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Introduction SEP-432 is a triple monoamine reuptake inhibitor of norepinephrine (NE), serotonin (5-HT), and dopamine (DA), based on in vitro binding studies. We sought evidence that SEP-432 engages these monoamine systems by measuring concentrations of monoamines and/or their main metabolites in cerebrospinal fluid (CSF) and plasma and comparing results to duloxetine, a dual reuptake inhibitor of NE and 5-HT. Methods Eighteen healthy normal subjects received either SEP-432 (300 mg/day), duloxetine (60 mg/day), or placebo for 14 days in-clinic (double blind) with CSF and plasma collections at baseline (single lumbar puncture) and Day 14 (24-h CSF and plasma collection). Concentrations of monoamines and their metabolites, as well as pharmacokinetic concentrations of SEP-432 and metabolite, were quantified by liquid chromatography-tandem mass spectrometry. Results Compared to placebo in the Day 14 area under the curve 24-h (AUC0-24 h) analysis, SEP-432 significantly ( P < 0.05) decreased the NE metabolite dihydroxyphenylglycol (DHPG) in CSF and plasma, decreased 5-HT in plasma, and did not affect DA metabolites, while duloxetine had significant effects on DHPG and 5-HT. Time-matched baseline to Day 14 biomarker comparisons confirmed these findings. Conclusion CSF monoamine biomarkers confirmed central NET activity for SEP-432 and duloxetine's dual reuptake inhibition. [ABSTRACT FROM AUTHOR]

Pteridine reductase (PTR1) is an NADPH-dependent short-chain reductase found in parasitic trypanosomatid protozoans. The enzyme participates in the salvage of pterins and represents a target for the development of improved therapies for infections caused by these parasites. A series of crystallographic analyses of Leishmania major PTR1 are reported. Structures of the enzyme in a binary complex with the cofactor NADPH, and ternary complexes with cofactor and biopterin, 5,6-dihydrobiopterin, and 5,6,7,8-tetrahydrobiopterin reveal that PTR1 does not undergo any major conformational changes to accomplish binding and processing of substrates, and confirm that these molecules bind in a single orientation at the catalytic center suitable for two distinct reductions. Ternary complexes with cofactor and CB3717 and trimethoprim (TOP), potent inhibitors of thymidylate synthase and dihydrofolate reductase, respectively, have been characterized. The structure with CB3717 reveals that the quinazoline moiety binds in similar fashion to the pterin substrates/products and dominates interactions with the enzyme. In the complex with TOP, steric restrictions enforced on the trimethoxyphenyl substituent prevent the 2,4-diaminopyrimidine moiety from adopting the pterin mode of binding observed in dihydrofolate reductase, and explain the inhibition properties of a range of pyrimidine derivates. The molecular detail provided by these complex structures identifies the important interactions necessary to assist the structure-based development of novel enzyme inhibitors of potential therapeutic value. [Copyright &y& Elsevier]

Many genetic (gene deletion, interruption or mutation), epigenetic (such as antisense or small interfering RNA) and immunological methods are being applied in ‘high-throughput target validation’ studies of the novel potential targets arising from whole genome sequencing. Such applications often focus on ‘loss of function’ approaches. However, target validation is most reliable when multiple orthogonal approaches are used. Initiating a target-based discovery project based on correlative evidence is faster than awaiting causative evidence. Indeed, the multiple tools needed to generate firm proof usually include methods and reagents only generated after starting a discovery project with little evidence beyond correlations. Robust and rigorous tests of whether a drug candidate is efficacious in vivo because of its effects on a specific molecular particular target are best made by simultaneously applying multiple orthogonal tools. Examples of the orthogonal tools approach will be discussed. [Copyright &y& Elsevier]

Describes the findings of a study conducted on the electrostatic guidance of catalysis by a conserved Glu58 in thymidylate synthase in Escherichia coli and dCMP hydroxymethylase in T4 bacteriophage.

Investigates the kinetic and equilibrium alpha-secondary tritium isotope effects on reactions catalyzed by deoxycytidylate (dCMP) hydroxymethylase (CH) from bacteriophage T4. Nucleotide analog 5-fluorodeoxuridylate's (FdUMP) inactivation of CH; FdUMP complexes formed.

Discusses the importance of genetic approaches to structure-function studies on the bacteriophage T4 lysozyme. Description of the T4 lysozyme; Biology and history; Systematic mutation; Functional importance and identification of conserved residues; Groups of residues; Role of hydrophobic strip residues; Catalytic role of Asp-20; Implications on the enzyme's catalytic machinery.

To interrogate whether altered function of the hippocampal-mPFC circuit underlies the deficit in fear extinction recall in rats subjected to single-prolonged stress (SPS), changes in brain region-specific metabolic rate were measured in male rats (control and SPS treated). Brain region metabolic rates were quantified using uptake of 14C-2-deoxyglucose (14C-2DG) during fear memory formation, fear memory extinction and extinction recall. Control and SPS rats had similar regional brain activities at baseline. During extinction recall, 14C-2DG uptake decreased in hippocampal regions in control rats, but not in SPS rats. SPS rats also exhibited a significant deficiency in fear extinction recall, replicating a previously reported finding. Reduced hippocampal activity during fear extinction recall in control animals may reflect reduction in fear overgeneralization, thereby enabling discrimination between distinct contexts. In contrast, persistent levels of hippocampal activity in SPS-exposed male animals during fear extinction recall may reflect the dysfunctional persistence of fear overgeneralization. Future studies in females can test gender-specificity of these effects, with appropriate attention to luteal dependent effects on extinction of fear learning. Detailed knowledge of regional brain activities underlying stress-induced deficits in extinction recall may help identify therapeutic targets in PTSD. [ABSTRACT FROM AUTHOR]

Reports on the parameters affecting the restoration of activity to inactive mutants of thymidylate synthase (TS) via subunit exchange. Effect of urea on the restoration of TS activity; Effect of temperature on the restoration of activity; Effect of pH on the restoration of TS activity; Effect of ethanol, ethylene glycol and glycerol.

Inhibitors of dopamine transporters (DAT), norepinephrine transporters (NET) and serotonin transporters (SERT) are effective treatments for neuropsychiatric diseases. Dasotraline [(1R,4 S)− 4-(3,4-dichlorophenyl)− 1,2,3,4-tetrahydro-1-naphthalenamine, also known as SEP-225289) was evaluated for its inhibitory potency at DAT, NET and SERT using in vitro and in vivo assays. In vitro radiometric functional uptake studies showed preferential inhibition by dasotraline of h DAT (IC 50 =3 nM) and h NET (IC 50 =4 nM relative to h SERT(IC 50 =15 nM). In mouse ex vivo occupancy studies, dasotraline demonstrated total plasma concentration-dependent occupancy at DAT, NET and SERT. Determination of the TO 50 (50% transporter occupancy) were 32, 109 and 276 ng/ml, respectively. In SPECT imaging studies in baboons, dasotraline (0.2 mg/kg iv) displaced radiotracer binding to DAT by 87% but only 20% at NET and SERT. Rat microdialysis studies were performed in prefrontal cortex and striatum. Dasotraline produced sustained (>4 h) increases in dopamine and norepinephrine concentrations. Dasotraline was also more potent at increasing synaptic dopamine in the striatum, and norepinephrine in the prefrontal cortex than serotonin in these regions. In summary, dasotraline preferentially inhibits DAT and NET relative to SERT. Together, the occupancy and neurochemical profile of dasotraline provide a mechanistic basis for the treatment of diseases that have an underlying causality involving dopamine and norepinephrine dysfunction. [ABSTRACT FROM AUTHOR]

The NMDAR (N-methyl-D-aspartate receptor) is a central regulator of synaptic plasticity and learning and memory. hDAAO (human D-amino acid oxidase) indirectly reduces NMDAR activity by degrading the NMDAR co-agonist D-serine. Since NMDAR hypofunction is thought to be a foundational defect in schizophrenia, hDAAO inhibitors have potential as treatments for schizophrenia and other nervous system disorders. Here, we sought to identify novel chemicals that inhibit hDAAO activity. We used computational tools to design a focused, purchasable library of compounds. After screening this library for hDAAO inhibition, we identified the structurally novel compound, 'compound 2' [3-(7-hydroxy-2-oxo-4-phenyl-2H-chromen-6-yl)propanoic acid], which displayed low nM hDAAO inhibitory potency (Ki =7 nM). Although the library was expected to enrich for compounds that were competitive for both D-serine and FAD, compound 2 actually was FAD uncompetitive, much like canonical hDAAO inhibitors such as benzoic acid. Compound 2 and an analog were independently co-crystalized with hDAAO. These compounds stabilized a novel conformation of hDAAO in which the active-site lid was in an open position. These results confirm previous hypotheses regarding active-site lid flexibility of mammalian D-amino acid oxidases and could assist in the design of the next generation of hDAAO inhibitors. [ABSTRACT FROM AUTHOR]

We characterized the mechanism andpharmacodynamics of five structurallydistinct inhibitors of d-amino acid oxidase. All inhibitorsbound the oxidized form of human enzyme with affinity slightly higherthan that of benzoate (Kd≈ 2–4μM). Stopped-flow experiments showed that pyrrole-based inhibitorspossessed high affinity (Kd≈ 100–200nM) and slow release kinetics (k< 0.01 s–1) in the presence of substrate, while inhibitors withpendent aromatic groups altered conformations of the active site lid,as evidenced by X-ray crystallography, and showed slower kineticsof association. Rigid bioisosteres of benzoic acid induced a closed-lidconformation, had slower release in the presence of substrate, andwere more potent than benzoate. Steady-state d-serine concentrationswere described in a PK/PD model, and competition for d-serinesites on NMDA receptors was demonstrated in vivo. DAAO inhibitionincreased the spatiotemporal influence of glial-derived d-serine, suggesting localized effects on neuronal circuits whereDAAO can exert a neuromodulatory role. [ABSTRACT FROM AUTHOR]

Abstract: The current work discloses a novel cyclohexylarylamine chemotype with potent inhibition of the serotonin, norepinephrine, and dopamine transporters and potential for treatment of major depressive disorder. Optimized compounds 1 (SERT, NET, DAT, IC50 =169, 85, 21nM) and 42 (SERT, NET, DAT IC50 =34, 295, 90nM) were highly brain penetrant, active in vivo in the mouse tail suspension test at 30mpk po and were not general motor stimulants. [Copyright &y& Elsevier]

Abstract: Novel chiral cyclohexylaryl amines were developed with potent reuptake inhibition against the serotonin, norepinephrine and dopamine transporters and activity at 10 and 30mpk PO in the mouse tail suspension test. Prototype compound 31 (SERT, NET, DAT IC50 ⩽1, 21, 28nM) was highly brain penetrant, had minimal CYP and hERG inhibition, and represents a previously undisclosed architecture with potential for treatment of major depressive disorder. [Copyright &y& Elsevier]

Abstract: The present work describes a series of novel tetrahydroquinoline amines that potently inhibit the in vitro reuptake of serotonin and dopamine (dual reuptake inhibitors). The compounds are structurally related to a series we disclosed previously, but are improved with respect to cytochrome P-450 enzyme (CYP) and potassium ion channel Kv11.1 (hERG) inhibition and synthetic accessibility. The detailed synthesis and in vitro activity and ADME profile of the compounds is described, which represent a previously undisclosed dual reuptake inhibitor chemotype. [Copyright &y& Elsevier]

Abstract: The present work describes a series of novel chiral amines that potently inhibit the in vitro reuptake of serotonin, norepinephrine and dopamine (triple reuptake inhibitors) and were active in vivo in a mouse model predictive of antidepressant like activity. The detailed synthesis and in vitro activity and ADME profile of compounds is described, which represent a previously undisclosed triple reuptake inhibitor chemotype. [ABSTRACT FROM AUTHOR]

Abstract: Trypanosoma cruzi, a protozoan parasite, is the causative agent of Chagas disease, a major cause of cardiovascular disease in many Latin American countries. There is an urgent need to develop an improved therapy due to the toxicity of existing drugs and emerging drug resistance. Cruzain, the primary cysteine protease of T. cruzi, is essential for the survival of the parasite in host cells and therefore is an important target for the development of inhibitors as potential therapeutics. A novel series of α-ketoamide-, α-ketoacid-, α-ketoester-, and aldehyde-based inhibitors of cruzain has been developed. The inhibitors were identified by screening protease targeted small molecule libraries and systematically optimizing the P1, P2, P3, and P1′ residues using specific structure-guided methods. A total of 20 compounds displayed picomolar potency in in vitro assays and three inhibitors representing different α-keto-based inhibitor scaffolds demonstrated anti-trypanosomal activity in cell culture. A 2.3Å crystallographic structure of cruzain bound with one of the α-ketoester analogs is also reported. The structure and kinetic assay data illustrate the covalent binding, reversible inhibition mechanism of the inhibitor. Information on the compounds reported here will be useful in the development of new lead compounds as potential therapeutic agents for the treatment of Chagas disease and as biological probes to study the role that cruzain plays in the pathology. This study also demonstrates the validity of structure-guided approaches to focused library design and lead compound optimization. [Copyright &y& Elsevier]

Pteridine reductase (PTR1) is a short-chain reductase (SDR) responsible for the salvage of pterins in parasitic trypanosomatids. PTR1 catalyzes the NADPH-dependent two-step reduction of oxidized pterins to the active tetrahydro-forms and reduces susceptibility to antifolates by alleviating dihydrofolate reductase (DHFR) inhibition. Crystal structures of PTR1 complexed with cofactor and 7,8-dihydrobiopterin (DHB) or methotrexate (MTX) delineate the enzyme mechanism, broad spectrum of activity and inhibition by substrate or an antifolate. PTR1 applies two distinct reductive mechanisms to substrates bound in one orientation. The first reduction uses the generic SDR mechanism, whereas the second shares similarities with the mechanism proposed for DHFR. Both DHB and MTX form extensive hydrogen bonding networks with NADP(H) but differ in the orientation of the pteridine. [ABSTRACT FROM AUTHOR]

Provides information on a study giving stereochemical and kinetic evidence that Leishmania major Pteridine reductase 1 belongs to the short chain dehydrogenase family. Methodology and materials used to conduct the study; Results of the study; Discussion on the results.