Inhibitors of b-arrestin

最近在GPCR信号中的发现阐明了β-拦阻蛋白的多个关键作用，它是调节GPCR内化和信号转导的支架蛋白。β-抑制素有望达到临床靶点，因为单个β-arrestin基因敲除的小鼠没有明显的表型，而双重敲除的β-arrestin1和β-arrestin2是致命的。此外，膀胱癌细胞表达高水平的β-抑制素2及其激活剂之一-血栓素β-gpr。这种增加与较差的临床预后相关，并提示β-抑制素2是膀胱癌的靶点。尽管GPCRs是一个主要的临床靶点，但到目前为止，还没有针对β-阻滞剂的小分子调节剂。利用NICS Kraken超级计算机上的高性能对接，我们对140万个化合物文库进行了虚拟筛选，并鉴定了约250个潜在的β-抑制素2抑制剂。然后，我们创建并验证了针对GPCR/B-arrestin2相互作用的高通量和高含量筛选，并测试了61种预测的抑制性化合物，其中15种使受体内化减少了40%，验证了我们的发现策略。我们将完成对所有预测抑制剂的筛选，并进行先导优化，以提高它们对β-抑制素2的效力和选择性。由此产生的第一个Betaarrestin2选择性抑制剂将用于在模型细胞系统中确定它们对下游GPCR信号事件的影响和选择性。我们还将使用基于细胞的膀胱癌和动物模型来测试我们的β-arrestin2抑制剂的抗癌活性。这项研究的成功将保证开发β-抑制素2抑制剂作为膀胱癌和其他与β-受体激动素介导的异常信号转导相关的疾病的治疗剂。

Recent discoveries in GPCR signaling have elucidated multiple critical roles of beta-arrestins which are scaffolding proteins that regulate GPCR internalization and signaling. Beta-arrestins are promising to clinical targets, because mice with a knockout of a single beta-arrestin have no overt phenotype, while dual knockout of beta-arrestin1 and beta-arrestin2 is lethal. Also, bladder cancer cells express elevated levels of beta-arrestin2 and one of its activators, the thromboxane-beta GPCR. This increase correlates with a poor clinical prognosis and suggests beta-arrestin2 as a bladder cancer target. Although GPCRs are a major clinical target, to date there are no small molecule regulators for beta-arrestins. Using high performance docking on the NICS Kraken supercomputer, we have conducted virtual screening of a 1.4 million compound library and identified ~250 potential beta-arrestin2 inhibitors. We then created and validated a high throughput and high-content screen targeting GPCR/ B-arrestin2 interactions and tested 61 predicted inhibitory compounds, 15 of which decreased receptor internalization by >40%, validating our discovery strategy. We will complete the screening of all the predicted inhibitors and carry out lead optimization to increase their potency and selectivity for beta-arrestin2. The resulting first selective inhibitors of betaarrestin2 will be used to determine their effect and selectivity on downstream GPCR signaling events in model cell systems. We will also test our beta-arrestin2 inhibitor for anticancer activity using cell based and animal models of bladder cancer. The success of this study will warrant the development of beta-arrestin2 inhibitors as therapeutic agents for bladder cancer and other diseases associated with aberrant betaarrestin- mediated signal transduction.