Reversible inhibitors of regulators of G-protein signaling identified in a high-throughput cell-based calcium signaling assay
Reversible inhibitors of regulators of G-protein signaling identified in a high-throughput cell-based calcium signaling assay
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DOI：
10.1016/j.cellsig.2013.09.007
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发表时间：
2013-12-01
影响因子：
4.8
通讯作者：
Neubig, Richard R.
中科院分区：
文献类型：
--
作者：
Storaska, Andrew J.;Mei, Jian P.;Neubig, Richard R.
G 蛋白信号传导 (RGS) 蛋白调节剂通过加速激活的异三聚体 G 蛋白 α 亚基上的 GTP 水解，有效抑制 G 蛋白偶联受体 (GPCR) 信号转导。 RGS4 在中枢神经系统中富集，被提议作为治疗神经病理状态（包括癫痫和帕金森病）的治疗靶点。因此，新型 RGS4 抑制剂的鉴定很有意义。采用稳定表达 M-3-毒蕈碱受体且具有多西环素调节的 RGS4 表达的 HEK293-FlpIn 细胞系来鉴定抑制 RGS4 介导的 M3-毒蕈碱受体信号传导抑制的化合物。筛选了超过 300,000 种在 RGS4 存在的情况下增强 G α(q) 介导的钙信号传导能力的化合物。没有寻找在不存在 RGS4 的情况下在反筛选中调节钙反应的化合物。在 1365 个 RGS4 依赖性初级筛选命中中，有 13 种化合物直接针对纯化系统中的 RGS-G 蛋白相互作用。所有 13 种化合物都失去了针对缺乏半胱氨酸的 RGS4 突变体的活性，表明 RGS4 上游离硫醇基团的共价修饰是一种常见机制。四种化合物在 100 muM 浓度下对 RGS4-G 蛋白结合产生 >85% 的抑制，但在 10 分钟内可逆性 >50%。这四种可逆化合物显着改变了 RGS4 的热解链温度，但没有改变 G 蛋白，表明抑制作用是通过与 RCS 蛋白相互作用而发生的。本研究采用的 HEK 细胞系提供了一个强大的工具，可在 GPCR 信号通路背景下有效识别 RCS 特异性调节剂。因此，几种新的可逆、细胞活性 RGS4 抑制剂已被确定用于未来的生物学研究。 (C) 2013 Elsevier Inc. 保留所有权利。
Regulator of G-protein signaling (RGS) proteins potently suppress G-protein coupled receptor (GPCR) signal transduction by accelerating GTP hydrolysis on activated heterotrimeric G-protein alpha subunits. RGS4 is enriched in the CNS and is proposed as a therapeutic target for treatment of neuropathological states includig epilepsy and Parkinson's disease. Therefore, identification of novel RGS4 inhibitors is of interest. An HEK293-FlpIn cell-line stably expressing M-3-muscarinic receptor with doxycycline-regulated RGS4 expression was employed to identify compounds that inhibit RGS4-mediated suppression of M3-muscarinic receptor signaling. Over 300,000 compounds were screened for an ability to enhance G alpha(q)-mediated calcium signaling in the presence of RGS4. Compounds that modulated the calcium response in a counter-screen in the absence of RGS4 were not pursued. Of the 1365 RGS4-dependent primary screen hits, thirteen compounds directly target the RGS-G-protein interaction in purified systems. All thirteen compounds lose activity against an RGS4 mutant lacking cysteines, indicating that covalent modification of free thiol groups on RGS4 is a common mechanism. Four compounds produce >85% inhibition of RGS4-G-protein binding at 100 mu M, yet are >50% reversible within a ten-minute time frame. The four reversible compounds significantly alter the thermal melting temperature of RGS4, but not G-protein, indicating that inhibition is occurring through interaction with the RCS protein. The HEK cell-line employed for this study provides a powerful tool for efficiently identifying RCS-specific modulators within the context of a GPCR signaling pathway. As a result, several new reversible, cell-active RGS4 inhibitors have been identified for use in future biological studies. (C) 2013 Elsevier Inc. All rights reserved.