Charles A. Glass

Surfen, a small molecule antagonist of heparan sulfate

In a search for small molecule antagonists of heparan sulfate, we examined the activity of bis-2-methyl-4-amino-quinolyl-6-carbamide, also known as surfen. Fluorescence-based titrations indicated that surfen bound to glycosaminoglycans, and the extent of binding increased according to charge density in the order heparin > dermatan sulfate > heparan sulfate > chondroitin sulfate. All charged groups in heparin (N-sulfates, O-sulfates, and carboxyl groups) contributed to binding, consistent with the idea that surfen interacted electrostatically. Surfen neutralized the anticoagulant activity of both unfractionated and low molecular weight heparins and inhibited enzymatic sulfation and degradation reactions in vitro. Addition of surfen to cultured cells blocked FGF2-binding and signaling that depended on cell surface heparan sulfate and prevented both FGF2- and VEGF165-mediated sprouting of endothelial cells in Matrigel. Surfen also blocked heparan sulfate-mediated cell adhesion to the Hep-II domain of fibronectin and prevented infection by HSV-1 that depended on glycoprotein D interaction with heparan sulfate. These findings demonstrate the feasibility of identifying small molecule antagonists of heparan sulfate and raise the possibility of developing pharmacological agents to treat disorders that involve glycosaminoglycan–protein interactions.

Metabolic engineering of Chinese hamster ovary cells: Towards a bioengineered heparin

Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS/heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS/heparin biosynthesis might be necessary.

A Disaccharide-Based Inhibitor of Glycosylation Attenuates Metastatic Tumor Cell Dissemination

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.

Bioengineering murine mastocytoma cells to produce anticoagulant heparin

Heparin (HP), an important anticoagulant polysaccharide, is produced in a complex biosynthetic pathway in connective tissue-type mast cells. Both the structure and size of HP are critical factors determining the anticoagulation activity. A murine mastocytoma (MST) cell line was used as a model system to gain insight into this pathway. As reported, MST cells produce a highly sulfated HP-like polysaccharide that lacks anticoagulant activity (Montgomery RI, Lidholt K, Flay NW, Liang J, Vertel B, Lindahl U, Esko JD. 1992. Stable heparin-producing cell lines derived from the Furth murine mastocytoma. Proc Natl Acad Sci USA 89:11327-11331). Here, we show that transfection of MST cells with a retroviral vector containing heparan sulfate 3-O-sulfotransferase-1 (Hs3st1) restores anticoagulant activity. The MST lines express N-acetylglucosamine N-deacetylase/N-sulfotransferase-1, uronosyl 2-O-sulfotransferase and glucosaminyl 6-O-sulfotransferase-1, which are sufficient to make the highly sulfated HP. Overexpression of Hs3st1 in MST-10H cells resulted in a change in the composition of heparan sulfate (HS)/HP and CS/dermatan sulfate (DS) glycosaminoglycans. The cell-associated HS/HP closely resembles HP with 3-O-sulfo group-containing glucosamine residues and shows anticoagulant activity. This study contributes toward a better understanding of the HP biosynthetic pathway with the goal of providing tools to better control the biosynthesis of HP chains with different structures and activities.

Abstract 2934: Novel approach for treating neural crest derived tumors: Selective inhibition of ganglioside biosynthesis with small molecules

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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