Synthesis, Biological Evaluation, and Computational Analysis of Biaryl Side-Chain Analogs of Solithromycin.

There is an urgent need for new antibiotics to mitigate the existential threat posed by antibiotic resistance. Within the ketolide class, solithromycin has emerged as one of the most promising candidates for further development. Crystallographic studies of bacterial ribosomes and ribosomal subunits complexed with solithromycin have shed light on the nature of molecular interactions (π-stacking and H-bonding) between from the biaryl side-chain of the drug and key residues in the 50S ribosomal subunit. We have designed and synthesized a library of solithromycin analogs to study their structure-activity relationships (SAR) in tandem with new computational studies. The biological activity of each analog was evaluated in terms of ribosomal affinity (Kd determined by fluorescence polarization), as well as minimum inhibitory concentration assays (MICs). Density functional theory (DFT) studies of a simple binding site model identify key H-bonding interactions that modulate the potency of solithromycin analogs.

State-dependent inhibition of BK channels by the opioid agonist loperamide.

Large-conductance Ca2+-activated K+ (BK) channels control a range of physiological functions, and their dysfunction is linked to human disease. We have found that the widely used drug loperamide (LOP) can inhibit activity of BK channels composed of either α-subunits (BKα channels) or α-subunits plus the auxiliary γ1-subunit (BKα/γ1 channels), and here we analyze the molecular mechanism of LOP action. LOP applied at the cytosolic side of the membrane rapidly and reversibly inhibited BK current, an effect that appeared as a decay in voltage-activated BK currents. The apparent affinity for LOP decreased with hyperpolarization in a manner consistent with LOP behaving as an inhibitor of open, activated channels. Increasing LOP concentration reduced the half-maximal activation voltage, consistent with relative stabilization of the LOP-inhibited open state. Single-channel recordings revealed that LOP did not reduce unitary BK channel current, but instead decreased BK channel open probability and mean open times. LOP elicited use-dependent inhibition, in which trains of brief depolarizing steps lead to accumulated reduction of BK current, whereas single brief depolarizing steps do not. The principal effects of LOP on BK channel gating are described by a mechanism in which LOP acts as a state-dependent pore blocker. Our results suggest that therapeutic doses of LOP may act in part by inhibiting K+ efflux through intestinal BK channels.

Novel compounds that reverse the disease phenotype in Type 2 Gaucher disease patient-derived cells.

Gaucher disease (GD) results from inherited mutations in the lysosomal enzyme ß-glucocerobrosidase (GCase). Currently available treatment options for Type 1 GD are not efficacious for treating neuronopathic Type 2 and 3 GD due to their inability to cross the blood-brain barrier. In an effort to identify small molecules which could be optimized for CNS penetration we identified tamoxifen from a high throughput phenotypic screen on Type 2 GD patient-derived fibroblasts which reversed the disease phenotype. Structure activity studies around this scaffold led to novel molecules that displayed improved potency, efficacy and reduced estrogenic/antiestrogenic activity compared to the original hits. Here we present the design, synthesis and structure activity relationships that led to the lead molecule Compound 31.

mTOR hyperactivity mediates lysosomal dysfunction in Gaucher's disease iPSC-neuronal cells.

Bi-allelic GBA1 mutations cause Gaucher's disease (GD), the most common lysosomal storage disorder. Neuronopathic manifestations in GD include neurodegeneration, which can be severe and rapidly progressive. GBA1 mutations are also the most frequent genetic risk factors for Parkinson's disease. Dysfunction of the autophagy-lysosomal pathway represents a key pathogenic event in GBA1-associated neurodegeneration. Using an induced pluripotent stem cell (iPSC) model of GD, we previously demonstrated that lysosomal alterations in GD neurons are linked to dysfunction of the transcription factor EB (TFEB). TFEB controls the coordinated expression of autophagy and lysosomal genes and is negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1). To further investigate the mechanism of autophagy-lysosomal pathway dysfunction in neuronopathic GD, we examined mTORC1 kinase activity in GD iPSC neuronal progenitors and differentiated neurons. We found that mTORC1 is hyperactive in GD cells as evidenced by increased phosphorylation of its downstream protein substrates. We also found that pharmacological inhibition of glucosylceramide synthase enzyme reversed mTORC1 hyperactivation, suggesting that increased mTORC1 activity is mediated by the abnormal accumulation of glycosphingolipids in the mutant cells. Treatment with the mTOR inhibitor Torin1 upregulated lysosomal biogenesis and enhanced autophagic clearance in GD neurons, confirming that lysosomal dysfunction is mediated by mTOR hyperactivation. Further analysis demonstrated that increased TFEB phosphorylation by mTORC1 results in decreased TFEB stability in GD cells. Our study uncovers a new mechanism contributing to autophagy-lysosomal pathway dysfunction in GD, and identifies the mTOR complex as a potential therapeutic target for treatment of GBA1-associated neurodegeneration.

High-Throughput Ca2+ Flux Assay To Monitor Cyclic Nucleotide-Gated Channel Activity and Characterize Achromatopsia Mutant Channel Function.

Cone photoreceptor cyclic-nucleotide gated channels (CNG) are tetrameric proteins composed of subunits from CNGA3 and CNGB3. These channels transduce light information into electrical signals carried by both Na+ and Ca2+ ions. More than 100 mutations in the CNGA3 gene are associated with the inherited retinal disorder, achromatopsia 2 (ACHM2), which results in attenuation or loss of color vision, daylight blindness, and reduced visual acuity. Classical techniques to measure CNG channel function utilize patch clamp electrophysiology measuring Na currents in the absence of divalent cations, yet intracellular Ca2+ regulates both light and dark adaptation in photoreceptors. We developed a fluorescence-based, high-throughput Ca2+ flux assay using yellow fluorescent protein (YFP) tagged CNGA3 channels expressed in HEK293 cells which allow monitoring for folding defects in mutant channels. The cell permeant cGMP analog, 8-(4-chlorophenylthio)-cGMP (CPT-cGMP), was used to activate Ca2+ flux. The assay was validated using wild-type CNGA3 homomeric and heteromeric channels and ACHM2-associated homomeric mutant CNG channels, CNGA3-R427C, CNGA3-E590K, and CNGA3-L633P. Additionally, we examined two naturally occurring canine mutations causing day-blindness previously studied by patch clamp. We compared the CPT-cGMP K0.5 values of the channels with patch clamp values from previous studies. The assay provides a screen for modulation of gating and/or rescue of trafficking and/or misfolding defects in ACHM2-associated CNG channels. Importantly, the calcium flux assay is advantageous compared to patch clamp as it allows the ability to monitor CNG channel activity in the presence of calcium.

Identification of Novel Inhibitors of DLK Palmitoylation and Signaling by High Content Screening.

After axonal insult and injury, Dual leucine-zipper kinase (DLK) conveys retrograde pro-degenerative signals to neuronal cell bodies via its downstream target c-Jun N-terminal kinase (JNK). We recently reported that such signals critically require modification of DLK by the fatty acid palmitate, via a process called palmitoylation. Compounds that inhibit DLK palmitoylation could thus reduce neurodegeneration, but identifying such inhibitors requires a suitable assay. Here we report that DLK subcellular localization in non-neuronal cells is highly palmitoylation-dependent and can thus serve as a proxy readout to identify inhibitors of DLK palmitoylation by High Content Screening (HCS). We optimized an HCS assay based on this readout, which showed highly robust performance in a 96-well format. Using this assay we screened a library of 1200 FDA-approved compounds and found that ketoconazole, the compound that most dramatically affected DLK localization in our primary screen, dose-dependently inhibited DLK palmitoylation in follow-up biochemical assays. Moreover, ketoconazole significantly blunted phosphorylation of c-Jun in primary sensory neurons subjected to trophic deprivation, a well known model of DLK-dependent pro-degenerative signaling. Our HCS platform is thus capable of identifying novel inhibitors of DLK palmitoylation and signalling that may have considerable therapeutic potential.

Patient-Derived Phenotypic High-Throughput Assay to Identify Small Molecules Restoring Lysosomal Function in Tay-Sachs Disease.

Tay-Sachs disease is an inherited lysosomal storage disease resulting from mutations in the lysosomal enzyme, ß-hexosaminidase A, and leads to excessive accumulation of GM2 ganglioside. Tay-Sachs patients with the infantile form do not live beyond 2-4 years of age due to rapid, progressive neurodegeneration. Enzyme replacement therapy is not a therapeutic option due to its inability to cross the blood-brain barrier. As an alternative, small molecules identified from high-throughput screening could provide leads suitable for chemical optimization to target the central nervous system. We developed a new high-throughput phenotypic assay utilizing infantile Tay-Sachs patient cells based on disrupted lysosomal calcium signaling as a monitor of diseased phenotype. The assay was validated in a pilot screen on a collection of Food and Drug Administration-approved drugs to identify compounds that could reverse or attenuate the disease. Pyrimethamine, a known pharmacological chaperone of ß-hexosaminidase A, was identified from the primary screen. The mechanism of action of pyrimethamine in reversing the defective lysosomal phenotype was by improving autophagy. This new high-throughput screening assay in patient cells will enable the screening of larger chemical compound collections. Importantly, this approach could lead to identification of new molecular targets previously unknown to impact the disease and accelerate the discovery of new treatments for Tay-Sachs disease.

Targeting CDK9 Reactivates Epigenetically Silenced Genes in Cancer.

Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.

Optimization of Novel Aza-benzimidazolone mGluR2 PAMs with Respect to LLE and PK Properties and Mitigation of CYP TDI.

Investigation of a novel amino-aza-benzimidazolone structural class of positive allosteric modulators (PAMs) of metabotropic glutamate receptor 2 (mGluR2) identified [2.2.2]-bicyclic amine 12 as an intriguing lead structure due to its promising physicochemical properties and lipophilic ligand efficiency (LLE). Further optimization led to chiral amide 18, which exhibited strong in vitro activity and attractive pharmacokinetic (PK) properties. Hypothesis-driven target design identified compound 21 as a potent, highly selective, orally bioavailable mGluR2 PAM, which addressed a CYP time-dependent inhibition (TDI) liability of 18, while maintaining excellent drug-like properties with robust in vivo activity in a clinically validated model of antipsychotic potential.

Ribosome-Templated Azide-Alkyne Cycloadditions: Synthesis of Potent Macrolide Antibiotics by In Situ Click Chemistry.

Over half of all antibiotics target the bacterial ribosome-nature's complex, 2.5 MDa nanomachine responsible for decoding mRNA and synthesizing proteins. Macrolide antibiotics, exemplified by erythromycin, bind the 50S subunit with nM affinity and inhibit protein synthesis by blocking the passage of nascent oligopeptides. Solithromycin (1), a third-generation semisynthetic macrolide discovered by combinatorial copper-catalyzed click chemistry, was synthesized in situ by incubating either E. coli 70S ribosomes or 50S subunits with macrolide-functionalized azide 2 and 3-ethynylaniline (3) precursors. The ribosome-templated in situ click method was expanded from a binary reaction (i.e., one azide and one alkyne) to a six-component reaction (i.e., azide 2 and five alkynes) and ultimately to a 16-component reaction (i.e., azide 2 and 15 alkynes). The extent of triazole formation correlated with ribosome affinity for the anti (1,4)-regioisomers as revealed by measured Kd values. Computational analysis using the site-identification by ligand competitive saturation (SILCS) approach indicated that the relative affinity of the ligands was associated with the alteration of macrolactone+desosamine-ribosome interactions caused by the different alkynes. Protein synthesis inhibition experiments confirmed the mechanism of action. Evaluation of the minimal inhibitory concentrations (MIC) quantified the potency of the in situ click products and demonstrated the efficacy of this method in the triaging and prioritization of potent antibiotics that target the bacterial ribosome. Cell viability assays in human fibroblasts confirmed 2 and four analogues with therapeutic indices for bactericidal activity over in vitro mammalian cytotoxicity as essentially identical to solithromycin (1).

Discovery of 5-aryl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-ones as positive allosteric modulators of metabotropic glutamate subtype-2 (mGlu2) receptors with efficacy in a preclinical model of psychosis.

Optimization of a benzimidazolone template for potency and physical properties revealed 5-aryl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-ones as a key template on which to develop a new series of mGlu2 positive allosteric modulators (PAMs). Systematic investigation of aryl-SAR led to the identification of compound 27 as a potent and highly selective mGlu2 PAM with sufficient pharmacokinetics to advance to preclinical models of psychosis. Gratifyingly, compound 27 showed full efficacy in the PCP- and MK-801-induced hyperlocomotion assay in rats at CSF concentrations consistent with mGlu2 PAM potency.

Dynamic mass redistribution analysis of endogenous ß-adrenergic receptor signaling in neonatal rat cardiac fibroblasts.

Label-free systems for the agnostic assessment of cellular responses to receptor stimulation have been shown to provide a sensitive method to dissect receptor signaling. ß-adenergic receptors (ßAR) are important regulators of normal and pathologic cardiac function and are expressed in cardiomyocytes as well as cardiac fibroblasts, where relatively fewer studies have explored their signaling responses. Using label-free whole cell dynamic mass redistribution (DMR) assays we investigated the response patterns to stimulation of endogenous ßAR in primary neonatal rat cardiac fibroblasts (NRCF). Catecholamine stimulation of the cells induced a negative DMR deflection resulting in a concentration-dependent pharmacological response that was competitively blocked by ßAR blockade and non-competitively blocked by irreversible uncoupling of Gs proteins. Pharmacological profiling of subtype-selective ßAR agonists and antagonists revealed a dominant role of ß2AR in mediating the DMR responses, consistent with the relative expression levels of ß2AR and ß1AR in NRCF. Additionally, ßAR-mediated cAMP generation was assessed via a fluorescence biosensor, revealing similar kinetics between DMR responses and cAMP generation. As such, ßAR-dependent DMR responses were enhanced via inhibition of cAMP degradation, as well as dynamin-mediated receptor internalization. Finally, we assessed G protein-independent ßAR signaling through epidermal growth factor receptor (EGFR). While inhibition of EGFR reduced the DMR response to ßAR stimulation, our results demonstrate that G protein-dependent signaling produces a majority of the biological response to ßAR stimulation in NRCF. Altogether, measurement of DMR responses in primary cardiac fibroblasts provides a sensitive readout for investigating endogenous ßAR signaling via both G protein-dependent and -independent pathways.

Differential effects of the mGluR5 positive allosteric modulator CDPPB in the cortex and striatum following repeated administration.

The glutamatergic hypofunction hypothesis of schizophrenia has led to the development of novel therapeutic strategies modulating NMDA receptor function. One of these strategies targets the activation of the metabotropic glutamate receptor 5 (mGlu5 receptor) using positive allosteric modulators (PAMs). Our goal was to evaluate the potential for repeated administration of the mGlu5 receptor PAM, CDPPB (3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide) (30 mg/kg) to induce tolerance to the anti-psychotic like effect using the amphetamine-induced hyperlocomotion rat model, and to produce receptor desensitization in mGlu5 receptor-enriched brain regions. CDPPB dose dependently reduced the locomotor response to amphetamine when administered acutely, and the same effect was observed following 7-day pre-treatment regime. In addition, 7-day dosing of CDPPB did not affect mGlu5 receptor density in the striatum, nor did it change mGlu5 receptor PAM-induced phosphorylation of NMDA, GluN1 and GluN2b, receptor subunits in striatum compared to the levels measured acutely. In contrast, in the frontal cortex, repeated administration of CDPPB decreased mGlu5 receptor density and resulted in a loss of its ability to increase GluN1 and GluN2b levels. Consistent with a reduction of cortical mGlu5 receptor density and phosphorylation, CDPPB (30 mg/kg) significantly affected sleep architecture as determined by cortical EEG at day one however by the seventh day of dosing all sleep changes were absent. Together these results suggest that the development of tolerance induced by the repeated treatment with the mGlu5 receptor PAM, CDPPB, may depend not only on the system being measured (sleep architecture vs psychostimulant induced hyperactivity), but also on the brain region involved with frontal cortex being a more susceptible region to receptor desensitization and internalization than striatum.

The M1 muscarinic receptor allosteric agonists AC-42 and 1-[1'-(2-methylbenzyl)-1,4'-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one bind to a unique site distinct from the acetylcholine orthosteric site.

Activation of M1 muscarinic receptors occurs through orthosteric and allosteric binding sites. To identify critical residues, site-directed mutagenesis and chimeric receptors were evaluated in functional calcium mobilization assays to compare orthosteric agonists, acetylcholine and xanomeline, M1 allosteric agonists AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride), TBPB (1-[1'-(2-methylbenzyl)-1,4'-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one), and the clozapine metabolite N-desmethylclozapine. A minimal epitope has been defined for AC-42 that comprises the first 45 amino acids, the third extracellular loop, and seventh transmembrane domain (Mol Pharmacol 61:1297-1302, 2002). Using chimeric M1 and M3 receptor constructs, the AC-42 minimal epitope has been extended to also include transmembrane II. Phe77 was identified as a critical residue for maintenance of AC-42 and TBPB agonist activity. In contrast, the functional activity of N-desmethylclozapine did not require Phe77. To further map the binding site of AC-42, TBPB, and N-desmethylclozapine, point mutations previously reported to affect activities of M1 orthosteric agonists and antagonists were studied. Docking into an M1 receptor homology model revealed that AC-42 and TBPB share a similar binding pocket adjacent to the orthosteric binding site at the opposite face of Trp101. In contrast, the activity of N-desmethylclozapine was generally unaffected by the point mutations studied, and the docking indicated that N-desmethylclozapine bound to a site distinct from AC-42 and TBPB overlapping with the orthosteric site. These results suggest that structurally diverse allosteric agonists AC-42, TBPB, and N-desmethylclozapine may interact with different subsets of residues, supporting the hypothesis that M1 receptor activation can occur through at least three different binding domains.

3-Aryl-5-phenoxymethyl-1,3-oxazolidin-2-ones as positive allosteric modulators of mGluR2 for the treatment of schizophrenia: Hit-to-lead efforts.

Hit to lead optimization of (5R)-5-hexyl-3-phenyl-1,3-oxazolidin-2-one as a positive allosteric modulator of mGluR2 is described. Improvements in potency and metabolic stability were achieved through SAR on both ends of the oxazolidinone. An optimized lead compound was found to be brain penetrant and active in a rat ketamine-induced hyperlocomotion model for antipsychotic activity.

Discovery of Oxazolobenzimidazoles as Positive Allosteric Modulators for the mGluR2 Receptor.

Novel oxazolobenzimidazoles are described as potent and selective positive allosteric modulators of the metabotropic glutamate receptor 2. The discovery of this class and optimization of its physical and pharmacokinetic properties led to the identification of potent and orally bioavailable compounds (20 and 21) as advanced leads. Compound 20 (TBPCOB) was shown to have robust activity in a PCP-induced hyperlocomotion model in rat, an assay responsive to clinical antipsychotic treatments for schizophrenia.

Dose-dependent effect of CDPPB, the mGluR5 positive allosteric modulator, on recognition memory is associated with GluR1 and CREB phosphorylation in the prefrontal cortex and hippocampus.

In the search for strategies to treat schizophrenia, attention has focused on enhancing NMDA receptor function. In vitro experiments show that metabotropic glutamate 5 receptor (mGluR5) activation enhances NMDA receptor activity, and in vivo experiments indicate that mGluR5 positive allosteric modulators (PAMs) are effective in preclinical assays measuring antipsychotic potential and cognition. Here we characterized the dose-effect function of CDPPB (3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide), an mGluR5 PAM, on novel object recognition memory in unimpaired Wistar Hannover rats (0, 10 or 30 mg/kg CDPPB) and animals with an MK-801-induced deficit (0, 3, 10, or 30 mg/kg CDPPB). In each experiment compound was given 30 min prior to the first exposure in order to affect acquisition/consolidation of the memory. In both cases, an inverted-U-shaped dose-effect function was observed, with lower doses improving recognition but higher doses having no effect. We then examined the effects of CDPPB (0, 3, 10, or 30 mg/kg) on markers of synaptic plasticity in prefrontal cortex and hippocampus, focusing on the expression and phosphorylation status of proteins involved in NMDA related signaling, including the NMDA receptor subunits NR1 and NR2B, the AMPA receptor subunit GluR1, alphaCa((2+))/CaM dependent Ser-Thr kinases II (alphaCaMKII), and the transcription factor CREB. Expression and phosphorylation of many of these proteins, particularly in the prefrontal cortex, were also characterized by an inverted-U-shaped dose-effect function. Taken together, these findings show that mGluR5 activation enhances NMDA receptor function and markers of neuronal plasticity commensurate with improvements in recognition memory. However, the effects of CDPPB are heavily dependent on dose, with higher doses being ineffective in improving recognition memory and producing downstream effects consistent with heightened NMDA receptor activation. These findings may have important implications for the development of mGluR5 PAMs to treat schizophrenia.

Discovery of GlyT1 inhibitors with improved pharmacokinetic properties.

Glycine transporter 1 (GlyT1) represents a novel target for the treatment of schizophrenia via the potentiation of glutamatergic NMDA receptors. The discovery of 4,4-disubstituted piperidine inhibitors of GlyT1 which exhibit improved pharmacokinetic properties, including oral bioavailability, is discussed.

Discovery of N-{[1-(propylsulfonyl)-4-pyridin-2-ylpiperidin-4-yl]methyl}benzamides as novel, selective and potent GlyT1 inhibitors.

Employing an iterative analogue library approach, novel potent and selective glycine transporter 1 (GlyT1) inhibitors containing a 4-pyridin-2-ylpiperidine sulfonamide have been discovered. These inhibitors are devoid of time-dependent CYP inhibition activity and exhibit improved aqueous solubility versus the corresponding 4-phenylpiperidine analogues.

The behavioral and neurochemical effects of a novel D-amino acid oxidase inhibitor compound 8 [4H-thieno [3,2-b]pyrrole-5-carboxylic acid] and D-serine.

Multiple studies indicate that N-methyl-D-aspartate (NMDA) receptor hypofunction underlies some of the deficits associated with schizophrenia. One approach for improving NMDA receptor function is to enhance occupancy of the glycine modulatory site on the NMDA receptor by increasing the availability of the endogenous coagonists D-serine. Here, we characterized a novel D-amino acid oxidase (DAAO) inhibitor, compound 8 [4H-thieno [3,2-b]pyrrole-5-carboxylic acid] and compared it with D-serine. Compound 8 is a moderately potent inhibitor of human (IC(50), 145 nM) and rat (IC(50), 114 nM) DAAO in vitro. In rats, compound 8 (200 mg/kg) decreased kidney DAAO activity by approximately 96% and brain DAAO activity by approximately 80%. This marked decrease in DAAO activity resulted in a significant (p < 0.001) elevation in both plasma (220% of control) and cerebrospinal fluid (CSF; 175% of control) D-serine concentration. However, compound 8 failed to significantly influence amphetamine-induced psychomotor activity, nucleus accumbens dopamine release, or an MK-801 (dizocilpine maleate)-induced deficit in novel object recognition in rats. In contrast, high doses of D-serine attenuated both amphetamine-induced psychomotor activity and dopamine release and also improved performance in novel object recognition. Behaviorally efficacious doses of D-serine (1280 mg/kg) increased CSF levels of D-serine 40-fold above that achieved by the maximal dose of compound 8. These findings demonstrate that pharmacological inhibition of DAAO significantly increases D-serine concentration in the periphery and central nervous system. However, acute inhibition of DAAO appears not to be sufficient to increase D-serine to concentrations required to produce antipsychotic and cognitive enhancing effects similar to those observed after administration of high doses of exogenous D-serine.