Bromosulfophthalein inhibition of 28-kDa Schistosoma japonicum glutathione transferase: Perspectives from experimental and molecular modelling studies
Date
2022
Authors
Pooe, Kagiso
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Abstract
Glutathione transferases are major detoxification enzymes vital for the survival and reproduction of schistosomes during infection of the human host. The parasite expresses two GST isoenzymes, the 26-kDa and 28-kDa SjGST, showing different substrate specificities and localisations inside the parasite’s tegument. Bromosulfophthalein (BSP), a diuretic compound with clinical history in liver diagnostics, has been identified and characterised as a potent Sj26GST inhibitor, showing promise as a lead molecule for the development of novel antischistosomal therapeutic drugs. This study characterises the functional, structural, and thermodynamic implications of BSP binding on Sj28GST; which has limited research attention. Enzyme kinetics show that BSP is a potent inhibitor of the enzyme, with a specific activity that decreases from 60.4 µmol min-1 mg-1 to 0.0742 µmol min-1 mg-1 and IC50 in the nanomolar range of 0.74 µM. Circular dichroism confirmed that purified Sj28GST follows a typical GST fold, predominantly alpha-helical. Extrinsic ANS (8-Anilino-1-naphthalene sulfonate) spectroscopy suggests that BSP binding occurs at a site distinct from the glutathione binding site (G-site). Isothermal titration calorimetry studies show that the binding of BSP to Sj28GST is exergonic ( ߡG = -34 kJ mol-1 and -31 kJ mol-1 ) and enthalpically-driven, with a reaction stoichiometry of 1 BSP molecule per enzyme dimer. Molecular docking studies and molecular dynamics simulations indicate that BSP shows preferential binding to a site at the enzyme’s dimeric interface, relative to the G-site and substrate binding site (H-site). The stability of Sj28GST (4.7 kcal mol-1 ) is notably lower than Sj26GST, owing to differences in the enzyme’s dimer interfaces. The findings of this study provide further insights into the binding and inhibitory behaviour of BSP on schistosome GSTs, as a potential candidate for the development of novel antischistosomal chemotherapeutics to attenuate the debilitating effects of Schistosomiasis.
Description
A dissertation submitted in fulfilment of the requirements for the degree of Master of Science to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2022