Elucidating the Complex Structural and Molecular Mechanisms Driving P-Glycoprotein-Mediated Transport of Cardiac Glycosides.

P-glycoprotein (Pgp) plays a significant role in the disposition of cardiac glycoside (CG) drugs across the cell membrane. The relatively narrow therapeutic indices of these drugs, coupled with the co-administration of drugs that inhibit Pgp's transport mechanism, often cause an increased level of CG in the patient's plasma, resulting in fatal arrhythmia. Therefore, understanding the underlying mechanism of the CG-Pgp interaction is necessary to circumvent Pgp-mediated transport and effectively design next-generation CGs. In this study, we conducted a comparative analysis to examine the interaction with Pgp and further understand the Pgp-mediated transport of digoxin, digitoxin, digoxigenin, and digitoxigenin. Through the drug-induced kinetic studies of Pgp, our findings suggest that each of the four drugs tested has a single binding site within Pgp. The CG-Pgp binding studies demonstrated that digoxin, digitoxin, and digoxigenin had relatively higher binding affinities. The CG-mediated conformational changes in Pgp indicated that each of the drugs shifts Pgp to an "outward-open" conformation in a nucleotide-dependent manner. STDD NMR indicated that the protons within the δ-lactone ring and the tri-D-digitoxose sugar moieties (glycones) predominantly interact with Pgp. Finally, a model was proposed for CG-induced Pgp-mediated ATP hydrolysis and transport by integrating our data with previously published Pgp-mediated CG transport results.