Generic Fluorescent HTS Assay for Oxidoreductases

描述（由申请人提供）：针对催化氧化和还原反应的酶进行治疗干预具有很高的药学兴趣。已证实的氧化还原酶靶点包括HMG-CoA还原酶和51-还原酶，前者是用于治疗高胆固醇血症的他汀类药物如立普妥的靶点，后者是用于治疗前列腺增生的药物的靶点。这类新出现的靶点包括在缺氧条件下生长的肿瘤细胞以及微生物和寄生病原体中的糖酵解脱氢酶。然而，由于缺乏适用于药物高通量筛选（HTS）实验室的稳健、通用的酶分析方法，新型氧化还原酶抑制剂的鉴定正在放缓。为了消除这一技术障碍，我们建议开发特异性荧光检测氧化和还原吡啶核苷酸，NAD(H)和NADP(H)的方法。根据吡啶核苷酸的氧化状态，将开发出特定的化学修饰方法。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质免疫检测。这些新的试剂和方法将在荧光极化免疫分析格式中用于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。适用于任何利用吡啶核苷酸辅助因子的氧化还原酶的强大的、基于荧光的检测方法的可用性将加速对这一经过验证的靶标类的探索，以用于广泛的疾病和失调。

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.