Pgp glo assay system

The σ₂ receptor ligand A011 was prepared as previously reported (Feng et al., 2019 (link)). KO143 was purchased from MedChemExpress (Monmouth Junction, NJ, United States). Cisplatin (DDP), adriamycin (ADR), paclitaxel, verapamil, mitoxantrone and rhodamine 123 (Rh123) were acquired from Solarbio (Beijing, China). Cell Counting Kit-8 (CCK-8) was bought from Dojindo Laboratories (Japan). Pgp-Glo™ Assay Systems were purchased from Promega (Madison, USA). Anti-P Glycoprotein antibody, ABCG2, GAPDH, Goat Anti-Rabbit IgG H&L Secondary Antibody (Alexa Fluor 488), Goat Anti-Rabbit IgG H&L Secondary Antibody and Goat Anti-Mouse IgG H&L Secondary Antibody were purchased from Abcam (Cambridge, United Kingdom). SlowFade^TM Gold antifade reagent was purchased from Thermo Fisher (MA, United States).

P-gp inhibitors block its efflux pumping activity via either covalent binding or inhibiting P-gp ATPase activity. Human recombinant membrane bound P-gp protein attached with ATPase subunit (Pgp-Glo™ Assay Systems, Promega Corporation, Madison, WI, USA) was used as previously described to determine the mechanism of P-gp inhibition via determining ATP consumption rate [18 (link),25 (link)]. Briefly, test compounds (10 µM) were incubated with Pgp-Glo™ Assay Systems according to manufacturer’s protocol. Rate of ATP consumption was calculated by measuring the luminescent signal of the unmetabolized ATP via a firefly luciferase system. Compounds whose covalents bind to P-gp substrate binding sites are supposed to stimulate ATPase subunits and increase ATP consumption, while ATPase inhibitor compounds would decrease ATPase subunit activity and decrease the ATP consumption rate. Verapamil and sodium vanadate were used as positive controls (binding site blocker and ATPase inhibitors, respectively). ATP consumption was expressed as remaining ATP concentration and normalized per P-gp protein concentration (p.mole ATP/µg P-gp protein).

The ABCB1-associated ATPase activity was measured using the Pgp-Glo™ Assay Systems (Promega). Briefly, ABCB1 membranes were incubated with gradient concentrations of verapamil, lapatinib or dFdCTP on the 37°C heat block for 5 minutes. Then 10 μl of 25 mM MgATP were added into each well and incubated at 37°C for 100 minutes. Then the reaction was terminated by adding 50 μl ATP Detection Reagent into all wells. Luminescence was then read on a multi-detection microplate reader (Biotek).

The P-gp ATPase activity was tested by Pgp-Glo™ Assay Systems (Promega). The impact of candidate compounds on P-gp ATPase activity were examined by comparing untreated samples and samples treated with Na₃VO₄ (sodium orthovanadate). The compounds could be assessed as stimulating, inhibiting, or having no effect on basal P-gp ATPase activity.

To reveal the mechanism of P-gp-inhibition induced by RES and DID, assessment of P-gp attached ATPase activity was performed using Pgp-Glo™ Assay Systems (Promega Corporation, Madison, Wisconsin, USA). Briefly, in 96-well plates, Pgp-Glo™ Assay Buffer was mixed with Na₃VO₄, VRP, RES, or DID. Recombinant human P-gp membrane fraction was incubated with the reaction mixture at 37 °C for about 5 minutes, and then the reaction was initiated by adding 10 μl of 25 mM MgATP and incubating for 40 minutes at 37 °C. An identical reaction mixture without drug treatment (NTC) was assayed in parallel. The reaction was stopped and the remaining un-consumed ATP was detected using luciferase firefly luminescent signal and ATP standard curve was plotted. The remaining concentrations of ATP were expressed as (p. mole/μg P-gp molecules). Sodium vanadate (Na₃VO₄) was used as selective inhibitor of P-gp related ATPase enzyme, and samples treated with Na₃VO₄ have greater luminescent signal than un-treated samples (NTC). In addition, VRP inhibits pgp molecules via covalent binding and competing with other substrates, hence, stimulating ATPase activity resulting in more consumption of ATP molecules compared to NTC.

Assessment of Pgp-attached ATPase activity was performed using Pgp-Glo^™ Assay Systems (Promega Corporation, Madison, Wisconsin, USA). Briefly, in 96-well plates, Pgp-Glo^™ Assay Buffer was mixed with sodium vanadate, VRP, or test compound (10 μM). Recombinant human Pgp membrane fraction was incubated with the reaction mixture at 37°C for about 5 minutes, and then the reaction was initiated by adding 10μl of 25mM MgATP and incubating for 40 minutes at 37°C. An identical reaction mixture without drug treatment (NTC) was assayed in parallel. The reaction was stopped and the remaining un-consumed ATP was detected using luciferase firefly luminescent signal and ATP standard curve was plotted. The remaining concentrations of ATP were expressed as (p. mole/μg Pgp molecules). Sodium vanadate (Na₃VO₄) was used as selective inhibitor of Pgp related ATPase enzyme, and samples treated with Na₃VO₄ have greater luminescent signal than un-treated samples (NTC). In addition, VRP inhibits pgp molecules via covalent binding and competing with other substrates, hence, stimulating ATPase activity resulting in more consumption of ATP molecules compared to NTC.