Molecular Cloning, Expression, And Characterization Of Glutathione-S-Transferase As A Novel Target In Anti-Malarial Drug Design And Discovery
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Date
2015-03
Authors
MAHMOD AL QATTAN, MOHAMMED NOORALDEEN
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Abstract
The Glutathione-S-transferases (GSTs) are group of detoxification enzymes. Plasmodium falciparum has a single isoform of GST (PfGST) that involves in heme detoxification. While other GSTs isoforms from human (hGSTP1) and mouse (mGSTM1) are involved in apoptotic stress kinase pathway and mediate cancer cell resistance to chemotherapy. The PfGST, hGSTP1 and mGSTM1 were successfully cloned and heterologously expressed in E. coli. Enzyme inhibition, kinetics and crystallization experiments were conducted to find potential lead compounds that inhibit PfGST. The GSTs substrates, glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB), as well as the known GSTs inhibitors of S-hexyl glutathione (GSX), cibacron blue (CB), ethacrynic acid (EA), hemin, protoporphyrin IX (protoIX) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were studied. The results revealed affinities, binding modes and possible interactions between the inhibitors and PfGST. Two binding sites were proposed for hemin in PfGST. However, CB, GSX, EA and HEPES were unable to compete with hemin binding, thus considered unsuitable leads to dislodge hemin from its binding site. Due to its high affinity and possible interaction with hemin, the crystal structure of CB in complex with PfGST was studied further using X-ray crystallography. As an alternative approach to obtain leads, molecules were computationally designed for targeting GSH binding site in PfGST and destabilize hemin binding. Leads based on GSH molecule
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and/or its binding pocket were searched using de novo approach. Three approaches were developed for reversible inhibitors using fragment-based and atom-based approaches, and one approach for irreversible inhibitors design. The isosteric-fragment replacement (IFR) and docked-fragment replacement (DFR) approaches successfully generated de novo ligands with free energy of binding and synthetic accessibility score were calculated for lead selection. Molecular dynamic simulation for selected IFR and DFR ligands showed that 1598-DFR maintain stable binding with free energy of binding of -16 kcal/mol and RMSD of less than 3 Å throughout simulation period of 7.5 ns. The third approach generated lead molecules by atomic assembly using Structure-Assisted Atom-based De novo molecular design (SAAD) which tailor made the molecular fragments to fit the binding pocket of γ-glutamyl moiety of GSH. In the last approach, irreversible specific inhibitors were designed to form covalent bond with PfGST by using its unique 3-dimensional arrangement of cysteine residues. The analogues were designed to establish covalent bond with Cys101 through electrophilic moieties that replaces α-amino of the γ-glutamyl of GSH. The designed lead molecules may open a new avenue for treating malaria by reversible and irreversible inhibition of PfGST. Plant extracts were screened as an alternative source for lead compounds. The highest PfGST inhibition was obtained using Cinnamomum iners bark, Terminalia catappa leaves and Phyllanthus watsonii leaves. The kinetic results suggest that these plants inhibited PfGST via competing with CDNB.
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Molecular Cloning, Expression, And Characterization Of Glutathione-S-Transferase As A Novel Target , In Anti-Malarial Drug Design And Discovery