Structural and biochemical characterisation of bacterial metallo-β-lactamases

One of the most prominent ways by which bacteria develop resistance to the widely used β-lactam antibiotics is by production of β-lactamases. Metallo-β-lactamases (MBLs) in particular pose an increasing public health risk due to the wide dissemination and broad substrate profile. The successful development of novel MBL inhibitors for clinical use is also dependent on sufficient understanding of the structural and functional properties of the target MBLs. From a medicinal chemistry point of view, the combination of biological diversity and structural similarity of MBLs presents a rather challenging task for the development of selective small molecules for inhibitors for bacterial MBLs. The progress made in this quest thus far is discussed at length in the introduction (Chapter 1). Robust and affordable high throughput screening platforms are indispensable in drug development. Chapter 2 of the thesis describes the identification of a novel cephalosporinbased substrates, CLS405, for MBL activity and inhibition assays. The CLS405-based assay is used in an exemplary screening of potential MBL inhibitors, resulting in the identification of N-hydroxythiazoles as novel MBL inhibitor scaffolds. An umbelliferone-derived cephalosporin substrate (FC-5) is also investigated for application in screening of MBL activity in vivo. Clinically useful MBL inhibitors must have a sufficient breadth of selectivity against multiple variants MBLs; this is particularly challenging due to the rapid MBL evolution. In Chapters 3 and 4, comparative studies of the activity, stability and structural properties of some of the clinically reported variants of the New Delhi metallo-β-lactamases (NDMs) are described. Nominal differences are noted in the kinetic parameters of the recombinant proteins, except for the apparent substrate inhibition observed with nitrocefin for variants containing the M154L substitution. Further investigations reveal significant differences in thermal stability of the NDM variants with the double mutants (NDM-5, NDM-7, NDM-8 and NDM-12) being the most stable variants, suggesting that protein stability may be a driver of MBL evolution. The Verona imipenemase metallo-β-lactamases (VIM) enzymes are among the most widely distributed MBLs with >40 variants reported (Chapter 5). Comparative studies on the biochemical and biophysical properties were carried out for five VIM variants: VIM-1, VIM- 2, VIM-4, VIM-5 and VIM-38. Clear differences in the thermal stabilities of the variants are observed, despite their similar β-lactamase activities, as previously shown for NDM variants. Interestingly, the variants show marked differences in the inhibition profile, with isoquionolone-based inhibitors selectively inhibiting VIM-5 and VIM-38 compared to VIM-1 and VIM-2. Overall, the work supports the proposal that protein stability may be an important factor in MBL evolution. Crucially, the study highlights the importance of screening MBL variants during inhibitor development programs.