Heterogeneous Antibody-Based Activity Assay for Lysine Specific Demethylase 1 (LSD1) on a Histone Peptide Substrate
Heterogeneous Antibody-Based Activity Assay for Lysine Specific Demethylase 1 (LSD1) on a Histone Peptide Substrate
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DOI：
10.1177/1087057114529156
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发表时间：
2014-07-01
影响因子：
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通讯作者：
Jung, Manfred
中科院分区：
文献类型：
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作者：
Schmitt, Martin L.;Ladwein, Kathrin I.;Jung, Manfred
组蛋白尾部的翻译后修饰对表观遗传基因调控具有重要意义。赖氨酸特异性去甲基化酶LSD1 （KDM1A/AOF2）在体外主要对组蛋白3 （H3K4）上的赖氨酸4单甲基化和二甲基化进行去甲基化，是药物发现的一个有希望的靶点。我们报告了一种基于异质抗体的检测方法，使用解离增强镧系荧光免疫分析法（DELFIA）检测LSD1活性。我们使用生物素化组蛋白3肽（氨基酸1-21）和单甲基化赖氨酸4 （H3K4me）作为底物，通过抗体介导的去甲基化产物定量检测LSD1活性。我们已经成功地使用该方法来测量参比抑制剂的效价。我们通过虚拟筛选确定了基于香豆素的LSD1抑制剂候选物，显示了异质格式的优势。它们在已建立的LSD1筛选试验中显示出良好的效力。新的异质分析将它们鉴定为假阳性，并使用质谱法进行验证。
Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine-specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody-based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody-mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. The advantage of the heterogeneous format is shown with cumarin-based LSD1 inhibitor candidates that we have identified using virtual screening. They had shown good potency in an established LSD1 screening assay. The new heterogeneous assay identified them as false positives, which was verified using mass spectrometry.