Engineering a Pseudo-26-kDa Schistosoma Glutathione Transferase from bovis/haematobium for Structure, Kinetics, and Ligandin Studies

Abstract

Glutathione transferases (GSTs) are the main detoxification enzymes in schistosomes. These parasitic enzymes tend to be upregulated during drug treatment, with Schistosoma haematobium being one of the species that mainly affect humans. There is a lack of complete sequence information on the closely related bovis and haematobium 26-kDa GST isoforms in any database. Consequently, we engineered a pseudo-26-kDa S. bovis/haematobium GST (Sbh26GST) to understand structure–function relations and ligandin activity towards selected potential ligands. Sbh26GST was overexpressed in Escherichia coli as an MBP-fusion protein, purified to homogeneity and catalyzed 1-chloro-2,4-dinitrobenzene-glutathione (CDNB-GSH) conjugation activity, with a specific activity of 13 μmol/min/mg. This activity decreased by ~95% in the presence of bromosulfophthalein (BSP), which showed an IC50 of 27 µM. Additionally, enzyme kinetics revealed that BSP acts as a non-competitive inhibitor relative to GSH. Spectroscopic studies affirmed that Sbh26GST adopts the canonical GST structure, which is predominantly α-helical. Further extrinsic 8-anilino-1-naphthalenesulfonate (ANS) spectroscopy illustrated that BSP, praziquantel (PZQ), and artemisinin (ART) might preferentially bind at the dimer interface or in proximity to the hydrophobic substrate-binding site of the enzyme. The Sbh26GST-BSP interaction is both enthalpically and entropically driven, with a stoichiometry of one BSP molecule per Sbh26GST dimer. Enzyme stability appeared enhanced in the presence of BSP and GSH. Induced fit ligand docking affirmed the spectroscopic, thermodynamic, and molecular modelling results. In conclusion, BSP is a potent inhibitor of Sbh26GST and could potentially be rationalized as a treatment for schistosomiasis.

Biochemical characterization and peptide mass fingerprinting of two glutathione transferases from Biomphalaria alexandrina snails (Gastropoda: Planorbidae)

“…Binding of GSH to the active site increases the interactions with the opposing subunit to 15 interactions, while only 8 interactions are found in the apoenzyme [ 42 ]. A similar result was reported for the increase in GST thermal stability in the presence of BSP and GSH [ 40 ].…”

“…The low-affinity-binding site(s) for BSP can simultaneously accommodates both BSP and the relatively small size electrophilic substrate, CDNB, and inhibits the enzyme non-competitively [ 39 ]. BSP also inhibits a 26-kDa Schistosoma GST from bovis/haematobium in a noncompetitive manner [ 40 ]. In the present investigation, BSP was found to be a competitive inhibitor for BaGST2 and mixed inhibitor for BaGST3.…”

Ligand based-design of potential schistosomiasis inhibitors through QSAR, homology modeling, molecular dynamics, pharmacokinetics, and DFT studies

“…S. mansoni GSTs (SmGST) bind and metabolize a diverse array of xenobiotic substances, including drugs, environmental toxins, and plant secondary metabolites. 16 , 17 , 18 Notably, SmGST has limited similarity to human GSTs, thus potentially enabling the development of selective inhibitors that target the parasite while preserving human GST enzymes, and enhancing drug safety. Numerous studies have emphasized the importance of phase I detoxification, and the central roles of glutathione-mediated detoxification and redox metabolism in the parasite.…”

Comparative structural analysis of the human and Schistosoma glutathione transferase dimer interface using selective binding of bromosulfophthalein

“…25,27,31,32 Several compounds have since demonstrated schistosome GST binding, causing varying degrees of enzyme inhibition. Among them are the liver test dye reagent bromosulfophthalein (BSP), 32,33 indanyloxyacetic acid, 32 artemether, 34 the polyphenol ellagic acid, 25 as well as anthraquinone dyes. 35 Much like praziquantel, 22 molecular modeling studies have confirmed the association of these species with non-substrate sites in the binding channel of the enzyme.…”