Pharmacological Characterization of [3H]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, [3H]3-chloro-N-((S)-((R)-1-methylpiperidin-2-yl)(thiophen- 3-yl)methyl)-4- (trifluoromethyl)picolinamide ([3H]CHIBA-3007), for studying GlyT-1 in the brain. The presence of a single saturable high-affinity binding component for [3H]CHIBA-3007 binding to the rat brain membranes was detected. Scatchard analysis revealed an apparent equilibrium dissociation constant (Kd) of 1.61±0.16 nM and a maximal number of binding sites (Bmax) of 692.8±22.8 fmol/mg protein (mean ± SEM, n = 3). The specific binding of [3H]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 [3H]CHIBA-3007 was significantly correlated with the potency of these inhibitors for inhibiting [14C]glycine uptake in the rat brain membranes. In contrast, the GlyT-2 inhibitor ALX1393 exhibited very weak for [3H]CHIBA-3007 binding. Furthermore, the regional distribution of [3H]CHIBA-3007 binding in the rat brain was similar to the previously reported distribution of GlyT-1. The present findings suggest that [3H]CHIBA-3007 would be a useful new radioligand for studying GlyT-1 in the brain.


Regional distribution of [ 3 H]CHIBA-3007 binding in the rat brain
The regional distribution of [ 3 H]CHIBA-3007 binding in the rat brain is shown in Figure 6. Specific [ 3 H]CHIBA-3007 binding was higher in the midbrain and lower in the cerebral cortex. The order of specific binding of [ 3 H]CHIBA-3007 in the rat brain was as follows: midbrain . pons . thalamus . cerebellum . striatum . hippocampus . cerebral cortex. The regional distribution of [ 3 H]CHIBA-3007 binding in the rat brain was similar to the distribution of GlyT-1 in the rat brain reported previously [6,9,10,32].

Discussion
The present study shows that [ 3 H]CHIBA-3007, a nonsarcosine-based GlyT-1 inhibitor, is a novel and excellent radioligand for studying the pharmacology and distribution of GlyT-1 in the rat brain in vitro. The major findings of the present study are summarized as follows. First, the potency (IC 50 = 21.4 nM) of CHIBA-3007 for inhibiting [ 14 C]glycine uptake in the rat brain was higher than that (IC 50 = 84.5 nM) of SSR504734, a potent, selective, and orally active GlyT-1 inhibitor [34]. Furthermore, CHIBA-3007 did not show any affinity (less than 50% at 1 mM) for a 28 standard target binding profile (Table S1), suggesting the high selectivity of CHIBA-3007 for GlyT-1. Second, the kinetic study showed that the specific binding of [ 3 H]CHIBA-3007 to rat brain membranes reached equilibrium rapidly (less than 5 min, data not shown). Furthermore, Scatchard analysis showed that [ 3 H]CHIBA-3007 selectively binds to rat brain with a high affinity (K d = 1.61 nM). Nonspecific binding of [ 3 H]CHIBA-3007 to rat brain membranes was very low (,10%).
Very recently, two non-sarcosine-based radioligands, [ 3 H]SB-733993 and [ 3 H]GSK931145, have been developed [33]. The potencies of SB-733993 (pIC 50 = 7.20) and GSK931145 (pIC 50 = 7.58) for inhibiting [ 3 H]glycine uptake were more potent than that of SSR504734 (pIC 50 = 6.52) [33]. [ 3 H]SB-733993 and [ 3 H]GSK931145 showed similar binding affinities for GlyT-1 and similar levels of specific binding. For both radioligands, the specific binding at concentrations around K d values (1-2 nM) represented .90% of total binding [33], indicating low non-specific binding. However, the B max values of both radioliagnds in the rat brain were higher (around 3000 fmol/mg protein) than the B max values (692.8 fmol/mg protein) using [ 3 H]CHIBA-3007. The reasons underlying this discrepancy are currently unclear. One possibility may be due to the differences in the methodology of sample preparation, and binding assay. Furthermore, non-sarcosine-based GlyT-1 inhibitors as well as glycine itself all showed competitive interactions with the binding of [ 3 H]SB-733993 and [ 3 H]GS-K931145, whereas the sarcosine-based GlyT-1 inhibitors (NFPS/ ALX5407 and Org25935) showed uncompetitive interactions with the bindings of both radioligands [33]. It is, therefore, likely that, similar to other non-sarcosine radioliagnds (e.g., [ 3
Previously, we reported that repeated administration of the NMDA receptor antagonist phencyclidine caused an increase of GlyT-1 protein as well as a reduction of extracellular glycine levels in the hippocampus, but not the frontal cortex [35]. The study suggests that increased GlyT-1 protein may play a role in removing the extracellular glycine in the synaptic cleft via GlyT-1, resulting in lower extracellular levels of glycine in the hippocampus [35]. To date, there has been no report about GlyT-1 density in the hippocampus of patients with schizophrenia, although it has been reported that GlyT-1 mRNA and protein levels were not altered in the prefrontal cortex and cerebellum of postmortem brain samples from patients with schizophrenia [36]. Therefore, it would be of interest to study whether levels of GlyT-1  In conclusion, the present study shows that [ 3 H]CHIBA-3007 binding sites are associated with GlyT-1 in the rat brain and that [ 3 H]CHIBA-3007 could be a highly and specific and selective radioligand for studying GlyT-1 function in the brain in vitro.

Synthesis of [ 3 H]CHIBA-3007
[ 3 H]CHIBA-3007 was synthesized by N-methylation of the desmethyl-CHIBA-3007 with [ 3 H]methyl iodide ( Figure S1). The 0.1 mL of [ 3 H]methyl iodide toluene solution (370 MBq) was added to an ice-cold reaction vessel containing desmethyl-CHIBA-3007 (4 mg) and potassium carbonate (1.5 mg) in N,N-dimethyl-formamide (DMF, 0.3 mL). The reaction vessel was stirred at 0uC for 30 min. The reaction mixture was applied to a high performance liquid chromatography (HPLC) using an YMC Pack ODS-A column (10 mm in inner diameter 6250 mm in length; YMC Co., Ltd., Kyoto, Japan), comprised of UV absorbance (270 nm). A mixture of CH 3 CN/50 mM CH 3 COONH 4 / CH 3 COOH (350/650/3) was used as the mobile phase at a flow rate of 4 mL/min. The column eluent was collected automatically by using a fraction collector (Model 2110; Bio-Rad Laboratories, K.K., Tokyo, Japan) directly into polypropylene tubes. The 10-mL of each collected fractions were sampled into glass vials with 4 ml of scintillation cocktail (ACS-II; GE Healthcare Japan K.K., Tokyo, Japan). The radioactivity was determined using a liquid scintillation counter (LS-6500; Beckman Coulter, Tokyo, Japan). The radioactive fraction, eluted with a retention time corresponding to that of the authentic standard by was collected into an evaporation flask and evaporated to dryness. The residue was redissolved with 2 ml of ethanol. Chemical and radiochemical purity of [ 3 H]CHIBA-3007 was analyzed by HPLC in a system consisting of a column (YMC-Pack Pro C18, 4.6 mm in inner diameter 6250 mm in length, YMC Co., Ltd., Kyoto, Japan), using CH 3 CN/50 mM CH 3 COONH 4 /CH 3 COOH (350/650/3) as a mobile phase at a flow rate of 1.0 ml/min.

Preparation of Rat Brain Membrane
Male Crl: CD (SD) SPF/VF rats (8-10 week olds, 180-200 g)(Japan Charles River Inc., Tokyo, Japan) were used for the experiments. All animal studies were approved by the Animal Care and Use Committee of Chiba University (Permit Number: 22-122). All experiments were performed according to the Guidelines for Animal Experimentation and also conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. All efforts were made to minimize suffering.
After sacrificing the rats by decapitation, the brains were rapidly removed from the skulls. Whole brains or seven specific cerebral regions -the cerebral cortex, striatum, hippocampus, thalamus, midbrain, cerebellum and pons -dissected on ice by the method of Glowinski and Iversen [39] were stored at 280uC until use for the assay.
For the [ 3 H]CHIBA-3007-binding assay, the tissues of whole brains or each specific brain region were homogenized in 15 volumes (w/v) of 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at pH 7.4 for 30 s on ice. The homogenate was centrifuged at 40,000 g for 15 min at 4uC. The supernatant was discarded and the pellet was re-suspended, homogenized and centrifuged as above. The membrane pellet was washed and resuspended in ice-cold HEPES buffer and was then centrifuged three times. The final pellet was re-suspended in 15 volumes of the buffer (120 mM NaCl, 2 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 10 mM HEPES, pH 7.5 at room temperature).
For [ 14 C]glycine uptake, whole brains were homogenized in 10 volumes (w/v) of 0.32 M sucrose, buffered with 10 mM HEPES (pH 7.4). The homogenate was centrifuged at 1,000 g for 10 min to remove nuclei and debris, and then the supernatant was centrifuged again at 20,000 g for 20 min (synaptosomal P 2 fraction). The pellet was washed and re-suspended in ice-cold 0.32 M sucrose, buffered with 10 mM HEPES (pH 7.4) and centrifuged again at 20,000 g for 20 min (washed P 2 fraction). The pellet was re-suspended in 10 volumes of assay buffer with the following composition: 10 mM HEPES buffer (pH 7.4) containing 140 mM NaCl, 5.5 mM KCl, 1.8 mM CaCl 2 , 0.8 mM MgSO 4 , 5 mM glucose and 5 mM L-alanine (HB). The protein concen- trations were measured by using a DC protein assay kit (Bio-Rad Laboratories Inc., Tokyo, Japan).

[ 3 H]CHIBA-3007 Binding Assay
Assays of the binding of [ 3 H]CHIBA-3007 to rat brain membranes were performed. Aliquots of the rat brain membrane suspension (200 mL) were added in duplicate to a reaction mixture containing [ 3 H]CHIBA-3007 and the indicated concentrations of test drug in a final volume of 0.5 mL. Non-specific binding was estimated in the presence of 10 mM SSR504734, a potent and selective GlyT-1 inhibitor [34].
[3H]CHIBA-3007 binding was allowed to occur for 120 min at room temperature for the equilibrium saturation and inhibition studies. The binding reaction was terminated by rapid vacuum filtration onto Whatman GF/B glass filters pretreated with 0.5% polyethyleneimine (Sigma-Aldrich Co.) using a 24-channel cell harvester (Brandell, Gaithersburg, MD). The filters were washed with 5 mL of icecold assay buffer 3 times, and placed in vials with 4 mL scintillation cocktail. The radioactivity trapped by the filters was determined using a liquid scintillation counter (Beckman LS-6500; Beckman Coulter K.K., Tokyo, Japan).

Inhibition of [ 14 C]glycine Uptake
The assay of [ 14 C]glycine uptake was started by adding 10 mM [ 14 C]glycine to 200 mL of rat brain membrane in HB. The inhibition of [ 14 C]glycine uptake by eight compoundsdesmethyl-CHIBA-3007, CHIBA-3007, SSR504734, NFPS/ ALX5407, LY2365109, Org24598, sarcosine and glycine-was performed for 15 min at 37uC as reported previously [34]. Nonspecific uptake was estimated in the presence of 30 mM SSR504734. The uptake of [ 14 C]glycine was terminated by rapid vacuum filtration onto Whatman GF/B glass filters pretreated with 0.5% polyethyleneimine. The filters were washed by buffer, and the radioactivity trapped by the filters was determined using a liquid scintillation counter as described above.

Statistical Analysis
The data are shown as the mean 6 standard error of the mean (S.E.M.). The dissociation constant (K d ) and maximal binding (B max ) values from saturation binding and the IC 50 values from binding displacement by each drug were determined using the program GraphPad Prism (GraphPad Software, San Diego, CA). The K i values were calculated from the IC 50 values using the formula of Chung and Prusoff, K i = IC 50 /(1+[L]/K d ) [40], where the IC 50 was the concentration that resulted in 50% inhibition of specific binding, [L] was the concentration of radioligand used and K d was the dissociation constant. Correlation was analyzed by Pearson's Correlation Coefficient (PASW Statistics 19, Tokyo, Japan). Significance was set at p,0.05.

Supporting Information
Table S1 Inhibition effect of CHIBA-3007 (1 mM) on radioligand binding to various receptors.