The fluorescence-based competitive counterflow assay developed for organic anion transporting polypeptides 1A2, 1B1, 1B3 and 2B1 identifies pentamidine as a selective OATP1A2 substrate.

Organic anion transporting polypeptides OATP1A2, OATP1B1, OATP1B3 and OATP2B1 are Na+ - and ATP-independent exchangers of large, organic compounds, encompassing structurally diverse xenobiotics, including various drugs. These OATPs influence intestinal absorption (OATP2B1), hepatic clearance (OATP1B1/3) and blood to brain penetration (OATP1A2, OATP2B1) of their drug substrates. Consequently, OATP-mediated drug or food interactions may lead to altered pharmacokinetics and toxicity. During drug development, investigation of hepatic OATP1B1 and OATP1B3 is recommended by international regulatory agencies. Most frequently, OATP-drug interactions are investigated in an indirect assay, i.e., by examining uptake inhibition of a radioactive or fluorescent probe. However, indirect assays do not distinguish between transported substrates and non-transported OATP inhibitors. To fill this hiatus, a novel assay, termed competitive counterflow (CCF) has been developed and has since been applied for several OATPs to differentiate between substrates and non-transported inhibitors. However, previous OATP CCF assays, with the exception of that for OATP1B1, used radioactive probes. In the current study, we demonstrate that sulforhodamine 101 or pyranine can be used as fluorescent probes in a CCF assay to identify transported substrates of OATP1A2, or OATPs 1B1, 1B3 and 2B1, respectively. With the help of the newly developed fluorescence-based CCF method, we identify the FDA-approved anti-protozoal drug, pentamidine as a unique substrate of OATP1A2. Furthermore, we confirm the selective, OATP1A2-mediated uptake of pentamidine in a cytotoxicity assay. Based on our results, OATP1A2 may be an important determinant of pentamidine transport through the blood-brain barrier.

Fluorescence-based methods for studying activity and drug-drug interactions of hepatic solute carrier and ATP binding cassette proteins involved in ADME-Tox.

In humans, approximately 70% of drugs are eliminated through the liver. This process is governed by the concerted action of membrane transporters and metabolic enzymes. Transporters mediating hepatocellular uptake of drugs belong to the SLC (Solute carrier) superfamily of transporters. Drug efflux either toward the portal vein or into the bile is mainly mediated by active transporters of the ABC (ATP Binding Cassette) family. Alteration in the function and/or expression of liver transporters due to mutations, disease conditions, or co-administration of drugs or food components can result in altered pharmacokinetics. On the other hand, drugs or food components interacting with liver transporters may also interfere with liver function (e.g., bile acid homeostasis) and may even cause liver toxicity. Accordingly, certain transporters of the liver should be investigated already at an early stage of drug development. Most frequently radioactive probes are applied in these drug-transporter interaction tests. However, fluorescent probes are cost-effective and sensitive alternatives to radioligands, and are gaining wider application in drug-transporter interaction tests. In our review, we summarize our current understanding about hepatocyte ABC and SLC transporters affected by drug interactions. We provide an update of the available fluorescent and fluorogenic/activable probes applicable in in vitro or in vivo testing of these ABC and SLC transporters, including near-infrared transporter probes especially suitable for in vivo imaging. Furthermore, our review gives a comprehensive overview of the available fluorescence-based methods, not directly relying on the transport of the probe, suitable for the investigation of hepatic ABC or SLC-type drug transporters.

Identifying Novel Inhibitors for Hepatic Organic Anion Transporting Polypeptides by Machine Learning-Based Virtual Screening.

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 (IC50 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 (IC50 values: 40 nM and 390 nM) comparable to the recently described estrone-based inhibitor (IC50 = 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.

Interactions of Mycotoxin Alternariol with Cytochrome P450 Enzymes and OATP Transporters.

Alternariol (AOH) is an emerging mycotoxin produced by Alternaria strains. The acute toxicity of the mycotoxin is low; however, chronic exposure to AOH may result in the development of endocrine disruptor and/or carcinogenic effects. The toxicokinetic properties of AOH have barely been characterized. Therefore, in this study, we aimed to investigate its interactions with CYP (1A2, 2C9, 2C19, 2D6, and 3A4) enzymes and OATP (1A2, 1B1, 1B3, and 2B1) transporters employing in vitro enzyme assays and OATP overexpressing cells, respectively. Our results demonstrated that AOH is a strong inhibitor of CYP1A2 (IC50 = 0.15 µM) and CYP2C9 (IC50 = 7.4 µM). Based on the AOH depletion assays in the presence of CYP enzymes, CYP1A2 is mainly involved, while CYP2C19 is moderately involved in the CYP-catalyzed biotransformation of the mycotoxin. AOH proved to be a strong inhibitor of each OATP transporter examined (IC50 = 1.9 to 5.4 µM). In addition, both direct and indirect assays suggest the involvement of OATP1B1 in the cellular uptake of the mycotoxin. These findings promote the deeper understanding of certain toxicokinetic interactions of AOH.

8-acetoxy-trisulfopyrene as the first activatable fluorogenic probe for add-and-read assessment of Organic anion-transporting polypeptides, OATP1B1, OATP1B3, and OATP2B1.

Organic anion-transporting polypeptides, OATP1B1, OATP1B3, and OATP2B1 are multispecific membrane proteins mediating the hepatocellular uptake of structurally diverse endo- and exogenous compounds, including various kinds of drugs. Co-administration of OATP1B/2B1 substrates may lead to altered pharmacokinetics or even toxicity. Therefore, the study of the interaction with these OATPs is essential in drug development and is recommended by international regulatory agencies, the FDA, EMA, and PMDA. In general, radiolabeled indicators are used to measure drug interactions of OATPs, and, lately, fluorescent probes are also gaining wider application in OATP tests. However, all of the currently available methods (either radioactive or fluorescence-based) comprise multiple steps, including the removal of the indicator in the end of the experiment. Hence, they are not ideally suited for high-throughput screening. In the current study, in order to find an indicator allowing real-time assessment of hepatic OATP function, we searched for an activatable fluorogenic OATP substrate. Here, we show that 8-acetoxypyrene-1,3,6-trisulfonate (Ace), a fluorogenic derivative of the hepatic OATP substrate pyranine (8-hydroxypyrene-1,3,6-trisulfonate) enters the cells via OATP1B1/3 or OATP2B1 function. In living cells, Ace is then converted into highly fluorescent pyranine, allowing "no-wash" measurement of OATP function and drug interactions. Furthermore, we demonstrate that Ace can be used in an indirect assay termed as competitive counterflow suitable to distinguish between transported substrates and inhibitors of OATP1B1. The fluorescence-based methods described here are unique and open the way toward high-throughput screening of interactions between new molecular entities and OATPs.

Data-Driven Ensemble Docking to Map Molecular Interactions of Steroid Analogs with Hepatic Organic Anion Transporting Polypeptides.

Hepatic organic anion transporting polypeptides-OATP1B1, OATP1B3, and OATP2B1-are expressed at the basolateral membrane of hepatocytes, being responsible for the uptake of a wide range of natural substrates and structurally unrelated pharmaceuticals. Impaired function of hepatic OATPs has been linked to clinically relevant drug-drug interactions leading to altered pharmacokinetics of administered drugs. Therefore, understanding the commonalities and differences across the three transporters represents useful knowledge to guide the drug discovery process at an early stage. Unfortunately, such efforts remain challenging because of the lack of experimentally resolved protein structures for any member of the OATP family. In this study, we established a rigorous computational protocol to generate and validate structural models for hepatic OATPs. The multistep procedure is based on the systematic exploration of available protein structures with shared protein folding using normal-mode analysis, the calculation of multiple template backbones from elastic network models, the utilization of multiple template conformations to generate OATP structural models with various degrees of conformational flexibility, and the prioritization of models on the basis of enrichment docking. We employed the resulting OATP models of OATP1B1, OATP1B3, and OATP2B1 to elucidate binding modes of steroid analogs in the three transporters. Steroid conjugates have been recognized as endogenous substrates of these transporters. Thus, investigating this data set delivers insights into mechanisms of substrate recognition. In silico predictions were complemented with in vitro studies measuring the bioactivity of a compound set on OATP expressing cell lines. Important structural determinants conferring shared and distinct binding patterns of steroid analogs in the three transporters have been identified. Overall, this comparative study provides novel insights into hepatic OATP-ligand interactions and selectivity. Furthermore, the integrative computational workflow for structure-based modeling can be leveraged for other pharmaceutical targets of interest.

Inhibitory Effects of Quercetin and Its Main Methyl, Sulfate, and Glucuronic Acid Conjugates on Cytochrome P450 Enzymes, and on OATP, BCRP and MRP2 Transporters.

Quercetin is a flavonoid, its glycosides and aglycone are found in significant amounts in several plants and dietary supplements. Because of the high presystemic biotransformation of quercetin, mainly its conjugates appear in circulation. As has been reported in previous studies, quercetin can interact with several proteins of pharmacokinetic importance. However, the interactions of its metabolites with biotransformation enzymes and drug transporters have barely been examined. In this study, the inhibitory effects of quercetin and its most relevant methyl, sulfate, and glucuronide metabolites were tested on cytochrome P450 (CYP) (2C19, 3A4, and 2D6) enzymes as well as on organic anion-transporting polypeptides (OATPs) (OATP1A2, OATP1B1, OATP1B3, and OATP2B1) and ATP (adenosine triphosphate) Binding Cassette (ABC) (BCRP and MRP2) transporters. Quercetin and its metabolites (quercetin-3'-sulfate, quercetin-3-glucuronide, isorhamnetin, and isorhamnetin-3-glucuronide) showed weak inhibitory effects on CYP2C19 and 3A4, while they did not affect CYP2D6 activity. Some of the flavonoids caused weak inhibition of OATP1A2 and MRP2. However, most of the compounds tested proved to be strong inhibitors of OATP1B1, OATP1B3, OATP2B1, and BCRP. Our data demonstrate that not only quercetin but some of its conjugates, can also interact with CYP enzymes and drug transporters. Therefore, high intake of quercetin may interfere with the pharmacokinetics of drugs.

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.

Chrysin is an abundant flavonoid in nature, and it is also contained by several dietary supplements. Chrysin is highly biotransformed in the body, during which conjugated metabolites chrysin-7-sulfate and chrysin-7-glucuronide are formed. These conjugates appear at considerably higher concentrations in the circulation than the parent compound. Based on previous studies, chrysin can interact with biotransformation enzymes and transporters; however, the interactions of its metabolites have been barely examined. In this in vitro study, the effects of chrysin, chrysin-7-sulfate, and chrysin-7-glucuronide on cytochrome P450 enzymes (2C9, 2C19, 3A4, and 2D6) as well as on organic anion-transporting polypeptides (OATPs; 1A2, 1B1, 1B3, and 2B1) and ATP binding cassette [P-glycoprotein, multidrug resistance-associated protein 2, and breast cancer resistance protein (BCRP)] transporters were investigated. Our observations revealed that chrysin conjugates are strong inhibitors of certain biotransformation enzymes (e.g., CYP2C9) and transporters (e.g., OATP1B1, OATP1B3, OATP2B1, and BCRP) examined. Therefore, the simultaneous administration of chrysin-containing dietary supplements with medications needs to be carefully considered due to the possible development of pharmacokinetic interactions. SIGNIFICANCE STATEMENT: Chrysin-7-sulfate and chrysin-7-glucuronide are the major metabolites of flavonoid chrysin. In this study, we examined the effects of chrysin and its conjugates on cytochrome P450 enzymes and on organic anion-transporting polypeptides and ATP binding cassette transporters (P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2). Our results demonstrate that chrysin and/or its conjugates can significantly inhibit some of these proteins. Since chrysin is also contained by dietary supplements, high intake of chrysin may interrupt the transport and/or the biotransformation of drugs.

Fluorescent probes for the dual investigation of MRP2 and OATP1B1 function and drug interactions.

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.