DNA Binding and Cleavage, Stopped-Flow Kinetic, Mechanistic, and Molecular Docking Studies of Cationic Ruthenium(II)...

This work aimed to evaluate in vitro DNA binding mechanistically of cationic nitrosyl ruthenium complex [RuNOTSP]⁺ and its ligand (TSPH₂) in detail, correlate the findings with cleavage activity, and draw conclusions about the impact of the metal center. Theoretical studies were performed for [RuNOTSP]⁺, TSPH₂, and its anion TSP^-2 using DFT/B3LYP theory to calculate optimized energy, binding energy, and chemical reactivity. Since nearly all medications function by attaching to a particular protein or DNA, the in vitro calf thymus DNA (ctDNA) binding studies of [RuNOTSP]⁺ and TSPH₂ with ctDNA were examined mechanistically using a variety of biophysical techniques. Fluorescence experiments showed that both compounds effectively bind to ctDNA through intercalative/electrostatic interactions via the DNA helix's phosphate backbone. The intrinsic binding constants (K_b), (2.4 ± 0.2) × 10⁵ M^-1 ([RuNOTSP]⁺) and (1.9 ± 0.3) × 10⁵ M^-1 (TSPH₂), as well as the enhancement dynamic constants (K_D), (3.3 ± 0.3) × 10⁴ M^-1 ([RuNOTSP]⁺) and (2.6 ± 0.2) × 10⁴ M^-1 (TSPH₂), reveal that [RuNOTSP]⁺ has a greater binding propensity for DNA compared to TSPH₂. Stopped-flow investigations showed that both [RuNOTSP]⁺ and TSPH₂ bind through two reversible steps: a fast second-order binding, followed by a slow first-order isomerization reaction via a static quenching mechanism. For the first and second steps of [RuNOTSP]⁺ and TSPH₂, the detailed binding parameters were established. The total binding constants for [RuNOTSP]⁺ (K_a = 43.7 M^-1, K_d = 2.3 × 10^-2 M^-1, ΔG⁰ = -36.6 kJ mol^-1) and TSPH₂ (K_a = 15.1 M^-1, K_d = 66 × 10^-2 M, ΔG⁰ = -19 kJ mol^-1) revealed that the relative reactivity is approximately ([RuNOTSP]⁺)/(TSPH₂) = 3/1. The significantly negative ΔG⁰ values are consistent with a spontaneous binding reaction to both [RuNOTSP]⁺ and TSPH₂, with the former being very favorable. The findings showed that the Ru(II) center had an effect on the reaction rate but not on the mechanism and that the cationic [RuNOTSP]⁺ was a more highly effective DNA binder than the ligand TSPH₂ via strong electrostatic interaction with the phosphate end of DNA. Because of its higher DNA binding affinity, cationic [RuNOTSP]⁺ demonstrated higher cleavage efficiency towards the minor groove of pBR322 DNA via the hydrolytic pathway than TSPH₂, revealing the synergy effect of TSPH₂ in the form of the complex. Furthermore, the mode of interaction of both compounds with ctDNA has also been supported by molecular docking.