D-Amino acid oxidase (DAAO) is an FAD-containing flavoenzyme that catalyzes with absolute stereoselectivity the oxidative deamination of all natural D-amino acids, the only exception being the acidic ones. This flavoenzyme plays different roles during evolution and in different tissues in humans. Its three-dimensional structure is well conserved during evolution: minute changes are responsible for the functional differences between enzymes from microorganism sources and those from humans. In recent years several investigations focused on human DAAO, mainly because of its role in degrading the neuromodulator D-serine in the central nervous system. D-Serine is the main coagonist of N-methyl D-aspartate receptors, i.e., excitatory amino acid receptors critically involved in main brain functions and pathologic conditions. Human DAAO possesses a weak interaction with the FAD cofactor; thus, in vivo it should be largely present in the inactive, apoprotein form. Binding of active-site ligands and the substrate stabilizes flavin binding, thus pushing the acquisition of catalytic competence. Interestingly, the kinetic efficiency of the enzyme on D-serine is very low. Human DAAO interacts with various proteins, in this way modulating its activity, targeting, and cell stability. The known properties of human DAAO suggest that its activity must be finely tuned to fulfill a main physiological function such as the control of D-serine levels in the brain. At present, studies are focusing on the epigenetic modulation of human DAAO expression and the role of post-translational modifications on its main biochemical properties at the cellular level.

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Human D-Amino Acid Oxidase: Structure, Function, and Regulation

Paeoniflorin, the main component of Xiaoyao Wan, presents low oral bioavailability and unclear antidepressant mechanism. To elucidate the potential reasons for the low bioavailability of paeoniflorin and explore its antidepressant mechanism from the perspective of the gut microbiota, here, a chronic unpredictable depression model and forced swimming test were firstly performed to examine the antidepressant effects of paeoniflorin. Then the pharmacokinetic study of paeoniflorin in rats was performed based on the gut microbiota; meanwhile, the gut microbiota incubated with paeoniflorin in vitro was used to identify the possible metabolites of paeoniflorin. Molecular virtual docking experiments together with the specific inhibitor tests were applied to investigate the mechanism of paeoniflorin metabolism by the gut microbiota. Finally, the intestinal microbiota composition was analyzed by 16S rRNA gene sequencing technology. The pharmacodynamics tests showed that paeoniflorin had significant antidepressant activity, but its oral bioavailability was 2.32%. Interestingly, we found paeoniflorin was converted into benzoic acid by the gut microbiota, and was mainly excreted through the urine with the gut metabolite benzoic acid as the prominent excreted form. Moreover, paeoniflorin could also regulate the composition of the gut microbiota by increasing the abundance of probiotics. Therefore, the metabolism effect of gut microbiota may be one of the main reasons for the low oral bioavailability of paeoniflorin. Additionally, paeoniflorin can be metabolized into benzoic acid via gut microbiota enzymes, which might exert antidepressant effects through the blood-brain barrier into the brain.

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Gut Microbiota-Based Pharmacokinetics and the Antidepressant Mechanism of Paeoniflorin.

One aspect of the present invention relates to a method of treating of drug addiction or drug dependence in a mammal, comprising the step of administering to a mammal in need thereof a therapuetically effective amount of a heterocyclic compound, e.g., a 3-substituted piperidine. In a preferred embodiment, the method of the present invention treats cocaine addiction or methamphetamine addiction.

Method of treating addiction or dependence using a ligand for a monoamine receptor or transporter

D-aspartate levels in the brain are regulated by the catabolic enzyme D-aspartate oxidase (DDO). D-aspartate activates NMDA receptors, and influences brain connectivity and behaviors relevant to schizophrenia in animal models. In addition, recent evidence reported a significant reduction of D-aspartate levels in the post-mortem brain of schizophrenia-affected patients, associated to higher DDO activity. In the present work, microdialysis experiments in freely moving mice revealed that exogenously administered D-aspartate efficiently cross the blood brain barrier and stimulates L-glutamate efflux in the prefrontal cortex (PFC). Consistently, D-aspartate was able to evoke L-glutamate release in a preparation of cortical synaptosomes through presynaptic stimulation of NMDA, mGlu5 and AMPA/kainate receptors. In support of a potential therapeutic relevance of D-aspartate metabolism in schizophrenia, in vitro enzymatic assays revealed that the second-generation antipsychotic olanzapine, differently to clozapine, chlorpromazine, haloperidol, bupropion, fluoxetine and amitriptyline, inhibits the human DDO activity. In line with in vitro evidence, chronic systemic administration of olanzapine induces a significant extracellular release of D-aspartate and L-glutamate in the PFC of freely moving mice, which is suppressed in Ddo knockout animals. These results suggest that the second-generation antipsychotic olanzapine, through the inhibition of DDO activity, increases L-glutamate release in the PFC of treated mice.

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Olanzapine, but not clozapine, increases glutamate release in the prefrontal cortex of freely moving mice by inhibiting D-aspartate oxidase activity

d-Amino-acid oxidase (DAO) catalyzes the oxidative deamination of d-amino acids. DAO is present in a wide variety of organisms and has important roles. Here, we review the distribution and physiological substrates of mouse DAO. Mouse DAO is present in the kidney, brain, and spinal cord, like DAOs in other mammals. However, in contrast to other animals, it is not present in the mouse liver. Recently, DAO has been detected in the neutrophils, retina, and small intestine in mice. To determine the physiological substrates of mouse DAO, mutant mice lacking DAO activity are helpful. As DAO has wide substrate specificity and degrades various d-amino acids, many d-amino acids accumulate in the tissues and body fluids of the mutant mice. These amino acids are d-methionine, d-alanine, d-serine, d-leucine, d-proline, d-phenylalanine, d-tyrosine, and d-citrulline. Even in wild-type mice, administration of DAO inhibitors elevates D-serine levels in the plasma and brain. Among the above d-amino acids, the main physiological substrates of mouse DAO are d-alanine and d-serine. These two d-amino acids are most abundant in the tissues and body fluids of mice. d-Alanine derives from bacteria and produces bactericidal reactive oxygen species by the action of DAO. d-Serine is synthesized by serine racemase and is present especially in the central nervous system, where it serves as a neuromodulator. DAO is responsible for the metabolism of d-serine. Since DAO has been implicated in the etiology of neuropsychiatric diseases, mouse DAO has been used as a representative model. Recent reports, however, suggest that mouse DAO is different from human DAO with respect to important properties.

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Mouse d-Amino-Acid Oxidase: Distribution and Physiological Substrates.

We characterized the mechanism and pharmacodynamics of five structurally distinct inhibitors of d-amino acid oxidase. All inhibitors bound the oxidized form of human enzyme with affinity slightly higher than that of benzoate (Kd ≈ 2–4 μM). Stopped-flow experiments showed that pyrrole-based inhibitors possessed high affinity (Kd ≈ 100–200 nM) and slow release kinetics (k < 0.01 s–1) in the presence of substrate, while inhibitors with pendent aromatic groups altered conformations of the active site lid, as evidenced by X-ray crystallography, and showed slower kinetics of association. Rigid bioisosteres of benzoic acid induced a closed-lid conformation, had slower release in the presence of substrate, and were more potent than benzoate. Steady-state d-serine concentrations were described in a PK/PD model, and competition for d-serine sites on NMDA receptors was demonstrated in vivo. DAAO inhibition increased the spatiotemporal influence of glial-derived d-serine, suggesting localized effects on neuronal circ...

Structural, Kinetic, and Pharmacodynamic Mechanisms of d-Amino Acid Oxidase Inhibition by Small Molecules

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.

Novel human D-amino acid oxidase inhibitors stabilize an active-site lid-open conformation.

Inhibition of d-amino acid oxidase (DAAO) activity is a potential target for the treatment of chronic pain. Here we characterized the effects of systemic administration of the DAAO inhibitor 4H-furo[3,2-b]pyrrole-5-carboxylic acid (SUN) in rat models of neuropathic and inflammatory pain. Oral administration of SUN dose dependently attenuated tactile allodynia induced by ligation of the L5 spinal nerve (SNL) and similarly reversed thermal hyperalgesia produced by chronic constriction injury. In addition, SUN was efficacious against complete Freund's adjuvant-induced thermal hyperalgesia. In these models, maximal reversal of pain-related behaviors corresponded with maximum rates of increase in brain and plasma d-serine concentrations, indicative of full inhibition of DAAO activity. To investigate the possible site(s) of action, we recorded spontaneous nerve activity and mechanically evoked responses of central spinal cord dorsal horn neurons and compared these with spontaneous activity of peripheral dorsal root filaments in anesthetized SNL model animals. Oral SUN reduced spontaneous activity in both central and peripheral recordings at doses and pretreatment times that corresponded to reduced mechanical allodynia in behavioral experiments. After intravenous administration of SUN, the onset of action for this central effect was rapid (maximal effects within 30 minutes), but was abolished by severing afferent inputs to the dorsal horn. Overall, these results indicate that inhibition of DAAO in peripheral afferent spinal circuits reduced spontaneous neuronal activity to attenuate pain-related behaviors in rat models of neuropathic and inflammatory pain.

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Pharmacodynamic Effects of a d-Amino Acid Oxidase Inhibitor Indicate a Spinal Site of Action in Rat Models of Neuropathic Pain

One aspect of the present invention relates to methods of synthesizing substituted piperidines. A second aspect of the present invention relates to stereoselective methods of synthesizing substituted piperidines. The methods of the present invention will find use in the synthesis of compounds useful for treatment of numerous ailments, conditions and diseases that afflict mammals, including but not limited to addiction and pain. An additional aspect of the present invention relates to the synthesis of combinatorial libraries of the substituted piperidines using the methods of the present invention. An additional aspect of the present invention relates to enantiomerically substituted pyrrolidines, piperidines, and azepines.

Michelle L. R. Heffernan

Papers

Structural, Kinetic, and Pharmacodynamic Mechanisms of d-Amino Acid Oxidase Inhibition by Small Molecules

Novel human D-amino acid oxidase inhibitors stabilize an active-site lid-open conformation.

Pharmacodynamic Effects of a d-Amino Acid Oxidase Inhibitor Indicate a Spinal Site of Action in Rat Models of Neuropathic Pain

Methods for the stereoselective synthesis of substituted piperidines