STRUCTURE BASED DESIGN OF RSH INHIBITORS

Persistence is one of the biological mechanisms by which bacteria can avoid to be killed by antibiotic treatment. This phenotypic variant is characterized by a slowdown of cell metabolism that promotes bacteria dormant state. The molecular mechanisms leading to persisters formation have not been elucidated, yet. One of the mechanisms that was thought to be involved in the persister formation is the stringent response, but this connection was retracted. However, the first step of the stringent response is the accumulation of (p)ppGpp (guanosine tetra or pentaphosphate), alarmone synthesised by a family of enzymes called RelA/SpoT Homologue (RSH), that has pleiotropic effect on the cell including the formation of persister cells.0 This PhD thesis is part of a multidisciplinary research project (ERC-StG ERACHRON, grant n. 758108) whose aim is hampering persister formation by blocking the stringent response at its early stage, inhibiting the RSH proteins synthetase activity. Specifically, the aim of this thesis was to identify, by in silico approaches, specific chemotypes able to interact with the synthetase active site of RelSeq, a RSH protein from Streptococcus equisimilis (RelSeq). Starting from the X-ray structure of RelSeq, virtual screening campaigns and molecular dynamics (MD) simulations were carried out. The identified chemotypes were then used to generate potential RelSeq ligands able to inhibit (p)ppGpp synthesis. In silico predictions and the activity of selected compounds were experimentally determined by thermal shift assays. Moreover, the role of GDP and Mn2+ in modulating the 3D conformation and the dynamic behaviour of RelSeq was also studied by means of molecular dynamics simulations.