RNA Aptamer-Based Screen for Selective Inhibitors of GRK2

描述（由申请人提供）：G蛋白偶联受体（GPCR）激酶（GRKs）的一个小家族通过磷酸化激活GPCR的胞质环和尾部中的多个位点来负调节异源三聚体G蛋白信号传导。通过这一过程，细胞适应作用于GPCR的持续刺激，并保护自己免受持续信号传导造成的损害。GRKs也可以在人类疾病中发挥适应不良的作用。GRK2在心力衰竭期间过表达，这不仅使心脏受体与中枢神经系统解偶联，而且还促进肾上腺释放过量的儿茶酚胺。通过转基因肽抑制GRK2可以预防小鼠模型中的心力衰竭，这表明GRK2是治疗心脏病的极好靶点。然而，选择性小分子抑制剂的GRKs尚未报道，可能是由于高同源性的活性位点的GRKs和其他AGC激酶。在过去的六年中，我们的实验室在理解GRKs的结构和功能方面取得了重大进展，我们目前正在研究高亲和力RNA适体选择性抑制GRK2的分子基础。我们对该复合物的初步晶体学研究表明，适体主要与激酶结构域的大叶结合，在那里它阻断了激酶结构域的核苷酸结合位点的入口。我们假设这种RNA适体可以用于置换试验，以鉴定与GRK2活性位点以外区域结合的小分子，这些区域对活性也至关重要。我们已经设计了原始RNA适体的改进版本，用于稳健的流式细胞术蛋白质相互作用测定，以筛选与RNA竞争结合GRK2的化合物。与密歇根大学化学基因组学中心合作，我们已经对约40，000种化合物进行了初步的HTS，并获得了出色的统计数据。使用基于活性的二次筛选，我们将确认来自该筛选的命中和来自在分子文库探针生产中心进行的筛选的命中直接结合GRK2并抑制激酶活性。将进一步表征这些化合物以确定膜渗透性、它们的抑制模式和它们对GRK2的选择性。尽管所有的活性分子都是感兴趣的，但不表现出与ATP竞争性抑制的小分子是特别重要的，因为它们可能代表用于治疗心脏病的新型和选择性治疗先导物。 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测定将使我们能够快速筛选大的小分子文库，目的是鉴定干扰选择性结合GRK2激酶结构域的高亲和力RNA适体的化合物。这些化合物具有与激酶结构域表面上的新位点相互作用的潜力，因此可作为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.