Design of a fluorescent ligand targeting the S-adenosylmethionine binding site of the histone methyltransferase MLL1.
Design of a fluorescent ligand targeting the S-adenosylmethionine binding site of the histone methyltransferase MLL1.
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DOI：
10.1039/c5ob01794g
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发表时间：
2016-01-14
影响因子：
3.2
通讯作者：
Zheng YG
中科院分区：
文献类型：
--
作者：
Luan Y;Blazer LL;Hu H;Hajian T;Zhang J;Wu H;Houliston S;Arrowsmith CH;Vedadi M;Zheng YG
组蛋白甲基转移酶MLL1与儿童白血病易位相关的基因融合有关，是一个有吸引力的药物靶点。MLL1甲基转移酶活性的高通量生化分析需要至少产生MLL1、RbBP5和WDR5的三聚体复合体，以获得强大的活性。高通量筛选所需的三聚体和高阶MLL1复合物的数量和可重复性对MLL1药物发现工作构成了重大障碍。我们在这里提出了一个小分子荧光配体（FL-NAH, 6），它能够以独立于相关复合物成员的方式结合MLL1的s -腺苷蛋氨酸（SAM）结合位点。我们使用FL-NAH开发了一种基于荧光偏振的SAM置换测定方法，该方法采用384孔格式，针对缺乏相关复合物成员的MLL1 SET结构域。FL-NAH与SAM竞争，并被其他具有与高阶配合物相似的Kdisp值的SAM结合位点配体从MLL1 SET结构域移位，但不受H3肽底物的影响。该方法能够筛选具有sam竞争性的MLL1抑制剂，而不需要使用三聚体或更高阶的MLL1复合物，从而大大减少了筛选时间和成本。
The histone methyltransferase MLL1 has been linked to translocation-associated gene fusion in childhood leukemias and is an attractive drug target. High-throughput biochemical analysis of MLL1 methyltransferase activity requires the production of at least a trimeric complex of MLL1, RbBP5 and WDR5 to elicit robust activity. Production of trimeric and higher order MLL1 complexes in the quantities and reproducibility required for high-throughput screening presents a significant impediment to MLL1 drug discovery efforts. We present here a small molecule fluorescent ligand (FL-NAH, 6) that is able to bind to the S-adenosylmethionine (SAM) binding site of MLL1 in a manner independent of the associated complex members. We have used FL-NAH to develop a fluorescence polarization-based SAM displacement assay in a 384-well format targeting the MLL1 SET domain in the absence of associated complex members. FL-NAH competes with SAM and is displaced from the MLL1 SET domain by other SAM-binding site ligands with Kdisp values similar to the higher-order complexes, but is unaffected by the H3 peptide substrate. This assay enables screening for SAM-competitive MLL1 inhibitors without requiring the use of trimeric or higher order MLL1 complexes, significantly reducing screening time and cost.