Activators of Adipocyte Fatty Acid Oxidation

说明(申请人提供)：肥胖是许多疾病的公认危险因素，包括2型糖尿病和冠心病。现有的治疗肥胖症的药物和饮食疗法只有一定的效果。一种很可能在治疗肥胖症方面有效且副作用很好的方法是一种直接作用于脂肪细胞中脂肪代谢的药物。AdipoGenix公司的使命是发现和开发在脂肪细胞水平上作用的新疗法，用于治疗肥胖、糖尿病和相关的代谢紊乱。肉毒碱棕榈酰转移酶I(CPT I)是细胞内脂肪酸代谢的关键控制点，是游离脂肪酸(FFA)氧化为CO2和酯化为甘油三酯(TG)之间的主要转换点。氧化和合成之间的平衡稍有变化，就可能对脂肪储存产生重大影响。丙二酰辅酶A是CPT I的变构抑制剂，调节FFA氧化，从而调节脂肪储存。干扰丙二酰辅酶A对CPT I影响的化合物可以促进CPT I的活性，从而促进FFA的氧化。我们建议表征CPT I亚型在人前脂肪细胞中的分化和脂库依赖的表达，并开发高通量筛选以确定解除丙二酰辅酶A对CPT I的抑制效应从而刺激氧化的化合物。采用相对定量RT-PCR和Western blotting方法分析人脂肪细胞分化过程中和不同解剖部位的CPT I亚型的表达。我们将建立一种高通量的检测方法，在有或没有丙二酰辅酶A抑制剂的情况下，测定分离的线粒体制剂和通透性的人脂肪细胞中CPT I的活性。同时，将开发一种高通量检测方法，使用基于氧气传感微板的系统来测量人类脂肪细胞的氧气消耗。被确定为最适合HTS的方法将被用于在第二阶段筛选化学库。这些目标的实现将建立一种可行的HTS方法和二次分析方法，用于鉴定激活CPT I和增加人类脂肪细胞氧化的化合物。这类化合物的开发可能导致有效调节脂肪代谢和治疗肥胖症的治疗干预。

DESCRIPTION (provided by applicant): Obesity is a well-established risk factor for a number of diseases, including type 2 diabetes and coronary heart disease. Existing drug and dietary treatments for obesity are only modestly effective. An approach that is very likely to be effective in treating obesity and with a good side effect profile is a drug that acts directly on lipid metabolism in the fat cell. The mission of AdipoGenix, Inc. is to discover and develop novel therapeutics acting at the level of the fat cell for treating obesity, diabetes, and related metabolic disorders. The enzyme carnitine palmitoyltransferase I (CPT I) is the critical control point for fatty acid metabolism in the cell and provides the main switch between free fatty acid (FFA) oxidation to CO2 and esterification to triglyceride (TG). A slight change in the balance between oxidation and synthesis could have a major impact on fat stores. Malonyl-CoA, an allosteric inhibitor of CPT I, regulates FFA oxidation and, consequently, fat storage. Compounds that interfere with the effect of malonyl-CoA on CPT I can promote CPT I activity and, thereby, FFA oxidation. We propose to characterize the differentiation- and fat depot-dependent expression of CPT I isoforms in human preadipocytes, and to develop high-throughput screens to identify compounds that relieve the inhibitory effect of malonyl-CoA on CPT I and thereby stimulate oxidation. Relative quantitative RTPCR and Western blotting will be used to analyze expression of CPT I isoforms in human adipocytes during differentiation and from different anatomical depots. A high-throughput assay to measure activity of CPT I in isolated mitochondrial preparations and in permeabilized human adipocytes in the presence and absence of the inhibitor malonyl-CoA will be developed. In parallel, a high-throughput assay to measure oxygen consumption in human adipocytes using an oxygen-sensing microplate-based system will be developed. The assay determined to be most suitable for HTS will be used to screen chemical libraries in Phase II. Achievement of these aims will establish a viable HTS assay and secondary assays for identifying compounds that activate CPT I and increase oxidation in human adipocytes. Development of such compounds may lead to therapeutic interventions that are effective for modulating lipid metabolism and treating obesity.