RNA Aptamer-Based Screen for Selective Inhibitors of GRK2

描述(由申请人提供)：G蛋白偶联受体(GPCRs)的一个小家族通过磷酸化激活的GPCRs的细胞质环和尾部的多个位点来负调控异源三聚体G蛋白信号。通过这个过程，细胞适应作用于GPCRs的持续刺激，并保护自己免受持续信号的损害。GRK也可以在人类疾病中扮演不适应的角色。GRK2在心力衰竭时过度表达，不仅使中枢神经系统的心脏受体解偶联，还促进肾上腺释放过量的儿茶酚胺。通过转基因多肽抑制GRK2可以防止小鼠心力衰竭，这表明GRK2是治疗心脏病的极佳靶点。然而，GRKs的选择性小分子抑制剂尚未见报道，可能是由于GRKs的活性部位与其他AGC激酶具有很高的同源性。在过去的六年里，我们的实验室在了解GRKs的结构和功能方面取得了重大进展，目前我们正在研究一种高亲和力RNA适体选择性抑制GRK2的分子基础。我们对该复合体的初步结晶学研究表明，适体主要与激酶域的大叶结合，在那里它阻止了激酶域的核苷酸结合位点的入口。我们假设这种RNA适配子可以用于置换试验，以确定与GRK2上活性部位以外的区域结合的小分子，这些区域对活性也是关键的。我们已经设计了原始RNA适配子的改进版本，用于强大的流式细胞术蛋白质相互作用分析，以筛选与GRK2结合的RNA竞争的化合物。在与密歇根大学化学基因组学中心的合作下，我们已经进行了大约40,000种化合物的初步HTS，并进行了出色的统计。使用基于活性的二级筛选，我们将确认来自该筛选和来自分子文库探针生产中心进行的筛选的哪些命中直接与GRK2结合并抑制激酶活性。这些化合物将被进一步表征以确定膜的通透性、它们的抑制模式以及它们对GRK2的选择性。尽管所有的活性分子都是有意义的，但不与ATP竞争抑制的小分子尤其重要，因为它们可能代表着治疗心脏病的新的和选择性的治疗线索。 公共卫生相关性：GRK2与心血管生理学和疾病密切相关。我们的流式细胞术蛋白质相互作用分析将使我们能够快速筛选大的小分子文库，目标是识别干扰高亲和力RNA适配子的化合物，该适配子选择性地结合到GRK2的激动域。这些化合物有可能与激酶结构域表面的新位点相互作用，从而成为GRK2的选择性抑制剂。

DESCRIPTION (provided by applicant): A small family of G protein-coupled receptor (GPCR) kinases (GRKs) negatively regulates heterotrimeric G protein signaling by phosphorylating multiple sites in the cytoplasmic loops and tails of activated GPCRs. Through this process, cells adapt to persistent stimuli that act at GPCRs and protect themselves from damage incurred by sustained signaling. GRKs can also play maladaptive roles in human disease. GRK2 is overexpressed during heart failure, which not only uncouples cardiac receptors from the central nervous system, but also promotes the release of excessive amounts of catecholamines from the adrenal gland. Inhibition of GRK2 by transgenic peptides prevents cardiac failure in mouse models, suggesting that GRK2 is an excellent target for the treatment of heart disease. However, selective small molecule inhibitors of GRKs have not been reported, perhaps due to high homology among the active sites of GRKs and other AGC kinases. Over the last six years, our lab has made significant progress in understanding the structure and function of GRKs, and we are currently investigating the molecular basis for the selective inhibition of GRK2 by a high affinity RNA aptamer. Our preliminary crystallographic studies of this complex demonstrate that the aptamer binds primarily to the large lobe of the kinase domain, where it blocks the entrance to the nucleotide binding site of the kinase domain. We hypothesize that this RNA aptamer can be used in a displacement assay to identify small molecules that bind to regions on GRK2 outside of its active site that are also critical for activity. We have designed improved versions of the original RNA aptamer for use in a robust flow cytometry protein interaction assay to screen for compounds that compete with RNA binding to GRK2. In collaboration with the Center for Chemical Genomics at the University of Michigan, we have conducted a preliminary HTS of ~40,000 compounds with excellent statistics. Using activity-based secondary screens, we will confirm which hits derived from this screen and those from screens conducted at a Molecular Libraries Probe Production Center bind directly to GRK2 and inhibit kinase activity. These compounds will be further characterized to establish membrane permeability, their mode of inhibition, and their selectivity for GRK2. Although all active molecules are of interest, small molecules that do not exhibit competitive inhibition with ATP are of particular importance because they would likely represent novel and selective therapeutic leads for the treatment of heart disease. PUBLIC HEALTH RELEVANCE: GRK2 is strongly linked to cardiovascular physiology and disease. Our flow cytometry protein interaction assay will allow us to rapidly screen large libraries of small molecules with the goal of identifying compounds that interfere with a high-affinity RNA aptamer that selectively binds to the kinase domain of GRK2. These compounds have the potential to interact with novel sites on the surface of the kinase domain and thus serve as selective inhibitors of GRK2.