Development of a Testing Funnel for Identification of Small-Molecule Modulators Targeting Secretin Receptors.
Development of a Testing Funnel for Identification of Small-Molecule Modulators Targeting Secretin Receptors.
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DOI：
10.1177/2472555220945284
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发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Sergienko EA
中科院分区：
文献类型：
--
作者：
Dengler DG;Sun Q;Holleran J;Pollari S;Beutel J;Brown BT;Shinoki Iwaya A;Ardecky R;Harikumar KG;Miller LJ;Sergienko EA
促胰液素受体（SCTR）是一种典型的B类G蛋白偶联受体（GPCR），主要通过与其内源性肽配体促胰液素结合后激活Gαs蛋白来发挥其作用。 SCTR 存在于跨物种的多种组织和器官中，包括胰腺、胃、肝脏、心脏、肺、结肠、肾脏和大脑。除此之外，SCTR介导的信号传导的调节具有治疗多种疾病的潜力，例如心力衰竭、肥胖和糖尿病。然而，除了促胰液素及其肽类似物之外，没有任何配体被描述可以调节 SCTR，这可能是由于 B 族 GPCR 药物发现中固有的挑战。在这里，我们报告了一个测试漏斗的创建，该漏斗允许定向检测 SCTR 小分子激活剂。为了遵循识别正变构调节剂 (PAM) 的策略，我们建立了一种独特的初级筛选测定法，采用三种正构刺激剂的混合物，并在测试 12,000 种小分子化合物的筛选活动中进行比较。除此之外，我们开发了一套全面的二级测定，例如无放射性标记的靶标结合测定和基于 NanoBiT（NanoLuc 二元技术）的方法来检测 β-arrestin-2 募集，所有这些都在高通量环境中可行，并且能够分析配体和命中对结合和受体功能的影响。这种方法的组合使得发现了五种有前途的支架，其中四种已经过验证并进一步表征了它们的变构活性。我们建议我们的结果可以作为开发第一个针对 SCTR 的体内活性小分子的起点。
The secretin receptor (SCTR), a prototypical class B G protein-coupled receptor (GPCR), exerts its effects mainly by activating Gαs proteins upon binding of its endogenous peptide ligand secretin. SCTRs can be found in a variety of tissues and organs across species including pancreas, stomach, liver, heart, lung, colon, kidney and brain. Beyond that, modulation of SCTR-mediated signaling has therapeutic potential for the treatment of multiple diseases, such as heart failure, obesity and diabetes. However, no ligands other than secretin and its peptide analogs have been described to regulate SCTRs, probably due to inherent challenges in family B GPCR drug discovery. Here we report creation of a testing funnel that allowed targeted detection of SCTR small molecule activators. Pursuing the strategy to identify positive allosteric modulators (PAMs), we established a unique primary screening assay employing a mixture of three orthosteric stimulators that was compared in a screening campaign testing 12,000 small molecule compounds. Beyond that, we developed a comprehensive set of secondary assays, such as a radiolabel-free target engagement assay and a NanoBiT (NanoLuc binary technology)-based approach to detect β-arrestin-2 recruitment, all feasible in a high-throughput environment as well as capable of profiling ligands and hits regarding their effect on binding and receptor function. This combination of methods enabled the discovery of five promising scaffolds, four of which have been validated and further characterized with respect to their allosteric activities. We propose that our results may serve as starting points for developing the first in vivo active small molecules targeting SCTRs.
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DOI：
10.2174/1875397301004010084
发表时间：
2010-12-21
期刊：
Current chemical genomics
影响因子：
--
作者：
Cheng Z;Garvin D;Paguio A;Stecha P;Wood K;Fan F
通讯作者：
Fan F
DOI：
10.1152/ajpgi.00371.2019
发表时间：
2020-04-01
影响因子：
4.5
作者：
Brandler, Justin;Miller, Laurence J.;Camilleri, Michael
通讯作者：
Camilleri, Michael
影响因子：
4.8
作者：
King, Klim;Lin, Nai-Pin;Chein, Rong-Jie
通讯作者：
Chein, Rong-Jie
影响因子：
4.6
作者：
Cottet M;Faklaris O;Zwier JM;Trinquet E;Pin JP;Durroux T
通讯作者：
Durroux T
B 类 G 蛋白偶联受体的变构调节剂。
影响因子：
5.3
作者：
Hoare SR
通讯作者：
Hoare SR