2.7. P-gp ATPase assay

The effect of compounds 1-5 on P-gp ATPase activity was measured using the Pgp-Glo™ assay system. Verapamil, a P-gp substrate and competitive inhibitor for other substrates, was used as a positive control. Sodium orthovanadate (Na₃VO₄), a selective inhibitor of P-gp ATPase, was used to account for P-gp-independent ATP consumption in the reactions. All compounds and controls were tested at either 40 μM or 200 μM (in 1% DMSO) as per the manufacturer's protocol. Sample luminescence was recorded after 50 min incubation with the ATP detection reagent using a luminometer (POLARstar Omega, BMG Labtech, Ortenberg, Germany). Change in luminescence in relative light units (RLU; RLU_Na3VO4–RLU_compound) is inversely proportional to ATP levels, which are negatively correlated with the activity of P-gp ATPase and therefore P-gp-mediated transport.