Characterization of Medically Relevant ABC Transporters for Targeted Drug Therapy

摘要

ABC Transporters, a ubiquitous class of membrane proteins, utilize ATP to transport a range of compounds out of or into a cell. Three ABC transporters were studied: P-glycoprotein (P-gp), Breast Cancer Resistance Protein (BCRP), and MsbA. P-gp and BCRP are mammalian proteins implicated in multidrug resistance. Inhibition of these enzymes in cancer cells leads to the re-sensitization of cells to anti-cancer drugs. MsbA is a prokaryotic lipid flippase, essential in gram-negative bacteria for proper membrane and might therefore be viewed as an antibiotic target. The fact that each of these ABC transporters can also transports a wide variety of drug-like compounds across the membrane, however, complicates the search for effective inhibitors. Computational screens were performed to search for drug-like compounds that effectively inhibit ATP hydrolysis but are not transport substrates. Identified compounds were purchased and tested in both in vivo and in vitro models to determine efficacy.;P-gp was cloned into and expressed in E. coli, but with low yield, likely due to inclusion bodies. BCRP expression in E. coli was inconclusive, therefore expression was tested in the P. pastoris eukaryotic system. Here, BCRP had high expression levels in membranes, and purification experiments are currently underway. Further experiments will focus on screening in silico identified inhibitors.;A His6-tagged MsbA was overexpressed and purified from E. coli and reconstituted into nanodiscs, where MsbA has been shown in the past to have high catalytic activity. ATP hydrolysis rates of MsbA were measured in the presence and absence of potential inhibitors using a coupled enzyme assay. E. coli growth assays in the presence of identified compounds were used as an in vivo model to evaluate potential growth inhibition. Preliminary results suggest that several compounds identified by the computational screening inhibited MsbA ATP hydrolysis activity. Further experiments will aim to better characterize and optimize these compounds for potential use as novel antibiotics.