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

Background 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. Essential methods 1. B2320 will be tested for anti-bacterial activity both against Pseudomonas lab strains and a collection of clinical isolates from cystic fibrosis patients. The mechanism of action of the compound will be investigated. 2. A Pa strain with rpsA conditional expression will be constructed in order to assess its essentiality. 3. A fluorescence-based assay to find inhibitors of S1-dependent translation initiation will be set up in E. coli, transferred in Pa and used to screen a collection of heterogeneous chemical compounds for translation inhibitors. Preliminary results 1. B2320 activity against PAO1 and PA14 lab strains has been tested in different growth conditions. Interestingly, the more virulent PA14 strain seems more sensitive to B2320 than PAO1 strain. 2. The mutant is under construction. We have mapped the 5’-end of rpsA transcript in Pa. This has been instrumental in designing the construct for rpsA mutation. 3. We have developed a simple whole-cell assay in E. coli that allows discriminating antibiotics inhibiting different translation steps. Expected results and their significance P. aeruginosa is the most common pathogen in CF 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. These could be identified both among PZA derivatives and by our whole-cell screening of libraries of chemical compounds. Moreover, if Pa S1 will result to be essential as expected, it would be a robust target for the design of new drugs.