Destabilizers of the thymidylate synthase homodimer accelerate its proteasomal degradation and inhibit cancer growth
- University of Modena and Reggio Emilia, Italy
- Åbo Akademi University, Finland
- University of Perugia, Italy
- University of Verona, Italy
- University of Siena, Italy
- University College London, United Kingdom
- University of Ferrara, Italy
- Heidelberg Institute for Theoretical Studies, Germany
- IBMC I3S, Portugal
- Amsterdam University Medical Centers, Netherlands
- University of California, San Francisco, United States
- 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.
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Crystal structure of human thymidylate synthase mutant Y202CProtein Data Bank, 4O1U.
Article and author information
Author details
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.
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.
Reviewing Editor
- Goutham Narla, University of Michigan, United States
Version history
- Preprint posted: March 8, 2021 (view preprint)
- Received: September 14, 2021
- Accepted: December 1, 2022
- Accepted Manuscript published: December 7, 2022 (version 1)
- 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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Destabilizers of the thymidylate synthase homodimer accelerate its proteasomal degradation and inhibit cancer growth
eLife 11:e73862.
https://doi.org/10.7554/eLife.73862
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