Manipulation of enzyme activity via substrate based control: a new paradigm in drug design

Abstract

This thesis documents our approach to the manipulation of enzyme activity in previously underexploited pathways through the design of artificial substrates. It was envisaged that this strategy would enable the discovery of new drugs targets. In particular, this thesis focused on two ancient enzymes– queuine insertase (QI) and elongation factor-P (EF-P). The QI enzyme is responsible for incorporating modified nucleobases into eukaryotic tRNA with the concomitant elimination of guanine. Previous reports by our research group described the design of a novel substrate for the QI enzyme - NPPDAG - which was found to have an antiproliferative effect on T-cells and reversed the symptoms of disease in a murine multiple sclerosis model. We, therefore, considered the development of new substrates for the enzyme-based on this pyrrolo[2,3-d]pyrimidine core. The compounds were evaluated in vitro and gratifyingly, were confirmed to act as substrates for the enzyme. Of note, was the emphasis the results of this study placed on the importance of an electron-withdrawing group at C-5 of the deazaguanine core. This information provided insight for future drug design in our research group. The emergence of bacterial strains resistant to treatment by antibiotics remains one of the biggest threats to global health today. The second part of this thesis aimed to develop a series of compounds which targeted a previously understudied bacterial pathway. EF-P is an enzyme exclusive to the bacterial kingdom, which alters the peptidyl transferase activity of the ribosome via the alleviation of polyproline induced stalling during protein translation. The post-translational modification of EF-P with a β-lysine moiety by the PoxA enzyme at a specific lysine residue has proven to be essential to the catalytic activity of this enzyme. This thesis focused on the design of novel substrates for the PoxA enzyme, specifically analogues of β-lysine in which the steric and electronic properties were systematically altered. This thesis reports the first examples of artificial substrates for PoxA; interestingly, a small sample of these substrates were found to alter the rate of bacterial growth