High-Throughput Screening Strategy Identifies Allosteric, Covalent Human D-Amino Acid Oxidase Inhibitor
| Autor: | Silvia Sacchi, Q. Kevin Fang, Kohtaroh Sugao, Ryan T. Terry-Lorenzo, Loredano Pollegioni, Michael A. Orsini, Keiki Masuda |
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| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
D-Amino-Acid Oxidase
High-throughput screening Allosteric regulation D-amino acid oxidase D-serine Biochemistry Analytical Chemistry High-Throughput Screening Assays medicine Humans Gene neuropathic pain Oxidase test covalent inhibitors schizophrenia Chemistry Intracellular Signaling Peptides and Proteins Mechanism of action Drug development Neuralgia Molecular Medicine medicine.symptom Carrier Proteins Allosteric Site Biotechnology |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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