Chemistry Department, Faculty of Science, Menoufia University, Shebin El-Kom 32512, Egypt.
Clinical Pathology Department, University Hospital, Menoufia University, Shebin El-Kom 32512, Egypt.
Molecules. 2023 Mar 28;28(7):3028. doi: 10.3390/molecules28073028.
This work aimed to evaluate in vitro DNA binding mechanistically of cationic nitrosyl ruthenium complex [RuNOTSP] and its ligand (TSPH) 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, and its anion TSP 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 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), (2.4 ± 0.2) × 10 M ([RuNOTSP]) and (1.9 ± 0.3) × 10 M (TSPH), as well as the enhancement dynamic constants (K), (3.3 ± 0.3) × 10 M ([RuNOTSP]) and (2.6 ± 0.2) × 10 M (TSPH), reveal that [RuNOTSP] has a greater binding propensity for DNA compared to TSPH. Stopped-flow investigations showed that both [RuNOTSP] and TSPH 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, the detailed binding parameters were established. The total binding constants for [RuNOTSP] (K = 43.7 M, K = 2.3 × 10 M, ΔG = -36.6 kJ mol) and TSPH (K = 15.1 M, K = 66 × 10 M, ΔG = -19 kJ mol) revealed that the relative reactivity is approximately ([RuNOTSP])/(TSPH) = 3/1. The significantly negative ΔG values are consistent with a spontaneous binding reaction to both [RuNOTSP] and TSPH, 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 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, revealing the synergy effect of TSPH in the form of the complex. Furthermore, the mode of interaction of both compounds with ctDNA has also been supported by molecular docking.
本工作旨在详细评估阳离子亚硝酰钌配合物[RuNOTSP]及其配体（TSPH）的体外 DNA 结合机制，将研究结果与切割活性相关联，并得出关于金属中心影响的结论。使用 DFT/B3LYP 理论对[RuNOTSP]、TSPH 及其阴离子 TSP 进行理论研究，以计算优化的能量、结合能和化学反应性。由于几乎所有药物都是通过附着在特定的蛋白质或 DNA 上来发挥作用的，因此通过使用多种生物物理技术，研究了[RuNOTSP]和 TSPH 与 ctDNA 的体外小牛胸腺 DNA（ctDNA）结合机制。荧光实验表明，这两种化合物都通过与 DNA 螺旋的磷酸骨架之间的静电相互作用有效地通过嵌入/静电相互作用结合到 ctDNA 上。内在结合常数（K），（2.4 ± 0.2）×10^M（[RuNOTSP]）和（1.9 ± 0.3）×10^M（TSPH），以及增强的动态常数（K），（3.3 ± 0.3）×10^M（[RuNOTSP]）和（2.6 ± 0.2）×10^M（TSPH），表明[RuNOTSP]与 DNA 的结合倾向大于 TSPH。停流研究表明，[RuNOTSP]和 TSPH 都通过两个可逆步骤结合：快速的二级结合，然后通过静态猝灭机制缓慢进行一级异构化反应。对于[RuNOTSP]和 TSPH 的第一和第二步，建立了详细的结合参数。[RuNOTSP]（K=43.7 M，K=2.3×10^M，ΔG=-36.6 kJ mol）和 TSPH（K=15.1 M，K=66×10^M，ΔG=-19 kJ mol）的总结合常数表明，相对反应性约为（[RuNOTSP]）/（TSPH）=3/1。显著的负ΔG 值表明，[RuNOTSP]和 TSPH 的结合反应都是自发的，前者非常有利。研究结果表明，Ru(II)中心对反应速率有影响，但对机制没有影响，并且阳离子[RuNOTSP]通过与 DNA 的磷酸末端的强静电相互作用，比配体 TSPH 更有效地结合 DNA。由于其更高的 DNA 结合亲和力，阳离子[RuNOTSP]通过水解途径对 pBR322 DNA 的小沟表现出比 TSPH 更高的切割效率，显示出 TSPH 以复合物形式的协同效应。此外，两种化合物与 ctDNA 的相互作用模式也得到了分子对接的支持。
