Molecular screening: MALDI-TOF scanning of peptides (RMI)

为响应RFA-RM-04-020，“Molecular Libraries Screening Instrumentation”，我们建议开发一个高度集成的硬件和软件平台，该平台可以对单个化合物处理的细胞提取物中的信号蛋白激活进行高复用高通量分析。目前的高通量筛选方法通过检测激酶、磷酸酶、蛋白酶和其他信号转导酶对底物的化学修饰，为在大的小分子文库中发现新的细胞信号转导调节剂提供了强大的工具。这种方法已经证明了其在发现致癌性酪氨酸激酶抑制剂方面的价值，导致临床重要药物的出现，如伊马替尼在CML中靶向Bcr-Abl，在GIST中靶向c-kit，吉非替尼在小细胞肺癌中靶向EGFR，以及一些正在研究的激酶拮抗剂，如SU11248在AML中靶向Flt3。然而，最先进的分子文库筛选在识别可能是特定信号通路的激动剂或拮抗剂的分子方面存在不足，但不一定针对特定的信号蛋白。在之前的工作中，我们开发了使用免疫和MALDI-TOF检测固定化肽和蛋白磷酸化的敏感检测方法，准确报告了Ph+白血病细胞系全细胞提取物中Bcr-Abl的活性和抑制作用。在这里，我们将扩展这项工作，并使用我们建立的共聚方法，通过光可切割的连接剂将多个酪氨酸激酶底物和同位素编码的对照肽固定在丙烯酰胺共聚物中。对单个酪氨酸激酶或特定类别显示高特异性的肽将以这种格式进行选择和验证。细胞提取物将以阵列形式应用于这些表面，其中每个点对应于用单一文库化合物处理过的细胞。孵育后，将表面洗净，并将释放的多肽用紫外光裂解。对每个点进行MALDI-TOF分析将得到与每个化合物相关的测试肽和对照肽的光谱。这些光谱将被归一化，并与参考光谱进行比较，以检测一个或多个信号通路中统计上显著的变化。激活或抑制细胞提取物中存在的一种或多种酪氨酸激酶的化合物将被标记为进一步分析。

DESCRIPTION (provided by applicant): In response to RFA-RM-04-020, "Molecular Libraries Screening Instrumentation", we propose to develop a highly integrated hardware and software platform that performs highly multiplexed high throughput assays for signaling protein activation in extracts from cells treated with individual compounds. Current high throughput screening methods offer powerful tools to discover novel modulators of cell signaling in large libraries of small molecules by detecting the chemical modification of substrates by kinases, phosphatase, proteases and other signaling enzymes. This approach has proven its value in discovering inhibitors of oncogenic tyrosine kinases, leading to clinically important drugs such as Imatinib to target Bcr-Abl in CML and c-kit in GIST, Gefitinib to target EGFR in small cell lung cancer and a number of investigational kinase antagonists such as SU11248 to target Flt3 in AML. However, the state-of-the-art in molecular library screening falls short in identifying molecules that may be agonists or antagonists of specific signaling pathways but that are not necessarily targeted at a particular signaling protein. In prior work, we have developed sensitive assays using immunologic and MALDI-TOF detection of the phosphorylation of immobilized peptides and proteins that accurately report Bcr-Abl activity and inhibition in whole cell extracts from Ph+ leukemia cell lines. Here, we will extend this work and use our established copolymerization methodology to immobilize multiple tyrosine kinase substrate and isotope-coded control peptides in an acrylamide copolymer via photo-cleavable linkers. Peptides that display high specificity for individual tyrosine kinases or specific classes will be selected and validated in this format. Cell extracts will be applied to these surfaces in an array format where each spot corresponds to cells treated with a single library compound. After incubation, the surface will be washed, and the peptides released by UV photo-cleavage. MALDI-TOF analysis of each spot will lead to a spectrum of test and control peptides associated with each compound. These spectra will be normalized and compared with reference spectra to detect statistically significant changes in one or more signaling pathways. Compounds that appear to activate or inhibit one or more tyrosine kinases present in the cell extracts will be flagged for further analysis.