High throughput screens for fatty acid uptake inhibitors

特定类别的长链脂肪酸与多种疾病有关，包括 肥胖、代谢综合征、糖尿病和心血管疾病。此外，还有一个很强的 膳食中饱和和反式不饱和脂肪酸摄入量与某些癌症的相关性 尤其包括结直肠和乳房。目前工作的重点是设计高吞吐量 鉴定选择性抑制细胞摄取脂肪酸的化合物的方法。具体来说，这项建议 解决计划公告编号PA-04-068所针对的目标，以开发HIGH检测方法 吞吐量药物筛选。脂肪酸摄取是一种蛋白质介导的事件，需要脂肪酸的运输 蛋白质(FATP)和酰辅酶A合成酶(ACSL)。我们的实验室已经识别并广泛识别了 缺乏这种摄取系统的酵母菌株的特征。酵母的运输缺陷得到了补充 由哺乳动物的酵母基因和哺乳动物运输系统在酵母中重组 目标为提出的抑制剂自动筛选方法的发展方向。《公约》的具体目标 建议是：[1]证明哺乳动物转运蛋白的敏感性和特异性 在酵母活细胞系统中表达，以96孔高通量格式进行分析；[2]进行证明 或对抑制脂肪酸摄取的化合物的多样化小分子库进行原则性筛选； 设计在培养中使用人类细胞的二次筛选。我们的最终目标是识别化合物 抑制细胞对脂肪酸的摄取而不产生细胞毒性作用。这类化合物预计将是有用的 防止肠道吸收膳食脂肪和/或吸收到其他类型的细胞，以防止细胞损伤 由过量的脂肪酸引起的，这会导致疾病。

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. This proposal specifically addresses goals targeted by program announcement number PA-04-068 to develop assays for high throughput drug screening. 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.