title: High-Throughput Screening Strategy Identifies Allosteric, Covalent Human D-Amino Acid Oxidase Inhibitor.
abstract: 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.
authors: Discovery and Preclinical Research Department, Sunovion Pharmaceuticals, Marlborough, MA, USA Ryan.Terry-Lorenzo@sunovion.com.
authors: Ryan T
authors: RT
authors: Terry-Lorenzo
authors: Genomic Science Laboratories, Sumitomo Dainippon Pharma (DSP), Osaka, Japan.
authors: Keiki
authors: K
authors: Masuda
authors: Genomic Science Laboratories, Sumitomo Dainippon Pharma (DSP), Osaka, Japan.
authors: Kohtaroh
authors: K
authors: Sugao
authors: Discovery and Preclinical Research Department, Sunovion Pharmaceuticals, Marlborough, MA, USA.
authors: Q Kevin
authors: QK
authors: Fang
authors: Discovery and Preclinical Research Department, Sunovion Pharmaceuticals, Marlborough, MA, USA.
authors: Michael A
authors: MA
authors: Orsini
authors: Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell'Insubria, Varese, Italy, and The Protein Factory, Politecnico di Milano and Università degli Studi dell'Insubria, Milano, Italy.
authors: Silvia
authors: S
authors: Sacchi
authors: Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell'Insubria, Varese, Italy, and The Protein Factory, Politecnico di Milano and Università degli Studi dell'Insubria, Milano, Italy.
authors: Loredano
authors: L
authors: Pollegioni
mesh_terms: D000495:Allosteric Site / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D003605:D-Amino-Acid Oxidase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D057166:High-Throughput Screening Assays; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D009437:Neuralgia / Q000188:drug therapy; D012559:Schizophrenia / Q000188:drug therapy*
chemical_list: D002352:Carrier Proteins; C475071:DAOA protein, human; D047908:Intracellular Signaling Peptides and Proteins; D003605:D-Amino-Acid Oxidase
keywords: D-serine; covalent inhibitors; neuropathic pain; schizophrenia