Novel Approaches to Fight Antimicrobial Resistance: a Structural Biology Perspective

Antimicrobial resistance is emerging as one of the worst threats to public health of our century. The use (and often misuse) of antimicrobials challenges bacterial pathogens to evolve molecular mechanisms that counteract the effect of bactericidal and bacteriostatic drugs. The SOS response is a widely conserved bacterial pathway activated in reply to genotoxic stress and is recognized as one of the main drivers of stress-induced (hyper)mutation and consequent acquisition of antimicrobial resistance. Its protein actors (RecA and LexA) regulate a large and species-specific number of genes and cellular functions, ranging from DNA repair and recombination to horizontal gene transfer, expression of virulence factors, biofilm organization and differentiation of persister bacterial populations. Pharmacological suppression of the SOS response has been suggested as a useful approach for the development of new, unconventional weapons in antimicrobial warfare, with the aim of prolonging the usability of currently available antibiotics and re-sensitizing resistant microbes. In this thesis, several efforts are presented to find inhibitors of the SOS response, screening large libraries of small drug-like molecules, cyclic peptides and antibody fragments (nanobodies). Deep biochemical and biophysical characterization of hit compounds is given in order to estimate binding affinities for the protein targets and inhibitory potency on isolated RecA and LexA, in vitro. Since anti-LexA nanobodies emerged as the most active SOS suppressors discovered so far, microbiological testing was focused on this class of molecular tools and revealed that they can significantly downregulate the expression of pro-mutagenic factors in bacterial cells undergoing to antibiotic-induced DNA stress. X-ray structures of LexA-nanobody complexes revealed a peculiar and previously unreported mechanism of inhibition on LexA and sustained the advancement of anti- LexA nanobodies (NbSOSs) by rational protein engineering. In particular, both the increase of NbSOSs affinity for Escherichia coli LexA and the tuning of their specificity towards Pseudomonas aeruginosa LexA have been investigated and preliminary results are presented. The latter task relied on the availability of structural information on P. aeruginosa LexA, whose atomic structure has been solved by x-ray crystallography and is described in detail. Towards a full comprehension of SOS response activation in P. aeruginosa, a high-resolution structure of P. aeruginosa RecA has also been obtained by cryoEM in this work. Last, another strategy to counteract antibiotic resistance has been pursued by developing inhibitors of NDM-1 metallo-b-lactamase, a major factor of resistance to b-lactam antibiotics. Among the tested compounds, potent enzymatic inhibitors were discovered, and one was able to strongly increase the bactericidal effect of a b-lactam. Once again, structural investigations on NDM-1 inhibitors were carried out by x-ray crystallography and disclosed the main binding determinants. Studies presented in this thesis pave the way for the development of novel classes of biopharmaceuticals that might reinforce the current arsenal of antimicrobial weapons. Moreover, evidence is given of the great impact that structural biology studies have in biotechnology and pharmaceutical chemistry, sustaining both the understanding of mechanisms underlying macromolecular machineries and the design of new functionalities.