Faculty of Science (ETDs)

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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.
Effects of Mg2+, Ni2+ and Ca2+ on ATP binding kinetics of nicotinate nucleotide adenylyltransferase from Klebsiella pneumoniae and Enterococcus faecium: insights from empirical and computational studies
(University of the Witwatersrand, Johannesburg, 2023-07) Van Deventer, Ruan; Achilonu, Ikechukwu Anthony
NNAT is an attractive target for drug development due to its crucial role in NAD+ synthesis. However, its characterisation in ESKAPE species, such as Klebsiella pneumoniae and Enterococcus faecium, remains limited. This study aimed to elucidate the binding mechanism of ATP, a pivotal substrate, to these NNAT species, focusing on the role of divalent metal ion cofactors. KpNNAT and EfNNAT enzymes were overexpressed and purified, yielding approximately 2 mg/ml for both. Various techniques were employed to investigate their properties, including far-UV CD, extrinsic ANS fluorescence, stopped-flow kinetic analyses, and MD simulations. The results revealed that KpNNAT could bind ATP independently of divalent metal ions, but catalytic activity required the presence of Mg2+. The kinetic analysis showed ka values of 5.99 μM-1 .sec-1 without divalent metal ions and 5.72 μM-1 .sec-1 in the presence of Mg2+. The "pseudo"-specific activity values were 0.005 μmol/min/mg without divalent metal ions and 0.374 μmol/min/mg in the presence of Mg2+. Conversely, recombinant EfNNAT exhibited limited ATP association, and the reasons for this remain unclear. Overall, this study shed light on the structural dynamics and functional kinetics of ATP association in both NNAT species. The findings contribute to our understanding of this druggable target and provide insights into the inactivity of EfNNAT, which warrants further investigation.
In Silico Exploration of Endocannabinoid Receptor–CB1 and CB2–Interactions Comparing Cannabidiol and Cannabidiol Diacetate: A Comprehensive Computational Study
(University of the Witwatersrand, Johannesburg, 2024-09) Soobben, Marushka; Achilonu, Ikechukwu Anthony; Sayed, Yasien
In the rapidly evolving field of cannabinoid research, acetylated phytocannabinoids such as cannabidiol diacetate (CBDDA) have shown prominence due to its enhanced effects compared to its natural counterpart, cannabidiol (CBD). Despite the growing popularity in the consumption of acetylated phytocannabinoids, in-depth research on its pharmacological impact, especially on CB1 and CB2 receptors, remains scarce. With rising reports of adverse reactions to acetylated phytocannabinoids, a molecular understanding of their interaction with endocannabinoid receptors (CBRs) is imperative. This study aimed to fill this knowledge gap by analysing receptor interactions of CBDDA in comparison with receptor interactions of CBD. The study showed that CBDDA forms stronger interactions with CBRs than CBD. Recognised for its heightened potency, the potential of CBDDA as a biopharmaceutical product was examined. CBR interactions with known endocannabinoids, agonists and inverse agonists validated the computational models used to determine the difference in conformational dynamics upon ligand binding. In this work, bioinformatics, molecular docking, and molecular dynamics (MD) simulations were used to determine the structural differences of CBRs when bound to CBD/CBDDA. Simulations in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and water environment successfully mimicked physiological conditions. Subsequent high-throughput virtual screening (HTVS) was conducted using CBDDA as a reference where ligands 142730975 and 21568811 were identified as the top scoring hits for CB1 and CB2 receptors, respectively. The identification of these ligands via HTVS highlights the therapeutic potential of targeting CBRs and the biopharmaceutical potential of CBDDA. This study elucidates the specific interactions of CBD and CBDDA with CB1 and CB2 receptors, laying a foundation for assessing the safety and efficacy of acetylated phytocannabinoids. Overall, the differential interaction of CBDDA compared to CBD with CBRs suggests that acetylation changes the conformational dynamics of CBRs thereby potentially affecting signalling. The identification of ligands 142730975 and 21568811 as strong interactors with the receptors may provide valuable leads for the development of new cannabinoid-based therapies.
Structure-activity relationship between Klebsiella pneumoniae β- Lactamase CTX-M-15 and selected β-lactam antibiotics: Evaluating the binding site promiscuity
(University of the Witwatersrand, Johannesburg, 2023-08) Esau, Veruschka Chloe-Zaan; Sayed, Yasien; Achilonu, Ikechukwu Anthony
Background: Nosocomial infections have become a major concern in Sub-Saharan Africa. Bacteria predominantly cause these infections due to resistance development attributed to lack of novel therapeutics. These resistant bacteria are classed as ESKAPE pathogens that gained resistance to most known antimicrobials; one such is the Gram-negative Klebsiella pneumoniae. K. pneumoniae developed resistant strategies against most known β-lactam antibiotics through the development of the enzyme, β-lactamase. Beta-lactamases hydrolyse the β-lactam ring of β-lactam antibiotics rendering them ineffective towards K. pneumoniae. This research aimed to investigate the structural and functional characteristics of K. pneumoniae β-lactamase and asses the conformational stability with penicillin and cefoperazone (CPZ). Methods: Overexpression of recombinant wild-type and mutant-(S70A) K. pneumoniae β-lactamase in E. coli T7 cells using a pET-28a vector and protein purification using Immobilised Metal Affinity Chromatography (IMAC), and enzyme activity assessed with nitrocefin. Secondary, tertiary, and quaternary structure studies were conducted with Far-UV CD, tryptophan fluorescence, ANS fluorescence and size exclusion HPLC respectively. The thermal stability and binding mechanisms were assessed with thermal shift assay and isothermal titration calorimetry (ITC). Results: The wild-type and mutant-(S70A) K. pneumoniae β-lactamase was successfully expressed and purified. S70A-KpBlac-1 showed no activity towards nitrocefin, and WT-KpBlac-1 was highly activity towards nitrocefin. The native structures were determined to be alpha-helical, but alpha-helical content is lost upon penicillin and CPZ binding to unorder and β-stranded conformations. The binding site was determined to be solvent exposed with one hydrophobic active site. The proteins were monomeric. CPZ induce thermal stability on S70A-KpBlac-1, whereas as penicillin binding had no effect on the thermal stability. Penicillin binding to S70A-KpBlac-1 was endothermic and the protein had low binding affinity for penicillin. CPZ binding was exothermic, and the protein had higher binding affinity for the substrate. Conclusion: Potential novel inhibitor design should be focused on CPZ. Through substrate-based drug discovery, potential drugs should confer a similar shape, size, or stereochemistry as CPZ. This would change the conformation of the protein, be tightly bound to the active site, and lower inhibitor concentration would be required. Therefore, these findings contribute, and provide insights on potential novel inhibitors against highly antibiotic resistant bacteria.
The ligandin activity of Schistosoma 26-kDa and 28-kDa glutathione transferases towards 17β-Hydroxyandrost-4-ene-3-one from a biophysical perspective
(University of the Witwatersrand, Johannesburg, 2023) Makumbe, Hattie Hope; Achilonu, Ikechukwu Anthony
Schistosomiasis, caused by helminth worms, ranks second amongst parasitic diseases and accounts for over 220 million fatalities globally. Statistics show that in South Africa, schistosomiasis (bilharzia) has infected approximately 4 million individuals. Currently, there are parasite resistance challenges with the sole available remedy. The World Health Organisation (WHO) acknowledges the need for new effective drugs. The 26-kDa Schistosoma bovis/haematobium (Sbh26GST) and 28-kDa Schistosoma haematobium (Sh28GST) are parasite Glutathione S-transferases (GSTs) which consist of two identical subunits that perform a vital role in mitigating the adverse effects of harmful electrophilic substances within the parasite since the parasite is devoid of the neutralizing cytochrome P-450. This automatically renders these parasite GSTs as potential therapeutic targets for schistosomiasis. Testosterone, the major hormone responsible for sexual characteristics and growth in males, can be repurposed as a drug target against schistosomiasis. In this study, we examined the structural, stability and functional interactions between the parasite GSTs and testosterone. After confirmation of inhibition, IC50 experiments were performed. The enzymes were overexpressed in Escherichia coli (E.coli) and then purified through a single-step nickel ion-immobilized metal affinity chromatography (IMAC). Extrinsic fluorescence spectroscopy was also done to provide evidence for the binding of the recombinant GSTs with testosterone. The GST activity was measured by employing 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate. Additionally, we investigated if the enzyme activity was influenced by the presence of testosterone. To analyse the stability of the enzymes, a SYPRO Orange-based thermal shift assay was used in the presence and absence of testosterone. In addition to empirical investigations, computational modelling, molecular docking, and molecular dynamic simulations were used to provide complementary insights to show binding affinities, prediction of binding modes and stability of the GST-testosterone complex. The secondary structural composition was found to be predominantly alpha-helical. Insights into tertiary structure analysis revealed the presence of buried solvent exposed tryptophan residues. The findings from spectroscopy with 8-anilino-1-naphthalenesulfonate (ANS) indicated that both Human GST-mu and parasite GSTs bound to ANS. Enzyme kinetic studies show that testosterone is a potent inhibitor of the parasite GSTs, with a specific activity that decreases from 16 μmol min-1mg-1 to 0.03 μmol min-1mg-1 and IC50 in the nanomolar range of 20 µM for Sh28GST. Sbh26GST exhibited a specific activity that decreased from 20 μmol min-1mg-1 to 0.14 μmol min-1mg-1, and a testosterone IC50 of 23 µM. The thermal stability assay confirmed Sh28GST to be more stable than Sbh26GST, and this stability of Sh28GST intensified when the enzyme bound to testosterone and GSH. Steady state kinetics towards glutathione (GSH) revealed a Km of 4.2mM and 6.6 mM for Sh28GST and Sbh26GST respectively. The present study has practical implications for novel application of the enzymes to serve as a basis for future studies aimed at development of inhibitors with potential therapeutic benefits through rational drug design.