A novel fluorescence‐based functional assay for human OATP1A2 and OATP1C1 identifies interaction between third‐generation P‐gp inhibitors and OATP1A2

Abstract

Organic anion‐transporting polypeptide 1A2 (OATP1A2), expressed in the human blood–brain barrier, promotes drug uptake from the blood and hence can be exploited for central nervous system‐targeted drug delivery. The thyroid transporter OATP1C1, expressed in the choroid plexus and in astrocytes, is also a potential pharmacological target. Based on their established pharmacological relevance, screening the drug interaction profile of OATP1A2 and OATP1C1 is highly desirable. However, drug interaction screens require suitable model systems and functional assays. In the current study, uptake of a set of cell‐impermeable fluorescent dyes was screened in HEK‐293 and A431 cell lines overexpressing OATP1A2 and OATP1C1. Based on the uptake of fluorescent dye substrates, a functional assay was developed, which was used to characterize OATP inhibitors/substrates. We identify Live/Dead Green (LDG), Live‐or‐Dye 488, and sulforhodamines 101, G, and B as novel fluorescent substrates of OATP1A2 and OATP1C1. We show that LDG uptake is proportional to OATP1A2/1C1 expression, allowing the isolation of cells expressing high transporter levels. Additionally, dye uptake can be used to characterize the drug interaction pattern of OATP1A2 and OATP1C1. We demonstrate that third‐generation P‐glycoprotein inhibitors elacridar, tariquidar, and zosuquidar inhibit OATP1A2 function. Increased toxicity of elacridar in OATP1A2‐expressing cells suggests that OATP1A2 may modulate the distribution of this compound. The fluorescence‐based assays developed in the current study are a good alternative of radioligand‐based tests and pave the way toward high‐throughput screens for OATP1A2/1C1 drug interaction studies.

Effects of Chrysin and Its Major Conjugated Metabolites Chrysin-7-Sulfate and Chrysin-7-Glucuronide on Cytochrome P450 Enzymes and on OATP, P-gp, BCRP, and MRP2 Transporters

“…Uptake of pyranine in A431 cell overexpressing OATPs, 1B1, 1B3, or 2B1, and the uptake of sulforhodamine 101 (OATP1A2) was measured in the presence of increasing concentrations of chrysin, C7S, or C7G (0-100 mM). Effect of BSP on CYP1A2 has been reported previously (Bakos et al, 2020). OATP-dependent transport was determined by extracting the fluorescence measured in mock transfected cells.…”

“…OATP Overexpressing Cell Lines and OATP Interaction Tests. A431 cells overexpressing human OATPs, 1A2, 1B1, 1B3 or 2B1, or their mock transfected controls were generated as previously described (Patik et al, 2018;Bakos et al, 2020). Briefly, overexpression of OATP2B1 was achieved by transposase-mediated genetic insertion of the OATP2B1 cDNA (BC041095.1, HsCD00378878), followed by puromycin selection (1 mg/ml for 2 weeks).…”

“…Regarding OATPs 1A2 (BC042452, HsCD00333163), 1B1 (Gene identifier: AB026257), and 1B3 (BC141525, HsCD00348132), overexpression was achieved by lentiviral transduction. To achieve maximal OATP expression, cells were further sorted based on Live/Dead Green uptake (Patik et al, 2018;Bakos et al, 2020). OATP expression was continuously monitored by functional measurements.…”

“…The interaction between chrysin and its conjugates was investigated in an indirect assay (Patik et al, 2018) employing two recently described fluorescent dye substrates, pyranine and sulforhodamine 101 (Bakos et al, 2020;Székely et al, 2020) as an indicator of OATPs' function. Briefly, A431 cells overexpressing OATPs, 1A2, 1B1, 1B3, or 2B1 or their mock transfected controls (Patik et al, 2018) were seeded on 96-well plates in a density of 8 Â 10 4 cells per well in 200 ml Dulbecco's modified Eagle's medium 1 day prior to the transport measurements.…”

“…Fluorescent substrates pyranine and sulforhodamine 101 have recently been demonstrated as good indicators of substrate-inhibitor interactions of OATPs (Bakos et al, 2020;Székely et al, 2020). Uptake of these dyes was determined in A431 cells, engineered to overexpress one of the multispecific OATPs (1A2, 1B1, 1B3, or 2B1), and bromosulfophthalein was used as a reference inhibitor (Patik et al, 2018;Bakos et al, 2020). Figure 4A represents that chrysin is not a potent inhibitor of OATP1A2: only 40% inhibition was achieved in the presence of 100 mM chrysin concentration.…”