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Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

Nature Chemical Biology volume 17, pages 954–963 (2021)Cite this article

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Abstract

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1’s active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.

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Fig. 1: Discovery of a covalent Pin1-binding fragment.
Fig. 2: Sulfopin engages Pin1 in cells and in vivo.
Fig. 3: Sulfopin is highly selective for Pin1 C113 in cells.
Fig. 4: Sulfopin shows a Pin1-dependent viability effect following long-term exposure.
Fig. 5: Sulfopin downregulates Myc transcription.
Fig. 6: Sulfopin abrogates neuroblastoma and PDAC growth in vivo.

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Data availability

Covalent fragment screen data are provided as an Excel file (Supplementary Dataset 1). CITe-Id and rdTOP proteomics data are provided as an Excel file (Supplementary Dataset 3). RNA-seq differential expression data analysis and Enrichr results are provided as an excel file (Supplementary Datasets 5 and 6) and have been deposited with GEO (GSE166786) All structural data have been deposited in the PDB (PDB code 6VAJ). Source data are provided with this paper.

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Acknowledgements

N.L. is the incumbent of the Alan and Laraine Fischer Career Development Chair. N.L. thanks the Israel Science Foundation for funding (grant no. 2462/19), The Rising Tide Foundation, The Israel Cancer Research Fund, the Israeli Ministry of Science and Technology (grant no. 3-14763) and the Moross integrated cancer center. N.L. is also supported by the Helen and Martin Kimmel Center for Molecular Design, Joel and Mady Dukler Fund for Cancer Research, the Estate of Emile Mimran and Virgin JustGiving and the George Schwartzman Fund. C.D. was supported by the Minerva Fellowship program of the Max Planck Society, funded by the German Federal Ministry for Education and Research. This work was supported in part by NIH grant no. R01CA205153 to K.P.L., N.S.G. and X.Z.Z. N.S.G. was also supported by the Hale Center for Pancreatic Research. Y.C. and C.W. thank the Computing Platform of the Center for Life Science at Peking University for supporting proteomic data analysis. S.D.-P acknowledges funding from the Linde Family Foundation. Part of this research was conducted at the Advanced Photon Source on the Northeastern Collaborative Access Team beamlines (no. NIGMS P41 GM103403), and SBGrid compiled software (no. eLife 2013;2:e01456). J.A.M. acknowledges support from the National Institutes of Health (nos. CA233800 and TR002933) and the Mark Foundation for Cancer Research. B.J.P. was supported by the Ruth L. Kirschstein NRSA Individual Predoctoral Fellowship (no. F31 CA225066), the Training Grant in Pharmacological Sciences (no. NIH 5 T32 GM007306), the Training Grant in Chemical Biology (no. NIH 5 T32 GM095450-04) and the Chleck Foundation (also to Z.M.D.). B.N. was supported by an American Cancer Society Postdoctoral Fellowship (no. PF-17-010-01-CDD) and the Katherine L. and Steven C. Pinard Research Fund (also to N.S.G.). L.C. is supported by the Cancer Research UK Program Grant (nos. C34648/A18339 and C34648/A14610). Y.J. is supported by a Children with Cancer UK Research Fellowship (no. 2014/176). R.C.S. acknowledges funding support from NCI R01s (nos. CA196228 and CA186241) and foundation support from The Brenden-Colson Center for Pancreatic Care. We thank I. Ulitski for help with RNA-seq analysis, M. Kostic for critical reading of the manuscript, T.-M. Salame for help with FACS analysis and P. Gehrtz and I. You for helping with compound characterization.

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Author notes

  1. Christopher M. Browne

    Present address: Discovery Biology, Discovery Sciences, Biopharmaceuticals R&D, AstraZeneca, Boston, MA, USA

  2. These authors contributed equally: Christian Dubiella, Benika J. Pinch.

Authors and Affiliations

  1. Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot, Israel

    Christian Dubiella, Daniel Zaidman, Efrat Resnick, Adi Rogel & Nir London

  2. Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA

    Benika J. Pinch, Theresa D. Manz, Behnam Nabet, Hyuk-Soo Seo, Guillaume Adelmant, Shabnam Sharifzadeh, Scott B. Ficarro, Zainab M. Doctor, Christopher M. Browne, Annan Yang, Nicholas E. Vangos, Ezekiel A. Geffken, Eriko Koide, Jarrod A. Marto, Sirano Dhe-Paganon & Nathanael S. Gray

  3. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    Benika J. Pinch, Theresa D. Manz, Behnam Nabet, Zainab M. Doctor, Christopher M. Browne, Eriko Koide & Nathanael S. Gray

  4. Department of Chemistry and Chemical Biology, Department of Chemical Biology, Harvard University, Cambridge, MA, USA

    Benika J. Pinch

  5. Department of Medicine, Division of Translational Therapeutics, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    Kazuhiro Koikawa, Xiaolan Lian, Shin Kibe, Shingo Kozono, Xiao Zhen Zhou & Kun Ping Lu

  6. Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    Kazuhiro Koikawa, Xiaolan Lian, Shin Kibe, Shingo Kozono, Xiao Zhen Zhou & Kun Ping Lu

  7. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Kazuhiro Koikawa, Xiaolan Lian, Shin Kibe, Shingo Kozono, Xiao Zhen Zhou & Kun Ping Lu

  8. Division of Clinical Studies, The Institute of Cancer Research, London, UK

    Evon Poon, Barbara Martins da Costa & Louis Chesler

  9. Department of Pharmaceutical and Medicinal Chemistry, Saarland University, Saarbruecken, Germany

    Theresa D. Manz

  10. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

    Shuning He, Mark W. Zimmerman & Thomas Look

  11. Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR, USA

    Ellen M. Langer, Colin J. Daniel & Rosalie C. Sears

  12. Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA

    Ellen M. Langer, Colin J. Daniel & Rosalie C. Sears

  13. College of Chemistry and Molecular Engineering, Peking University, Beijing, China

    Ying Chen & Chu Wang

  14. Department of Oncologic Pathology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

    Guillaume Adelmant, Shabnam Sharifzadeh, Scott B. Ficarro & Jarrod A. Marto

  15. Blais Proteomics Center, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

    Guillaume Adelmant, Shabnam Sharifzadeh, Scott B. Ficarro & Jarrod A. Marto

  16. Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA

    Guillaume Adelmant, Shabnam Sharifzadeh, Scott B. Ficarro & Jarrod A. Marto

  17. Division of Radiotherapy and Imaging, The Institute of Cancer Research, London, UK

    Yann Jamin

  18. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    Annan Yang

  19. Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel

    Liat Stoler-Barak & Ziv Shulman

  20. Department of Medicine, Division of Hematology and Medical Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Richa B. Shah & Samuel Sidi

  21. Department of Cell, Developmental and Regenerative Biology, The Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Richa B. Shah & Samuel Sidi

  22. Department of Veterinary Resources, The Weizmann Institute of Science, Rehovot, Israel

    Roni Oren

  23. Division of Pediatric Hematology/Oncology Boston Children’s Hospital, Boston, MA, USA

    Thomas Look

  24. Brenden-Colson Center for Pancreatic Care, Oregon Health & Science University, Portland, OR, USA

    Rosalie C. Sears

  25. Department of Chemical and Systems Biology, Chem-H and Stanford Cancer Institute, Stanford School of Medicine, Stanford University, Stanford, CA, USA

    Nathanael S. Gray

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  1. Christian Dubiella
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C.D., B.J.P., N.L. and N.S.G. wrote the manuscript with input from all authors. C.D. and T.D.M. undertook organic synthesis and design. C.D. performed covalent labeling studies. E.R. performed the covalent fragment screen. Chemoproteomics was carried out by Y.C., G.A., S. Sharifzadeh, S.B.F., C.M.B., C.W. and J.A.M. Crystallography was performed by H.-S.S., N.E.V., E.A.G. and S.D.-P. Pin1 biochemical assays were done by X.L., S. Kibe., S. Kozono. and B.J.P., led by X.Z.Z. and K.P.L. Pin1 cellular studies were performed by B.J.P., A.R., E.R., Z.M.D., E.K., T.D.M. and B.N. The PDAC mouse model is credited to K.K., led by X.Z.Z. and K.P.L. The Myc reporter is credited to E.M.L., C.J.D. and R.C.S. Neuroblastoma zebrafish models and cell lines are credited to S.H., M.W.Z. and T.L. Neuroblastoma mouse models are credited to E.P., Y.J., B.M.d.C. and L.C. Molecular modeling was performed by D.Z. The mouse PD study was conducted by A.Y. The mouse toxicity assay was performed by R.O. Radiation resensitization was carried out by R.B.S and S. Sidi. Germinal center studies were performed by L.S.-B. and Z.S. N.L. and N.S.G. conceived of and led this study.

Corresponding authors

Correspondence to Nathanael S. Gray or Nir London.

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Competing interests

N.S.G. is a Scientific Founder and member of the Scientific Advisory Board (SAB) of C4, Jengu, Inception, Larkspur, Syros, Soltego, Gatekeeper and Petra Pharmaceuticals and has received research funding from Novartis, Astellas, Taiho and Deerfield. N.L. is a member of the SAB of Totus medicines and Monte Rosa Therapeutics and has received research support from Teva and Pfizer. J.A.M. has received support through sponsored research agreements with AstraZeneca and Vertex. J.A.M. serves on the SAB of 908 Devices. C.M.B. is an employee of AstraZeneca. C.D., B.J.P., D.Z., S.H., X.L., K.P.L., X.Z.Z., T.L., N.S.G. and N.L. are inventors on a patent application related to the inhibitors described in this manuscript (no. PCT/IL2020/050043).

Additional information

Peer review information Nature Chemical Biology thanks Timothy Chi-Wai Cheng, Yan Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Chemoproteomic approaches to establish Sulfopin’s selectivity.

a, Schematic depiction of Covalent Inhibitor Target-Site Identification (CITe-Id) workflow, showing hypothetical results. CiTe-Id identifies Sulfopin-DTB modified sites across the proteome, and profiles competitively labeled cysteine residues following dose-response treatment with Sulfopin in live PATU-8988T cells. b, Schematic depicting the rdTOP-ABPP experimental workflow to assess Sulfopin proteomic selectivity in MDA-MB-231 cells.

Extended Data Fig. 2 Sulfopin phenocopies Pin1 knockout phenotypes.

a, HeLa cells were treated with either DMSO, Sulfopin, or Go6976 (a Chk1 inhibitor) and exposed to 7.5 Gy IR 1 h after drug treatment. Viability was assessed 3 days post-IR. Sulfopin shows a dose dependent sensitization of the cells to irradiation (n=3; data are represented as mean values with standard deviation). b, Western blot analysis was performed 24 h post-IR, showing Sulfopin blocked phosphorylation of Thr209 of IRAK1. c, A shorter exposure shows that Sulfopin inhibits IRAK1 phosphorylation already at concentrations of 0.1 μM. d, A scheme for testing the effect of Sulfopin in vivo on germinal center B cells in response to immunization. e, Representative flow cytometric plots with Vehicle and Sulfopin (left) and quantification (right) of FASHi CD38− germinal center (GC) cells in WT mice 11 days after immunization with NP-OVA. ** p<0.01, two tailed Student’s t test.

Source data

Extended Data Fig. 3 Sulfopin affects PATU-8988T cell cycle.

PATU-8988T cells were treated in triplicates or more for 4 days with either DMSO (0.1%), Sulfopin (2.5 M) or the non-covalent control Sulfopin-AcA (2.5 M). Cell cycle analysis was performed by BRDU and Propidium-Iodide staining, followed by FACS analysis. Sulfopin treatment reduces the % of cells in S-phase and in turn more cells are found in G1, while the non-covalent Sulfopin-AcA doesn’t show this effect. Representative FACS analysis graphs and a quantification of the results SD of two independent experiments are presented (A. n=3; B. n=4). Statistical significance was calculated using one-tailed Student’s t test (** = p < 0.01, *** = p < 0.001).

Extended Data Fig. 4 Sulfopin treatment does not induce apoptosis in cells.

a, PATU-8988T cells were treated for 5 or 6 days with either DMSO (0.1%), Sulfopin (1 μM, 2.5 μM) or the non-covalent control Sulfopin-AcA (2.5 μM). The cells were lysed and activation of caspase 3 and Pin1 levels were analysed by Western blot. As a positive control for caspase 3 activation the cells were treated with Staurosporin (1 μM, 4h; STS). See Supplementary Fig. 13a for the results of an additional independent experiment. Caspase 3 was not activated and Pin1 levels were not changed by the treatment with Sulfopin. b, PATU-8988T cells were treated in triplicates for 6 days with either DMSO (0.1%), Sulfopin (1 M, 2.5 M) or the non-covalent control Sulfopin-AcA (2.5 M). The cells were then stained with AnnexinV-FITC/ 7AAD and analysed by FACS. Staurosporin treatment (1 M, 4h) was used as a positive control for apoptosis. Representative FACS analysis graphs and a quantification of the results (n=3; data are represented as mean values with standard deviation). See Supplementary Fig. 13b for the results of an additional independent experiment. Live cells were defined as AnnexinV-/7AAD-, early apoptosis AnnexinV+/7AAD- and late apoptosis AnnexinV+/ 7AAD+.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–15, Tables 1–4 and Note (synthetic procedures and analytical data).

Reporting Summary

Supplementary Datasets

Supplementary Datasets 1–6.

Source data

Source Data Fig. 2

Unprocessed immunolots.

Source Data Extended Data Fig. 2

Unprocessed immunolots.

Source Data Extended Data Fig. 4

Unprocessed immunolots.

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Dubiella, C., Pinch, B.J., Koikawa, K. et al. Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo. Nat Chem Biol 17, 954–963 (2021). https://doi.org/10.1038/s41589-021-00786-7

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