Molecular Cloning, Expression, And Characterization Of Glutathione-Stransferase As A Novel Target In Antimalarial Drug Design And Discovery

Glutation-S-transferase (GSTs) adalah sekumpulan enzim detoksifikasi. Plasmodium falciparum mempunyai isoform tunggal GST (PfGST) yang terlibat dalam bagi detoksifikasi heme. 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

Structural and Functional Characterisation of Glutathione-S-Transferases to Combat Multiple Herbicide Resistance in Black Grass (Alopecurus myosuroides)

Black grass (Alopecurus myosuroides) is one of the most common weeds in Western Europe. With the increasing use of herbicides, in response to pressure to produce higher crop yields, incidences of multiple herbicide resistance have been widely reported. Previous work in this area has identified a phi class glutathione-s-transferase, AmGSTF1, as playing a causative role in this multiple herbicide resistance. Two classes of inhibitor have previously been identified: the first CNBF, a multi drug resistance drug; and the second a group of flavone compounds identified from ligand fishing experiments. These have been shown to have activity in vitro inhibiting the action of AmGSTF1 and in vivo against multiple herbicide resistant black grass. However, little was known about their mode of action. This work has focussed on investigating the interactions between these inhibitors and AmGSTF1 using crystallographic, biochemical and complementary biophysical techniques. Apo AmGSTF1 has successfully been crystallised, with the structure solved to 1.5 Å. In addition a structure has been solved to 2.0 Å with CNBF covalently modifying the Cys120 residue. For both these structures the crystal packing results in loops in the active site region being disordered as well as preventing small molecule binding within the active site. In order to determine the complete structure, a series of mutants were designed to alter crystal packing. The structure of these were determined, and they adopt a different packing arrangement which results in the previously disordered loops being ordered, as well as exposing the binding site. The structure of the F122T mutant was used for in silico modelling to determine the likely binding site for flavonoid ligands. The mutants were subsequently used for seeding and soaking experiments which allowed for a complete structure of wild type AmGSTF1 to be determined in complex with a glutathione conjugate of CNBF in the active site. This structure gives a significantly better understanding of the mode of action of these inhibitors, in addition to allowing for the possibility of future development of the inhibitors using structure based design

Ligandability Assessment of Human Glutathione Transferase M1-1 Using Pesticides as Chemical Probes

Glutathione transferases (GSTs; EC 2.5.1.18) form a group of multifunctional enzymes that are involved in phase II of the cellular detoxification mechanism and are associated with increased susceptibility to cancer development and resistance to anticancer drugs. The present study aims to evaluate the ligandability of the human GSTM1-1 isoenzyme (hGSTM1-1) using a broad range of structurally diverse pesticides as probes. The results revealed that hGSTM1-1, compared to other classes of GSTs, displays limited ligandability and ligand-binding promiscuity, as revealed by kinetic inhibition studies. Among all tested pesticides, the carbamate insecticide pirimicarb was identified as the strongest inhibitor towards hGSTM1-1. Kinetic inhibition analysis showed that pirimicarb behaved as a mixed-type inhibitor toward glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). To shine a light on the restricted hGSTM1-1 ligand-binding promiscuity, the ligand-free crystal structure of hGSTM1-1 was determined by X-ray crystallography at 1.59 angstrom-resolution. Comparative analysis of ligand-free structure with the available ligand-bound structures allowed for the study of the enzyme's plasticity and the induced-fit mechanism operated by hGSTM1-1. The results revealed important structural features of the H-site that contribute to xenobiotic-ligand binding and specificity. It was concluded that hGSTM1-1 interacts preferentially with one-ring aromatic compounds that bind at a discrete site which partially overlaps with the xenobiotic substrate binding site (H-site). The results of the study form a basis for the rational design of new drugs targeting hGSTM1-1

In vitro metabolism of tetrazole aminoquinolines and derivatives of metergoline and fusidic acid

Includes bibliographical references.Drug metabolism is recognised as a key component of the drug discovery and development process. It exerts an influence on the action, duration of action and toxicity of a drug in vivo. The integration of drug metabolism studies is therefore crucial to compound progression through the various stages of the development process. This work details the in vitro metabolism work conducted during the early development of aminoquinoline tetrazoles, and derivatives of metergoline and fusidic acid as potential antiplasmodial and/or antimycobacterial agents

Structure-based inhibitor design and validation : application to Plasmodium falciparum glutathione S-transferase

The primary aim of this study was to use a computational structure-based ligand design strategy in finding novel ligands that could act as inhibitors of PfGST as basis for future antimalarial drug development. Since there is only one PfGST isoenzyme present in the parasite and the architecture of the binding site differs significantly from its human counter part, PfGST is considered a highly attractive drug target. Inhibition of PfGST is expected to interfere at more than one metabolic site in synergy: it is likely to disrupt the glutathione-dependent detoxification process, which will lead to an increase in the cytotoxic peroxide concentration and most likely lead to an increase in the levels of ferriprotoporphyrin IX and hemin as well. S-hexyl glutathione was co-crystallized with PfGST (Harwaldt et al., 2004), consequently it was seen as one of the most important lead compounds in the development of PfGST inhibitors. The first step in the rational drug design strategy was to modify GTX, concentrating on its ability to bind competitively to the G site and the hydrocarbon chain protrudes into the H site as well. Considering the 3D structure of the enzyme, modifications to GTX were made by LUDI and NEWLEAD, resulting in a library of active site binding ligands ranked by AutoDock according to their ability to optimally bind to PfGST. Additionally, the ligands were ranked according to their affinity for binding to PfGST produced by AutoDock, LUDI and XScore. Once all the compounds were ranked by these in silico methods they were screened for acquisition or synthetic accessibility and those available were experimentally screened for activity against recombinantly expressed PfGST. Based on in silico predictions NDA was the best inhibitor followed by LAP and EDP. From the biological assay and Lineweaver-Burk analysis the order of inhibition was NDA as the best inhibitor tested, followed by LAP and EDP. EDP and LAP showed competitive inhibition but the inhibition constant values were signi_cantly lower than GTX. With respect to GSH and CDNB, NDA was found to be a non-competitive inhibitor. It was suggested therefore that NDA binds to a non-substrate Summary 93 binding site that may lead to conformational change of the enzyme and hence lead to a loss in enzyme activity. This data leads to the conclusion that the H site should be better exploited in order to find more potent inhibitors or non-substrate binding sites. It was concluded that the experimental results add confidence to the discriminative power of the structure-based ligand design strategy and that these inhibitors could form scaffolds for future antimalarial drug development.Dissertation (MSc (Bioinformatics))--University of Pretoria, 2008.Biochemistryunrestricte

Computational Study on Protein Arginine methyl-transferases (PRMTs)

Protein arginine methyltransferases (PRMTs) are essential epigenetic players in living cells. The dysregulation of PRMTs is closely related to many diseases, including cancer. Based on previously reported PRMT1 inhibitors bearing the diamidine pharmacophore, a combinatorial high throughput screening strategy led to compound K313, which possesses a biochemical IC50 value of 0.84 µM against PRMT1. Histone code is the post-translational modification patterns appear at histone, which regulates transcription and many other cellular events. H4R3 is one of the important substrates for both PRMT1 and PRMT5. PRMTs are important in establishing histone code. They are also regulated by the histone code. In this study, we explored the mechanism of how the post-translational modifications on H4 tail peptide affect the activity of PRMTs

Glutathione transferases: probing for isoform specificity using dynamic combinatorial chemistry

Cytosolic glutathione transferases (GSTs) are a large family of enzymes that play an important role in detoxification of xenobiotics. They catalyse the conjugation of the glutathione tripeptide (GSH) to a wide range of toxic electrophilic acceptors. The overall 3D folds and architectures of the catalytic sites of many GSTs are conserved. They are composed of a well conserved glutathione binding site (G-site) and a promiscuous hydrophobic binding site (H-site). The 3D structure and ligand specificity has allowed the sub-classification of the multiple isoforms within the soluble GST superfamily. GSTs are involved in the drug detoxification and so are the target of medicinal chemistry programmes but it has proven difficult to generate isoform-specific inhibitors due to their inherent promiscuity. In this project, Venughopal Bhat (University of Edinburgh, laboratory of Dr. Mike Greaney) and I have explored a new platform to probe enzyme specificity. Protein-directed dynamic combinatorial chemistry (DCC) allows the assembly and amplification of a ligand within the confines of a binding site. DCC was used as a tool to explore the promiscuous H-site of four eukaryotic GSTs. I purified recombinant forms of SjGST, hGST P1-1, mGST M1-1 and mGST A4-4 from E. coli and assayed them with the universal, synthetic GST substrate 1-chloro-2,4-dinitrobenzene (CDNB). Venughopal Bhat prepared a ten-member, thermodynamically-controlled, dynamic combinatorial library (DCL) of acyl hydrazones from a 1-chloro-2-nitrobenzene aldehyde and ten acylhydrazides. This DCL was incubated with each of the four GST isozymes (spanning diverse classes) and distinct amplification effects were observed for SjGST and hGST P1-1. I subsequently carried out several biophysical experiments in an attempt to rank each of the ligands. These experiements, coupled with molecular modelling, provided insight into the basis of the observed selectivity. Bacterial GSTs are thought to play a role in primary metabolism and display a different GSH-conjugation mechanism compared to the eukaryotic GSTs. A recombinant form of the beta-class GST from the pathogenic bacterium Burkholderia cenocepacia was isolated, purified and biochemically characterised. The same ten-member acylhydrazone DCL was interfaced with the bacterial GST which was shown to amplify a hydrophobic library member that shared structural features with the known substrate 2-hydroxy-6-oxo-6-phenyl-2,4-dienoate (HOPDA). With the collaboration of Venughopal Bhat, I attempted to explore the putative active site of a GST-like protein with an unknown function using the same DCL. Although no amplification was observed, a new aldehyde template was suggested for future DCC experiments on this protein. GSTs are widely employed in biotechnology as protein fusion tags to enhance target protein solubility coupled with a facile enzyme assay. Manish Gupta and Juan Mareque-Rivas (University of Edinburgh) used the N-terminal, hexahistidine-tagged SjGST to demonstrate that quantum dots (QDs) coated with nitrilotriacetic acid (NTA) bound to Ni2+ ions can be used to reversibly and selectively bind, purify, and fluorescently label a His6-tagged GST in one step with retention of enzymatic activity. For this prupose, I purified and characterized both the untagged and hexahistidinetagged – SjGST prior to their experiments

WISDOM-II: Screening against multiple targets implicated in malaria using computational grid infrastructures

<p>Abstract</p> <p>Background</p> <p>Despite continuous efforts of the international community to reduce the impact of malaria on developing countries, no significant progress has been made in the recent years and the discovery of new drugs is more than ever needed. Out of the many proteins involved in the metabolic activities of the <it>Plasmodium </it>parasite, some are promising targets to carry out rational drug discovery.</p> <p>Motivation</p> <p>Recent years have witnessed the emergence of grids, which are highly distributed computing infrastructures particularly well fitted for embarrassingly parallel computations like docking. In 2005, a first attempt at using grids for large-scale virtual screening focused on plasmepsins and ended up in the identification of previously unknown scaffolds, which were confirmed in vitro to be active plasmepsin inhibitors. Following this success, a second deployment took place in the fall of 2006 focussing on one well known target, dihydrofolate reductase (DHFR), and on a new promising one, glutathione-S-transferase.</p> <p>Methods</p> <p>In silico drug design, especially vHTS is a widely and well-accepted technology in lead identification and lead optimization. This approach, therefore builds, upon the progress made in computational chemistry to achieve more accurate <it>in silico </it>docking and in information technology to design and operate large scale grid infrastructures.</p> <p>Results</p> <p>On the computational side, a sustained infrastructure has been developed: docking at large scale, using different strategies in result analysis, storing of the results on the fly into MySQL databases and application of molecular dynamics refinement are MM-PBSA and MM-GBSA rescoring. The modeling results obtained are very promising. Based on the modeling results, <it>In vitro </it>results are underway for all the targets against which screening is performed.</p> <p>Conclusion</p> <p>The current paper describes the rational drug discovery activity at large scale, especially molecular docking using FlexX software on computational grids in finding hits against three different targets (PfGST, PfDHFR, PvDHFR (wild type and mutant forms) implicated in malaria. Grid-enabled virtual screening approach is proposed to produce focus compound libraries for other biological targets relevant to fight the infectious diseases of the developing world.</p

Investigating the chemical space and metabolic bioactivation of natural products and cross-reactivity of chemical inhibitors in CYP450 phenotyping

Includes bibliographical references.Natural products have been exploited by humans as the most consistently reliable source of medicines for hundreds of years. Owing to the great diversity in chemical scaffolds they encompass, these compounds provide an almost limitless starting point for the discovery and development of novel semi-synthetic or wholly synthetic drugs. In Africa, and many other parts of the world, natural products in the form of herbal remedies are still used as primary therapeutic interventions by populations far removed from conventional healthcare facilities. However, unlike conventional drugs that typically undergo extensive safety studies during development, traditional remedies are often not subjected to similar evaluation and could therefore harbour unforeseen risks alongside their established efficacy. A comparison of the ‘drug-like properties’ of 335 natural products from medicinal plants reported in the African Herbal Pharmacopoeia with those of 608 compounds from the British Pharmacopoeia 2009 was performed using in silico tools. The data obtained showed that the natural products differed significantly from conventional drugs with regard to molecular weight, rotatable bonds and H-bond donor distributions but not with regard to lipophilicity (cLogP) and H-bond acceptor distributions. In general, the natural products were found to exhibit a higher degree of deviation from Lipinski’s ‘Rule-of-Five’. Additionally, these compounds possessed a slightly greater number of structural alerts per molecule compared to conventional drugs, suggesting a higher likelihood of undergoing metabolic bioactivation