Development of High Throughput Assays for N-type Calcium Channels

N 型钙通道高通量检测的开发

• 批准号: 7345651
• 负责人: EDWARD PEREZ-REYES
• 金额: $ 16.55万
• 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/7345651
• 关键词: Absence of pain sensation; Acquired Immunodeficiency Syndrome; Adverse effects; Affect; Afferent Neurons; American; Amlodipine; Analgesics; Binding; Binding Sites; Biological Assay; Brain; Calcium; Calcium Channel; Cardiac; Cardiovascular system; Cell Line; Cells; Chromosome Pairing; Clinical Treatment; Clinical Trials; Complex; Conotoxin; Data; Dependence; Development; Dihydropyridines; Drug Delivery Systems; Dyes; Electrophysiology (science); Family; Fluo 4; Fluorescent Dyes; Funding; Genes; Goals; Grant; Image; Infusion procedures; Intractable Pain; Knock-out; Ligation; Malignant Neoplasms; Measures; Mediating; Membrane; Membrane Potentials; Messenger RNA; Molecular Conformation; N-Type Calcium Channels; Neurons; Nicardipine; Nociception; Pain; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Pharmacology; Play; Presynaptic Terminals; Property; Protein Isoforms; Proteins; Public Health; RNA Splicing; Reader; Recombinants; Reporting; Research; Research Personnel; Rest; Ribosomes; Role; Safety; Signal Transduction; Smooth Muscle; Spinal cord posterior horn; Spinal nerve structure; Structure; Symptoms; Synapses; Testing; Therapeutic; Tissues; Toxin; Variant; analog; base; chronic pain; concept; dihydropyridine; expression vector; high throughput screening; hypertension treatment; member; neurotransmitter release; novel; novel therapeutics; painful neuropathy; patch clamp; programs; protein expression; small molecule; stable cell line; therapeutic target; tool; vector; voltage; voltage gated channel; ziconotide

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.

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