Intestinal G protein-coupled receptors (GPCRs): characterising gut GPCR-signalling with potential to treat colitis

游离脂肪酸受体(FFAR)是已知在肠道内分泌细胞中表达的GPCRs，因此可能参与哺乳动物胃肠道的营养和微生物代谢物信号传递。FFA1和FFA4被长链脂肪酸(LCFA)共同激活，而FFA2和FFA3优先被微生物衍生的短链脂肪酸(SCFAs)共同激活。这对GPCR之间的非选择性被其他与脂肪酸有亲和力的GPCR进一步复杂化。例如，SCFA激活Gpr109a(也被称为Hca2；Husted等人2017年)以及嗅觉受体Olf78和Olf558(Bellono等人2017年)，由于缺乏选择性配体，使得特定的ffars的功能(S)具有挑战性。因此，就胃肠道健康和疾病而言，这个GPCR组的作用仍然不清楚。然而，利用商业上可用的工具，Cox等人已经部分表征了健康GI粘膜中的FFA1-4信号，并确定了它们对小鼠GI转运的影响(Forbes等人2015年；Moodaley等人2017年；Tough等人2018年)。这是通过结合已证实的体外电生理学方法(平行测量矢量上皮离子转运和屏障功能)和测量上、下胃肠道转运的变化来实现的(Tough等人，2011年)。我们对胃肠道内神经上皮信号的深入理解也是至关重要的。值得注意的是，我们在正常小鼠和人类结肠中看到了类似的FFA2和FFA3活性的药理学和细胞机制(Tough等人2018年)，表明这一方法的翻译。选择性的、强有力的FFAR配体现在对于继续取得进展至关重要。对于Heptares，我们对GI信号的综合理解使我们能够描述由急性GI炎症引起的粘膜功能障碍。为此，我们将包括博士生埃文斯(在考克斯实验室)目前正在展示的轻度结肠炎葡聚糖硫酸钠(DSS)模型，概括了在人类中观察到的结肠炎病理方面，使我们能够测试新配体在小鼠结肠炎中的保护潜力。Hephtares通过其专有的GPCR靶向STARR技术(https://soseiheptares.com/about-sosei-heptares)生产了结构多样化的一流的FFA4和GPR109a配体以及选择性的FFA1、FFA2和FFA3合成激动剂，具有作为胃肠道免疫反应调节剂的治疗潜力(van Daal等人，2021年)。然而，确定的胃肠道区域的相对药物效力尚不清楚。这个项目将利用所选择的配体来确定它们在小鼠胃肠道中的作用，从而使它们的靶标GPCR的明确功能意义得以确定。Heptares与一家CRO(位于克罗地亚萨格勒布的Selvita)合作，后者为不同的IBD啮齿动物模型提供病理学验证。我们从这种已建立的关系中受益，Evans(Lido三年级学生)将在Heptares工作，以完成低剂量DSS诱导的粘膜炎症的评分。我们的建议将在体内建立胃肠道疗效的治疗效用和配基生物利用度，从而有助于理解一种新的潜在疗法，以满足溃疡性结肠炎治疗的需求。目的：i)确定胃肠道(Gi)G蛋白偶联受体(GPCRs)的一流化合物的有效性，以及ii)在小鼠模型中表征保护和/或逆转结肠炎的途径。

Free fatty acid receptors (FFARs) are GPCRs known to be expressed in enteroendocrine cells and therefore likely to be involved in nutrient- and microbial metabolite signaling in the mammalian GI tract. FFA1 and FFA4 are co-activated by long-chain fatty acids (LCFAs) while FFA2 and FFA3 are preferentially co-stimulated by microbial-derived short chain fatty acids (SCFAs). The nonselectivity between these pairs of GPCRs is complicated further by other GPCRs with affinity for fatty acids. For example, SCFAs activate GPR109A (also known as HCA2; Husted et al 2017) as well as olfactory receptors Olf78 and Olf558 (Bellono et al 2017) making specific FFARs' function(s) challenging due to lack of selective ligands. Thus, the roles of this GPCR group remain obscure in terms of GI health and disease. Using commercially available tools, Cox et al have however partially characterized FFA1-4 signaling in healthy GI mucosae and established their effects on GI transit in mice (Forbes et al 2015; Moodaley et al 2017; Tough et al 2018). This has been achieved using a combination of proven electrophysiological in vitro methods (measuring vectorial epithelial ion transport and barrier function in parallel) and measuring changes in upper and lower GI transit (Tough et al 2011). Our in-depth understanding of neuro-epithelial signaling within the GI tract, has been critical too. Notably, we see similar pharmacology and cellular mechanisms for FFA2 & FFA3 activities in normal mouse and human colon (Tough et al 2018) indicating translation of this approach. Selective, potent FFAR ligands are now critical for continued progress. With Heptares, our combined understanding of GI signalling is allowing us to characterise mucosal dysfunctions induced by acute GI inflammation. To this end we will include the mild colitis dextran sulphate sodium (DSS) model which PhD student, Evans (in Cox lab) is showing currently, recapitulates aspects of colitis pathology observed in man, allowing us to test the protective potential of novel ligands in murine colitis. Heptares have via their proprietary GPCR-targeted STaRR technology (https://soseiheptares.com/about-sosei-heptares) produced structurally diverse, first-in-class FFA4 and GPR109A ligands as well as selective FFA1, FFA2 and FFA3 synthetic agonists, with therapeutic potential as modulators of GI immune responses (van Daal et al 2021). However, the relative drug potencies in defined GI regions are unknown. This project will utilise chosen ligands to establish their efficacies in mouse GI tract allowing clear functional significance of their target GPCR to be identified. Heptares collaborate with a CRO (Selvita, in Zagreb, Croatia) who provide pathological validation of different rodent animal models of IBD. We benefit from this established relationship and Evans (LiDO student in year 3) will be working in Heptares to complete the scoring of low dose DSS-induced mucosal inflammation. Our proposal will establish therapeutic utility and ligand bioavailability for GI efficacy in vivo, thus contributing understanding of a new potential therapy for the unmet need of ulcerative colitis treatments. AIMS: i) to establish the efficacy of first-in-class compounds for gastrointestinal (GI) G protein-coupled receptors (GPCRs) and, ii) to characterise pathways that protect and/or reverse colitis in a mouse model.