Expanding the DNA Damaging Potential of Artificial

Metallo-Nucleases with Click Chemistry

Alex Gibney 1

Margareth Sidarta 2

Sriram KK 2

Obed Akwasi Aning 2

Lily Arrué 1

Francisca Figueiredo 1

Pierre Mesdom 1

Kevin Cariou 1

Pegah Johansson 1

Shayon Bhattacharya 1

Damien Thompson 1

Vickie McKee 1

Michaela Wenzel 2

Gilles Gasser 1

Fredrik Westerlund 2

Andrew Kellett 1✉ Emailandrew.kellett@dcu.ie

1 Centre for Pharmaceuticals, School of Chemical Sciences Dublin City University Dublin 9 Glasnevin Ireland

2 Department of Life Sciences Chalmers University of Technology Gothenburg Sweden

3 Centre for Antibiotic Resistance Research in Gothenburg (CARe) Gothenburg Sweden

Centre for Pharmaceuticals, Department of Physics University of Limerick Ireland

5 [e] Chimie ParisTech, Institute of Chemistry for Life and Health Sciences PSL Université, CNRS Paris France

6 Department of Clinical Chemistry Sahlgrenska University Hospital, Region Vastra Gotaland Gothenburg Sweden

7 Department of Laboratory Medicine, Institute of Biomedicine Sahlgrenska Academy at University of Gothenburg Sweden

8 Department of Physics, Chemistry and Pharmacy University of Southern Denmark Campusvej 55 5230 Odense Denmark

Alex Gibney,^a Margareth Sidarta,^b,c Sriram KK,^b Obed Akwasi Aning,^b,c Lily Arrué,^d Francisca Figueiredo,^e Pierre Mesdom,^e Kevin Cariou,^e Pegah Johansson,^f,g Shayon Bhattacharya,^d Damien Thompson,^d Vickie McKee,^a,h Michaela Wenzel,^b,c Gilles Gasser,^e Fredrik Westerlund,^b,c and Andrew Kellett^a*

[a] Research Ireland Centre for Pharmaceuticals, School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland. andrew.kellett@dcu.ie

[b] Department of Life Sciences, Chalmers University of Technology, Gothenburg, Sweden.

[c] Centre for Antibiotic Resistance Research in Gothenburg (CARe), Gothenburg, Sweden

[d] Research Ireland Centre for Pharmaceuticals, Department of Physics, University of Limerick, Ireland.

[e] Chimie ParisTech, PSL Université, CNRS, Institute of Chemistry for Life and Health Sciences, Paris, France.

[f] Department of Clinical Chemistry, Sahlgrenska University Hospital, Region Vastra Gotaland, Gothenburg, Sweden.

[g] Department of Laboratory Medicine, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Sweden.

[h] Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.

Abstract

The preparation of new metallodrugs targeting DNA is of key therapeutic interest. Recently, the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) "click chemistry" reaction 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 prepared a library of Tri-Click ligands incorporating systematic cyclic and acyclic N-, O-, and S-donors and evaluated 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 characterised 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.

Introduction

The copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) reactions were recognised by the 2022 Nobel prize in chemistry as fundamental advancements in functional chemistry.^1–5 In medicinal chemistry, the CuAAC reaction primarily serves as a fast and efficient method for creating complex molecules.^6–10 However, the 1,2,3-triazole generated during this reaction has distinctive properties, including sp²-hybridised nitrogen atoms capable of forming coordination bonds with transition metals. Click chemistry therefore serves as a valuable tool for the development of novel coordinating ligands.^11–13 We recently demonstrated that click chemistry could be used to efficiently prepare new metallodrug candidates from simple, inert starting materials.^14–17 This concept is exemplified by the Tri-Click (TC) ligands (Fig. 1a), characterised for their ability to coordinate up to three copper(II) ions that promote DNA damage. These agents belong to a class of metal complex that oxidatively cleaves DNA, known as artificial metallo-nucleases (AMNs), which offer therapeutic potential due to their metallo-bleomycin-like activity.^18,19 Our first study reporting the discovery of TC-1 revealed the positioning of the secondary donor relative to the 1,2,3-triazole group was vital to copper binding and DNA reactivity.¹⁴ Recent work then identified TC-Thio as a promising ligand that introduced aromaticity and Cu(I) sensitivity through a sulfur donor, ethynylthiophene.¹⁵ However, the influence of different alkyne donors on metal binding, DNA recognition, and oxidative DNA damage remains poorly understood. Here, we report a library of aromatic and aliphatic N-, O- and S- donors into the Tri-Click scaffold with the aim of identifying properties favourable to copper-sensitised DNA binding and reactivity in biological systems with particular focus on anticancer applications.

Results

Library design and preparation

New TC ligands were designed to contain a secondary donor proximal to the 1,2,3-triazole to create suitable metal ion chelators. Previous studies show that a three-bond spacer between the terminal alkyne and the secondary donor, such as that found in propargyl amine or 2-ethynyl thiophene (Fig. 1a), is suited for this purpose. Therefore, we selected a diverse range of propargyl and heteroaromatic 2-ethynyl starting materials for CuAAC coupling with a tris(azidomethyl)-mesitylene (triazide) core (Figure S1). In each case, we introduced both weak and strong donors while adjusting the steric and planarity properties. Commercially available alkyne starting materials were selected from: propargyl alcohol, propiolic acid, propiolamide, 2- ethynylpyrimidine, 2-ethynylpyridine, and 2-ethynylbenzothiazole. Each of the alkyne donors were then reacted via CuAAC with the triazide core, resulting in TC-OH, TC-Acid, TC-Amide, TC-Pyrm, TC-Py and TC-Benzo respectively (Fig. 1b). All ligand syntheses proceeded readily in benign solvents, with high yields that required simple, non-chromatographic purification (Figures S2-S15).

Copper binding analysis

Electrospray ionisation-mass spectrometry (ESI-MS) was used to characterise the Cu(II) binding properties of each TC ligand (Fig. 1c and Table S2). Spectra of samples containing TC ligands with three molar equivalents of Cu(II) nitrate trihydrate in a 50:50 water:DMF solution were recorded and compared to the theoretical patterns expected for an assumed molecular structure with the general formula [Cu₃(TC)(NO₃)₆] (M). Trinuclear complexes were identified by the isotope patterns for M + and M + 2 peak ratios arising from the natural ⁶³Cu / ⁶⁵Cu abundances. All spectra indicated the presence of trinuclear Cu(II) complexes, except for the TC-Amide sample, which was not further investigated. The spectra of heteroaromatic ligands TC-Pyrm and TC-Benzo showed mono-cationic [M-NO₃]⁺ formation due to the loss of a single nitrate anion, while TC-Pyrm showed additional evidence for the loss of a second nitrate [M-2(NO₃)]²⁺. The TC-OH and TC-Acid spectra required more complex analysis and the compounds likely exist as an equilibrium mixture between the free ligand and complex. Mono- and di-cationic ions of Cu₃-TC-Py, [M-NO₃]⁺ and [M-2(NO₃)]²⁺, were detected along with hydrogen and nitric acid adducts ([M + H]⁺ and [M + HNO₃]⁺), which were not observed for any other TC sample, indicating potentially improved solution stability of this complex. The ESI-MS spectrum for Cu₃-TC-Py provided no evidence of mono- or di-copper(II) complexes forming in tandem with the trinuclear complex.

Fig. 1

Molecular structures, ESI-MS profiles with copper(II) ions, and DNA recognition properties of new TC ligands. (a) Molecular structures of earlier reported TC-1 and TC-Thio ligands. (b) Molecular structures of six new TC ligands reported in this study where the alkyne donors give rise to a variety of aliphatic and heteroaromatic copper(II) binding groups. (c) TC ligands form trinuclear Cu complexes as identified using electrospray ionisation-mass spectrometry (ESI-MS) analyses. Identifiable fragments are shown in blue and the full list of fragments, their masses and comparisons of calculated versus found masses are available Table S2. Inset is a simple molecular model of the expected structure less counterions. [M] indicates the assumed molecular ion. (d) Competitive DNA binding and quenching experimental results with calf thymus DNA (ctDNA). C₅₀, Q_Hoechst, and Q_MG, are the concentrations of Cu₃-TC complex required to reduce the respective fluorescence of bound ethidium bromide (EtBr), Hoechst 34580, and methyl green (MG) by 50%. EtBr was used in excess to calculate the apparent DNA binding constant (K_app), while limited concentrations of Hoechst 34580 and MG were employed to identify preferential binding mode.

DNA recognition

Next, the DNA binding properties of the Cu₃-TC complexes were investigated using 1) a competition assay with the DNA intercalating fluorophore ethidium bromide (EtBr), and 2) quenching experiments using limited bound fluorophores Hoechst-34580 (Hoechst) and methyl green (MG),²⁰ which bind in the minor and major grooves of DNA, respectively (Figs. 1d and S16). In the competition study, EtBr was added in excess to calf thymus DNA (ctDNA) and titrated Cu₃-TC complexes were observed to efficiently displace EtBr with C₅₀ values (the concentration required to reduce fluorescence by 50%) ranging from 2–30 µM (EtBr concentration was 12.5µM). The C₅₀ values were then used to calculate apparent DNA binding constants via a derivative of the Cheng-Prusoff equation: K_app = (8.8 x 10⁶ M^− 1)(12.5/C₅₀), where 8.8 x 10⁶ M^− 1 is the binding constant of EtBr, 12.5 is the micromolar concentration of EtBr, and K_app is the apparent binding constant of the analyte.²¹ The K_app values ranged from 3.6 x 10⁶ to 5.8 x 10⁷ M^− 1. Within these results, both Cu₃-TC-Pyrm and Cu₃-TC-Py stood out at the upper end of the series. Next, fluorescence quenching assays with Hoechst and MG, conducted under limited bound conditions with ctDNA, showed that the minor groove-binding Hoechst was more efficiently quenched compared to the major groove-binding MG; in all cases, the Q_MG value (where Q is the analyte concentration required to reduce the intrinsic fluorescence by 50%) was approximately double that of Q_Hoechst. Cu₃-TC-Py most efficiently displaced both fluorophores, closely followed by the structurally similar Cu₃-TC-Pyrm. Overall, these data suggest that the Cu₃-TC complexes are selective for the minor groove of duplex DNA and that heteroaromatic N,N donors provide enhanced DNA recognition properties.

DNA hairpin analysis

Due to the favourable results obtained for Cu₃-TC-Py in both the ESI-MS and DNA binding experiments, this complex was selected for in-depth analysis using microscale thermophoresis (MST) and Förster resonance energy transfer (FRET) melting analysis. Here, palindromic DNA hairpins containing the Dickerson-Drew (DD) consensus sequence separated by an internal five-nucleotide adenine (A) loop were designed (Figure S17). The first hairpin (F-DDH) contained a 5′-Alexa Fluor™ 647N modification that facilitated MST measurements, while the second hairpin (FRET-DDH) contained a FRET pair consisting of 3′-Iowa Black® and 5′-Alexa Fluor™ 647 labels. MST measures changes in the movement of an analyte along a microscopic temperature gradient (thermophoresis) due to changes in size, shape, charge, or hydration shell of the fluorescently labelled target upon binding.^22–24 Analysis of F-DDH in the presence of increasing concentrations of Cu₃-TC-Py showed a typical MST profile with clear unbound and bound MST trace populations at low titrant concentration (Fig. 2a). However, at high concentrations the MST traces lost resolution and became irregular. Examination of the initial intensity values (the fluorescence of the sample prior to heating) showed a clear dose-dependent decrease, indicating that Cu₃-TC-Py was likely condensing the hairpin. An overlay of the normalised MST trace values at t = 5 s (FNorm₅) and the initial intensity values allowed us to clearly differentiate the binding and the non-specific condensation phases (Fig. 2a). The FNorm₅ plot gradually increases with titrated Cu₃-TC-Py, before a sharp inflection point and subsequent decrease that aligns well with the condensation profile shown in the initial intensity plot. The shared inflection point at 1.8 equivalents of Cu₃-TC-Py suggests a binding site of approximately 6 base pairs. Nonlinear regression of the binding region gave a K_d of 430 nM and that of the condensation region gave an EC₅₀ of 9 µM. FRET melting showed a similar trend to that of the MST experiments. Here, the normalised melting curves show two distinct phases, which were assigned to discrete binding and condensation phases; the first phase represents increasing thermal stability of the hairpin upon complex binding, while the second phase represents dissolution of larger DNA condensates. Plotting the observed T_m values clearly demonstrates these two phases (Fig. 2b). The binding region here shows saturation at r ≈ 1.8 (where r = [complex]/[DNA]), and a K_d of 690 nM, in strong agreement with the MST data. Fitting the MST data with the Bard equation (Figure S18) returned an intrinsic binding constant (K_b) of 1.9 x 10⁷ M^− 1 and a 2:1 Cu₃-TC-Py:F-DDH binding stoichiometry.²⁵ Applying this same model to the FRET melting data returned a K_b = 9.2 x 10⁶ M^− 1 (Figure S19) with the same binding stoichiometry as the MST analysis suggesting Cu₃-TC-Py occupies a binding site of 6 base pairs. Finally, both K_b values are in general agreement with the earlier calculated K_app value and corroborate Cu₃-TC-Py as high-affinity DNA binding agent.

Single-molecule DNA analysis

In order to directly probe structural changes imposed on DNA by Cu₃-TC-Py, single-molecule images of ~ 50 kbp long λ-DNA molecules exposed to the complex were taken using fluorescence microscopy for DNA confined in nanofluidic channels.²⁶ By comparing DNA molecules in samples with increasing Cu₃-TC-Py, trends related to DNA length and fluorescence intensity could be plotted to yield relative distribution profiles. The DNA was visualised by incubating the DNA with the Cu₃-TC-Py prior to addition of YOYO-1, followed by fluorescence imaging in the nanofluidic channels. YOYO-1 is a bis-intercalating fluorescent dye that extends the length of the DNA helix.^27–29 Saturating the DNA molecules with YOYO-1 therefore ensures DNA molecules are maximally stretched, with the highest possible fluorescence intensity. Change in either DNA extension or intensity can then be directly monitored upon exposure to an analyte, such as Cu₃-TC-Py. Staining of λ-DNA, pretreated with 2.5 µM Cu₃-TC-Py decreased the average pixel intensity observed relative to the untreated control, indicating that Cu₃-TC-Py competes with YOYO-1 binding at this concentration (Fig. 2c). The intensity values then increase somewhat in samples treated with 5 and 10 µM Cu₃-TC-Py before reaching a minimum at 25 µM of Cu₃-TC-Py. It appears the initial reduction in intensity is due to displacement of YOYO-1 via direct competitive DNA binding by the complex, while recovery at 5 and 10 µM occurs due to non-competitive binding events that cause the DNA to contract, increasing the YOYO-1 density per pixel. Saturating λ-DNA with 25 µM of the complex causes complete DNA condensation and a dramatic decrease in YOYO-1 emission. This is supported by DNA extension plots which show a steady decrease in λ-DNA molecule length with increasing Cu₃-TC-Py and complete condensation at 25 µM and also total fluorescence plots (figure S20) that show a sharp decrease (indicating YOYO-1 ejection) at 2.5µM Cu₃-TC-Py which remains constant up to 10 µM Cu3-TC-Py. The single molecule data are in excellent agreement with earlier hairpin and quenching analysis suggesting two distinct DNA interaction phases: minor groove binding, and non-specific electrostatic effects resulting in DNA condensation.

In-silico Binding Studies

Molecular docking

To evaluate the minor groove binding properties further, molecular docking studies of Cu₃-TC-Py with the Dickerson-Drew dodecamer (DDD, PDB code 1BNA) were performed. We began by generating a model of Cu₃-TC-Py using classical mechanics in Avogadro and optimising this to an energy minimum. The resulting structure was then added to a grid box that encapsulated the entire DDD target for docking. Here, Cu₃-TC-Py was found to bind predominantly in the minor groove with eight of the nine docking output poses showing minor groove residency and one pose showing a major groove binding (Figure S21). We next sought to investigate the saturation characteristics of Cu₃-TC-Py binding by taking the top ranked pose from the docking with 1BNA and treating this entire complex as a rigid macromolecule for a second round of docking with another molecule of Cu₃-TC-Py (Figure S22). All output poses placed the second Cu₃-TC-Py molecule in the major groove of the duplex.

Molecular dynamics

To provide greater depth on the binding mode of Cu₃-TC-Py, MD simulations of the DDD were undertaken using the highest affinity major and minor groove docking poses as starting positions. Figures 2d and 2e show still frames of the minor and major groove simulations respectively. First, both MD simulations show that the Cu₃-TC-Py complex remains bound within the starting groove of the duplex, supporting earlier findings that the complex is a high-affinity DNA binder. Plotting the coulombic, van der Waals (VdW), and total energies versus time (Fig. 2f and g) then revealed that the major contributing binding force is electrostatic or coulombic interactions. Therefore, it appears that the binding interaction is driven largely by the highly cationic nature of the complex towards anionic DNA. However, although the coulombic interactions in both simulations were broadly similar, the minor groove simulation showed a significant increase in vdW interactions that supports our earlier finding of Cu₃-TC-Py binding with some selectively for the narrower minor groove. Mechanistically, two arms of the complex remain within each of the grooves while the third is ejected and interacts with the phosphate backbone.

Fig. 2

Single-molecule nucleic acid interactions and molecular dynamic simulations of Cu₃-TC-Py. (a) Two-population MST traces indicating Cu₃-TC-Py DNA binding. Plotting of FNorm₅ and initial intensity allowed for K_d and C₅₀ values to be calculated. (b) Fluorescence melting of hairpin DNA indicate gradual increases in the melting temperature upon exposure to the complex. Plotting the melting temperature (T_m) versus Cu₃-TC-Py equivalents enabled the K_d value to be directly calculated. (c) Nanofluidic single-molecule imaging employed to identify trends in the average pixel intensity and molecule length of YOYO-1-stained λ-DNA molecules. (d) Still frames taken from molecular dynamics simulations of Cu₃-TC-Py bound in the minor groove of duplex DNA (PDB: 1BNA). (e) Still frames taken from molecular dynamics simulations of Cu₃-TC-Py bound in the major groove duplex DNA (PDB: 1BNA). (f) Time-course binding energies taken from the molecular dynamics simulation of Cu₃-TC-Py bound in the minor groove of B-DNA shown in (d). (g) Time-course binding energies taken from the molecular dynamics simulation of Cu₃-TC-Py bound in the major groove of B-DNA shown in (e).

NCI-60 screening and Cu Internalisation

To compare TC-Py’s broad-spectrum anticancer properties with earlier TC compounds, the free ligand was submitted alongside TC-1 and TC-Thio to the U.S. National Cancer Institute’s (NCI) Developmental Therapeutics Program (DTP) 60 human cancer cell line screen. At the time of submission, conducting an NCI-60 screen of the copper complexes was not feasible due to the acceptance criteria of small molecules being limited to organic compounds. However, screening the free ligands remains important, as it may offer insights into potential prodrug activity, where the ligand could interact with bioavailable copper to promote activity. The cytotoxic effects were identified initially at one-dose (10 µM) shown as a heat map in Fig. 3. The panel consists of cell lines from a variety of cancers including leukemia, non-small cell lung, colon, central nervous system, melanoma, ovarian, renal, prostate, and breast cancers. TC-1 was inactive and was therefore not analysed further. However, both TC-Thio and TC-Py ligands displayed selectivity against several cell lines including triple-negative breast cancer MDA-MB-231 and MDA-MB-468, renal cancer (RXF 393), melanoma (SK-MEL-2), along with some ovarian, CNS, and non-small cell lung cancers. Both compounds were then examined using five-dose analysis within the NCI-60 panel where additional analyses to identify 50% growth inhibition (GI₅₀) were mapped (Supplementary Table S3 and Figure S23). Overall, both ligands were least active against the leukaemia panel but display greatest activity against ovarian cancer lines (except OVCAR-5) with heightened activity toward IGROV1, OVCAR-4, and SK-OV-3. The five-dose analysis also confirmed sensitivity towards triple-negative breast cancers MDA-MB-231 and MDA-MB-468 with GI₅₀ values ranging between 7.24 µM – 20.42 µM for both ligands. Since TC-Py was designed as an AMN, its ability to internalise copper³⁰ and access the nucleus of eukaryotic cells was next assessed using the triple-negative breast cancer cell line MDA-MB-231 (Fig. 4a). Here, inductively coupled plasma mass spectrometry (ICP-MS) studies were undertaken by treating the cells with 20 µM of Cu3-TC-Py for 48 h. MDA-MB-231 cells were then harvested to identify both the total and nuclear uptake of Cu arising from the complex. Data here shows significant uptake within the total cell population along with notable nuclear Cu accumulation.

Fig. 3

NCI-60 anticancer growth inhibition data of TC-1, TC-Thio, and TC-Py. Decreased % growth is visualised as orange while increases are shown as blue.

DNA damage assessment

pUC19 relaxation

To assess the artificial nuclease activity of Cu₃-TC-Py, electrophoresis experiments with supercoiled pUC19 DNA were undertaken. Damage to one strand of supercoiled DNA causes relaxation to the open circular conformation, while double strand breaks (DSBs)—including two proximate nicking events on the opposite strands of the helix—cause relaxation to the linear form. Since each of these forms has different topological states, damaged molecules can be distinguished and quantified by resolving their mobility using agarose electrophoresis. Triplicate experiments involving supercoiled pUC19 DNA exposed to increasing concentrations of Cu₃-TC-Py in the presence of added reductant, sodium-L-ascorbate, were performed. DNA damage was then visualised and quantified using band densitometry (Fig. 4b). Here, the gradual conversion from supercoiled to open circular, followed by linear forms was identified as a function of increasing Cu₃-TC-Py concentration. Notably, the formation of linear form occurs before the complete degradation of the supercoiled form (Figure S24). This profile is a classic indication of independent double strand break formation. To confirm DSB formation, Freifelder-Trumbo analysis was then performed (Fig. 4c). The Freifelder-Trumbo model (Eq. 1–3) correlates the number of single strand nicks (n_nick) and the number of linearisation events (n_lin) per plasmid molecule in each sample based on the relative presence of the three forms. A plot of n_lin versus n_nick can then be compared to the theoretical nicking only plot to identify independent DSB formation. The value of H in this model represents the minimum number of base pairs within which two nicking events must occur for the plasmid to revert to its linear form. This parameter has been a subject of some debate, with calculated values ranging from 16 to 50 base pairs.³¹ To ensure that any deviation from the theoretical plots was genuinely indicative of DSBs we calculated theoretical plots with H values of 16 and 50. The plot of n_lin versus n_nick for Cu₃-TC-Py in Fig. 4c significantly departs from both theoretical plots and strongly indicates the complex cleaves DNA through a combination of nicking and independent DSBs, with the former predominating.

$\:{\text{F}}_{\text{l}\text{i}\text{n}\text{e}\text{a}\text{r}}=\:{\text{n}}_{\text{l}\text{i}\text{n}\:}{\text{e}}^{{\text{n}}_{\text{l}\text{i}\text{n}}}\:\:\:(Eq.\:1)$

$\:{\text{F}}_{\text{s}\text{u}\text{p}\text{e}\text{r}\text{c}\text{o}\text{i}\text{l}\text{e}\text{d}}=\:{\text{e}}^{-({\text{n}}_{\text{n}\text{i}\text{c}\text{k}}+\:{\text{n}}_{\text{l}\text{i}\text{n}})}\:\:\:(Eq.\:2)$

$\:{\text{n}}_{\text{l}\text{i}\text{n}}=\frac{{{\text{n}}_{\text{n}\text{i}\text{c}\text{k}}}^{2}(2\text{H}+1)}{4\text{L}}\:\:\:(Eq.\:3)$

To probe the cleavage mechanism, experiments in the presence of non-covalent DNA binding agents and antioxidants were undertaken (Figure S24).^32,33 First, the presence of major groove binding methyl green (MG) produced a significant increase in damage while activity was completely inhibited by netropsin—a known minor groove binding agent. Next, antioxidant experiments revealed that cleavage was maximally inhibited by tiron, a superoxide scavenger, and N,N-dimethyl thiourea (DMTU), a peroxide scavenger. Taken together these results indicate Cu₃-TC-Py mediates oxidative DNA cleavage within the minor groove using a Fenton / Haber-Weiss type catalytic cycle.³⁴

Repair-assisted damage detection

We next assessed the ability of Cu₃-TC-Py to induce intracellular DNA damage using a single-molecule repair-assisted damage detection (RADD) protocol (Fig. 4d).³⁵ Here, peripheral blood monoclonal cells (PBMCs)—selected as models of healthy somatic cells—were treated with Cu₃-TC-Py or left untreated. In parallel PBMC were pre-treated with different antioxidants prior to Cu₃-TC-Py treatment as indicated on Fig. 3d. Genomic DNA was extracted from the cells and incubated with DNA repair enzymes. Next, fluorescently labelled deoxynucleotide triphosphate (dNTP) aminoallyl-dUTP-ATTO-647N was added along with a processive DNA polymerase, and the mixture was counterstained with YOYO-1. Finally, individual DNA molecules stretched on functionalised coverslips were imaged using fluorescence microscopy. Individual RADD events were then quantified due to the appearance of red foci arising from the incorporation of ATTO-647N labelled dNTPs. We conducted experiments with a DNA repair cocktail (APE1, Endo III, Endo IV, Endo VIII, Fpg, and AAG). Cu₃-TC-Py treatment alone generated a significant increase in the number of DNA lesions relative to the untreated sample, confirming the ability of the complex to access and damage intracellular genomic DNA (Fig. 3d). Pre-treatment of the PBMCs with antioxidants, tiron, L-histidine, and D-mannitol, reduced the observed number of lesions to the same level as the untreated sample, implicating superoxide, singlet oxygen, and hydroxyl radicals, respectively, in lesion formation. Additionally, we found that sodium pyruvate, and L-methionine had little effect on the number of lesions observed, suggesting peroxide and hypochlorous acid are not involved in the DNA damage mechanism of Cu₃-TC-Py. This data distinguishes Cu₃-TC-Py from earlier Cu₃-TC-1 and Cu₃-TC-Thio complexes which act predominantly via a superoxide- and peroxide-dependent mechanisms.

Fig. 4

DNA damaging experiments. (a) Band densitometry analysis obtained from pUC19 cleavage experiments with Cu₃-TC-Py. Individual values shown as dots. (b) Freifelder-Trumbo analysis of the Cu₃-TC-Py cleavage profile. Blue and green plots are theoretical plots for nicking-only agents where H (the number of base pairs within which two nicking events occur) is set to 16 or 50 bp. Orange dots represent values calculated from densitometry values. (c) ICP-MS measurements of intracellular Cu localisation in MDA-MB-231 cells after exposure to 20 µM of Cu₃-TC-Py for 48 hours. Blue bars indicate untreated samples, and green bars indicate samples treated with Cu₃-TC-Py. (d) RADD experiments with peripheral blood monoclonal cells (PBMCs). Untreated represents the density of lesions observed in untreated PBMCs, control is that in cells treated with Cu₃-TC-Py only. All other bars are indicative of samples treated with Cu₃-TC-Py where the indicated antioxidant was prophylactically incubated with PBMCs. Statistical significance was calculated using one-way ANOVA in GraphPad Prism. (e) Microscopy images of Bacillus subtilis treated with 750 µM Cu₃-TC-Py for 10- and 30-min. Composite image is an overlay of DAPI and RecA-GFP images. (f) DNA compaction ratio analysis where B. subtilis was treated with 750 µM of Cu₃-TC-Py, 3 µM ciprofloxacin, or 268 µM nitrofurantoin for 30 minutes. Individual values represented as dots, mean values shown as black lines. Triplicate experiments are shown as individual groups within the sample group plot (g) Single-molecule profile analysis of DNA extracted from B. subtilis along with cells treated with nitrofurantoin or Cu₃-TC-Py. Inset images are representative microscope images from each experiment.

Cytological profiling

To probe the structural changes imposed on DNA by Cu₃-TC-Py, the AMN activity was probed in bacterial cells using a combination of cytological profiling using phase contrast microscopy and DAPI DNA staining, functional profiling using GFP-tagged RecA (which is an essential protein for maintaining and repairing DNA in bacteria) and single-molecule analysis. We first treated Bacillus subtilis with Cu₃-TC-Py for 10 and 30 min before staining and imaging via phase contrast and fluorescence microscopy (Fig. 4e). DNA targeted agents can be expected to cause a change (compaction or relaxation) of the bacterial nucleoid while a DNA damaging agent such as an AMN may be expected to increase recruitment of RecA and a resultant increase in RecA foci. Ciprofloxacin and nitrofurantoin were used as controls in these experiments. Ciprofloxacin inhibits DNA gyrase and topoisomerase IV causing defects in DNA replication and nucleoid separation resulting in clear nucleoid compaction and recruitment of the RecA protein to single-stranded DNA arising from strand breaks.³⁶ Nitrofurantoin is a prodrug that is activated by cellular nitroreductases leading to the formation of reactive species that damage cellular macromolecules, most prominently DNA, causing nucleoid relaxation and, at high doses, destruction of the entire nucleoid.³⁷ Relative to the positive controls treated with ciprofloxacin and nitrofurantoin, Cu₃-TC-Py resulted in an apparent loss of DAPI staining together with limited recruitment of GFP-RecA foci (Figure S26 and S27). Given the earlier evidence of combined AMN and DNA condensation activity by Cu₃-TC-Py, we hypothesised this cytological profile may arise due to near-total degradation of the genetic material. Therefore, we conducted image analysis to identify the DNA compaction ratio within imaged cells (Fig. 4f) where the compaction ratio is an expression of the nucleoid volume relative to the total cell volume. Theoretically, DNA degradation causes a decrease in the DNA compaction ratio as the nucleoid is dispersed, while compaction would have the inverse effect. Data here showed that after 30 min, Cu₃-TC-Py significantly decreased the DNA compaction ratio, producing a similar profile to nitrofurantoin, thus demonstrating Cu₃-TC-Py damages and disperses the genetic material. Confirmation of this mechanism was then sought using a combination of gel electrophoresis and single-molecule analysis. Gel electrophoresis experiments involved treating cells in an identical manner to those used for the image analysis presented in Fig. 4f. Thereafter, the total DNA content was extracted and visualised using pulse-field agarose gel electrophoresis where changes in DNA molecule sizes are clearly identifiable. Untreated samples contained relatively uniform DNA molecules while those treated with Cu₃-TC-Py demonstrated reduced overall DNA content together with fragmentation patterns indicative of DNA ablation (Figure S28). Tandem single-molecule analysis experiments were then performed using the same treatment and extraction steps. Here, DNA was stained using YOYO-1, stretched on cover slides and measured (Fig. 4g). Shortening of DNA molecules was evident as untreated samples contained high counts of molecules in the 40–80 µm range, while samples treated with Cu₃-TC-Py and nitrofurantoin contained limited numbers of molecules above 40 µm and a significantly increased density of molecules below 20 µm.

Discussion

Click chemistry, including the copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) reaction, is a staple of modern synthetic chemistry and continues to provide functionality to new fields. Here, we applied the CuAAC reaction to diversify and expand the DNA damaging potential of new polynuclear copper metallodrug candidates. Six new ligands of the Tri-Click (TC) class were prepared and their ability to coordinate three copper(II) ions was determined using electrospray ionisation-mass spectrometry (ESI-MS). The data shows that N,N-donor systems of TC-Py (Pyridine), TC-Pyrm (Prymidine), and TC-Benzo (Benzothiazole) form trinuclear complexes, while N,O- systems such as TC-OH (Hydroxide) and TC-Acid (Carboxylic acid) produce mixtures of complexes with varying nuclearity. Competitive fluorescence displacement experiments indicate that the N,N-complexes Cu₃-TC-Py and Cu₃-TC-Pyrm possess exceptionally high DNA recognition properties, surpassing earlier reported TC-1 and TC-Thio complexes.^14,15 Next, fluorescence quenching probes revealed preferential minor groove binding with the N,N-donor complexes outperforming other agents in this screen. Competition and quenching data were combined to select Cu₃-TC-Py as the leading candidate, and to better understand its DNA recognition mode, advanced analysis involving microscale thermophoresis (MST), Förster resonance energy transfer (FRET) melting, and single-molecule DNA imaging experiments were performed. These techniques revealed a bi-phasic interaction mode consisting of high-affinity DNA binding characterised by stabilisation of duplex DNA, followed by molecular condensation associated with charge neutralisation. In-silico docking and molecular dynamics corroborate the complex preferentially binding within the minor groove; it appears the agent is predominantly stabilised by electrostatic forces augmented by van der Waals interactions that are otherwise lacking when the complex resides in the major groove.

Broad-spectrum NCI-60 screening of TC-Py and TC-Thio identified both ligands had promising activity against a range of human cancer cell lines including MDA-MB-231. This result contrasts with the earlier reported TC-1 scaffold and supports the role of heteroaromatic groups in promoting cytotoxicity. The uptake of copper within MDA-MB-231 cells was then probed upon exposure to Cu₃-TC-Py. Here, both the total and nuclear uptake increased significantly indicating an ability by the ligand to intracellularly traffic copper ions. Favourable NCI-60 and ICP-MS indications prompted our investigation into the DNA damaging properties of Cu₃-TC-Py on native DNA and within cellular models. Firstly, native DNA damaging studies showed Cu₃-TC-Py cleaves pUC19 through a mixture of single and double strand breaks with high efficiency compared to other copper AMN systems.¹⁷ The intracellular DNA damage repair response triggered by Cu₃-TC-Py was then probed using single-molecule DNA imaging of primary blood monoclonal cells (PBMCs). Here, DNA lesions characteristic of oxidised purine and pyrimidine bases were identified with further analysis revealing intracellular ROS associated with superoxide, singlet oxygen, and hydroxyl radicals chiefly mediate lesion formation. To investigate these effects further, functional assays revealed that Cu₃-TC-Py disrupts normal DNA packing in bacterial cells, resulting in DNA dispersion.

The discovery of Cu₃-TC-Py from the broader series demonstrates the use of click chemistry in metallodrug discovery and points to a conserved set of structural features adapted to maximise artificial metallo-nuclease activity. While the TC scaffold serves as a strong foundation for designing DNA-damaging candidates, advancing TC-Py to the clinic will require investigation into its in vivo biological stability when complexed with bioavailable copper. Given recent developments in the study of metallothionein and its impediment of oxygen activators,³⁸ the strategic inclusion of pyridine donor groups within the TC scaffold may enhance intracellular stability and improve clinical viability.

Acknowledgement

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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Supplementary Material 1

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