Exploring pyrazinamide derivatives as novel Pseudomonas aeruginosa inhibitors: unexploited antibacterial molecules for a new antibiotics target

Background and Rationale P. aeruginosa is the most common pathogen in cystic fibrosis lung infection with a high negative impact on lung functionality and patients’ mortality. The increasing diffusion of multi-resistant strains demands for the development of new anti-Pa agents. The ribosomal protein S1 (encoded by rpsA) is a promising target for new antibacterial drugs. In Escherichia coli, S1 has an essential role in translation. S1 is highly conserved among Gram negative bacteria and absent in mammalian cells. Recently, it has been found that pyrazinamide (PZA), a first-line tuberculosis drug, targets S1 protein. PZA derivatives were developed by Bracco SpA in the early ‘60s and roughly characterized for antibacterial activity; interestingly, in preliminary tests, the derivative B2320 seemed to be active against Pseudomonas aeruginosa (Pa). B2320 target in Pa is currently unknown. Hypothesis and objectives 1. Characterizing the anti-Pa activity of B2320. 2. Exploring Pa S1 as a potential target for new antibacterials. 3. Setting-up an E. coli biosensor strain for the screening of S1 inhibitors. Results and their significance 1. B2320 has been tested for anti-bacterial activity both against Pseudomonas lab strains and a collection of clinical isolates from CF patients in different growth conditions. We found that although only high concentrations of B2320 impaired aerobic growth of most P. aeruginosa strains, the sensitivity to B2320 was increased by stress conditions and in vivo, in the infection model Galleria mellonella. The genome of a clinical isolate particularly sensitive to B2320 and of a B2320-resistant derivative were sequenced. 2. rpsA essentiality in P. aeruginosa has been addressed by site-directed mutagenesis. Our data strongly support rpsA essentiality in P. aeruginosa. 3. We have set up a fluorescence-based assay to find inhibitors of S1-dependent translation initiation in E. coli. The screen allows to identify compounds interfering with an essential and bacteria specific step of gene expression, discriminating from molecules with nonspecific cell toxicity. Moreover, as we have adapted the assay to P. aeruginosa, cell penetration properties of the active compounds identified in the primary screening could be easily assessed. Since our screen is technically very simple and appears to be robust, it is potentially adaptable to High-Throughput-Screening campaigns.