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Cellularly active N-hydroxyurea FEN1 inhibitors block substrate entry to the active site

Abstract

The structure-specific nuclease human flap endonuclease-1 (hFEN1) plays a key role in DNA replication and repair and may be of interest as an oncology target. We present the crystal structure of inhibitor-bound hFEN1, which shows a cyclic N-hydroxyurea bound in the active site coordinated to two magnesium ions. Three such compounds had similar IC50 values but differed subtly in mode of action. One had comparable affinity for protein and protein–substrate complex and prevented reaction by binding to active site catalytic metal ions, blocking the necessary unpairing of substrate DNA. Other compounds were more competitive with substrate. Cellular thermal shift data showed that both inhibitor types engaged with hFEN1 in cells, and activation of the DNA damage response was evident upon treatment with inhibitors. However, cellular EC50 values were significantly higher than in vitro inhibition constants, and the implications of this for exploitation of hFEN1 as a drug target are discussed.

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Figure 1: Compounds used in this study and crystal structure of hFEN1-336Δ in complex with compound 1.
Figure 2: Differences in inhibition characteristics of the compounds.
Figure 3: Effect of inhibitors on substrate binding assessed by fluorescence anisotropy and FRET.
Figure 4: N-Hydroxyurea inhibitors prevent FEN1 reaction by blocking substrate unpairing.
Figure 5: Cellular engagement and activity of hFEN1 inhibitors 1 and 4.

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Acknowledgements

This work was supported by U.K. Biotechnology and Biological Sciences Research Council grants BB/K009079/1 and BB/M00404X/1 (both to J.A.G.) and AstraZeneca. J.C.E. thanks the U.K. Engineering and Physical Sciences Research Council and AstraZeneca for a studentship. The authors thank C. Phillips for assistance with submissions of the crystallographic data and T. McGuire for synthetic support.

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C.D.J. designed and synthesized inhibitors. J.C.E., M.J.T., L.D.F. and S.J.S. carried out kinetic and biophysical experiments. J.C.E., M.J.T., L.D.F., C.M., J.W.M.N., W.M.A. and J.A.G. designed experiments and analyzed this data. J.D. and J.W.M.N. obtained and analyzed structures. C.L.B.S. and D.M.M. performed the CETSA assays. T.A.W. carried out other cellular experiments, and T.A.W. and S.T.D. analyzed data. All authors contributed to the preparation of the manuscript.

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Correspondence to J Willem M Nissink, Stephen T Durant or Jane A Grasby.

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W.M.A., C.M., J.D., J.W.M.N. and S.T.D. are employees of AstraZeneca. C.D.J. and T.A.W. were employees of AstraZeneca at the time of writing. C.L.B.S. and D.M.M. are employees of Pelago Bioscience AB.

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Supplementary Results, Supplementary Tables 1–6 and Supplementary Figures 1–20. (PDF 8959 kb)

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Exell, J., Thompson, M., Finger, L. et al. Cellularly active N-hydroxyurea FEN1 inhibitors block substrate entry to the active site. Nat Chem Biol 12, 815–821 (2016). https://doi.org/10.1038/nchembio.2148

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