Abstract

Cyclin-dependent kinase 9 (CDK9), an important regulator of transcriptional elongation, is a promising target for cancer therapy, particularly for cancers driven by transcriptional dysregulation. We characterized NVP-2, a selective ATP-competitive CDK9 inhibitor, and THAL-SNS-032, a selective CDK9 degrader consisting of a CDK-binding SNS-032 ligand linked to a thalidomide derivative that binds the E3 ubiquitin ligase Cereblon (CRBN). To our surprise, THAL-SNS-032 induced rapid degradation of CDK9 without affecting the levels of other SNS-032 targets. Moreover, the transcriptional changes elicited by THAL-SNS-032 were more like those caused by NVP-2 than those induced by SNS-032. Notably, compound washout did not significantly reduce levels of THAL-SNS-032-induced apoptosis, suggesting that CDK9 degradation had prolonged cytotoxic effects compared with CDK9 inhibition. Thus, our findings suggest that thalidomide conjugation represents a promising strategy for converting multi-targeted inhibitors into selective degraders and reveal that kinase degradation can induce distinct pharmacological effects compared with inhibition.

  • Compound

    N-(5-(((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazol-2-yl)piperidine-4-carboxamide

  • Compound

    N-(5-(((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazol-2-yl)-1-(14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxo-6,9,12-trioxa-3-azatetradecyl)piperidine-4-carboxamide

  • Compound

    4-(((5'-chloro-2'-(((1R,4r)-4-(((R)-1-methoxypropan-2-yl)amino)cyclohexyl)amino)-[2,4'-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile

  • Compound

    2-(((1r,4r)-4-((5'-chloro-6-(((4-cyanotetrahydro-2H-pyran-4-yl)methyl)amino)-[2,4'-bipyridin]-2'-yl)amino)cyclohexyl)amino)-N-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)acetamide

  • Compound

    4-(((5'-chloro-2'-(((1r,4r)-4-((6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)amino)cyclohexyl)amino)-[2,4'-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile

  • Compound

    N-(6-(((1r,4r)-4-((5'-chloro-6-(((4-cyanotetrahydro-2H-pyran-4-yl)methyl)amino)-[2,4'-bipyridin]-2'-yl)amino)cyclohexyl)amino)hexyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)acetamide

  • Compound

    4-(((1r,4r)-4-((5'-chloro-6-(((4-cyanotetrahydro-2H-pyran-4-yl)methyl)amino)-[2,4'-bipyridin]-2'-yl)amino)cyclohexyl)amino)-N-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)butyl)butanamide

  • Compound

    4-fluoroisobenzofuran-1,3-dione

  • Compound

    2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione

  • Compound

    tert-butyl (2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)carbamate

  • Compound

    tert-butyl 2-(4-((5-(((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazol-2-yl)carbamoyl)piperidin-1-yl)acetate

  • Compound

    4-(aminomethyl)tetrahydro-2H-pyran-4-carbonitrile

  • Compound

    4-(((6-bromopyridin-2-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile

  • Compound

    4-(((5'-chloro-2'-fluoro-[2,4'-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile

  • Compound

    (S)-1-methoxypropan-2-ol

  • Compound

    (S)-1-methoxypropan-2-yl 4-methylbenzenesulfonate

  • Compound

    4-(((2'-(((1r,4r)-4-aminocyclohexyl)amino)-5'-chloro-[2,4'-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile

  • Compound

    ethyl ((1r,4r)-4-((5'-chloro-6-(((4-cyanotetrahydro-2H-pyran-4-yl)methyl)amino)-[2,4'-bipyridin]-2'-yl)amino)cyclohexyl)glycinate

  • Compound

    N-(tert-butoxycarbonyl)-N-((1r,4r)-4-((5'-chloro-6-(((4-cyanotetrahydro-2H-pyran-4-yl)methyl)amino)-[2,4'-bipyridin]-2'-yl)amino)cyclohexyl)glycine

  • Compound

    4-(((2'-(((1r,4r)-4-((6-aminohexyl)amino)cyclohexyl)amino)-5'-chloro-[2,4'-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile

  • Compound

    ethyl 4-(((1r,4r)-4-((5'-chloro-6-(((4-cyanotetrahydro-2H-pyran-4-yl)methyl)amino)-[2,4'-bipyridin]-2'-yl)amino)cyclohexyl)amino)butanoate

  • Compound

    4-((tert-butoxycarbonyl)((1r,4r)-4-((5'-chloro-6-(((4-cyanotetrahydro-2H-pyran-4-yl)methyl)amino)-[2,4'-bipyridin]-2'-yl)amino)cyclohexyl)amino)butanoic acid

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Acknowledgements

We thank D. Buckley, E. Wang, J. Paulk, and members of the Gray and Bradner laboratories for helpful discussions. This work was supported by the US National Institutes of Health (CA179483-03 to N.K. and N.S.G., and T32GM007753 to Z.Z.), the Koch Institute and Dana-Farber/Harvard Cancer Center Bridge Grant (N.K., N.S.G., R.A.Y., C.M.O.),

Author information

Affiliations

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

    • Calla M Olson
    • , Baishan Jiang
    • , Yanke Liang
    • , Zainab M Doctor
    • , Zinan Zhang
    • , Tinghu Zhang
    • , Nicholas Kwiatkowski
    • , Eric S Fischer
    •  & Nathanael S Gray
  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.

    • Calla M Olson
    • , Baishan Jiang
    • , Yanke Liang
    • , Zainab M Doctor
    • , Zinan Zhang
    • , Tinghu Zhang
    • , Nicholas Kwiatkowski
    • , Eric S Fischer
    •  & Nathanael S Gray
  3. Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

    • Michael A Erb
    • , James E Bradner
    •  & Georg E Winter
  4. Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.

    • Michael A Erb
    • , James E Bradner
    •  & Georg E Winter
  5. Massachusetts General Hospital Cancer Center and Department of Medicine, Harvard Medical School, Charlestown, Massachusetts, USA.

    • Myriam Boukhali
    •  & Wilhelm Haas
  6. ActivX Biosciences, La Jolla, California, USA.

    • Jennifer L Green
    •  & Tyzoon Nomanbhoy
  7. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.

    • Richard A Young
  8. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Richard A Young
  9. Novartis Institutes for Biomedical Sciences (NIBR), Cambridge, Massachusetts, USA.

    • James E Bradner
  10. Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

    • Georg E Winter

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Contributions

N.S.G., C.M.O., N.K. and T.Z. conceived the project. N.S.G., Y.L., B.J. and T.Z. conceived and directed chemistry effort. Chemical synthesis and small-molecule structure determination was performed by T.Z., Y.L. and Z.Z. R.A.Y., N.S.G., C.M.O., M.A.E., G.E.W. 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. W.H. and M.B. designed and executed the proteomics efforts. E.S.F. designed and executed statistical analysis of proteomics. T.N. and J.G. designed and executed the Kinativ efforts. M.A.E. designed and performed genomics data analyses. C.M.O., N.K. and N.S.G. wrote the manuscript. All of the authors edited the manuscript.

Competing interests

N.S.G. is a Scientific Founder and member of the Scientific Advisory Board of C4 Therapeutics, Syros Pharmaceuticals and Petra Pharmaceuticals and is the inventor on intellectual property licensed to these entities. J.E.B. is a Scientific Founder of Syros Pharmaceuticals, SHAPE Pharmaceuticals, Acetylon Pharmaceuticals, Tensha Therapeutics (now Roche) and C4 Therapeutics and is the inventor on intellectual property licensed to these entities. J.E.B. is now an executive and shareholder in Novartis AG. R.A.Y. is a Scientific Founder of Syros Pharmaceuticals and Marauder Therapeutics.

Corresponding author

Correspondence to Nathanael S Gray.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Results, Supplementary Tables 1–2, Supplementary Figures 1–9

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Note 1

    Chemical Synthesis of THAL-SNS-032, NVP-2, THAL-NVP-2-02-099, THAL-NVP-2- 3 03-069, HAL-NVP-2-03-099, THAL-NVP-2-03-084, THAL-NVP-2-03-105.

Excel files

  1. 1.

    Supplementary Dataset 1

    Lysate Kinativ of SNS-032, THAL-SNS-032, and NVP-2

  2. 2.

    Supplementary Dataset 2

    Proteomics of THAL-SNS-032 treatment

  3. 3.

    Supplementary Dataset 3

    Selectivity of NVP-2 by Ambit profiling

  4. 4.

    Supplementary Dataset 4

    Live cell Kinativ of SNS-032 and THAL-SNS-032

  5. 5.

    Supplementary Dataset 5

    Live cell Kinativ of NVP-2

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DOI

https://doi.org/10.1038/nchembio.2538