High throughput screens for fatty acid uptake inhibitors

高通量筛选脂肪酸摄取抑制剂

基本信息

批准号：
7487712
负责人：
Concetta C. DiRusso
金额：
$ 3.06万
依托单位：
ORDWAY RESEARCH INSTITUTE, INC.
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-01 至 2009-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7487712
关键词：
Address Adipose tissue Biological Assay Blood Vessels Breast Caco-2 Cells Cardiomyopathies Cardiovascular Diseases Carrier Proteins Cell Membrane Permeability Cell physiology Cells Class Coenzyme A Ligases Colon Adenocarcinoma Colorectal Complement Defect Developed Countries Developing Countries Diabetes Mellitus Dietary Fats Dietary intake Disease Doctor of Philosophy Event Fatty Acids Fluorescence Genes Goals Human Human Cell Line Hyperphagia Intestinal Absorption Laboratories Lead Libraries Life Link Malignant Neoplasms Mediating Metabolic Metabolic syndrome Methods Monounsaturated Fatty Acids Morbidity - disease rate NIH Program Announcements Nonesterified Fatty Acids Normal Cell Numbers Obesity Orthologous Gene Pancreatitis Permeability Pharmaceutical Preparations Polyunsaturated Fatty Acids Preclinical Drug Evaluation Prevention Primary carcinoma of the liver cells Property Proteins Purpose Reader Research Personnel Robotics Screening procedure Sensitivity and Specificity System Systems Analysis Testing Therapeutic Intervention Tissues Toxic effect Toxicity Tests Trypan Blue United States Unsaturated Fatty Acids Work Yeasts base cell injury cell type chemical property cytotoxic cytotoxicity test fatty acid analog fatty acid-transport protein high throughput screening inhibitor/antagonist lipid metabolism long chain fatty acid method development mortality mutant prevent protein function reconstitution research study small molecule libraries uptake

项目摘要

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.

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，还有一个强大的饱和脂肪酸和反式不饱和脂肪酸的膳食摄入量与某些癌症之间的相关性尤其包括结直肠癌和乳腺癌。目前工作的重点是设计高通量鉴定选择性抑制细胞摄取脂肪酸的化合物的方法。该提案具体解决了计划公告号 PA-04-068 所针对的目标，以开发高浓度检测方法通量药物筛选。脂肪酸摄取是蛋白质介导的事件，需要脂肪酸运输蛋白质（FATP）和酰基辅酶A合成酶（ACSL）。我们的实验室已鉴定并广泛鉴定了缺乏该摄取系统的酵母菌株。酵母运输缺陷得到补充通过酵母基因的哺乳动物直系同源物和在酵母中重建的哺乳动物运输系统所提出的自动抑制剂筛选方法开发的目标。该计划的具体目标建议是： [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，用于以 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进行多样化小分子库的原理筛选；和[3] 设计在培养物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脂肪酸摄入细胞，而无细胞毒性作用。此类化合物预计可用于防止肠道吸收膳食脂质和/或摄取其他细胞类型以防止细胞损伤由脂肪酸浓度过高引起，从而导致疾病。