An HTS Assay for the Discovery of Specific Inhibitors of Adipocyte FABP

描述(申请人提供)：脂肪酸结合蛋白(FABP)的分子功能在发现近35年后仍不清楚。最近对基因敲除小鼠的研究表明，FABP在代谢综合征和某些癌症的发展中发挥关键作用，使它们成为这些疾病的潜在治疗靶点。具体地说，脂肪细胞FABP(A-FABP)与胰岛素抵抗、动脉粥样硬化和脂肪肝疾病有关。抑制脂肪酸与A-FABP的结合将为研究其影响代谢的分子功能提供一种有用的方法，但目前还没有A-FABP结合的抑制剂。为了满足这一需求，将开发一种高通量筛选(HTS)方法，以使用荧光标记的A-FABP来发现A-FABP结合的特定抑制剂。本项目的目的是(1)优化用于HTS的A-FABP探针；(2)筛选一个小分子文库，以验证该方法在鉴定A-FABP结合的特异性抑制剂方面的有效性。为了实现这些目标，将改变A-FABP测定的条件以优化Z‘因子，将开发快速二次筛选以识别改变观察到的荧光的光学伪影并确认与A-FABP的结合，筛选结果将通过定量测定抑制剂/FABP结合亲和力和膜通透性来评估。有效的抑制剂将用于后续研究计划，以研究FABP在脂质代谢中的分子功能，并可能导致开发治疗II型糖尿病、冠状动脉疾病和某些形式癌症的药物。最近的研究表明，脂肪细胞脂肪酸结合蛋白(A-FABP)在胰岛素抵抗、动脉粥样硬化和脂肪肝疾病中起重要作用。抑制脂肪酸与A-FABP的结合将为研究其影响代谢的分子功能提供一种有用的方法，但目前还没有A-FABP结合的抑制剂。为了满足这一需求，将开发一种高通量筛选试验来发现特定的A-FABP抑制剂，这些抑制剂可用于研究A-FABP的功能，并可能作为治疗II型糖尿病和冠状动脉疾病的先导。

DESCRIPTION (provided by applicant): The molecular functions of fatty acid binding proteins (FABPs) remain obscure nearly 35 years after their discovery. Recent studies with knock-out mice indicate that FABPs play critical roles in the development of the metabolic syndrome and certain cancers making them potential therapeutic targets for these diseases. Specifically, the adipocyte FABP (A-FABP) has been linked to insulin resistance, atherosclerosis, and fatty liver disease. Inhibition of fatty acid binding to A-FABP should provide a useful method to investigate the molecular functions that underlie its observed effects on metabolism, but no inhibitors of A-FABP binding are currently available. To address this need, a high throughput screening (HTS) assay will be developed to discover specific inhibitors of A-FABP binding using a fluorescently labeled A-FABP. The aims of this project are to (1) optimize the A-FABP probe for HTS and (2) screen a small molecular library to validate the effectiveness of the assay for the identification of specific inhibitors of A-FABP binding. To accomplish these aims, the conditions of the A-FABP assay will be varied to optimize the Z'-factor, rapid secondary screens will be developed to identify optical artifacts that alter the observed fluorescence and to confirm binding to A-FABP, and screening results will be assessed by quantitative determination of inhibitor / FABP binding affinities and membrane permeability. Effective inhibitors will be used in a follow-up research program to study the molecular functions of FABPs in lipid metabolism and could lead to the development of therapeutic agents for such diseases as type II diabetes, coronary artery disease, and certain forms of cancer. Recent studies indicate that adipocyte fatty acid binding protein (A-FABP) plays a critical role in insulin resistance, atherosclerosis, and fatty liver disease. Inhibition of fatty acid binding to A-FABP should provide a useful method to investigate the molecular functions that underlie its observed effects on metabolism, but no inhibitors of A-FABP binding are currently available. To address this need, a high throughput screening assay will be developed to discover specific A-FABP inhibitors that can be used to study A-FABP function and may serve as therapeutic leads for the treatment of type II diabetes and coronary artery disease.