| Intestinal Co-culture System to Study TGR5 Agonism and Gut Restriction
|
|
Author:
Date:
2021-03-20
[Abstract] The activation of the Takeda G-protein receptor 5 (TGR5, also known as the G protein-coupled bile acid receptor 1, GPBAR1) in enteroendocrine L-cells results in secretion of the anti-diabetic hormone Glucagon-Like Peptide 1 (GLP-1) into systemic circulation. Consequently, recent research has focused on identification and development of TGR5 agonists as type 2 diabetes therapeutics. However, the clinical application of TGR5 agonists has been hampered by side effects of these compounds that primarily result from their absorption into circulation. Here we describe an in vitro screening protocol to evaluate the TGR5 agonism, GLP-1 secretion, and gut-restricted properties of small molecules. The protocol involves differentiating gut epithelial and endocrine cells together in transwells to ...
[摘要] [摘要]肠内分泌L细胞中的Takeda G蛋白受体5（TGR5，也称为G蛋白偶联胆汁酸受体1，GPBAR1）的激活导致抗糖尿病激素胰高血糖素样肽1的分泌。 （GLP-1）进入全身循环。因此，最近的研究集中于鉴定和开发TGR5激动剂作为2型糖尿病治疗剂。但是，TGR5激动剂的临床应用受到这些化合物的副作用的阻碍，这些副作用主要是由于它们吸收进入循环系统所致。这里我们描述一个体外 筛选协议以评估TGR5激动剂，GLP-1分泌和小分子的肠道限制性特性。该协议涉及在跨孔中将肠道上皮细胞和内分泌细胞一起分化，以评估TGR5激动剂的药效学和化合物对肠道单层的毒性。作为概念的证明，我们证明了该协议在评估有效的TGR5激动剂自然存在的胆汁酸代谢物的性质中的应用。该协议改编自Chaudhari等人。（202 1 ）。
[背景和d ] GI道的肠壁是由几个不同类型的细胞，每一个特定的和，有时独有的功能的（阿莱尔等人。，2018） ...
|
|
|
| Assessing Gαq/15-signaling with IP-One: Single Plate Transfection and Assay Protocol for Cell-Based High-Throughput Assay
|
|
Author:
Date:
2020-08-20
[Abstract] Cell-based functional assays are an important part of compound screening and drug lead optimization, and they can also play a crucial role in the determination of the residues involved in ligand binding and signaling for a particular G-protein-coupled receptor. Conventional methods used for Gαq/15-coupled receptors rely on the use of fluorescent probes for Ca++ sensing (such as Fura-2 and Fluo-4) or on the incorporation of [3H]-inositol into inositol 1,4,5- triphosphate (IP3). However, these methods are not suitable for screening large libraries of compounds or for screening several mutants of the same receptor. In contrast, the IP-One assay by Cisbio is a TR-FRET assay suitable for large compound library screening when using stable cell lines that express ...
[摘要] [摘要 ] 基于细胞的功能测定法是化合物筛选和药物先导物优化的重要组成部分，并且它们可以也参与配体结合和信令残留量的测定起到至关重要的作用为一个特定的G蛋白偶联受体。用于Gα常规方法的q / 15 -偶联受体依赖于使用用于钙荧光探针++ 感测（如的Fura-2和的Fluo-4）或上掺入[的3 H] - 肌醇成肌醇1,4- ，5-三磷酸（IP3）。然而，这些方法不适合用于筛选大文库的化合物或用于筛选相同的受体的几个突变体。相反，IP-一个测定由Cisbio公司是TR-FRET测定适合大型化合物文库使用的稳定细胞株的筛选时，表达一个特定7TMR 。但是，当使用瞬时转染的7TMR突变体时，此检测方法并不理想，因为它需要两步操作进行细胞培养。因此，我们已经优化了IP-One的测定使用协议的在384孔反向转染方法的板。这为先前用于筛选Gαq / 15 偶联7TMR 的几种突变体的两步法提供了一种省时和省资源的替代方案。
[背景 ] 七跨膜受体（7TMR），也被称为G蛋白偶联受体，是超家族参与信号转导的最重要的跨膜蛋白。它们是临床批准药物的30％至50％的目标（Overington 等，2006）。Gαq / 15 偶联的7TMR激活磷脂酶Cβ（PLCβ），并产生D-肌醇1,4,5-三磷酸（IP3）和二酰基甘油（DAG）。IP3触发细胞内Ca ++ ...
|
|
|