Fellow: Venkata Krishnan Ramaswamy
Supervisor: Paolo Ruggerone (University of Cagliari)
Our computational research is focused on the efflux pumps of the resistance-nodulation-division (RND) family, the major transporter superfamily responsible for the appearance of multi drug resistance. We are performing a comparative study of transporters of the RND family showing different substrate specificity, i.e., AcrD and AcrB of E. coli and MexY and MexB of P. aeruginosa. For instance, AcrD and MexY transport aminoglycosides but AcrB and MexB do not. The results of this comparative investigation will offer insights into the microscopic details of the functioning of the efflux systems and, when mapped onto the chemical structures of the compounds considered in the present study, will possibly help the design of molecules able to escape and/or to inhibit the efflux systems.
University of Cagliari together with Jacobs University and Goethe University has performed MD and state-of-the-art docking studies on AcrB addressing also the impact of a point mutation on AcrB activity. Additionally, UCA, in collaboration with BPI, has investigated the recognition mechanism of imipenem and meropenem by MexB. Still unclear are several issues, such as to what extent the functional rotation, i.e., the specific series of sequential conformational changes, is essential for the drug extrusion and whether cooperativity effects are also involved. Thus, molecular details of the mechanism, recognition and uptake for AcrB and MexB require further investigations that will be performed by University of Cagliari in collaboration with Jacobs University and Goethe University. Inhibitors, used in combination with antibiotics, expand the spectrum of antibacterial activity, reverse resistance and dramatically reduce the rates of resistance development, but the molecular details of their action are still elusive. The ‘non-specificity’ of the transporters asks for the role of the pump’s putative affinity in resistance and inhibition. Additionally, insights on possible allosteric sites in the efflux pumps will be gained by extended MD simulations and indicate sites to be targeted by inhibitors.
Venkata Krishnan is carrying out a very thorough computational study of the RND transporters of E. coli and P. aeruginosa, comparison of the interaction pattern of compounds that are affected differently by different efflux systems; and also comparison of interaction pattern of compounds that are poor and good substrates of a specific efflux system.
The research requires a series of steps:
- Homology modeling. For two transporters, namely AcrD and MexY, no crystal structures are to date available, although the purification of AcrD has already been completed and crystallization is ongoing within the ITN Translocation Consortium (Prof Klaas Martinus Pos, Goethe University). Structures of AcrD and MexY have to be built by homology modeling using the crystal structures of AcrB and MexB as template followed by model validation with bioinformatics tools.
- MD simulations of the selected models. Equilibration of the models is performed by MD simulations with the transporters inserted in the membrane, in the presence of the solvent and at physiological temperature. The resulting trajectories will be used to characterize the key features of the dynamics of the systems, i.e., interaction with solvents, relevant interactions stabilizing the proteins, structural features of key regions and domains. The comparison with results obtained by Cagliari’s group on AcrB and MexB will be helpful to select putative relevant regions in AcrD and MexY.
- Molecular Docking. Docking runs of ceftobiprole and kanamycin, two compounds known to be extruded by MexY, have already been performed using the homology model of the transporter. Furthermore, the extensive trajectories of Point b will be exploited to identify the relevant conformations explored by the systems during the simulation time. These representatives will be used for docking runs to analyse the binding of selected compounds to dynamical structures. Similar procedure will be adopted for AcrD.
- Interaction pattern. The poses extracted by the docking runs of Point c will undergo validation and equilibration by MD simulations. The trajectories will be analysed to extract key interaction patterns of the different compounds with the transporters. The interaction with the solvent in terms of residence time of the water molecules in specific regions will be also examined. Finally, the affinity of the compounds will be dissected in residue contributions to highlight hotspots of the interaction.
- MD simulations of AcrB. Simulations of AcrB already performed in Cagliari will be extended and analysed in tune with the strategy highlighted in Point b. Behaviour of waters, relevant motions of domains and regions will be identified. This will offer insights into possible coupling between key parts of the transporter. Additionally, the interactions of compounds that are known as poor substrate of AcrB but are affected by AcrD will be studied in their corresponding docked complexes. Analogous research will be done for MexB