Exploring vasomotor mechanisms using new PKG inhibitors

描述(申请人提供)：cGMP依赖的蛋白激酶(PKG) 在调节血管平滑肌张力方面起着中心作用。 PKG的激活导致细胞内钙离子的变化，进而， 影响多种细胞信号通路。然而，在这方面的进展 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。我们已经开发出 新型细胞渗透性PKG特异性多肽抑制剂，具有前所未有的 通过融合来自组合多肽的效价和激酶专一性 含有膜转位信号(MTS)多肽的文库。这些融合 多肽在抑制PKG方面具有非凡的协同作用。 在拟议的实验中，这些PKG选择性抑制剂将进一步 开发、改进并用于研究PKG在体内的特定功能角色 血管平滑肌。特定目标1将开发新的多肽文库 基于结合和竞争文库的设计策略。这种方法 应该会产生比目前可用的更具摩尔效力的新的PKG抑制剂。 特定目标2将探索MTS序列相对于以下各项的变异 它们用于细胞内递送，以及它们与 文库衍生的多肽抑制剂。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抑制剂对动脉内径的影响 以及单个血管平滑肌细胞的离子通道活性。追寻 为了实现这项提案的目标，将采用多方面的办法，包括 最先进的技术：1)筛选和合成选择性PKG 使用组合文库方法的抑制物肽，2)递送多肽 使用膜转位多肽序列的抑制剂并监测 使用激酶活性分析这些化合物在细胞内的积累， 荧光光谱和共聚焦显微镜，以及3)评估疗效 使用离子通道活性(Patch)功能分析的PKG抑制剂 钳夹)和血管收缩能力(肌造影术)。以上概述的研究将 提供新的、选择性的和有效的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。从这些研究中发现的抑制剂的应用不会 仅说明PKG在血管张力调节中的重要作用 阻力动脉，还能显著推进蛋白激酶领域 总体来说，这是一种信号。这项工作也可能为治疗提供新的靶点。 对高血压和感染性休克等血管疾病的干预。

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.