ABSTRACT
The concept of substrate inhibition to prevent its phosphorylation has potential in drug discovery and is envisioned to treat the autoimmune disorder multiple sclerosis (MS). Glia maturation factor-ß (GMF-ß) Ser83 phosphorylation by protein kinase A (PKA) is pivotal in the activation of GMF-ß-p38MAPK-NFκB biochemical pathway towards proinflammatory response induction in experimental autoimmune encephalomyelitis (EAE). Using structure-based drug design, we identified the small molecule inhibitor 1-H-indazole-4yl methanol (GMFBI.1) that specifically blocked Ser83 phosphorylation site on GMF-ß substrate. Using in vitro and in vivo techniques, molecular mechanism of action of GMFBI.1's direct interaction with GMF-ß substrate and prevention of its Ser83 phosphorylation was established. GMFBI.1 down regulated p38MAPK phosphorylation and NFκB expression essential for proinflammatory response. Further, GMFBI.1 administration at peak of EAE reversed clinical symptoms, immunopathology, proinflammatory cytokine response and up regulated the anti-inflammatory cytokines. Present strategy of substrate inhibition against the key immunomodulatory target has immense therapeutic potential in MS.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/drug therapy , Glia Maturation Factor/metabolism , Multiple Sclerosis/drug therapy , Small Molecule Libraries/administration & dosage , Amino Acid Motifs , Animals , Drug Design , Encephalomyelitis, Autoimmune, Experimental/metabolism , Female , Glia Maturation Factor/chemistry , Humans , Immunologic Factors/chemistry , Immunologic Factors/metabolism , Mice, Inbred C57BL , Multiple Sclerosis/metabolism , Phosphorylation/drug effects , Small Molecule Libraries/chemistryABSTRACT
Quinine dihydropteridinereductase (QDPR) is involved in the synthesis of tetradihydrobiopteridine (BH4) that serve as cofactor for many aromatic hydroxylases including induced nitric oxide synthase (NOS) leading to NO production. Increased activity of QDPR has been associated with decrease levels of TGF-ß, a cytokine that regulates the immune response and that elevated levels of NO has been associated with neurodegenerative diseases. Thus, expression of QDPR in astrocytes is essential to study the pathological changes observed in many neurodegenerative disorders. We have expressed QDPR in astrocytes and generated stably expressing clones that overexpresses QDPR. We further verified the specificity of QDPR expression using immunofluorescence and immunoblotting. To further confirm, we purified QDPR using Ni-NTA column and subjected the purified fraction to immunoblotting using anti-QDPR antibody and identified two major protein products of QDPR resolving at 25 and 17â¯kDa as reported in the literature. In order to further assess the significance of QDPR expression, we verified the expression of iNOS in QDPR over expressing cells. We show for the first time statistically significant up regulation of iNOS in QDPR overexpressing astrocytes. Increased expression of iNOS associated with astrocyte pathology seen in many neurodegenerative disorders may have implications in autoimmune neurodegenerative disorders.
