Phenanthroline polyazamacrocycles as G-quadruplex DNA binders.

Area of Science:

• Medicinal Chemistry
• Molecular Biology
• Drug Design

Background:

• G-quadruplex (G4) DNA structures are attractive targets for anti-cancer drug development.
• Small molecules can modulate G4 stability and function, offering therapeutic potential.
• Understanding ligand-G4 interactions is crucial for designing effective G4-targeting agents.

Purpose of the Study:

• To investigate the G4 binding affinity and stabilization capabilities of four phenanthroline polyazamacrocycles.
• To evaluate the inhibitory effects of these ligands on helicase activity and DNA polymerase extension.
• To assess the potential of these compounds as anti-cancer agents by targeting G4 structures like c-MYC and telomeric G4s.

Main Methods:

• Fluorescence resonance energy transfer (FRET) melting assays to determine thermal stabilization (ΔTm) and G4 selectivity.
• Circular dichroism (CD) spectroscopy to confirm G4 structure formation.
• Fluorescent intercalator displacement (FID) assays to quantify G4 stabilization (DC50 values).
• Fluorescence titrations to determine binding constants (KSV).
• Inhibition assays using Taq polymerase and Pif1 helicase to assess biological activity.

Main Results:

• Ligands [32]phen2N4 and [16]phenN4 demonstrated significant thermal stabilization of c-MYC and 22AG G4s (ΔTm up to 20.3 °C).
• Competitive FRET assays confirmed G4 selectivity over duplex DNA for [32]phen2N4 and [16]phenN4.
• FID assays showed high stabilization of c-MYC and 22AG G4s by [32]phen2N4 (DC50 0.87-1.24 μM).
• Moderate binding constants (KSV 10^5–10^6 M⁻¹) were observed, with [16]phenN4 showing slightly higher affinity for telomeric 22AG G4.
• Both [16]phenN4 and [32]phen2N4 inhibited Taq polymerase and affected Pif1 helicase activity, confirming biological relevance.

Conclusions:

• Phenanthroline polyazamacrocycles, particularly [32]phen2N4, are effective stabilizers of G-quadruplex DNA structures.
• The hexyl chain appears to play a key role in modulating the G4 stabilization properties of these compounds.
• These ligands exhibit promising biological activity, including inhibition of DNA extension and helicase unwinding, supporting their potential as anti-cancer agents targeting G4s.