P-glycoprotein (P-gp) is a multidrug resistance (MDR) transporter with unknown structural details. This macromolecule is normally responsible for extruding xenobiotics from normal cells. Overexpression of P-gp in tumor cells is a major obstacle in cancer chemotherapy. In this study, human 3D model of P-gp was built by homology modeling based on mouse P-gp crystallographic structure and stabilized through 1 ns molecular dynamics (MD) simulation. Stabilized human P-gp structure was used for flexible docking of 80 drugs into the putative active site of P-gp. Accordingly, digoxin, itraconazole, risperidone, ketoconazole, prazosin, verapamil, cyclosporine A, and ranitidine were selected for further in vitro assay. Subsequently, cell-based P-gp inhibition assay was performed on Caco-2 cells while 99m Tc-methoxyisobutylisonitrile (MIBI) was used as a P-gp efflux substrate for calculating IC50 values. Results of the 99m Tc-MIBI uptake in drug-treated Caco-2 cells were in agreement with the previously reported activities. This study for the first time described the relation between molecular dynamics and flexible docking with cellular experiments using 99m Tc-MIBI radiotracer for evaluation of potencies of P-gp inhibitors. Finally, results showed that our radiotracer-cell-based assay is an accurate and fast screening tool for detecting P-gp inhibitors and non-inhibitors in drug development process.
Keywords: 99mTc-MIBI; Caco-2; P-glycoprotein; flexible docking; homology modeling.
© 2018 John Wiley & Sons A/S.