TY - JOUR T1 - Direct measurement of kinetic parameters of ABCG2-dependent transport of natural flavonoids using a fluorogenic substrate JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther DO - 10.1124/jpet.119.261347 SP - jpet.119.261347 AU - Michal Rozanski AU - Maciej Studzian AU - Lukasz Pulaski Y1 - 2019/01/01 UR - http://jpet.aspetjournals.org/content/early/2019/09/09/jpet.119.261347.abstract N2 - Flavonoids are an important part of the human diet as plant-derived polyphenols and the mechanisms governing their pharmacokinetics are important both due to their own nutriceutical activity and due to the potential for food-drug interactions. A central determinant of absorption and distribution of flavonoids in the human body is the ABC transporter ABCG2, expressed in gut epithelium and in 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 which allows the direct measurement of kinetic constants for ABCG2-mediated cellular efflux of natural flavonoids thanks to the application of fluorogenic 2-aminoethyl diphenylborinate (APB) which reacts with intracellular flavonoids forming a fluorescent, non-membrane-permeable conjugate, thus making it possible to measure the intracellular substrate concentration throughout the experiment. Our studies were performed in MDCKII-derived 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 pre-loaded cells - we obtained Km values of 137 μM for quercetin, 36 μM for kaempferol and 348 μM for luteolin. Our method allows also direct verification of transport inhibition mechanism and potentially 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. ER -