Structural and functional characterization of novel inhibitors targeting the β-lactamase NDM-1

The worldwide increase in antimicrobial resistance (AMR) poses a major public health concern. In this regard, β-lactamases play a crucial role as they confer bacteria resistance to β-lactam antibiotics as penicillin, monobactam and carbapenems. Among them, New Dehli Metal-β-lactamase-1 (NDM-1), first identified in New Dehli, has also been reported in countries around the world, including United States, Japan, Australia and the United Kingdom. The NDM-1 gene was identified for the first time in 2009 in Escherichia coli and Klebsiella pneumoniae and has since spread among diverse bacterial species via horizontal gene transfer. This thesis project aims to investigate nanobodies as potential inhibitors of NDM-1, with a particular focus on structural characterization and binding determinants. Nanobodies derived from a phage display library, were produced in E. coli and subsequently purified. Their binding to NDM-1 was assessed through analytic size-exclusion chromatography, which enabled observing the complex formation, by thermal fluorescence and by grating-coupled interferometry (GCI), which provided thermodynamic and kinetic parameters. The structure of NDM-1 and nanobodies were analyzed by X-ray crystallography.