Electrochemical quantification based on the interactions of nucleoside analog cladribine with dsDNA via experimental and in-silico studies

Cladribine is a deoxyadenosine analog prodrug originally developed to treat hairy-cell leukemia and other lymphoproliferative diseases. However, it is now primarily used in the treatment of relapsing types of multiple sclerosis (MS). Understanding how medications interact with dsDNA is crucial for developing more effective and efficient medications. This study aims to examine the binding behavior of cladribine with dsDNA via various analytical methods, such as heat denaturation, UV spectroscopy, fluorescence spectroscopy, electrochemistry, and viscosity tests. The binding constant (K_b) of cladribine with dsDNA has been estimated to be 2.41 × 10⁴ ± 0.20 at 298 K using the Benesi-Hildebrand plot. Molecular docking simulations were employed to explore the dsDNA-cladribine interactions quantitatively at the molecular level. Molecular Dynamic simulations were performed to follow the stability of drug-bound DNA for 50 ns. The simulations revealed that cladribine binds to dsDNA via the minor groove region of DNA by forming hydrogen bonds mainly with Guanine's DNA bases. The post-MD analyses enabled us to follow the stability of DNA and cladribine complex. Additionally, two methods based on the electrochemical approach were developed in this study for low-level cladribine assessment using differential pulse voltammetry (DPV). The first method relies on cladribine oxidation in pH 2 phosphate buffer, while the second method uses deoxyguanosine oxidation signals resulting from cladribine and dsDNA binding in pH 4.80 acetate buffer. The analytical efficacy of the two methods was verified using cladribine concentrations ranging from 2 to 25 μM, with a limit of detection (LOD) of 0.30 and 0.92 μM, respectively. Furthermore, the study conducted percent recovery tests by employing pharmaceutical injection using both established methodologies.