Aspergillus flavus glutathione transferase as a potential approach for synthesis of epothilone-glutathione conjugates with enhanced anticancer activity for the drug-resistant cells: characterization and molecular docking analysis

Abstract Epothilone is one of the lead anticancer compounds, with unique binding affinity to the cellular β-tubulin, however, the solubility and efficiency towards the drug-resistant tumors are the challenges. Glutathionylation has been recently used as a sophisticated tool to enhance the solubility and efficiency of the bioactive compounds. Consequently, the purpose of this study was to purify and assess the potency glutathione transferase as one-pot approach for glutathione-epothilone complex synthesis. Glutathione transferase (GT) was purified from Aspergillus flavus EFBL-SKH (PV263561) to its homogeneity by ion exchange, and affinity chromatography, with an overall maximum activity 72.22 μmol/mg/min, at 37–42 °C, pH 7.0–7.5, with 28 kDa under SDS-PAGE. The highest affinity (K m ) of A. flavus GT was reported for CDNB (0.11 mM), Epothilone (0.31 mM) and MBHC (0.37 mM) as co-substrates with glutathione. The utmost catalytic efficiency (K cat /K m ) were reported for CDNB (13.4 mM−1 s−1), MBHC (2.25 mM−1 s-1) and Epothilone (2.39 mM−1 s−1). The GT mediated reaction of glutathione—epothilone conjugate was purified and checked by LC/MS, with 823.9 m/z. The glutathione-epothilone complex has an elevated molecular flexibility, higher solubility via the stronger interactions with aqueous environments and biological targets, with moderate lipophilicity. From the ESOL Log S values, the solubility of epothilone was increased by 100 folds via glutathione conjugation. From the SwissADME, the accessibility of glutathione-epothilone conjugate was completely diminished as a P-glycoprotein substrate, unlike to the higher feasibility of epothilone, ensuring the molecular structural change of the compound by Glutathionylation. The docking simulation gave a binding affinity -8.0 kcal/ mol for epothilone with the GT binding pocket. The chemical conjugation of glutathione-Epothilone was confirmed from the XRD analysis. The energy of binding of glutathione-epothilone B complex and epothilone with β-tubulin was -7.7–8.0 kcal/mol. The anticancer activity of the glutathione-Epothilone conjugate towards the HepG-2 was 1.9 and 1.45 μM, at molar ratios 1:2 and 1:4 M/M, respectively, by 2.5 higher activity folds, compared to the Epothilone individually. The selectivity indices of GSH-EPT complex was 5.8–6.2 folds, compared to the EPT (2.1- 2.6 folds), to HepG-2 over the normal cells.