Assessing Lipid Activation of Human P‐Glycoprotein for ATP Hydrolysis Analyses in the Presence of Novel Inhibitors

Abstract

P‐glycoprotein (P‐gp) belongs to the ATP‐Binding Cassette (ABC) transporter family of protein. This class of membrane proteins is strongly linked with multidrug resistance (MDR) in tumors where overexpression of these proteins often nullifies the effects of chemotherapeutics by removing the therapeutics from the cells. In hopes of creating co‐therapeutics to re‐sensitize tumors to chemotherapeutics, we previously used high‐throughput in silico ligand docking studies and identified drug‐like compounds that reversed MDR in human cancer cells in culture. The effects on P‐gp ATPase hydrolysis activity were used as an initial biochemical screen for inhibitor effectiveness followed by assessments of MDR‐reversal in various P‐gp overexpressing cancer cell lines. For the initial biochemical analyses, P‐glycoprotein (MDR3) from mouse was used, which is closely related to the human (MDR1) protein. To evaluate the mechanism of inhibition also in the human P‐protein, we have isolated human P‐gp from the yeast Pichia pastoris using mixed detergent micelles. Lipid activation of ABC transporters has been shown previously to be a vital step in ensuring best enzyme activity for the analysis of such proteins. In our pursuit to optimize the P‐gp ATPase activity, we have varied amounts and combinations of different lipids including phosphatidylcholine (PC), E. coli lipids and cholesterol and assessed the resulting ATPase hydrolysis activities in coupled enzyme assays. Preliminary results showed P‐gp activity in the presence of PC was significantly higher than when E. coli lipids were used. Here we report our efforts to achieve the best protein:lipid ratios as well as assess various techniques to achieve the high ATP hydrolysis activity of P‐gp to test our newly found drug‐like molecules in their effectiveness as P‐gp inhibitors.Support or Funding InformationThis work was supported by NIH NIGMS [R15GM094771‐02] to PDV and JGW, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas. The authors would like to thank Dr. Ina L. Urbatsch, Texas Tech University Health Science Center, for providing the P. pastoris cells expressing the human wild‐type MDR1 used in this work.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.