The Interaction of the Chemotherapeutic Drug Chlorambucil with Human Glutathione Transferase A1-1: Kinetic and Structural Analysis

Abstract

Glutathione transferases (GSTs) are enzymes that contribute to cellular detoxification by catalysing the nucleophilic attack of glutathione (GSH) on the electrophilic centre of a number of xenobiotic compounds, including several chemotherapeutic drugs. In the present work we investigated the interaction of the chemotherapeutic drug chlorambucil (CBL) with human GSTA1-1 (hGSTA1-1) using kinetic analysis, protein crystallography and molecular dynamics. In the presence of GSH, CBL behaves as an efficient substrate for hGSTA1-1. The rate-limiting step of the catalytic reaction between CBL and GSH is viscosity-dependent and kinetic data suggest that product release is rate-limiting. The crystal structure of the hGSTA1-1/CBL-GSH complex was solved at 2.1 Å resolution by molecular replacement. CBL is bound at the H-site attached to the thiol group of GSH, is partially ordered and exposed to the solvent, making specific interactions with the enzyme. Molecular dynamics simulations based on the crystal structure indicated high mobility of the CBL moiety and stabilization of the C-terminal helix due to the presence of the adduct. In the absence of GSH, CBL is shown to be an alkylating irreversible inhibitor for hGSTA1-1. Inactivation of the enzyme by CBL followed a biphasic pseudo-first-order saturation kinetics with approximately 1 mol of CBL per mol of dimeric enzyme being incorporated. Structural analysis suggested that the modifying residue is Cys112 which is located at the entrance of the H-site. The results are indicative of a structural communication between the subunits on the basis of mutually exclusive modification of Cys112, indicating that the two enzyme active sites are presumably coordinated.

Synthesis of unsymmetrical disulfanes bearing 1,2,4-triazine scaffold and their in vitro screening towards anti-breast cancer activity

“…Chlorambucil, as the commonly used chemotherapeutic drug, interacts with the human glutathione transferases (GSTs) and the human GSTs are the natural targets to form active complexes with chlorambucil [41, 42]. …”

Silencing TAK1 alters gene expression signatures in bladder cancer cells

“…CYP3A5 and GSTA1 are associated with the detoxification of chemical stimuli, electrophilic compounds or other damage stimuli (21,22). They were also enriched in the term metabolism of xenobiotics by cytochrome P450, which plays important roles in detoxification (23).…”

Regulation of Glutathione in Health and Disease with Special Emphasis on Chronic Alcoholism and Hyperglycaemia Mediated Liver Injury: A Brief Perspective

“…Glutathione S-transferases (GSTs) are involved in drug detoxification through the conjugation of reactive drug metabolites to GSH [34,58,85]. The GSTs catalyze the nucleophilic attack of GSH on the electrophilic centre of a number of xenobiotic compounds, including several chemotherapeutic drugs [58,85].…”

“…Glutathione S-transferases (GSTs) are involved in drug detoxification through the conjugation of reactive drug metabolites to GSH [34,58,85]. The GSTs catalyze the nucleophilic attack of GSH on the electrophilic centre of a number of xenobiotic compounds, including several chemotherapeutic drugs [58,85]. Apart from conjugation of xenobiotics to GSH, many other activities are also associated with the GSTs, including steroid and leukotriene biosynthesis, peroxide degradation, double-bond cis-trans isomerization, dehydroascorbate reduction, Michael addition, and noncatalytic 'ligandin' activity (ligand binding and transport) [85].…”

“…In the presence of GSH, the chemotherapeutic drug chlorambucil (CBL) behaves as an efficient substrate for human GSTA1-1 (hGSTA1-1). In the absence of GSH, CBL acts as an alkylating irreversible inhibitor for hGSTA1-1 [58]. The GSTs are known to be overexpressed in tumor, and naturally occurring isothiocyanates, such as benzyl isothiocyanate (BITC), are effective cancer chemopreventive compounds [95].…”