Swensen Andrew M, Niforatos Wende, Vortherms Timothy A, Perner Richard J, Li Tao, Schrimpf Michael R, Scott Victoria E, Lee Lance, Jarvis Michael F, McGaraughty Steve
Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois 60064-6118, USA.
Assay Drug Dev Technol. 2012 Dec;10(6):542-50. doi: 10.1089/adt.2011.437. Epub 2012 Mar 19.
Ca(V)2.2 (N-type) calcium channels are key regulators of neurotransmission. Evidence from knockout animals and localization studies suggest that Ca(V)2.2 channels play a critical role in nociceptive transmission. Additionally, ziconotide, a selective peptide inhibitor of Ca(V)2.2 channels, is clinically used to treat refractory pain. However, the use of ziconotide is limited by its low therapeutic index, which is believed, at least in part, to be a consequence of ziconotide inhibiting Ca(V)2.2 channels regardless of the channel state. Subsequent efforts have focused on the discovery of state-dependent inhibitors that preferentially bind to the inactivated state of Ca(V)2.2 channels in order to achieve an improved safety profile relative to ziconotide. Much less attention has been paid to understanding the binding kinetics of these state-dependent inhibitors. Here, we describe a novel electrophysiology-based assay on an automated patch platform designed to differentiate Ca(V)2.2 inhibitors based on their combined state dependence and kinetics. More specifically, this assay assesses inactivated state block, closed state block, and monitors the kinetics of recovery from block when channels move between states. Additionally, a use-dependent assay is described that uses a train of depolarizing pulses to drive channels to a similar level of inactivation for comparison. This use-dependent protocol also provides information on the kinetics of block development. Data are provided to show how these assays can be utilized to screen for kinetic diversity within and across chemical classes.
Ca(V)2.2（N型）钙通道是神经传递的关键调节因子。基因敲除动物实验和定位研究的证据表明，Ca(V)2.2通道在伤害性传递中起关键作用。此外，齐考诺肽是一种Ca(V)2.2通道的选择性肽类抑制剂，临床上用于治疗顽固性疼痛。然而，齐考诺肽的使用受到其低治疗指数的限制，这至少部分被认为是齐考诺肽无论通道状态如何都抑制Ca(V)2.2通道的结果。随后的研究致力于发现状态依赖性抑制剂，这些抑制剂优先结合Ca(V)2.2通道的失活状态，以相对于齐考诺肽获得更好的安全性。人们对齐考诺肽这类状态依赖性抑制剂的结合动力学了解甚少。在此，我们描述了一种基于电生理学的新型检测方法，该方法在自动化膜片平台上进行，旨在根据Ca(V)2.2抑制剂的状态依赖性和动力学特性对其进行区分。更具体地说，该检测方法评估失活状态阻断、关闭状态阻断，并监测通道在不同状态间转换时从阻断中恢复的动力学过程。此外，还描述了一种使用依赖性检测方法，该方法使用一系列去极化脉冲将通道驱动到相似的失活水平进行比较。这种使用依赖性方案还提供了阻断发展动力学的信息。文中提供的数据展示了如何利用这些检测方法在化学类别内部和之间筛选动力学多样性。
