Discovering potential inhibitors for Plasmodium falciparum and vivax glutathione transferases through systematic integration of empirical with theoretical studies

Abstract

Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) are parasites that have progressively resisted existing antimalarial drugs by evading their current therapeutic mode of action. This has thereby necessitated research into novel therapeutics with a distinct and unique mode of action. The rapid development of resistance to prophylactic and interventional malaria drugs has undeniably been a thing of concern to researchers and healthcare practitioners. Glutathione S-Transferase (GST) as an adept enzyme has been brought to the limelight as a potential target for developing antimalarial drugs. This is connected to GST’s significant involvement in cellular processes leading to metabolizing or detoxifying carcinogens, chemotherapeutic drugs, environmental pollutants, and oxidative stress products. Hence, the accelerated effort to target GST as a potential drug target in Plasmodium falciparum and Plasmodium vivax, are parasites which had progressively resisted existing antimalarial drugs. Although, the artemisinin derivatives antimalarials known as Artemisinin Combination Therapy (ACT) are currently one of the World Health Organization (WHO) approved drugs against Pf and Pv. Artemisinin-based combination therapies are currently used as the frontline treatment for malaria, thanks to their rapid action and high efficacy against Plasmodium parasites. However, the growing problem of drug resistance necessitates the study of ACTs in the context of protein-ligand interactions to discover their activity towards the drug target under consideration. It’s also to gain new antimalarial strategies and enhance existing therapies. The seven selected ACTs used in this study were based on their reported efficacy and availability. Both Plasmodium falciparum and Plasmodium vivax possess a versatile enzyme which are respectively known as Plasmodium falciparum Glutathione S-Transferase (PfGST) and Plasmodium vivax Glutathione S-Transferase (PvGST). This class of enzyme exogenously and endogenously inactivates toxic molecules, metabolizing, and detoxifying carcinogens, and chemotherapeutic drugs. This has made these two enzymes catch the attention of many researchers for drug discovery. This is because the inhibition of PfGST and PvGST will reduce both Plasmodium falciparum's and Plasmodium vivax’s detoxification abilities and weakened their antioxidant ability. Hence, this makes these targets represent a potential target for developing antimalarial drugs. The biochemistry of the protective role (detoxification of xenobiotics) being exerted by PfGST and PvGST was thus explored as a potential target for antimalarial drug development. In this study, the interactions, binding, stability, and inhibition potentials by seven selected antimalarials, bromosulfophthalein (BSP) and natural products (flavonoids) are in this study insightfully considered as potential inhibitors. Computational studies novelly support all the biophysical characterizations employed in this study. Spectroscopic analysis of the selected antimalarial drugs for biotransformation by GSH conjugation of the targets revealed that all the antimalarials are GSH-glutathionylated and GST biotransformed. Expectedly, while the GSH-CDNB conjugation assay showed that the antimalarials weakly inhibited the enzymatic activities of PfGST and PvGST, the ANS extrinsic fluorescence spectrophotometric analysis also revealed the weak binding to the targets. The quantitative polymerase chain reaction (qPCR) thermal shift stability elucidation shows that the antimalarial drugs moderately cause thermal instability of the targets. The ligandin properties of PfGST and PvGST towards the steady-state kinetics and molecular modelling-induced fit docking all showed that BSP binds more strongly to PfGST and PvGST than the selected ACTs. Steady state kinetics evaluation and all the post-dynamic analysis from molecular dynamics simulations of PfGST and PvGST with BSP are in tandem in demonstrating BSP’s drug-likeness. All the spectroscopic and spectrofluorometric techniques showed that the selected antimalarials were less effective as an inhibitor compared to BSP. The various results of molecular dynamics simulations and other computational studies reveal that BSP impacts the compactness of PfGST and PvGST in a decreasing manner and decreases their conformational dynamics accordingly more than the selected antimalarials. On the other hand, the high-throughput virtual computational studies screening selected baicalin and 5,7,3'-Trihydroxy-6,4',5'-trimethoxyflavone to outperform BSP and ellagic acid in binding and a better inhibitor of PfGST and PvGST. This study insightfully elucidated and positioned BSP’s ability as a potential drug candidate to be further developed as an efficacious antimalarial and advanced a further study on two flavonoids, baicalin and 5,7,3'- Trihydroxy-6,4',5'- imethoxyflavone as a better inhibitor of PfGST and PvGST respectively.