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In Silico Identification of Potential Cholestasis-Inducing Agents Via Modeling of Na+-Dependent Taurocholate Cotransporting Polypeptide Substrate Specificity

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In Silico Identification of Potential Cholestasis-Inducing Agents Via Modeling of Na+-Dependent Taurocholate Cotransporting Polypeptide Substrate Specificity TOXICOLOGICAL SCIENCES 129(1), 35–48 (2012) doi:10.1093/toxsci/kfs188 Advance Access publication May 28, 2012 In Silico Identification of Potential Cholestasis-Inducing Agents via Modeling of Na+-Dependent Taurocholate Cotransporting Polypeptide Substrate Specificity Rick Greupink,*,1 Sander B. Nabuurs,†,2 Barbara Zarzycka,† Vivienne Verweij,* Mario Monshouwer,‡ Maarten T. Huisman,‡ and Frans G. M. Russel* Downloaded from https://academic.oup.com/toxsci/article/129/1/35/1629030 by guest on 29 September 2021 *Department of Pharmacology and Toxicology, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands; †Centre for Molecular and Biomolecular Informatics, Radboud University Nijmegen, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands; and ‡Department of Drug Metabolism and Pharmacokinetics, Janssen Pharmaceutical Companies of Johnson and Johnson, Beerse, Belgium 1To whom correspondence should be addressed at Department of Pharmacology and Toxicology 149, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Fax: +(31) 24 36 14214. E-mail: [email protected]. 2Present address: Lead Pharma Medicine, Nijmegen, The Netherlands. Received March 23, 2012; accepted May 16, 2012 The liver plays an important role in bile acid homeostasis. At Na+-dependent taurocholate cotransporting polypeptide (NTCP, physiological pH, bile acids do not effectively cross cell mem- SLC10A1) is the main transporter facilitating the hepatic uptake branes by passive diffusion because they exist for an important of bile acids from the circulation. Consequently, the interaction of part in the anionic form. Hepatocellular uptake of bile acids xenobiotics, including therapeutic drugs, with the bile acid bind- ing pocket of NTCP could lead to impairment of hepatic bile acid from portal blood and subsequent excretion into bile, therefore, uptake. We pursued a 3D-pharmacophore approach to model the completely depends on a coordinated function of transport NTCP substrate and inhibitor specificity and investigated whether proteins located in the basolateral and canalicular membranes it is possible to identify compounds with intrinsic NTCP inhibi- of liver cells (Kosters and Karpen, 2008). As a consequence, tory properties. Based on known endogenous NTCP substrates, a any (xenobiotic-mediated) functional disruption of these trans- 3D-pharmacophore model was built, which was subsequently used port processes may lead to cholestasis (Pauli-Magnus and to screen two virtual libraries together containing the structures of Meier, 2006). 10 million compounds. Studies with Chinese hamster ovary cells The Na+-dependent taurocholate cotransporting polypeptide overexpressing human NTCP, human hepatocytes, ex vivo per- (NTCP, SLC10A1) is the major transporter that takes up bile fused rat livers, and bile duct–cannulated rats were conducted to acids from the portal blood, which represents the first step in validate the activity of the virtual screening hits. Modeling yielded hepatocellular bile acid handling. Hence, intact NTCP functioning a 3D-pharmacophore, consisting of two hydrogen bond acceptors and three hydrophobic features. Six out of 10 structurally diverse is considered important for the maintenance of the enterohepatic compounds selected in the first virtual screening procedure signifi- circulation of bile acids and hepatocyte homeostasis (Ho et al., cantly inhibited taurocholate uptake in the NTCP overexpressing 2004). In humans, it has been estimated that approximately cells. For the most potent inhibitor identified, an anthraquinone 80% of the hepatic uptake of conjugated bile acids occurs in a derivative, this finding was confirmed in human hepatocytes and sodium-dependent manner, whereas the remainder is taken up perfused rat livers. Subsequent structure and activity relationship through sodium-independent pathways, e.g., via organic anion studies with analogs of this derivative indicated that an appropri- transporting polypeptide 1B1 (OATP1B1, SLCO1B1) (Kosters ate distance between hydrogen bond acceptor features and pres- and Karpen, 2008). Under physiological conditions, bile ence of one or two negative charges appear critical for a successful acids are mainly confined to the enterohepatic circulation, but NTCP interaction. In conclusion, pharmacophore modeling was impaired uptake of bile acids from the portal blood will lead to an successfully used to identify compounds that inhibit NTCP. Our increase in bile acid concentrations in the systemic circulation. approach represents an important first step toward the in silico flagging of potential cholestasis-inducing molecules. During cholestasis, a 20- to 100-fold increase in systemic bile Key Words: NTCP; cholestasis; pharmacophore modeling; acid concentrations have been reported, which is associated with drug-induced toxicity. morbidity (Makino et al., 1969). For instance, hypercholanemia has been associated with extrahepatic adverse effects, a © The Author 2012. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected] 36 GREUPINK ET AL. well-known example being pruritus (Kuiper et al., 2010). Also, database, Accelrys, San Diego, CA) by screening the databases against the asymptomatic hypercholanemia during pregnancy, which is pharmacophore model. Once identified in silico, the selected chemicals were characterized by specifically elevated maternal serum levels of purchased from the registered chemical suppliers indicated in these databases. Both database resources contain only commercially available molecules bile acids > 40µM, has been associated with a higher incidence and can be found through http://cococo.unimore.it and http://accelrys.com. of preterm labor, fetal asphyxia, and third trimester intrauterine Chemical structures of the compounds are provided in this article. In addition, death (Abu-Hayyeh et al., 2010; Glantz et al., 2004; Williamson full chemical names are listed in Supplementary table 1. Prior to conducting in et al., 2004). Studies suggest bile acid–induced damage of vitro experiments, the purchased chemicals were dissolved in dimethylsulfox- 3 cell membranes as a potential biochemical mechanism of the ide (DMSO), unless indicated otherwise. H-taurocholic acid (specific activity 4.6 Ci/mmol) was obtained from Perkin Elmer (Boston, MA). L-Glutamine and observed toxicity during cholestasis, whereas, additionally, bile sodium pyruvate were obtained from Invitrogen, whereas DMSO and all other acids have also been shown to cause mitochondrial dysfunction reagents were of the highest obtainable grade available and were purchased and modulate second messenger pathways involved in the from local suppliers. regulation of cellular metabolism (Bouscarel et al., 1995; Experimental animals. Male Sprague Dawley rats (Harlan, Horst, The Downloaded from https://academic.oup.com/toxsci/article/129/1/35/1629030 by guest on 29 September 2021 Schölmerich et al., 1984; Spivey et al., 1993). Netherlands) weighing 220–240 g (in vivo experiments) or 240–300 g (liver Recently, some drugs and other xenobiotics have been iden- perfusions) were housed under a 12-h dark/light cycle at constant humidity tified as NTCP substrates, based on data obtained in isolated and temperature. Animals had free access to tap water and standard lab chow. hepatocytes and NTCP recombinant overexpression systems. Prior to conducting experiments, animals were allowed to acclimatize for 1 week after arrival. All experiments were approved by the local committees for For example, various statins, the liver function diagnostics bro- care and use of experimental animals and were performed according to strict mosulphophtalein and indocyanine green, as well as the antifun- governmental and international guidelines for the use of experimental animals. gal drug micafungin are transported by NTCP (Greupink et al., Generation of a common feature pharmacophore and database screen- 2011; Ho et al., 2006; Yanni et al., 2010). Hilgendorf et al. inves- ing. Molecular modeling studies were performed with the Discovery Studio tigated the hepatic mRNA concentration of basolateral and cana- Software package version 2.5.5 (Accelrys). A common feature, ligand-based licular transporters and found that NTCP expression is similar to pharmacophore model was built based on the molecular structure of five estab- that of the well-known and clinically relevant basolateral drug lished NTCP substrates, namely the unconjugated bile acid cholic acid, the uptake transporter OATP1B1, which is among the most abun- conjugated bile acids taurocholic acid, tauroursodeoxycholic acid and gly- cocholic acid, and the non–bile acid estrone-3-sulphate. The 3D chemical dantly expressed transporters in human liver (Hilgendorf et al., structures of the molecules were obtained from the PubChem online database 2007). Studies in human hepatocytes demonstrated that NTCP (National Center for Biotechnology Information, Bethesda, MD). Due to the can indeed contribute substantially to the overall uptake of drugs conformational freedom of the studied molecules, functional groups within in these cells (Ho et al., 2006; Yanni et al., 2010). This suggests molecules will not have fixed positions in 3D space. To account for the effect that next to cholestasis, xenobiotic-induced inhibition of NTCP of rotational freedom, for each molecule up to 255 conformers were gener-
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