DNA Binding and Cleavage, Stopped-Flow Kinetic, Mechanistic, and Molecular Docking Studies of Cationic Ruthenium(II) Nitrosyl Complexes Containing “NS 4 ” Core

Highlights Theoretical studies were performed on [RuNOTSP] + , TSPH 2 , and its anion TSP 2− using the DFT/B3LYP method. Cationic complex [RuNOTSP] + and TSPH 2 were investigated mechanistically for ctDNA interaction. Spontaneous ctDNA binding via a static mechanism with two steps was reported. Detailed kinetic data are reported and relative reactivity is [RuNOTSP] + /TSPH 2 = 3/1. The ruthenium effect on affinity and mechanism is reported. The ruthenium center improves the reaction rate through coordination affinity, but does not change its mechanism. Molecular docking was used to predict the binding between [RuNOTSP] + and TSPH 2 and the receptors. DNA cleavage studies are correlated with kinetic data. Abstract This work aimed to evaluate in vitro DNA binding mechanistically of cationic nitrosyl ruthenium complex [RuNOTSP] + and its ligand (TSPH 2 ) in detail, correlate the findings with cleavage activity, and draw conclusions about the impact of the metal center. Theoretical studies were performed for [RuNOTSP] + , TSPH 2 , 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 TSPH 2 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) × 10 5 M −1 ([RuNOTSP] + ) and (1.9 ± 0.3) × 10 5 M −1 (TSPH 2 ), as well as the enhancement dynamic constants (K D ), (3.3 ± 0.3) × 10 4 M −1 ([RuNOTSP] + ) and (2.6 ± 0.2) × 10 4 M −1 (TSPH 2 ), reveal that [RuNOTSP] + has a greater binding propensity for DNA compared to TSPH 2 . Stopped-flow investigations showed that both [RuNOTSP] + and TSPH 2 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 TSPH 2 , 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 , ΔG 0 = −36.6 kJ mol −1 ) and TSPH 2 (K a = 15.1 M −1 , K d = 66 × 10 −2 M, ΔG 0 = −19 kJ mol −1 ) revealed that the relative reactivity is approximately ([RuNOTSP] + )/(TSPH 2 ) = 3/1. The significantly negative ΔG 0 values are consistent with a spontaneous binding reaction to both [RuNOTSP] + and TSPH 2 , 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 TSPH 2 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 TSPH 2 , revealing the synergy effect of TSPH 2 in the form of the complex. Furthermore, the mode of interaction of both compounds with ctDNA has also been supported by molecular docking.