High throughput screening of ligands of TRP channels

描述（由申请人提供）：瞬时受体电位（TRP）通道已成为各种内部和外部信号的细胞传感器。典型TRP （TRPC）通道参与受体和储存操作的Ca2+进入，这是磷脂酶c激活的Ca2+信号级联的重要组成部分。在静息电位下，这些通道也引导Na+内流，允许细胞以受体操作的方式去极化。TRPC通道参与了许多功能的调节，包括血管中的平滑肌和血管张力、内皮通透性、受精、唾液分泌、突触传递和神经突生长。我们的长期目标是了解细胞内Ca2+信号的调节机制及其在人类健康中的作用。然而，由于缺乏特异性的激动剂和拮抗剂，严重阻碍了对不同TRP通道生理功能表征和调控机制的研究。生物研究界迫切需要针对不同TRP成员的药理学工具。此外，一些色氨酸通道与人类疾病有关。针对TRP通道的特异性药物的开发也可能具有治疗意义。当前提案的目标是开发基于微孔板的检测方法来监测TRP通道功能，并将其配置为高通量筛选。重点将放在TRPC通道与使用基于荧光膜电位测定。选定数量的TRPV和TRPM通道也将用于反筛。细胞内Ca2+测量将用作二次筛选，全细胞电压钳实验将用于活性化合物的最终验证。提出了两个具体目标。目的：我将探索在基于微孔板的荧光膜电位测定中，产生HEK293细胞中稳定表达的TRPC通道受体诱导激活的最佳信噪比、高一致性和最佳再现性的条件。AIM II将配置高通量筛选的分析。TRPC5和TRPC6的中试筛选将使用2-氨基乙氧基二苯硼酸盐和经典l型Ca2+通道阻滞剂的类似物进行。配置的分析条件和细胞系将在分子文库筛选中心网络中用于筛选TRPC通道的配体。

DESCRIPTION (provided by applicant): Transient receptor potential (TRP) channels have emerged as cellular sensors of various internal and external cues. The canonical TRP (TRPC) channels are involved in receptor and store-operated Ca2+ entry, an important component of phospholipase C-activated Ca2+ signaling cascade. Under resting potentials, these channels also conduct Na+ influx, allowing cells to be depolarized in a receptor-operated manner. TRPC channels have been implicated in the regulation of many functions, including smooth muscle and vascular tones in blood vessels, endothelial permeability, fertilization, saliva secretion, synaptic transmission, and neurite outgrowth. Our long-term objective is to understand the mechanism of regulation of Ca2+ signal inside cells and its roles in human health. However, the lack of specific agonists and antagonists has seriously hindered the studies on the characterization of physiological functions as well as the mechanisms of regulation of different TRP channels. The biological research community has an urgent need for pharmacological tools that are specifically targeted at different TRP members. Moreover, several TRP channels have been implicated in human diseases. The development of specific drugs for TRP channels could also have therapeutic implications. The goals of the current proposal are to develop microplate-based assays to monitor TRP channel function and to configure them for high throughput screening. Emphasis will be placed on TRPC channels with the use of fluorescence-based membrane potential assays. A selected number of TRPV and TRPM channels will also be used for counter-screens. Intracellular Ca2+ measurement will be used as secondary screens and whole cell voltage clamp experiments will be used for final verification of the active compounds. Two specific aims are proposed. AIM I will explore conditions that give rise to the best signal-to-noise ratio, high consistency, and optimal reproducibility of receptor-induced activation of TRPC channels stably expressed in HEK293 cells in microplate-based fluorescence membrane potential assays. AIM II will configure the assays for high throughput screening. Pilot screens on TRPC5 and TRPC6 will be performed using analogs of 2-aminoethoxydiphenyl borate and classical L-type Ca2+ channel blockers. The configured assay conditions and cell lines are to be used in the Molecular Libraries Screening Centers Network to screen for ligands of TRPC channels.