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

使用 FLIPR 开发人类脂肪酸转运蛋白 4 的 HTS 平台

• 批准号: 7171606
• 负责人: Xinmin Simon Xie
• 金额: $ 20.64万
• 依托单位: SRI INTERNATIONAL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-15 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7171606
• 关键词: NIH Roadmap Initiative tag; biotechnology; chemical registry /resource; chemical synthesis; drug discovery /isolation; fatty acid transport; fatty acids; high throughput technology; inhibitor /antagonist; technology /technique development

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.

描述（由申请人提供）：在西方饮食中，长链脂肪酸（LCFA）占热量含量的40%。LCFA是重要的代谢产物，有助于许多细胞结构和功能，但过量的血清脂肪酸水平与肥胖，胰岛素脱敏和2型糖尿病有关。LCFA跨细胞膜摄取主要由脂肪酸转运蛋白（FATP）介导。在人类中，该转运蛋白家族的六个成员，命名为hsFATP 1 -6，已被表征并显示在整个身体中差异表达。FATP 4亚型构成小肠中的主要FATP，并且优先摄取长链脂肪酸（>10个碳原子）而不是短链脂肪酸。选择性抑制该亚型FATP可有效降低LCFA吸收。大规模化合物库的高通量筛选（HTS）将是鉴定新型FATP亚型选择性抑制剂的第一步。目前使用放射性标记的脂肪酸或荧光脂肪酸类似物来测量鼠3 T3-L1脂肪细胞中LCFA摄取的方法不适合HTS来鉴定针对人FATP的亚型选择性抑制剂。为了响应RAF-RM-06-004：“高重复性分子筛选的测定开发”，我们建议使用荧光成像酶标仪（FLIPRZ）和新开发的QBT（TM）脂肪酸摄取测定试剂盒开发和配置HTS平台，以通过稳定的hsFATP 4表达HEK 293细胞来定性LCFA摄取动力学。我们的具体目标是：1.针对原代HTS的hsFATP 4开发FLIPR 96孔或384孔格式试验。将hsFATP 4表达HEK 293细胞中的LCFA摄取动力学与载体对照HEK 293细胞和3 T3-L1脂肪细胞进行比较，以确定HTS试验的信噪比、特异性和重现性。通过检测试剂盒优化，我们将为主要HTS配置检测试剂盒。2.通过初步筛选评价并验证HTS检测试剂盒。一组由天然长链和短链脂肪酸、合成脂肪酸衍生物和小分子组成的数百种化合物将针对hsFATP 4进行筛选，作为初步评估。我们开发的HTS检测可用于分子库筛选中心网络的初步筛选，并将作为后续开发其他FATP家族成员检测的通用原型。二次筛选将使用针对相同靶标的标准放射性标记方法进行命中确认，并使用针对另一种FATP亚型的FLIPR测定进行选择性反筛选。这些亚型选择性的FATP抑制剂将为FATP生物学的进一步研究提供新的药理学工具。此外，它们可以形成开发用于治疗代谢紊乱的新疗法的基础。