Structure based inhibition of GMF and#946; activity and its in vitro and in vivo validation in animal model of multiple sclerosis

Abstract

Current treatment strategies for multiple sclerosis (MS) are aimed at modifying cellular immune responses without addressing the reversal of autoimmunity induced by proinflammatory modulatory proteins at a fundamental level.Glia maturation factor-and#946; (GMF-and#946;) is one such protein that functions at a fundamental level as an immune modulator in the CNS. GMF-and#946; is activated by phosphorylation, leading to the induction of downstream proinflammatory pathway. GMF-and#946; is overexpressed in Experimental Autoimmune Encephalomyelitis (EAE) - the animal model of MS and that knockout mice developed only a very mild form of EAE. Thus, in an effort to bring in new and effective molecule against the activation of GMF-and#946; and controlling the local immune surge in CNS, we utilised a novel concept of substrate specific inhibition by which the specific phosphorylation sites of GMF-and#946; is blocked. Using structure-based drug design strategies, a novel small molecule inhibitor 1-H indazole, 4yl-methanol (GMFBI.1) against GMF-and#946; substrate was developed. GMFBI.1 blocked the protein kinase A (PKA) mediated phosphorylation of GMF-and#946; in-silico by binding to Ser-83 aminoacid residue of the protein. The Ser-83 phosphorylation is critical for the direct activation of GMF-and#946; p38MAPK-NFand#954;B axis. Subsequently, the GMFBI.1and#8223;s direct binding and molecular mechanism in blocking the key Ser-83 phosphorylation site of GMF-and#946; that mediates p38MAPK activation was validated using in-vitro and in-vivo techniques. Incubation of GMFBI.1 with astrocytes overexpressing GMF-and#946; down regulated p38MAPK and NFand#954;B expression. Treatment of EAE animals with GMFBI.1 also reversed the paralytic symptoms at the peak of disease. These results were further corroborated with the significant reduction in the number of mononuclear cell infiltration, suppression of proinflammatory cytokines levels (IFN-and#947;, IL-1and#946;, IL-6, TNF-and#945;, IL-17A, GM-CSF) and up regulation of anti-inflammatory cytokines (TGF-and#946; and IL-10). In addition,treatment with GMFBI.1showed remyelination potential and reduced mast cell..