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
复制标题
高通量筛选策略鉴定变构共价人 D-氨基酸氧化酶抑制剂
DOI：
10.1177/1087057115600413
复制
发表时间：
2015
影响因子：
--
通讯作者：
L. Pollegioni
中科院分区：
文献类型：
--
作者：
Ryan T. Terry;K. Masuda;Kohtaroh Sugao;Q. Kevin Fang;M. Orsini;S. Sacchi;L. Pollegioni
全基因组关联研究已经将基因G72的多态与几个人类群体中的精神分裂症风险联系起来。尽管存在争议，但生化实验表明，G72与精神分裂症的机制联系是由于G72蛋白产物pLG72对人类D-氨基酸氧化酶(HDAAO)活性起调节作用。为了寻找新的作用机制，我们设计了一种适合于高通量筛选(HTS)的pLG72导向的hDAAO活性测定方法。在实验过程中，我们证实了pLG72是hDAAO的抑制剂。因此，我们的检测采用了足够高的IC20 pLG72浓度来允许动态的pLG72-hDAAO复合体形成，但在HTS期间具有足够的剩余hDAAO活性来测量。在进行了大约150,000种化合物的HTS之后，我们进一步表征了一类化合物点击，这些化合物在pLG72存在时hDAAO抑制剂的效力较低。主要集中在化合物2[2-(2，5-dimethylphenyl)-6-fluorobenzo[d]isothiazol-3(2H)-on]，，我们证明了这些化合物通过变构共价机制抑制hDAAO。虽然人们对化合物2及其类似物的治疗潜力非常感兴趣，但在治疗药物开发过程中，需要解决它们对还原剂的敏感性以及它们与半胱氨酸共价结合的能力。
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.