An artificial model of ATP-binding cassette mediated transport.

Active drug efflux is a critical bottleneck in multidrug-resistance (MDR) in cancers, bacterial infections and in neurological disease. Preclinical acellular permeation assays do not predict efflux (active) transport, and cell-based assays may be confounded by the complexity and heterogeneity of cells which can make efflux data analysis challenging. We report here a cell-free platform as a model for functional multidrug resistance (MDR) efflux pump that mimics the inner membrane of Escherichia coli (E. coli). The system comprises a PE:PG:CL (67:23.2:9.8 mol%) lipid bilayer reconstituted with ABC (ATP-Binding Cassette) transporter BmrA, that is suspended over gold and PDMS microcavity pore arrays. Using real-time electrochemical impedance spectroscopy, adsorption and intercalation of six antibiotics-doxorubicin, daunorubicin, epirubicin, moxifloxacin, lomefloxacin, and vancomycin-were detected in E. coli bilayers with and without reconstituted BmrA, each producing a characteristic decrease in membrane admittance. Upon activation of BmrA with Mg2 +/ATP, all six drugs displayed ATP-dependent efflux, manifested as increases in membrane admittance. The extent of drug-membrane adsorption and active transport was quantified by fitting the impedance responses to kinetic association-dissociation models. Transporter mediated efflux was then validated using a direct assay format, by tracking the metal-enhanced fluorescence (MEF) of intrinsic doxorubicin fluorescence in real time. Inhibition studies revealed that verapamil suppressed ATP-dependent transport for all tested drugs except epirubicin. These results establish the microcavity-supported lipid bilayer (MSLB) platform as a robust, biomimetic, and label-free system capable of resolving active versus passive drug transport across biomembranes. The approach offers a powerful tool for mechanistic interrogation of ABC transporters and may accelerate the discovery of antibiotics and efflux pump inhibitors.