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（ASK 1）在几种生理和病理生理过程中起重要作用。特别是，遗传研究表明，这种丝氨酸/苏氨酸激酶的损失改善了类风湿性关节炎，心脏肥大和神经退行性疾病的小鼠模型中表现出的许多表型。值得注意的是，ASK 1是一个非常容易处理的靶标，ASK 1的选择性小分子抑制剂将提供有价值的探针，以进一步表征ASK 1介导的途径。然而，在专利文献之外，学术界没有可获得的ASK 1抑制剂。我们的研究提案符合PAR-12-058的所有规范，标题为“高通量筛选（HTS）发现化学探针的试验征集”，我们将进行HTS活动，并将实施严格的研究操作计划，包括生物化学和基于细胞的试验，以识别，确认和验证选择性小分子ASK 1抑制剂。具体来说，我们利用我们最近的活性ASK 1复合物的酶促研究，开发了一种新的384孔相容性，同质ASK 1生化测定。此外，我们还使用Sigma-LOPAC（药理活性化合物库）库验证了该试验。在Aim 1的研究中，我们将把该测定方法扩展到1536格式，并对斯克里普斯研究所（TSRI）的650，000小分子化合物库进行HTS活动。在目标2中，将确认并进一步评估在初级筛选中识别的“命中”，以：i）分类假阳性; ii）使用 正交生物化学测定，iii）基于抑制50%（IC 50）ASK 1激酶活性所需的浓度，对“命中物”的生物化学活性进行排序;和iv）在基于细胞的测定分析之前，对支架进行分类以获得化学易处理性。在目标3中，我们将使用底物磷酸化测定来测试我们的ASK 1抑制剂的细胞效力，该测定特异性地定量MKK 6（ASK 1的直接底物）的磷酸化状态。然后将使用细胞凋亡测定法评估细胞渗透剂分子的细胞毒性，并在多参数、功能性、基于细胞的成像测定法中测试无毒化合物，以确定先导ASK 1抑制剂是否确实可以防止ASK 1介导的细胞死亡。最后，我们将评估我们的顶级先导抑制剂对一组300种激酶的激酶选择性（Reaction Biology Corp）。我们在TSRI的Scripps佛罗里达校区的Multi-PI研究团队拥有进行这些实验的专业知识和基础设施，我们认为我们研究的成功完成将确定一系列新的，有效的和选择性的分子探针，这将有助于确定ASK 1在正常生理过程中以及疾病状态中所起的作用。

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.