G-protein-coupled receptors (GPCRs) are modulated by many marketed drugs, and as such, they continue to be key targets for drug discovery and development. Many GPCR targets at Merck Research Laboratories (MRL) are profiled using homogenous time-resolved fluorescence (HTRF) inositol monophosphate (IP-1) cell-based functional assays using adherent cells in 384-well microplates. Due to discrepancies observed across several in vitro assays supporting lead optimization structure-activity relationship (SAR) efforts, different assay paradigms were evaluated for removing growth medium from the assay plates prior to compound addition and determination of IP-1 accumulation. Remarkably, employing the noncontact centrifugation BlueWasher method leads to left-shifted potencies across multiple structural classes and rescues "false negatives" relative to the traditional manual evacuation method. Further, assay performance is improved, with the minimum significant ratio of challenging chemotypes dropping from ~5-6 to <3. While the impact of BlueWasher on a broad range of our GPCR targets remains to be determined, for highly protein-bound small molecules, it provides a path toward improving assay reproducibility across scientists and sites as well as reducing replicates in SAR assay support.

中文翻译：

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基于细胞的体外测定自动化：平衡技术和数据可重复性/可预测性。

G蛋白偶联受体（GPCR）受许多市售药物调节，因此，它们仍继续是药物发现和开发的主要目标。默克研究实验室（MRL）的许多GPCR靶标均使用均质时间分辨荧光（HTRF）肌醇单磷酸酯（IP-1）细胞功能分析，在384孔微孔板中使用贴壁细胞进行了分析。由于在支持铅最优化结构-活性关系（SAR）努力的几个体外测定中观察到差异，因此在添加化合物和确定IP-1积累之前，对不同的测定范例进行了评估，以从测定板上去除生长培养基。值得注意的是，采用非接触式离心BlueWasher方法可在多个结构类别中产生左移效价，并挽救“假阴性” 相对于传统的手动疏散方法。此外，测定性能得以提高，挑战性化学型的最小显着比例从〜5-6降至<3。尽管BlueWasher对我们广泛的GPCR靶点的影响尚待确定，但对于高度结合蛋白质的小分子而言，它为提高科学家和站点之间的测定重现性以及减少SAR测定支持中的重复提供了一条途径。