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Fluorescent probes for the dual investigation of MRP2 and OATP1B1 function and drug interactions
Virág Székely1, Izabel Patik2, Orsolya Ungvári2
1Membrane protein research group, Institute of Enzymology, Research Centre for Natural Sciences, H-1117 Budapest, Hungary; Doctoral School of Molecular Medicine, Semmelweis University, H-1085 Budapest, Hungary.
Abstract:
Detoxification in hepatocytes is a strictly controlled process, in which the governed action of membrane transporters involved in the uptake and efflux of potentially dangerous molecules has a crucial role. Major transporters of hepatic clearance belong to the ABC (ATP Binding Cassette) and Solute Carrier (SLC) protein families. Organic anion-transporting polypeptide OATP1B1 (encoded by the SLCO1B1 gene) is exclusively expressed in the sinusoidal membrane of hepatocytes, where it mediates the cellular uptake of bile acids, bilirubin, and also that of various drugs. The removal of toxic molecules from hepatocytes to the bile is accomplished by several ABC transporters, including P-glycoprotein (ABCB1), MRP2 (ABCC2) and BCRP (ABCG2). Owing to their pharmacological relevance, monitoring drug interaction with OATP1B1/3 and ABC proteins is recommended. Our aim was to assess the interaction of recently identified fluorescent OATP substrates (various dyes used in cell viability assays, pyranine, Cascade Blue hydrazide (CB) and sulforhodamine 101 (SR101)) (Bakos et al., 2019; Patik et al., 2018) with MRP2 and ABCG2 in order to find fluorescent probes for the simultaneous characterization of both uptake and efflux processes. Transport by MRP2 and ABCG2 was investigated in inside-out membrane vesicles (IOVs) allowing a fast screen of the transport of membrane impermeable substrates by efflux transporters. Next, transcellular transport of shared OATP and ABC transporter substrate dyes was evaluated in MDCKII cells co-expressing OATP1B1 and MRP2 or ABCG2. Our results indicate that pyranine is a general substrate of OATP1B1, OATP1B3 and OATP2B1, and we find that the dye Live/Dead Violet and CB are good tools to investigate ABCG2 function in IOVs. Besides their suitability for MRP2 functional tests in the IOV setup, pyranine, CB and SR101 are the first dual probes that can be used to simultaneously measure OATP1B1 and MRP2 function in polarized cells by a fluorescent method.
Insights
Researchers identified fluorescent probes to simultaneously monitor drug uptake and efflux transporters in liver cells. These probes, including pyranine, Cascade Blue hydrazide, and sulforhodamine 101, aid in understanding drug interactions and liver detoxification processes.
Area of Science:
- Hepatology and Pharmacology
- Molecular Biology and Biochemistry
Background:
- Hepatocyte detoxification relies on membrane transporters like ATP Binding Cassette (ABC) and Solute Carrier (SLC) proteins.
- Organic anion-transporting polypeptide 1B1 (OATP1B1) mediates drug uptake, while ABC transporters (e.g., MRP2, ABCG2) handle efflux into bile.
- Monitoring drug interactions with these transporters is crucial for pharmacological relevance.
Purpose of the Study:
- To identify fluorescent probes for simultaneous characterization of hepatic uptake (OATP) and efflux (ABC) transporter functions.
- To assess the interaction of novel fluorescent OATP substrates with MRP2 and ABCG2 transporters.
Main Methods:
- Transport assays using inside-out membrane vesicles (IOVs) to screen efflux transporter activity.
- Transcellular transport studies in MDCKII cells co-expressing OATP1B1 with MRP2 or ABCG2.
- Evaluation of fluorescent dyes including pyranine, Cascade Blue hydrazide (CB), and sulforhodamine 101 (SR101).
Main Results:
- Pyranine was identified as a general substrate for OATP1B1, OATP1B3, and OATP2B1.
- Live/Dead Violet and CB demonstrated utility for investigating ABCG2 function in IOVs.
- Pyranine, CB, and SR101 serve as the first dual probes for simultaneously measuring OATP1B1 and MRP2 function in polarized cells.
Conclusions:
- Novel fluorescent probes enable simultaneous assessment of hepatic drug uptake and efflux transporter activity.
- These probes facilitate a deeper understanding of drug interactions and liver detoxification mechanisms.
- The developed fluorescent methods offer a valuable tool for pharmacological research and drug development.
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