Screen:Blockers of a CaV2.2-Mint-PDZ1 association (RMI)

筛选：CaV2.2-Mint-PDZ1 关联 (RMI) 的阻断剂

• 批准号: 6879432
• 负责人: Ilya B Bezprozvanny
• 金额: $ 7.8万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 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/6879432
• 关键词: biotechnology; calcium channel blockers; chemical registry /resource; chimeric proteins; chronic pain; drug discovery /isolation; fluorescence resonance energy transfer; high throughput technology; peptide chemical synthesis; protein binding; protein localization; protein protein interaction; small molecule; technology /technique development; voltage gated channel

DESCRIPTION (provided by applicant): The broad, long-term objectives of the project are to setup and optimize a high-throughput screen (HTS) for small molecule inhibitors of association between carboxy-terminal region of N-type calcium channel and Mintl-PDZ1 domain. N-type Ca2+ channels play a predominant role in the sensation of pain. Opening of presynaptic N-type (CaV2.2) Ca2+ channels results in Ca2+ influx into presynaptic terminal, synaptic vesicle fusion and secretion of a neurotransmitter. In previous biochemical experiments we discovered the association of carboxy-terminal regions of CaV2.2 subunit with Mintl-PDZ1 domain and demonstrated that association of CaV2.2 with Mintl-PDZ1 is important for synaptic targeting and synaptic function of N-type Ca2+ channels. Based on these results we proposed that small molecule inhibitors of association between CaV2.2 and Mintl-PDZ1 may be used to treat chronic pain (neuropathic pain, inflammatory pain, cancer pain). To identify such inhibitors, we intend to: Use scintillation-proximity assay (SPA, Aim 1) and homogeneous time-resolved fluorescence resonance energy transfer assay (HTRF, Aim 2) to screen for small-molecule inhibitors of association between CaV2.2carboxy-terminal fragment (NC peptide) and Mintl-PDZ1 domain expressed as GST-fusion protein (GSTMintl-PDZ1). The proposed SPA and HTRF HTS screens will be optimized with the test library of 8,000compounds to yield a Z factor of at least 0.5 (Aim 3).Assay that yields most robust results (SPA or HTRF) will be used in HTS screen with a full library of150,000-500,000 compounds. Biological activity of compounds identified in the full HTS screen will be tested in whole animal pain assays (formalin, hot plate, tail flick).

描述（由申请人提供）：项目的广泛，长期目标是设置和优化一个高通量屏幕（HTS），用于在N型钙通道和Mintl-PDZ1领域的羧基末端区域之间关联的小分子抑制剂。 N型Ca2+通道在疼痛的感觉中起主要作用。 突触前N型（CAV2.2）Ca2+通道的开放会导致Ca2+流入到突触前末端，突触囊泡融合和神经递质的分泌。 在先前的生化实验中，我们发现Cav2.2亚基与MINTL-PDZ1结构域的羧基末端区域的关联，并证明Cav2.2与Mintl-PDZ1的关联对于N型Ca2+通道的突触靶向和突触功能很重要。 基于这些结果，我们提出，Cav2.2和Mintl-PDZ1之间关联的小分子抑制剂可用于治疗慢性疼痛（神经性疼痛，炎症性疼痛，癌症疼痛）。 为了识别这种抑制剂，我们打算： 使用闪烁 - 抗性测定（SPA，AIM 1）和均匀的时间分辨荧光共振能量转移测定（HTRF，AIM 2），以筛选CAV2.2Carboxy末端末端片段（NC肽）和Mintl-PDZ1 Domain Domain Domain domain domain Expecte aS GSt-Fiuse的小分子抑制剂（cav2.2Carboxy-boxy末端）之间的关联抑制剂。 所提出的SPA和HTRF HTS屏幕将通过8,000个组合的测试库进行优化，以产生至少0.5的Z系数（AIM 3）.-串在HTS屏幕中使用最强大的结果（SPA或HTRF），其完整库中的完整库为150,000-500,000，000，000，000，000年。 在全HTS筛选中鉴定出的化合物的生物学活性将在全动物疼痛测定中进行测试（福尔马林，热板，尾部轻弹）。