High Throughput Screening to Discover Chemical Probes of ASK1

描述（由申请人提供）：应激激活的凋亡信号调节激酶（ASK1）在许多生理和病理生理过程中起重要作用。特别是，遗传学研究表明，这种丝氨酸/苏氨酸激酶的缺失改善了许多在类风湿性关节炎、心脏肥大和神经退行性疾病小鼠模型中表现出来的表型。值得注意的是，ASK1是一个非常容易处理的靶点，ASK1的选择性小分子抑制剂将为进一步表征ASK1导向的途径提供有价值的探针。然而，在专利文献之外，学术界没有可用的ASK1抑制剂。我们的研究计划符合PAR-12-058的所有规范，题为“高通量筛选（HTS）检测以发现化学探针的招标”，我们将执行HTS活动，并将实施严格的研究操作计划，包括生化和基于细胞的检测，以识别、确认和验证选择性小分子ASK1抑制剂。具体来说，我们利用了我们最近对活性ASK1复合物的酶促研究，开发了一种新的384孔兼容的、均匀的ASK1生化检测方法。此外，我们使用Sigma-LOPAC （Library of pharmacological Active Compounds）文库验证了该分析。在Aim 1的研究中，我们将把这个实验缩小到1536格式，并对斯克里普斯研究所（TSRI）的65万个小分子化合物文库进行HTS测试。在目标2中，在主屏幕中识别的“命中”将被确认并进一步评估，以：i)分类假阳性；Ii)使用an确认“命中”

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.