccharides that includes HS. Several GAGs, such as heparin (a highly sulfated analog of HS), are used to treat various health indications, including COVID-19. This review is focused on current research on the involvement of HS in SARS-CoV-2 infection, implications of viral mutations, as well as the use of GAGs and other sulfated polysaccharides as antiviral agents. Graphical Unlabelled Image

Interactions between heparin and SARS-CoV-2 spike glycoprotein RBD from omicron and other variants.

Heparan sulfate (HS) acts as a co-receptor of angiotensin-converting enzyme 2 (ACE2) by interacting with severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) spike glycoprotein (SGP) facilitating host cell entry of SARS-CoV-2 virus. Heparin, a highly sulfated version of heparan sulfate (HS), interacts with a variety of proteins playing key roles in many physiological and pathological processes. In this study, SARS-CoV-2 SGP receptor binding domain (RBD) wild type (WT), Delta and Omicron variants were expressed in Expi293F cells and used in the kinetic and structural analysis on their interactions with heparin. Surface plasmon resonance (SPR) analysis showed the binding kinetics of SGP RBD from WT and Delta variants were very similar while Omicron variant SGP showed a much higher association rate. The SGP from Delta and Omicron showed higher affinity (K D ) to heparin than the WT SGP. Competition SPR studies using heparin oligosaccharides indicated that binding of SGP RBDs to heparin requires chain length greater than 18. Chemically modified heparin derivatives all showed reduced interactions in competition assays suggesting that all the sulfo groups in the heparin polysaccharide were critical for binding SGP RBDs with heparin. These interactions with heparin are pH sensitive. Acidic pH (pH 6.5, 5.5, 4.5) greatly increased the binding of WT and Delta SGP RBDs to heparin, while acidic pH slightly reduced the binding of Omicron SGP RBD to heparin compared to binding at pH 7.3. In contrast, basic pH (pH 8.5) greatly reduced the binding of Omicron SGP RBDs to heparin, with much less effects on WT or Delta. The pH dependence indicates different charged residues were present at the Omicron SGP-heparin interface. Detailed kinetic and structural analysis of the interactions of SARS-CoV-2 SGP RBDs with heparin provides important information for designing anti-SARS-CoV-2 molecules.

Platelet factor 4 polyanion immune complexes: heparin induced thrombocytopenia and vaccine-induced immune thrombotic thrombocytopenia.

BACKGROUND: This is a review article on heparin-induced thrombocytopenia, an adverse effect of heparin therapy, and vaccine-induced immune thrombotic thrombocytopenia, occurring in some patients administered certain coronavirus vaccines. MAIN BODY/TEXT: Immune-mediated thrombocytopenia occurs when specific antibodies bind to platelet factor 4 /heparin complexes. Platelet factor 4 is a naturally occurring chemokine, and under certain conditions, may complex with negatively charged molecules and polyanions, including heparin. The antibody-platelet factor 4/heparin complex may lead to platelet activation, accompanied by other cascading reactions, resulting in cerebral sinus thrombosis, deep vein thrombosis, lower limb arterial thrombosis, myocardial infarction, pulmonary embolism, skin necrosis, and thrombotic stroke. If untreated, heparin-induced thrombocytopenia can be life threatening. In parallel, rare incidents of spontaneous vaccine-induced immune thrombotic thrombocytopenia can also occur in some patients administered certain coronavirus vaccines. The role of platelet factor 4 in vaccine-induced thrombosis with thrombocytopenia syndrome further reinforces the importance the platelet factor 4/polyanion immune complexes and the complications that this might pose to susceptible individuals. These findings demonstrate, how auxiliary factors can complicate heparin therapy and drug development. An increasing interest in biomanufacturing heparins from non-animal sources has driven a growing interest in understanding the biology of immune-mediated heparin-induced thrombocytopenia, and therefore, the development of safe and effective biosynthetic heparins. SHORT CONCLUSION: In conclusion, these findings further reinforce the importance of the binding of platelet factor 4 with known and unknown polyanions, and the complications that these might pose to susceptible patients. In parallel, these findings also demonstrate how auxiliary factors can complicate the heparin drug development.

Facile fabrication of antibacterial and antiviral perhydrolase-polydopamine composite coatings.

In situ generation of antibacterial and antiviral agents by harnessing the catalytic activity of enzymes on surfaces provides an effective eco-friendly approach for disinfection. The perhydrolase (AcT) from Mycobacterium smegmatis catalyzes the perhydrolysis of acetate esters to generate the potent disinfectant, peracetic acid (PAA). In the presence of AcT and its two substrates, propylene glycol diacetate and H2O2, sufficient and continuous PAA is generated over an extended time to kill a wide range of bacteria with the enzyme dissolved in aqueous buffer. For extended self-disinfection, however, active and stable AcT bound onto or incorporated into a surface coating is necessary. In the current study, an active, stable and reusable AcT-based coating was developed by incorporating AcT into a polydopamine (PDA) matrix in a single step, thereby forming a biocatalytic composite onto a variety of surfaces. The resulting AcT-PDA composite coatings on glass, metal and epoxy surfaces yielded up to 7-log reduction of Gram-positive and Gram-negative bacteria when in contact with the biocatalytic coating. This composite coating also possessed potent antiviral activity, and dramatically reduced the infectivity of a SARS-CoV-2 pseudovirus within minutes. The single-step approach enables rapid and facile fabrication of enzyme-based disinfectant composite coatings with high activity and stability, which enables reuse following surface washing. As a result, this enzyme-polymer composite technique may serve as a general strategy for preparing antibacterial and antiviral surfaces for applications in health care and common infrastructure safety, such as in schools, the workplace, transportation, etc.

Characterization of heparin and severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) spike glycoprotein binding interactions.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and continues to spread around the globe at an unprecedented rate. To date, no effective therapeutic is available to fight its associated disease, COVID-19. Our discovery of a novel insertion of glycosaminoglycan (GAG)-binding motif at S1/S2 proteolytic cleavage site (681-686 (PRRARS)) and two other GAG-binding-like motifs within SARS-CoV-2 spike glycoprotein (SGP) led us to hypothesize that host cell surface GAGs may interact SARS-CoV-2 SGPs to facilitate host cell entry. Using a surface plasmon resonance direct binding assay, we found that both monomeric and trimeric SARS-CoV-2 SGP bind more tightly to immobilized heparin (KD = 40 pM and 73 pM, respectively) than the SARS-CoV and MERS-CoV SGPs (500 nM and 1 nM, respectively). In competitive binding studies, the IC50 of heparin, tri-sulfated non-anticoagulant heparan sulfate, and non-anticoagulant low molecular weight heparin against SARS-CoV-2 SGP binding to immobilized heparin were 0.056 µM, 0.12 µM, and 26.4 µM, respectively. Finally, unbiased computational ligand docking indicates that heparan sulfate interacts with the GAG-binding motif at the S1/S2 site on each monomer interface in the trimeric SARS-CoV-2 SGP, and at another site (453-459 (YRLFRKS)) when the receptor-binding domain is in an open conformation. The current study serves a foundation to further investigate biological roles of GAGs in SARS-CoV-2 pathogenesis. Furthermore, our findings may provide additional basis for further heparin-based interventions for COVID-19 patients exhibiting thrombotic complications.