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着核心作用。它们在受体介导的激活过程中扮演辅助因子的角色，并在向下游靶点的信号传递中发挥直接作用。在许多系统中积累了大量的数据，超出了多少？信号具有病理性后果，而操纵？亚单位信号转导可能是治疗心力衰竭和其他疾病的一种有效策略。我们开发了一种新的靶向策略来选择性地操纵G蛋白？？亚基信号通路通过使用小分子选择性地阻断亚基与功能蛋白伙伴的结合相互作用。在之前的资助期间，我们通过表面等离子体共振(SPR)结合定点突变确定了几种化合物的结合模式，并解决了M201与G？热点结合的共晶结构。这些数据证实了与G？结合的直接机制？这影响了蛋白质之间的相互作用，并支持我们的总体假设，即小分子通过与G？上不同的亚位点结合来选择性地调节下游效应器的信号传递。热点。此外，我们还发表了这些化合物在心力衰竭、炎症和吗啡依赖止痛的细胞和动物模型中的有效性和特异性的结果。在本申请中提出的实验中，我们将继续探索这些物质的结合和选择性的基本机制。结合化合物。具体目标1将重点放在诱变和X射线结晶学上，以确定G？有助于选择性的热点。具体目标2将探索化合物依赖G？的机制。亚基激活。特异靶3将探索在完整细胞中的特异性和作用机制。这些实验的成功完成将导致对一系列新的靶向G23信号的分子的作用机制的彻底理解，这些分子在剖析GPCR刺激信号的作用机制方面具有潜在的用途，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.