Elisa Sassetti

• Antibiotic resistance has become a world-wide problem as the number of resistant and multi-resistant bacteria has dramatically increased over the last years, especially for Gram-negative bacteria. The PhD project, part of INTEGRATE, a multidisciplinary Marie Curie Educational Training Network (ETN), concerned the identification and characterization of small molecule modulators of the enzyme Caseinolytic protease proteolytic subunit (ClpP). ClpP is a serine protease and it has been proposed as an antibacterial target because of its central roles in many essential bacterial cellular processes. The main aim of this work is the identification of inhibitors directed against ClpP from Gram-negative bacteria, using E. coli as model organism. An in vitro target-centric approach was adopted to identify novel hits by screening collections of small molecules, including diverse compounds. Selected compounds were profiled in biochemical assays and confirmed in biophysical readouts. For the most promising compounds, potential cytotoxicity against selected human cell lines was evaluated. The compound efficacy as an anti-bacterial in the presence and absence of selected stress condition and the hypothetical binding mode (using in silico approaches) were determined. In parallel, an ongoing structural biology effort is ongoing to reveal information on the interaction between selected compounds and ClpP and validate the computational models. With this PhD thesis, further validation of the existence of phenotypic ClpP-related effects in E. coli can be found and new evidence provided for the role of ClpP as a valid target in Escherichia coli and Gram-negative bacteria antibiotic research. This thesis reports several compounds active in vitro in the low micromolar or sub-micromolar range, with an acceptable safety profile and with possible ClpP-related activity in bacteria. This study must be seen as an important starting point for further development inhibitors in follow on studies.