A Fluorescent HTS Assay for the Discovery of FABP(RMI)

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。抑制脂肪酸与FABP的结合应该为研究FABP对代谢的影响提供了一种有用的分子功能，但目前还没有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FABP的ADI Fab来发现FABP结合的抑制剂。本项目的目的是(1)优化用于HTS的ADI Fab方法，(2)筛选一个小分子文库，以验证该方法在鉴定FABP结合抑制剂方面的有效性。为了实现这些目标，将改变ADI Fab分析的条件以优化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，并可能导致胰岛素抵抗、动脉粥样硬化和某些形式的癌症的治疗药物的开发。

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. Inhibition of fatty acid binding to FABPs should provide a useful method to investigate the molecular functions that underlie their observed effects on metabolism, but no inhibitors of FABP binding are currently available. To address this need, a high throughput screening (HTS) assay will be developed to discover inhibitors of FABP binding using ADI FAB, a fluorescently labeled FABP. The aims of this project are to (1) optimize the ADI FAB assay for HTS and (2) screen a small molecule library to validate the effectiveness of the assay for the identification of inhibitors of FABP binding. To accomplish these aims, conditions of the ADI FAB 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 FABPs, 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 insulin resistance, atherosclerosis, and certain forms of cancer.