The role of molecular complexes in overcoming multidrug resistance (MDR) caused by efflux pumps

Multidrug resistance [MDR] is an ever increasing problem in bacterial cells and cancer cells. One mechanism of resistance occurs due to the presence of efflux pumps. One way of combating MDR is by inhibiting the efflux pump activity via binding of inhibitors to the binding sites of efflux pumps or via substrate-escort molecule complexation. This study aims to develop an in silico screening method to find escort molecules and to provide experimental evidence of substrate-escort molecule complex formation. A similarity search was performed to find potential escort molecules that might modulate norfloxacin and tetracycline against MDR strains of Staphylococcus aureus. Molecular modelling experiments were used to calculate the interaction energies of antibiotics with selected drugs and natural products. Evaluating the interaction energies of the complexes found that many of the drugs and natural products selected from the Super Drug and Super Natural databases could be potential escort molecules as they exhibited interaction energies that were ≤ -9.0 kcal/mol. It was found that the major interaction involved in complexation was aromatic pi-pi interactions and that the change in physicochemical properties found upon complexation would increase the permeability of the antibiotics. The computational studies found that resveratrol, fisetin, apomorphine, chlorpromazine, nordihydroguaiaretic acid and equilin were the best candidates as escort molecules. Apomorphine, chlorpromazine, nordihydroguaiaretic acid and resveratrol did modulate norfloxacin and tetracycline against MDR strains of S. aureus. Checkerboard assays indicated that this was due to a synergistic interaction. Nuclear magnetic resonance (NMR) spectroscopy, ultraviolet (UV) spectroscopy and mass spectrometry were able to successfully detect complex formation between these escort molecules and the antibiotics norfloxacin and tetracycline. Molecular modelling experiments were performed using mitoxantrone and 123 structurally diverse breast cancer resistance protein [BCRP] modulators. It was found that the potentiation [modulation] of mitoxantrone could be correlated qualitatively with interaction energies and experimentally the presence of mitoxantrone-escort molecule complexation was detected using NMR spectroscopy and mass spectrometry techniques. To study other mechanisms for overcoming MDR, molecular docking experiments were carried out using the permeability glycoprotein [P-gp] efflux pump and anticancer agents and inhibitors of P-gp. It was found that first and second generation inhibitors bind to P-gp in the same binding space as anticancer agents creating competition. Therefore, it is possible that these inhibitors act via competitive inhibition. Additionally, two taxol analogues were synthesised, however these were found to be less cytotoxic than paclitaxel against MCF-7 human breast cancer cells. Overall, this study has been able to provide experimental evidence of substrate-escort molecule complexation and shown that escort molecules can restore the activity of antibiotics and anticancer agents making complex formation a mechanism that can modulate the activity of the efflux pumps. An in silico screening method was proposed to find potential escort molecules for antibiotics; however this should be subject to further development to encompass other criteria in the search for MDR inhibitors.