High Throughput Screening to Discover Ulk1 Kinase Inhibitors

 描述（由申请人提供）：自噬是细胞的主要再循环中心，其中大量细胞质物质和受损的细胞器被双膜囊泡吞没，所述双膜囊泡形成自噬体，然后与溶酶体融合，所述溶酶体降解其货物并回收构建块和能量，作为稳态机制和应激期间的生存策略。自噬途径的缺陷可导致多种病理，包括退行性疾病和癌症。重要的是，使用遗传方法，我们已经表明，抑制自噬途径禁用几种类型的难治性人类恶性肿瘤，并增强辐射和癌症治疗的疗效。自噬途径的选择性调节剂显然是需要的，但还不存在。自噬途径是由一个保守的丝氨酸/苏氨酸激酶控制的，该激酶被称为Ulk 1（ULK-51-like kinase-1）。我们的Multi-PI研究团队已经证明：（1）Ulk 1激酶活性对于激活自噬途径至关重要;（2）Atg 13是Ulk 1的真正底物;（3）Ulk 1指导的S318上Atg 13磷酸化对于自噬至关重要。鉴于Ulk 1作为靶点的重要性，我们开发了一系列高通量兼容检测方法，现在正在进行HTS活动，我们将实施严格的研究操作计划，包括生物化学和基于细胞的检测，以识别，确认和验证选择性小分子Ulk 1抑制剂。具体来说，我们使用我们的新研究工具开发了一种384孔兼容的同质Ulk 1生化检测。此外，我们使用Sigma-LOPAC（药理活性化合物库）库验证了该测定。在目标1的研究中，我们将把该测定方法扩展到1536孔格式，并对斯克里普斯研究所的650，000个小分子化合物库进行HTS活动。在Aim 2中，将确认并进一步评估在初步筛选中鉴定的“命中”，以：（i）分类假阳性;（ii）使用正交生物化学测定法确认“命中”;（iii）基于抑制50% Ulkl激酶活性所需的浓度（IC 50）对“命中”的生物化学活性进行排序;以及（iv）在基于细胞的测定法分析之前分类支架的化学易处理性。在目标3中，将使用一系列经验证的后续试验评估先导化合物。首先，将使用定量的、基于细胞的Atg 13 S318磷酸化测定来确定确认的HTS“命中”的细胞效力。其次，我们将定义铅的抑制剂作用机制（ATP竞争性，底物竞争性等）。第三，我们将确定针对包括高度相关的激酶Ulk 2在内的456种激酶（DiscoveRx）的组的顶部引线的选择性。最后，我们将在基于细胞的功能测定中测试我们最有活性的先导物，其中我们将：（i）使用经验证的β-内酰胺酶-LC 3、β-内酰胺酶-p62/SQSTM 1和mCherry-GFP-LC 3自噬通量测定来确认先导物分子抑制自噬;和（ii）评估先导抑制剂在原代野生型和Ulk 1- an Atg 7-用激活自噬途径的试剂处理的缺陷小鼠胚胎成纤维细胞和骨髓细胞。

 DESCRIPTION (provided by applicant): Autophagy is the principle recycling center of the cell, where bulk cytoplasmic material and damaged organelles are engulfed by double membrane vesicles coined autophagosomes that then fuse with lysosomes, which degrade their cargo and recoup building blocks and energy, both as a homeostatic mechanism and as a survival strategy during times of stress. Defects in the autophagy pathway can lead to several pathologies including degenerative diseases and cancer. Importantly, using genetic approaches, we have shown that inhibiting the autophagy pathway disables several types of refractory human malignancies and augments the efficacy of irradiation and cancer therapeutics. Selective modulators of the autophagy pathway are clearly needed yet none exist. 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: (1) Ulk1 kinase activity is essential for activating the autophagy pathway; (2) Atg13 is a bona fide substrate of Ulk1; and (3) that Ulk1-directed phosphorylation of Atg13 on S318 is essential for autophagy. Given the importance of Ulk1 as a target we developed a series of high-throughput compatible assays and are now positioned to perform a HTS-campaign, where we will implement a rigorous research operating plan comprised of biochemical and cell-based assays to identify, confirm and validate selective small molecule Ulk1 inhibitors. Specifically, we have used our novel research tools to develop a 384-well compatible, homogenous Ulk1 biochemical assay. Further, we have validated this assay using the Sigma-LOPAC (Library of Pharmacologically Active Compounds) library. In the studies of Aim 1, we will miniaturize this assay to a 1536-well format and perform a HTS campaign against the 650,000 small molecule compound library of The Scripps Research Institute. In Aim 2 'hits' identified in the primary screen will be confirmed and further evaluated to: (i) triage false positives; (ii) confirm 'hits' using an orthogonal biochemical assay (iii) rank order the biochemical activity of 'hits' based upon the concentration needed to inhibit 50% (IC50) of Ulk1 kinase activity; and (iv) triage scaffolds for chemical tractability prior to cel-based assay analyses. In Aim 3, lead compounds will be assessed using a series of validated follow-up assays. First, cellular potency of confirmed HTS 'hits' will be determined using a quantitative, cell-based, Atg13S318 phosphorylation assay. Second, we will define the mechanism of inhibitor action of leads (ATP competitive, substrate competitive, etc.). Third, we will determine the selectivity of top leads against a panel of 456 kinases (DiscoveRx) including the highly related kinase Ulk2. Finally, our most active leads will be tested in cell-based functional assays, where we will: (i) confirm that lead molecules inhibit autophagy using validated luciferase-LC3, luciferase-p62/SQSTM1 and mCherry-GFP-LC3 autophagic flux assays; and (ii) assess the biological effects of lead inhibitors in primary wild type and Ulk1- an Atg7-deficient mouse embryo fibroblast and myeloid cells treated with agents that activate the autophagy pathway.