G45 and V386 in Transmembrane Domains 1 and 8 Are Critical for the Activation of OATP1B3-Mediated E17ßG Uptake by Clotrimazole.

Organic anion-transporting polypeptides (OATPs) 1B1 and 1B3 are two highly homologous transport proteins. However, OATP1B1- and 1B3-mediated estradiol-17ß-glucuronide (E17ßG) uptake can be differentially affected by clotrimazole. In this study, by functional characterization on chimeric transporters and single mutants, we find that G45 in transmembrane domain 1 (TM1) and V386 in TM8 are critical for the activation of OATP1B3-mediated E17ßG uptake by clotrimazole. However, the effect of clotrimazole on the function of OATP1B3 is substrate-dependent as clotrimazole does not stimulate OATP1B3-mediated uptake of 4',5'-dibromofluorescein (DBF) and rosuvastatin. In addition, clotrimazole is not transported by OATP1B3, but it can efficiently permeate the plasma membrane due to its lipophilic properties. Homology modeling and molecular docking indicate that E17ßG binds in a substrate binding pocket of OATP1B3 through hydrogen bonding and hydrophobic interactions, among which its sterol scaffold forms hydrophobic contacts with V386. In addition, a flexible glycine residue at position 45 is essential for the activation of OATP1B3. Finally, clotrimazole is predicted to bind at an allosteric site, which mainly consists of hydrophobic residues located at the cytoplasmic halves of TMs 4, 5, 10, and 11.

The Concise Guide to PHARMACOLOGY 2023/24: Transporters.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and over 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16182. Transporters are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Molecular Mechanisms of Organic Anion Transporting Polypeptide-Mediated Organic Anion Clearance at the Blood-Cerebrospinal Fluid Barrier.

The blood-cerebrospinal fluid barrier (BCSFB), formed by the choroid plexus epithelial (CPE) cells, plays an active role in removing drugs and metabolic wastes from the brain. Recent functional studies in isolated mouse choroid plexus (CP) tissues suggested the presence of organic anion transporting polypeptides (OATPs, encoded by SLCOs) at the apical membrane of BCSFB, which may clear large organic anions from the cerebrospinal fluid (CSF). However, the specific OATP isoform involved is unclear. Using quantitative fluorescence imaging, we showed that the fluorescent anions sulforhodamine 101 (SR101), fluorescein methotrexate (FL-MTX), and 8-fluorescein-cAMP (fluo-cAMP) are actively transported from the CSF to the subepithelial space in CP tissues isolated from wild-type mice. In contrast, transepithelial transport of these compounds across the CPE cells was abolished in Oatp1a/1b-/- mice due to impaired apical uptake. Using transporter-expressing cell lines, SR101, FL-MTX, and fluo-cAMP were additionally shown to be transported by mouse OATP1A5 and its human counterpart OATP1A2. Kinetic analysis showed that estrone-3-sulfate and SR101 are transported by OATP1A2 and OATP1A5 with similar Michaelis-Menten constants (Km). Immunofluorescence staining further revealed the presence of OATP1A2 protein in human CP tissues. Together, our results suggest that large organic anions in the CSF are actively transported into CPE cells by apical OATP1A2 (OATP1A5 in mice), then subsequently effluxed into the blood by basolateral multidrug resistance-associated proteins (MRPs). As OATP1A2 transports a wide array of endogenous compounds and xenobiotics, the presence of this transporter at the BCSFB may imply a novel clearance route for drugs and neurohormones from the CSF. SIGNIFICANCE STATEMENT: Drug transporters at the blood-cerebrospinal fluid (CSF) barrier play an important but understudied role in brain drug disposition. This study revealed a functional contribution of rodent organic anion transporting polypeptide (OATP) 1A5 towards the CSF clearance of organic anions and suggested a similar role for OATP1A2 in humans. Delineating the molecular mechanisms governing CSF organic anion clearance may help to improve the prediction of central nervous system (CNS) pharmacokinetics and identify drug candidates with favorable CNS pharmacokinetic properties.

Palmitoylation of solute carriers.

Post-translational modifications are an important mechanism in the regulation of protein expression, function, and degradation. Well-known post-translational modifications are phosphorylation, glycosylation, and ubiquitination. However, lipid modifications, including myristoylation, prenylation, and palmitoylation, are poorly studied. Since the early 2000s, researchers have become more interested in lipid modifications, especially palmitoylation. The number of articles in PubMed increased from about 350 between 2000 and 2005 to more than 600 annually during the past ten years. S-palmitoylation, where the 16-carbon saturated (C16:0) palmitic acid is added to free cysteine residues of proteins, is a reversible protein modification that can affect the expression, membrane localization, and function of the modified proteins. Various diseases like Huntington's and Alzheimer's disease have been linked to changes in protein palmitoylation. In humans, the addition of palmitic acid is mediated by 23 palmitoyl acyltransferases, also called DHHC proteins. The modification can be reversed by a few thioesterases or hydrolases. Numerous soluble and membrane-attached proteins are known to be palmitoylated, but among the approximately 400 solute carriers that are classified in 66 families, only 15 found in 8 families have so far been documented to be palmitoylated. Among the best-characterized transporters are the glucose transporters GLUT1 (SLC2A1) and GLUT4 (SLC2A4), the three monoamine transporters norepinephrine transporter (NET; SLC6A2), dopamine transporter (DAT; SLC6A3), and serotonin transporter (SERT; SLC6A4), and the sodium-calcium exchanger NCX1 (SLC8A1). While there is evidence from recent proteomics experiments that numerous solute carriers are palmitoylated, no details beyond the 15 transporters covered in this review are available.

Investigating the interactions of flavonoids with human OATP2B1: inhibition assay, IC50 determination, and structure-activity relationship analysis.

Human organic anion transporting polypeptide 2B1 (OATP2B1) is a crucial transporter for the absorption and disposition of many drugs. Its inhibition by small molecules may alter the pharmacokinetic profile of its substrate drugs. In this study, the interactions of 29 common flavonoids with OATP2B1 were explored using the fluorescent substrate 4',5'-dibromofluorescein and structure-activity relationship analysis. Our results showed that flavonoid aglycones interact with OATP2B1 more strongly than their 3-O- and 7-O-glycoside counterparts, as hydrophilic and bulky groups at these two sites are detrimental to flavonoids' binding with OATP2B1. In contrast, hydrogen-bond forming groups at the C-6 position of ring A and the C-3' and C-4' positions of ring B could strengthen the interaction of flavonoids with OATP2B1. However, a hydroxyl or sugar moiety at the C-8 position of ring A is unfavorable. Our results also indicated that flavones usually interact more strongly with OATP2B1 than their 3-hydroxyflavones (flavonols). The obtained information could be useful for the prediction of additional flavonoids for their interaction with OATP2B1.

Using a competitive counterflow assay to identify novel cationic substrates of OATP1B1 and OATP1B3.

OATP1B1 and OATP1B3 are two drug transporters that mediate the uptake of multiple endo- and xenobiotics, including many drugs, into human hepatocytes. Numerous inhibitors have been identified, and for some of them, it is not clear whether they are also substrates. Historically radiolabeled substrates or LC-MS/MS methods were needed to test for transported substrates, both of which can be limiting in time and money. However, the competitive counterflow (CCF) assay originally described for OCT2 and, more recently, for OCT1, OATP2B1, and OATP1A2 does not require radiolabeled substrates or LC-MS/MS methods and, as a result, is a more cost-effective approach to identifying substrates of multidrug transporters. We used a CCF assay based on the stimulated efflux of the common model substrate estradiol-17ß-glucuronide (E17ßG) and tested 30 compounds for OATP1B1- and OATP1B3-mediated transport. Chinese Hamster Ovary (CHO) cells stably expressing OATP1B1 or OATP1B3 were preloaded with 10 nM [3H]-estradiol-17ß-glucuronide. After the addition of known substrates like unlabeled estradiol-17ß-glucuronide, estrone-3-sulfate, bromosulfophthalein, protoporphyrin X, rifampicin, and taurocholate to the outside of the preloaded CHO cells, we observed efflux of [3H]-estradiol-17ß-glucuronide due to exchange with the added compounds. Of the tested 30 compounds, some organic cation transporter substrates like diphenhydramine, metformin, and salbutamol did not induce [3H]-estradiol-17ß-glucuronide efflux, indicating that the two OATPs do not transport them. However, 22 (for OATP1B1) and 16 (for OATP1B3) of the tested compounds resulted in [3H]-estradiol-17ß-glucuronide efflux, suggesting that they are OATP substrates. Among these compounds, we further tested clarithromycin, indomethacin, reserpine, and verapamil and confirmed that they are substrates of the two OATPs. These results demonstrate that the substrate spectrum of the well-characterized organic anion transporting polypeptides includes several organic cations. Furthermore, as for other drug uptake transporters, the CCF assay is an easy-to-use screening tool to identify novel OATP substrates.

Free Cholesterol Affects the Function and Localization of Human Na+/Taurocholate Cotransporting Polypeptide (NTCP) and Organic Cation Transporter 1 (OCT1).

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are associated with obesity. They are accompanied by increased levels of free cholesterol in the liver. Most free cholesterol resides within the plasma membrane. We assessed the impact of adding or removing free cholesterol on the function and localization of two hepatocellular uptake transporters: the Na+/taurocholate cotransporting polypeptide (NTCP) and the organic cation transporter 1 (OCT1). We used a cholesterol-MCD complex (cholesterol) to add cholesterol and methyl-ß-cyclodextrin (MCD) to remove cholesterol. Our results demonstrate that adding cholesterol decreases NTCP capacity from 132 ± 20 to 69 ± 37 µL/mg/min and OCT1 capacity from 209 ± 66 to 125 ± 26 µL/mg/min. Removing cholesterol increased NTCP and OCT1 capacity to 224 ± 65 and 279 ± 20 µL/mg/min, respectively. In addition, adding cholesterol increased the localization of NTCP within lipid rafts, while adding or removing cholesterol increased OCT1 localization in lipid rafts. These results demonstrate that increased cholesterol levels can impair NTCP and OCT1 function, suggesting that the free cholesterol content of the liver can alter bile acid and drug uptake into the liver. This could explain the increased plasma bile acid levels in NAFLD and NASH patients and potentially lead to altered drug disposition.

The Importance of Val386 in Transmembrane Domain 8 for the Activation of OATP1B3 by Epigallocatechin Gallate.

Estrone-3-sulfate (E3S) uptake mediated by organic anion transporting polypeptide 1B3 (OATP1B3) can be activated by epigallocatechin gallate (EGCG). In this study, by using chimeric transporters and site-directed mutagenesis, we found that Val386 in transmembrane domain 8 (TM8) is essential for OATP1B3's activation by EGCG. Kinetic studies showed that the loss of activation of 1B3-TM8 and 1B3-V386F in the presence of EGCG is due to their decreased substrate binding affinity and reduced maximal transport rate. The overall transport efficiencies of OATP1B3, 1B3-TM8, and 1B3-V386F in the absence and presence of EGCG are 8.6 ± 0.7 vs 15.9 ± 1.4 (p < 0.05), 11.2 ± 2.1 vs 2.7 ± 0.3 (p < 0.05), and 10.2 ± 1.0 vs 2.5 ± 0.3 (p < 0.05), respectively. While 1B3-V386F cannot be activated by EGCG, its transport activity for EGCG is also diminished. OATP1B3's activation by EGCG is substrate-dependent as EGCG inhibits OATP1B3-mediated pravastatin uptake. Furthermore, the activation of OATP1B3-mediated E3S uptake by quercetin 3-O-α-l-arabinopyranosyl(1 → 2)-α-l-rhamnopyranoside is not affected by TM8 and V386F. Taken together, the activation of OATP1B3 by small molecules is substrate- and modulator-dependent, and V386 in TM8 plays a critical role in the activation of OATP1B3-mediated E3S uptake by EGCG.

Structural Plasticity Is a Feature of Rheostat Positions in the Human Na+/Taurocholate Cotransporting Polypeptide (NTCP).

In the Na+/taurocholate cotransporting polypeptide (NTCP), the clinically relevant S267F polymorphism occurs at a "rheostat position". That is, amino acid substitutions at this position ("S267X") lead to a wide range of functional outcomes. This result was particularly striking because molecular models predicted the S267X side chains are buried, and thus, usually expected to be less tolerant of substitutions. To assess whether structural tolerance to buried substitutions is widespread in NTCP, here we used Rosetta to model all 19 potential substitutions at another 13 buried positions. Again, only subtle changes in the calculated stabilities and structures were predicted. Calculations were experimentally validated for 19 variants at codon 271 ("N271X"). Results showed near wildtype expression and rheostatic modulation of substrate transport, implicating N271 as a rheostat position. Notably, each N271X substitution showed a similar effect on the transport of three different substrates and thus did not alter substrate specificity. This differs from S267X, which altered both transport kinetics and specificity. As both transport and specificity may change during protein evolution, the recognition of such rheostat positions may be important for evolutionary studies. We further propose that the presence of rheostat positions is facilitated by local plasticity within the protein structure. Finally, we note that identifying rheostat positions may advance efforts to predict new biomedically relevant missense variants in NTCP and other membrane transport proteins.

Perfluoroalkyl Carboxylic Acids Interact with the Human Bile Acid Transporter NTCP.

Na+/taurocholate cotransporting polypeptide (NTCP) is important for the enterohepatic circulation of bile acids, which has been suggested to contribute to the long serum elimination half-lives of perfluoroalkyl substances in humans. We demonstrated that some perfluoroalkyl sulfonates are transported by NTCP; however, little was known about carboxylates. The purpose of this study was to determine if perfluoroalkyl carboxylates would interact with NTCP and potentially act as substrates. Sodium-dependent transport of [3H]-taurocholate was measured in human embryonic kidney cells (HEK293) stably expressing NTCP in the absence or presence of perfluoroalkyl carboxylates with varying chain lengths. PFCAs with 8 (PFOA), 9 (PFNA), and 10 (PFDA) carbons were the strongest inhibitors. Inhibition kinetics demonstrated competitive inhibition and indicated that PFNA was the strongest inhibitor followed by PFDA and PFOA. All three compounds are transported by NTCP, and kinetics experiments revealed that PFOA had the highest affinity for NTCP with a Km value of 1.8 ± 0.4 mM. The Km value PFNA was estimated to be 5.3 ± 3.5 mM and the value for PFDA could not be determined due to limited solubility. In conclusion, our results suggest that, in addition to sulfonates, perfluorinated carboxylates are substrates of NTCP and have the potential to interact with NTCP-mediated transport.

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: Transporters.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15543. Transporters are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Why is elevation of serum cholesterol associated with exposure to perfluoroalkyl substances (PFAS) in humans? A workshop report on potential mechanisms.

Serum concentrations of cholesterol are positively correlated with exposure to perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) in humans. The associated change in cholesterol is small across a broad range of exposure to PFOA and PFOS. Animal studies generally have not indicated a mechanism that would account for the association in humans. The extent to which the relationship is causal is an open question. Nonetheless, the association is of particular importance because increased serum cholesterol has been considered as an endpoint to derive a point of departure in at least one recent risk assessment. To gain insight into potential mechanisms for the association, both causal and non-causal, an expert workshop was held Oct 31 and Nov 1, 2019 to discuss relevant data and propose new studies. In this report, we summarize the relevant background data, the discussion among the attendees, and their recommendations for further research.

A clinically relevant polymorphism in the Na+/taurocholate cotransporting polypeptide (NTCP) occurs at a rheostat position.

Conventionally, most amino acid substitutions at "important" protein positions are expected to abolish function. However, in several soluble-globular proteins, we identified a class of nonconserved positions for which various substitutions produced progressive functional changes; we consider these evolutionary "rheostats". Here, we report a strong rheostat position in the integral membrane protein, Na+/taurocholate (TCA) cotransporting polypeptide, at the site of a pharmacologically relevant polymorphism (S267F). Functional studies were performed for all 20 substitutions (S267X) with three substrates (TCA, estrone-3-sulfate, and rosuvastatin). The S267X set showed strong rheostatic effects on overall transport, and individual substitutions showed varied effects on transport kinetics (Km and Vmax) and substrate specificity. To assess protein stability, we measured surface expression and used the Rosetta software (https://www.rosettacommons.org) suite to model structure and stability changes of S267X. Although buried near the substrate-binding site, S267X substitutions were easily accommodated in the Na+/TCA cotransporting polypeptide structure model. Across the modest range of changes, calculated stabilities correlated with surface-expression differences, but neither parameter correlated with altered transport. Thus, substitutions at rheostat position 267 had wide-ranging effects on the phenotype of this integral membrane protein. We further propose that polymorphic positions in other proteins might be locations of rheostat positions.

Functional Consequences of Pravastatin Isomerization on OATP1B1-Mediated Transport.

Pravastatin acid (PVA) can be isomerized to its inactive metabolite 3'α-iso-pravastatin acid (3αPVA) under acidic pH conditions. Previous studies reported interindividual differences in circulating concentrations of PVA and 3αPVA. This study investigated the functional consequences of PVA isomerization on OATP1B1-mediated transport. We characterized 3αPVA inhibition of OATP1B1-mediated PVA uptake into human embryonic kidney 293 cells expressing the four different OATP1B1 proteins (*1a, *1b, *5, and *15). 3αPVA inhibited OATP1B1-mediated PVA uptake in all four OATP1B1 gene products but with lower IC50/Ki values for OATP1B1*5 and *15 than for the reference proteins (*1a and *1b). PVA and 3αPVA were transported by all four OATP1B1 proteins. Kinetic experiments revealed that maximal transport rates (Vmax values) for OATP1B1 variants *5 and *15 were lower than for *1a and *1b for both substrates. Apparent affinities for 3αPVA transport were similar for all four variants. However, the apparent affinity of OATP1B1*5 for 3αPVA was higher (lower Km value) than for PVA. These data confirm that PVA conversion to 3αPVA can have functional consequences on PVA uptake and impacts OATP1B1 variants more than the reference protein, thus highlighting another source variation that must be taken into consideration when optimizing the PVA dose-exposure relationship for patients. SIGNIFICANCE STATEMENT: 3'α-iso-pravastatin acid inhibits pravastatin uptake for all OATP1B1 protein types; however, the IC50 values were significantly lower in OATP1B1*5 and *15 transfected cells. This suggests that a lower concentration of 3'α-iso-pravastatin is needed to disrupt OATP1B1-mediated pravastatin uptake, secondary to decreased cell surface expression of functional OATP1B1 in variant-expressing cells. These data will refine previous pharmacokinetic models that are utilized to characterize pravastatin interindividual variability with an ultimate goal of maximizing efficacy at the lowest possible risk for toxicity.

Rheostat positions: A new classification of protein positions relevant to pharmacogenomics.

To achieve the full potential of pharmacogenomics, one must accurately predict the functional out comes that arise from amino acid substitutions in proteins. Classically, researchers have focused on understanding the consequences of individual substitutions. However, literature surveys have shown that most substitutions were created at evolutionarily conserved positions. Awareness of this bias leads to a shift in perspective, from considering the outcomes of individual substitutions to understanding the roles of individual protein positions. Conserved positions tend to act as "toggle" switches, with most substitutions abolishing function. However, nonconserved positions have been found equally capable of affecting protein function. Indeed, many nonconserved positions act like functional dimmer switches ("rheostat" positions): This is revealed when multiple substitutions are made at a single position. Each substitution has a different functional outcome; the set of substitutions spans arange of outcomes. Finally, some nonconserved positions appear neutral, capable of accommodating all amino acid types without modifying function. This manuscript reviews the currently-known properties of rheost at positions, with examples shown for pyruvate kinase, organic anion transporting polypeptide 1B1, the beta-lactamase inhibitory protein, and angiotensin-converting enzyme 2. Outcomes observed for rheostat positions have implications for the rational design of drug analogs and allosteric drugs. Furthermore, this new framework - comprising three types of protein positions - provides a new approach to interpreting disease and population-based databases of amino acid changes. In conclusion, although a full understanding of substitution out comes at rheostat positions poses a challenge, utilization of this new frame of reference will further advance the application of pharmacogenomics.

OATP1B3 Expression and Function is Modulated by Coexpression with OCT1, OATP1B1, and NTCP.

Organic anion transporting polypeptide (OATP) 1B3 is a drug transporter expressed at the basolateral membrane of human hepatocytes. Along with other transporters, including OATP1B1, Na+/taurocholate cotransporting polypeptide (NTCP), and organic cation transporter (OCT) 1, it is responsible for the uptake of endo- and xenobiotics into hepatocytes. Our previous studies demonstrated that OATP1B3 can form hetero-oligomers with OATP1B1 in human embryonic kidney 293T (HEK293) cells and with NTCP in both HEK293 cells and frozen human liver sections. To further characterize the hetero-oligomerization of OATP1B3, we investigated OCT1 as a potential interacting partner and determined the functional consequences of OATP1B3 hetero-oligomerization. We demonstrated interactions between OATP1B3 and OCT1 by coimmunoprecipitation with an anti-OATP1B3 antibody from human hepatocytes. In addition, we visualized the interaction using the proximity ligation assay in both HEK293 cells and in frozen human liver sections. We investigated the functional consequences of OATP1B3 hetero-oligomerization by measuring the OATP1B3 plasma membrane expression and the uptake of the OATP1B3 selective substrate cholecystokinin-8 (CCK-8) in the absence and presence of OATP1B1, NTCP, and OCT1. A significant decrease of OATP1B3 plasma membrane expression was observed after coexpression with OCT1, whereas coexpression with OATP1B1 or NTCP resulted in an increase of plasma membrane expression. With respect to transport, coexpression of OCT1 increased the apparent turnover rate of OATP1B3, whereas coexpression of OATP1B1 or NTCP decreased it. These findings demonstrated that coexpression of OATP1B3 with OATP1B1, NTCP, and OCT1 in HEK293 cells results in a transporter-dependent modification of OATP1B3-mediated CCK-8 transport and suggest that functional results obtained in single transporter overexpressing cell lines over- or underestimate OATP1B3 function in human hepatocytes. SIGNIFICANCE STATEMENT: Coexpression of organic anion transporting polypeptide (OATP) 1B3 with organic cation transporter (OCT) 1, Na+/taurocholate cotransporting polypeptide, or OATP1B1 in human embryonic kidney 293T cells affects its expression level and function. When OCT1 is knocked down in human hepatocytes, function of OATP1B3 goes up. These results suggest that protein-protein interactions can affect the expression and function of the involved proteins, and thus single transporter expression systems might lead to over- or underestimation of drug-drug interactions.

Protein-protein interactions of drug uptake transporters that are important for liver and kidney.

Drug uptake transporters are membrane proteins responsible for the trans-membrane transport of endo- and xenobiotics, including numerous drugs. They are important for the uptake of drugs into target tissues or into organs for metabolism and excretion. Many drug uptake transporters have a broad spectrum of structural-independent substrates, which make them vulnerable to drug-drug interactions. Recent studies have shown more and more complex pharmacokinetics involving transporters, and regulatory agencies now require studies to be performed to measure the involvement of transporters in drug development. A better understanding of the factors affecting the expression of transporters is needed. Despite many efforts devoted to the functional characterization of different drug uptake transporters, transporter in vitro to in vivo extrapolations are far from predicting the behavior under physiological conditions. There is an increasing number of uptake transporters demonstrated to form protein-protein interactions or to oligomerize. This raises the possibility that these interactions between or among transporters could help explaining the gap between in vitro and in vivo measurement of drug transporters. In this review, we summarized protein-protein interactions of drug uptake transporters that are important for pharmacokinetics, especially those in the liver and the kidneys.

A Pharmacokinetic Natural Product-Disease-Drug Interaction: A Double Hit of Silymarin and Nonalcoholic Steatohepatitis on Hepatic Transporters in a Rat Model.

Patients with nonalcoholic steatohepatitis (NASH) exhibit altered hepatic protein expression of metabolizing enzymes and transporters and altered xenobiotic pharmacokinetics. The botanical natural product silymarin, which has been investigated as a treatment of NASH, contains flavonolignans that inhibit organic anion-transporting polypeptide (OATP) transporter function. The purpose of this study was to assess the individual and combined effects of NASH and silymarin on the disposition of the model OATP substrate pitavastatin. Male Sprague Dawley rats were fed a control or a methionine- and choline-deficient diet (NASH model) for 8 weeks. Silymarin (10 mg/kg) or vehicle followed by pitavastatin (0.5 mg/kg) were administered intravenously, and the pharmacokinetics were determined. NASH increased mean total flavonolignan area under the plasma concentration-time curve (AUC0-120 min) 1.7-fold. Silymarin increased pitavastatin AUC0-120 min in both control and NASH animals approx. 2-fold. NASH increased pitavastatin plasma concentrations from 2 to 40 minutes, but AUC0-120 min was unchanged. The combination of silymarin and NASH had the greatest effect on pitavastatin AUC0-120 min, which increased 2.9-fold compared with control vehicle-treated animals. NASH increased the total amount of pitavastatin excreted into the bile 2.7-fold compared with control animals, whereas silymarin decreased pitavastatin biliary clearance approx. 3-fold in both control and NASH animals. This double hit of NASH and silymarin on hepatic uptake transporters is another example of a multifactorial pharmacokinetic interaction that may have a greater impact on drug disposition than each hit alone. SIGNIFICANCE STATEMENT: Multifactorial effects on xenobiotic pharmacokinetics are within the next frontier for precision medicine research and clinical application. The combination of silymarin and NASH is a probable clinical scenario that can affect drug uptake, liver concentrations, biliary elimination, and ultimately, efficacy and toxicity.

Gly45 and Phe555 in Transmembrane Domains 1 and 10 Are Critical for the Activation of Organic Anion Transporting Polypeptide 1B3 by Epigallocatechin Gallate.

Organic anion transporting polypeptides (OATPs) 1B1 and 1B3 are two highly homologous transporters expressed in the human liver. However, epigallocatechin gallate (EGCG), which is the most predominant catechin in green tea, has opposite effects on the function of OATP1B1 and OATP1B3. In the present study, the critical structural domains and amino acid residues for the activation of OATP1B3 by EGCG have been determined by characterizing the function of a series of OATP1B3-derived chimeric transporters, site-directed mutagenesis, and kinetic studies. Our results showed that G45 and F555 in transmembrane domains 1 and 10 are the most important amino acid residues for OATP1B3 activation. Kinetic studies showed that the activation of OATP1B3 by EGCG at a low substrate concentration was due to its increased substrate binding affinity. However, EGCG caused increased Km and decreased Vmax for 1B3-G45A and 1B3-F555H. The flexibility at position 45 and aromaticity at position 555 might be important for OATP1B3 activation. While 1B3-G45A and 1B3-F555H could not be activated by EGCG, their transport activity for EGCG was comparable to that of wild-type OATP1B3. In conclusion, the present study elucidated the molecular mechanism for OATP1B3 activation by EGCG.