Structural and kinetic studies on beta-lactamase mechanism and inhibition

Beta-lactamases constitute one of the most prevalent identified mechanisms of bacterial resistance to the beta-lactam antibiotics. These enzymes are broadly divided into two mechanistic subclasses, the serine-beta-lactamases and the metallo-beta-lactamases. The metallo-beta-lactamases constitute an important subclass of beta-lactamases on account of their ability to hydrolyse almost all classes of beta-lactam, in particular the carbapenems, which are considered to be beta-lactams of last resort. At present inhibitors of the serine-beta-lactamases are clinically available for the treatment of resistant bacterial infections; these include clavulanic acid and related compounds, and more recently avibactam. However, there are currently no clinically validated inhibitors of the metallo-beta-lactamases.
Structural and mechanistic study of the beta-lactamases will aid in the development of novel inhibitor scaffolds with clinical potential in the treatment of resistant bacterial infections. In this work, the ability of the metallo-beta-lactamases to promiscuously bind and employ transition metals, other than their native zinc, in catalysis is described in the context of ferrous iron. The effects of metal substitution on enzyme structure, mechanism and susceptibility to inhibitors are investigated. These studies demonstrate that the metallo-beta-lactamases are able to employ iron, a transition metal of relatively high bioavailability, in catalysis with only small changes in catalytic efficiency.
In an increasing number of cases, bacteria are found to exhibit resistance to beta-lactams mediated by both serine- and metallo-beta-lactamases, simultaneously. With this likely to be an important issue in the future, the utility of developing molecules with the capability to inhibit both serine- and metallo-beta-lactamases cannot be understated. In the work described herein, cyclic boronic acids were explored as a chemotype with inhibitory activity against the two mechanistic classes of beta-lactamase and are shown to be potent inhibitors of both. Structural characterisation of the cyclic boronic acids in complex with beta-lactamases reveals that their inhibitory activity likely derives from their mimicking of a tetrahedral anionic intermediate common to both mechanistic classes of beta-lactamase. The activity of these molecules against clinically derived resistant bacterial strains is also explored.
While the metallo-beta-lactamases are largely studied on account of their involvement in antibiotic resistance, the metallo-beta-lactamase protein fold can facilitate a diverse range of chemistry from small molecule hydrolysis, to DNA and RNA processing, to oxidoreductase reactions. Moreover, a number of human enzymes share the metallo-beta-lactamase fold. Thus the study of these human enzymes and development of activity assays for their functions will likely prove useful in the discovery of bacterial metallo-beta-lactamase inhibitors with low toxicity in human patients. In this thesis, efforts made in the development of a novel high throughput assay for the unusual metallo-beta-lactamase persulfide dioxygenase ETHE1 via the fluorescent detection of sulfite are described.
Overall, the work of this thesis explores the mechanism of beta-lactamases as a framework for the discovery of novel beta-lactamase inhibitors, and makes efforts in the development of novel assays for human metallo-beta-lactamase fold enzymes to expedite the development of such molecules.

Advisor: Schofield, C