An in-depth study of clofarabine's binding mechanism to DNA: A thorough experimental and theoretical investigation

Abstract

The interaction of medicinal compounds with nucleic acids is a critical challenge in drug development. This research focused on the binding dynamics of clofarabine (CLO), an antineoplastic agent, with fish sperm DNA (dsDNA) under conditions that simulate the physiological environment at a pH of 7.4. The investigations employed multiple techniques, including fluorescence and UV spectroscopy, thermal denaturation analysis, viscosity measurements, electrochemical assessments, and molecular docking studies to elucidate these binding interactions. The binding constant (K_b) for the interaction between CLO and dsDNA, as determined from the Benesi-Hildebrand plot, was found to be 2.74 × 10⁴ at a temperature of 298 K. The entropy (∆S) and enthalpy (∆H) changes associated with this binding interaction were measured as + 43.08 J mol⁻¹ K⁻¹ and –12.44 kJ mol⁻¹, respectively. These values indicate that the predominant forces driving the binding interaction are primarily due to hydrogen bonding. Studies using the ethidium bromide and the Hoechst probe showed that CLO does not bind to dsDNA intercalatively. Findings obtained through UV-Vis absorption spectroscopy, competitive binding assays, and viscosity assessments indicated that CLO associates with dsDNA by binding within the minor groove. Molecular docking analyses demonstrated that CLO is accommodated within the AT-rich segment of the minor groove, with significant hydrogen bonding interactions occurring between CLO and dsDNA. These findings may offer valuable perspectives for elucidating the mechanisms underlying the toxicity, resistance, and adverse effects associated with CLO.