Abstract
Translation initiation plays a critical role in cellular homeostasis, proliferation, differentiation and malignant transformation. Consistently, increasing the abundance of the eIF2–GTP–tRNAiMet translation initiation complex transforms normal cells and contributes to cancer initiation and the severity of some anemias. The chemical modifiers of the eIF2–GTP–tRNAiMet ternary complex are therefore invaluable tools for studying its role in the pathobiology of human disorders and for determining whether this complex can be pharmacologically targeted for therapeutic purposes. Using a cell-based assay, we identified N,N′-diarylureas as unique inhibitors of ternary complex accumulation. Direct functional-genetic and biochemical evidence demonstrated that the N,N′-diarylureas activate heme-regulated inhibitor kinase, thereby phosphorylating eIF2α and reducing the abundance of the ternary complex. Using tumor cell proliferation in vitro and tumor growth in vivo as paradigms, we demonstrate that N,N′-diarylureas are potent and specific tools for studying the role of eIF2–GTP–tRNAiMet ternary complex in the pathobiology of human disorders.
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Acknowledgements
We thank D.C. Tosteson (Harvard Medical School), mentor and role model to authors, for encouragement; J.J. Chen for providing the HRI expression plasmid; and M. Tosteson for critical comments on the manuscript. This work was supported in part by NIH grant NCDDG 5 U19 CA87427 to J.A.H., Susan B. Komen Foundation for Cancer Research grant BCTR0707713, US Department of Defense grant no. W81ZWH-05-1-0096 and US National Institutes of Health grant no. R21AG032546 to B.H.A.
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T.C. characterized hits compounds, carried out experiments to determine the mechanism of action of hit compounds, analyzed the data and contributed to writing the paper; D.O. generated the screening assay and helped initial compound characterization; Y.Q. generated and characterized PC-3 cell lines; F.H. carried out screening campaign; L.C. carried out cell growth experiments; S.D. carried out small scale resynthesis of compounds; X.H., N.Z. and J.G.S. developed and validated an LC-MS method and analyzed mouse plasma samples; M.C. supervised synthesis and contributed to writing the paper; J.A.H. discussed the data and contributed to writing the manuscript; B.H.A. conceived, designed and supervised all aspects of research, analyzed the data and wrote the manuscript.
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Supplementary Dataset 1
Confirmation of initial hits with the ternary complex assay. Initial hits were confirmed in the ternary complex assay by testing in triplicate at 10 and 5 or 2.5 μM concentrations. Chemical structures can be obtained by entering the NSC number at http://dtp.nci.nih.gov/dtpstandard/ChemData/index.jsp. (XLS 78 kb)
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Chen, T., Ozel, D., Qiao, Y. et al. Chemical genetics identify eIF2α kinase heme-regulated inhibitor as an anticancer target. Nat Chem Biol 7, 610–616 (2011). https://doi.org/10.1038/nchembio.613
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DOI: https://doi.org/10.1038/nchembio.613
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