Enabling Lead Discovery for Histone Lysine Demethylases by High-Throughput RapidFire Mass Spectrometry
Enabling Lead Discovery for Histone Lysine Demethylases by High-Throughput RapidFire Mass Spectrometry
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DOI：
10.1177/1087057111416660
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发表时间：
2012-01-01
影响因子：
--
通讯作者：
Argyrou, Argyrides
中科院分区：
文献类型：
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作者：
Hutchinson, Sue E.;Leveridge, Melanie V.;Argyrou, Argyrides
一个高通量RapidFire质谱分析描述了铁（2+），O（2），和α-酮戊二酸依赖性组蛋白赖氨酸脱甲基酶的JMJD 2家族。该测定采用短氨基酸肽底物，其对应于组蛋白H3的前15个氨基酸残基，但在两个位置突变以增加测定灵敏度。该测定监测从三甲基化-Lys 9肽底物直接形成二甲基化-Lys 9产物。还可以监测单甲基化和去甲基化肽产物的形成。使用已知的组蛋白赖氨酸脱甲基酶抑制剂（包括2，4-吡啶二羧酸和α-酮戊二酸类似物）验证了该测定法。RapidFire技术以每孔7秒的采样率，允许使用两种仪器在10天内针对JMJD 2C对101226种化合物进行单浓度筛选，通常给出0.75至0.85的Z'值。鉴定了8-羟基喹啉化学型的几种化合物，这是已知的一系列Lys 9特异性组蛋白脱甲基酶的抑制剂。该肽还可作为JMJD 2A、JMJD 2D和JMJD 2 E的底物，从而能够开发所有3种酶的测定方法，以监测化合物选择性的进展。该测定代表了用于表观遗传学靶点的RapidFire质谱测定的第一份报告。
A high-throughput RapidFire mass spectrometry assay is described for the JMJD2 family of Fe(2+), O(2), and alpha-ketoglutarate-dependent histone lysine demethylases. The assay employs a short amino acid peptide substrate, corresponding to the first 15 amino acid residues of histone H3, but mutated at two positions to increase assay sensitivity. The assay monitors the direct formation of the dimethylated-Lys9 product from the trimethylated-Lys9 peptide substrate. Monitoring the formation of the monomethylated and des-methylated peptide products is also possible. The assay was validated using known inhibitors of the histone lysine demethylases, including 2,4-pyridinedicarboxylic acid and an a-ketoglutarate analogue. With a sampling rate of 7 s per well, the RapidFire technology permitted the single-concentration screening of 101 226 compounds against JMJD2C in 10 days using two instruments, typically giving Z' values of 0.75 to 0.85. Several compounds were identified of the 8-hydroxyquinoline chemotype, a known series of inhibitors of the Lys9-specific histone demethylases. The peptide also functions as a substrate for JMJD2A, JMJD2D, and JMJD2E, thus enabling the development of assays for all 3 enzymes to monitor progress in compound selectivity. The assay represents the first report of a RapidFire mass spectrometry assay for an epigenetics target.