Structural and functional studies on members of the Aldo-Keto reductase family: Identifying molecular determinants responsible for substrate selectivity and inhibitor potency

The aldo-keto reductase (AKR) superfamily encompasses more than 150 enzymes that catalyse the NADP(H)-dependent reduction of several endogenous and xenobiotic aldehydes and ketones. Human AKRs have been implicated in the development of diabetes, cancer chemotherapeutic drug resistance, tobacco carcinogenesis and other hormone-dependent cancers. As such, there is a growing need to understand the molecular basis for their involvement in disease progression and develop selective inhibitors that could be used as therapeutics. The research project presented in this thesis originates from the need to study the tertiary structures of new members of the AKR family and correlate their unique structural features to enzyme function. Three members of the AKR family, namely AKR1C1, AKR1C21 and AKR1B14 were investigated in this thesis. Human AKR1C1 enzyme regulates activity of progesterone receptors by catalysing the reductive inactivation of progesterone into 20α-hydroxyprogesterone. Aberrant AKR1C1 activity has been associated with several types of cancers, end ometriosis and obesity-related metabolic disorders. In section A of this thesis, we present a new class of salicylic acid based inhibitors of AKR1C1 identified from high throughput virtual screening of the NCI database. These compounds were developed further into potent and selective inhibitors of AKR1C1 using a structure - based drug design approach. Section B of this thesis is dedicated to structure- function analysis of mouse AKR1C21 which is the only enzyme known to exhibit 17α-reductase activity. The role of active site residues in dictating 17α- stereospecificity of this enzyme was investigated by determining the crystal structures of AKR1C21 and its mutants. The mode of binding of substrates and inhibitors in the active site of AKR1C21 was explored through molecular modelling and docking analysis. In section C, we present the first crystal structure of rat AKR1B14 which exhibits significant prostaglandin F2α synthase activity and reduces reactive products derived from lipid peroxidation and glycation with lower Km values than other members of the AKR1B subfamily. Residues involved in binding of coenzyme, bile-acid activator and aldose reductase inhibitors were investigated through molecular modelling and docking analysis. The overall aim of this study was to characterise the mode of binding of substrates and inhibitors within the active site of these enzymes in order to ascertain the role of active site residues in dictating substrate selectivity and inhibitor potency. The structural information obtained from solving the crystal structures of these enzymes was used for high throughput database screening to identify new ligands that could be developed into potent drug-like inhibitors with potential for therapeutic use through a structure based drug design process.