Biochemical Evaluation of Drug Inhibition of ABC Transporters

Multidrug resistant (MDR) cancers often become resistant to pharmacologically and structurally diverse drugs by overexpressing members of the family of ATP Binding Cassette (ABC) transporters. These membrane pumps protect normal cells by actively exporting a large range of toxins out of the cells. Classic MDR is often correlated with the overexpression of three well-studied mammalian ABC transporters: P-glycoprotein (P-gp), Breast Cancer Resistant Protein (BCRP), and Multidrug Resistant Protein 1 (MRP1). P-gp is the most studied ABC transporter, and the search for P-gp inhibitors as co-therapeutics to combat MDR has had little success after decades of studies. We previously used high-throughput in silico ligand docking studies and identified drug-like compounds that reversed MDR in human cancers cell lines. Other compounds were more recently found that also reversed MDR in cancer cells, but interestingly, not all of them were recognized in the biochemical ATPase inhibition assays 1, 2. One reason why the biochemical screens did not identify these compounds may be that the mouse P-gp homolog used in the biochemical test may be sufficiently different from human P-gp, making the mouse protein non-representative. We have established methods to successfully incorporate P-gp into membrane nanodiscs with high recovery yields and increases in activity and stability. The human MDR1-clone initially available in our lab exhibited low protein yield during purification making it undesirable for further experimentation. A newly constructed MDR1 clone, codon-optimized for expression in P. pastoris, was transformed into regular (GS115) and high expression (PichiaPinkTM) Pichia pastoris systems in attempts to obtain higher protein yield to proceed with further experiments. Both new P. pastoris strains containing human MDR1 failed to express P-gp. Finally, P. pastoris cells expressing the human wild-type MDR1 P-gp were provided by Dr. Ina L. Urbatsch (Texas Tech University Health Science Center). The enzyme was successfully purified in our lab. To improve the overall enzymatic activity of P-gp, several lipid activation tests and assembly into nanodisc with different lipid content were tested for the optimized activity improvement of the protein. P-gp reconstituted into nanodiscs were used to evaluate the potential of our newly discovered drug-like compound to serve as inhibitors of human P-gp ATP hydrolysis activity. Here we also reported our attempts to optimize the expression and purification of BCRP from Pichia pastoris cells