Abstract
Recently, copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) click chemistry has emerged as a promising approach for designing new artificial metallo-nucleases (AMNs) with DNA-damaging properties. By functionalising a central organic azide with three alkyne donors, Tri-Click (TC) ligands capable of chelating three copper ions through the donor group and triazole linker can be generated. However, the versatility of this approach along with the influence of specific donors on metal binding, DNA recognition, and cellular DNA damage in an anticancer context remains poorly understood. Here, we prepare a series of Tri-Click ligands incorporating systematic cyclic and acyclic N-, O-, and S-donors and evaluate their AMN activities. Screening experiments pinpoint planar N-donor ligands as high value agents. Among these, the copper complex of Tri-Click-Pyridine (Cu3-TC-Py) displays significant potential. We characterise its activity using single-molecule imaging, microscale thermophoresis, FRET-based binding assays, molecular dynamics, and intracellular DNA interaction studies in human and functional bacterial cells. We report the emergence of Cu3-TC-Py as a lead AMN with high reactivity for DNA damage applications central to anticancer therapy.
Data availability
All molecular dynamics simulation data supporting the findings of this study, including structure, topology, parameter, and trajectory files for the Cu₃-TC-Py–DNA systems, are publicly available on Zenodo at https://zenodo.org/records/17143195 (DOI: 10.5281/zenodo.17143194). All other data is presented in the supplementary information or the source data file provided. Source data are provided with this paper.
Code availability
The data analysis involving extraction of kymographs from multi-TIFF files and measurement of end-to-end lengths from the obtained kymographs were done using a custom written MATLAB code publicly available on GitHub at https://github.com/dnadevcode/lldev (DOI: 10.5281/zenodo.17652641).
References
Kolb, H. C., Finn, M. G. & Sharpless, K. B. Click chemistry: diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. Engl. 40, 2004–2021 (2001).
Rostovtsev, V. V., Green, L. G., Fokin, V. V. & Sharpless, K. B. A stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed. Engl. 41, 2596–2599 (2002).
Tornøe, C. W., Christensen, C. & Meldal, M. Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 67, 3057–3064 (2002).
Meldal, M. & Diness, F. Recent fascinating aspects of the CuAAC click reaction. Trends Chem. 2, 569–584 (2020).
Agard, N. J., Prescher, J. A. & Bertozzi, C. R. A strain-promoted [3 + 2] azide−alkyne cycloaddition for covalent modification of biomolecules in living systems. J. Am. Chem. Soc. 126, 15046–15047 (2004).
Liang, L. & Astruc, D. The copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) “click” reaction and its applications. An overview. Coord. Chem. Rev. 255, 2933–2945 (2011).
Wang, X., Huang, B., Liu, X. & Zhan, P. Discovery of bioactive molecules from CuAAC click-chemistry-based combinatorial libraries. Drug Discov. Today 21, 118–132 (2016).
Hennessy, J. et al. A click chemistry approach to targeted DNA crosslinking with cis-platinum(II)-modified triplex-forming oligonucleotides. Angew. Chem. Int. Ed. Engl. 61, e202110455 (2022).
McGorman, B. et al. Enzymatic synthesis of chemical nuclease triplex-forming oligonucleotides with gene-silencing applications. Nucleic Acids Res. 50, 5467–5481 (2022).
Hennessy, J. et al. Thiazole orange-carboplatin triplex-forming oligonucleotide (TFO) combination probes enhance targeted DNA crosslinking. RSC Med. Chem. 15, 485–491 (2024).
Crowley, J. D. & McMorran, D. A. “Click-Triazole” coordination chemistry: exploiting 1,4-disubstituted-1,2,3-triazoles as ligands. in Topics in Heterocyclic Chemistry 31–83 (Springer Berlin Heidelberg, Berlin, 2012).
Ahmad, M., Balamurali, M. M. & Chanda, K. Click-derived multifunctional metal complexes for diverse applications. Chem. Soc. Rev. https://doi.org/10.1039/d3cs00343d (2023).
Mindt, T. L. et al. “Click to chelate”: synthesis and installation of metal chelates into biomolecules in a single step. J. Am. Chem. Soc. 128, 15096–15097 (2006).
McStay, N. et al. Click and Cut: a click chemistry approach to developing oxidative DNA damaging agents. Nucleic Acids Res. 49, 10289–10308 (2021).
Gibney, A. et al. A click chemistry-based artificial metallo-nuclease. Angew. Chem. Int. Ed. Engl. 62, e202305759 (2023).
Kellett, A. & McStay, N. Metallodrug therapeutic compounds and prodrugs of metallodrug therapeutic compounds. US Patent Application US20240376129A1 (2024).
Gibney, A. & Kellett, A. Gene editing with artificial DNA scissors. Chem. Eur. J. 30, e202401621 (2024).
Chen, J. & Stubbe, J. Bleomycins: towards better therapeutics. Nat. Rev. Cancer 5, 102–112 (2005).
Poole, S. et al. Design and in vitro anticancer assessment of a click chemistry-derived dinuclear copper artificial metallo-nuclease. Nucleic Acids Res. 53, gkae1250 (2025).
Kellett, A., Molphy, Z., Slator, C., McKee, V. & Farrell, N. P. Molecular methods for assessment of non-covalent metallodrug–DNA interactions. Chem. Soc. Rev. 48, 971–988 (2019).
Cheng, Y. & Prusoff, W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099–3108 (1973).
Jerabek-Willemsen, M. et al. MicroScale thermophoresis: interaction analysis and beyond. J. Mol. Struct. 1077, 101–113 (2014).
Jerabek-Willemsen, M., Wienken, C. J., Braun, D., Baaske, P. & Duhr, S. Molecular interaction studies using microscale thermophoresis. Assay Drug Dev. Technol. 9, 342–353 (2011).
Seidel, S. A. I. et al. Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods 59, 301–315 (2013).
Carter, M. T., Rodriguez, M. & Bard, A. J. Voltammetric studies of the interaction of metal chelates with DNA. 2. Tris-chelated complexes of cobalt(III) and iron(II) with 1,10-phenanthroline and 2,2’-bipyridine. J. Am. Chem. Soc. 111, 8901–8911 (1989).
Travers, A. Michael Waring-A scientific life in DNA. Biopolymers 112, e23408 (2021).
Prieto Otoya, T. D. et al. Probing a major DNA weakness: resolving the groove and sequence selectivity of the diimine complex Λ-[Ru(phen)2 phi]2. Angew. Chem. Int. Ed Engl. 63, e202318863 (2024).
Zeglis, B. M., Pierre, V. C. & Barton, J. K. Metallo-intercalators and metallo-insertors. Chem. Commun. 44, 4565–4579 (2007).
Prieto Otoya, T. D. et al. Re-pairing DNA: binding of a ruthenium phi complex to a double mismatch. Chem. Sci. 15, 9096–9103 (2024).
Walt, D. R. Optical methods for single molecule detection and analysis. Anal. Chem. 85, 1258–1263 (2013).
Rye, H. S. et al. Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications. Nucleic Acids Res. 20, 2803–2812 (1992).
Perkins, T. T., Smith, D. E., Larson, R. G. & Chu, S. Stretching of a single tethered polymer in a uniform flow. Science 268, 83–87 (1995).
Sischka, A. et al. Molecular mechanisms and kinetics between DNA and DNA binding ligands. Biophys. J. 88, 404–411 (2005).
Hanwell, M. D., Curtis, D. E., Lonie, D. C. & Vandermeersch, T. Avogadro: an advanced semantic chemical editor, visualization, and analysis platform. J Cheminformatics 4, 17 (2012).
Messina, M. S. et al. A histochemical approach to activity-based copper sensing reveals cuproplasia-dependent vulnerabilities in cancer. Proc. Natl. Acad. Sci. USA 122, e2412816122 (2025).
Kastan, M. B. & Bartek, J. Cell-cycle checkpoints and cancer. Nature 432, 316–323 (2004).
Gałczyńska, K. et al. Copper(II) complex with 1-allylimidazole induces G2/M cell cycle arrest and suppresses A549 cancer cell growth by attenuating Wnt, JAK-STAT, and TGF-β signaling pathways. J. Inorg. Biochem. 264, 112791 (2025).
Molinaro, C. et al. A novel copper(II) indenoisoquinoline complex inhibits topoisomerase I, induces G2 phase arrest, and autophagy in three adenocarcinomas. Front. Oncol. 12, 837373 (2022).
Hilbert, B. J., Hayes, J. A., Stone, N. P., Xu, R.-G. & Kelch, B. A. The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain. Nucleic Acids Res 45, 3591–3605 (2017).
Zuin Fantoni, N. et al. Polypyridyl-based copper phenanthrene complexes: a new type of stabilized artificial chemical nuclease. Chem. Eur. J. 25, 221–237 (2019).
Molphy, Z. et al. Copper phenanthrene oxidative chemical nucleases. Inorg. Chem. 53, 5392–5404 (2014).
Detinis Zur, T., Deek, J. & Ebenstein, Y. Single-molecule approaches for DNA damage detection and repair: a focus on repair assisted damage detection (RADD). DNA Repair 129, 103533 (2023).
Su’etsugu, M. & Errington, J. The replicase sliding clamp dynamically accumulates behind progressing replication forks in Bacillus subtilis cells. Mol. Cell 41, 720–732 (2011).
Santoro, A. et al. The glutathione/metallothionein system challenges the design of efficient O2-activating copper complexes. Angew. Chem. Int. Ed Engl. 59, 7830–7835 (2020).
Rigaku Oxford Diffraction, CrysAlisPro Software system, Version 1.171.43.115a, Rigaku Corporation, Wroclaw, Poland (2024).
Hübschle, C. B. ShelXle: a Qt graphical user interface for SHELXL. Acta Crystallogr. A Found. Adv. 75, a187–a187 (2019).
Dolomanov, O. V., Bourhis, L. J., Gildea, R. J., Howard, J. & Puschmann, H. OLEX2: a complete structure solution, refinement and analysis program. J. Appl. Crystallogr. 42, 339–341 (2009).
Sheldrick, G. M. SHELXT - integrated space-group and crystal-structure determination. Acta Crystallogr. A Found. Adv. 71, 3–8 (2015).
Sheldrick, G. M. Crystal structure refinement with SHELXL. Acta Crystallogr. C Struct. Chem. 71, 3–8 (2015).
McCann, M. et al. A new phenanthroline-oxazine ligand: synthesis, coordination chemistry and atypical DNA binding interaction. Chem. Commun. 49, 2341–2343 (2013).
Frykholm, K., Müller, V., Sriram, K. K. Dorfman, K. D. & Westerlund, F. DNA in nanochannels: theory and applications. Q. Rev. Biophys. 55, e12 (2022).
Sriram, K. K. et al. Fluorescence microscopy of nanochannel-confined DNA. Methods Mol. Biol. 2694, 175–202 (2024).
Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455–461 (2010).
Abraham, M. J. et al. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2, 19–25 (2015).
Van Der Spoel, D. et al. GROMACS: fast, flexible, and free. J. Comput. Chem. 26, 1701–1718 (2005).
Huang, J. et al. CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nat. Methods 14, 71–73 (2017).
Wang, J., Wang, W., Kollman, P. A. & Case, D. A. Automatic atom type and bond type perception in molecular mechanical calculations. J. Mol. Graph. Model. 25, 247–260 (2006).
Salentin, S., Schreiber, S., Haupt, V. J., Adasme, M. F. & Schroeder, M. PLIP: fully automated protein-ligand interaction profiler. Nucleic Acids Res. 43, W443–W447 (2015).
Acknowledgements
We acknowledge funding from Research Ireland (12/RC/2275_P2), the Irish Research Council (IRCLA/2022/3815), and the Novo Nordisk Foundation (NNF19OC0056845). We also acknowledge the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 861381 (NATURE-ETN). We are grateful for financial support from the ANR (ANR-20-CE07-0035), the ERC Consolidator Grant PhotoMedMet to G.G. (GA 681679), the program “Investissements d’ Avenir” launched by the French Government and implemented by the ANR with the reference ANR-10-IDEX-0001-02 PSL (G.G.). F.W. acknowledges funding from the European Research Council (ERC consolidator, grant no 866238), the Swedish Research Council (grant no. 2020–03400), the Swedish Cancer Foundation (grant no. 201145 PjF) and the Swedish Child Cancer Foundation (grant no. PR2022-001). The nanofluidic devices used in this study were fabricated at MyFab Chalmers cleanroom facility. P.J. acknowledges funding from the Swedish Child Cancer Foundation (grant no. 2022-0010), Jubileumsklinikens Cancerfond (2023:504).
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A.G., M.S., E.D., P.M., L.A., S.K.K., O.A.A., H.H., F.F. and K.C. conducted experiments. A.G. and A.K. wrote the manuscript and prepared figures. V.M., P.J., S.B., D.T., M.W., G.G., F.W. and A.K. provided supervision. All authors assisted with manuscript review and revision. A.G. and A.K. conceptualised the study.
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Gibney, A., Sidarta, M., Delahunt, E. et al. Expanding the DNA damaging potential of artificial metallo-nucleases with click chemistry. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68911-5
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DOI: https://doi.org/10.1038/s41467-026-68911-5
