Therapeutic rescue of misfolded mutants: validation of primary high throughput screens for identification of pharmacoperone drugs.
Therapeutic rescue of misfolded mutants: validation of primary high throughput screens for identification of pharmacoperone drugs.
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对错误折叠突变体的治疗救助：对原发性高吞吐量筛选的验证，用于鉴定药物药物。
DOI：
10.1371/journal.pone.0022784
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Conn PM
中科院分区：
文献类型：
--
作者：
Janovick JA;Park BS;Conn PM
小非肽分子对错误折叠突变受体的功能性拯救已被证明。这些小目标特异性分子（药理学伴侣或“pharmacoperones”）充当分子模板，促进正确折叠，并允许错误折叠的突变体通过细胞质量控制系统（QCS）的审查，并在质膜（PM）上表达，其功能与野生型（WT）蛋白类似。就促性腺激素释放激素受体 (GnRHR) 而言，拯救一种突变体的药物通常可以拯救许多突变体，即使突变位于较远的位点（细胞外环、细胞内环、跨膜螺旋）。这增加了这些药物的价值。这些药物通常是事后从旨在检测拮抗剂或激动剂的屏幕中的“命中”中识别出来的。由于缺乏能够鉴定Pharmaperones本身的筛选，因此Pharmaperones的治疗效用受到限制。我们描述了一种通用的 pharmaperone 药物初步筛选方法，该方法基于稳定表达两种不同模型 G 蛋白偶联受体 (GPCR)（hGnRHR[E90K] 或 hV2R[L83Q]）突变体的稳定 HeLa 细胞中的活性增益测量。在四环素及其类似物存在的情况下，这些细胞关闭感兴趣的受体突变基因的表达，这提供了识别假阳性的便捷方法。本文描述和表征的方法为用于检测拯救特定受体的 GPCR 突变体的药物的 pharmaperones 的新型初级筛选提供了基础。这种方法将识别在旨在仅选择激动剂或拮抗剂的筛选中被遗漏的结构。非拮抗性药用哌酮具有治疗优势，因为它们不会与内源性激动剂竞争，并且一旦发生救援并且在被内源性或外源性激动剂激活之前可能不必被洗掉。
Functional rescue of misfolded mutant receptors by small non-peptide molecules has been demonstrated. These small, target-specific molecules (pharmacological chaperones or “pharmacoperones”) serve as molecular templates, promote correct folding and allow otherwise misfolded mutants to pass the scrutiny of the cellular quality control system (QCS) and be expressed at the plasma membrane (PM) where they function similarly to wild type (WT) proteins. In the case of the gonadotropin releasing hormone receptor (GnRHR), drugs that rescue one mutant typically rescue many mutants, even if the mutations are located at distant sites (extracellular loops, intracellular loops, transmembrane helices). This increases the value of these drugs. These drugs are typically identified, post hoc, from “hits” in screens designed to detect antagonists or agonists. The therapeutic utility of pharmacoperones has been limited due to the absence of screens that enable identification of pharmacoperones per se. We describe a generalizable primary screening approach for pharmacoperone drugs based on measurement of gain of activity in stable HeLa cells stably expressing the mutants of two different model G-protein coupled receptors (GPCRs) (hGnRHR[E90K] or hV2R[L83Q]). These cells turn off expression of the receptor mutant gene of interest in the presence of tetracycline and its analogs, which provides a convenient means to identify false positives. The methods described and characterized here provide the basis of novel primary screens for pharmacoperones that detect drugs that rescue GPCR mutants of specific receptors. This approach will identify structures that would have been missed in screens that were designed to select only agonists or antagonists. Non-antagonistic pharmacoperones have a therapeutic advantage since they will not compete for endogenous agonists and may not have to be washed out once rescue has occurred and before activation by endogenous or exogenous agonists.
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影响因子：
--
作者：
Conn, P Michael;Leanos-Miranda, Alfredo;Janovick, Jo Ann
通讯作者：
Janovick, Jo Ann
影响因子：
5.8
作者：
Janovick, JA;Maya-Nunez, G;Conn, PM
通讯作者：
Conn, PM
影响因子：
4.8
作者：
Zhang, XM;Wang, XT;Guggino, SE
通讯作者：
Guggino, SE
影响因子：
4.2
作者：
Amaral, MD
通讯作者：
Amaral, MD
DOI：
10.1124/jpet.102.048454
发表时间：
2003-05-01
影响因子：
3.5
作者：
Janovick, JA;Goulet, M;Conn, PM
通讯作者：
Conn, PM