Discovery of potent, and selective allosteric inhibitors of GSK-3b

描述(申请人提供)：丝氨酸/苏氨酸激酶糖原合成酶-3β(GSK-3b)是已知的多种细胞通路的主要调节者，包括胰岛素信号和糖原合成、神经营养因子信号和Wnt信号。因此，这种酶在新陈代谢、转录、发育和神经元功能中起着关键作用，并与多种人类疾病有关，包括阿尔茨海默病、双相情感障碍、非胰岛素依赖型糖尿病、心脏肥厚和癌症。GSK-3b如何在许多细胞过程的十字路口有效地维持其通路特异性尚不清楚。这种调节可能涉及这种复杂的激酶不同结构域中的变构位点。现有的大多数化学抑制剂竞争GSK-3b的ATP结合部位并抑制额外的激酶，而已知的两个结构系列的ATP非竞争性抑制剂效价低，药理性质不佳，限制了它们的使用。靶向GSK-3b变构位点的小分子有可能提供高度特异性的GSK-3b抑制剂，这可能有助于阐明GSK-3b在不同细胞通路中的功能和调节。因此，我们提出了一种初级筛选和二级生化和细胞分析的级联方法来鉴定GSK-3b的ATP非竞争性变构抑制剂。这种探针预期的高特异性可能导致依赖于GSK-3b的不同细胞通路的选择性调节。这一探针开发计划包括：1)一种初级激酶活性测定，它将确定影响GSK-3b激酶活性的ATP竞争性和非竞争性抑制剂。2)基于ADP的时间分辨荧光(HTRF)检测的次级激酶活性测定，可以区分变构调节剂和ATP竞争性抑制物。3)基于结合的分析将更准确地定义化合物的作用模式。4)重要的是，我们已经建立了许多有助于区分变构抑制剂和识别特定途径的GSK-3b抑制剂的细胞检测方法。最终，我们计划优化途径特定的变构GSK-3b抑制剂的药代动力学特性，并在已建立的情绪和记忆啮齿动物模型中测试它们。 公共卫生相关性：糖原合成酶-3β是多个细胞信号通路的主要调节者，与多种人类疾病有关。我们建议对MLPCN文库进行筛选，以确定GSK-3b的有效和选择性变构调节子。与MLPCN和Stanley Center的化学家合作，我们将优化HIT化合物以产生化学探针，以阐明不同细胞途径中的GSK-3b生物学。

DESCRIPTION (provided by applicant): The serine/threonine kinase glycogen synthase kinase-3 beta (GSK-3b) is a known master regulator for multiple cellular pathways that include insulin signaling and glycogen synthesis, neurotrophic factor signaling, and Wnt signaling. Consequently, this enzyme has a critical role in metabolism, transcription, development, and neuronal functions and has been implicated in multiple human disorders including Alzheimer's disease, bipolar disorder, noninsulin-dependent diabetes mellitus, cardiac hypertrophy, and cancer. Precisely how GSK-3b maintains its pathway specificity efficiently at the crossroads of many cellular processes is unclear. This regulation may involve allosteric sites within distinct structural domains of this complex kinase. The majority of the existing chemical inhibitors compete for the ATP-binding site of GSK-3b and inhibit additional kinases, while the two known structural series of ATP-noncompetitive inhibitors have low potency and suboptimal pharmacological properties that limit their use. Small molecules targeting the allosteric sites of GSK-3b could have the potential to provide highly specific GSK-3b inhibitors that may help elucidate GSK-3b function and regulation in distinct cellular pathways. Hence we propose a primary screen and a cascade of secondary biochemical and cellular assays to identify ATP-non competitive, allosteric inhibitors of GSK-3b. The anticipated high specificity of such probes could lead to the selective modulation of distinct cellular pathways dependent on GSK-3b. This probe development plan includes: 1) A primary kinase activity assay that will identify ATP-competitive as well as ATP- noncompetitive inhibitors that affect GSK-3b kinase activity. 2) A secondary kinase activity assay based on time resolved fluorescence (HTRF) detection of ADP that will distinguish allosteric modulators and ATP-competitive inhibitors. 3) Binding-based assays that will more precisely define a compound's mode of action. 4) Importantly, we have established a number of cellular assays that will help differentiate allosteric inhibitors and identify pathway-specific GSK-3b inhibitors. Ultimately, we plan to optimize the pharmacokinetic properties of pathway-specific, allosteric GSK-3b inhibitors, and test them in the established rodent models of mood and memory. PUBLIC HEALTH RELEVANCE: Glycogen synthase kinase-3 beta is a master regulator of multiple cell signaling pathways and is implicated in multiple human disorders. We propose to screen the MLPCN library to identify potent and selective allosteric modulators of GSK-3b. Collaborating with MLPCN and Stanley Center chemists, we will optimize the hit compounds to generate chemical probes to elucidate GSK-3b biology in distinct cellular pathways.