Molecular Genetics and Metabolism 83 (2004) 184–187

www.elsevier.com/locate/ymgme

T0901317 is a dual LXR/FXR agonist

Keith A. Houcka, Kristen M. Borcherta, Christopher D. Heplera, JeVrey S. Thomasb, Kelli S. Bramlettb,c, Laura F. Michaelb, Thomas P. Burrisb,c,¤

a RTP Laboratories, Eli Lilly & Company, Research Triangle Park, NC 27709, USAb Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA

c Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Received 26 April 2004; received in revised form 8 July 2004; accepted 8 July 2004Available online 26 August 2004

Abstract

We characterize the ability of the liver X receptor (LXR� [NR1H3] and LXR� [NR1H2]) agonist, T0901317, to activate thefarnesoid X receptor (FXR [NR4H4]). Although T0901317 is a much more potent activator of LXR than FXR, this ligand actuallyactivates FXR more potently than a natural bile acid FXR ligand, chenodeoxycholic acid. Thus, the FXR activity of T0901317 mustbe considered when utilizing this agonist as a pharmacological tool to investigate LXR function. 2004 Elsevier Inc. All rights reserved.

Keywords: Bile acid; Oxysterol; Nuclear receptor

Introduction

The liver X receptors (LXR� [NR1H3] and LXR�[NR1H2]), members of the nuclear receptor superfamily,function as receptors for oxidized cholesterol andregulate a variety of physiological processes includingcholesterol metabolism and transport, lipogenesis, glu-coneogenesis, and inXammation. Synthetic LXR agon-ists have been proposed to have potential utility intreatment of disorders such as dyslipidemia, atheroscle-rosis, and diabetes [1]. To this end, high aYnity LXRagonists, such as T0901317 and GW3965, have beendescribed, which have allowed for the identiWcation andcharacterization of many physiological processes regu-lated by LXR [2,3].

Since T0901317 is often used as a speciWc LXR ago-nist “tool” to deWne the physiological eVects of thisreceptor both in vitro and in vivo, we tested this ligandfor activity against related receptors. Using cell-based

* Corresponding author. Fax: +1 317 276 1414.E-mail address: burris_thomas_p@lilly.com (T.P. Burris).

1096-7192/$ - see front matter 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.ymgme.2004.07.007

transfection and biochemical ligand sensing assays, wefound that T0901317 also acts as an FXR agonist. LikeLXR, the farnesoid X receptor (FXR [NR1H4]) wasoriginally identiWed as an orphan member of the nuclearreceptor superfamily [4,5]. FXR was later identiWed asthe physiological receptor for bile acids and shown toregulate a feedback loop for bile acid transport and syn-thesis as well as modulating additional functions in lipidmetabolism [6].

Materials and methods

Cell culture and transfections

A Gal4 DNA-binding domain–human FXR ligand-binding domain fusion (Gal4DBD-FXRLBD) transfec-tion assay was utilized as previously described with severalmodiWcations [7]. HEK293 cells were cultured in 3:1DMEM:F-12 containing 10% fetal bovine serum andsupplemented with 1% penicillin and streptomycin, 1%L-glutamine, and 20 mM Hepes. Forty-eight hours

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before transfection, cells were seeded at 6 £ 106 cells/T225 Xask in 30 ml growth media. Cells were trans-fected with Fugene transfection reagent (Roche, India-napolis, IN) according to the Fugene protocol with9.5 �g Gal4-FXRLBD and 500 ng pSG5-luc [6] and10 �L Fugene per 106 cells. Growth media were replacedduring transfection with 3:1 DMEM:F-12 containing10% charcoal/dextran treated, heat-inactivated fetalbovine serum and supplemented with 1% penicillin andstreptomycin, 1% L-glutamine, and 20 mM Hepes. After24 h, cells were harvested and plated into 96-wellwhite plates at 50,000 cells/well in 90 �l complete trans-fection media, allowed to attach for 2 h, then treatedwith 10�l of 10£ compound and DMSO controls.After 24 h, cells were lysed and assayed for luciferaseactivity. CDCA and T0901317 were obtained fromSigma (St. Louis, MO) and Cayman Chemicals (AnnArbor, MI), respectively.

Coactivator interaction assay

Interaction between nuclear receptor and the coacti-vators hSRC-1 or hSRC-2 was assayed using Alpha-Screen (ampliWed luminescent proximity homogenousassay) technology (Perkin–Elmer Life Sciences) as previ-ously described modiWed for use with hFXR [8]. Theassay was performed in white, low volume, 384-wellplates utilizing a Wnal volume of 15�l containing Wnalconcentrations of 20 nM of His-tagged Escherichia coliexpressed FXRLBD protein, 5 nM of GST-SRC-2 orGST-SRC-1 protein that contained the entire nuclearreceptor interacting domain of the coactivator proteinfused to GST and 10�g/ml of both Ni2+ chelate donorbeads and anti-GST acceptor beads (Perkin–Elmer LifeSciences). The assay buVer contained 25 mM Hepes (pH7.0), 100 mM NaCl, 0.1% BSA, and 2 mM DTT. Allmanipulations involving assay beads were done in ambi-ent light. Assay plates were covered with a clear seal andincubated in the dark for 2 h after which the plates wereread for 1 s/well in a Perkin–Elmer Fusion microplateanalyzer using the manufacturer’s standard AlphaScreendetection protocol.

Cell culture and FXR target gene activation

The human hepatocellular carcinoma cell line,Huh7, was maintained in DMEM/F12 3:1 in 5% FBS inmonolayer culture at 37 °C in 5% CO2. Cells wereplated at 50% conXuence and were grown overnight.Compounds were administered at the concentrationsindicated for 16 h. RNA was isolated using the ABIPrism 6100 Nucleic Acid PrepStation reagents, andcDNA was synthesized using ABI High CapacityArchive kit reagents (Applied Biosystems, Foster City,CA). Quantitative PCR was performed as previouslydescribed [9].

Results

As shown in Fig. 1A, HEK293 cells transfected with aGal4 DNA-binding domain (DBD)–FXR ligand-bind-ing domain (LBD) chimeric receptor along with a Gal4-responsive luciferase reporter responded in the expectedfashion when treated with the bile acid ligand, chenode-oxycholic acid (CDCA). CDCA activated the chimericreceptor with the expected EC50 in the range of 40�M;surprisingly however, we also noted that T0901317 acti-vated FXR with an EC50 of »5�M. Although the FXRpotency of T0901317 is considerably less than thatdescribed for LXR (»50 nM; [2]) it is approximately 10-fold more potent than the natural FXR ligand CDCA.To determine if T0901317 was acting as a direct ligandfor FXR we utilized a biochemical ligand sensing assayin which we assessed the ability of T0901317 to induce aconformational change in the FXRLBD suYcient tocause recruitment of coactivator proteins, SRC-1 orSRC-2. Consistent with the cell-based transfection assay,T0901317 induced FXR recruitment of either SRC-1(Fig. 1B) or SRC-2 (Fig. 1C) with greater potency thanthe natural FXR ligand, CDCA. EC50 values for CDCAwere 37 and 18�M for SRC-1 and SRC-2, respectively,while the values for T0901317 were 7 and 4�M for thetwo coactivators. Maximal eYcacies for the two ligandswere similar. The activity of a structurally unique LXRligand, GW3965 [3], was also assessed in these assaysand found to be inactive (<10% eYcacy at 10�M) indi-cating that the dual LXR/FXR activity was speciWc forT0901317 (data not shown).

To determine if T0901317 exhibited FXR agonist activ-ity the context of a natural FXR target gene, we examinedthe ability of this compound to induce expression of eitherthe bile salt export protein (BSEP) [10] or the short hetero-dimer partner (SHP) [11,12] in Huh7 cells. As illustrated inFigs. 2A and B, CDCA induces the expression of bothBSEP and SHP mRNA in a dose-dependent manner.Consistent with our previous data, T0901317 alsoincreases the expression of both FXR target genes in adose-dependent manner with maximal eYcacy similar tothat of CDCA (Figs. 2C and D). Thus, our data demon-strate that the high aYnity LXR ligand T0901317 alsoexhibits FXR agonist activity albeit at a lower potency.

Discussion

LXR was initially identiWed as an orphan member ofthe nuclear receptor superfamily in 1995 [13]; however, itsrole as the physiological receptor for oxidized metabolitesof cholesterol was rapidly elucidated [14,15]. The naturaloxysterol ligands were not extremely useful for character-ization of LXR function due to their relatively low aYnityand pleiotrophic actions. The discovery of the Wrst highaYnity synthetic LXR agonist, T0901317, provided a

186 K.A. Houck et al. / Molecular Genetics and Metabolism 83 (2004) 184–187

critical tool for characterization of the biological signiW-cance of this receptor indicating a critical role in regula-tion of cholesterol transport [16] and lipogenesis [2,17].

Like LXR, characterization of the physiological path-ways regulated by FXR was signiWcantly hampered untilthe discovery of high aYnity ligands for this bile acid

receptor [18]. It has been demonstrated using both phar-macological tools and genetic models that LXRs andFXR coordinately regulate triglyceride and lipoproteinmetabolism as well as reverse cholesterol transport anddisplay signiWcant overlap in the array of genes that theymodulate either directly or indirectly [6,19].

Fig. 1. The LXR ligand, T0901317, acts as an FXR agonist. (A) HEK293 cells were transfected with vectors directing the expression of a Gal4DBD-FXRLBD along with a Gal4-responsive luciferase reporter as previously described [6]. EC50 values along with 95% conWdence intervals in bracketsare 45 �M [31 �M, 64 �M] for CDCA and 5 �M [3 �M, 7 �M] for T0901317. (B) His-tagged FXRLBD and GST-tagged SRC-1 were expressed inE. coli and used in an AlphaScreen assay (biochemical ligand sensing assay) as previously described [8]. EC50 values along with 95% conWdence inter-vals in brackets are 37 �M [27 �M, 47 �M] for CDCA and 7 �M [5 �M, 9 �M] for T0901317. (C) AlphaScreen assay as described in (B) above utilizingbacterially expressed SRC-2 as previously described [6] values along with 95% conWdence intervals in brackets are 18 �M [16 �M, 19 �M] for CDCAand 4 �M [3 �M, 5 �M] for T0901317. Analysis of the dose–response curves was performed using Graphpad Prism (Monrovia, CA).

Fig. 2. The LXR ligand, T0901317, induces FXR target genes BSEP and SHP. (A) Induction of BSEP expression in Huh7 cells by the bile acidCDCA. (B) Induction of SHP expression in Huh7 cells by the bile acid CDCA. (C) Induction of BSEP expression in Huh7 cells by T0901317. (D)Induction of SHP expression in Huh7 cells by T0901317. Expression of BSEP and SHP was measured using quantitative PCR as previouslydescribed [9] and was normalized to 18S rRNA. Expression values are reported as fold induction relative to untreated control expression levels.

K.A. Houck et al. / Molecular Genetics and Metabolism 83 (2004) 184–187 187

Initial biological characterization of T0901317 indi-cated that it was speciWc for LXR [2] or retained slightFXR activity relative to LXR [16]. In the latter study,which indicated some limited FXR activity, no potencydata were provided. Our results indicate that T0901317acts as an FXR agonist with eYcacy similar to a naturalbile acid ligand, CDCA. The EC50 for T0901317 rangedfrom 4 to 7�M within the various FXR assays, which iswithin a range that is signiWcant given that 1�M con-centrations are often used as a standard in various invitro assays assessing LXR activity. Since T0901317 hasbeen the primary pharmacological tool for elucidatingthe physiological role of the LXRs, it is apparent thatthe concentration of this ligand must be carefullymonitored so as to avoid FXR activation and conclu-sions that may be erroneous due to activation of bothreceptors.

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