OBJECTIVE AND DESIGN: Elucidate the mechanism of action of the small molecule inhibitor of protein binding to glycosaminoglycans, RX-111 and assay its anti-inflammatory activity in animal models of inflammatory disease. MATERIALS: The glycosaminoglycan, heparin, was used in the mechanism of action study of RX-111. Human T lymphocytes and umbilical vein endothelial cells were used to assay the in vitro activity of RX-111. Mouse and rat models of disease were used to assay the anti-inflammatory activity of RX-111 in vivo. METHODS: Circular dichroism and UV/Vis absorption spectroscopy were used to study the binding of RX-111 to the glycosaminoglycan, heparin. T lymphocyte rolling on endothelial cells under shear flow was used to assay RX-111 activity in vitro. Delayed-type hypersensitivity (DTH) and tri-nitrobenzene sulfonic acid (TNBS)-induced colitis in mice and experimental autoimmune encephalomyelitis (EAE) in rats were used to assay anti-inflammatory activity of RX-111 in vivo. RESULTS: RX-111 was shown to bind directly to heparin. It inhibited leukocyte rolling on endothelial cells under shear flow and reduced inflammation in the mouse model of DTH. RX-111 was efficacious in the mouse model of inflammatory bowel disease, TNBS-induced colitis and the rat model of multiple sclerosis, EAE. CONCLUSIONS: RX-111 exercises its broad spectrum anti-inflammatory activity by a singular mechanism of action, inhibition of protein binding to the cell surface GAG, heparan sulfate. RX-111 and related thieno[2,3-c]pyridine derivatives are potential therapeutics for the treatment of inflammatory and autoimmune diseases.

Biomolecular Self Assembly Group Institute of Materials and Environmental Chemistry Research Centre for Natural Sciences Budapest Hungary

Department of Immunology Rappaport Institute Rappaport Faculty of Medicine Technion Haifa Israel

Institute of Animal Physiology Slovak Academy of Sciences 04001 Kosice Slovakia

Institute of Animal Physiology Slovak Academy of Sciences 04001 Kosice Slovakia GYN FIV a s Záhradnícka 42 821 085 Bratislava Slovakia

Institute of Animal Physiology Slovak Academy of Sciences 04001 Kosice Slovakia Institute of Animal Physiology and Genetics of the ASCR v v i Rumburská 89 277 21 Liběchov Czech Republic

Rimonyx Pharmaceuticals Ltd Rabin Science Park 70400 Ness Ziona Israel

Rimonyx Pharmaceuticals Ltd Rabin Science Park 70400 Ness Ziona Israel Ephraim Katzir Department of Biotechnology Engineering ORT Braude Academic College of Engineering 21982 Karmiel Israel

Rimonyx Pharmaceuticals Ltd Rabin Science Park 70400 Ness Ziona Israel GISMO Therapeutics Inc A253 ASTECC UK Lexington KY 40506 USA

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