Allosteric Inhibition of P-Glycoprotein-Mediated Efflux by DMH1

Background/Objectives: P-glycoprotein (P-gp), an ATP-binding cassette (ABC) transporter, plays a key role in multidrug resistance by actively exporting chemotherapeutic agents and xenobiotics from cells. Overexpression of P-gp significantly reduces intracellular drug accumulation and compromises treatment efficacy. Despite extensive research, clinically approved P-gp inhibitors remain elusive due to toxicity, poor specificity, and limited efficacy. This study investigates DMH1, a selective type I BMP receptor inhibitor, as a novel P-gp inhibitor. Methods: DMH1 cytotoxicity was assessed in P-gp-overexpressing (PC3-TxR, K562/Dox) and P-gp-deficient (PC3) cell lines using MTT assays. P-gp inhibition was evaluated using calcein AM retention and daunorubicin (DNR) accumulation assays. Kinetic analysis determined DMH1's effect on P-gp-mediated transport (Vmax and Km). ATPase activity assays were performed to assess DMH1's impact on ATP hydrolysis. Preliminary molecular docking (CB-Dock2) was used to predict DMH1's binding site on the human P-gp structure (PDB ID: 6QEX). Results: DMH1 showed no cytotoxicity in P-gp-overexpressing or deficient cells. It significantly enhanced intracellular accumulation of Calcein AM and DNR, indicating effective inhibition of P-gp function. Kinetic data revealed that DMH1 reduced Vmax without affecting Km, consistent with noncompetitive, allosteric inhibition. DMH1 also inhibited ATPase activity in a dose-dependent manner. Docking analysis suggested DMH1 may bind to an allosteric site in the transmembrane domain, potentially stabilizing the inward-facing conformation. Conclusions: DMH1 is a promising noncompetitive, allosteric P-gp inhibitor that enhances intracellular drug retention without cytotoxicity, supporting its potential as a lead compound to overcome multidrug resistance and improve chemotherapeutic efficacy.