High-throughput assay development for molecular probes targeting the ULK1 kinase

描述(申请人提供)：自噬途径引导细胞的主要循环中心，在那里，长寿命的蛋白质、大量的细胞质材料和受损的细胞器(例如线粒体)被产生的双膜小泡吞噬，然后自噬小体与降解这些货物的溶酶体融合，在压力或营养缺乏的时候恢复积木和能量。因此，自噬对于细胞内稳态是必要的，而该途径的缺陷会导致各种病理，包括神经退行性疾病和肌病。重要的是，我们已经证明，损害自噬可以增强抗癌药物的疗效，并可以克服耐药性。尽管人们对产生自噬途径特定成分的拮抗剂和激动剂很感兴趣，但目前还缺乏这类药物。自噬途径是由一个保守的丝氨酸/苏氨酸激酶Ulk1(UNC-51样激酶-1)控制的。我们的多PI研究小组已经证明，Ulk1激酶的活性对于控制自噬途径是必不可少的，Atg13是Ulk1的真正底物，而Ulk1指导的S318上Atg13的磷酸化对于自噬是必不可少的。重要的是，我们已经开发了一些试剂，可以监测ULK1对Atg13的磷酸化、细胞内Ulk1激酶的活性、自噬通量的速率以及自噬小体与溶酶体的成熟和融合。鉴于Ulk1作为靶标的重要性，我们将启用、开发和优化一套有针对性的生化和基于细胞的分析方法，以识别和表征Ulk1的小分子抑制剂。在具体目标1中，我们将基于全长Ulk1对全长人Atg13的磷酸化建立一种新的均相高通量筛选(HTS)兼容方法。这项检测使用了一种基于珠粒的邻近形式，非常适合小型化和高通量筛查(HTS)。一旦得到验证，该分析将被提交给分子库生产中心网络(MLPCN)，通过对分子库小分子资料库(MLSMR)化合物集合执行HTS活动来识别Ulk1的探针。识别和确认的“命中”将根据一组精选的激酶进行反筛选，以对化合物进行分类，以推动药物化学努力。为了评估铅声明的分子，在特定的目标2中，我们将开发一种基于细胞的检测方法，测量Atg13的磷酸化，这将提供细胞内效力的定量测量。这项测试将在MLCPN支持的后续几轮药物化学中驱动Ulk1细胞抑制物活性的结构-活性关系(SAR)。在具体目标3中，我们将开发一种新的基于细胞的分析方法，使人们能够实时确定活细胞中的自噬通量的速率。这项化验将使我们能够 量化我们的顶级Ulk1抑制剂对激活自噬途径的不同信号的反应效果。总而言之，这些研究将提供一套全面的决策工具，极大地促进有效的、选择性的、细胞穿透的Ulk1抑制剂的开发，这些抑制剂可用于询问Ulk1在正常和病理状态下所扮演的生物学角色(S)。 与公共健康相关：我们的研究表明，破坏自噬途径--细胞的主要回收中心，在压力或营养缺乏时提供积木和能量--会增加癌细胞对传统疗法的敏感性。然而，缺乏抑制自噬途径的靶向药物，我们已经证明ULK1(UNC-51-like kinase1)丝氨酸/苏氨酸激酶对于几种形式的自噬是必不可少的，就像ULK1指导的底物ATG13的磷酸化一样。我们的多PI研究团队将开发、验证和提供高通量兼容的生化和基于细胞的分析，以识别和优化ULK1选择性抑制剂，这些抑制剂可以用作分子探针来询问ULK1的功能，并最终可以在临床上提炼成显示出对多种恶性肿瘤耐药肿瘤类型的活性的药物。

DESCRIPTION (provided by applicant): The autophagy pathway directs the principle recycling center of the cell, where long-lived proteins, bulk cytoplasmic material and damaged organelles (e.g., mitochondria) are engulfed by double-membraned vesicles coined autophagosomes, which then fuse with the lysosome that degrades this cargo to recoup building blocks and energy under times of stress or nutrient deprivation. Accordingly, autophagy is necessary for cellular homeostasis, and defects in the pathway leads to various pathologies, including neurodegenerative diseases and myopathies. Importantly, we have shown that impairing autophagy augments the efficacy of anti-cancer drugs and can overcome resistance. Though there is much interest in generating antagonists and agonists of specific components of the autophagy pathway, currently such agents are lacking. The autophagy pathway is controlled by a conserved serine/threonine kinase coined Ulk1 (UNC-51-like kinase-1). Our Multi-PI research team has shown that Ulk1 kinase activity is essential for control of the autophagy pathway that Atg13 is a bona fide substrate of Ulk1 and that Ulk1-directed phosphorylation of Atg13 on S318 is essential for autophagy. Importantly, we have developed reagents that allow us to monitor Atg13 phosphorylation by ULK1, intracellular Ulk1 kinase activity, rates of autophagic flux and the maturation and fusion of autophagosomes with lysosomes. Given the importance of Ulk1 as a target, we will enable, develop and optimize a focused set of biochemical and cell-based assays to identify and characterize small molecule inhibitors of Ulk1. In Specific Aim 1 we will develop a novel homogenous high-throughput screening (HTS) compatible assay based on the phosphorylation of full-length human Atg13 by full-length Ulk1. This assay uses a bead-based proximity format ideally suited for miniaturization and high-throughput screening (HTS). Once validated, this assay will be submitted to the Molecular Libraries Production Centers Network (MLPCN) to identify probes of Ulk1 by performing a HTS-campaign against the Molecular Libraries Small Molecule Repository (MLSMR) compound collection. Identified and confirmed 'hits' will be counter-screened against a panel of select kinases to triage compounds to drive medicinal chemistry efforts. To assess lead declared molecules, in Specific Aim 2 we will develop a cell-based assay that measures Atg13 phosphorylation and that will provide a quantitative measure of intracellular potency. This assay will drive structure activity relationshi (SAR) of Ulk1 cell-based inhibitor activity in subsequent rounds of medicinal chemistry supported by the MLCPN. In Specific Aim 3 we will develop a novel cell-based assay that allows one to determine rates of autophagic flux in real time in living cells. This assay will allow us to quantify effects of our top Ulk1 inhibitors in response to different cues that activate the autophagy pathway. Collectively, these studies will provide a comprehensive set of decision making tools that will greatly facilitate the development of potent and selective, cell-penetrant Ulk1 inhibitors that can be used to interrogate the biological role(s) that Ulk1 plays in normal an pathological states. PUBLIC HEALTH RELEVANCE: Our studies have shown that impairing the autophagy pathway, the principle recycling center of the cell that provides building blocks and energy during times of stress or nutrient deprivation, augments the sensitivity of cancer cells to conventional therapeutics. However, targeted agents that inhibit the autophagy pathway are lacking, and we have shown that the ULK1 (UNC-51-like kinase- 1) serine/threonine kinase is essential for several forms of autophagy, as is ULK1-directed phosphorylation of its substrate ATG13. Our Multi-PI research team will develop, validate and deliver high-throughput compatible biochemical and cell-based assays that will identify and optimize ULK1-selective inhibitors, which can be used as molecular probes to interrogate ULK1 function and which can ultimately be clinically refined into agents that show activity against resistant tumor types acros a broad spectrum of malignancies.