Establishment and Validation of Competitive Counterflow as a Method To Detect Substrates of the Organic Anion Transporting Polypeptide 2B1

The organic anion transporting polypeptide (OATP) 2B1 is ubiquitously expressed and known to facilitate cellular entry. It is widely accepted that transport proteins play a pivotal role in pharmacokinetics. Consequently, testing for interaction with drug transporters became an important part in the...

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Vydané v:Molecular pharmaceutics Ročník 15; číslo 12; s. 5501
Hlavní autori: Schäfer, Anima M, Bock, Thomas, Meyer Zu Schwabedissen, Henriette E
Médium: Journal Article
Jazyk:English
Vydavateľské údaje: United States 03.12.2018
Predmet:
Animals
Chemistry, Pharmaceutical - instrumentation
Chemistry, Pharmaceutical - methods
Dogs
Drug Interactions
Estrone - analogs & derivatives
Estrone - pharmacokinetics
HeLa Cells
Hep G2 Cells
Humans
Madin Darby Canine Kidney Cells
Organic Anion Transporters - genetics
Organic Anion Transporters - metabolism
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Small Molecule Libraries - pharmacokinetics
Substrate Specificity
organic anion transport
competitive counterflow
drug transporter
substrate identification
OATP2B1
ISSN:1543-8392, 1543-8392
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Shrnutí:The organic anion transporting polypeptide (OATP) 2B1 is ubiquitously expressed and known to facilitate cellular entry. It is widely accepted that transport proteins play a pivotal role in pharmacokinetics. Consequently, testing for interaction with drug transporters became an important part in the assessment of new molecular entities in order to predict and prevent drug-drug interactions. Recently, competitive counterflow (CCF), an indirect method allowing the identification of substrates, was successfully applied to the organic cation transporter 2. It was the aim of this study to test whether CCF can be used to identify substrates of OATP2B1. A protocol for CCF experiments using estrone 3-sulfate (E S) as the driven compound in expression-verified MDCKII-OATP2B1 cells was established. The protocol was tested using a substance library, which was prior screened for inhibition of OATP2B1-mediated transport accounting for both E S-binding sites. In CCF experiments, all previously reported OATP2B1 substrates significantly reduced the amount of E S in equilibrium, classifying them as substrates. In addition, we identified and verified novel substrates of OATP2B1, namely, astemizole and domperidone. Results of the CCF were complemented with cytotoxicity assays or cell-based reporter gene assays to validate the finding of etoposide and teniposide or hyperforin being substrates of OATP2B1, respectively. Our study indicates that the method of CCF can be used to identify substrates of OATP2B1, irrespective, whether interacting with binding site A or A and B, but is limited by solubility issues or the amount of transporter that is expressed in the used cellular system.
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ISSN:1543-8392
1543-8392
DOI:10.1021/acs.molpharmaceut.8b00631