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: 6688276
• 负责人: WOLFGANG R DOSTMANN
• 金额: $ 37.88万
• 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/6688276
• 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.

性状（由申请方提供）：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的激活导致细胞内Ca 2+的改变，这反过来， 影响多种细胞信号通路。然而， 了解PKG在血管平滑肌中的特定功能作用 由于缺乏特异性PKG抑制剂而受到阻碍。我们已经开发 新的，细胞渗透性PKG特异性肽抑制剂，具有前所未有的 通过融合衍生自组合的肽的效力和激酶特异性 用膜易位信号（MTS）肽构建文库。这些融合 肽导致关于PKG抑制的显著协同作用。 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在 血管平滑肌Specific Aim 1将开发新型肽库 基于绑定库和竞争库的设计策略。这种方法 应该导致新的和摩尔有效的PKG抑制剂比目前可用。 具体目标2将探索MTS序列的变化， 它们用于细胞内递送的用途以及它们与细胞内递送的协同作用的能力。 文库衍生的肽抑制剂。具体目标3将决定 PKG在完整阻力血管张力调节中的作用 通过测量PKG抑制剂对动脉直径的影响， 以及单个血管平滑肌细胞中的离子通道活性。追求 为了实现这一建议的目标，将采用多方面的方法，包括 现有技术：1）筛选和合成选择性PKG 使用组合文库方法的抑制剂肽，2）递送肽 抑制剂使用膜转位肽序列，并监测 使用激酶活性测定这些化合物的细胞内积累， 荧光光谱和共聚焦显微镜，以及3）评估功效 使用离子通道活性的功能测定（贴片） 钳夹）和血管收缩性（肌造影）。上述研究将 提供新的、选择性的和有效的PKG抑制剂，其将用于 揭示PKG在动脉调节中的基本生理作用， 语气从这些研究中产生的抑制剂的应用将不会 仅证明PKG在调节血管张力中的重要作用， 阻力动脉，而且还大大推进蛋白激酶领域 信号一般。这项工作也可能为治疗提供新的靶点。 高血压和感染性休克等血管疾病的干预措施。