ANTIVIRULENCE STRATEGIES AGAINST PSEUDOMONAS AERUGINOSA

Abstract

The long-term use of antibiotics has dramatically accelerated the emergence of multi-drug resistant (MDR) bacterial pathogens worldwide, leading to an alarming increase of difficult-to-treat infections. In parallel, only a handful of new antibiotics have been approved by the U.S. Food and Drug Administration (FDA) in the last decades, with many companies considering the R&D for new antibiotics a less attractive asset compared to more rewarding therapeutic areas. The serious health and economic problems caused by MDR pathogens, alongside the progresses in understanding the antibiotic resistance and virulence mechanisms in many bacterial pathogens, have fostered research not only into new antibiotics, but also into antibiotic adjuvants and antivirulence drugs. Antibiotic adjuvants target antibiotic resistance determinants (e.g., efflux pumps, antibiotic inactivating enzymes), thus decreasing bacterial resistance to conventional antibiotics. Antivirulence drugs target bacterial virulence processes rather than growth; consequently, they are expected to reduce bacterial adaptability to the host environment with lower selective pressure for the emergence of resistance compared to antibiotics. Pseudomonas aeruginosa is a model organism for studies focused on antibiotic adjuvants and antivirulence drugs. This Gram-negative bacterium is one of the most dreaded nosocomial pathogens and the main cause of death in cystic fibrosis patients, and has recently been included by the World Health Organization in the list of pathogens for which new therapeutic options are urgently needed (Priority 1: Critical; http://www.who.int/en/news-room/detail/27-02-2017-who publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed). The active efflux of antibiotics via efflux pumps contributes to the MDR phenotype of P. aeruginosa. Hence, inhibitors of P. aeruginosa efflux pumps hold promise to overcome P. aeruginosa antibiotic resistance. Moreover, the notion that efflux pumps could play a role in P. aeruginosa infection is emerging, thus implying that efflux pumps inhibitors (EPIs) could also be endowed with antivirulence properties. Nevertheless, EPIs are usually considered only for their antibiotic adjuvant activity, while their antivirulence potential is seldom taken into account. On this basis, during the first part of this PhD thesis, the effect of the model EPI Phe-Arg-β-naphthylamide (PAβN, also named MC-207,110) on P. aeruginosa transcriptome and virulence was evaluated. Quorum sensing (QS) is an intercellular communication system that plays a key role in controlling the expression of key virulence determinants in a number of bacterial pathogens, including P. aeruginosa. P. aeruginosa QS defective strains show decreased virulence in animal and plant infection models, therefore QS has received attention as a potential therapeutic target for the development of antivirulence agents against P. aeruginosa. Over the last decade, a number of compounds inhibiting the P. aeruginosa QS circuitry have been identified, most of which are active on the las QS system. Recently, few studies have described inhibitors also of the pqs QS system. This complex QS circuit is required for full virulence and is active during P. aeruginosa infections in humans. Unfortunately, most of the QS inhibitors identified so far are cytotoxic or display unfavourable pharmacological properties, thus limiting their transfer to the clinical practice. The discovery and development of new drugs for use in humans is a challenging task that usually requires decade-long laboratory experimentation followed by extensive clinical trials. Searching for off-target activities in drugs already approved for use in humans represents a potential shortcut to develop new therapeutic options. As compared with de novo drug discovery, drug-repurposing (or drug-repositioning) is expected to reduce the time and costs generally associated to standard drug discovery processes. Also in silico approaches have been proved as valid aids to conventional drug discovery programmes. In particular, virtual screenings carried out through molecular docking simulations allow selecting promising drug candidates in vast libraries of molecules. On this basis, during the second part of this PhD thesis the drug-repurposing and the in silico screening approaches were used to identify FDA-approved drugs able to reduce P. aeruginosa virulence potential by targeting different elements of the pqs QS system.