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）： 电压门控钙通道是已确定的药物靶点。然而，治疗上有用的药物仅针对 Ca2+ 通道家族的 10 个成员之一，即心血管 L 型通道 (Cav1.2)。大量证据支持这样的假设：N 型通道小分子阻断剂 (Cav2.2) 可有效治疗慢性疼痛。感觉神经元中的 N 型通道介导钙流入突触前末梢，从而触发神经递质释放到脊髓背角的神经元上。敲除编码 N 型通道 a1 亚基 (?12.2) 的基因可降低疼痛敏感性，并减少脊神经结扎后神经性疼痛症状的发展。最后，齐考诺肽，一种对N型通道具有高度选择性的肽毒素，在治疗癌症和艾滋病患者鞘内输注后顽固性疼痛的临床试验中证明了安全性和有效性。这些研究证实了口服小分子 N 型通道阻滞剂将成为慢性疼痛治疗的重大进展。阻碍此类药物开发的两大障碍：一是N型通道在许多突触处介导神经递质释放，因此阻滞剂可能会产生许多副作用；第二，缺乏筛选候选化合物的高通量测定方法。最近的研究表明，伤害性神经元表达特定的剪接变体亚型 ?12.2。因此，这种异构体的选择性和状态依赖性阻断剂可能会产生镇痛作用而没有副作用。这笔资助的目标是开发重组 N 型通道的稳定细胞系，该细胞系可用于高通量筛选。将使用全细胞钳电生理学测试细胞系的通道表达，并使用荧光染料测定来测试它们在筛选中的有用性以测量钙流入。最终目标是测试 N 型通道变体是否具有独特的药理学特征。 慢性疼痛仍然是一个主要的公共卫生问题，影响着 4000 万美国人，目前的药物几乎无法缓解疼痛。通过针对疼痛途径中的重要蛋白质，这笔资助资助的研究将提供可用于在新型镇痛药开发过程中筛选候选化合物的工具。