Generic Fluorescent HTS Assay for Oxidoreductases

描述(由申请人提供)：在治疗干预中，对催化氧化和还原反应的酶有很高的药学兴趣。经过验证的氧化还原酶靶标包括HMG-CoA还原酶和51-还原酶，HMG-CoA还原酶是立普妥等用于治疗高胆固醇血症的他汀类药物的靶标，51-还原酶是用于治疗良性前列腺增生的药物的靶标。这类新出现的靶点包括在低氧条件下生长的肿瘤细胞中的糖酵解脱氢酶，以及微生物和寄生虫病原体中的糖酵解脱氢酶。然而，由于缺乏适用于制药高通量筛选(HTS)实验室的可靠、通用的酶分析方法，氧化还原酶新抑制剂的鉴定工作正在放缓。为了消除这一技术障碍，我们建议开发特异的、荧光检测氧化和还原的吡啶核苷酸NAD(H)和NADP(H)的方法。将开发基于其氧化状态的吡啶核苷酸的特定化学修饰方法。同时，抗体和荧光示踪剂将被开发用于未修饰的吡啶核苷酸的均相免疫检测。这些新的试剂和方法将以荧光偏振免疫分析的形式验证脱氢酶和还原酶活性的均相检测，并作为HTS检测试剂盒商业化。强大的、基于荧光的分析方法适用于任何利用吡啶核苷酸辅因子的氧化还原酶，这将加速探索这一针对广泛疾病和紊乱的有效目标类别。 催化氧化和还原反应的酶构成了一类主要的蛋白质，已成功地用作药物靶标。我们正在提议开发方法和试剂，使制药研究人员能够加快针对这些酶的药物发现，以治疗包括癌症、心血管疾病和糖尿病在内的广泛疾病。

DESCRIPTION (provided by applicant): There is a high level of pharmaceutical interest in targeting enzymes that catalyze oxidation and reduction reactions for therapeutic intervention. Validated redox enzyme targets include HMG-CoA reductase, the target of statin drugs like Lipitor that are used to treat hypercholesterolemia, and 51-reductase, the target of drugs used to treat benign prostatic hyperdysplasia. Emerging targets in this class include glycolytic dehydrogenases in tumor cells growing under hypoxic conditions and in microbial and parasitic pathogens. However, the identification of novel inhibitors for redox enzymes is being slowed by the lack of robust, generic enzyme assay methods that are suitable for use in pharmaceutical high throughput screening (HTS) laboratories. To remove this technical hurdle, we are proposing to develop methods for specific, fluorescent detection of oxidized and reduced pyridine nucleotides, NAD(H) and NADP(H). Methods will be developed for specific chemical modification of pyridine nucleotides based on their oxidation state. Comcomitantly, antibodies and fluorescent tracers will be developed for homogenous immunodetection of unmodified pyridine nucleotides. These novel reagents and methods will be validated for homogenous detection of dehydrogenase and reductase enzyme activity in a fluorescence polarization immunoassay format and commercialized as HTS assay kits. The availability of robust, fluorescence based assay methods applicable to any redox enzyme that utilizes a pyridine nucleotide cofactor will accelerate the exploration of this validated target class for a broad range of diseases and disorders. Enzymes that catalyze oxidation and reduction reactions constitute a major class of proteins that have been successfully exploited as drug targets. We are proposing to develop methods and reagents that will enable pharmaceutical researchers to accelerate drug discovery targeting these enzymes for a broad range of diseases including cancer, cardiovascular disease, and diabetes.