Uncovering the pharmacology of the G protein-coupled receptor GPR40

G蛋白偶联受体(GPCRs)既是人类最大的跨膜信号蛋白家族，也是治疗活性小分子药物最成功的靶向作用部位。尽管如此，激活许多GPCRs的配体要么仍不为人所知，要么特征不佳。最近，一组名为GPR40-GPR43的GPCR被证明对循环在血液中的中长链脂肪酸产生反应。GPR40由产生和分泌胰岛素的胰岛β细胞高度表达。众所周知，在短期内，血液中脂肪酸水平的升高会增加胰岛素的分泌。然而，从长远来看，血液中脂肪酸的升高对β细胞功能是有害的，众所周知，肥胖糖尿病患者血液中的脂肪酸浓度会升高。目前，还没有GPCR靶向药物用于治疗糖尿病。然而，我们最近表明，一组临床使用的药物，称为格列酮，激活GPR40，尽管它们被接受的作用模式是通过激活另一组完全不同的非GPCR，即被称为过氧化体增殖物激活的伽马亚型受体(PPARGamma)的核受体。尽管格列酮用于临床，但它们的副作用限制了它们的使用，而且因为它们需要大量的时间才能产生益处，所以它们的一些副作用可能至少反映了它们激活GPR40的能力。最近，与中链脂肪酸相关的分子如何与其目标GPCR结合的模型已经发表。将这些模型与GPR40序列进行比较，为脂肪酸如何与GPR40相互作用提供了分子基础上的假设，我将使用突变和我们最近开发的新方法来测量GPR40的活性来测试这些假设。我还将测试这些突变是否会干扰格列酮和一组最近报道的激活GPR40的小分子化学物质对GPR40的激活。激活GPR40的脂肪酸与激活GPR41和GPR43的脂肪酸之间的主要区别是分子的长度。GPR40与GPR41和GPR43序列的比较表明，可能控制这种选择性的关键氨基酸。这也将通过突变和功能测试相结合的方式进行测试。可以假设，一种激活PPAR伽马但抑制GPR40的分子将在糖尿病治疗中有用。因此，我还将评估与活性格列酮相关但不激活PPARγ的一系列其他配体的活性，以了解它们激活或阻止GPR40激活的能力。这种配体与GPR40结合和活性GPR40要求的映射将为这种GPCR是如何调节的提供新的见解，从长远来看，可能有助于进一步开发能够激活或抑制这种受体功能的小分子。

G protein-coupled receptors (GPCRs) are both the largest family of transmembrane signalling proteins in man and the most successfully targetted as sites of action by therapeutically active small molecule medicines. Despite this, the ligands that activate many GPCRs remain either unknown or poorly characterised. Recently, the group of GPCRs named GPR40-GPR43 were shown to respond to medium- and long-chain fatty acids that circulate in the bloodstream. GPR40 is highly expressed by pancreatic beta-cells that produce and secrete insulin. It is known that, in the short term, elevation of blood fatty acid levels increase insulin secretion. However, in the longer term elevated blood fatty acids are detrimental to beta-cell function and it is known that the concentration of fatty acids is elevated in the blood of obese diabetics. At the moment, no GPCR targeted medicines are used to treat diabetes. However, we have recently shown that a group of clinically used medicines, called glitazones, activate GPR40 although their accepted mode of action is via activation of a completely different group of non-GPCR, nuclear receptors called Peroxisome Proliferator-Activated Receptors of the gamma subtype (PPARgamma). Although glitazones are used clinically they have side effects that limit their use and because they require to be used for significant amounts of time to produce benefits it is possible that at least some of their side effects reflect their ability to activate GPR40. Recently, models of how molecules related to medium-chain length fatty acids bind to their target GPCRs have been published. Comparing these models with the sequence of GPR40 has provided hypotheses on the molecular basis of how fatty acids interact with GPR40 and I will use combinations of mutagenesis and novel assays we have recently developed to measure activation of GPR40 to test these. I will also test if these mutations interfere with the activation of GPR40 by glitazones and by a group of small molecule chemicals also recently reported to activate GPR40. The major difference between fatty acids that activate GPR40 and those that active GPR41 and GPR43 is the length of the molecule. Comparisons of the sequences of GPR40 with GPR41 and GPR43 suggest key amino acids that may control this selectivity. This will also be tested by combinations of mutagenesis and functional assay. It can be hypothesised that a molecule that activates PPARgamma but inhibits GPR40 would be useful in the treatment of diabetes. I will thus also assess the activity of a range of other ligands that are related to the active glitazones but do not activate PPARgamma, for their ability to either activate or block activation of GPR40. Such mapping of the requirements for ligands to bind and to active GPR40 will provide new insights into how this GPCR is regulated and may, in the longer term help with further development of small molecules able to either activate or inhibit the function of this receptor.