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 (Δ T m values) of c-MYC and 22AG G4s are 17.2 and 20.3 °C, respectively, for the ligand [32]phen 2 N 4 followed by [16]phenN 4 (11.3 and 15.0 °C, for c-MYC and 22AG, respectively). Competitive FRET assays show that [32]phen 2 N 4 and [16]phenN 4 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]phenN 4 and [32]phen 2 N 4 are the most stabilizing ligands with a Δ T 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]phen 2 N 4 furnishes very low DC 50 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 10 6 M −1 in which [16]phenN 4 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]phenN 4 and [32]phen 2 N 4 against the c-MYC G4. In addition, ligands [32]phen 2 N 4 and [16]phenN 4 affect the unwinding activity of Pif1 in the presence of DNA systems harboring c-MYC and telomeric G4 motifs. Several phenanthroline polyazamacrocycles are proposed as ligands for c-MYC and telomeric G-quadruplex structures.