Small molecule stabilizers of RGS protein expression

描述：包括蛋白质-蛋白质相互作用在内的许多生物过程的抑制剂的合理设计已经取得了巨大的进展。然而，增加蛋白质的活性往往是可取的。阻断负调控通路是直接针对感兴趣蛋白质的另一种选择。泛素-蛋白酶体机制是通过快速和可控的降解来调节蛋白质功能的关键机制，但目前的蛋白酶体抑制剂特异性较低。有600多种不同的E3泛素连接酶，在靶向蛋白质降解方面有很大的特异性。作为一个确定选择性药物靶向蛋白质降解策略的模型，我们将研究两个G蛋白信号蛋白的调节蛋白，RGS2和RGS4，它们被不同的E3连接酶机制降解。RGS2表达减少见于心血管疾病(高血压和心力衰竭)，也在焦虑和癌症生物学中发挥作用。RGS4与心力衰竭有关，在精神分裂症患者和侵袭性人类乳腺癌株的大脑中发现了低水平的RGS4。因此，增加RGS2或4的表达可能会产生广泛的药理学影响。RGS4被单链N-末端规则E3连接酶机制降解。我们最近发现，RGS2的降解利用了独特的Cullin-Ring-Ligase E3机制。泛素-蛋白酶体分子机器的复杂性和规律性使得很难准确预测可用药的“软点”可能在哪里，因此广泛的筛选方法提供了一种检测新的作用部位的方法。通过比较RGS2和RGS4的调控，我们将探索E3靶向的机制和特异性。使用强大的、HTS兼容的、基于细胞的检测RGS2或RGS4蛋白水平的方法，我们将：1)鉴定RGS细胞表达的小分子增强剂，2)在反筛选中检查它们作用的特异性，3)获得初步的结构-活性关系 化合物和4)通过确定直接与RGS蛋白或相关E3连接酶组分结合的作用来评估化合物的作用机制。这项工作的长期目标是确定选择性药物靶向蛋白质降解的策略。这项工作的成功完成将提供一系列具有机械特征的化合物，增强RGS2和RGS4蛋白的表达和功能。这些化合物将作为蛋白质降解的化学探针，阐明RGS上调在生物学以及神经精神、心血管和恶性疾病中的影响。

DESCRIPTION: Tremendous advances have been made in the rational design of inhibitors of many biological processes including protein-protein interactions. However it is often desirable to increase the activity of a protein. Blocking negative regulation pathways is an alternative to directly targeting the protein of interest. The ubiquitin- proteasome mechanism is a key regulator of protein function through rapid and controlled degradation but current proteasome inhibitors have low specificity. With over 600 distinct E3 ubiquitin ligases there is potential for great specificity in targeting protein degradation. As a model to define strategies for selective pharmacological targeting of protein degradation, we will study two Regulator of G protein Signaling proteins, RGS2 and RGS4 that are degraded by different E3 ligase mechanisms. Reduced RGS2 expression is seen in cardiovascular pathologies (hypertension and heart failure) and also plays a role in anxiety and cancer biology. RGS4 has been implicated in heart failure and low levels have been found in brains of schizophrenia patients and in aggressive human breast cancer lines. Thus increasing RGS2 or 4 expressions could have broad pharmacological ramifications. RGS4 is degraded by a single-chain N-end rule E3 ligase mechanism. We recently found that RGS2 degradation utilizes the distinct Cullin-Ring-Ligase E3 mechanism. The complexities and regulation of ubiquitin-proteasome molecular machines make it hard to predict exactly where the druggable "soft-spots" are likely to be, so broad screening approaches provide a way to detect novel sites of action. By comparing RGS2 and RGS4 regulation, we will explore mechanisms and specificity in E3 targeting. Using a robust, HTS- compatible, cell-based assay to detect RGS2 or RGS4 protein levels we will: 1) identify small molecule enhancers of RGS cellular expression, 2) examine the specificity of their actions in counter- screens, 3) obtain initial structure-activity relationships with commercially available compounds and 4) assess compound mechanisms of action by defining the role of direct compound binding to RGS proteins or to the relevant E3 ligase components. The long term goal of this work is to define strategies for the selective pharmacological targeting of protein degradation. Successful completion of this work will provide mechanistically characterized compound series that enhance RGS2 and RGS4 protein expression and function. Such compounds will serve as chemical probes of protein degradation that would illuminate effects of RGS upregulation in biology and in neuropsychiatric, cardiovascular, and malignant diseases.