Optimization and Characterization of Small Molecule GPR17 Antagonists for Multiple Sclerosis

Project Summary: MS is a severe neurological disease characterized by autoimmune-mediated demyelination of neurons and oligodendrocyte damage. The resulting axonal degeneration impairs rapid nerve conduction, which leads to neurological disability if not repaired through remyelination. The remyelination process requires proliferation and maturation of oligodendrocyte precursor cells (OPCs) into myelin producing mature oligodendrocytes. GPR17 is an orphan GPCR that has been identified as a negative regulator of oligodendrocyte maturation and is predominantly expressed in OPCs. GPR17-/- mice have increased CNS myelination and GPR17 overexpressing mice showed lack of myelin sheath formation in the CNS, similar to that seen in myelinating disorders. Addition of the GPR17 agonist probe MDL29,951 to cultures from GPR17+/- mice resulted in reduced differentiation of oligodendrocytes, along with a decrease in myelin basic protein (MBP) and dendrite formation. In addition, studies using an MS mouse model (experimental autoimmune encephalomyelitis, EAE) showed an upregulation of GPR17 in CNS regions where demyelination was occurring. Collectively, these data suggest the involvement of GPR17 in CNS demyelination upon activation. Hence, novel small molecule antagonists that can selectively modulate GPR17 functions will be invaluable tools to study GPR17 biology and lead to new drug discovery opportunities to treat severe demyelinating diseases such as MS. To date, very few small molecule antagonists have been reported in the literature and pharmacological studies with the existing non-selective and less potent antagonists have been challenging. Our preliminary work led to the identification of a novel small molecule lead compound SN-191 (GPR17 IC50 = 413 nM, CysLT1, IC50 > 10,000 nM) through structural modification of Pranlukast, a non-selective CysLT1 antagonist (CysLT1 IC50 = 2 nM, GPR17 IC50 = 588 nM). This novel lead compound exhibits >5000-fold reduced potency at CysLT1 compared to Pranlukast and >20-fold preference for GPR17 over CysLT1. SN-191 showed minimal cross reactivity to CysLT2 and P2Y1 (<15 % inhibition), exhibiting selectivity over phylogenetically closely related CysLT and P2Y1 receptors. In addition, our early selective lead SN-70 (GPR17 IC50 = 938 nM, CysLT1, IC50 > 10,000 nM) promoted oligodendrocyte differentiation (induction of MBP expression at 10 µM concentration) in a rat OPC differentiation assay suggesting the ability of GPR17 antagonists to promote oligodendrocyte differentiation and potential for myelin repair. SN-191 therefore serves as an excellent lead to identify potent and selective GPR17 antagonists. In Specific Aim 1 of the proposed R21 project, we will synthesize at least 100 novel SN-191 analogs through structural modification. In Aim 2, we will evaluate the synthesized compounds for GPR17 potency and selectivity over CysLT1. In Specific Aim 3, selected analogs will be evaluated for oligodendrocyte myelination using in vitro OPC differentiation/myelination assays. Preliminary ADME properties will also be evaluated. These Aims will be accomplished through a collaboration of chemistry and in vitro pharmacology.