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

Genome-wide association studies have linked polymorphisms in the gene G72 to schizophrenia risk in several human populations. Although controversial, biochemical experiments have suggested that the mechanistic link of G72 to schizophrenia is due to the G72 protein product, pLG72, exerting a regulatory effect on human D-amino acid oxidase (hDAAO) activity. In an effort to identify hDAAO inhibitors of novel mechanism of action, we designed a pLG72-directed hDAAO activity assay suitable for high-throughput screening (HTS). During assay development, we confirmed that pLG72 was an inhibitor of hDAAO. Thus, our assay employed an IC20 pLG72 concentration that was high enough to allow dynamic pLG72-hDAAO complexes to form but with sufficient remaining hDAAO activity to measure during an HTS. After conducting an approximately 150,000-compound HTS, we further characterized a class of compound hits that were less potent hDAAO inhibitors when pLG72 was present. Focusing primarily on compound 2: [2-(2,5-dimethylphenyl)-6-fluorobenzo[d]isothiazol-3(2H)-on], we demonstrated that these compounds inhibited hDAAO via an allosteric, covalent mechanism. Although there is significant interest in the therapeutic potential of compound 2: and its analogues, their sensitivity to reducing agents and their capacity to bind cysteines covalently would need to be addressed during therapeutic drug development.

https://journals.sagepub.com/doi/pdf/10.1177/1087057115600413

High-Throughput Screening Strategy Identifies Allosteric, Covalent Human D-Amino Acid Oxidase Inhibitor.

A practical and mild method for the 5-exo cyclization of N-substituted α-alkynyl hydroxamic acids into isoxazolidin-3-ones catalyzed by Au(PPh3)SbF6 was developed. Under optimized conditions, the cyclization shows a high Z-selectivity for the newly formed exocyclic double bond.

Gold(I)-catalyzed heterocyclization of β -alkynyl hydroxamic acids: synthesis of isoxazolidin-3-ones

Schizophrenia is a psychiatric disorder affecting about 1% of the world's population and manifests itself as positive symptoms (eg. hallucinations), negative symptoms (eg. social withdrawal) in conjunction with cognitive impairments (eg. working memory). Evidence suggests that schizophrenia is, in part, a heritable disease. Candidate susceptibility genes implicate the glutamatergic neurotransmitter system, reinforcing clinical observations of N-methyl-D-aspartate receptor (NMDAR) hypofunction in schizophrenia. One of those genes, D-amino acid oxidase (DAO), is genetically associated with schizophrenia and its function includes degradation of D-serine, the NMDAR co-agonist. DAO is a candidate gene of high interest due to schizophrenic patients' manifestation of reduced D-serine levels, increased DAO expression and increased DAO activity.
Despite mounting evidence for DAO involvement in schizophrenia, its regulation is poorly understood. Characterization of DAO may lead to a more thorough understanding of its function and biology and ultimately to the identification of novel targets for this disorder. To this end, DAO-specific antibodies were utilized to identify DAO-interacting proteins through co-immunoprecipitation from rat cerebellum, where DAO expression is especially high. Subsequent mass spectroscopy analysis of associating proteins yielded twenty-four putative DAO interactors. The most abundant and interesting interactors include known presynaptic active zone members such as bassoon (BSN) and piccolo (PCLO). The DAO interaction with BSN was confirmed through co-immunoprecipitation and both proteins were shown to localize in the presynaptic junction. These data suggest that BSN is a novel DAO interactor and defines a previously unappreciated localization of DAO perhaps as a result of a physiologically important interaction with BSN. Furthermore, BSN was found to partially inhibit DAO enzymatic activity in transiently transfected human embryonic kidney (Hek293) cells. Collectively these novel findings suggest that synaptic D-serine concentration may be under tight regulation by BSN via proximally localized DAO. DAO may thus play a role in modulating the functions of the presynaptic active zone via its interaction with BSN.

Characterization of the D-amino acid oxidase interactome

Methods for increasing D-Serine concentration and reducing concentration of the toxic products of D- Serine oxidation, for enhancing learning, memory and/or cognition, or for treating schizophrenia, Alzheimer's disease, ataxia, or neuropathic pain, or preventing loss in neuronal function characteristic of neurodegenerative diseases involve administering to a subject in need of treatment a therapeutic amount of a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof: I wherein Z1 is N or CR3; Z2 is N or CR4; Z3 is O or S; A is hydrogen, alkyl or M+; M is aluminum, calcium, lithium, magnesium, potassium, sodium, zinc or a mixture thereof; R1, R2, R3 and R4 are independently selected from hydrogen, alkyl, hydroxy alkoxy, aryl, acyl, halo, cyano, haloalkyl, NHCOOR5 and SO2NH2; R5 is aryl, arylalkyl, heteroaryl or heteroarylalkyl; at least one of R1, R2, R3 and R4 is other than hydrogen; and at least one of Z1 and Z2 is other than N.

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Benzo[d]isoxazol-3-ol daao inhibitors