Substrate-dependent breast cancer resistance protein (Bcrp1/Abcg2)-mediated interactions: consideration of multiple binding sites in in vitro assay design.
In vitro assays are frequently used for the screening of substrates and inhibitors of transporter-mediated efflux. Examining directional flux across Madin-Darby canine kidney (MDCK) II cell monolayers that overexpress a transporter protein is particularly useful in identifying whether or not a candidate compound is an inhibitor or substrate for that transport system. Studies that use a single substrate or inhibitor in competition assays can be challenging to interpret because of the possible multiple mechanisms involved in substrate/inhibitor-protein interactions. During our previous studies of substrate-inhibitor-transporter interactions, we observed differences in breast cancer resistance protein (BCRP) inhibition, depending on the substrate and the inhibitor. Therefore, we investigated BCRP-mediated interactions with a 4 x 4 matrix of substrates and inhibitors using monolayers formed from MDCKII cells transfected with murine BCRP (Bcrp1/Abcg2). The selective BCRP inhibitor 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino [1',2':1,6] pyrido [3,4-b]indol-3-yl)-propionic acid tert-butyl ester (Ko143) effectively inhibited the Bcrp1-mediated transport of all substrates examined. However, N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), nelfinavir, and Pluronic P85 exhibited differences in inhibition depending on the substrate examined. Our findings support recent reports suggesting that the interactions of substrate molecules with BCRP involve multiple binding regions in the protein. The nucleoside substrates zidovudine and abacavir seem to bind to a region on BCRP that may have little or no overlap with the binding regions of either prazosin or imatinib. In conclusion, the choice of substrate or inhibitor molecules for an in vitro assay system can be crucial for the optimal design of experiments to evaluate transporter-mediated drug-drug interactions.
Giri
Nagdeep
N
Department of Pharmaceutics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Agarwal
Sagar
S
Shaik
Naveed
N
Pan
Guoyu
G
Chen
Ying
Y
Elmquist
William F
WF
D000166:Acridines / Q000494:pharmacology; D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D054659:Diketopiperazines; D004285:Dogs; D006576:Heterocyclic Compounds, 4 or More Rings; D019888:Nelfinavir / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020442:Poloxamer / Q000494:pharmacology; D011485:Protein Binding; D044005:Tetrahydroisoquinolines / Q000494:pharmacology
C541506:3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino(1',2'-1,6)pyrido(3,4-b)indol-3-yl)propionic acid tert-butyl ester; D000166:Acridines; D054659:Diketopiperazines; D006576:Heterocyclic Compounds, 4 or More Rings; D009363:Neoplasm Proteins; D044005:Tetrahydroisoquinolines; D020442:Poloxamer; D019888:Nelfinavir; D000241:Adenosine; C083501:Elacridar