Conn P Michael, Smith Emery, Hodder Peter, Janovick Jo Ann, Smithson David C
Divisions of Reproductive Sciences and Neuroscience, Oregon National Primate Research Center, Beaverton, OR 97006, USA.
J Biomol Screen. 2013 Sep;18(8):930-7. doi: 10.1177/1087057113483559. Epub 2013 May 2.
Pharmacoperone drugs correct the folding of misfolded protein mutants and restore function (i.e., "rescue") by correcting the routing of (otherwise) misrouted mutants. Assays for pharmacoperones have not been applied to screen large libraries previously. Currently, most pharmacoperones possess intrinsic agonist or antagonist activities since these were identified using high-throughput screens aimed at discovering direct agonists or antagonists. Here we describe an ultra-high-throughput compatible no-wash assay system designed to specifically identify pharmacoperones of the vasopressin type 2 receptor (V2R). Development of such assays is important and novel since useful chemical structures with the ability to control cellular trafficking but lacking intrinsic agonist or antagonist properties have not likely been identified using existing screens. In the described assay, the level of functional human V2R (hV2R) (mutant) present in each test well is quantitated by stimulation with saturating levels of agonist followed by use of a luminescent-based cyclic adenosine monophosphate assay. This allows the assay to identify compounds that increase the trafficking of mutant hV2R[L(83)Q] in our model system.
药物伴侣分子药物可纠正错误折叠的蛋白质突变体的折叠，并通过纠正（否则）错误转运的突变体的转运途径来恢复功能（即“挽救”）。此前，尚未应用药物伴侣分子的检测方法来筛选大型文库。目前，大多数药物伴侣分子具有内在的激动剂或拮抗剂活性，因为这些是通过旨在发现直接激动剂或拮抗剂的高通量筛选鉴定出来的。在此，我们描述了一种超高通量兼容的免洗检测系统，该系统旨在特异性鉴定血管加压素2型受体（V2R）的药物伴侣分子。开发此类检测方法既重要又新颖，因为使用现有筛选方法不太可能鉴定出具有控制细胞转运能力但缺乏内在激动剂或拮抗剂特性的有用化学结构。在所描述的检测方法中，通过用饱和水平的激动剂刺激，然后使用基于发光的环磷酸腺苷检测法，对每个测试孔中存在的功能性人V2R（hV2R）（突变体）水平进行定量。这使得该检测方法能够鉴定出在我们的模型系统中增加突变体hV2R[L(83)Q]转运的化合物。
