SYNTHESIS AND BIOLOGICAL EVALUATION OF NEW INHIBITORS OF ENZYMES INVOLVED IN c-di-GMP METABOLISM

Recently, much attention has been focused on the need for new antimicrobial agents with new targets or mechanisms of action against multidrug-resistant bacteria. Heavy antibiotic use and person to person spread of bacteria have greatly increased antibiotic resistance due to genetic mutation and this problem is continually increasing in severity. Biofilm-forming bacteria, resistant to antibiotics, cause over 65% of hospital infections. Biofilms are structural communities of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inhert or living surface. In the biofilm, bacteria are 1000-times more resistant to conventional antibiotic treatment. Despite the central role that bacterial biofilm plays in infection, there is currently no anti-biofilm drugs in clinical use. Therefore, the development of original compounds that specifically target the formation of biofilm is of great need in view of a rational use of antibiotics. Cyclic di-GMP (c-di-GMP) is a second messenger unique of bacteria that plays a central role in biofilm formation and also in the expression of virulence traits. Synthesis of c-di-GMP occurs via diguanylate cyclase enzymes (DCGs), while degradation of c-di-GMP occurs via phosphodiesterase enzymes (PDE). Small molecules interfering with c-di-GMP metabolism could potentially inhibit biofilm formation and virulence in a variety of bacteria. Inhibition of bacteria virulence rather than growth is an alternative strategy that allows to combat bacterial infections without exerting strong selective pressure for the bacteria to evolve resistance mechanisms. The exact details of c-di-GMP signaling is currently being studied by several laboratories and it is expected that analogs of c-di-GMP or other small molecules able to inhibit the enzymes involved in the c-di-GMP metabolism will become useful as either antivirulence or antibiofilm drugs. To date, only few molecular scaffolds have been identified and new small molecules that are able to prevent or destroy biofilm formation are needed. Based on these findings new c-di-GMP analogs have been synthesized and their ability to inhibit both PDE or DCG enzymes has been evaluated using an innovative approach to follow the enzymatic c-di-GMP formation and degradation in real-time.(1) The enzymes assayed are RocR and the cytoplasmatic portion of PA1120 from P. aeruginosa (PDE and DCG, respectively) and PleD from C. crescentus, as a reference of DCGs. The newly synthesized compounds showing the highest activity in vitro are currently being analyzed for their ability to inhibit c-di-GMP signaling, biofilm formation and/or virulence factors production in vivo, using the human pathogen P. aeruginosa as model bacterium. Results of these studies will be discussed. (1) Stelitano, V, et al. Nucleic Acids Res. 2013, 41, e79