Peters Matthew F, Vaillancourt François, Heroux Madeleine, Valiquette Manon, Scott Clay W
Lead Generation Department, AstraZeneca Pharmaceuticals LP, Wilmington, Delaware 19810, USA.
Assay Drug Dev Technol. 2010 Apr;8(2):219-27. doi: 10.1089/adt.2009.0232.
The diversity and impact of label-free technologies continues to expand in drug discovery. Two classes of label-free instruments, using either an electrical impedance-based or an optical-based biosensor, are now available for investigating the effects of ligands on cellular targets. Studies of GPCR function have been especially prominent with these instruments due to the importance of this target class in drug discovery. Although both classes of biosensors share similar high sensitivity to changes in cell shape and structure, it is unknown whether these biosensors yield similar results when comparing the same GPCR response. Furthermore, since cell morphology changes induced by GPCRs differ depending on which G-protein is activated, there is potential for these instruments to have differential sensitivities to G-protein signaling. Here 1 impedance (CellKey)- and 2 optical-based instruments (BIND and Epic) are compared using Gi-coupled (ACh M2), Gq-coupled (ACh M1), and Gs-coupled (CRF1) receptors. All 3 instruments were robust in agonist and antagonist modes yielding comparable potencies and assay variance. Both the impedance and optical biosensors showed similar high sensitivity for detecting an endogenous D1/D5 receptor response and a melanocortin-4 receptor inverse agonist (agouti-related protein). The impedance-based biosensor was uniquely able to qualitatively distinguish G-protein coupling and reveal dual signaling by CRF1. Finally, responses with a ligand-gated ion channel, TRPV1, were similarly detectable in each instrument. Thus, despite some differences, both impedance- and optical-based platforms offer robust live-cell, label-free assays well suited to drug discovery and typically yield similar pharmacological profiles for GPCR ligands.
无标记技术在药物发现中的多样性和影响力持续扩大。目前有两类无标记仪器可供使用，一类基于电阻抗，另一类基于光学生物传感器，用于研究配体对细胞靶点的作用。由于这类靶点在药物发现中的重要性，利用这些仪器对GPCR功能的研究尤为突出。尽管这两类生物传感器对细胞形状和结构的变化都具有相似的高灵敏度，但在比较相同的GPCR反应时，这些生物传感器是否会产生相似的结果尚不清楚。此外，由于GPCR诱导的细胞形态变化因激活的G蛋白不同而有所差异，这些仪器对G蛋白信号传导的灵敏度可能存在差异。本文使用Gi偶联（ACh M2）、Gq偶联（ACh M1）和Gs偶联（CRF1）受体对1种基于阻抗的仪器（CellKey）和2种基于光学的仪器（BIND和Epic）进行了比较。所有3种仪器在激动剂和拮抗剂模式下都表现出稳健性，产生了相当的效价和测定方差。基于阻抗和光学的生物传感器在检测内源性D1/D5受体反应和黑皮质素-4受体反向激动剂（刺鼠相关蛋白）时都表现出相似的高灵敏度。基于阻抗的生物传感器能够独特地定性区分G蛋白偶联，并揭示CRF1的双重信号传导。最后，在每种仪器中都能类似地检测到配体门控离子通道TRPV1的反应。因此，尽管存在一些差异，但基于阻抗和光学的平台都提供了强大的活细胞无标记检测方法，非常适合药物发现，并且通常为GPCR配体产生相似的药理学特征。
