Studies on the Modulation of the Enzymatic Activity of Hypoxia-Inducible Factor Hydroxylases

Abstract

This thesis describes the work investigating the use of different chemical approaches to modulate the enzymatic activity of human hypoxia-inducible factor (HIF) prolyl hydroxylase-domain containing enzyme 2 (PHD2). PHD2 is a member of the non-heme Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenases, which catalyses the hydroxylation of the α-subunit of HIF (HIF-α) by utilising 2OG and molecular oxygen as co-substrates. PHD2 regulates the stability of HIF in normoxia and it is considered the most important oxygen sensor in humans. PHD2 is a current inhibition target for the treatment of a number of diseases including cancer, anemia, ischemia, and stroke. Hence, the ability to modulate the activity of PHD2 by the use of chemical inhibitors and activators is of significant interest from both biology and medicinal chemistry perspectives. In chapter 2, the modulation of PHD2 activity by carbon monoxide, an endogenously produced gasotransmitter, was investigated. Carbon monoxide-releasing molecule 2 (CORM-2) was used as an in situ donor of CO. The results showed that CO is an inhibitor of PHD2, presumably by competing with molecular oxygen for the binding to the PHD2 active site. By using different peptidyl substrates, the ability of CO to inhibit PHD2 was found to be substrate-dependent. Previous studies using cellular models have shown that CO may stabilise HIF in normoxia. This work helps provide a chemical rationale towards the observations that were reported in the previous studies. Chapter 3 focused on the screening of new inhibitors against PHD2. Most inhibitors of PHD2 that have been reported to date are structural mimics of 2OG. As the active site of 2OG oxygenases are highly conserved, these inhibitors lack selectivity. Two classes of potential PHD2 inhibitors were investigated in this study. These include compounds that are mostly used as ligands for organometallic anticancer drugs and small molecule compounds that were obtained through high-throughput virtual screening against the peptidyl substrate binding site. The results showed that PHD2 was inhibited by metal chelators. However, their mechanism of actions were through metal sequestration rather than binding to the enzyme. In Chapter 4, the use of metals of medical interest to modulate the activity of PHD2 was investigated. The metals that were studied included ruthenium, gold, and gadolinium. It was found that Ru(III) and Au(III) are inhibitors of PHD2 with half-maximal inhibitory concentration (IC50) values in the micromolar range. Interestingly, Gd(III) was found to act as an activator of PHD2. It was found that the native Fe(II) could be replaced by Gd(III) although its ability to activate PHD2 was found to be ~100-fold lower than that of Fe(II). This finding unveils the possibility of exploring other lanthanide metals or actinide metals for their ability to activate 2OG oxygenases.