The project envisages a multidisciplinary approach, targeting a specific bacterial survival enzyme that, when activated, increases significantly the intracellular levels of a signalling molecule called (p)ppGpp (an “alarmone”). We aim to design small molecules ad hoc to inhibit the accumulation of the alarmone, therefore hampering bacterial persisters formation.
We initially performed computational studies and obtained a catalytically competent model of the enzyme. We performed virtual screening of large and diversified fragment libraries (imagine collections of different Lego bricks) into the catalytic site of the enzyme, identifying three preferred chemotypes (i.e. the best fitting Lego brick shapes into a pocket) that were subsequently modified to obtain the first generation of ligands (i.e. adding small pieces to the core shape). We synthesised a selected set of molecules and performed interaction studies with the target enzyme, finding several ligands with affinities comparable to those of the natural substrates or better. Affinity measurements do not reveal where the binding takes place (i.e. where the Lego brick sticks to the protein). For this reason, we can either assess their effect on the enzyme activity (its inhibition is our end goal) or adopt structural approaches to identify their binding site.
We tried several techniques to quantify the formation of the alarmone, such as nuclear magnetic resonance (NMR), liquid chromatography (HPLC), electrochemistry, and fluorescence measurements with little success. Recently, we have been implementing a coupled enzymatic assay that could provide an easy readout of the reaction.
From the structural point of view, we have been working towards the achievement of the protein-ligand complexes X-ray crystal structures (in collaboration with another research group) and towards the assignment of the protein nuclear resonances in solution.
These results have been disseminated over the years by all involved group members mostly in the form of poster or oral communication at conferences.
