Development of High Throughput Assays for HVA CA Channels

HVA CA 通道高通量检测的开发

• 批准号: 7049771
• 负责人: EDWARD PEREZ-REYES
• 金额: $ 17.11万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7049771
• 关键词: afferent nerve; analgesics; binding sites; biotechnology; calcium channel; calcium channel blockers; calcium flux; cell line; chronic pain; conotoxin; dihydropyridines; drug screening /evaluation; fluorescent dye /probe; fluorimetry; high throughput technology; neuropharmacology; protein structure function; recombinant proteins; sensory neuropathy; small molecule; spinal ganglion; technology /technique development; transfection /expression vector; voltage /patch clamp; voltage gated channel

DESCRIPTION (provided by applicant): Voltage-gated calcium channels are an established drug target. Nevertheless, therapeutically useful drugs only target one of the ten members of the Ca2+ channel family, the cardiovascular L-type channel (Cav1.2). Considerable evidence supports the hypothesis that a small molecule blocker of N-type channels (Cav2.2) would be effective in the treatment of chronic pain. N-type channels in sensory neurons mediate calcium influx into presynaptic terminals, thereby triggering neurotransmitter release onto neurons in the dorsal horn of the spinal cord. Knock-out of the gene encoding the a1 subunits of N-type channels (?12.2) diminishes pain sensitivity and reduces the development of neuropathic pain symptoms after spinal nerve ligation. Finally, ziconotide, a peptide toxin that is highly selective for N-type channels, demonstrated safety and efficacy in clinical trials for the treatment of intractable pain in cancer and AIDS patients after intrathecal infusion. These studies establish the proof-of-concept that an orally available small molecule blocker of N-type channels would be a major therapeutic advance for chronic pain. Two major obstacles have hampered the development of such drugs: one, N-type channels mediate neurotransmitter release at many synapses, so a blocker might have many side effects; and two, the lack of a high throughput assay to screen candidate compounds. Recent studies demonstrate that nociceptive neurons express a specific splice variant isoform of ?12.2. Therefore, a selective and state-dependent blocker of this isoform might produce analgesia without side effects. The goal of this grant is to develop stable cell lines of recombinant N-type channels that will be useful in high throughput screening. The cell lines will be tested for channel expression using whole cell clamp electrophysiology, and their usefulness in a screen will be tested using a fluorescent dye assay to measure calcium influx. A final goal is to test whether the N-type channel variants have unique pharmacological profiles. Chronic pain continues to be a major public health problem, affecting 40 million Americans, with little relief from current drugs. By targeting an important protein in the pain pathway, the research funded by this grant will provide tools that can be used to screen candidate compounds during the development of novel analgesics.

描述（由申请人提供）： 电压门控钙通道是既定的药物靶标。然而，治疗上有用的药物仅针对Ca2+通道家族的十个成员之一，即心血管L型通道（CAV1.2）。大量证据支持以下假设：N型通道的小分子阻滞剂（CAV2.2）将有效治疗慢性疼痛。感觉神经元中的N型通道介导钙涌入突触前末端，从而触发神经递质释放到脊髓背角的神经元上。编码N型通道A1亚基的基因（？12.2）的敲除可以减轻疼痛的敏感性，并减少脊柱神经结扎后神经性疼痛症状的发展。最后，Ziconotide是一种对N型通道具有高度选择性的肽毒素，在临床试验中表现出安全性和功效，用于治疗肠内输注后癌症和AIDS患者的可治性疼痛。这些研究确定了概念概念，即N型通道的口服小分子阻滞剂将是慢性疼痛的主要治疗疗法。两个主要障碍阻碍了此类药物的发展：一种，N型通道介导许多突触的神经递质释放，因此阻断剂可能会产生许多副作用。第二，缺乏对筛选候选化合物的高吞吐量测定。最近的研究表明，伤害感受神经元表达特定的剪接变体同工型为12.2。因此，该同工型的选择性和状态依赖性阻滞剂可能会产生镇痛，而无需副作用。该赠款的目的是开发重组N型通道的稳定细胞系，这将在高吞吐量筛选中有用。将使用全细胞夹电生理学测试细胞系的通道表达，并将使用荧光染料测定法测量筛网中的有用性来测量钙涌入。最终目标是测试N型通道变体是否具有独特的药理概况。 慢性疼痛仍然是一个主要的公共卫生问题，影响了4000万美国人，而目前的药物几乎没有缓解。通过靶向疼痛途径中的重要蛋白质，该赠款资助的研究将提供可用于筛选候选化合物在新型镇痛药过程中使用的工具。