G protein-coupled receptor (GPCR) signaling in the mouse and human gastrointestinal (GI) tract.

目的：建立GPR35信号在小鼠和人类胃肠道中的机制。REMIT：这项建议属于BBSRC战略“生物科学促进健康”和“食品、营养和健康”的职权范围内。背景：GPR35是一种孤儿受体，缺乏内源性配体，但在哺乳动物胃肠道，尤其是结肠中高表达，与炎症性肠病(IBD)的发病有关。如果没有选择性激动剂，S的作用机制仍然不清楚。一些研究表明，来自微生物代谢的芳香酸激活了GPR35，暗示了GPCR的微生物传感器作用。在高剂量的临床验证的药物，铬甘油酸钠也激活GPR35，似乎保护胃肠道粘膜免受炎症。因此，GPR35对我们的合作伙伴Heptares治疗有限公司来说是高度优先的，他们已经合成了一系列结构多样化、有效的GPR35激动剂。我们将评估它们的有效性与铬甘酸盐和芳香酸，犬尿酸(KA)，一种色氨酸的微生物代谢产物。阐明健康GI组织中特定的GPR35信号将有助于研究炎性粘膜和受体在IBD模型中的保护潜力。学生将使用已建立的方法比较新型GPR35激动剂、糖化铬和KA在正常结肠粘膜中的疗效(COX实验室)。确定粘膜反应是发光导向的还是基础导向的，对于预测未来的配方、传递和暴露效应以支持YR 2-4的工作将是重要的。体内转运试验将确定是否ip。在赫普塔斯，学生将利用突变的GPR35(在赫普塔斯产生)来建立一个高置信度同源模型，以阐明配体结合和信号传递的分子机制。体外GI组织的放射自显影分析将识别口服或腹腔注射的靶细胞。[~3H]-GPR35配体，补充YR-1功能研究。优化的GPR35配基剂量(口服或腹腔注射)将测试所用药物是否对给定的炎症挑战具有保护作用，并保持粘膜健康以及胃肠道和结肠传输的正常比率。最后，学生将把他们的结果整合到GPR35信号的预测模型中，i)健康的小鼠和人类组织共同的信号，ii)发炎的结肠和正在进行的确定GPR35是否以及希望在哪里是治疗IBD的治疗靶点。

AIM: To establish the mechanisms of GPR35 signaling in the mouse and human GI tract.REMIT: This proposal falls within the BBSRC strategy 'Bioscience for Health', and remit of 'Food, nutrition & health'. BACKGROUND: GPR35 is an orphan receptor that lacks an endogenous ligand but is highly expressed in mammalian GI tract, particularly in the colon, and is implicated in the onset of inflammatory bowel disease (IBD). Without selective agonists GPR35's mechanisms of action remain obscure. Some studies suggest that aromatic acids derived from microbial metabolism activate GPR35, implicating a microbial sensor role for the GPCR. At high doses the clinically validated drug, sodium chromoglycate also activates GPR35 and appears to protect GI mucosa from inflammation. GPR35 is therefore a high priority for our partner Heptares Therapeutics Ltd, who have synthesised a range of structurally diverse, potent GPR35 agonists. We will evaluate their efficacies alongside chromoglycate and the aromatic acid, kynurenic acid (KA) a microbial metabolite of tryptophan. Elucidation of specific GPR35 signaling in healthy GI tissues will inform investigations of inflamed mucosae and the receptor's protective potential in models of IBD. To this end we will include a mild colitis model refined in mice during a recent BBSRC Sparking Impact project (to Cox) with alternate models that most closely recapitulate human IBD, collectively tackling an unmet need for improved translational biology.PLANYr 1 Cellular GPR35 signaling in epithelia, enteric nerves, enteroendocrine or mast cells will be investigated using proven strategies in murine and human colon. The student will compare the efficacies of novel GPR35 agonists, chromoglycate and KA in normal colonic mucosae using established methods (Cox lab). Determining whether mucosal responses are luminally or basally-directed will be important for predicting future formulation, delivery and exposure effects to support work in Yr 2-4. In vivo transit assays will establish if i.p. or oral drug alters GI transit in normal mice.Yr 2 At Heptares the student will utilise mutated GPR35 (generated at Heptares) to establish a high confidence homology model to elucidate the molecular mechanisms of ligand binding and signaling. Autoradiographic analysis of ex vivo GI tissue will identify the cells targetted by oral or i.p. [3H]-GPR35 ligand, complementing Yr 1 functional studies. Inflamed GI tissues from IBD models e.g. dextran sulphate sodium, or oxazolone-induced colitis, will be assessed functionally (at KCL) and for GPR35 expression and other key genes implicated in mucosal changes that most closely recapitulate those in human IBD.Yr 3-4 Using chosen IBD models the student (in KCL) will treat mice with GPR35 ligand, monitoring the onset of GI inflammation by measuring epithelial integrity functionally and histologically (scoring the degree of inflammation and stool consistency) and comparing murine mucosal erosion and dysplasia with that in human IBD colon. Optimised GPR35 ligand dosing (oral or i.p.) will test whether administered drug protects against a given inflammatory challenge and maintains mucosal health and normal rates of GI and colonic transit. Finally, the student will integrate their results into a predictive model of GPR35 signaling, i) common to healthy mouse and human tissue, ii) in inflamed colon and in-so-doing determine whether, and hopefully where, GPR35 is a therapeutic target for treating IBD.