In vitro activity: MDL-29951 is a potent glycine antagonist of NMDA (N-methyl-D-aspartate) receptor activation with Ki value of 0.14 μM in an in vitro and in vivo [3H]glycine binding assay. MDL 100,748 and MDL 29,951 are approximately 2000-fold selective for the glycine binding site relative to the glutamate recognition sites.The MDL29951-activated receptor exhibits other activities associated with GPCR-mediated signaling, including G protein-dependent activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and recruitment of β-arrestin. MDL29951 did not activate any of the known uracil or adenine nucleotide-activated P2Y receptors or cysteinyl leukotriene receptors. Gαi- and Gαq-dependent signaling responses also were observed in primary rat oligodendrocytes in the presence of MDL29951. Moreover, MDL29951 diminished myelination in primary oligodendrocytes isolated from heterozygous mice but had no effect on myelination in oligodendrocytes from GPR17 knockout mice. Effects of a small-molecule GPR17 agonist observed during oligodendrocyte differentiation support the idea that development of antagonists of GPR17 is a rational goal for elaboration of pharmacotherapies in demyelinating diseases.

Kinase Assay: MDL 100,748 and MDL 29,951 are approximately 2000-fold selective for the glycine binding site relative to the glutamate recognition sites. MDL-29951 is found to inhibit the human F16Bpase under these conditions (IC50=2.5 μM). MDL-29951 inhibits the human liver (IC50=2.5 μM), porcine kidney (IC50=1.0 μM), and rabbit liver (IC50=0.21 μM) isoforms of the enzyme, but is significantly less potent against the rat liver isoform (IC50=11 μM). The MDL29951-activated receptor exhibits other activities associated with GPCR-mediated signaling, including G protein-dependent activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and recruitment of β-arrestin. [3H]JCPP (30.7 Ci/mmol) binding assays are conducted in minivials, incubated for 15 mm at 25°C in 1 mL of 50 mM Tris-HC1 (pH 7.4) containing 10 nM [3H]JCPP, 200 g of membrane protein and unlabeled ligands as indicated. Nonspecific binding is defined using 1 mM L-glutamate. Bound ligand is separated by centrifugation. Specific binding accounted for approximately 80% of total binding.

Cell Assay: As with recombinant cell systems, MDL29951 promotes Ca2+ signaling responses and inhibition of cyclic adenosine monophosphate (cAMP) accumulation in rat oligodendrocyte precursor cells during the period of peak GPR17 abundance. Effects of MDL29951 are markedly reduced in cells with low GPR17 abundance and are blocked by pranlukast.