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

描述（由申请人提供）：自噬途径指导细胞的主要再循环中心，其中长寿命的蛋白质、大量细胞质物质和受损的细胞器（例如，线粒体）被双膜囊泡（自噬体）吞噬，然后与溶酶体融合，溶酶体降解这种货物，以在压力或营养缺乏的情况下回收构建模块和能量。因此，自噬是细胞稳态所必需的，并且该途径中的缺陷导致各种病理，包括神经退行性疾病和肌病。重要的是，我们已经证明，削弱自噬可以增强抗癌药物的疗效，并可以克服耐药性。虽然在产生自噬途径的特定组分的拮抗剂和激动剂方面有很大的兴趣，但目前缺乏这样的试剂。自噬途径由一种保守的丝氨酸/苏氨酸激酶Ulk 1（UNC-51样激酶-1）控制。我们的Multi-PI研究团队已经表明，Ulk 1激酶活性对于控制自噬途径至关重要，Atg 13是Ulk 1的真正底物，并且Ulk 1指导的S318上Atg 13磷酸化对于自噬至关重要。重要的是，我们已经开发出试剂，使我们能够监测Atg 13的磷酸化ULK 1，细胞内ULK 1激酶活性，自噬流量和成熟率和融合的自噬体与溶酶体。鉴于Ulk 1作为靶点的重要性，我们将启用、开发和优化一套集中的生化和基于细胞的检测方法，以鉴定和表征Ulk 1的小分子抑制剂。在特定目标1中，我们将开发一种基于全长Ulk 1对全长人Atg 13的磷酸化的新型均相高通量筛选（HTS）兼容测定。该检测使用基于微珠的邻近格式，非常适合小型化和高通量筛选（HTS）。验证后，该检测试剂盒将提交至分子库生产中心网络（MLPCN），以通过对分子库小分子储存库（MLSMR）化合物集合进行HTS活动来鉴定Ulk 1探针。已识别和确认的“命中”将针对一组选定的激酶进行反筛选，以筛选化合物，以推动药物化学工作。为了评估铅申报分子，在特定目标2中，我们将开发一种基于细胞的测定方法，该方法可测量Atg 13磷酸化，并提供细胞内效价的定量测量。本试验将在MLCPN支持的后续药物化学轮次中驱动基于Ulk 1细胞的抑制剂活性的构效关系（SAR）。在具体目标3中，我们将开发一种新的基于细胞的测定方法，该方法允许在活细胞中真实的时间内确定自噬通量的速率。这项检测将使我们能够 量化我们的顶级Ulk 1抑制剂对激活自噬途径的不同线索的响应效果。总的来说，这些研究将提供一套全面的决策工具，这将大大促进开发有效的和选择性的细胞渗透性Ulk 1抑制剂，可用于询问Ulk 1在正常和病理状态下发挥的生物学作用。

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.