Sakurai Masaaki, Rose Nathan R, Schultz Lena, Quinn Amy M, Jadhav Ajit, Ng Stanley S, Oppermann Udo, Schofield Christopher J, Simeonov Anton
NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-3370, USA.
Mol Biosyst. 2010 Feb;6(2):357-64. doi: 10.1039/b912993f. Epub 2009 Oct 8.
2-Oxoglutarate- and Fe(ii)-dependent oxygenases are a major class of N(epsilon)-methyl lysine demethylases that are involved in epigenetic regulation. Assays suitable for implementation in a high-throughput manner have been lacking for these enzymes. Here, we describe the design and implementation of a robust and miniaturized high-throughput kinetic assay for inhibitors of JMJD2E using a formaldehyde dehydrogenase-coupled reaction with real-time fluorescence detection. Reactant compatibility studies resulted in simplification of the assay scheme to the mixing of two reagent solutions, both of which were stable overnight. The assay was miniaturized to a 4 microL volume in 1536-well format and was used to screen the library of pharmacologically active compounds (LOPAC(1280)). Inhibitors identified by the screen were further characterized in secondary assays including FDH counterscreen and demethylation assays that monitored demethylation by MALDI-TOF MS. The assay developed here will enable the screening of large compound libraries against the Jumonji demethylases in a robust and automated fashion.
2-氧代戊二酸和Fe（II）依赖性加氧酶是一类主要的N（ε）-甲基赖氨酸去甲基化酶，参与表观遗传调控。目前缺乏适用于高通量检测这些酶的方法。在此，我们描述了一种针对JMJD2E抑制剂的稳健且小型化的高通量动力学检测方法的设计与实施，该方法采用甲醛脱氢酶偶联反应并进行实时荧光检测。反应物兼容性研究简化了检测方案，只需混合两种试剂溶液，且这两种溶液均可稳定保存过夜。该检测方法被小型化为1536孔板形式的4微升体积，并用于筛选药理活性化合物文库（LOPAC(1280)）。通过筛选鉴定出的抑制剂在包括FDH反筛选和通过基质辅助激光解吸电离飞行时间质谱监测去甲基化的去甲基化检测等二级检测中进一步表征。此处开发的检测方法将能够以稳健且自动化的方式针对Jumonji去甲基化酶筛选大型化合物文库。
