Abstract

Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12–cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.

  • Compound

    (R,E)-N-(4-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)piperidine-1-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide

  • Compound

    N-((E)-20-((4-((R)-3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)piperidine-1-carbonyl)phenyl)amino)-16-methyl-13,20-dioxo-3,6,9-trioxa-12,16-diazaicos-18-en-1-yl)-6-(5-methyl-2-oxoimidazolidin-4-yl)hexanamide

  • Compound

    (R)-N-(4-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)piperidine-1-carbonyl)phenyl)-4-(dimethylamino)butanamide

  • Compound

    (S,E)-N-(4-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)piperidine-1-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide

  • Compound

    (R)-5-chloro-4-(1-(phenylsulfonyl)-1H-indol-3-yl)-N-(piperidin-3-yl)pyrimidin-2-amine

  • Compound

    (R)-(3-((5-chloro-4-(1-(phenylsulfonyl)-1H-indol-3-yl)pyrimidin-2-yl)amino)piperidin-1-yl)(4-nitrophenyl)methanone

  • Compound

    (R)-(4-aminophenyl)(3-((5-chloro-4-(1-(phenylsulfonyl)-1H-indol-3-yl)pyrimidin-2-yl)amino)piperidin-1-yl)methanone

  • Compound

    (R,E)-N-(4-(3-((5-chloro-4-(1-(phenylsulfonyl)-1H-indol-3-yl)pyrimidin-2-yl)amino)piperidine-1-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide

  • Compound

    N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-6-(5-methyl-2-oxoimidazolidin-4-yl)hexanamide

  • Compound

    N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-6-(5-methyl-2-oxoimidazolidin-4-yl)hexanamide

  • Compound

    tert-butyl methyl(22-(5-methyl-2-oxoimidazolidin-4-yl)-3,17-dioxo-7,10,13-trioxa-4,16-diazadocosyl)carbamate

  • Compound

    6-(5-methyl-2-oxoimidazolidin-4-yl)-N-(5-oxo-9,12,15-trioxa-2,6-diazaheptadecan-17-yl)hexanamide

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Gene Expression Omnibus

NCBI Reference Sequence

Protein Data Bank

Referenced accessions

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Acknowledgements

We thank R. George, K. Wong, P. Hammerman, J. Bradner and members of the Gray and Young laboratories for helpful discussions. We thank P. Wisniewski and C. Zollo from the Whitehead FACS facility for help with FACS instruments. We thank J. Love, T. Volkert, and S. Gupta from the Whitehead Genome Core for help with genomics experiments. The authors would like to thank Diamond Light Source for beamtime (proposal mx8421), and the staff of Beamline I03 for assistance with data collection. We thank W. Massefski from the Dana–Farber NMR lab for help in collecting NMR data for the manuscript. M.G. is a member of the DFG excellence cluster ImmunoSensation. This work was supported by the National Institutes of Health (HG002668 and CA109901 to R.A.Y. and 5 R01 CA179483-02 C to N.S.G.), the Koch Institute and Dana–Farber/Harvard Cancer Center Bridge Grant (N.K., N.S.G., R.A.Y.), the DFG (GE 976/9-1 to M.G.), the Hope Funds for Cancer Research Grillo-Marxuach Family Fellowship (B.J.A.), and a NDM Prize Studentship, funded in part by the Medical Research Council (S.E.D.-C.). The SGC is a registered charity (no. 1097737) that receives funds from AbbVie, Bayer, Boehringer Ingelheim, Genome Canada through Ontario Genomics Institute Grant OGI-055, GlaxoSmithKline, Janssen, Lilly Canada, the Novartis Research Foundation, the Ontario Ministry of Economic Development and Innovation, Pfizer, Takeda, and Wellcome Trust Grant 092809/Z/10/Z.

Author information

Author notes

    • Tinghu Zhang
    • , Nicholas Kwiatkowski
    •  & Calla M Olson

    These authors contributed equally to this work.

Affiliations

  1. Department of Cancer Biology, Dana–Farber Cancer Institute, Boston, Massachusetts, USA.

    • Tinghu Zhang
    • , Nicholas Kwiatkowski
    • , Calla M Olson
    • , Scott B Ficarro
    • , Yanke Liang
    • , Theresa Manz
    • , Mingfeng Hao
    • , Jarrod A Marto
    •  & Nathanael S Gray
  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.

    • Tinghu Zhang
    • , Nicholas Kwiatkowski
    • , Calla M Olson
    • , Scott B Ficarro
    • , Yanke Liang
    • , Mingfeng Hao
    • , Jarrod A Marto
    •  & Nathanael S Gray
  3. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.

    • Nicholas Kwiatkowski
    • , Brian J Abraham
    • , Nancy M Hannett
    •  & Richard A Young
  4. Structural Genomics Consortium, University of Oxford, Oxford, UK.

    • Sarah E Dixon-Clarke
    • , Jonathan M Elkins
    •  & Alex N Bullock
  5. Department of Structural Immunology, Institute of Innate Immunity, University of Bonn, Bonn, Germany.

    • Ann K Greifenberg
    •  & Matthias Geyer
  6. Center of Advanced European Studies and Research, Bonn, Germany.

    • Ann K Greifenberg
    •  & Matthias Geyer
  7. Blais Proteomics Center, Dana–Farber Cancer Institute, Boston, Massachusetts, USA.

    • Scott B Ficarro
    •  & Jarrod A Marto
  8. Pharmaceutical and Medicinal Chemistry, Department of Pharmacy, Saarland University, Saarbrücken, Germany.

    • Theresa Manz
  9. Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA.

    • Bartlomiej Bartkowiak
    •  & Arno L Greenleaf
  10. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Richard A Young

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Contributions

N.S.G., R.A.Y., N.K., and T.Z. conceived the project. N.S.G. and T.Z. conceived and directed chemistry effort with input from Y.L. Chemical synthesis and small molecule structure determination was performed by T.Z., M.H., and T.M. R.A.Y., N.S.G., B.J.A. and N.K. conceived genomics effort. N.K. and C.M.O. designed and executed cellular biological experimental research with input from N.S.G. and R.A.Y.S.E.D.-C. solved co-crystal structure of CDK12–cyclin K with THZ531 with guidance from J.M.E. and A.N.B. A.K.G. designed and executed CDK in vitro kinase assays with input from M.G. S.B.F. designed and performed protein mass spectrometry on THZ531–CDK12 adducts with input from J.A.M. N.M.H. provided cloning expertise. B.B. and A.L.G. developed and provided CDK12 and CDK13 antibodies. B.J.A. designed and performed genomics data analyses. N.K., T.Z., N.S.G. and R.A.Y. co-wrote the paper. All authors edited the manuscript.

Competing interests

N.S.G., T.Z., N.K. are inventors on patent applications covering THZ531 (WO 1421 2015/058126, WO/2014/063068, WO 2015/058140), which are licensed to a company Syros co-founded by N.S.G. and R.A.Y.

Corresponding authors

Correspondence to Richard A Young or Nathanael S Gray.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Results, Supplementary Figures 1–11 and Supplementary Tables 1–3.

  2. 2.

    Supplementary Note 1

    Synthetic Procedures

Excel files

  1. 1.

    Supplementary Data Set 1

    Mass spectrometry identifies CDK12-cyclin K and CDK13–cyclin K complexes as major targets of bioTHZ531 in Jurkat cell lysates.

  2. 2.

    Supplementary Data Set 2

    In vitro Ambit™ binding assay shows THZ531 potently inhibits CDK13.

  3. 3.

    Supplementary Data Set 3

    Gene expression microarray data of THZ531, Flavopiridol, THZ1-treated cells.

  4. 4.

    Supplementary Data Set 4

    Jurkat enhancers and super–enhancers identified by H3K27Ac ChIP–seq.

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DOI

https://doi.org/10.1038/nchembio.2166

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