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Integration of statistical learning methods with structure-based modeling approaches is a contemporary strategy to identify novel lead compounds in drug discovery. Hepatic organic anion transporting polypeptides (OATP1B1, OATP1B3, and OATP2B1) are classical off-targets, and it is well recognized that their ability to interfere with a wide range of chemically unrelated drugs, environmental chemicals, or food additives can lead to unwanted adverse effects like liver toxicity and drug-drug or drug-food interactions. Therefore, the identification of novel (tool) compounds for hepatic OATPs by virtual screening approaches and subsequent experimental validation is a major asset for elucidating structure-function relationships of (related) transporters: they enhance our understanding about molecular determinants and structural aspects of hepatic OATPs driving ligand binding and selectivity. In the present study, we performed a consensus virtual screening approach by using different types of machine learning models (proteochemometric models, conformal prediction models, and XGBoost models for hepatic OATPs), followed by molecular docking of preselected hits using previously established structural models for hepatic OATPs. Screening the diverse REAL drug-like set (Enamine) shows a comparable hit rate for OATP1B1 (36% actives) and OATP1B3 (32% actives), while the hit rate for OATP2B1 was even higher (66% actives). Percentage inhibition values for 44 selected compounds were determined using dedicated in vitro assays and guided the prioritization of several highly potent novel hepatic OATP inhibitors: six (strong) OATP2B1 inhibitors (IC 50 values ranging from 0.04 to 6 μM), three OATP1B1 inhibitors (2.69 to 10 μM), and five OATP1B3 inhibitors (1.53 to 10 μM) were identified. Strikingly, two novel OATP2B1 inhibitors were uncovered ( C7 and H5 ) which show high affinity (IC 50 values: 40 nM and 390 nM) comparable to the recently described estrone-based inhibitor (IC 50 = 41 nM). A molecularly detailed explanation for the observed differences in ligand binding to the three transporters is given by means of structural comparison of the detected binding sites and docking poses.

Special attention is required when pharmacological treatment is indicated for a pregnant woman. P-glycoprotein (MDR1) is a well-known transporter localized in the maternal blood-facing apical membrane of placental syncytiotrophoblast and is considered to play an important role in protecting the developing fetus. Maraviroc, a MDR1 substrate that is registered for treatment of HIV infection, shows a low toxicity profile, suggesting favorable tolerability also if administered to pregnant women. Nevertheless, there is only poor understanding to date regarding the extent to which it permeates across the placental barrier and what are the transport mechanisms involved. Endeavoring to clarify the passage of maraviroc across placenta, we used in this study the method of closed-circuit perfusion of maraviroc across human placental cotyledon. The data obtained confirmed slight involvement of MDR1, but they also suggest possible interaction with other transport system(s) working in the opposite direction from that of MDR1. Complementary in vitro studies, including cellular experiments on choriocarcinoma BeWo cells as well as transporter-overexpressing MDCKII and A431 cell lines and accumulation in placental fresh villous fragments, revealed maraviroc transport by MRP1, OATP1A2, and OATP1B3 transporters. Based on mRNA expression data in the placental tissue, isolated trophoblasts, and fetal endothelial cells, especially MRP1 and OATP1A2 seem to play a crucial role in cooperatively driving maraviroc into placental tissue. By the example of maraviroc, we show here the important interplay of transporters in placental drug handling and its possibility to overcome the MDR1-mediated efflux., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

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 IC 50 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.