HTS Assay for Ca 3 T-type Channels Using FLIPR (RMI)

使用 FLIPR (RMI) 进行 Ca 3 T 型通道的 HTS 测定

• 批准号: 6879398
• 负责人: Xinmin Simon Xie
• 金额: $ 9.99万
• 依托单位: SRI INTERNATIONAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6879398
• 关键词: biotechnology; calcium channel; calcium channel blockers; calcium flux; chemical registry /resource; computer data analysis; drug discovery /isolation; fluorescent dye /probe; fluorimetry; high throughput technology; tissue /cell culture; voltage /patch clamp

DESCRIPTION (provided by applicant): T-type Ca2+ channels, encoded by three genes (Cav3.1, 3.2 and 3.3, or alpha1-G, H, and I), are heterogeneously expressed in the brain and many peripheral organs such as the heart and vascular smooth muscle. They play a role as the pacemaker that regulates spontaneous neuronal activity, cardiac rhythm and vascular tone. T-channels have been a drug target for absence epilepsy and hypertension. The conventional anti-absence epilepsy drug ethosuximide produces weak inhibition of the T-channel. The anti-hypertensive drug, mibefradil, potently blocks the T channel but was withdrawn from the market due to drug interaction. Inhibition of T-channels by 619C89 and SB-209712 may contribute to the neuroprotective effects of the compounds; however, these drugs are non-specific and produce many side effects. Phenotypic studies on the Cav3.1 or 3.2 gene knockout mice have shed light on the biological role of T-channels and suggest the T-channel could be a potential target for therapeutic intervention in pathological pain and cardiovascular diseases. There is no doubt that subtype-selective and potent Cav3 modulators should produce more specific pharmacological actions with fewer side effects. High throughput screening (HTS) of a large compound library is the initial step to identify such a novel compound. Standard electrophysiological techniques are not suitable for primary HTS. A recent study using single cell Ca2+ imaging techniques has demonstrated that manipulation of extracellular Ca2+ significantly changes the intracellular Ca2+ ([Ca2+]j) in the HEK293 cells expressing human Cav3.2 channels (Cav3.2-HEK293). Detection of [Ca2+]j indirectly measures Ca2+ entry through spontaneous opening of the Cav3.2 channels (i.e., window currents). This method is amenable to HTS. In a pilot study we tested the feasibility using the Ca2+ sensitive dye fluo-4/AM and measured with a fluorometric imaging plate reader (FLIPRR) in a 96-well format. In this proposal, our specific aims are to (1) develop a 384-well HTS format through optimization of assay conditions and automation of procedures such as the integration of a robotic system and automated data analysis, and (2) validate this fluorometric method by comparison of the potency of a large number of ion channel inhibitors determined using both the FLIPR assay and whole-cell voltage-clamp recording techniques. The proposed fluorometric method will provide a high throughput and quantitative assay for the primary screening of compounds against three subtypes of Cav3 T-type channels.

描述（由申请人提供）：t型Ca2+通道由三个基因（Cav3.1, 3.2和3.3，或alpha1-G， H和I）编码，在大脑和许多外周器官如心脏和血管平滑肌中异质表达。它们起着调节自发神经元活动、心律和血管张力的起搏器的作用。t通道已成为治疗癫痫和高血压的药物靶点。传统的抗癫痫药物乙thosuximide对t通道产生微弱的抑制作用。降压药米贝替拉能有效阻断T通道，但由于药物相互作用而退出市场。619C89和SB-209712对t通道的抑制作用可能与这两种化合物的神经保护作用有关；然而，这些药物是非特异性的，而且会产生许多副作用。对Cav3.1或3.2基因敲除小鼠的表型研究揭示了t通道的生物学作用，并提示t通道可能是病理性疼痛和心血管疾病治疗干预的潜在靶点。