Direct Measurement of Kinetic Parameters of ABCG2-Dependent Transport of Natural Flavonoids Using a Fluorogenic Substrate

Abstract

Flavonoids are an important part of the human diet since plantderived polyphenols and the mechanisms governing their pharmacokinetics are important both due to their own nutriceutical activity and the potential for food-drug interactions. A central determinant of absorption and distribution of flavonoids in the human body is the ATP-binding cassette transporter ABCG2, expressed in gut epithelium and other barrier tissues. While flavonoids were previously identified as substrates and/or inhibitors of this protein, precise enzyme kinetic calculations of affinity and activity parameters are rare due to the lack of suitable experimental models. We present a novel method that allows the direct measurement of kinetic constants for ABCG2-mediated cellular efflux of natural flavonoids thanks to the application of fluorogenic 2-aminoethyl diphenylborinate, which reacts with intracellular flavonoids forming a fluorescent, nonmembranepermeable conjugate, thus making it possible to measure the intracellular substrate concentration throughout the experiment. Our studies were performed in Madin-Darby canine kidney IIderived cell lines expressing human ABCG2 and involve substrate efflux from whole, unmodified cells, precluding the need for plasma membrane vesicle preparation. We present methods for calculation of enzyme kinetic constants by measuring substrate concentration at efflux-influx equilibrium or during efflux from preloaded cells, and we obtained K m values of 137 mM for quercetin, 36 mM for kaempferol, and 348 mM for luteolin. Our method also allows direct verification of the transport inhibition mechanism and potentially the structure-activity relationship in substrates. SIGNIFICANCE STATEMENT The study presents the first direct calculation of kinetic constants for enzyme-mediated active transport of natural flavonoids in a whole-cell assay, using a fluorogenic compound to measure intracellular substrate concentrations at specific time points. It has implications for nutriceutical use of polyphenols, mechanisms of food-drug interactions, and studies on absorption/ distribution-determining membrane transporters, allowing a quantitative approach to pharmacokinetics of flavonoid transport across barrier tissues.

Inhibitory Effects of Quercetin and Its Main Methyl, Sulfate, and Glucuronic Acid Conjugates on Cytochrome P450 Enzymes, and on OATP, BCRP and MRP2 Transporters

“…In monolayers, selective inhibitors were applied to show the protein specified inhibitions [ 38 , 40 , 74 ]. Based on blood–brain barrier penetration and monolayer cell experiments, Q seems to be transported by BCRP and MRP2 [ 40 , 41 , 43 , 74 , 75 ]. Although the interactions of certain Q conjugates with BCRP and MRP2 have been reported [ 27 , 30 , 42 , 44 , 72 , 76 , 77 ], the characterization of inhibition/transport is far from complete.…”

Review of the pharmacokinetics of French maritime pine bark extract (Pycnogenol®) in humans

“…Besides passive diffusion across bilayer membranes, uptake or efflux via membrane transporter molecules is of high significance for compound absorption, distribution and elimination ( 14 – 16 ). Individual plant-derived polyphenols have been identified as transporter substrates ( 17 , 18 ). Likewise, a Pycnogenol ® metabolite was observed to be subjected to facilitated uptake via GLUT-1 transporters into erythrocytes and endothelial cells ( 19 , 20 ).…”