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.

描述（由申请人提供）：环状GMP依赖性蛋白激酶（PKG） 在调节血管平滑肌张力中起着核心作用。 PKG的激活导致细胞内Ca2+的改变，这反过来又改变了 影响多个细胞信号通路。但是，进展 了解PKG在血管平滑肌中的特定功能作用 由于缺乏特定的PKG抑制剂而受到阻碍。我们已经发展了 新颖的，可渗透的PKG特异性肽抑制剂具有前所未有的 通过融合的肽的效力和激酶特异性 带有膜易位信号（MTS）肽的文库。这些融合 肽导致有关PKG抑制作用的非凡协同作用。 在提出的实验中，这些PKG选择性抑制剂将进一步 开发，完善和用于研究PKG在 血管平滑肌。特定目标1将发展新型肽库 设计基于绑定和竞争库的策略。这种方法 应该导致与目前可用的新型和摩尔有效的PKG抑制剂。 特定的目标2将探索MTS序列的变化 它们用于细胞内交付以及与 库得出的肽抑制剂。特定目标3将确定 PKG在完整耐药性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AS来动脉 以及单血管平滑肌细胞中的离子通道活性。追求 该提案的目的，将使用多方面的方法，包括 最先进的技术：1）屏幕和合成选择性PKG 使用组合库方法的抑制剂肽，2）输送肽 使用膜易位肽序列的抑制剂并监测 使用激酶活性测定的这些化合物的细胞内积累， 荧光光谱和共聚焦显微镜，3）评估功效 使用离子通道活性的功能测定（斑块）的PKG抑制剂 夹具）和血管收缩力（metraphy）。上面概述的研究将 提供新的，有选择性和有效的PKG抑制剂，这将在 揭示PKG在动脉调节中的基本生理作用 语气。从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 电阻动脉，但也显着推进了蛋白激酶的领域 一般信号传导。这项工作还可能暗示治疗的新目标 高血压和败血性休克等血管疾病的干预措施。