In vitro Characterization of a small molecule inhibitor of the alanine serine cysteine transporter -1 (SLC7A10)

Authors


Abstract

NMDA receptor hypofunction is hypothesized to contribute to cognitive deficits associated with schizophrenia. Since direct activation of NMDA receptors is associated with serious adverse effects, modulation of the NMDA co-agonists, glycine or D-serine, represents a viable alternative therapeutic approach. Indeed, clinical trials with glycine and D-serine have shown positive results, although concerns over toxicity related to the high-doses required for efficacy remain. Synaptic concentrations of D-serine and glycine are regulated by the amino acid transporter alanine serine cysteine transporter-1 (asc-1). Inhibition of asc-1 would increase synaptic D-serine and possibly glycine, eliminating the need for high-dose systemic D-serine or glycine treatment. In this manuscript, we characterize Compound 1 (BMS-466442), the first known small molecule inhibitor of asc-1. Compound 1 selectively inhibited asc-1 mediated D-serine uptake with nanomolar potency in multiple cellular systems. Moreover, Compound 1 inhibited asc-1 but was not a competitive substrate for this transporter. Compound 1 is the first reported selective inhibitor of the asc-1 transporter and may provide a new path for the development of asc-1 inhibitors for the treatment of schizophrenia.

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Enhancing NMDA function by increasing synaptic D-serine is a proposed therapeutic approach for the treatment of schizophrenia. Synaptic D-serine levels are regulated by the alanine, serine, cysteine transporter 1 (asc-1). The following study describes the first novel asc-1 inhibitor, Compound 1 (BMS-466442), and provides a path forward for the development of additional asc-1 inhibitors.

Abbreviations used
NMDA

N-methyl-d-aspartate

asc-1

Na+-independent alanine–serine–cysteine transporter 1

ASCT-1 and -2

Na+-dependent alanine–serine–cysteine transporter 1 and 2

LAT-1 and 2

large amino acid transporter

AIB

α-aminoisobutyric acid

Schizophrenia is a complex neuropsychiatric disorder characterized by positive symptoms, negative symptoms, and cognitive deficits (Brown et al. 2010). While current antipsychotic medications are effective at alleviating positive symptoms, treatment of negative symptoms and cognitive deficits remain an unmet medical need. Cognitive deficits are hypothesized to results from an under-activation or hypofunction of NMDA receptors. This hypothesis is based on data in human subjects, demonstrating that the pharmacological blockade of NMDA receptors produces cognitive deficits that parallel those seen in schizophrenia patients (Malhotra et al. 1996; Adler et al. 1999). These findings are supported by pre-clinical data demonstrating that inhibition of NMDA receptor function disrupts measures of cognition (Jentsch et al. 1997a,b; Stefani and Moghaddam 2005). Based on these data, development of newer medications has largely focused on improving NMDA receptor function. However, since direct pharmacological activation of NMDA receptors leads to over-excitation and seizures, indirect modulation of NMDA receptors at one of the many regulatory sites may provide an attractive alternative to addressing NMDA receptor hypofunction.

Activation of NMDA receptors requires the binding of both glutamate as an agonist and either glycine or D-serine as a co-agonist. Recent clinical trials have shown beneficial effects of adjunctive D-serine and glycine treatment on both cognitive and negative symptom domains of schizophrenia (Javitt et al. 1994; Heresco-Levy et al. 1996, 2005). For example, Tsai et al. showed that adjunctive D-serine treatment improved positive, negative, and cognitive symptoms associated with schizophrenia (Tsai et al. 1998). Using the MATRICS cognition battery, Kantrowitz et al. showed that doses of adjunctive D-serine up to 120 mg/kg/day improved the overall composite score on the MATRICS battery (Kantrowitz et al. 2010). A recent meta analysis of clinical data supports the conclusion that both adjunctive glycine and D-serine treatment showed significant improvement in negative and total symptom scores (Singh and Singh 2011). Although adjunctive high-dose glycine or D-serine treatment has produced clinical improvements in schizophrenia patients, concerns over the high doses required, off target effects and toxicity remain. For example, in rat models high-dose D-serine treatment can result in irreversible nephrotoxicity (Williams and Lock 2004; Orozco-Ibarra et al. 2007), an effect that was not seen in mice (Balu et al. 2013), suggesting a possible species selective nephrotoxicity. However, in clinical studies with chronic D-serine treatment, reversible nephrotoxic patterns were seen in one patient treated with the highest dose of D-serine (Kantrowitz et al. 2010).

An alternative approach for increasing synaptic concentrations of glycine and D-serine is to inhibit the normal uptake processes which are involved in clearing these NMDA co-agonists. Indeed, inhibitors of the glycine transporter l (GlyT1) have shown to be effective in preclinical models and are currently in clinical trials for the treatment of schizophrenia (Black et al. 2009; Nikiforuk et al. 2011; Harada et al. 2012). In contrast, no small molecule inhibitors of D-serine transporters have been identified.

Within the brain, synaptic concentrations of amino acids are regulated by multiple redundant amino acid transporter systems. The primary amino acid transporters for D-serine include the sodium-dependent alanine, serine, cysteine, theronine transporter 2 (ASCT-2) and the sodium-independent alanine, cysteine, serine transporter-1 (asc-1). Of these two transporters, the asc-1 is the predominant regulator of extracellular D-serine as shown by studies where deletion of the asc-1 transporter eliminated 70% of the D-serine uptake into synaptosomes (Rutter et al. 2007). In addition, enhancing extracellular D-serine using a competitive amino acid substrate of asc-1, D-isoleucine, enhances the long-term potentiation in rodent hippocampal slices directly linking asc-1, D-serine and NMDA receptor function (Rosenberg et al. 2013). The asc-1 transporter is encoded by the SLC7A10 gene and belongs to a larger family of glycoprotein associated transporters which are unique in that they require association with a glycoprotein, are sodium-independent and often have overlapping amino acid substrates (for review, see Verrey et al. 2004). Inhibition of asc-1 may provide some advantages over Glyt1 inhibitors. Specifically, D-serine and asc-1 show overlapping distribution and are both localized to the cortex and hippocampus, regions critically involved with cognition. (Schell et al. 1997; Helboe et al. 2003; Oliet and Mothet 2006). In addition, unlike the glycine transporter which is relatively selective for glycine, asc-1 transporters have multiple substrates including D-serine and glycine. Therefore, inhibition of asc-1 may provide the opportunity to increase both glycine and D-serine in critical brain regions; however, the impact of altering uptake of other amino acids transported by asc-1 and/or increase seizure liability as seen in asc-1 knockout mice (Xie et al. 2005) should also be considered. While speculative, the lack of an available tool compound to inhibit asc-1 prevents the direct testing of this hypothesis. The current studies reported here use multiple heterologous and endogenous asc-1 systems to characterize the first selective, small molecule inhibitor of the asc-1 transporter BMS-466442, designated as Compound 1.

Methods

Materials

Sprague–Dawley rats were obtained from Charles River Laboratory and were housed in groups of four and maintained on a 12 h light:dark cycle (0600 : 1800) with food and water provided ad libitum. All animal use and procedures were conducted under approval of the Bristol-Myers Squibb IACUC guidelines and all efforts were taken to minimize pain and discomfort. All experiments were conducted in accordance with ARRIVE guidelines.

Synthesis of compound 1

Compound 1 (BMS-466442) was synthesized by Neuroscience Discovery Chemistry at Bristol Myers-Squibb. All reagents were purchased from Aldrich (Sigma-Aldrich Corporation Saint Louis, MO, USA). To a stirred solution of 6-benzyloxy-5-methoxy-1H-indole-2-carboxylic acid (0.31 g, 1.05 mmol), (S)-methyl 2-amino-3-(1-benzyl-1H-imidazol-4-yl)propanoate, 2 HCl (0.35 g, 1.05 mmol) and (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) (0.44 g, 1.16 mmol) in dimethylformamide (20 mL) at 0°C was added N,N-diisopropylethylamine (0.37 mL, 2.11 mmol). The solution was warmed to 25°C and stirred for 1.5 h. The reaction mixture was quenched with ethyl acetate and washed with saturated aqueous sodium bicarbonate followed by brine. The organic layer was dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by reverse phase preparative HPLC (30 × 100 mm Xterra column; gradient: 30–100% B over 26 min. (Solvent A: 90% water, 10% Methanol, 0.1% trifluoroacetic acid (TFA); Solvent B: 10% Water, 90% Methanol, 0.1% TFA)). The required fractions were concentrated to obtain (S)-methyl 3-(1-benzyl-1H-imidazol-4-yl)-2-(6-benzyloxy-5-methoxy-1H-indole-2-carboxamido)propanoate (0.32 g, 0.57 mmol, 54% yield) as a brown oil. Liquid chromatography-mass spectroscopy (electrospray ionization) m/e 539.1 [(M+H)+, calculated for C31H31N4O5 539.2]; 1H NMR (400 MHz, CDCl3) δ ppm 9.51 (s, 1 H), 8.19 (d, = 7.6 Hz, 1 H), 7.49 (d, = 1.0 Hz, 1 H), 7.46 (d, = 7.3 Hz, 2 H), 7.32 - 7.38 (m, 2 H), 7.25–7.31 (m, 4 H), 7.08 (dd, = 7.4, 1.9 Hz, 2 H), 7.04 (s, 1 H), 6.90 (s, 2 H), 6.67 (d, = 1.0 Hz, 1 H), 5.18 (s, 2 H), 5.02 (s, 2 H), 4.93 (dt, = 7.4, 5.1 Hz, 1 H), 3.90 (s, 3 H), 3.60 (s, 3 H), 3.13 (qd, = 14.8, 5.0 Hz, 2 H).

Stable cell-line generation

Stable cell lines expressing the full-length asc-1 open reading frame (proceeded by Kozak consensus sequence) for human asc-1 (hasc-1; accession number NM019849) and large amino acid transporter -2 (LAT- 2; accession number BC052250) were generated by transfecting HEK293 cells using Lipofectamine Plus reagent and the asc-1 and LAT2 cDNAs cloned into the pIRESpuro2 (Clonetech) plasmid. Stable pools of cells were attained by selection and maintenance in MEM media (#11095; Invitrogen, Carlsbad, CA, USA) containing 2 mM glutamine, 10% fetal bovine serum, 1 μg/mL puromycin. Cells were plated at appropriate densities in white, Poly-D-Lysine coated, 96-well plates one day prior to assay in growth media.

Generation of primary cortical cultures for uptake assays

Primary cortical cultures were isolated from cortices dissected from E19 rat embryos. Tissue was dissociated and processed using the Papain Dissociation System (Worthington Biochemical Corporation, Lakewood, NJ, USA) as described by the manufacturer. Cells were plated at 100 000 cells/well in white, clear bottom, poly-d-lysine coated 96-well plates (BD, Biocoat) in Neurobasal medium plus 1X B27 supplement, 1X penicillin–streptomycin, and 0.5 mM glutamax (Invitrogen). All experiments were performed using 6-8 day in vitro cultures.

[3H]-amino-acid uptake assay

On the day of the assay, media was removed and cells were washed three times with sodium-free assay buffer (120 mM choline chloride, 25 mM triethylammonium bicarbonate, 1.5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 1.2 mM KH2PO4, 10 mM glucose, 10 mM HEPES, pH 7.4 with KOH) for asc-1 and LAT-2 uptake or sodium containing buffer (120 mM sodium chloride, 25 mM sodium bicarbonate, 1.5 mM KCl,1.2 mM CaCl2, 1.2 mM MgCl2, 1.2 mM KH2PO4, 10 mM glucose, 1 mM HEPES, pH 7.4 with KOH) for ASCT-2. After the final wash, either sodium-containing or sodium-free buffer was added to each well followed by the addition of test-compound or non-labeled amino acid. Cells were pre-incubated with test compounds for 10 min. Uptake was initiated by the addition of [3H]-amino acid ([3H]D-serine Perkin Elmer 21.2 Ci/mmol [asc-1 or ASCT-2]; [3H]-glycine 15 Ci/mmol [asc-1], [3H]-L-serine Perkin Elmer 30.9 Ci/mmol [LAT-2]) at a final assay concentration of 100 nM. Final assay volume was 50 μL. Non-specific uptake was defined by the addition of 10 mM D-serine. Cells were incubated at 25°C for 5 min (asc-1 and ASCT-2), 4 min (LAT-2) or 60 minutes (primary cultures) and the reaction was terminated by the addition of 100 μl of ice-cold assay buffer followed by three rapid washes. After the final washing, all buffer was removed from wells and 200 μL scintillant was added per well and plates were read on a Wallac Trilux Microbeta scintillation counter (Perkin Elmer, Waltham, MA, USA). IC50 values were determined from three to five independent experiments.

Rat synaptosomal uptake assay

Rat cortical synaptosomes were generated from male Sprague–Dawley rats as previously described (Haughey et al. 2000). Briefly, rat cortex was homogenized in 10 volumes of 0.32 M sucrose and centrifuged for 15 min at 600 g at 4°C. The supernatant was then centrifuged for 20 min at 20 000 g at 4°C. The resulting P2 pellet was resuspended at a concentration of 50 mg wet weight per ml sodium-free buffer. For [3H]D-serine uptake, 40 μL of synaptosomal sample was added to a deep-well 96-well plate followed by cold-amino acid or test compounds and the mixture was incubated for 10 minutes at 25°C. The uptake reaction was initiated by addition of [3H]D-serine (100 nM final concentration, 100 μL final assay volume). The mixture was incubated for 3 min at 25°C and the reaction was terminated by rapid filtration (Brandell 96-well harvester) onto glass GF/B filter paper soaked for 1 h in 0.3% polyethyleneimine. Samples were washed three times in ice-cold sodium free buffer and collected in 5 mL of scintillation fluid, incubated at 25°C for 8 h, and counted in a scintillation counter.

Glycine efflux assay

HEK cells expressing the human asc-1 cDNA were pre-loaded with [3H]-glycine as described above with the minor modification that the incubation time was increased to 60 min. After washing cells three times, the buffer was replaced with fresh sodium free buffer (50 μL final volume) and test compound or non-labeled amino acid was added and cells incubated at 25°C for 20 min. After 20 min, the supernatant was removed and placed in scintillation vials containing 200 μL of scintillation fluid to capture extracellular [3H]-glyine. To the remaining cells, 200 μL of scintillation fluid was added and the cells collected and placed into scintillation vials to capture non-effluxed (remaining) [3H]-glycine. Percent release was calculated by dividing the amount of [3H]-glycine effluxed by the total amount of glycine pre-loaded (effluxed plus remaining). Data presented are representative of three independent experiments.

Statistics

All values are shown as the mean ± STDEV unless otherwise indicated. Data were analyzed using GraphPad Prism software (GraphPad Software, Inc. La Jolla, CA, USA). Multiple comparisons were made using an anova followed by Newman–Keuls post hoc analysis. In all cases, p < 0.05 was considered to be statistically significant.

Results

Compound 1 was identified as an asc-1 inhibitor following a high-throughput screen of ~ 1 million compounds from the BMS chemical library (Fig. 1). To evaluate the effects of Compound 1 on asc-1 function, three different cellular systems were used: HEK cells stably transfected with human asc-1 cDNA, primary rat cortical cultures, and rat brain synaptosomes. In the absence of sodium, mock-transfected HEK cells showed negligible uptake of D-serine while HEK cells stably expressing the human asc-1 showed robust D-serine uptake that could be blocked by D-serine and α-aminoisobutyric acid (AIB, an amino acid analog and substrate for asc-1; Fukasawa et al. 2000) (Fig. 2a and b). To determine if D-serine inhibited uptake with similar potency in asc-1 expressing HEK cells and primary cultures, the potency of non-labeled D-serine was determined using a concentration response curve. Non-labeled D-serine inhibited [3H] D-serine uptake in human asc-1 HEK cells and primary cultures with IC50 values of 25 ± 8.2 and 34 ± 13.3 μM, respectively (determined from five independent experiments). Incubation of both human asc-1 expressing HEK cells or primary cortical cultures with 1 mM AIB completely inhibited [3H] D-serine uptake, (Fig. 2b). Amino acid selectivity profiles have been used to characterize various amino acid transporters including the large amino acid transporter-1 (LAT-1), LAT-2, y+LAT-2, ASCT-2 and asc-1 (Utsunomiya-Tate et al. 1996; Pineda et al. 1999; Bröer et al. 2000; Fukasawa et al. 2000; Nakauchi et al. 2000 and Yanagida et al. 2001). Amino acid selectivity profiles in stable human asc-1 and primary cultures confirm that, in sodium-free conditions, asc-1 is the primary amino acid transporter responsible for [3H] D-serine uptake (Fig. 2c).

Figure 1.

Structure of Compound 1 (BMS-466442) identified following a high-throughput screen for inhibitors of alanine serine cysteine transporter-1 (asc-1) function.

Figure 2.

(a) HEK cells were stably transfected with the human alanine serine cysteine transporter-1 (asc-1) transporter and D-serine uptake assay was performed as described in 'Methods'section. [3H] D-serine uptake was measured in sodium-free buffer in the absence and presence of 10 mM non-labeled D-serine. Mock-transfected HEK cells were used to define non-specific uptake. (b) [3H] D-serine uptake was performed in HEK asc-1 cells or primary cortical cultures in the presence or absence (totals) of 1 mM α-aminoisobutyric acid (AIB) or D-serine. Values represent the average fmoles in cells ± 1 SEM. *indicates p < 0.05 versus totals. (c) [3H] D-serine uptake was performed in HEK asc-1 cells or primary cortical cultures in the presence or absence of 1 mM non-labeled amino acid. Percent inhibition was defined using 10 mM non-labeled D-serine (100% inhibition) and no addition of non-labeled amino acids (0% inhibition). Bars represent average percent inhibition ± 1 SEM.

To evaluate the potency of Compound 1 on asc-1 function, concentration–response curves were tested in both stable human asc-1 cell-lines and cultures. Representative data presented in Fig. 3 demonstrate that Compound 1 dose-dependently inhibited asc-1 activity in human asc-1 expressing cells and primary cultures with IC50 values of 36.8 ± 11.6 nM and 19.7 ± 6.7 nM, respectively (Fig. 3. Actual IC50 values determined from 5 independent experiments). To determine if Compound 1 inhibited [3H] D-serine uptake in an ex vivo system, a synaptosomal [3H] D-serine uptake assay was developed and validated. Again, amino acid selectivity profiles were consistent with that seen in primary cultures and HEK expressing human asc-1 (Fig. 4). In addition non-labeled D-serine inhibited D-serine uptake with an IC50 values of 10 ± 4.2 μM, similar to that seen in HEK asc-1 expressing cells and rat primary cultures. Using this system, Compound 1 inhibited [3H] D-serine uptake into rat brain synaptosomes with an IC50 value of 400 ± 110 nM (determined from three independent experiments).

Figure 3.

Compound 1 inhibited D-serine uptake into human alanine serine cysteine transporter-1 (asc-1)expressing HEK cells and rat primary cortical cultures. Full inhibition was defined as uptake in the presence of 10 mM non-labeled D-serine. Points represent the average percent inhibition ± SEM for each concentration run in triplicate.

Figure 4.

3H D-serine uptake was performed in rat brain synaptosomal preparations in the presence or absence of 1 mM non-labeled amino acid. Percent inhibition was defined using 10 mM non-labeled D-serine (100% inhibition) and no addition of non-labeled amino acids (0% inhibition). Bars represent average percent inhibition ± 1 SEM.

To determine the selectivity of Compound 1 for asc-1, two additional amino acid transporters were evaluated, LAT-2 and ASCT-2. To confirm the activity of LAT-2 and ASCT-2 in these cellular systems, amino acid profiles were evaluated. Data presented in Fig. 5 demonstrate that D-serine and AIB (both asc-1 substrates) showed minimal inhibition of [3H] L-serine or [3H] D-serine reflecting LAT-2 (Fig. 5a) and ASCT-2 (Fig. 5b) activity respectively. Data presented in Fig. 6 demonstrate that Compound 1 was ≥ 1000 fold selective for asc-1 versus LAT-2 and ASCT-2 (IC50 values: 11 nM for asc-1, ≥ 10 000 nM for LAT-2 and, ≥ 10 000 nM for ASCT-2). The selectivity of Compound 1 was further tested by evaluation against > 40 targets from the in-house BMS selectivity panel. Compound 1 showed IC50s > 10 μM at all targets except for the dopamine transporter (IC50 = 420 nM) and Adrenergic α1D receptor (IC50 = 6.1 μM).

Figure 5.

Uptake was performed using stable hLAT-2 cells in sodium-free buffer with [3H] L-serine as the ligand (5A) or in non-transfected HEK cells using sodium-containing buffer with [3H] D-serine as the ligand to capture alanine, serine, cysteine, theronine transporter 2 (ASCT-2) activity (5B). One-hundred percent inhibition was defined using 10 mM non-labeled D-serine (ASCT-2) or L-serine (hLAT-2) and no addition of non-labeled amino acids (0% inhibition). Bars represent average percent inhibition ± 1 SEM.

Figure 6.

Effect of Compound 1 on alanine serine cysteine transporter-1 (asc-1), LAT2 and alanine, serine, cysteine, theronine transporter 2 (ASCT-2) activity. Uptake was performed as described in 'Methods' section. Points represent the average percent inhibition ± 1 SEM for each concentration run in triplicate.

The asc-1 transporter functions as an amino acid exchange protein. To determine if Compound 1 causes direct inhibition of transporter function or serves as a competitive substrate similar to AIB, an efflux assay was developed. Cells were preloaded with glycine and exogenous amino acids or compound was added. Data presented in Fig. 7a demonstrate that the asc-1 substrate D-serine causes a dose-dependent increase in [3H]-glycine-mediated efflux, while the non-asc-1 substrate glutamine does not. The concentration of D-serine required to increase [3H]-glycine efflux by 50% (EC50 26 μM) was similar to that reported for the inhibition of [3H] D-serine uptake (IC50 37 μM). Evaluation of other amino acid substrates confirmed the selectivity of asc-1 in mediating [3H]-glycine efflux as only asc-1 substrates (D-serine, glycine, and AIB) and not other amino acids (tyrosine, glutamate, leucine, or glutamine), increased transporter-mediated efflux of [3H]-glycine (Fig. 7b). In contrast to amino acid substrates, Compound 1 inhibited basal [3H]-glycine efflux, indicating that Compound 1 is not a substrate for the asc-1 transporter.

Figure 7.

Compound 1 is a non-competitive substrate inhibitor of the alanine serine cysteine transporter-1 (asc-1) transporter. [3H] glycine efflux was performed as described in 'Methods' section. (a) Concentrations response curves were run using an asc-1 substrate D-serine or a low affinity/non-substrate glutamine. Points represent the average fold increase in [3H] glycine efflux over control (no cold amino acid addition) ± 1 SEM. Each point was run in triplicate. (b) Effect of asc-1 amino acids (1 mM final) and Compound 1 (50 μM final) on 3H-glycine-mediated efflux. Bars represent the average fold change in [3H] glycine efflux over control (no amino acid treatment) ± 1 SEM. *p < 0.05 versus control.

Discussion

Compound 1(BMS-466442) was identified through a high-throughput screen as an inhibitor of the asc-1 transporter. The activity and selectivity of Compound 1 was evaluated in multiple cellular systems. Since multiple amino acid transporters exist, some with overlapping amino acid substrates, it was important to first validate the activity of each transporter in these cellular systems. This was done using amino acid selectivity profiles (Utsunomiya-Tate et al. 1996; Pineda et al. 1999; Bröer et al. 2000; Fukasawa et al. 2000; Nakauchi et al. 2000 and Yanagida et al. 2001), altering assay conditions to capture sodium-dependent and sodium-independent activity, and evaluating the potency of D-serine to inhibit [3H] D-serine uptake across the multiple cellular systems. Asc-1 transports small amino acids such as serine, threonine, cysteine, alanine, and glycine with high affinity. However, large acidic or polar amino acids (e.g. glutamate, glutamine or aspartate) are less efficiently transported by asc-1. The amino-acid profile of HEK cells expressing the human asc-1 showed that only small amino acids inhibited [3H] D-serine uptake by greater than 80%, whereas basic, acidic, large polar, and non-polar amino acids showed less than 80% inhibition at 1 mM concentrations (Fig. 2c). A similar pattern was seen in rat primary cultures (Fig. 2c) and rat brain synaptosomes (Fig. 4) suggesting that asc-1 is the primary transporter of [3H] D-serine in these systems under these assay conditions. This conclusion is further supported by the finding that [3H] D-serine uptake was seen in sodium-independent conditions which differentiates asc-1 activity from the sodium-dependent D-serine uptake mediated by ASCT-2. Moreover, the potency of D-serine to inhibit uptake into both HEK human asc-1 cells, rat primary cultures and synaptosomes, was similar to the reported Km for D-serine at the asc-1 transporter (Nakauchi et al. 2000; Helboe et al. 2003). Collectively, these data support the conclusion that asc-1 is the primary [3H] D-serine transporter in these endogenous and heterologous systems under the current assay conditions and validates their use as models to evaluate the potency and selectivity of Compound 1.

The fact that Compound 1 inhibited D-serine uptake in both HEK h-asc-1 cells and rat primary cortical cultures with similar potency supports the conclusion that Compound 1 is equipotent at rat and human asc-1 transporters and that transfected HEK cells mimic the activity of the endogenously expressed transporter.

Of note to these findings is that the primary cultures in the current studies were used at 6–8 days in culture. One potential caveat related to the use of younger cultures is that neurons may not yet be fully mature and this may impact overall uptake or processing of D-serine. However, expression profiling of primary cortical cultures at 7 and 14 days in culture showed only a 1.2 fold increase in mRNA for the asc-1 gene and no change in expression of serine racemase (unpublished data). Moreover, the amino acid profiles and potency of D-serine was similar between primary cultures and HEK cells expressing the human asc-1 transporter.

Compound 1 inhibited glycine uptake into human asc-1 expressing HEK cells (data not shown) suggesting that the inhibition of asc-1 is not selective for D-serine but also inhibits transport of other asc-1 substrates. Since both glycine and D-serine are co-agonists of the NMDA receptors, inhibition of asc-1 may provide added clinical benefit by increasing both D-serine and glycine. Compound 1 also inhibited D-serine uptake in an ex vivo synaptosomal system, similar to the inhibition in human asc-1 expressing HEK cells and primary cultures. However the potency was right shifted in the synaptosomal preparation. Possible explanations for this finding include the presence of endogenous amino acids in the synaptosomal preparation, binding of Compound 1 to proteins and or lipids present in the synaptosomal preparation and/or alternatively, a yet unidentified, sodium-independent D-serine transport mechanism present within synaptosomal preparations.

Compound 1 was ≥ 1000-fold selective for inhibition of asc-1 over LAT-2 and ASCT-2. LAT-2 shares many similarities to asc-1. For example, both transporters are sodium-independent and utilize an accessory protein, 4F2 (Pineda et al. 1999). In addition, LAT-2 shows the closest overall amino acid sequence homology (vs. other amino acid transporters) to the asc-1 transporter and is able to transport L- but not D-serine (Verrey et al., 2004). The ASCT-2 transporter is a sodium-dependent transporter that is also able to transport D-serine albeit with lower (Km ~ 2 mM) affinity (Gliddon et al. 2009). The fact that Compound 1 was selective for asc-1 over LAT-2 and ASCT-2 suggests that Compound 1 does not inhibit asc-1 by simply disrupting the disulfide linkage between the 4F2 protein and the amino acid transporters, in which case one would expect inhibition of LAT-2 to be seen as well. Moreover, Compound 1 did not inhibit L-serine (LAT-2) or D-serine (ASCT-2) uptake suggesting that the mechanism of inhibition is not related to a non-specific interaction with the amino acid substrate, a non-selective inhibition of D/L-serine uptake or a generalized cellular toxicity.

Studies were also conducted to evaluate the mechanism of inhibition of the asc-1 transporter by Compound 1. As stated previously the asc-1 transporter, like most sodium-independent transporters, functions as an amino acid exchanger. The previously identified inhibitor of asc-1, AIB, is actually a substrate for asc-1 (Fukasawa et al. 2000) and its apparent inhibition results from competitive substrate inhibition. Therefore, we developed an assay to differentiate between competitive substrate inhibitors and non-substrate inhibitors. Data presented in (Fig. 7) demonstrates that only asc-1 substrates and the competitive substrate inhibitor AIB increased [3H]-glycine efflux from HEK cells expressing asc-1 confirming the efflux of glycine was asc-1 mediated. Interestingly, Compound 1 did not increase, but actually decreased glycine efflux. These data suggest that Compound 1 acts as an inhibitor, and not as a competitive substrate of the asc-1 transporter. Thus Compound 1 may represent a superior inhibitor of asc-1 versus amino acid analogs, which would be rapidly cleared from the synapse by a transporter mediated mechanism. Interestingly, Rosenberg et al. (2013) recently demonstrated that D-isoleucine enhances D-serine release via asc-1 and this, in-turn, enhances NMDA receptor function, again implicating a link between asc-1, D-serine and NMDA receptor function. The authors of this study hypothesize that asc-1 blockers may serve to decrease NMDA receptor activation by decreasing non-vesicular, asc-1 mediated, D-serine release whereas activators of asc-1 may be useful for conditions of NMDA hypofunction such as schizophrenia. While studies by Rutter et al. (2007) have shown that the asc-1 primary transporter mediating D-serine uptake, the functional consequence of asc-1 modulation (activation and/or inhibition) remains to be determined. In this respect, Compound 1 may provide a useful tool to address this question.

While Compound 1 represents an advance in our understanding of asc-1 inhibitors, there are limitations. For example, the biophysical and biochemical properties of the molecule, such as a large molecular weight and presence of an ester within the molecule make this compound unsuitable for in vivo studies. While extensive SAR studies are needed to address these issues, Compound 1 serves as an initial structural starting point for larger SAR efforts. Moreover, Compound 1 serves as an in vitro tool molecule to evaluate the effects of asc-1 inhibition in both heterologous over-expression systems as well as endogenous primary culture preparations.

Acknowledgements

This study was conducted at, and funded by, Bristol-Myers Squibb Research Laboratories Wallingford, CT, in compliance with the ARRIVE guidelines. No additional conflicts of interest exist. The authors would like to thank Ted Molski for his editorial review of this manuscript.

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