Development of HTS Platform for Human Fatty Acid Transport Protein 4 Using FLIPR

描述(申请人提供)：饮食中的长链脂肪酸(LCFA)贡献了西方饮食中高达40%的卡路里含量。低碳脂肪酸是重要的代谢产物，有助于许多细胞结构和功能，但血清脂肪酸水平过高与肥胖、胰岛素脱敏和2型糖尿病有关。LCFA跨细胞膜的摄取主要由脂肪酸转运蛋白(FATPs)介导。在人类中，这个转运蛋白家族的六个成员，被指定为hsFATP1-6，已经被鉴定并被证明在整个身体中差异表达。FATP4亚型构成了小肠中的主要FATP，并优先摄取长-(&gt；10个碳原子)而不是短链脂肪酸。选择性抑制这一亚型FATP可能有效地减少LCFA的吸收。对大型化合物文库的高通量筛选(HTS)将是鉴定新型FATPs亚型选择性抑制剂的第一步。目前使用放射性标记脂肪酸或荧光脂肪酸类似物来测量小鼠3T3-L1脂肪细胞摄取LCFA的方法不适用于HTS来识别针对人类FATP的亚型选择性抑制剂。为响应RAF-RM-06-004：“高通量分子筛选的分析开发”，我们建议开发和配置一个HTS平台，该平台使用荧光成像平板阅读器(FLIPRZ)和新开发的QBT(TM)脂肪酸摄取分析试剂盒，以提高稳定表达hsFATP4的HEK293细胞摄取LCFA的动力学质量。我们的具体目标是：1.建立一种针对hsFATP4的FLIPR 96孔或384孔格式的检测方法。在表达hsFATP4的HEK293细胞中摄取LCFA的动力学将与载体对照的HEK293细胞和3T3-L1脂肪细胞进行比较，以确定HTS检测的信噪比、特异性和重复性。通过化验优化，我们将配置针对初级HTS的化验。2.通过初步筛选对HTS检测进行评估和验证。作为初步评估，将针对hsFATP4筛选数百种化合物，其中包括天然长链和短链脂肪酸、合成脂肪酸衍生物和小分子。我们开发的HTS方法可用于分子文库筛选中心网络的初步筛选，并将作为后续开发其他FATP家族成员的分析的通用原型。二次筛查将针对相同的靶点使用标准的放射性标记方法以确认命中，并使用FLIPR分析针对另一种FATP亚型进行选择性的反筛查。所鉴定的亚型选择性FATP抑制剂将为FATP生物学的进一步研究提供新的药理工具。此外，它们可能成为开发治疗代谢紊乱的新疗法的基础。

DESCRIPTION (provided by applicant): Dietary long-chain fatty acids (LCFAs) contribute up to 40% of the caloric content in western diets. LCFAs are important metabolites and contribute to many cellular structures and functions, but excessive serum fatty acid levels are linked to obesity, insulin desensitization, and type 2 diabetes. LCFA uptake across cell membranes is mediated principally by fatty acid transport proteins (FATPs). In humans, six members of this transporter family, designated as hsFATP1-6, have been characterized and shown to be differentially expressed throughout the body. The FATP4 subtype constitutes the major FATP in the small intestine and preferentially uptakes long- (>10 carbon atoms) but not short-chain fatty acids. Selective inhibition of this subtype FATP may effectively reduce LCFA absorption. High-throughput screening (HTS) of a large compound library will be the initial step toward identifying novel subtype-selective inhibitors of FATPs. Current methods using radiolabeled fatty acids or fluorescent fatty acid analogs to measure LCFA uptake in murine 3T3-L1 adipocytes are not suitable for HTS to identify subtype-selective inhibitors against human FATPs. In response to RAF-RM-06-004: "Assay Development for High Throughput Molecular Screening," we propose to develop and configure an HTS platform using a fluorometric imaging plate reader (FLIPRZ) with the newly developed QBT(TM) Fatty Acid Uptake Assay Kit to quality LCFA uptake kinetics by the stable hsFATP4-expressing HEK293 cells. Our specific aims are to: 1. Develop a FLIPR 96- or 384-well format assay against the hsFATP4 for primary HTS. LCFA uptake kinetics in hsFATP4-expressing HEK293 cells will be compared with the vector control HEK293 cells and 3T3-L1 adipocytes to determine signal-to-noise ratios, specificity, and reproducibility for the HTS assay. Through assay optimization, we will configure the assay for primary HTS. 2. Evaluate and validate the HTS assay through an initial screening. A panel of several hundred compounds consisting of natural long- and short-chain fatty acids, synthetic fatty acid derivatives, and small molecules will be screened against the hsFATP4 as an initial evaluation. The HTS assay we develop could be used in the Molecular Library Screening Center Network for primary screens and will serve as a general prototype for subsequent development of assays for other FATP family members. Secondary screens will use the standard radiolabeled method against the same target for hit confirmation and the FLIPR assay against another FATP subtype in counter-screening for selectivity. The identified subtype-selective FATP inhibitors will provide a novel pharmacologic tool for further studies of FATP biology. Furthermore, they may form a basis for developing novel therapeutics for the treatment of metabolic disorders.