Barsyte-Lovejoy Dalia, Li Fengling, Oudhoff Menno J, Tatlock John H, Dong Aiping, Zeng Hong, Wu Hong, Freeman Spencer A, Schapira Matthieu, Senisterra Guillermo A, Kuznetsova Ekaterina, Marcellus Richard, Allali-Hassani Abdellah, Kennedy Steven, Lambert Jean-Philippe, Couzens Amber L, Aman Ahmed, Gingras Anne-Claude, Al-Awar Rima, Fish Paul V, Gerstenberger Brian S, Roberts Lee, Benn Caroline L, Grimley Rachel L, Braam Mitchell J S, Rossi Fabio M V, Sudol Marius, Brown Peter J, Bunnage Mark E, Owen Dafydd R, Zaph Colby, Vedadi Masoud, Arrowsmith Cheryl H
Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada M5G 1L7;
Biomedical Research Centre, University of British Columbia, Vancouver, BC, Canada V6T1Z3;
Proc Natl Acad Sci U S A. 2014 Sep 2;111(35):12853-8. doi: 10.1073/pnas.1407358111. Epub 2014 Aug 18.
SET domain containing (lysine methyltransferase) 7 (SETD7) is implicated in multiple signaling and disease related pathways with a broad diversity of reported substrates. Here, we report the discovery of (R)-PFI-2-a first-in-class, potent (Ki (app) = 0.33 nM), selective, and cell-active inhibitor of the methyltransferase activity of human SETD7-and its 500-fold less active enantiomer, (S)-PFI-2. (R)-PFI-2 exhibits an unusual cofactor-dependent and substrate-competitive inhibitory mechanism by occupying the substrate peptide binding groove of SETD7, including the catalytic lysine-binding channel, and by making direct contact with the donor methyl group of the cofactor, S-adenosylmethionine. Chemoproteomics experiments using a biotinylated derivative of (R)-PFI-2 demonstrated dose-dependent competition for binding to endogenous SETD7 in MCF7 cells pretreated with (R)-PFI-2. In murine embryonic fibroblasts, (R)-PFI-2 treatment phenocopied the effects of Setd7 deficiency on Hippo pathway signaling, via modulation of the transcriptional coactivator Yes-associated protein (YAP) and regulation of YAP target genes. In confluent MCF7 cells, (R)-PFI-2 rapidly altered YAP localization, suggesting continuous and dynamic regulation of YAP by the methyltransferase activity of SETD7. These data establish (R)-PFI-2 and related compounds as a valuable tool-kit for the study of the diverse roles of SETD7 in cells and further validate protein methyltransferases as a druggable target class.
含SET结构域（赖氨酸甲基转移酶）7（SETD7）参与多种信号传导和疾病相关途径，其报道的底物种类繁多。在此，我们报告了（R）-PFI-2的发现——一种一流的、强效（表观抑制常数Ki（app）=0.33 nM）、具有选择性且具有细胞活性的人SETD7甲基转移酶活性抑制剂——及其活性低500倍的对映体（S）-PFI-2。（R）-PFI-2通过占据SETD7的底物肽结合槽（包括催化赖氨酸结合通道）并与辅因子S-腺苷甲硫氨酸的供体甲基直接接触，表现出一种不寻常的辅因子依赖性和底物竞争性抑制机制。使用（R）-PFI-2的生物素化衍生物进行的化学蛋白质组学实验表明，在经（R）-PFI-2预处理的MCF7细胞中，其与内源性SETD7的结合存在剂量依赖性竞争。在小鼠胚胎成纤维细胞中，（R）-PFI-2处理模拟了Setd7缺陷对Hippo信号通路的影响，通过调节转录共激活因子Yes相关蛋白（YAP）以及YAP靶基因的调控。在汇合的MCF7细胞中，（R）-PFI-2迅速改变YAP的定位，表明SETD7的甲基转移酶活性对YAP进行持续和动态的调节。这些数据确立了（R）-PFI-2及相关化合物作为研究SETD7在细胞中多种作用的有价值工具，并进一步验证了蛋白质甲基转移酶作为可成药靶标的类别。
