Molyneaux, Carrie-Anne; (2017) Developing novel antibacterials targeting the glyoxalase system using kinetic target guided synthesis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Antibiotic multidrug resistant infections are increasingly challenging to treat. To avoid entering a post antibiotic era novel antibiotics acting via new or underexploited mechanisms are required. In the human body bacteria are exposed to numerous stresses, e.g. methylglyoxal, and detoxification is essential for bacterial survival and infection progression. Glutathione and the glyoxalase (Glx) system are essential to this detoxification process. We hypothesise that inhibitors of bacterial GlxI and/or GlxII will disrupt this protective pathway leading to bactericidal activity, and may act alone or synergistically with existing antibiotics and the host immune system. To identify potent and selective chemical tools to investigate our hypothesis kinetic target guided synthesis (kTGS), where the enzyme generates its own inhibitors by acting as a catalyst to ‘click’ fragments together within the binding site, was employed in 2 ways; 1) a biased and 2) an unbiased approach. The chemistry used was a Huisgen [3+2] cycloaddition between azides and alkynes to form triazoles and hit compounds were identified by LC-MS/MS. In the biased approach an azido derivative of glutathione (GAz) was designed and synthesised. kTGS with GlxI and GlxII using 96 alkyne fragments to probe the hydrophobic pocket resulted in hit rates up to 28%. The unbiased approach used azide and alkyne fragment libraries to identify non-peptidic inhibitors and to investigate the potential of kTGS as a tool for fragment based drug discovery. Using 2 azide and 2 alkyne fragments and the corresponding fragment library resulted in hit rates up to 11%. Identified peptidic and non-peptidic hits were synthesised and biophysical evaluation demonstrated binding to and inhibition of the glyoxalase enzymes, thereby demonstrating the potential of kTGS for lead discovery and the opportunity to develop these hits into novel antibacterial agents.

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