High throughput screens for fatty acid uptake inhibitors

描述(由申请人提供)：特定类别的长链脂肪酸与促进各种疾病有关，包括肥胖、代谢综合征、糖尿病和心血管疾病。此外，饮食中饱和脂肪酸和反式不饱和脂肪酸的摄入量与某些癌症有很强的相关性，特别是结直肠癌和乳腺癌。目前工作的重点是设计高通量的方法来鉴定选择性地抑制细胞摄取脂肪酸的化合物。脂肪酸摄取是一种蛋白质介导的事件，需要脂肪酸转运蛋白(FATP)和酰辅酶A合成酶(ACSL)。我们的实验室已经鉴定了缺乏这种摄取系统的酵母菌株，并对其进行了广泛的表征。酵母的运输缺陷被酵母基因的哺乳动物同源基因所补充，而在酵母中重组的哺乳动物运输系统是所提出的抑制物自动筛选方法的目标。该建议的具体目的是：[1]展示在酵母活细胞系统中表达的哺乳动物运输蛋白的敏感性和特异性，以便以96孔高通量格式进行分析；[2]为抑制脂肪酸摄取的化合物进行多样化的小分子文库的证明或原则性筛选；以及[3]设计利用人类细胞进行培养的二级筛选。我们的最终目标是找出抑制脂肪酸进入细胞而不产生细胞毒性作用的化合物。这些化合物预计将有助于防止肠道吸收膳食脂肪和/或吸收到其他类型的细胞，以防止过量脂肪酸导致的细胞损害，从而导致疾病。

DESCRIPTION (provided by applicant): Specific classes of long chain fatty acids have been implicating in promoting various diseases including obesity, metabolic syndrome, diabetes, and cardiovascular disease. Additionally, there is a strong correlation between dietary intake of saturated and trans-unsaturated fatty acids with certain cancers including especially, colorectal and breast. The focus of the present work is to devise high throughput methods to identify compounds that selectively inhibit fatty acid uptake into cells. Fatty acid uptake is a protein-mediated event requiring a fatty acid transport protein (FATP) and an acyl-CoA synthetase (ACSL). Our laboratory has identified and extensively characterized yeast strains deficient in this uptake system. The transport defect of yeast is complemented by mammalian orthologs of the yeast genes and the mammalian transport system reconstituted in yeast is the target for the proposed automated inhibitor screening method development. The specific aims of the proposal are to: [1] demonstrate the sensitivity and specificity of the mammalian transport proteins expressed in the yeast live cell system for analysis in a 96-well high throughput format; [2] to conduct a proof or principle screen of a diversified small molecule library for compounds that inhibit fatty acid uptake; and [3] to devise secondary screens which employ human cells in culture. Our ultimate goal is to identify compounds that inhibit fatty acid uptake into cells without cytotoxic effects. Such compounds are expected to be useful to prevent intestinal absorption of dietary lipid and/or uptake into other cell types to prevent cell damage caused by excessive concentrations of fatty acids, which lead to disease.