Biochemical evaluation of drug inhibition of human MDR1 P‐glycoprotein

Abstract

P‐glycoprotein (P‐gp) overexpression is often correlated with multidrug‐resistance (MDR) of cancer. To search for P‐gp inhibitors as co‐therapeutics to combat MDR, we had previously used high‐throughput in silico ligand docking studies to identify drug‐like compounds and their effects on P‐gp ATPase hydrolysis activity, followed by assays of MDR‐reversal of a prostate and ovarian cancer cell lines. More recently, additional computationally identified compounds were shown to reverse MDR in cancer cells. Biochemical assays using purified P‐gp provide important information regarding the mechanism of inhibition and have been used as primary screens in our drug finding process. Some of these newly found compounds were observed to be inhibitors of ATP hydrolysis activity of a cysteine‐less mouse MDR3 homolog of the human MDR1 P‐glycoprotein. Even though human and mouse P‐gp share about 87% identity in amino acid sequence, there may still be sufficient differences between the two proteins that may prevent MDR3 from faithfully reporting on inhibitor action on the human protein. High expression of human P‐gp in Pichia pastoris in a biologically active form was previously reported and the purified P‐gp was used on our recent assays. Lipid activation of ABC transporters, as shown by others, is a requirement to achieve optimal enzyme activity, but in our hands has proven to be more challenging than expected. The best P‐gp activation was achieved using L‐α‐phosphatidyl choline. The resulting lipid‐activated P‐gp showed low basal activity (~8.5 nmol NADH/min/mg P‐gp), but more than 30‐fold activation by the P‐gp transport substrate, verapamil (~306 nmol NADH/min/mg P‐gp). Here we report our optimized strategy to lipid‐activate wild‐type human P‐gp as well as the evaluation of the potential of our previously discovered drug‐like compounds to serve as inhibitors of human P‐gp ATP hydrolysis activity.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.