Novel approaches to identify small molecules modulating TolC protein function of E. coli
Fellow: Alessia Gilardi
Supervisor:Dr Philip Gribbon (IME Fraunhofer Institut)
Co-supervisor: Mathias Winterhalter (Jacobs University)
In the frame of the ITN Translocation project the main goal is a better understanding of the mechanisms involved in gram-negative bacteria antibiotic resistance and the finding of reliable solutions to overcome this problem. Down-regulation of porin expression and increasing of translocation of molecules through efflux systems are the key mechanisms used by bacteria to reduce the presence of toxic compounds, such as antibiotics, in their intracellular and periplasmic space. Within the project developed at the Fraunhofer IME ScreeningPort, the efflux system AcrAB-TolC present in E. coli has been selected as a target, focusing major attention on the outer membrane protein TolC of this tripartite complex.
TolC is a homotrimer of ~53 KDa, presenting an alpha/beta-barrel structure, that spanning from the periplasm to the outer membrane, creates a channel through which the molecules can be transported to the extracellular space. In its structure 3 main domains can be identified: an outer membrane domain, consisting in a rigid beta-barrel inserted in the outer membrane; an equatorial domain, where TolC can essentially get in contact with the periplasmic adaptor protein AcrA; and a periplasmic domain, site of interaction with AcrB, catalytic protomer of the complex. The periplasmic site seems to have an important role in the control of TolC functionality, in fact it presents two Aspartates ring (two residues for each monomer) are interacting together to keep a close conformation of the protein channel. So far only few compounds have been identified showing inhibitory activity against this protein (all toxic) and a molecule of hexaamminecobalt has been co-crystallized with TolC, interacting with the aspartate rings present in the periplasmic site.
Based on these studies a strategy was developed for the identification of small molecules that could act on the periplasmic site of TolC and specifically with aspartate rings, keeping the protein structure in its closing position. A virtual screening has been set up using different software and tools to prepare the protein structure as well as a deep analysis of the target site, virtual compound library preparation and docking parameters. At the same time an assay was established to characterize functional and electrophysiological features of TolC, confirming previous literature data in order to have a robust system where screen compound hits resulting from the in silico screening. Alternative approaches have been planned to target the AcrAB-TolC complex. Recent publications have shown how the interaction between AcrA and TolC could be fundamental for TolC to switch to its opening state and complete its function. For this reason it would be interesting to investigate which regions of the proteins are involved in the cited interaction and conduct an in silico and in vitro campaign to identify compounds able to interact in that area.
The X-ray structure of the wild type TolC (PDB ID: 1EK9) and the co-crystallized structure with the hexaamminecobalt bound (PDB ID: 1TQQ) have been deeply analyzed and prepared for the virtual screening campaign. In first place, the structures have been protonated using the “protonate 3D” function of the MOE suite, while the energy minimization step was done using the SYBYL tool, after a testing process of different parameters for the identification of the best conditions. The structures were then analyzed using validation tools such as Procheck and Prosa. A database has been prepared starting from the Clean Leads subset (ZINC – is not commercial – library): from the original group of about 6 million compounds, a first selection has been done discarding the molecules without any positive charged amino group and following the accordance with drug likeness (Lipinski’s rule of 5).
300000 compounds have been docked using GOLD docking software (parameters selected after a test on a set of benchmark proteins, data not shown), based on a Lamarckian Genetic Algorithm to predict the position of interaction between protein and ligand. The best ranked poses have been selected for a post-docking analysis. Almost 3000 compounds have been re-docked to obtain more precise information about protein-ligand interaction and Ligand Efficiency (LE). Interesting its will be selected for in vitro screening.
Figure 1: Structure TolC (PDB ID: 1EK9) with highlighted site targeted for the virtual screening (left). Interactions between a docked compound with amino acid residues of the target site (right)
TolC functional characterization
Functionality and conductance of TolC have been investigated in the past, showing and proving its closing state in physiological conditions and not complexed to AcrA/B.
On biophysical cell-free based assay, using the Black Lipid Bilayer (BLM) method, in a single insertion experiments, TolC shows specific features confirming data presented previously in literature.
The conductance value is about 80 pS at low-medium voltages (±10-100 mV), while shows an increase at higher voltages (100-160 mV). From this we can generally prove that its opening state is voltage-dependent, while further validation on pH influences are under process.
In presence of hexaamminecobalt at nM concentration, TolC shows decreased conductance values (about 30 pS) and blockage events visible in recorded traces. The same compound has been also co-crystallized bound to the periplasmic site of the protein, proving its interaction with the aspartate rings, involved in the control of the opening/closing state.These functional studies are the starting point for in vitro screening of compound hits from the computational studies