Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation

Abstract

PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases through clinical genetics and gene disruption in mice. New selective PAD4 inhibitors binding a calcium-deficient form of the PAD4 enzyme have validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation for, to our knowledge, the first time. The therapeutic potential of PAD4 inhibitors can now be explored.

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Figure 1: Structure and biochemical characterization of PAD4 inhibitors.
Figure 2: GSK199 binds to a reordered PAD4 active site.
Figure 3: Inhibition of mouse NETs.

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Acknowledgements

This work was supported in part by US National Institutes of Health grants GM079357 (to P.R.T.) and R01 HL102101 (to D.D.W.). We thank the following colleagues for their medicinal and synthetic chemistry contributions to this manuscript: D. Amans, H. Diallo, C. Douault, N. Garton, K. Jones, J. Renaux, T. Shipley, A. Walker, B. Watson and C. Wellaway. We also gratefully acknowledge S. Ready for diligently preparing and quantifying S. aureus for neutrophil studies. For the chemoproteomics data, we would like to thank M. Boesche and Cellzome's tissue culture and target validation teams for technical expertise and M. Bantscheff for stimulating discussion. We thank M. Neu and P. Homes for assistance for structural studies and B. Nolte for advice on crystallography.

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H.D.L., J.L. and J.E.C. led the project, designed the research, interpreted data and drafted the manuscript with input from all authors. S.J.A., M.D.B., M.C., R.J.S. and D.M.W. synthesized or designed key compounds. K.E.L., C.E.R. and G.Y. performed and analyzed the screening, which identified the PAD4 inhibitors. K.L.B., R.P.B., P.D.C., K.L. and D.J.S. designed the biochemical assays and deduced the mechanism of action of compounds. D.E., G.J. and G.D. generated chemoproteomic data. Y.H.C., R.P.D., D.E., C.M., K.M., C.P. and M.R. performed cellular assays. For structures, O.P. purified protein; J.T. crystallized protein, soaked crystals and collected data; B.D.B. refined structures; and P.T. performed sequence and structural analysis. C.-w.C. D.D.W., P.R.T., R.K.P. and D.M.W. guided aspects of this work. C.M., S.J.A., C.-w.C. and H.D.L. also contributed invaluably to the revision and formatting of the final manuscript.

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Correspondence to Huw D Lewis.

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Competing interests

P.R.T. is a co-founder and consultant to Padlock Therapeutics. The majority of the other authors are pharmaceutical industry employees and shareholders.

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Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–19 and Supplementary Tables 1–6. (PDF 9590 kb)

Supplementary Note

General Procedures (PDF 1514 kb)

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Lewis, H., Liddle, J., Coote, J. et al. Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation. Nat Chem Biol 11, 189–191 (2015). https://doi.org/10.1038/nchembio.1735

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