Development of High Throughput Assays for N-type Calcium Channels

描述（由申请人提供）： 电压门控钙通道是一个既定的药物靶点。然而，治疗上有用的药物仅针对Ca 2+通道家族的十个成员之一，心血管L型通道（Cav1.2）。大量证据支持N型通道的小分子阻断剂（Cav2.2）将有效治疗慢性疼痛的假设。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中的神经元上。敲除编码N型通道α 1亚基的基因（？12.2）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，齐考诺肽是一种对N型通道具有高度选择性的肽毒素，在鞘内输注后治疗癌症和艾滋病患者顽固性疼痛的临床试验中证明了其安全性和有效性。这些研究建立了概念验证，即口服N型通道的小分子阻滞剂将是慢性疼痛的主要治疗进展。两个主要障碍阻碍了此类药物的开发：一是N型通道介导许多突触的神经递质释放，因此阻断剂可能有许多副作用;二是缺乏高通量试验来筛选候选化合物。最近的研究表明，伤害性神经元表达一个特定的剪接变体异构体？12.2.因此，这种亚型的选择性和状态依赖性阻断剂可能会产生镇痛而无副作用。这项资助的目标是开发重组N型通道的稳定细胞系，这将有助于高通量筛选。将使用全细胞钳电生理学测试细胞系的通道表达，并且将使用荧光染料测定法测试其在筛选中的有用性以测量钙内流。最终的目标是测试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，目前的药物几乎没有缓解。通过靶向疼痛通路中的一种重要蛋白质，这项研究将提供可用于在开发新型镇痛药期间筛选候选化合物的工具。

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.