We show that SARS-CoV-2 spike protein interacts with cell surface heparan sulfate and angiotensin converting enzyme 2 (ACE2) through its Receptor Binding Domain. Docking studies suggest a putative heparin/heparan sulfate-binding site adjacent to the domain that binds to ACE2. In vitro, binding of ACE2 and heparin to spike protein ectodomains occurs independently and a ternary complex can be generated using heparin as a template. Contrary to studies with purified components, spike protein binding to heparan sulfate and ACE2 on cells occurs codependently. Unfractionated heparin, non-anticoagulant heparin, treatment with heparin lyases, and purified lung heparan sulfate potently block spike protein binding and infection by spike protein-pseudotyped virus and SARS-CoV-2 virus. These findings support a model for SARS-CoV-2 infection in which viral attachment and infection involves formation of a complex between heparan sulfate and ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin may represent new therapeutic opportunities.Funding: This work was supported by RAPID grant 2031989 from the National Science Foundation and Project 3 of NIH P01 HL131474 to J.D.E.; The Alfred Benzon Foundation to T.M.C; NIH R01 AI146779 and a Massachusetts Consortium on Pathogenesis Readiness MassCPR grant to A.G.S.; DOD grant W81XWH-20-1-0270 and Fluomics/NOSI U19 AI135972 to S.K.C; a Career Award for Medical Scientists from the Burroughs Wellcome Fund to A.F.C.; Bill and Melinda Gates Foundation grant OPP1170236 to A.B.W.; COVID19 seed funding from the Huck Institutes of the Life Sciences and Penn State start-up funds to J.J.; and T32 training grants GM007753 for B.M.H. and T.C and AI007245 for J.F.; J.P. received funding from the Innovation Fund Denmark and VAR2 Pharmaceuticals.Conflict of Interest: J.D.E. is a co-founder of TEGA Therapeutics. J.D.E. and The Regents of the University of California have licensed a University invention to and have an equity interest in TEGA Therapeutics. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. C.A.G and B.E.T are employees of TEGA Therapeutics.Ethical Approval: The collection of human tissue in this study abided by the Helsinki Principles. This work included postmortem human tissue, collected at the University Hospital, at the University of Copenhagen in Denmark. The patient provided informed consent for the tissue to be used for research purposes. All samples were completely deidentified before transfer to the researchers and this did not need specific IRB approval.
Abstract Purpose: The binding of hematogenously borne malignant cells that express the carbohydrate sialyl Lewis X (sLeX) to selectin adhesion receptors on leukocytes, platelets, and endothelial cells facilitates metastasis. The glycosylation inhibitor, per-O-acetylated GlcNAcβ1,3Galβ-O-naphthalenemethanol (AcGnG-NM), inhibits the biosynthesis of sLeX in tumor cells. To evaluate the efficacy of AcGnG-NM as an antimetastatic agent, we examined its effect on experimental metastasis and on spontaneous hematogenous dissemination of murine Lewis lung carcinoma and B16BL6 melanoma cells. Experimental Design: Tumor cells were treated in vitro with AcGnG-NM, and the degree of selectin ligand inhibition and experimental metastasis was analyzed in wild-type and P-selectin-deficient mice. Conditions were developed for systemic administration of AcGnG-NM, and the presence of tumor cells in the lungs was assessed using bromodeoxyuridine labeling in vivo. The effect of AcGnG-NM on inflammation was examined using an acute peritonitis model. Results: In vitro treatment of Lewis lung carcinoma cells with AcGnG-NM reduced expression of sLeX- and P-selectin-dependent cell adhesion to plates coated with P-selectin. Treatment also reduced formation of lung foci when cells were injected into syngeneic mice. Systemic administration of the disaccharide significantly inhibited spontaneous dissemination of the cells to the lungs from a primary s.c. tumor, whereas an acetylated disaccharide not related to sLeX in structure had no effect. AcGnG-NM did not alter the level of circulating leukocytes or platelets, the expression of P-selectin ligands on neutrophils, or sLeX-dependent inflammation. Conclusion: Taken together, these data show that AcGnG-NM provides a targeted glycoside-based therapy for the treatment of hematogenous dissemination of tumor cells.
Glycosaminoglycans (GAGs) are a class of highly negatively charged membrane-associated and extracellular matrix polysaccharides involved in the regulation of myriad biological functions, including cell adhesion, migration, signaling, and differentiation, among others. GAGs are typically attached to core proteins, termed proteoglycans (PGs), and can engage >500 binding proteins, making them prominent relays for sensing external stimuli and transducing cellular responses. However, their unique substructural protein-recognition domains that confer their binding specificity remain elusive. While the emergence of glycan arrays has rapidly enabled the profiling of ligand specificities of a range of glycan-binding proteins, their adaptation for the analysis of GAG-binding proteins has been considerably more challenging. Current GAG microarrays primarily employ synthetically defined oligosaccharides, which capture only a fraction of the structural diversity of native GAG polysaccharides. Augmenting existing array platforms to include GAG structures purified from tissues or produced in cells with engineered glycan biosynthetic pathways may significantly advance the understanding of structure–activity relationships in GAG–protein interactions. Here, we demonstrate an efficient and tunable strategy to mimic cellular proteoglycan architectures by conjugating biologically derived GAG chains to a protein scaffold, defined as neoproteoglycans (neoPGs). The use of a reactive fluorogenic linker enabled real-time monitoring of the conjugation reaction efficiency and tuning of the neoPG valency. Immobilization of the reagents on a 96-well array platform allowed for efficient probing of ligand binding and enzyme–substrate specificity, including growth factors and the human sulfatase 1. The neoPGs can also be used directly as soluble probes to evaluate GAG-dependent growth factor signaling in cells.
Abstract Neural crest derived tumors express high levels of a unique class of lipid linked glycan known as gangliosides. Gangliosides are involved in growth factor signaling by regulating complexes in lipid rafts. Genetic studies show that through aberrant expression of gangliosides, these tumors acquire aggressive growth properties. Prior to this research, the only ganglioside inhibitors identified were non-specific and broadly blocked virtually all glycolipid classes. These non-specific glycolipid inhibitors demonstrated anti-cancer activity in animal models of neural crest tumors. However, due to substantial off-target dose-limiting toxicity from lack of specificity for the ganglioside sub-class, they effectively cannot be used in humans for cancer treatment. Selective inhibition of gangliosides without affecting other glycan classes could potentially avoid these problems and provide an effective treatment for neural crest and other ganglioside-dependent tumors. To identify the first known selective inhibitors of gangliosides, we developed a novel molecular screening strategy for identifying selective small-molecule ganglioside inhibitors. This platform identified the first drug-like selective inhibitors of gangliosides. ZP10395, a lead compound, selectively and dose-dependently reduces gangliosides in multiple tumor cell lines and is 10-15 fold more potent than the existing non-specific inhibitors. Importantly, it does not inhibit other glycolipid classes associated with dose-limiting toxicity. Administering ZP10395 to a mouse xenograph melanoma model significantly reduced ganglioside production and slowed tumor growth in the presence of a reduced T-cell response. These results demonstrate the potential utility of specific ganglioside inhibitors for treating ganglioside dependent tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2934. doi:10.1158/1538-7445.AM2011-2934
Abstract We show that SARS-CoV-2 spike protein interacts with cell surface heparan sulfate and angiotensin converting enzyme 2 (ACE2) through its Receptor Binding Domain. Docking studies suggest a putative heparin/heparan sulfate-binding site adjacent to the domain that binds to ACE2. In vitro, binding of ACE2 and heparin to spike protein ectodomains occurs independently and a ternary complex can be generated using heparin as a template. Contrary to studies with purified components, spike protein binding to heparan sulfate and ACE2 on cells occurs codependently. Unfractionated heparin, non-anticoagulant heparin, treatment with heparin lyases, and purified lung heparan sulfate potently block spike protein binding and infection by spike protein-pseudotyped virus and SARS-CoV-2 virus. These findings support a model for SARS-CoV-2 infection in which viral attachment and infection involves formation of a complex between heparan sulfate and ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin may represent new therapeutic opportunities.
