Inside-out-oriented membrane vesicles are useful tools to investigate whether a compound can be an inhibitor of efflux transporters such as multidrug resistance-associated protein 2 (MRP2). However, because of technical limitations of substrate diffusion and low dynamic uptake windows for interacting drugs used in the clinic, estradiol-17-glucuronide (E17G) remains the probe substrate that is frequently used in MRP2 inhibition assays. Here we recapitulated the sigmoidal kinetics of MRP2-mediated transport of E17G, with apparent Michaelis-Menten constant () and values of 170 ±17M and 1447 ± 137 pmol/mg protein/min, respectively. The Hill coefficient (2.05 ± 0.1) suggests multiple substrate binding sites for E17G transport with cooperative interactions. Using E17G as a probe substrate, 51 of 97 compounds tested (53%) showed up to 6-fold stimulatory effects. Here, we demonstrate for the first time that coproporphyrin-I (CP-I) is a MRP2 substrate in membrane vesicles. The uptake of CP-I followed a hyperbolic relationship, adequately described by the standard Michaelis-Menten equation (apparent and values were 7.7 ± 0.7 M and 48 ± 11 pmol/mg protein/min, respectively), suggesting the involvement of a single binding site. Of the 47 compounds tested, 30 compounds were inhibitors of human MRP2 and 8 compounds (17%) stimulated MRP2-mediated CP-I transport. The stimulators were found to share the basic backbone structure of the physiologic steroids, which suggests a potential in vivo relevance of in vitro stimulation of MRP2 transport. We concluded that CP-I could be an alternative in vitro probe substrate replacing E17G for appreciating MRP2 interactions while minimizing potential false-negative results for MRP2 inhibition due to stimulatory effects.
P-glycoprotein (P-gp) is a plasma membrane efflux transporter belonging to ATP-binding cassette superfamily, responsible for multidrug resistance in tumor cells. Over-expression of P-gp in cancer cells limits the efficacy of many anticancer drugs. A clear understanding of P-gp substrate binding will be advantageous in early drug discovery process. However, substrate poly-specificity of P-gp is a limiting factor in rational drug design. In this investigation, we report a dynamic trans-membrane model of P-gp that accurately identified the substrate binding residues of known anticancer agents. The study included homology modeling of human P-gp based on the crystal structure of C. elegans P-gp, molecular docking, molecular dynamics analyses and binding free energy calculations. The model was further utilized to speculate substrate propensity of in-house anticancer compounds. The model demonstrated promising results with one anticancer compound (NSC745689). As per our observations, the molecule could be a potential lead for anticancer agents devoid of P-gp mediated multiple drug resistance. The in silico results were further validated experimentally using Caco-2 cell lines studies, where NSC745689 exhibited poor permeability (P app 1.03 ± 0.16 × 10(-6) cm/s) and low efflux ratio of 0.26.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.