Substrate Specific Inhibitor Designed against the Immunomodulator GMF-beta Reversed the Experimental Autoimmune Encephalomyelitis

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-β (GMF-β) Ser83 phosphorylation by protein kinase A (PKA) is pivotal in the activation of GMF-β-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-β substrate. Using in vitro and in vivo techniques, molecular mechanism of action of GMFBI.1's direct interaction with GMF-β 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. Multiple sclerosis is a chronic autoimmune, demyelinating, neurodegenerative disorder of the central nervous system (CNS) affecting 2.5 million people globally 1,2. Despite different disease-modifying therapies adopted to mitigate the inflammatory milieu of MS using different drugs 3-5 , patient's progress from an acute phase to a stage with considerable neurological disabilities. Current therapies modulate disease differently as they target activated T cells, antigen presenting cells or prevent the egress of T cells into brain. Importantly, none of these drugs control molecules that modulate the proinflammatory response at a fundamental level following infiltration of activated T and B lymphocytes 6,7. Activated lymphocytes undergo clonal expansion assisted by the cytokines produced mainly by astrocytes and microglia and glia maturation factor-β (GMF-β) plays an instrumental role in cytokine induction 8,9. Thus, targeting GMF-β could signify a novel approach in controlling the immune response in the brain. Over expression of GMF-β in response to immune challenge up regulates p38MAPK and NFκB expression in astrocytes leading to increased GM-CSF production by astrocytes and microglial generation of TNF-α, IL1-β, IL-6 and IFN-γ, augmenting proinflammatory response 10-12. In the well-established EAE animal model of MS,

Translational Significance of GMF-β Inhibition by Indazole-4-yl-methanol in Enteric Glial Cells for Treating Multiple Sclerosis

“…Miglyol) by western blotting technique using anti-GMF-β/anti-GMF-β Ser-83 phosphorylation-specific antibodies. 4 As expected, despite total GMF-β levels remaining the same, there were statistically significant differences in levels of Ser-83 phosphorylated GMF-β between EAE and GMFBI.1 p.o. 40 mg/kg (p.o.…”

“…Thus, our initial objective is to develop a biocompatible carrier to increase the retention time of GMFBI.1 in circulation due to its high clearance when administered in saline. 4 Following identification of Miglyol 812N/ethanol (hereinafter referred to as Miglyol) as the best GMFBI.1 carrier, we addressed whether administration of GMFBI.1 by different routes [subcutaneous (s.c.) and p.o.] at different doses with differential bioavailability of the compound in the gut and brain would throw light on the significance of local bioavailability as an index of EAE reversal.…”

“…delivery of GMFBI.1 in saline could be circumvented (Figure 2A,C). 4 Absence of a carrier thus makes it clear quickly from circulation. This is evident in 12 mg/kg administered s.c. in saline as control, wherein high clearance of the GMFBI.1 compound was observed and can only be detected in kidneys (Figure S4).…”

Discovery of Anticancer Hybrid Molecules by Supervised Machine Learning Models and in Vitro Validation in Drug Resistant Chronic Myeloid Leukemia Cells

“…The molecular docking technique predicts the binding affinity of a ligand toward its target. , The hybrid molecules with a good binding affinity toward the targets were selected based on their docking pose and score to understand its molecular mechanism of interactions. The hybrid molecules showing docking scores greater than −30 kcal/mol towards the active sites of BCR-ABL and HDAC were selected.…”

“…Molecular docking techniques are widely used in chemoinformatic studies to predict the best possible biologically significant binding conformation of a molecule to its target and thereby decipher the molecular mechanism of action. , Thus, FP7 and FP10 hybrid molecules were docked using CDOCKER tool of BIOVIA Discovery Studio software along with the existing known FDA approved drugs ponatinib, imatinib, and vorinostat to BCR-ABL and HDAC. These two hybrid molecules were docked at the active sites of wild type BCR-ABL, Thr315Ile mutant BCR-ABL, and HDAC crystal structures.…”