Kinase experiment:

Association and dissociation kinetic analysis of [3H]ORG24598 to hGlyT1 and ratforebrain membranes is performed. [3H]ORG24598 binding experiments are performed using membranes from CHO cells expressing hGlyT1b and also in membranes from mouse, rat, monkey, and dogforebrains. Saturation isotherms are determined by adding [3H]ORG24598 to rat, mouse, monkey, and dog forebrain membranes (40 μg/well) and cell membranes (10 μg/well) in a total volume of 500 μL for 3 h at room temperature. Saturation binding experiments are analyzed by an Excel-based curve-fitting program using the Michaelis-Menten equation derived from the equation of a bimolecular reaction and the law of mass action:B=(Bmax×[F])/(Kd+[F]), where B is the amount of ligand bound at equilibrium, Bmax the maximum number of binding sites, [F] the concentration of free ligand, and Kd the ligand dissociation constant. For inhibition experiments, membranes are incubated with 3 nM [3H]ORG24598 and 10 concentrations of Bitopertin for 1 h at room temperature. Schild analysis is performed in the presence of increasing concentrations of [3H]ORG24598 (1-300 nM). IC50 values are derived as described above. Ki values are calculated according to the following equation: Ki=IC50/(1+[L]/Kd)[1].

Animal experiment:

Mice[1] Male NMRI mice (20-30 g) are treated with Bitopertin (0.3, 3, 1, and 10 mg/kg p.o.) or vehicle (p.o.). After 1 min, L-687,414 (50 mg/kg s.c.) or vehicle is given. After 15 min of habituation in the activity chambers, horizontal activity is recorded for 60 min. The time course of Bitopertin effects on L-678,414-induced hyperactivity is also examined; locomotor activity is assessed 2.5, 4.5, and 24 h after administration of Bitopertin (L-678,414 is always given 15 min before the activity procedure). In addition, the effect of subchronic Bitopertin is investigated. Mice receive vehicle or Bitopertin (1 mg/kg p.o.) for 4 consecutive days and L-678,414-induced hyperactivity is evaluated on day 5. Rats[1] Wistar rats receive a 14-day treatment of PCP HC1 (5 mg/kg) or vehicle (NaCl 0.9%, 5 mL/kg i.p.). 24 h following the last injection, rats (6-18 per group) are allowed to individually habituate to the test boxes for 30 min. Rats then received Bitopertin (1, 3, 10 mg/kg p.o.) or vehicle (Polysorbate 80, HEC, Methyl- and Propylparaben pH 6.0; 5 mL/kg p.o.), followed after 1 h by 1 mg/kg D-amphetamine or vehicle i.p. Horizontal activity is recorded directly after the administration of Bitopertin until 120 min after dosing with amphetamine. Data are analyzed by ANOVA supplemented by Fischer's least significant difference post hoc test.

Bitopertin, also known as RG1678, is a potent and selective inhibitor of GlyT1 with an EC50 of 30nM. [1]
Abnormal signaling through the N-methyl-D–aspartate (NMDA) receptor has been hypothesized to be a key factor underlying many signs and symptoms of schizophrenia. Increasing NMDA receptor function pharmacologically is thought to compensate for dysfunctional receptor signaling and is a promising approach for the treatment of schizophrenia. Targeting the NMDA receptor allosteric glycine site has been assumed as an approach to enhance NMDA receptor functioning and thus normalize glutamate transmission, which can be achieved by glycine agonists and also by preventing synaptic clearance of glycine through the inhibition of the glycine transporter type 1 (GlyT1). Bitopertin selectively inhibits GlyT1, thus increasing the synaptic level of glycine, an obligatory coagonist at the NMDA receptor, and consequently enhancing NMDA signaling. [1, 2]
Bitopertin was found to possess a high in vitro GlyT1 inhibitory activity with EC50 of 30nM, and a favorable hERG activity with IC50 of 17μM. Moreover, Bitopertin also showed excellent selectivity over GlyT2, as well as a panel of 86 other targets including transmembrane and soluble receptors, enzymes, ion channels, and monoamine transporters. Notwithstanding Bitopertin showed low aqueous solubility, it exhibited a satisfactory solubility characteristics in both fasted state simulated intestinal fluid (20 μg/mL) and fed state simulated intestinal fluid (60 μg/mL). Additionally, by parallel artificial membrane permeation assay it exhibited excellent membrane permeability.  [1]
In a phase II RCT study, placebo and different doses of Bitopertin were added to standard antipsychotic treatment for 8 weeks, which revealed that Bitopertin groups had significantly higher response rates and trends towards improved function. Bitopertin inhibiting glycine reuptake is likely to be a new treatment for schizophrenia with negative symptoms. [2]
References:
1.Pinard E, Alanine A, Alberati D, et al. Selective GlyT1 inhibitors: discovery of [4-(3-fluoro-5-trifluoromethylpyridin-2-yl) piperazin-1-yl][5-methanesulfonyl-2-((S)-2, 2, 2-trifluoro-1-methylethoxy) phenyl] methanone (RG1678), a promising novel medicine to treat schizophrenia[J]. Journal of medicinal chemistry, 2010, 53(12): 4603-4614.
2.Umbricht D, Alberati D, Martin-Facklam M, et al. Effect of bitopertin, a glycine reuptake inhibitor, on negative symptoms of schizophrenia: a randomized, double-blind, proof-of-concept study[J]. JAMA psychiatry, 2014.