Preventing the adhesion of pathogens to host cells provides an innovative approach to tackling multidrug-resistant bacteria. In this regard, the identification of outer membrane protein A (OmpA) as a key bacterial virulence factor has been a major breakthrough. The use of virtual screening helped us to identify a cyclic hexapeptide AOA-2 that inhibits the adhesion of Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli to host cells and the formation of biofilm, thereby preventing the development of infection in vitro and in a murine sepsis peritoneal model. Inhibition of OmpA offers a strategy as monotherapy to address the urgent need for treatments for infections caused by Gram-negative bacilli.

Deciphering conformational dynamics is crucial for understanding the biological functions of proteins and for designing compounds targeting them. In particular, providing an accurate description of µs-ms motions opens up the opportunity for regulating protein-protein interactions (PPIs) by modulating dynamics of one interacting partner. Here we analyzed the conformational dynamics of prolyl oligopeptidase (POP) and the effects of active site-directed inhibitors on the dynamics. For this purpose, we used an integrated structural biology approach based on NMR spectroscopy and SAXS experiments complemented by MD simulations. Our results found that POP is in a slow equilibrium in solution between open and closed conformations, and that inhibitors effectively prevented this equilibrium by stabilizing the enzyme in a closed conformation.

Prolyl oligopeptidase (POP) is a large 80 kDa protease, which cleaves oligopeptides at the C-terminal side of proline residues and constitutes an important pharmaceutical target. Despite the existence of several crystallographic structures, there is an open debate about migration (entrance and exit) pathways for ligands, and their coupling with protein dynamics. Recent studies have shown the capabilities of molecular dynamics and classical force fields in describing spontaneous binding events and nonbiased ligand migration pathways. Due to POP's size and to the buried nature of its active site, an exhaustive sampling by means of conventional long enough molecular dynamics trajectories is still a nearly impossible task. Such a level of sampling, however, is possible with the breakthrough protein energy landscape exploration technique. Here, we present an exhaustive sampling of POP with a known inhibitor, Z-pro-prolinal. In >3000 trajectories Z-pro-prolinal explores all the accessible surface area, showing multiple entrance events into the large internal cavity through the pore in the β-propeller domain. Moreover, we modeled a natural substrate binding and product release by predicting the entrance of an undecapeptide substrate, followed by manual active site cleavage and nonbiased exit of one of the products (a dipeptide). The product exit shows preference from a flexible 18-amino acid residues loop, pointing to an overall mechanism where entrance and exit occur in different sites.

After decades of work, the correct determination of the binding mode of a small molecule into a target protein is still a challenging problem, whose difficulty depends on: (i) the sizes of the binding site and the ligand; (ii) the flexibility of both interacting partners, and (iii) the differential solvation of bound and unbound partners. We have evaluated the performance of standard rigid(receptor)/flexible(ligand) docking approaches with respect to last-generation fully flexible docking methods to obtain reasonable poses in a very challenging case: soluble Epoxide Hydrolase (sEH), a flexible protein showing different binding sites. We found that full description of the flexibility of both protein and ligand and accurate description of solvation leads to significant improvement in the ability of docking to reproduce well known binding modes, and at the same time capture the intrinsic binding promiscuity of the protein.