Mechanism of action and efficacy of RX-111, a thieno[2,3-c]pyridine derivative and small molecule inhibitor of protein interaction with glycosaminoglycans (SMIGs), in delayed-type hypersensitivity, TNBS-induced colitis and experimental autoimmune encephalomyelitis.

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.

Small molecule inhibitors of protein interaction with glycosaminoglycans (SMIGs), a novel class of bioactive agents with anti-inflammatory properties.

BACKGROUND: Small molecule inhibitors of biologically important protein-glycosaminoglycan (GAG) interactions have yet to be identified. METHODS: Compound libraries were screened in an assay of L-selectin-IgG binding to heparin (a species of heparan sulfate [HS-GAG]). Hits were validated, IC-50s established and direct binding of hits to HS-GAGs was investigated by incubating compounds alone with heparin. Selectivity of inhibitors was assessed in 11 different protein-GAG binding assays. Anti-inflammatory activity of selected compounds was evaluated in animal models. RESULTS: Screening identified a number of structurally-diverse planar aromatic cationic amines. Scaffolds similar to known GAG binders, chloroquine and tilorone, were also identified. Inhibitors displayed activity also against bovine kidney heparan sulfate. Direct binding of compounds to GAGs was verified by incubating compounds with heparin alone. Selectivity of inhibitors was demonstrated in a panel of 11 heparin binding proteins, including selectins, chemokines (IL-8, IP-10), Beta Amyloid and cytokines (VEGF, IL-6). A number of selected lead compounds showed dose-dependent efficacy in peritonitis, paw edema and delayed type hypersensitivity. CONCLUSIONS: A new class of compounds, SMIGs, inhibits protein-GAG interaction by direct binding to GAGs. Although their IC-50s were in the low micro-molar range, SMIGs binding to HS-GAGs appeared to be stable in physiological conditions, indicating high avidity binding. SMIGs may interfere with major checkpoints for inflammatory and autoimmune events. GENERAL SIGNIFICANCE: SMIGs are a class of structurally-diverse planar aromatic cationic amines that have an unusual mode of action - inhibiting protein-GAG interactions via direct and stable binding to GAGs. SMIGs may have therapeutic potential in inflammatory and autoimmune disorders.