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Structural analysis of human KDM5B guides histone demethylase inhibitor development

Abstract

Members of the KDM5 (also known as JARID1) family are 2-oxoglutarate- and Fe2+-dependent oxygenases that act as histone H3K4 demethylases, thereby regulating cell proliferation and stem cell self-renewal and differentiation. Here we report crystal structures of the catalytic core of the human KDM5B enzyme in complex with three inhibitor chemotypes. These scaffolds exploit several aspects of the KDM5 active site, and their selectivity profiles reflect their hybrid features with respect to the KDM4 and KDM6 families. Whereas GSK-J1, a previously identified KDM6 inhibitor, showed about sevenfold less inhibitory activity toward KDM5B than toward KDM6 proteins, KDM5-C49 displayed 25–100-fold selectivity between KDM5B and KDM6B. The cell-permeable derivative KDM5-C70 had an antiproliferative effect in myeloma cells, leading to genome-wide elevation of H3K4me3 levels. The selective inhibitor GSK467 exploited unique binding modes, but it lacked cellular potency in the myeloma system. Taken together, these structural leads deliver multiple starting points for further rational and selective inhibitor design.

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Figure 1: Domain organization of full-length human KDM5B, and construct design and substrate kinetics for KDM5B deletion constructs.
Figure 2: Structure determination of human KDM5B.
Figure 3: The KDM5B structures reveal hybrid features with respect to the KDM4 and KDM6 families.
Figure 4: The active site of human KDM5B is amenable to selective-inhibitor development and can accommodate distinct inhibitor chemotypes.
Figure 5: KMD5-C70 is active in MM.1S myeloma cells.
Figure 6: KDM5-C70 increases H3K4me3 levels in myeloma cells.

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Acknowledgements

Research in our laboratories is supported by funding from Arthritis Research UK (program grant number 20522 to U.O.), the NIHR Oxford Biomedical Research Unit (U.O.), Sarcoma UK (N.A.), the Bone Cancer Research Trust (N.A.), the Rosetrees Trust (N.A. and U.O.), and Cancer Research UK (grants C8717/A18245 (to C.J.) and 300/A13058 (to N.B.L.) and CRUK Oxford Development Fund to U.O. and C.J.). The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, the Canada Foundation for Innovation, Genome Canada, GlaxoSmithKline, Janssen, Lilly Canada, Merck & Co., the Novartis Research Foundation, the Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation–FAPESP, Takeda, and the Wellcome Trust (092809/Z/10/Z). We thank Diamond Light Source for beamtime (proposal mx10619) and the staff of beamlines I02 and I03 for assistance with crystal testing and data collection. We are grateful to R. Rambo for help with SAXS data collection and analysis at the Diamond Light Source beamline B21, and to R. Klose (Department of Chemistry, Oxford University, Oxford, UK) for the human KDM5A construct and the FDH expression clone. We also acknowledge a sample of GSK467 kindly provided by GlaxoSmithKline.

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Authors and Affiliations

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Contributions

C.J. and U.O. designed experiments, analyzed data, supervised the study and wrote the manuscript. Data collection and structure refinements were done by S.V., R.P.N. and J.K. Construct design was done by C.J., H.S., U.E. and V.B. Cloning, mutagenesis and expression trials were done by C.S.-D., A.S., M.P. and N.B.-B. Purification, crystallization and optimization were done by C.J., S.V., A.S. and C.G. Enzymology was done by A.T. Cell culture experiments were done by N.W., E.S.H., S.M., N.A. and N.B.L. Compounds were provided by R.P., S.W., A.N., J.B. and P.E.B. All authors approved the final version of the manuscript.

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Correspondence to Catrine Johansson or Udo Oppermann.

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Competing interests

H.S., U.E., V.B., S.W., J.B. and R.P. are pharmaceutical industry employees and shareholders.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–18 and Supplementary Tables 1–6. (PDF 21199 kb)

Supplementary Note

Synthetic Procedures (PDF 450 kb)

Supplementary Data Set 1

Annotated list of differentially H3K4me3 methylated peaks in multiple myeloma MM1S cell line, treated with KDM5-C70. (XLSX 4200 kb)

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Johansson, C., Velupillai, S., Tumber, A. et al. Structural analysis of human KDM5B guides histone demethylase inhibitor development. Nat Chem Biol 12, 539–545 (2016). https://doi.org/10.1038/nchembio.2087

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