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多种疾病有关，包括肥胖、代谢综合征、糖尿病和心血管疾病。此外，饮食中摄入饱和脂肪酸和反不饱和脂肪酸与某些癌症有很强的相关性，尤其是结肠直肠癌和乳腺癌。目前工作的重点是设计高通量方法来鉴定选择性抑制脂肪酸摄取进入细胞的化合物。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）和酰基辅酶a合成酶（ACSL）。我们的实验室已经确定并广泛表征了酵母菌株缺乏这种摄取系统。酵母的转运缺陷可以通过哺乳动物的酵母基因同源物来弥补，在酵母中重组的哺乳动物转运系统是所提出的自动化抑制剂筛选方法开发的目标。该提案的具体目标是：b[1]证明在酵母活细胞系统中表达的哺乳动物转运蛋白的敏感性和特异性，以便在96孔高通量格式下进行分析；[2]对抑制脂肪酸摄取的化合物的多样化小分子文库进行验证或原理筛选；b[3]设计二次筛选，利用培养的人类细胞。我们的最终目标是鉴定出抑制脂肪酸进入细胞而不产生细胞毒性作用的化合物。预计这类化合物可用于防止肠道吸收膳食脂质和/或吸收到其他细胞类型，以防止脂肪酸浓度过高引起的细胞损伤，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.