Pharmacological Characterization of [(3)H]CHIBA-3007 Binding to Glycine Transporter 1 in the Rat Brain

Glycine transporter-1 (GlyT-1) in glial cells regulates extracellular levels of glycine, which acts as an obligatory co-agonist at the N-methyl-D-aspartate (NMDA) receptors in the brain. In the present study, we developed a novel radioligand, [(3)H]3-chloro-N-((S)-((R)-1-methylpiperidin-2-yl)(thiophen- 3-yl)methyl)-4- (trifluoromethyl)picolinamide ([(3)H]CHIBA-3007), for studying GlyT-1 in the brain. The presence of a single saturable high-affinity binding component for [(3)H]CHIBA-3007 binding to the rat brain membranes was detected. Scatchard analysis revealed an apparent equilibrium dissociation constant (K(d)) of 1.61±0.16 nM and a maximal number of binding sites (B(max)) of 692.8±22.8 fmol/mg protein (mean ± SEM, n = 3). The specific binding of [(3)H]CHIBA-3007 was inhibited by a number of GlyT-1 inhibitors, such as CHIBA-3007, desmethyl-CHIBA-3007, CHIBA-3008, SSR504734, NFPS/ALX5407, LY2365109 and Org24598, consistent with the pharmacological profiles of GlyT-1 inhibitors. Interestingly, the potency of eight GlyT-1 inhibitors (CHIBA-3007, desmethyl-CHIBA-3007, NFPS/ALX5407, LY2365109, Org24598, SSR504734, sarcosine, and glycine) for blocking in vitro specific binding of [(3)H]CHIBA-3007 was significantly correlated with the potency of these inhibitors for inhibiting [(14)C]glycine uptake in the rat brain membranes. In contrast, the GlyT-2 inhibitor ALX1393 exhibited very weak for [(3)H]CHIBA-3007 binding. Furthermore, the regional distribution of [(3)H]CHIBA-3007 binding in the rat brain was similar to the previously reported distribution of GlyT-1. The present findings suggest that [(3)H]CHIBA-3007 would be a useful new radioligand for studying GlyT-1 in the brain.