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Novel in Vitro Method Reveals Drugs That Inhibit Organic Solute Transporter Alpha/Beta (OST?/?).

ABSTRACT: Drug interactions with the organic solute transporter alpha/beta (OST?/?) are understudied even though OST?/? is an important transporter that is expressed in multiple human tissues including the intestine, kidneys, and liver. In this study, an in vitro method to identify novel OST?/? inhibitors was first developed using OST?/?-overexpressing Flp-In 293 cells. Incubation conditions were optimized using previously reported OST?/? inhibitors. A method including a 10 min preincubation step with the test compound was used to screen for OST?/? inhibition by 77 structurally diverse compounds and fixed-dose combinations. Seven compounds and one fixed-dose combination (100 ?M final concentration) inhibited OST?/?-mediated dehydroepiandrosterone sulfate (DHEAS) uptake by >25%. Concentration-dependent OST?/? inhibition was evaluated for all putative inhibitors (atorvastatin, ethinylestradiol, fidaxomicin, glycochenodeoxycholate, norgestimate, troglitazone, and troglitazone sulfate). Ethinylestradiol, fidaxomicin, and troglitazone sulfate yielded a clear concentration-inhibition response with IC50 values <200 ?M. Among all tested compounds, there was no clear association between physicochemical properties, the severity of hepatotoxicity, and the degree of OST?/? inhibition. This study utilized a novel in vitro method to identify OST?/? inhibitors and, for the first time, provided IC50 values for OST?/? inhibition. These data provide evidence that several drugs, some of which are associated with cholestatic drug-induced liver injury, may impair the function of the OST?/? transporter.

SUBMITTER: Malinen MM

PROVIDER: S-EPMC6465078 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Novel in Vitro Method Reveals Drugs That Inhibit Organic Solute Transporter Alpha/Beta (OSTα/β).

Malinen Melina M MM Kauttonen Antti A Beaudoin James J JJ Sjöstedt Noora N Honkakoski Paavo P Brouwer Kim L R KLR

Molecular pharmaceutics 20181214 1

Drug interactions with the organic solute transporter alpha/beta (OSTα/β) are understudied even though OSTα/β is an important transporter that is expressed in multiple human tissues including the intestine, kidneys, and liver. In this study, an in vitro method to identify novel OSTα/β inhibitors was first developed using OSTα/β-overexpressing Flp-In 293 cells. Incubation conditions were optimized using previously reported OSTα/β inhibitors. A method including a 10 min preincubation step with the ...[more]

PMID: 30481467

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Project description:Organic solute transporter alpha/beta (OSTα/β) is a heteromeric solute carrier protein that transports bile acids, steroid metabolites and drugs into and out of cells. OSTα/β protein is expressed in various tissues, but its expression is highest in the gastrointestinal tract where it facilitates the recirculation of bile acids from the gut to the liver. Previous studies established that OSTα/β is upregulated in liver tissue of patients with extrahepatic cholestasis, obstructive cholestasis, and primary biliary cholangitis (PBC), conditions that are characterized by elevated bile acid concentrations in the liver and/or systemic circulation. The discovery that OSTα/β is highly upregulated in the liver of patients with nonalcoholic steatohepatitis (NASH) further highlights the clinical relevance of this transporter because the incidence of NASH is increasing at an alarming rate with the obesity epidemic. Since OSTα/β is closely linked to the homeostasis of bile acids, and tightly regulated by the nuclear receptor farnesoid X receptor, OSTα/β is a potential drug target for treatment of cholestatic liver disease, and other bile acid-related metabolic disorders such as obesity and diabetes. Obeticholic acid, a semi-synthetic bile acid used to treat PBC, under review for the treatment of NASH, and in development for the treatment of other metabolic disorders, induces OSTα/β. Some drugs associated with hepatotoxicity inhibit OSTα/β, suggesting a possible role for OSTα/β in drug-induced liver injury (DILI). Furthermore, clinical cases of homozygous genetic defects in both OSTα/β subunits resulting in diarrhea and features of cholestasis have been reported. This review article has been compiled to comprehensively summarize the recent data emerging on OSTα/β, recapitulating the available literature on the structure-function and expression-function relationships of OSTα/β, the regulation of this important transporter, the interaction of drugs and other compounds with OSTα/β, and the comparison of OSTα/β with other solute carrier transporters as well as adenosine triphosphate-binding cassette transporters. Findings from basic to more clinically focused research efforts are described and discussed.

Project description:Organic solute transporter (OST) α/β is a key bile acid transporter expressed in various organs, including the liver under cholestatic conditions. However, little is known about the involvement of OSTα/β in bile acid-mediated drug-induced liver injury (DILI), a major safety concern in drug development. This study investigated whether OSTα/β preferentially transports more hepatotoxic, conjugated, primary bile acids and to what extent xenobiotics inhibit this transport. Kinetic studies with OSTα/β-overexpressing cells revealed that OSTα/β preferentially transported bile acids in the following order: taurochenodeoxycholate > glycochenodeoxycholate > taurocholate > glycocholate. The apparent half-maximal inhibitory concentrations for OSTα/β-mediated bile acid (5 µM) transport inhibition by fidaxomicin, troglitazone sulfate, and ethinyl estradiol were: 210, 334, and 1050 µM, respectively, for taurochenodeoxycholate; 97.6, 333, and 337 µM, respectively, for glycochenodeoxycholate; 140, 265, and 527 µM, respectively, for taurocholate; 59.8, 102, and 117 µM, respectively, for glycocholate. The potential role of OSTα/β in hepatocellular glycine-conjugated bile acid accumulation and cholestatic DILI was evaluated using sandwich-cultured human hepatocytes (SCHH). Treatment of SCHH with the farnesoid X receptor agonist chenodeoxycholate (100 µM) resulted in substantial OSTα/β induction, among other proteomic alterations, reducing glycochenodeoxycholate and glycocholate accumulation in cells+bile 4.0- and 4.5-fold, respectively. Treatment of SCHH with troglitazone and fidaxomicin together under cholestatic conditions resulted in increased hepatocellular toxicity compared with either compound alone, suggesting that OSTα/β inhibition may accentuate DILI. In conclusion, this study provides insights into the role of OSTα/β in preferential disposition of bile acids associated with hepatotoxicity, the impact of xenobiotics on OSTα/β-mediated bile acid transport, and the role of this transporter in SCHH and cholestatic DILI.

Project description:Organic solute transporter α/β (OSTα/β) is a bidirectional bile acid transporter localized on the basolateral membrane of hepatic, intestinal, and renal epithelial cells. OSTα/β plays a critical role in intestinal bile acid reabsorption and is upregulated in hepatic diseases characterized by elevated bile acids, whereas genetic variants in SLC51A/B have been associated with clinical cholestasis. OSTα/β also transports and is inhibited by commonly used medications. However, there is currently no high-resolution structure of OSTα/β, and structure-function data for OSTα, the proposed substrate-binding subunit, are lacking. The present study addressed this knowledge gap and identified amino acids in OSTα that are important for bile acid transport. This was accomplished using computational modeling and site-directed mutagenesis of the OSTα subunit to generate OSTα/β mutant cell lines. Out of the 10 OSTα/β mutants investigated, four (S228K, T229S, Q269E, Q269K) exhibited decreased [3H]-taurocholate (TCA) uptake (ratio of geometric means relative to OSTα/β wild type (WT) of 0.76, 0.75, 0.79, and 0.13, respectively). Three OSTα/β mutants (S228K, Q269K, E305A) had reduced [3H]-TCA efflux % (ratio of geometric means relative to OSTα/β WT of 0.86, 0.65, and 0.79, respectively). Additionally, several OSTα/β mutants demonstrated altered expression and cellular localization when compared with OSTα/β WT. In summary, we identified OSTα residues (Ser228, Thr229, Gln269, Glu305) in predicted transmembrane domains that affect expression of OSTα/β and may influence OSTα/β-mediated bile acid transport. These data advance our understanding of OSTα/β structure/function and can inform future studies designed to gain further insight into OSTα/β structure or to identify additional OSTα/β substrates and inhibitors. SIGNIFICANCE STATEMENT: OSTα/β is a clinically important transporter involved in enterohepatic bile acid recycling with currently no high-resolution protein structure and limited structure-function data. This study identified four OSTα amino acids (Ser228, Thr229, Gln269, Glu305) that affect expression of OSTα/β and may influence OSTα/β-mediated bile acid transport. These data can be utilized to inform future investigation of OSTα/β structure and refine molecular modeling approaches to facilitate the identification of substrates and/or inhibitors of OSTα/β.

Project description:The heteromeric steroid transporter organic solute transporter ?/? (OST?/?, SLC51A/B) was discovered over a decade ago, but its physiological significance in the liver remains uncertain. A major challenge has been the lack of suitable models expressing OST?/?. Based on observations first reported here that hepatic OST?/? is upregulated in nonalcoholic steatohepatitis, the aim of this research was to develop an in vitro model to evaluate OST?/? function and interaction with drugs and bile acids. OST?/? expression in human liver tissue was analyzed by quantitative RT-PCR, Western blotting, and immunofluorescence. Radiolabeled compounds were used to determine OST?/?-mediated transport in the established in vitro model. The effect of bile acids and drugs, including those associated with cholestatic drug-induced liver injury, on OST?/?-mediated transport was evaluated. Expression of OST?/? was elevated in the liver of patients with nonalcoholic steatohepatitis and primary biliary cholangitis, whereas hepatocyte expression of OST?/? was low in control liver tissue. Studies in the novel cell-based system showed rapid and linear OST?/?-mediated transport for all tested compounds: dehydroepiandrosterone sulfate, digoxin, estrone sulfate, and taurocholate. The interaction study with 26 compounds revealed novel OST?/? inhibitors: a biomarker for cholestasis, glycochenodeoxycholic acid; the major metabolite of troglitazone, troglitazone sulfate; and a macrocyclic antibiotic, fidaxomicin. Additionally, some drugs (e.g., digoxin) consistently stimulated taurocholate uptake in OST?/?-overexpressing cells. Our findings demonstrate that OST?/? is an important transporter in liver disease and imply a role for this transporter in bile acid-bile acid and drug-bile acid interactions, as well as cholestatic drug-induced liver injury. NEW & NOTEWORTHY The organic solute transporter OST?/? is highly expressed in hepatocytes of liver tissue obtained from patients with nonalcoholic steatohepatitis and primary biliary cholangitis. OST?/? substrates exhibit rapid, linear, and concentration-driven transport in an OST?/?-overexpressing cell line. Drugs associated with hepatotoxicity modulate OST?/?-mediated taurocholate transport. These data suggest that hepatic OST?/? plays an essential role in patients with cholestasis and may have important clinical implications for bile acid and drug disposition.

Dataset Information

Novel in Vitro Method Reveals Drugs That Inhibit Organic Solute Transporter Alpha/Beta (OST?/?).

Publications

Novel in Vitro Method Reveals Drugs That Inhibit Organic Solute Transporter Alpha/Beta (OSTα/β).

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