Structure-Based Design of RSH inhibitors to Hamper persistence

Among the mechanisms that stimulate and regulate chronic infections, bacterial persistence plays an important role. Many efforts have been already done to hamper the persistence, but none of them stop definitively the process. One of the most studied pathways involved in persister formation is the stringent response, that is activated by stress conditions such as: nutrient starvation, darkness, antibiotic treatment, etc. In this signaling cascade, the first event is the hyper-activation of RSH (RelA/SpoT homolog) proteins in forming (p)ppGpp (guanosine tetra- or petaphosphate). Based on this, the aim of this study is to find out potential inhibitors of RSH enzymes, selective for the synthetase site. To this purpose, by using the crystalized structure of bifunctional RSH from Steptococcus disgalactiae subs. equisimilis (pdb entry 1VJ7), we performed: i) virtual screening (VS) campaigns of eight fragment libraries, by using Glide v.8.0; ii) a chemotype expansion, used for the following docking simulations, of the best ranked scaffolds selected from the VS, using Pubchem; and finally iii) molecular dynamics simulations of chosen fragments, by using Desmond driver version 2.3, to evaluate the binding stability of fragments to RSH protein. The promiscuity of selected scaffolds was also evaluated by using the BADAPPLE tool. As a result, the affinity of a small set of fragments for RSH proteins was tested in vitro. Experimental data showed that most of them present binding activity for the synthetase site of RSH and can be considered promising for the design of new ligands.