Exploring vasomotor mechanisms using new PKG inhibitors

使用新型 PKG 抑制剂探索血管舒缩机制

• 批准号: 6422573
• 负责人: WOLFGANG R DOSTMANN
• 金额: $ 35.25万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6422573
• 关键词: Escherichia coli; SDS polyacrylamide gel electrophoresis; Sf9 cell line; biological signal transduction; cGMP dependent protein kinase; calcium channel; chemical kinetics; combinatorial chemistry; confocal scanning microscopy; enzyme inhibitors; enzyme mechanism; fluorescent dye /probe; intracellular transport; membrane permeability; membrane potentials; peptide chemical synthesis; peptide library; potassium channel; radiotracer; tissue /cell culture; vascular endothelium; vascular smooth muscle; vasoconstriction; voltage /patch clamp; western blottings

DESCRIPTION (provided by applicant): Cyclic GMP-dependent protein kinase (PKG) plays a central role in the regulation of vascular smooth muscle tone. Activation of PKG leads to alterations in intracellular Ca2+, which in turn, effects multiple cellular signaling pathways. However, progress in understanding the specific functional roles of PKG in vascular smooth muscle has been hampered by the lack of specific PKG inhibitors. We have developed novel, cell-permeable PKG specific peptide inhibitors with unprecedented potency and kinase specificity by fusion of peptides derived from combinatorial libraries with membrane translocation signal (MTS) peptides. These fusion peptides result in an extraordinary synergism with respect to PKG inhibition. In the proposed experiments, these PKG selective inhibitors will be further developed, refined and used to study specific functional roles of PKG in vascular smooth muscle. Specific Aim 1 will develop novel peptide library design strategies based on binding- and competition libraries. This approach should lead to new and mole potent PKG inhibitors than are currently available. Specific Aim 2 will explore variations of the MTS sequences with respect to their use for intracellular delivery and their ability to synergize with the library derived peptide inhibitors. Specific Aim 3 will determine the contributions of PKG in the regulation of vascular tone in intact resistance arteries by measuring the effects of PKG inhibitors on arterial diameter as well as ion channel activity in single vascular smooth muscle cells. To pursue the aims of this proposal, a multi-faceted approach will be used, including state-of-the-art techniques to: 1) screen and synthesize selective PKG inhibitor peptides using combinatorial library approaches, 2) deliver peptide inhibitors using membrane translocation peptide sequences and monitor the intracellular accumulation of these compounds using kinase activity assays, fluorescence spectroscopy and confocal microscopy, and 3) assess the efficacy of the PKG inhibitors using functional assays of ion channel activity (patch clamp) and vascular contractility (myography). The studies outlined above will provide new, selective, and potent inhibitors of PKG which will be useful in revealing the fundamental physiological roles of PKG in regulation of arterial tone. The application of the inhibitors that emerge from these studies will not only demonstrate the essential role of PKG in regulation of vascular tone in resistance arteries, but also significantly advance the field of protein kinase signaling in general. This work may also suggest novel targets for therapeutic interventions in vascular diseases such as hypertension and septic shock.

描述（由申请人提供）：环 GMP 依赖性蛋白激酶（PKG） 在血管平滑肌张力的调节中起核心作用。 PKG 的激活会导致细胞内 Ca2+ 的变化，进而， 影响多种细胞信号通路。然而，进展 了解 PKG 在血管平滑肌中的具体功能作用 由于缺乏特异性 PKG 抑制剂，该研究受到阻碍。我们开发了 新型、细胞渗透性 PKG 特异性肽抑制剂，具有前所未有的 通过组合衍生的肽融合来提高效力和激酶特异性 具有膜易位信号 (MTS) 肽的文库。这些融合 肽在 PKG 抑制方面产生非凡的协同作用。 在拟议的实验中，这些 PKG 选择性抑制剂将进一步 开发、完善并用于研究 PKG 的特定功能作用 血管平滑肌。具体目标1将开发新型肽库 基于结合库和竞争库的设计策略。这种做法 应该会产生比目前可用的新的和摩尔有效的 PKG 抑制剂。 具体目标 2 将探索 MTS 序列的变化 它们在细胞内递送的用途以及它们与 文库衍生的肽抑制剂。具体目标 3 将决定 PKG 在完整抵抗中血管张力调节中的贡献 通过测量 PKG 抑制剂对动脉直径的影响来测量动脉 以及单个血管平滑肌细胞中的离子通道活性。追求 为了实现该提案的目标，将采用多方面的方法，包括 最先进的技术：1) 筛选和合成选择性 PKG 使用组合库方法抑制肽，2) 递送肽 使用膜易位肽序列的抑制剂并监测 使用激酶活性测定检测这些化合物的细胞内积累， 荧光光谱和共焦显微镜，3) 评估功效 使用离子通道活性的功能测定（补丁 钳）和血管收缩力（肌电图）。上述研究将 提供新的、选择性的、有效的 PKG 抑制剂，这将有助于 揭示 PKG 在动脉调节中的基本生理作用 语气。这些研究中出现的抑制剂的应用不会 仅证明 PKG 在调节血管张力中的重要作用 阻力动脉，而且还显着推进了蛋白激酶领域 信号一般。这项工作还可能提出新的治疗靶点 高血压和感染性休克等血管疾病的干预措施。