The molecular basis of G4 recognition by CX-5461 and MTR-106 is elucidated, and a structural framework for the rational development of next-generation G4-targeted therapeutics with improved selectivity and efficacy is provided.
Abstract
Abstract Small-molecule stabilization of G-quadruplexes (G4s) has become an active focus in nucleic acid-targeted drug discovery. The mechanisms of the ligands’ binding selectivity and biological activities are critical for rational drug design. CX-5461, the first clinically advanced G4-targeting agent, has shown notable efficacy in DNA repair–deficient cancers. Its close analogue MTR-106 also displayed comparable anti-proliferative effects on cancer cells. However, the molecular basis of their interaction with G4s remains elusive. Here, through differential scanning calorimetry, complementary biophysical assays, and solution NMR technique, we probed their recognition modes with G4s. Although both ligands induced stronger thermal stabilization of the parallel G4s, the MYT1L quadruplex–duplex hybrid (QDH) formed the most homogeneous ligand-bound complexes in solution, highlighting the G4–duplex junction as a potential recognition site. Solution structural determination at high resolution demonstrated that both CX-5461 and MTR-106 inserted into the G4–duplex junction pocket. Their rigid polyaromatic scaffolds stacked with the 3′-end G-tetrad of G4, while their flexible side chains extended into the groove of QDH to enable spatial recognition. These findings elucidated the molecular basis of G4 recognition by CX-5461 and MTR-106, and provided a structural framework for the rational development of next-generation G4-targeted therapeutics with improved selectivity and efficacy.
G-quadruplex (G4) structures within oncogene promoters represent promising therapeutic targets, particularly in the context of transcriptional regulation. Here, we report that the red box, a fully organic macrocyclic cationic receptor, exhibits high affinity and selectivity for G4 structures. Biophysical analyses reveal that the red box binds the c-MYC G4 with nanomolar affinity. Notably, the red box displays greater selectivity for c-MYC over duplex DNA compared to the well-characterized porphyrin TMPyP4, and its macrocyclic architecture is essential for this activity, as its acyclic analogue displays negligible G4 stabilization. In cellular assays, treatment with non-cytotoxic concentrations of the red box selectively downregulated c-MYC expression in HEK293T cells, with minimal effects on c-KIT and hTERT. Unlike previously reported G4-selective ligands based on Pd(ii)/Pt(ii) metallacycles, the red box achieves nanomolar affinity and selectivity through a fully metal-free, hydrazone-based scaffold, addressing key limitations related to cost, biodegradability, and long-term biocompatibility. Furthermore, c-MYC downregulation was consistently observed across multiple cell lines, including tumour-derived cellular models, underscoring the translational relevance of this new class of organic G4 binders.
Paula Novo, Iván Montes de Oca, C. García-Delgado et al.· RSC Chemical Biology· 0 citations
G-quadruplexes (G4s) are non-canonical nucleic acid structures formed by guanine-rich sequences that assemble into stacked guanine tetrads. These unusual RNA or DNA structures are implicated in essential regulatory processes in both human and viral genomes. In recent years, compounds that bind and stabilise G4s have emerged as promising antiviral agents against several viruses such as HIV-1, HCV or HPV. We report the design, synthesis, and biophysical characterization of a novel class of G4-stabilizing ligands based on a positively charged dibenzoacridinium (DBA) core. A combination of FRET-melting assays, fluorescence quenching assay (FQA), circular dichroism spectroscopy, native mass spectrometry, NMR titration, and molecular dynamics simulations confirmed that DBA ligands bind selectively to G4s, with thermal stabilization (ΔTm) values reaching +18 °C and dissociation constants (Kd) as low as 0.14 μM. In cellular assays, DBA4 and DBA5 compounds exhibited antiviral activity against HIV-1, with IC50 values of 1 μM and 3 μM, respectively. These results identify DBAs as a new class of G-quadruplex ligands with anti-HIV properties.
Aman Kabbara, C. Buré, A. Guédin et al.· RSC Medicinal Chemistry· 0 citations
Abstract CX-5461 (pidnarulex) is the most clinically advanced small-molecule ligand associated with G-quadruplex (G4) targeting and shows activity in DNA repair–deficient tumors, yet its clinical use is associated with dose-limiting phototoxicity. Here, we repurpose this intrinsic photoreactivity to investigate CX-5461 as a G4-associated, light-activated antitumor scaffold. In solution, CX-5461 produces both Type I and Type II reactive oxygen species (ROS), whereas complexation with G4 DNA markedly decreases detectable singlet oxygen (1O2) formation and favors radical-associated Type I oxidation. This G4-modulated photochemical shift drives oxidative remodeling and destabilization of G4-containing DNA. In cancer cells, photoactivation enhances cytotoxicity by up to two orders of magnitude relative to the dark state and is accompanied by elevated intracellular ROS, increased 8-oxoG formation, γ-H2AX accumulation, and changes in BG4-detected nuclear G4 structures. In vivo, light-activated CX-5461 suppresses tumor growth and extends survival in immunocompetent syngeneic models, eliciting hallmarks consistent with immunogenic genome stress. Collectively, these findings reposition CX-5461 as a clinically relevant scaffold for G4-associated photogenomic cancer therapy, while supporting a model in which broader oxidative DNA damage may also contribute to the biological response.
Jakub Trojnar, Maria V Cottini, Marta Dudek et al.· Nucleic Acids Research· 0 citations
The urgent need for novel glioblastoma (GBM) therapies has motivated the exploration of EGFR G-quadruplex (G4) targeting, which suppresses oncogene transcription independently of kinase activity. However, existing EGFR G4 ligands lack subtype selectivity. Through computer-aided design and systematic bioisosteric modifications of the 1H-imidazo[4,5-f][1,10]phenanthroline scaffold, we synthesized a series of derivatives. We evaluated their G4 interactions by molecular docking, UV-Vis, FRET, ITC, and CD. Compound 3, bearing a 6-bromopiperonyl group, exhibited nanomolar affinity for EGFR G4 (Kd = 102 nM) with ~146-fold selectivity over K-Ras G4, stabilizing it via end-stacking. It potently inhibited U87-MG glioblastoma cell proliferation (IC50 = 0.49 μM), with a wide safety margin relative to normal HMC3 microglia (safety index = 14.18). Mechanistically, compound 3 triggered DNA damage, mitochondrial oxidative stress, and sequential multi-organelle damage to mitochondria, lysosomes, and the endoplasmic reticulum, while impairing cell migration and invasion. These findings establish compound 3 as a uniquely selective EGFR G4 stabilizer, offering a promising multi-organelle-damage strategy for glioblastoma therapy.
Mengjiao She, Ao Yu, Xiao-Zhan Qiu et al.· Antioxidants· 0 citations
G-quadruplexes are structurally diverse nucleic acid motifs with important biological properties as well as emerging applications in biotechnology and therapeutics. Here, we investigate whether a double-headed bis-guanine (G) nucleotide, GG-a substantial deviation from canonical nucleic acid architecture-can be accommodated within G-quadruplex structures and how its effects depend on sequence context and topology. Using a combination of circular dichroism (CD) spectroscopy, thermal denaturation analysis, UV thermal differential spectroscopy (TDS), fluorescence light-up assays, polyacrylamide gel electrophoresis (PAGE) analysis, and molecular dynamics (MD) simulations, we show that GG can substitute two consecutive guanosines in G-quadruplex-forming oligonucleotides (ONs) and directly participate in G-tetrad formation, but with strong sequence- and position-dependent consequences. GG incorporation is best tolerated in parallel G-quadruplexes and at G-tetrad steps with low native torsional twist, where local unwinding can be accommodated. In a tetramolecular TG4T system, GG positioned near the 3'-end of the G-stack preserved parallel G-quadruplex topology while providing significant thermal stabilization (+21°C). In the antiparallel thrombin-binding aptamer (TBA), a single GG incorporation yielded exceptional stabilization (+34°C) but was accompanied by altered topological signatures. Together, the results presented herein establish GG as a powerful but context-dependent G-quadruplex stabilizer and define design principles for its use in engineered G-quadruplexes and aptamer development.
Krista Urup, Peter Reinholdt, Kasper M. Beck et al.· Chemistry· 0 citations
Abstract G-quadruplexes (G4s) are non-canonical nucleic acids involved in gene regulation and have attracted interest as potential therapeutic targets. However, developing ligands that selectively recognize specific G4 structures remains challenging. Here, we report the rational design of chimeric peptides that combine a de novo selected peptide (Pep11) with a native peptide (Rhau18) derived from the endogenous DHX36 protein. The chimeric peptides bound RNA G4s (rG4s) with nanomolar affinity, representing a 10-fold enhancement over the individual modules, while retaining selectivity for rG4s over DNA G-quadruplexes (dG4s) and non-G4 RNA structures. These chimeric peptides inhibit telomerase activity in vitro and suppress the translation of an rG4-containing reporter gene in living cells. Live-cell imaging further supports their ability to recognize rG4 structures in a cellular context. Molecular dynamics (MD) simulations of the peptide–rG4 complexes were consistent with the experimental observations and suggested that peptide fusion promoted a more extensive and persistent peptide–rG4 interface, providing a possible structural basis for the enhanced binding affinity. Overall, our study suggests that chimeric peptide design provides a useful strategy for developing selective rG4-targeting ligands and modulating functional nucleic acid structures.
Xi Mou, Jiuming Wang, Yanruisheng Shao et al.· Nucleic Acids Research· 0 citations