Merck
  • Home
  • Search Results
  • High-Throughput Screening Strategy Identifies Allosteric, Covalent Human D-Amino Acid Oxidase Inhibitor.

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

Journal of biomolecular screening (2015-08-25)
Ryan T Terry-Lorenzo, Keiki Masuda, Kohtaroh Sugao, Q Kevin Fang, Michael A Orsini, Silvia Sacchi, Loredano Pollegioni
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium chloride solution, 0.85%
Sigma-Aldrich
Sodium chloride-35Cl, 99 atom % 35Cl
Sigma-Aldrich
Sodium chloride, BioUltra, for molecular biology, ≥99.5% (AT)
Sigma-Aldrich
Hydrogen peroxide solution, 34.5-36.5%
Sigma-Aldrich
Sodium chloride solution, BioUltra, for molecular biology, ~5 M in H2O
Sigma-Aldrich
Sodium chloride, BioXtra, ≥99.5% (AT)
Sigma-Aldrich
Sodium chloride solution, 0.9% in water, BioXtra, suitable for cell culture
Sigma-Aldrich
Sodium chloride solution, 5 M in H2O, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
Sodium chloride, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
Sigma-Aldrich
Sodium chloride, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99%
Sigma-Aldrich
Hydrogen peroxide solution, purum p.a., ≥35% (RT)
Sigma-Aldrich
Hydrogen peroxide solution, contains ~200 ppm acetanilide as stabilizer, 3 wt. % in H2O
Sigma-Aldrich
Hydrogen peroxide solution, 50 wt. % in H2O, stabilized
Sigma-Aldrich
Hydrogen peroxide solution, contains inhibitor, 30 wt. % in H2O, ACS reagent
Sigma-Aldrich
Hydrogen peroxide solution, contains inhibitor, 35 wt. % in H2O
Sigma-Aldrich
Hydrogen peroxide solution, contains inhibitor, 30 wt. % in H2O, meets USP testing specifications
SAFC
Sodium chloride solution, 5 M
Sigma-Aldrich
Resorufin, 95%
Sigma-Aldrich
Hydrogen peroxide solution, 30 % (w/w) in H2O, contains stabilizer
Sigma-Aldrich
Potassium sodium tartrate solution, BioUltra, 1.5 M in H2O
Sigma-Aldrich
Dimethyl sulfoxide, meets EP testing specifications, meets USP testing specifications
Sigma-Aldrich
Dimethyl sulfoxide, Hybri-Max, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7%
Sigma-Aldrich
Dimethyl sulfoxide, ≥99.5% (GC), suitable for plant cell culture
Sigma-Aldrich
Dimethyl sulfoxide, for molecular biology
Sigma-Aldrich
Dimethyl sulfoxide, anhydrous, ≥99.9%
Sigma-Aldrich
8-Octanoyloxypyrene-1,3,6-trisulfonic acid trisodium salt, suitable for fluorescence, ≥90% (HPCE)
Sigma-Aldrich
Dimethyl sulfoxide, PCR Reagent
Sigma-Aldrich
L-Glutathione reduced, ≥98.0%
Sigma-Aldrich
L-Glutathione reduced, BioXtra, ≥98.0%
Sigma-Aldrich
L-Glutathione reduced, suitable for cell culture, BioReagent, ≥98.0%, powder