Destabilizers of the thymidylate synthase homodimer accelerate its proteasomal degradation and inhibit cancer growth

  1. Luca Costantino
  2. Stefania Ferrari
  3. Matteo Santucci
  4. Outi MH Salo-Ahen
  5. Emanuele Carosati
  6. Silvia Franchini
  7. Angela Lauriola
  8. Cecilia Pozzi
  9. Matteo Trande
  10. Gaia Gozzi
  11. Puneet Saxena
  12. Giuseppe Cannazza
  13. Lorena Losi
  14. Daniela Cardinale
  15. Alberto Venturelli
  16. Antonio Quotadamo
  17. Pasquale Linciano
  18. Lorenzo Tagliazucchi
  19. Maria Gaetana Moshella
  20. Remo Guerrini
  21. Salvatore Pacifico
  22. Rosaria Luciani
  23. Filippo Genovese
  24. Stefan Henrich
  25. Silvia Alboni
  26. Nuno Santarem
  27. Anabela da Silva Cordeiro
  28. Elisa Giovannetti
  29. Godefridus J Peters
  30. Paolo Pinton
  31. Alessandro Rimessi
  32. Gabriele Cruciani
  33. Robert M Stroud
  34. Rebecca C Wade
  35. Stefano Mangani
  36. Gaetano Marverti
  37. Domenico D'Arca  Is a corresponding author
  38. Glauco Ponterini  Is a corresponding author
  39. Maria Paola Costi  Is a corresponding author
  1. University of Modena and Reggio Emilia, Italy
  2. Åbo Akademi University, Finland
  3. University of Perugia, Italy
  4. University of Verona, Italy
  5. University of Siena, Italy
  6. University College London, United Kingdom
  7. University of Ferrara, Italy
  8. Heidelberg Institute for Theoretical Studies, Germany
  9. IBMC I3S, Portugal
  10. Amsterdam University Medical Centers, Netherlands
  11. University of California, San Francisco, United States
  12. University of Heidelberg, Germany

Abstract

Drugs that target human thymidylate synthase (hTS), a dimeric enzyme, are widely used in anti-cancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces over-expression of this protein and development of drug resistance. We thus pursued an alternative strategy that led us to the discovery of TS-dimer destabilizers. These compounds bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. A structural, spectroscopic, and kinetic investigation has provided evidence and quantitative information on the effects of the interaction of these small molecules with hTS. Focusing on the best among them, E7, we have shown that it inhibits hTS in cancer cells and accelerates its proteasomal degradation, thus causing a decrease in the enzyme intracellular level. E7 also showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-resistant cancers.

Data availability

Diffraction data have been deposited in PDB under the accession codes PDB-ID 4O1U humanTS mutant Y202C and PDB-ID 4O1X humanTS double mutant C195SY202C. All other data generated or analyzed during this study are included in the manuscript and as Supplementary files and Source data.

The following data sets were generated

Article and author information

Author details

  1. Luca Costantino

    Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
    Competing interests
    The authors declare that no competing interests exist.
  2. Stefania Ferrari

    Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
    Competing interests
    The authors declare that no competing interests exist.
  3. Matteo Santucci

    Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4331-5566
  4. Outi MH Salo-Ahen

    Faculty of Science and Engineering, Pharmacy/Biochemistry, Åbo Akademi University, Turku, Finland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0725-126X
  5. Emanuele Carosati

    Department of Chemistry, Biology and Biotechnology, University of Perugia, Perugia, Italy
    Competing interests
    The authors declare that no competing interests exist.
  6. Silvia Franchini

    Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
    Competing interests
    The authors declare that no competing interests exist.
  7. Angela Lauriola

    Department of Biotechnology, University of Verona, Verona, Italy
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5286-8627
  8. Cecilia Pozzi

    Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italy
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2574-3911
  9. Matteo Trande

    Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
    Competing interests
    The authors declare that no competing interests exist.
  10. Gaia Gozzi

    Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
    Competing interests
    The authors declare that no competing interests exist.
  11. Puneet Saxena

    Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
    Competing interests
    The authors declare that no competing interests exist.
  12. Giuseppe Cannazza

    Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
    Competing interests
    The authors declare that no competing interests exist.
  13. Lorena Losi

    Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
    Competing interests
    The authors declare that no competing interests exist.
  14. Daniela Cardinale

    Great Ormond Street Institute of Child Health, University College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  15. Alberto Venturelli

    Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
    Competing interests
    The authors declare that no competing interests exist.
  16. Antonio Quotadamo

    Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
    Competing interests
    The authors declare that no competing interests exist.
  17. Pasquale Linciano

    Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
    Competing interests
    The authors declare that no competing interests exist.
  18. Lorenzo Tagliazucchi

    Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
    Competing interests
    The authors declare that no competing interests exist.
  19. Maria Gaetana Moshella

    Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
    Competing interests
    The authors declare that no competing interests exist.
  20. Remo Guerrini

    Department of Chemical and Pharmaceutical Science, University of Ferrara, Ferrara, Italy
    Competing interests
    The authors declare that no competing interests exist.
  21. Salvatore Pacifico

    Department of Chemical and Pharmaceutical Science, University of Ferrara, Ferrara, Italy
    Competing interests
    The authors declare that no competing interests exist.
  22. Rosaria Luciani

    Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
    Competing interests
    The authors declare that no competing interests exist.
  23. Filippo Genovese

    Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
    Competing interests
    The authors declare that no competing interests exist.
  24. Stefan Henrich

    Molecular and Cellular Modeling Group, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  25. Silvia Alboni

    Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
    Competing interests
    The authors declare that no competing interests exist.
  26. Nuno Santarem

    IBMC I3S, Porto, Portugal
    Competing interests
    The authors declare that no competing interests exist.
  27. Anabela da Silva Cordeiro

    IBMC I3S, Porto, Portugal
    Competing interests
    The authors declare that no competing interests exist.
  28. Elisa Giovannetti

    Department of Medical Oncology, Amsterdam University Medical Centers, Amsterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  29. Godefridus J Peters

    Department of Medical Oncology, Amsterdam University Medical Centers, Amsterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  30. Paolo Pinton

    Department of Medical Sciences, University of Ferrara, Ferrara, Italy
    Competing interests
    The authors declare that no competing interests exist.
  31. Alessandro Rimessi

    Department of Medical Sciences, University of Ferrara, Ferrara, Italy
    Competing interests
    The authors declare that no competing interests exist.
  32. Gabriele Cruciani

    Department of Chemistry, Biology and Biotechnology, University of Perugia, Perugia, Italy
    Competing interests
    The authors declare that no competing interests exist.
  33. Robert M Stroud

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  34. Rebecca C Wade

    Center for Molecular Biology, University of Heidelberg, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5951-8670
  35. Stefano Mangani

    Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italy
    Competing interests
    The authors declare that no competing interests exist.
  36. Gaetano Marverti

    Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy
    Competing interests
    The authors declare that no competing interests exist.
  37. Domenico D'Arca

    Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy
    For correspondence
    domenico.darca@unimore.it
    Competing interests
    The authors declare that no competing interests exist.
  38. Glauco Ponterini

    Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
    For correspondence
    glauco.ponterini@unimore.it
    Competing interests
    The authors declare that no competing interests exist.
  39. Maria Paola Costi

    Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
    For correspondence
    mariapaola.costi@unimore.it
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0443-5402

Funding

Associazione Italiana per la Ricerca sul Cancro (IG25785)

  • Maria Paola Costi

Associazione Italiana per la Ricerca sul Cancro (IG16977)

  • Maria Paola Costi

Associazione Italiana per la Ricerca sul Cancro (14422 Start-Up)

  • Elisa Giovannetti

CCA foundation grant (CCA2015-1-19)

  • Rebecca C Wade

National Institute of General Medical Sciences (GM24485)

  • Robert M Stroud

Associazione Italiana per la Ricerca sul Cancro (IG23670)

  • Paolo Pinton

Ministero dell'Istruzione, dell'Università e della Ricerca (2017XA5J5N)

  • Alessandro Rimessi

Academy of Finland (137918)

  • Outi MH Salo-Ahen

European Commission (GA037852)

  • Maria Paola Costi

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Goutham Narla, University of Michigan, United States

Ethics

Animal experimentation: Animal experiments on orthotropic mice were approved by the Committees on Animal Experiments of the VU University Amsterdam, The Netherlands and of the University of Pisa, Pisa, Italy, and were performed in accordance with institutional guidelines and international law and policies.--------Pharmacokinetic studies on BALB/c mice of E5 and E7. ': All animal experiments were carried out in accordance with the IBMC.INEB Animal Ethics Committees and the Portuguese National Authorities for Animal Health guidelines, according to the statements on the Directive 2010/63/EU of the European Parliament and of the Council. NS and ACS have an accreditation for animal research given by the Portuguese Veterinary Direction (Ministerial Directive 1005/92).----------Experiments performed in Ferrara University (IT)In vivo experiments will be carried out by competent staff members, with a scientific education relevant to the experimental work proposals, including the ability to manipulate and taking care of laboratory animals. All the procedures will be performed in accordance with Institutional Animal Care and Use Committee (IACUC), in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Italian Ministry of Health. The experimental protocol will be approved by the Animal Ethics Committee at the University of Ferrara and by the Italian Ministry of Health. Authorization number n{degree sign} 1098/2020-PR, rilasciata ai sensi dell'art. 31 del D.lgs. 26/2014.

Version history

  1. Preprint posted: March 8, 2021 (view preprint)
  2. Received: September 14, 2021
  3. Accepted: December 1, 2022
  4. Accepted Manuscript published: December 7, 2022 (version 1)
  5. Version of Record published: January 10, 2023 (version 2)

Copyright

© 2022, Costantino et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Luca Costantino
  2. Stefania Ferrari
  3. Matteo Santucci
  4. Outi MH Salo-Ahen
  5. Emanuele Carosati
  6. Silvia Franchini
  7. Angela Lauriola
  8. Cecilia Pozzi
  9. Matteo Trande
  10. Gaia Gozzi
  11. Puneet Saxena
  12. Giuseppe Cannazza
  13. Lorena Losi
  14. Daniela Cardinale
  15. Alberto Venturelli
  16. Antonio Quotadamo
  17. Pasquale Linciano
  18. Lorenzo Tagliazucchi
  19. Maria Gaetana Moshella
  20. Remo Guerrini
  21. Salvatore Pacifico
  22. Rosaria Luciani
  23. Filippo Genovese
  24. Stefan Henrich
  25. Silvia Alboni
  26. Nuno Santarem
  27. Anabela da Silva Cordeiro
  28. Elisa Giovannetti
  29. Godefridus J Peters
  30. Paolo Pinton
  31. Alessandro Rimessi
  32. Gabriele Cruciani
  33. Robert M Stroud
  34. Rebecca C Wade
  35. Stefano Mangani
  36. Gaetano Marverti
  37. Domenico D'Arca
  38. Glauco Ponterini
  39. Maria Paola Costi
(2022)
Destabilizers of the thymidylate synthase homodimer accelerate its proteasomal degradation and inhibit cancer growth
eLife 11:e73862.
https://doi.org/10.7554/eLife.73862

Share this article

https://doi.org/10.7554/eLife.73862

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