Phenanthroline polyazamacrocycles as G-quadruplex DNA binders

Targeting quadruplex DNA structures with small molecules is a promising strategy for anti-cancer drug design. Four phenanthroline polyazamacrocycles were studied for their binding affinity, thermal stabilization, inhibitory effect on the activity of helicase towards human telomeric 22AG and oncogene promoter c-MYC G-quadruplexes (G4s), and their ability to inhibit Taq polymerase-mediated DNA extension. The fluorescence resonance energy transfer (FRET) melting assay indicates that the melting temperature increases (ΔT_m values) of c-MYC and 22AG G4s are 17.2 and 20.3 °C, respectively, for the ligand [32]phen₂N₄ followed by [16]phenN₄ (11.3 and 15.0 °C, for c-MYC and 22AG, respectively). Competitive FRET assays show that [32]phen₂N₄ and [16]phenN₄ exhibit G4 selectivity over duplex DNA. Different G4s were compared; no considerable selectivity of the ligands for a specific G4 was found. Circular dichroism (CD) confirms the formation of G4 structures and the melting experiments show that [16]phenN₄ and [32]phen₂N₄ are the most stabilizing ligands with a ΔT_m of 19.3 °C and 15.1 °C, respectively, at 5 molar equivalents for the c-MYC G4. The fluorescent intercalator displacement (FID) assay also demonstrates that ligand [32]phen₂N₄ furnishes very low DC₅₀ values (0.87-1.24 μM), indicating high stabilization of c-MYC and 22AG G4s. These results suggest that the hexyl chain in these compounds plays an important role in regulating the stabilization of these G4s. Binding constants, determined by fluorescence titrations, indicate a moderate ligand-G4 binding with K_SV between 105 and 10⁶ M^-1 in which [16]phenN₄ has a slightly higher apparent binding constant for telomeric 22AG G4 than that for the c-MYC G4. The ligand's ability to inhibit Taq polymerase confirms the biological activity of [16]phenN₄ and [32]phen₂N₄ against the c-MYC G4. In addition, ligands [32]phen₂N₄ and [16]phenN₄ affect the unwinding activity of Pif1 in the presence of DNA systems harboring c-MYC and telomeric G4 motifs.