Balancing Affinity, Selectivity, and Cytotoxicity of Hydrazone-Based G-Quadruplex Ligands for Activation of Interferon β Genes in Cancer Cells

Abstract

G-quadruplex (G4) ligands are investigated to discover new anticancer drugs with increased cell-killing potency. These ligands can induce genome instability and activate innate immune genes at non-cytotoxic doses, opening the discovery of cytostatic immune-stimulating ligands. However, the interplay of G4 affinity/selectivity with cytotoxicity and immune gene activation is not well-understood. We investigated a series of closely related hydrazone derivatives to define the molecular bases of immune-stimulation activity. Although they are closely related to each other, such derivatives differ in G4 affinity, cytotoxicity, genome instability, and immune gene activation. Our findings show that G4 affinity of ligands is a critical feature for immune gene activation, whereas a high cytotoxic potency interferes with it. The balance of G4 stabilization versus cytotoxicity can determine the level of immune gene activation in cancer cells. Thus, we propose a new rationale based on low cell-killing potency and high immune stimulation to discover effective anticancer G4 ligands.

Exploring the G-quadruplex binding and unwinding activity of the bacterial FeS helicase DinG

“…Most of the work addressing the cellular function of DinG suggests a role in the resolution of R-loops 57 . However, this is very compatible with a parallel role in G4 resolution, as there is growing evidence of a widespread www.nature.com/scientificreports/ interplay between G4s and R-loops 52,[58][59][60] indicating that G4s are structurally compatible with R-loops and they can form simultaneously in vitro and in vivo, thus suggesting they act synergistically 59,61 .…”

“…The different behaviour of the helicase on c-KIT1 compared to c-MYC G4, whose topological arrangement is very similar, suggests a correlation between the unwinding and the thermodynamic stability of the G4, with c-MYC G4 exhibiting a higher www.nature.com/scientificreports/ melting temperature than c-KIT1. The higher the stability, the lower the unwinding and vice versa [52][53][54] . At a more general level this result suggests that helicases display a selectivity that goes beyond the simple distinction between intramolecular and intermolecular G4s, or among the different G4 topologies; the possibility of a selective recognition/activity towards some but not other G4s should thus be taken into account when assessing the activity of a helicase towards G4s.…”

Exploring the Dispersion and Electrostatic Components in Arene–Arene Interactions between Ligands and G4 DNA to Develop G4-Ligands

“…1 H NMR (400 MHz, DMSO-d 6 ): δ (ppm) 9.92 (s, 1H), 8.12 (d, J = 8.2 Hz, 1H), 7.81 (t, J = 7.7 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H), 6.82 (s, 1H), 2.82 (s, 3H), 2.34 (s, 6H). 13 (26). A vial was charged with 15b (200 mg, 0.929 mmol).…”

“…The design and discovery of small organic molecules that target G4 structures is an attractive area of research. ,,− Known G4 ligands often contain permanently charged species or multiple basic amine residues, , and the advances of G4 ligands into selective G4 binders with satisfactory pharmacokinetic properties are still scarce. , This hampers further elucidations of G4s as drug targets and can be linked to the lack of detailed descriptors of the interaction between G4 DNA and G4-ligands. Current guidelines in G4 ligand design propose that rigid aromatic systems can engage in stacking interactions with the guanines on the G4 surface, electron-withdrawing groups can enhance binding, and cationic groups can engage in electrostatic interactions with the phosphate backbone. , Gaining a deeper and more detailed understanding of these binding interactions is essential to advance the development of G4 ligands with properties suitable to explore G4 DNA as drug targets. , …”

“… 15 , 16 , 22 − 25 Known G4 ligands often contain permanently charged species or multiple basic amine residues, 16 , 22 and the advances of G4 ligands into selective G4 binders with satisfactory pharmacokinetic properties are still scarce. 25 , 26 This hampers further elucidations of G4s as drug targets and can be linked to the lack of detailed descriptors of the interaction between G4 DNA and G4-ligands. Current guidelines in G4 ligand design propose that rigid aromatic systems can engage in stacking interactions with the guanines on the G4 surface, electron-withdrawing groups can enhance binding, and cationic groups can engage in electrostatic interactions with the phosphate backbone.…”