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.
-
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.
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.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Destabilizers of the thymidylate synthase homodimer accelerate its proteasomal degradation and inhibit cancer growth
eLife 11:e73862.
https://doi.org/10.7554/eLife.73862
Further reading
-
- Biochemistry and Chemical Biology
N 6,2’-O-dimethyladenosine (m6Am) is a modified nucleotide located at the first transcribed position in mRNA and snRNA that is essential for diverse physiological processes. m6Am mapping methods assume each gene uses a single start nucleotide. However, gene transcription usually involves multiple start sites, generating numerous 5’ isoforms. Thus, gene-level annotations cannot capture the diversity of m6Am modification in the transcriptome. Here, we describe CROWN-seq, which simultaneously identifies transcription-start nucleotides and quantifies m6Am stoichiometry for each 5’ isoform that initiates with adenosine. Using CROWN-seq, we map the m6Am landscape in nine human cell lines. Our findings reveal that m6Am is nearly always a high stoichiometry modification, with only a small subset of cellular mRNAs showing lower m6Am stoichiometry. We find that m6Am is associated with increased transcript expression and provide evidence that m6Am may be linked to transcription initiation associated with specific promoter sequences and initiation mechanisms. These data suggest a potential new function for m6Am in influencing transcription.
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.
