The biophysical characterization of recombinant glutathione transferase from Plasmodium berghei, a murine model of human malaria: Insights from empirical and computational methods.

Malaria remains a major global health challenge, exacerbated by the rise of drug-resistant Plasmodium species despite decades of eradication efforts. Glutathione S-transferase (GST), an enzyme that protects parasites from oxidative stress and mediates drug detoxification, has emerged as a promising target for novel antimalarial therapies. Natural and synthetic compounds, including ellagic acid (ELA) and bromosulfophthalein (BSP), have shown potential as GST inhibitors by disrupting parasite survival mechanisms. In this study, and for the first time, we biophysically characterize Plasmodium berghei GST (PbGST), a rodent model with physiological and life-cycle similarities to human-infective Plasmodium species. This thus provides a well-established nexus to an in vivo platform for investigating malaria pathogenesis and therapeutic interventions. PbGST was recombinantly overexpressed and purified to homogeneity, with Far-UV circular dichroism analysis revealing a predominantly alpha-helical structure. Fluorescence studies indicated that both ELA and BSP interact with PbGST's hydrophobic and catalytic sites, displacing 8-anilino-1-naphthalenesulfonate (ANS) probe and inducing conformational changes. Enzyme kinetics confirmed mixed-type inhibition, with BSP exhibiting stronger inhibition (IC50 = 1.89 μM) than ELA (IC50 = 7.05 μM). Size-exclusion HPLC revealed ligand-dependent oligomeric transition, with glutathione (GSH) stabilizing PbGST in the monomeric form. Thermal stability assays and computational studies further revealed that while GSH enhances PbGST stability, BSP destabilizes it, and ELA has minimal effect on its compactness. These findings establish PbGST as a valuable model for GST-targeted antimalarial strategies, providing insights into the inhibitory potential of ELA and BSP, as well as comprehensive structural and functional insights toward the design of next-generation antimalarials.