Selective Targeting of G Protein beta gamma Subunits with Small Molecules

描述（由申请人提供）：G蛋白？亚基在G蛋白偶联受体（GPCR）介导的信号转导中起核心作用。它们在受体介导的激活过程中作为辅因子，并在向下游靶点的信号传递中发挥直接作用。大量的数据已经积累在系统的数量超过？信号有病理后果和操纵？亚基信号传导可能是心力衰竭以及其他疾病的有效治疗策略。我们开发了一种新的靶向策略选择性操纵G蛋白？通过选择性阻断亚基信号通路？- 使用小分子与功能蛋白质伴侣的亚基结合相互作用。在之前的资助期间，我们通过表面等离子体共振（SPR）结合定点诱变确定了几种化合物的结合模式，并解决了与G？热点结合的M201的共晶结构。这些数据证实了一个直接的机制结合G？影响蛋白质-蛋白质相互作用，并支持我们的整体假设，即小分子选择性地调节下游效应信号的结合不同的亚位点上的G？热点此外，我们发表的结果证明了这些化合物在心力衰竭、炎症和吗啡依赖性镇痛的细胞和动物模型中的功效和特异性。在本申请中提出的实验中，我们将继续探索这些结合和选择性的基本机制。结合化合物具体目标1将集中在诱变和X射线晶体学，以确定多种结合模式内的G？有助于选择性热点。具体目标2将探讨化合物依赖性G？亚基活化具体目标3将探索完整细胞中的特异性和作用机制。成功完成所提出的实验将导致对靶向G23信号传导的新分子家族的作用机制的透彻理解，所述新分子家族在剖析GPCR刺激的信号传导的作用机制中具有潜在用途，并为新的治疗方法提供基础。

DESCRIPTION (provided by applicant): G proteins ¿? subunits play a central role in G-protein coupled receptor (GPCR)-mediated signal transduction. They act as cofactors in the receptor-mediated activation process as well as playing direct roles in signal transfer to downstream targets. Considerable data has accumulated in number of systems that excess ¿? signaling has pathological consequences and that manipulation of ¿? subunit signaling could be an effective therapeutic strategy in heart failure as well as other diseases. We developed a novel targeting strategy for selective manipulation of G protein ¿? subunit signaling pathways by selectively blocking ¿? -subunit binding interactions with functional protein partners using small molecules. In the previous funding period we defined the binding modes for several compounds by surface plasmon resonance (SPR) coupled with site directed mutagenesis and solved the co-crystal structure of M201 bound to the hot spot of G¿?. These data confirmed a direct mechanism for binding to G¿? that influences protein-protein interactions and support our overall hypothesis that small molecules selectively modulate downstream effectors signaling by binding to different subsites on the G¿? hotspot. Additionally, we published results demonstrating efficacy and specificity of these compounds in cellular and animal models of heart failure, inflammation and morphine- dependent analgesia. In the experiments proposed in this application we will continue to explore the fundamental mechanisms underlying binding and selectivity of these ¿? binding compounds. Specific aim 1 will focus on mutagenesis and x-ray crystallography to identify multiple binding modes within the G¿? hotspot that contribute to selectivity. Specific aim 2 will explore the mechanism for compound-dependent G¿? subunit activation. Specific aim 3 will explore specificity and mechanism of action in intact cells. Successful completion of the proposed experiments will lead to a thorough understanding of a the mechanism of action of a new family of molecules that target G23 signaling that have potential uses in dissecting the mechanisms of action of GPCR stimulated signaling and providing the basis for novel therapeutic approaches.