Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2017/05/23/dmd.117.075523.DC1

1521-009X/45/8/974–976$25.00 https://doi.org/10.1124/dmd.117.075523 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 45:974–976, August 2017 Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics Short Communication

Rosuvastatin and Atorvastatin Are Ligands of the Human Constitutive Androstane Receptor/Retinoid X Receptor a Complex s

Received February 21, 2017; accepted May 17, 2017

ABSTRACT Statins are well known lipid lowering agents that inhibit the enzyme models of CAR/RXRa-LBD were constructed by ligand-based and – 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase. They also structure-based in silico modeling. Experiments and computational Downloaded from activate drug metabolism but their exact receptor-mediated action modeling show that atorvastatin and rosuvastatin bind to the human has not been proven so far. We tested whether atorvastatin and CAR/RXRa-LBD heterodimer, suggesting both can modulate the rosuvastatin are direct ligands of human constitutive androstane activity of CAR through direct interaction with the LBD of this receptor (CAR). We measured binding activities of atorvastatin and receptor. We confirm that atorvastatin and rosuvastatin are direct rosuvastatin to the human constitutive androstane receptor/retinoid ligands of CAR. The clinical consequences of CAR activation by X receptor a ligand-binding domain (CAR/RXRa-LBD) heterodimer statins are in their potential drug-drug interactions, and changes in dmd.aspetjournals.org with surface plasmon resonance (SPR). Additionally, three-dimensional glucose and energy metabolism.

Introduction many other pathways (Ueda et al., 2002; Rezen et al., 2009). Therefore, – Statins, which have been recognized as drugs of choice for use in the clinical relevance of statin CAR interactions is not only in potential drug-drug interactions but also in changes in glucose and energy patients at high risk for developing cardiovascular disease, also may at ASPET Journals on September 29, 2021 have some side effects, including an increased risk for diabetes (Sattar metabolism induced by statins. et al., 2014). Transcriptome analyses have revealed that rosuvastatin and In this study we have combined computational and experimen- atorvastatin affect expression of many metabolic and signaling pathways in tal approaches to determine whether rosuvastatin and atorvastatin are a a the liver, including drug metabolism (Hafner et al., 2011). Several statins ligands of the CAR/retinoid X receptor (RXR ) heterodimer. Using have also been identified as inducers of drug metabolism and potential surface plasmon resonance (SPR) we measured the binding of atorvastatin, agonists for the constitutive androstane receptor (CAR, NR1I3) (Kocarek rosuvastatin, and 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde a et al., 2002; Kobayashi et al., 2005; Monostory et al., 2009). However, O-(3,4-dichlorobenzyl)oxime (CITCO) to the CAR/RXR ligand-binding discrepancies among the studies exist. For example, a study using cell-based domain (LBD) heterodimer. We also used ligand and structure-based reporter assay and CAR inverse agonist identified fluvastatin, atorvastatin, modeling of different statins to CAR and the pregnane X receptor (PXR, simvastatin, and cerivastatin as human CAR (hCAR) activators (Kobayashi NR1I2), another drug metabolism-inducing nuclear receptor. et al., 2005; Monostory et al., 2009). However, in a study using a ligand- binding assay none of the tested statins (fluvastatin, atorvastatin, simva- Materials and Methods statin) were confirmed to be activators of hCAR (Howe et al., 2011). In addition, atorvastatin did not activate hCAR using two-hybrid assembly The materials and methods are described in more detail in the Supplemental Data. S (Hoffart et al., 2012). Thus, although studies have suggested that statins The glutathione -transferase (GST)-hCAR (gift from Jean Marc Pascussi, INSERM, France) and GST-hRXRa-LBD (Addgene, Cambridge, MA) plasmids were trans- might be ligands of this nuclear receptor, experimental evidence measuring formed into the BL21 (DE3) Escherichia coli strain. Briefly, the overnight culture direct binding of statins to CAR has been lacking so far. was grown in Lysogeny broth medium with ampicillin, then protein expression was CAR is a member of the nuclear receptor superfamily and was initially induced with the addition of isopropyl b-D-1-thiogalactopyranoside (IPTG). Cell discovered as a xenosensor regulating drug metabolism (Honkakoski supernatant was loaded on a GST affinity column (Clontech, Palo Alto, CA) and et al., 1998; Sueyoshi et al., 1999). CAR also was implicated in the concentrated using a Microcon YM-30 centrifugal filter (Millipore, Bedford, MA). regulation of lipid, glucose, and energy metabolism, proliferation, and The purity of the isolated GST-hCAR and GST-hRXRa-LBD were checked by SDS-PAGE and Western blot analysis. The interaction of rosuvastatin, atorvastatin, and CITCO was measured using a This work was supported by the Slovenian Research Agency [Grant P1-0104, Biacore X analytical system and CM5 sensor chip (Biacore/GE Healthcare, P1-0390], and was part of the doctoral thesis of M.H. Uppsala, Sweden). We prepared 10 mM stock solution of each analyte in T.R. and M.H. contributed equally to this work. dimethylsulfoxide, which then was diluted into a series of concentrations with https://doi.org/10.1124/dmd.117.075523. the running buffer to detect direct binding with immobilized GST-hCAR/

s This article has supplemental material available at dmd.aspetjournals.org. GST-hRXRa-LBD. The equilibrium dissociation constant (KD) values were

ABBREVIATIONS: CAR, constitutive androstane receptor; CITCO, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4- dichlorobenzyl)oxime; GST, glutathione S-transferase; hCAR, human CAR; KD, equilibrium dissociation constant; LBD, ligand-binding domain; PXR, pregnane X receptor; RXRa, retinoid X receptor a; SPR, surface plasmon resonance; SRC-1, steroid receptor coactivator 1.

974 Statins Are Ligands of Human CAR/RXRa Complex 975

Fig. 1. SPR analysis of atorvastatin, rosuvastatin, and CITCO binding to hCAR/hRXRa-LBD hetero- dimer. Different concentrations (1.56, 3.125, 12.5, 25, and 50 mM for CITCO and 6, 12.5, 25, 50, 75, 100, and 150 mM for atorvastatin and rosuvastatin) of analytes were tested for the binding (left panels). The binding curves (right panels) were generated by fitting the steady-state response levels at the end of the association phase versus the concentration of the injected analyte. The KD values were obtained by fitting the data to the steady-state affinity model. For each analyte six or seven independent titration experiments were performed. Downloaded from dmd.aspetjournals.org determined by fitting the data to the steady-state affinity model using BIAevaluation The measured KD indicated that rosuvastatin binds to the CAR/RXR software (Biacore/GE Healthcare). Previously generated pharmacophores for PXR or heterodimer with approximately 2-fold higher affinity than atorvastatin, CAR agonists or Bayesian models (Ekins et al., 2009) generated with steroids or but approximately 3-fold lower affinity than CITCO (Table 1). A com- diverse compounds were employed to generate predictions for the statins tested in this parison of measured maximal response with the theoretical maximal study. The crystal structures of PXR and CAR were obtained from the Protein Data response indicated that several molecules of atorvastatin and CITCO Bank (www.rcsb.org) with codes 1M13 and 1XVP, respectively, and used for bind to the hCAR/hRXRa-LBD complex (Table 1). By applying SPR docking.

we show for the first time that atorvastatin and rosuvastatin directly bind at ASPET Journals on September 29, 2021 to the hCAR/RXR-LBD heterodimer. Binding of statins to hCAR is consistent with our previous transcriptome Results and Discussion study,inwhichweshowedthatmanygenes were differentially expressed Because it was still not known whether atorvastatin and rosuvastatin in human primary hepatocytes treated with atorvastatin and rosuvastatin are binding to human CAR, we measured the interaction of rosuvastatin, and are under direct regulation of CAR (Hafner et al., 2011). Another study atorvastatin, and the positive control CITCO with hCAR using the SPR used surface plasmon resonance to test binding of atorvastatin and hCAR technique. The structures of the molecules are shown in Supplemental Fig. 1. with coactivator (Hoffart et al., 2012). They preincubated atorvastatin or Recombinant proteins GST-hCAR and GST-hRXRa-LBD were isolated and CITCO with CAR-LBD protein and injected it onto immobilized steroid their purity assessed by SDS-PAGE and Western blot analysis (Supplemental receptor coactivator 1–receptor interaction domain (SRC-1-RID) protein. Fig. 2). The SDS-PAGE analyses of recombinant proteins revealed high Under these conditions atorvastatin did not induce binding of hCAR-LBD purity, and the Western blot analyses confirmed the presence of GST proteins. with coactivator SRC-1. Further, they did not observe the assembly of The GST-hCAR/GST-hRXRa-LBD heterodimer was covalently human CAR-LBD with coactivators in COS1 cells. attached to the surface of the SPR sensor chip and assayed with at least The difference in results obtained by SPR between our study and that of four different concentrations of compounds. Each concentration was Hoffart et al. (2012) was probably due to the different CAR dimerization injected 3 times to test the reproducibility. All binding responses and KD partner and steps used. We used RXR as a dimerization partner, and studies values were reproducible, which indicates that immobilized complex did show that RXRa mayalsohavearoleinstabilizingCAR-LBDinitsactive not lose activity over time. conformation via direct interactions across the heterodimer interface (Xu As expected, CITCO, which is known to be a direct ligand for hCAR, et al., 2004). RXR is also important for recruitment of coactivators such as showed specific binding to the complex. We also observed specific SRC-1 (steroid receptor coactivator 1) (Dussault et al., 2002; Pavlin et al., concentration-dependent binding of both statins of this study (Fig. 1). 2014). Also, we tested the binding of atorvastatin to the CAR/RXR dimer

TABLE 1

KD values and theoretical and measured maximal responses of atorvastatin, rosuvastatin, and CITCO binding to hCAR/hRXRa-LBD heterodimer as measured by surface plasmon resonance and by scores of ligand-based (CAR pharmacophore) and structure-based (CAR score docking) in silico calculations

CAR Maximal Response Compound KD (mM) Score Docking Pharmacophore Theoretical Measured Atorvastatin 0.225 6 0.06 73.18 2.07 84 251 Rosuvastatin 0.144 6 0.03 67.09 — 70 86 CITCO 0.045 6 0.04 63.02 — 30 74 976 Rezen et al. whereas Hoffart et al. (2012) tested binding of the atorvastatin–CAR pair to Acknowledgments the SRC-1 coactivator. It is important to note that none of the described The authors thank Biovia for providing Discovery Studio. experiments published on statins used physiologic concentrations of the drugs. All used micromolar concentrations, but in humans in serum only Authorship Contributions nanomolar concentrations were measured; certainly concentrations in the Participated in research design: Rezen, Rozman. liver have not been determined, so it is unclear how the in vitro observations Conducted experiments: Hafner, Hodnik. relate to the in vivo situation. Contributed new reagents or analytic tools: Ekins, Kortagere.  Our computational predictions using a structure-based or ligand-based Performed data analysis: Rezen, Hafner, Hodnik, Rozman, Ekins, Kortagere. Wrote or contributed to the writing of the manuscript:  approach indicated that statins are good potential ligands for CAR and Rezen, Hafner, Kortagere, Ekins, Hodnik, Rozman. PXR with little difference in potential between them. Furthermore, statins docked in CAR produced scores that correlate with the KD values determined by SPR. Our data suggested variability in binding to these References different models (Table 1, Supplemental Table 1, Supplemental Fig. 3). Based on the docking results, the statins appeared to be good ligands for Dussault I, Lin M, Hollister K, Fan M, Termini J, Sherman MA, and Forman BM (2002) A structural model of the constitutive androstane receptor defines novel interactions that mediate both CAR and PXR. Atorvastatin had the highest score against CAR ligand-independent activity. Mol Cell Biol 22:5270–5280. (73.18), and rosuvastatin was lower (67.09) (Table 1). Similarly, the Ekins S, Kortagere S, Iyer M, Reschly EJ, Lill MA, Redinbo MR, and Krasowski MD (2009) Challenges predicting ligand-receptor interactions of promiscuous proteins: the nuclear receptor pharmacophores and Bayesian models suggest that all other statins will PXR. PLOS Comput Biol 5:e1000594. Downloaded from likely bind to CAR and PXR (Supplemental Table 1). These results broadly Hafner M, Juvan P, Rezen T, Monostory K, Pascussi J-M, and Rozman D (2011) The human primary hepatocyte transcriptome reveals novel insights into atorvastatin and rosuvastatin action. agree with the published data, confirming that the majority of statins are Pharmacogenet Genomics 21:741–750. CAR/PXR activators, although the manner of this activation awaits Hoffart E, Ghebreghiorghis L, Nussler AK, Thasler WE, Weiss TS, Schwab M, and Burk O (2012) Effects of atorvastatin metabolites on induction of drug-metabolizing enzymes and membrane experimental confirmation in the future. When comparing these scores with transporters through human pregnane X receptor. Br J Pharmacol 165:1595–1608. other studies using the same in silico approaches, we observed that the Honkakoski P, Zelko I, Sueyoshi T, and Negishi M (1998) The nuclear orphan receptor CAR- retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the calculated scores for statins are within the same range as for known hCAR CYP2B gene. Mol Cell Biol 18:5652–5658. activators (Kortagere et al., 2009; Lynch et al., 2013). Interestingly, we and Howe K, Sanat F, Thumser AE, Coleman T, and Plant N (2011) The statin class of HMG-CoA dmd.aspetjournals.org others have predicted that pravastatin is also a good potential ligand for CAR reductase inhibitors demonstrate differential activation of the nuclear receptors PXR, CAR and FXR, as well as their downstream target genes. Xenobiotica 41:519–529. and PXR, although this statin has never been shown to have any ability to Kobayashi K, Yamanaka Y, Iwazaki N, Nakajo I, Hosokawa M, Negishi M, and Chiba K (2005) induce these two nuclear receptors or drug metabolism (Howe et al., 2011). Identification of HMG-CoA reductase inhibitors as activators for human, mouse and rat con- stitutive androstane receptor. Drug Metab Dispos 33:924–929. In conclusion, these findings are important for understanding the full Kocarek TA, Dahn MS, Cai H, Strom SC, and Mercer-Haines NA (2002) Regulation of CYP2B6 biologic consequences of statin exposure due to molecular interaction and CYP3A expression by hydroxymethylglutaryl coenzyme A inhibitors in primary cultured human hepatocytes. Drug Metab Dispos 30:1400–1405. of statins with nuclear receptors. We showed in silico that statins are Kortagere S, Chekmarev D, Welsh WJ, and Ekins S (2009) Hybrid scoring and classification – potentially ligands of CAR and PXR and confirmed in vitro that approaches to predict human pregnane X receptor activators. Pharm Res 26:1001 1011. at ASPET Journals on September 29, 2021 Lynch C, Pan Y, Li L, Ferguson SS, Xia M, Swaan PW, and Wang H (2013) Identification of novel atorvastatin and rosuvastatin directly bind to the CAR/RXRa dimer. As activators of constitutive androstane receptor from FDA-approved drugs by integrated compu- CAR and PXR regulate not only drug metabolism but also glucose and tational and biological approaches. Pharm Res 30:489–501. MonostoryK,PascussiJM,SzabóP,TemesváriM,KöhalmyK,AcimovicJ,KocjanD,KuzmanD, energy metabolism, this had led us to the assumption that they are Wilzewski B, Bernhardt R, et al. (2009) Drug interaction potential of 2-((3,4-dichlorophenethyl) potentially involved in the statins’ unwanted side effects such as the (propyl)amino)-1-(pyridin-3-yl)ethanol (LK-935), the novel nonstatin-type cholesterol-lowering agent. Drug Metab Dispos 37:375–385. increase in developing type 2 diabetes in humans. Additionally, we Pavlin MR, Brunzelle JS, and Fernandez EJ (2014) Agonist ligands mediate the transcriptional conclude that computational approaches may be useful for assessing statin response of nuclear receptor heterodimers through distinct stoichiometric assemblies with coactivators. J Biol Chem 289:24771–24778. binding to CAR and PXR and that SPR can provide valuable insights on Rezen T, Tamasi V, Lövgren-Sandblom A, Björkhem I, Meyer UA, and Rozman D (2009) Effect binding to validate these predictions. of CAR activation on selected metabolic pathways in normal and hyperlipidemic mouse livers. BMC Genomics 10:384. Centre for Functional Genomics and TADEJA REzEN Sattar NA, Ginsberg H, Ray K, Chapman MJ, Arca M, Averna M, Betteridge DJ, Bhatnagar D, Bilianou E, Carmena R, et al. (2014) The use of statins in people at risk of developing diabetes Bio-Chips, Institute of Biochemistry, MATEJA HAFNER mellitus: evidence and guidance for clinical practice. Atheroscler Suppl 15:1–15. Faculty of Medicine (T.R., M.H., SANDHYA KORTAGERE Sueyoshi T, Kawamoto T, Zelko I, Honkakoski P, and Negishi M (1999) The repressed nuclear receptor CAR responds to phenobarbital in activating the human CYP2B6 gene. J Biol Chem D.R.), and Department of Biology, SEAN EKINS 274:6043–6046. Biotechnical Faculty (V.H.), VESNA HODNIK Ueda A, Hamadeh HK, Webb HK, Yamamoto Y, Sueyoshi T, Afshari CA, Lehmann JM, and Negishi M (2002) Diverse roles of the nuclear orphan receptor CAR in regulating hepatic University of Ljubljana, Ljubljana, DAMJANA ROZMAN genes in response to phenobarbital. Mol Pharmacol 61:1–6. Slovenia; Department of Xu RX, Lambert MH, Wisely BB, Warren EN, Weinert EE, Waitt GM, Williams JD, Collins JL, Moore LB, Willson TM, et al. (2004) A structural basis for constitutive activity in the human Microbiology and Immunology, CAR/RXRa heterodimer. Mol Cell 16:919–928. College of Medicine, Drexel University, Philadelphia, Address correspondence to: Dr. Damjana Rozman, Centre for Functional Genomics Pennsylvania (S.K.); Collaborations and Bio-Chips, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Pharmaceuticals, Inc., Fuquay Vrazov trg 2, SI-1000 Ljubljana, Slovenia. E-mail: [email protected] Varina, North Carolina (S.E.) DMD # 75523

Drug Metabolism and Disposition

Supplemental Data

Short Communication

Rosuvastatin and Atorvastatin are Ligands of the Human CAR/RXRα Complex

Tadeja Režen, Mateja Hafner, Sandhya Kortagere, Sean Ekins, Vesna Hodnik, and

Damjana Rozman

Center for Functional Genomics and Bio-Chips. Institute of Biochemistry, Faculty of

Medicine, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia

TR, MH, DR

Department of Microbiology and Immunology, Drexel University College of Medicine,

Philadelphia, PA 19129, USA

SK

Collaborations Pharmaceuticals, Inc, 5616 Hilltop Needmore Road, Fuquay Varina,

NC27526, USA

SE

Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101,

Ljubljana, Slovenia

VH

TR and MH contributed equally to this work.

DMD # 75523

MATERIALS AND METHODS

Chemicals

Dimethyl sulfoxide (DMSO) and CITCO were purchased from Sigma Chemie GmbH

(Deisenhofen, Germany), atorvastatin and rosuvastatin was kind gift from Lek

Pharmaceuticals (Ljubljana, Slovenia).

Protein Expression and Purification

The pGEX-4T-GST-hCAR (gift from Jean Marc Pascussi, INSERM, France) and pGEX-

2TK-GST-hRXRa-LBD (Addgene, Cambridge, MA, USA) plasmids were transformed into the BL21 (DE3) E. coli strain. The overnight culture was grown for 12 h in Lysogeny broth

(LB) media containing 100 µg/mL ampicillin with shaking (180 rpm) at 37°C. The culture was inoculated in 6 x 0.25 L of LB media with 100 µg/mL ampicillin and grown at the same conditions until the optical density at 600 nm reached the value of 0.6-0.9. The protein expression was induced with the addition of 0.1 mM IPTG (Isopropyl β-D-1- thiogalactopyranoside) and bacteria were grown for additional 4 h. The cells were harvested by centrifugation (20 min, 4000 rpm, 4°C). The cell pellet (1 part) was homogenized in precooled mortar and pestle together with Alumina (Sigma-Aldrich, St.

Louis, MO, USA) (2.5 part) until the composition of the mixture was pasta-like and then re-suspended in extraction buffer (140 mM NaCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.5). Cell debris was removed by centrifugation (20 min, 11400 rpm, 4°C). The cleared supernatant was loaded on a GST affinity column (Clontech, Palo Alto, CA, USA) according to the manufacturer instruction. Column fractions were pooled and

TR and MH contributed equally to this work.

DMD # 75523 concentrated/dialyzed with PBS buffer using Microcon YM-30 centrifugal filter (Millipore,

Bedford, MA, USA).

SDS-PAGE and Western blotting

Purity of isolated GST-hCAR and GST-hRXRα-LBD were checked by SDS-PAGE and western blot analysis. Firstly, proteins were loaded onto 10% polyacrylamide gel, electrophoreticaly separated and dyed with Coomassie Blue. Secondly, proteins were loaded, separated and electrophoretically transferred onto polyvinylidene flouride membrane (Immobilon-P, Millipore, Bedford, MA, USA). After transfer, the nitrocellulose membrane was treated with blocking solution (5% non-fat milk in PBS-Tween buffer).

Blotted proteins were probed with mouse anti-glutathione-S-transferase-antibody (Sigma-

Aldrich, St. Louis, MO, USA) or anti-CAR-antibody (R&D Systems, Minneapolis, MN,

USA) both at 1:1000 dilution over night at 4°C. The membrane was washed 3 times with

PBS-Tween buffer (PBST) and incubated for 1h at room temperature with goat anti- mouse HRP-conjugated secondary antibody at dilution 1:5000 (Jackson Immunoresearch

Laboratories, West Grove, PA, USA). The membrane was washed 3 times with PBST and once with PBS and specific bands were detected by enhanced chemiluminiscence

ECL (Pierce, Rockford, IL, USA) and signal was read using LAS-3000 (FUJIFILM, Tokyo, japan)

Surface Plasmon Resonance

The interaction of rosuvastatin, atorvastatin and CITCO was measured using a Biacore

X analytical system and CM5 sensor chip (Biacore, GE Healthcare, Uppsala, Sweden).

TR and MH contributed equally to this work.

DMD # 75523

All measurements were performed at 25°C. The system was primed twice with running buffer (100 mM TRIS, 140 mM NaCl, 0.005% surfactant P20, pH 8.0). The running buffer was filtered through 0.22 µm filter and degassed prior to use. The carboxymethyl dextran surface of CM5 sensor chip was activated with a 7 min injection of a 1:1 (vol/vol) mixture of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide

(NHS). Proteins GST-hCAR and GST-hRXRα-LBD were mixed in 1.5:1 mg ratio and protein complex was concentrated on Amicon ultracentrifugal filter (Millipore, Billerica,

MA, USA). Protein complex (CAR and RXR) was diluted to final concentration 150 µg/µL in 10 mM Na-acetate (pH 5.0) and applied on the second flow cell. Protein complex was injected in three short pulses (2x 2 min and 1x 4 min) to reach the immobilization level around 8300 response units using standard amine coupling (Johnsson et al., 1991). The surface of both flow cells was deactivated with 7 min injection of ethanolamine. The first flow cell served as a reference cell for subtraction of non-specific binding.10 mM stock solution of each analyte was prepared in DMSO and diluted into a series of concentrations with the running buffer to detect direct binding with immobilized GST-hCAR/GST-hRXRα-

LBDs. DMSO was added to running buffer to diminish the difference in a refractive index between the samples and running buffer. Final concentration of DMSO for each analyte solution was adjusted to 1% vol/vol. The association lasted for 120 s and dissociation was monitored for additional 60 s at a flow rate 10 µL/min. No regeneration was needed. Each analyte was tested in three parallel titrations using different concentrations depending on their solubility. The equilibrium binding responses were determined from the binding levels 5 s before the end of sample injection. KD values were determined by the fitting of the data to the steady state affinity model using BIAevaluation software.

TR and MH contributed equally to this work.

DMD # 75523

In silico modeling: Ligand-based

The computational molecular modeling studies were carried out using Discovery Studio

2.5.5 (Biovia, San Diego, CA). Pharmacophore models attempt to describe the arrangement of key features that are important for biological activity and their generation has been widely described (Ekins et al., 2007). Previously generated pharmacophores for Pregnane X receptor (PXR) agonists (Ekins and Erickson, 2002; Bachmann et al.,

2004; Ekins et al., 2007) were employed to generate predictions for statins tested in this study. These pharmacophores represent 1.) the original published PXR pharmacophore using 12 diverse ligands with EC50 data from competition binding assays or CV1 cells, which has previously been used to predict affinity of imidazoles (Ekins and Erickson,

2002; Bachmann et al., 2004; Ekins et al., 2007), 2.) a pharmacophore using 30 steroids with EC50 data from HepG2 cells using a reporter assay that may define a unique site within PXR (Ekins et al., 2007), and 3.) a pharmacophore using 31 diverse ligands (Ekins et al., 2007) with EC50 data from HepG2 cells using a reporter assay (Sinz et al., 2006),

4.) A preliminary CAR pharmacophore published previously was also used(Ekins et al.,

2002). The structures of 9 FDA approved statins (atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin) were downloaded from ChemSpider (www.chemspider.com) and exported as sdf files. In

Catalyst, the 3-D molecular structures were produced using up to 255 conformers with the best conformer generation method, allowing a maximum energy difference of 20 kcal/mol. The pharmacophores were then applied to screen the compounds using the

Best mapping method and rigid search using the ligand pharmacophore mapping

TR and MH contributed equally to this work.

DMD # 75523 protocol. The quality of the molecule mapping to the pharmacophore was determined by the FitValue which is dependent on the proximity of a compound to the pharmacophore feature centroids and the weights assigned to each centroid, where a higher FitValue represents a better fit. Bayesian machine learning models for PXR were either generated with steroids (Ekins et al., 2009) or diverse compounds (Kortagere et al., 2009) and run with the ‘calculate molecular properties’ protocol in Discovery Studio.

In silico modeling: Structure-based

The Crystal structure of PXR and CAR were obtained from the Protein DataBank with codes 1M13 and 1XVP respectively (Watkins et al., 2003; Xu et al., 2004). The crystal structures were prepared for docking studies by adding hydrogen atoms and refining the structures using energy minimization techniques adopted in the molecular modeling program MOE (Chemical Computing Group, Montreal, Quebec, Canada). The ligands were modeled using the builder module and the compounds were optimized for geometry.

Further all nine compounds were docked to the LBD of CAR and PXR using the program

GOLD (ver 4.1, The Cambridge Crystallographic Data Centre, UK) as previously described (Willett and Glen, 1995; Jones et al., 1997; Kortagere et al., 2009).

REFERENCES

Bachmann K, Patel H, Batayneh Z, Slama J, White D, Posey J, Ekins S, Gold D, and

Sambucetti L (2004) PXR and the regulation of apoA1 and HDL-cholesterol in

rodents. Pharmacol Res 50:237–46.

Ekins S, Chang C, Mani S, Krasowski MD, Reschly EJ, Iyer M, Kholodovych V, Ai N,

TR and MH contributed equally to this work.

DMD # 75523

Welsh WJ, Sinz M, Swaan PW, Patel R, and Bachmann K (2007) Human pregnane

X receptor antagonists and agonists define molecular requirements for different

binding sites. Mol Pharmacol 72:592–603.

Ekins S, and Erickson JA (2002) A pharmacophore for human pregnane X receptor

ligands. Drug Metab Dispos 30:96–9.

Ekins S, Kortagere S, Iyer M, Reschly EJ, Lill MA, Redinbo MR, and Krasowski MD

(2009) Challenges predicting ligand-receptor interactions of promiscuous proteins:

the nuclear receptor PXR. PLoS Comput Biol 5:e1000594.

Ekins S, Mirny L, and Schuetz EG (2002) A ligand-based approach to understanding

selectivity of nuclear hormone receptors PXR, CAR, FXR, LXRalpha, and LXRbeta.

Pharm Res 19:1788–800.

Johnsson B, Löfås S, and Lindquist G (1991) Immobilization of proteins to a

carboxymethyldextran-modified gold surface for biospecific interaction analysis in

surface plasmon resonance sensors. Anal Biochem 198:268–77.

Jones G, Willett P, Glen RC, Leach AR, and Taylor R (1997) Development and

validation of a genetic algorithm for flexible docking. J Mol Biol 267:727–48.

Kortagere S, Chekmarev D, Welsh WJ, and Ekins S (2009) Hybrid scoring and

classification approaches to predict human pregnane X receptor activators. Pharm

Res 26:1001–11.

Sinz M, Kim S, Zhu Z, Chen T, Anthony M, Dickinson K, and Rodrigues AD (2006)

Evaluation of 170 xenobiotics as transactivators of human pregnane X receptor

TR and MH contributed equally to this work.

DMD # 75523

(hPXR) and correlation to known CYP3A4 drug interactions. Curr Drug Metab

7:375–88.

Watkins RE, Maglich JM, Moore LB, Wisely GB, Noble SM, Davis-Searles PR, Lambert

MH, Kliewer SA, and Redinbo MR (2003) 2 . 1 Å Crystal Structure of Human PXR

in Complex with the St . John ’ s Wort Compound Hyperforin. Biochemistry

42:1430–1438.

Willett P, and Glen C (1995) Molecular Recognition of Receptor Sites using a Genetic

Algorithm with a Description of Desolvation. 43–53.

Xu RX, Lambert MH, Wisely BB, Warren EN, Weinert EE, Waitt GM, Williams JD,

Collins JL, Moore LB, Willson TM, and Moore JT (2004) A structural basis for

constitutive activity in the human CAR/RXRalpha heterodimer. Mol Cell 16:919–28.

TR and MH contributed equally to this work.

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Supplemental Figure Legend

Supplemental Figure 1. Molecular structures of atorvastatin, rosuvastatin and CITCO.

Supplemental Figure 2. SDS-PAGE (A) and Western blot analysis (B) of isolated GST- hCAR and GST-hRXRα-LBD. (A) Isolated proteins were separated on gel electrophoresis and dyed using Coomassie blue. Lane 1: GST-hCAR with marker; Lane 2: GST-hRXRα-

LBD with marker. (B) Western blot analyses of isolated proteins. Lane 1: GST-hCAR detected by anti-CAR-Ab; M: marker; Lane 2: GST-hCAR detected by anti-GST-Ab;

Lane3: GST-hRXRα-LBD detected by anti-GST-Ab.

Supplemental Figure 3. Atorvastatin mapped to the CAR pharmacophore.

TR and MH contributed equally to this work.

DMD # 75523

Atorvastatin Rosuvastatin CITCO

Supplemental Figure 1.

TR and MH contributed equally to this work.

DMD # 75523

Supplemental Figure 2.

TR and MH contributed equally to this work.

DMD # 75523

Supplemental Figure 3.

TR and MH contributed equally to this work.

DMD # 75523

Supplemental Table 1. Summary of ligand-based and structure-based results

Molecule PXR-score CAR- Bachman - Krasowski BMS PXR CAR Bayesian PXR diverse Bayesian PXR - docking score - PXR model diverse pharmacophore pharmacophore steroidal docking steroidal PXR pharmacophore

Atorvastatin 74.8 73.18 3.39 4.57 2.07 FALSE TRUE

Cerivastatin 61.03 68.23 4.29 0.48 FALSE TRUE

Fluvastatin 60.91 60.86 4.76 2.42 FALSE TRUE

Lovastatin 52.46 61.59 8.73 4.9 3.72 TRUE - lovastatin is in TRUE the model

Mevastatin 57.49 59.23 9.35 4.52 3.64 TRUE TRUE

Pitavastatin 56.11 65.32 4.12 1.61 FALSE TRUE

Pravastatin 58.55 68.87 6.6 5.4 3.26 TRUE TRUE

Rosuvastatin 63.57 67.09 4.41 FALSE TRUE

Simvastatin 52.4 64.04 10.52 4.74 3.76 TRUE TRUE

CITCO 54.26 63.02

TR and MH contributed equally to this work.