Identification of novel cell-impermeant fluorescent substrates for testing the function and drug interaction of Organic Anion-Transporting Polypeptides, OATP1B1/1B3 and 2B1

Abstract

Organic Anion-Transporting Polypeptides are multispecific membrane proteins that regulate the passage of crucial endobiotics and drugs across pharmacological barriers. OATP1B1 and OATP1B3 have been described to play a major role in the hepatic uptake of statins, antivirals and various chemotherapeutics; whereas the pharmacological role of the ubiquitously expressed OATP2B1 is less well characterized. According to current industry standards, in vitro testing for susceptibility to OATP1B1 and 1B3 mediated transport is recommended for drug candidates that are eliminated in part via the liver. Here we show that human OATP1B1, 1B3 and 2B1 transport a series of commercially available viability dyes that are generally believed to be impermeable to intact cells. We demonstrate that the intracellular accumulation of Zombie Violet, Live/Dead Green, Cascade Blue and Alexa Fluor 405 is specifically increased by OATPs. Inhibition of Cascade Blue or Alexa Fluor 405 uptake by known OATP substrates/inhibitors yielded IC50 values in agreement with gold-standard radioligand assays. The fluorescence-based assays described in this study provide a new tool for testing OATP1B/2B1 drug interactions.

Pd-Catalyzed microwave-assisted synthesis of phosphonated 13α-estrones as potential OATP2B1, 17β-HSD1 and/or STS inhibitors

“…The influence of the basic (13αE1OH, 13αE1OMe and 13αE1OBn) and new compounds 8 – 13 on the transport function of OATP2B1 was investigated by measuring Cascade Blue uptake ( Table 3 ) [ 31 ]. 13α-Estrone (13αE1OH) itself displayed weak inhibitory action with an IC 50 value above 50 μM.…”

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

“…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.…”

Structural similarities between some common fluorophores used in biology and marketed drugs, endogenous metabolites, and natural products

“…Rhodamine 123 and 6G also [103] served as substrates. All shown to be direct substrates, and uptake inhibited by known transporter inhibitors [124] Str u ctu r a l sim ila r ity (or th e a ssessm en t of p r op er ties b a sed sim p ly on a n a lyzin g str u ctu r es) is a n elu sive con cep t (e.g. [125]), b u t a s ju d ged b y a sta n da r d en cod in g (RDKit p a tter n ed ) th er e is con sid er a b le sim ila r ity in str u ctu r e b etw een a lm ost a ll of ou r ch osen flu or op h or es a n d a t lea st on e d r u g, w h eth er th is is ju d ged b y th eir d escr ip tor -or fin ger p r in t-b a sed p r op er ties (Figs 1-3), b y ob ser va tion (Figs 4, 6), or ( Fig 5) via th e QED [74] m ea su r e.…”