High Throughput Screening to Discover Ulk1 Kinase Inhibitors

 描述(申请人提供)：自噬是细胞的主要循环中心，在这里，大量细胞质物质和受损的细胞器被双膜囊泡吞噬，产生自噬小体，然后与溶酶体融合，溶酶体降解货物并回收积木和能量，这既是一种体内平衡机制，也是压力时期的一种生存策略。自噬途径的缺陷可能导致几种病理现象，包括退行性疾病和癌症。重要的是，使用遗传方法，我们已经证明，抑制自噬途径可以禁用几种类型的人类难治性恶性肿瘤，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必需的，但目前还不存在。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：(1)Ulk1激酶活性对于激活自噬途径是必不可少的；(2)Atg13是Ulk1的真正底物；(3)Ulk1指导的S318上Atg13的磷酸化对于自噬是必不可少的。鉴于Ulk1作为目标的重要性，我们开发了一系列高通量兼容分析，现在可以进行HTS活动，我们将实施严格的研究操作计划，包括生化和基于细胞的分析，以识别、确认和验证选择性小分子Ulk1抑制剂。具体地说，我们使用我们的新研究工具开发了一种384兼容的、均一的Ulk1生化分析。此外，我们已经使用Sigma-Lopac(药理活性化合物文库)库验证了这一方法。在目标1的研究中，我们将把这一分析小型化到1536孔格式，并针对斯克里普斯研究所的650,000个小分子化合物文库进行HTS活动。在AIM中，将确认并进一步评估在初级筛查中确定的两个‘HITS’：(I)对假阳性进行分类；(Ii)使用正交生化分析来确认‘HITS’；(Iii)基于抑制50%Ulk1激酶活性所需的浓度(IC50)对‘HITS’的生化活性进行排序；以及(Iv)在基于CEL的分析分析之前对支架进行化学可处理性分类。在目标3中，将使用一系列有效的后续分析对先导化合物进行评估。首先，确认的HTS‘HITS’的细胞效力将通过基于细胞的Atg13S318磷酸化定量分析来确定。其次，我们将明确Leads的抑制作用机制(ATP竞争性、底物竞争性等)。第三，我们将确定顶部导联相对于456个激酶(DiscoveRx)的选择性，其中包括高度相关的激酶Ulk2。最后，我们将在基于细胞的功能分析中测试我们最活跃的铅，其中我们将：(I)使用经验证的荧光素酶-LC3、荧光素酶-P62/SQSTM1和mCherry-GFP-LC3自噬通量分析证实铅分子抑制自噬；以及(Ii)评估铅抑制剂在原代野生型和Ulk1-ATG7缺失的小鼠胚胎成纤维细胞和髓系细胞中的生物学效应，这些细胞和细胞经激活自噬途径的制剂处理。

 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.