High Throughput Screening to Discover Chemical Probes of ASK1

高通量筛选发现 ASK1 化学探针

基本信息

批准号：
8419212
负责人：
Derek Ronald Duckett
金额：
$ 43.69万
依托单位：
SCRIPPS FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-01-01 至 2015-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8419212
关键词：
Academia Apoptosis Biochemical Biological Assay Biology Cardiovascular Diseases Cardiovascular system Cell Death Cells Cellular Stress Chemicals Chemistry Cherry - dietary Complex Critical Pathways Detection Development Development Plans Disease Ensure Family Florida Foundations Future Generations Genetic Goals Heart Hypertrophy Human Image Inflammatory Inhibitory Concentration 50 Investigation Knockout Mice Laboratories Lead Legal patent Length Libraries Literature MAP2K6 gene Measurement Measures Mediating Mitogen-Activated Protein Kinases Molecular Probes Monitor Neurodegenerative Disorders Pathway interactions Pharmaceutical Chemistry Phenotype Phosphorylation Phosphotransferases Physiological Physiological Processes Physiology Play Positioning Attribute Process Protein-Serine-Threonine Kinases Radioactive Reaction Research Research Infrastructure Research Institute Research Personnel Research Proposals Rheumatoid Arthritis Role Running Scheme Series Signal Transduction Stress Talents Testing Triage base cytotoxic cytotoxicity design drug discovery follow-up high throughput screening human disease inhibitor/antagonist inorganic phosphate lead series meetings member miniaturize mouse model novel novel therapeutics programs research study scaffold screening small molecule small molecule libraries tool

项目摘要

DESCRIPTION (provided by applicant): The stress-activated, apoptosis signal-regulating kinase (ASK1) plays important roles in several physiological and pathophysiological processes. In particular, genetic studies have shown that loss of this serine/threonine kinase ameliorates many of the phenotypes manifest in mouse models of rheumatoid arthritis, cardiac hypertrophy and neurodegenerative disorders. Notably, ASK1 is an eminently tractable target and selective small molecule inhibitors of ASK1 would provide valuable probes to further characterize ASK1-directed pathways. However, outside of the patent literature no ASK1 inhibitors are available to academia. Our research proposal fulfills all of the specifications of PAR-12-058, entitled "Solicitation of Assays for High Throughput Screening (HTS) to Discover Chemical Probes", where we will perform a HTS-campaign and will implement a rigorous research operating plan comprised of biochemical and cell-based assays to identify, confirm and validate selective small molecule ASK1 inhibitors. Specifically, we have exploited our recent enzymatic studies of the active ASK1 complex to develop a new 384-well compatible, homogenous ASK1 biochemical assay. Furthermore, 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 1536- format and perform a HTS campaign against The Scripps Research Institute's (TSRI) 650,000 small molecule compound library. 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 ASK1 kinase activity; and iv) triage scaffolds for chemical tractability prior to cell-based assay analyses. In Aim 3, we will test the cellular potency of our ASK1 inhibitors using a substrate phosphorylation assay that specifically quantifies the phosphorylation state of MKK6, a direct substrate of ASK1. Cell penetrant molecules will then be assessed for cytotoxicity using an apoptosis assay and nontoxic compounds will be tested in multi-parametric, functional, cell-based imaging assays to determine if lead ASK1 inhibitors can indeed protect against ASK1-mediated cell death. Finally, we will assess the kinase selectivity of our top lead inhibitors against a panel of 300 kinases (Reaction Biology Corp). Our Multi-PI research team at the Scripps Florida campus of TSRI has both the expertise and infrastructure to perform these experiments, and we submit that the successful completion of our studies will identify a series of new, potent and selective molecular probes that will help define the role(s) that ASK1 plays in normal physiological processes as well as in disease states.

描述（由申请人提供）：应激激活的凋亡信号调节激酶（ASK1）在多种生理和病理生理过程中发挥重要作用。特别是，遗传学研究表明，这种丝氨酸/苏氨酸激酶的缺失可以改善类风湿性关节炎、心脏肥大和神经退行性疾病小鼠模型中表现出的许多表型。值得注意的是，ASK1 是一个非常容易处理的靶标，ASK1 的选择性小分子抑制剂将为进一步表征 ASK1 导向的通路提供有价值的探针。然而，除了专利文献之外，学术界还没有可用的 ASK1 抑制剂。我们的研究计划满足 PAR-12-058 的所有规范，题为“高通量筛选 (HTS) 检测化学探针的征集”，我们将开展 HTS 活动并实施严格的研究运营计划，其中包括生物化学和基于细胞的测定法来识别、确认和验证选择性小分子 ASK1 抑制剂。具体来说，我们利用最近对活性 ASK1 复合物的酶学研究，开发了一种新的 384 孔兼容、均质 ASK1 生化检测。此外，我们还使用 Sigma-LOPAC（药理活性化合物库）库验证了该测定法。在目标 1 的研究中，我们将将此测定小型化为 1536 格式，并针对斯克里普斯研究所 (TSRI) 的 650,000 个小分子化合物库进行 HTS 活动。在目标 2 中，将确认主筛选中确定的“命中”并进一步评估： i) 对误报进行分类； ii) 使用正交生化测定，iii) 根据抑制 50% (IC50) ASK1 激酶活性所需的浓度对“命中”的生化活性进行排序； iv) 在基于细胞的测定分析之前对化学易处理性进行分类支架。在目标 3 中，我们将使用底物磷酸化测定来测试 ASK1 抑制剂的细胞效力，该测定专门量化 ASK1 的直接底物 MKK6 的磷酸化状态。然后使用细胞凋亡测定评估细胞渗透分子的细胞毒性，并在多参数、功能性、基于细胞的成像测定中测试无毒化合物，以确定先导 ASK1 抑制剂是否确实可以防止 ASK1 介导的细胞死亡。最后，我们将评估我们的顶级先导抑制剂针对一组 300 种激酶（Reaction Biology Corp）的激酶选择性。我们位于 TSRI 佛罗里达斯克里普斯校区的多 PI 研究团队拥有进行这些实验的专业知识和基础设施，我们认为，成功完成我们的研究将确定一系列新的、有效的、选择性的分子探针，这些探针将有助于定义ASK1 在正常生理过程以及疾病状态中发挥的作用。