DEVELOPMENT OF SMALL MOLECULES AS INHIBITORS OF FTSZ AND RNPA ACTING AS POTENTIAL ANTIMICROBIAL AGENTS

The antimicrobial resistance (AMR) is nowadays one of the most worrying threats of modern medicine. Recently, AMR has been also called “the silent pandemic” because is as worrying as the COVID-19 outbreak, but without receiving the same public attention. The most important organizations such as World Health Organization (WHO) and Centres for Disease Control and Prevention (CDC) pointed out how the academic work on the development of new chemical entities as antimicrobials able to interact with new molecular targets remains one of the key actions to fight antimicrobial resistance (World Health Organization, 2015). Within the present work, two innovative bacterial targets have been considered to develop new potential inhibitors: FtsZ and RnpA. FtsZ is a 40 kDa protein with 40 -50% of conservation among all bacteria and archaea. This protein represents the main actor of the bacterial cell division cycle, the essential process that allows the parent cell to divide through binary fission into two daughter cells. At the beginning of the cycle, FtsZ polymerizes at the centre of the cell, constituting the “Z-ring”. This step serves as guide for the continuation of the process, therefore the inhibition of FtsZ leads to cell filamentation and death. For these reasons, FtsZ arose as a potential target for the development of new antimicrobials. In the first part of this work, starting from the insights gained in the last years, new families of benzodioxane-benzamides have been designed, synthesized, purified and evaluated as potential antimicrobials on S. aureus, B. subtilis and E. coli. Moreover, FtsZ was validated as the target of these compounds. In particular, S. aureus and B. subtilis FtsZs were validated as targets through specific cellular assays, while the capability of these compounds to interact with E. coli FtsZ was demonstrated through an in vitro study on the isolated protein. Moreover, the cytotoxicity of all the derivatives was evaluated, and most of the derivatives are characterized by low to non-detectable cytotoxicity on human cells. RnpA is a small protein of S. aureus essential for two cellular processes: mRNA degradation and precursor tRNA (ptRNA) maturation. Indeed, RnpA alone can catalyse the degradation of bulk mRNA thus promoting mRNA turnover among different growth phases (exponential and stationary). Furthermore, RnpA is able to associate with a ribozyme to form RNase P, a riboprotein complex that promotesthe removal of the 5’ leader sequence from precursor tRNA, inducing tRNA maturation. Shortly after the discovery of the essentiality of RnpA, the investigation of RnpA inhibitors as potential anti-staphylococcal compounds started. Within the second main body of this work, the structure-activity relationship (SAR) of two classes of known RnpA inhibitors was evaluated. Modifications of the structures of the two main RnpA inhibitors, JC2 and RNPA2000, have been designed and applied. The resulting compounds were tested as antimicrobials on S. aureus, both methicillin-sensitive and methicillin-resistant. Also in this case, the in vitro profile of inhibition of RnpA as well as the cytotoxicity of the obtained derivatives were assessed. With the results obtained in the present work, the understanding of both FtsZ and RnpA inhibitors was significantly increased, speeding up the development of these important tools to fight antimicrobial resistance.