Glutathione transferases: probing for isoform specificity using dynamic combinatorial chemistry

Abstract

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.