Chirality affects biological activity and interactions of azacryptands with alternative DNA structures

Abstract

Azacryptands represent a promising class of ligands targeting alternative DNA structures. Their presumed primary targets are DNA three-way junctions (3WJs), whose stabilization in human cancer cells induces DNA damage and subsequent cell death. Although azacryptands exhibit some affinity for G-quadruplexes (G4), their cellular effects differ from those of conventional G4 binders. Importantly, all azacryptands explored so far have been achiral compounds. Here, we investigated three enantiomeric pairs of chiral azacryptands, 4,4 ′- TrisBP, and TrisPOB, prepared from a chiral valine-based derivative of tris(2-aminoethyl)amine (TREN), for their binding affinity and selectivity toward DNA 3WJs, bulges, and G4s. All compounds selectively stabilized the fully base-paired DNA 3WJ, even in the presence of competing DNA structures, indicating preferential binding within the central cavity of the junction. Only the 4,4-TrisBP, 3,3 ′-TrisBP enantiomers stabilized 3WJs containing unpaired nucleotides and DNA bulges. Notably, the (R,R)-enantiomers of 4,4 ′-TrisBP and TrisPOB exhibited stronger stabilization of 3WJs than the (S,S)-enantiomers. All azacryptands exhibited weak affinity toward G4-DNA. Nevertheless, (R,R)-4,4 ′-TrisBP arrested DNA polymerization on templates containing G4-forming sequences, presumably due to its specific interaction within the G4 grooves. Biological evaluation revealed markedly enhanced antiproliferative activity of (R,R)-4,4 ′-TrisBP against human cancer cells compared with its enantiomer, while maintaining low toxicity toward normal cells. At equitoxic concentrations, both enantiomers of 4,4-TrisBP triggered DNA damage and mildly suppressed c-MYC expression. However, when applied at equimolar concentrations, the (S,S)-enantiomer lost its activity. These findings demonstrate that chirality strongly influences DNA interactions and biological activity of azacryptands, underscoring its importance in DNA-targeted drug design.