Dual targeting of higher-order DNA structures by azacryptands induces DNA junction-mediated DNA damage in cancer cells

Abstract

DNA is intrinsically dynamic and folds transiently into alternative higher-order structures such as G-quadruplexes (G4s) and three-way DNA junctions (TWJs). G4s and TWJs can be stabilised by small molecules (ligands) that have high chemotherapeutic potential, either as standalone DNA damaging agents or combined in synthetic lethality strategies. While previous approaches have claimed to use ligands that specifically target either G4s or TWJs, we report here on a new approach in which ligands targeting both TWJs and G4s in vitro demonstrate cellular effects distinct from that of G4 ligands, and attributable to TWJ targeting. The DNA binding modes of these new, dual TWJ-/G4-ligands were studied by a panel of in vitro methods and theoretical simulations, and their cellular properties by extensive cell-based assays. We show here that cytotoxic activity of TWJ-/G4-ligands is mitigated by the DNA damage response (DDR) and DNA topoisomerase 2 (TOP2), making them different from typical G4-ligands, and implying a pivotal role of TWJs in cells. We designed and used a clickable ligand, TrisNP-α, to provide unique insights into the TWJ landscape in cells and its modulation upon co-treatments. This wealth of data was exploited to design an efficient synthetic lethality strategy combining dual ligands with clinically relevant DDR inhibitors.

Dinuclear nickel(ii) supramolecular helicates down-regulate gene expression in human cells by stabilizing DNA G-quadruplexes formed in the promoter regions

“…10 Presented results widen the spectrum of non-B DNA targets for cylinders and suggest that selective interaction with alternative structures in nucleic acids might greatly contribute to the biological activity of the cylinders. Moreover, the ability of cylinders to reach cellular DNA and stabilize DNA G4s and TWJs opens opportunities for the application of cylinders for triggering DNA damage in cancer cells, 46 possibly in combination with DNA repair inhibitors in a chemically induced synthetic lethality strategy. 47 In any case, metallo-supramolecular cylinders [M 2 L 3 ] 4+ should be regarded as multitargeted agents due to their ability to target various alternative nucleic acid structures.…”

Interactions of small molecules with DNA junctions

“…Studies were also performed to investigate whether TWJ ligands trigger DNA damage in dividing cells. This was firmly established via the immunodetection of ␥ H2AX by both optical imaging and flow cytometry: after a thorough characterization of their TWJ-interacting properties in vitro (affinity and specificity via a panel of different techniques), two azacryptands (TrisPOB, TrisNP) were studied in cells and shown to trigger an accumulation of DNA strand breaks (and particularly DSBs) (Figures 3C, 8C and 9) (155,156). When combined with the established ability of TWJ ligands to prevent polymerase activity in vitro (194), these results thus provided a strong rationale for a mechanistic model in which ligand-stabilized TWJs act as roadblocks that hamper proper processivity of DNA-related enzymes, slowing or even stopping their motion along the genomic duplex, which is recognized as a DNA damage and trigger the DDR and repair machineries.…”

“…Stabilised TWJs might thus act as physical roadblocks to polymerases, which is recognized and coped with as a form of DNA damage by the cellular surveillance machinery (60)(61)(62). Therefore, the cellular activity of TWJ-targeting agents could be potentiated by DNA repair inhibitors (vide infra) (155,156).…”

“…The exquisite recognition of TWJs by helicates was further demonstrated by another series of X-ray structure analyses (172), and confirmed by results collected via alternative techniques, including PAGE (173) and NMR (174). These results thus gave strong impetus to the use of sterically demanding small molecules, displaying shapes and volumes suited to 'fill' the TWJ cavity, including helical metallopeptides (studied by circular dichroism (CD), NMR and PAGE) (175)(176)(177), polyazamacrocyles such as TACN-Q (Figure 3C, by CD, PAGE, UV-and fluorescence resonance energy transfer (FRET)-melting assays) (178,179), porphyrins (by UVand FRET-melting assays) (180), triptycenes (Figure 3C, by UV-melting and fluorescence quenching assays) (181,182), azacryptands such as TrisNP (Figure 3C) (155,156,179) and azacyclophanes (183,184) (by PAGE, FRET-melting and fluorescence quenching assays, and electrospray ionization mass spectrometry (ESI-MS) investigations), metallocages (by fluorescence titrations, fluorescence quenching assays and PAGE) (183)(184)(185), and calix [3]carbazoles (Figure 3C, by fluorescence titrations, CD, UV-melting assay and PAGE) (186).…”

Requirements for MRN endonuclease processing of topoisomerase II-mediated DNA damage in mammalian cells

“…The rejoining reaction can also be disrupted by small molecule inhibitors of the reaction (Li and Liu, 2001); these small molecules have found clinical uses as both antibacterial and anticancer agents (Nitiss, 2009b;Drlica et al, 2009;McKie et al, 2021). Structural alterations in DNA, including DNA damage and non B-DNA structures, can also alter the enzyme cleavage/resealing cycle (Wilstermann and Osheroff, 2001;Nitiss et al, 2019;Zell et al, 2021;Pommier et al, 2022).…”