The purpose of this study was to extensively explore in vitro protein binding to [Ru (NO)(Et2NpyS4)]Br (RuNOTSP) and its ligand (TSP), correlate the findings with antibacterial and cytotoxic effects, and try to answer the question: Is the metal center always important for binding? With the aid of stopped‐flow and other spectroscopic and computational methods, the interaction between RuNOTSP and TSP and bovine serum albumin (BSA) was studied and a mechanism was proposed. From UV–Vis and fluorescence experiments, a static quenching mechanism and binding constants at a single site demonstrated the higher stability of the RuNOTSP‐BSA system at 298 K compared with TSP‐BSA. Stopped‐flow experiments indicated that binding of RuNOTSP and TSP to BSA is accomplished mainly via a two‐step mechanism: a fast second‐order binding followed by a slow first‐order isomerization process. The first reaction step is reversible with coordinate affinity Ka1 190 (RuNOTSP) and 50 M−1 (TSP). The second step for RuNOTSP is a reversible reaction with Ka2 of 76.4 M−1, whereas an irreversible step that was observed with a K2 of 0.18 M−1 for TSP indicates that TSP‐BSA is kinetically more stable than RuNOTSP‐BSA. The results revealed that the ruthenium center not only improves reaction rate through coordination affinity but also changes the binding process. Mode and strength of interaction of RuNOTSP and TSP with protein have also been supported by molecular docking and showed that RuNOTSP is located in IA pocket (Trp134) with a binding affinity (−7.27 kcal/mol), a slightly lower than TSP (−8.05 kcal/mol), and these results are in agreement with the binding constants. Furthermore, antibacterial testing revealed that RuNOTSP and TSP had high antibacterial activity against both Gram‐positive and Gram‐negative bacteria. They are also highly cytotoxic to HepG2 human liver cancer cells. Because the TSP‐BSA complex is kinetically more stable than that of RuNOTSP, the former illustrated better antibacterial and cytotoxic activities.

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 (Kb), (2.4 ± 0.2) × 105 M−1 ([RuNOTSP]+) and (1.9 ± 0.3) × 105 M−1 (TSPH2), as well as the enhancement dynamic constants (KD), (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]+ (Ka = 43.7 M−1, Kd = 2.3 × 10−2 M−1, ΔG0 = −36.6 kJ mol−1) and TSPH2 (Ka = 15.1 M−1, Kd = 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.

The current study describes the preparation of chitosan nanoparticles (CNPs) using hydroxychloroquine (HCQ), widely used in traditional medicine due to its diverse phar-macological and medicinal uses. This work aims to combine the HCQ drug with CS NPs to generate a novel nanocomposite with improved characteristics and bioavailability. HCQ@CS NPs is roughly shaped like roadways and has a smooth surface with an average size of 159.3±7.1 nm, a PdI of 0.224±0.101, and a zeta potential of +46.6±0.8 mV. To aid in the development of pharmaceutical systems for use in cancer therapy, the binding mech-anism and affinity of the interaction between HCQ and HCQ@CS NPs and BSA were ex-amined using stopped-flow, other spectroscopic approaches, supplemented by molecular docking analysis. HCQ and HCQ@CS NPs binding with BSA is driven by a ground-state complex formation that may be accompanied by a non-radiative energy transfer process, and binding constants indicated that HCQ@CS NPs-BSA was more stable than HCQ-BSA. The stopped-flow analysis demonstrated that, in addition to increasing BSA affinity, the nano formulation HCQ@CS NPS changes the binding process and may open up new routes for interaction. Docking experiments verified the development of the HCQ-BSA complex, with HCQ binding to the site I on the BSA structure, primarily with the amino acids Thr 578, Gln 579, Gln 525, Tyr 400, and Asn 404. Furthermore, the nano-formulation HCQ@CS NPS not only increased cytotoxicity against the A549 lung cancer cell line (IC50 = 28.57±1.72 g/ml) compared to HCQ (102.21±0.67) g/ml), but also exhibited higher anti-bacterial activity against both Gram-positive and Gram-negative bacteria when compared to HCQ and chloramphenicol which in agreement with the binding constants. The nano formulation developed in this study may offer a viable therapy option for A549 lung cancer.