Browsing by Author "Oyiogu, Blessing Oluebube"

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Biophysical evaluation of the kinetics, thermodynamics, and structure-stability relationship of Wuchereria bancrofti glutathione transferase in comparison with human µ and π glutathione transferases
(University of the Witwatersrand, Johannesburg, 2024-06) Oyiogu, Blessing Oluebube; Achilonu, Ikechukwu Anthony
Lymphatic filariasis is an endemic disease caused mainly by the Wuchereria bancrofti parasite and has been classified as a major neglected tropical disease. The emergence of drug-resistant strains of W. bancrofti and the limited efficacy of the available drugs on adult worms threatens the eradication of the disease. W. bancrofti glutathione S-transferase (WbGST) is a homodimeric enzyme central to detoxifying electrophilic compounds in the parasite due to its lack of cytochrome P-450. Therefore, WbGST is a potential therapeutic target for lymphatic filariasis. Bromosulphophthalein (BSP) and epigallocatechin gallate (EGCG) were previously shown to inhibit glutathione S-transferase activity. In this study, the interaction of WbGST with BSP and EGCG in comparison with human glutathione S-transferase P1-1 (hGSTP1-1) and human glutathione S-transferase M1-1 (hGSTM1-1) isoforms was investigated. Soluble WbGST, hGSTP1-1 and hGSTM1-1 were recombinantly produced and purified successfully to homogeneity. Glutathione and 1-chloro-2,4-dinitrobenzene conjugation assay was employed to analyse the enzyme activity, kinetics and inhibitory potency of the compounds. Spectroscopic studies were employed to investigate the functional and structural impact of ligand binding to the enzymes. Both thermal and chemical stability studies were performed, and binding energetics were analysed using isothermal titration calorimetry. The activity of WbGST was predominantly inhibited, with IC50 values of 5 μM for BSP and 12 μM for EGCG. The EGCG displayed uncompetitive and mixed modes of inhibition towards WbGST with respect to glutathione and hydrophobic binding sites, respectively. Whereas BSP showed a mixed type of inhibition for both active sites of WbGST. Ligands reduced the turnover rates (kcat) and the catalytic efficiencies (kcat/KM) of the enzymes. Upon ligand binding, 8-anilino-1-napthalene sulphonate was displaced from WbGST and hGSTM1-1 by 67%(BSP), 24%(EGCG) and 72%(BSP), 5%(EGCG), respectively; suggesting that the ligands bind to the 8-anilino-1-napthalene sulphonate binding site. Stability studies indicate that WbGST is the least stable of the three enzymes and that glutathione increases its stability. Isothermal titration calorimetry showed that BSP binds to multiple sites in WbGST with binding at site-1 (S1) and site-2 (S2), which are entropically and enthalpically driven, respectively. S1 showed a higher affinity for BSP than S2. EGCG binding to WbGST was entropically driven. BSP had a higher affinity for the enzymes than EGCG. All the results indicated that the ligands significantly impact WbGST more than the human GSTs. Further investigations, such as crystallography and molecular dynamics simulations, will shed more light on the ligan-protein interactions on a molecular level. Overall, this study suggests that BSP and EGCG are efficient inhibitors of WbGST that probably bind to both L and H-sites of WbGST, altering catalytic activity of the enzyme. The unique properties of the L-site are particularly suitable for rational drug design. Therefore, both ligands can be repurposed as new-generation therapeutics against filariasis.
Ellagic acid-mediated inhibition of schistosoma japanicum glutathione transferase: enzyme kinetics and structural perspectives
(2018) Oyiogu, Blessing Oluebube
Schistosoma japonicum glutathione S-transferase (SjGST) is a homodimeric enzyme central to detoxification of electrophilic compounds in the parasite due to its lack of cytochrome P-450. Hence, SjGST is a potential therapeutic target for schistosomiasis. Akin to most cytosolic GSTs, SjGST is composed of two active sites per monomer, which are the glutathione binding site (G-site) and the hydrophobic binding site (H-site). Ellagic acid (EA), a polyphenolic compound ubiquitous in fruits and other plant-based products has been identified as a therapeutic agent against cancer, heart and liver diseases. The interaction between SjGST and EA was characterised in this study. Using 1-chloro-2,4 dinitrobenzene (CDNB) and glutathione (GSH) as SjGST substrates, EA was shown to inhibit SjGST activity, with an IC50 of 2.4 μM. The observed mode of inhibition with respect to the G-site and H-site was non-competitive and uncompetitive, respectively. The turnover rate of SjGST was reduced from 0.002 ± 0.0001 sec1 to 0.0006 ± 0.00001 sec-1 in the presence of 9 μM EA, while the catalytic efficiency (kcat/KM) was reduced from 0.02 ± 0.0002 M-1s-1 to 0.009 ± 0.0001 M-1s-1. Using ANS as an extrinsic fluorescence probe, SjGST bound to ANS in a concentration-dependent manner in the absence (Kd = 40.6 ± 2.6 μM) or presence of EA (Kd = 30.3 ± 1.4 μM). This observation was further probed by the displacement of ANS by EA, which suggest that about 64.5% of ANS was displaced by EA. The ANS studies indicated that EA binds to the H-site of the enzyme. Secondary and quaternary structure analyses indicate that EA does not alter the secondary and tertiary structures of SjGST. Thermodynamic parameters obtained by isothermal titration calorimetry (ITC) indicate that the interaction between SjGST and EA is enthalpically-driven and compensated by a negative entropy change (-T∆S = 20.40 ± 0.08 kJ/mol) because of enthalpy-entropy compensation. The free energy change (∆G = -29.88 ± 0.07 kJ/mol) is also favoured by entropic contributions; therefore, indicating a strong hydrophobic interaction. A stoichiometry of 4 molecule of EA per mole of the dimeric enzyme was obtained. Molecular modelling studies suggests that EA binds more to the dimer interface compared with the Hsite. Therefore, it is possible that EA may bind to two different sites in the enzyme, the H-site and the dimer interface.