0022-3565/03/3071-291–296$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 307, No. 1 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 52852/1090865 JPET 307:291–296, 2003 Printed in U.S.A.

A Natural Product Ligand of the Oxysterol Receptor, Liver X Receptor

KELLI S. BRAMLETT, KEITH A. HOUCK, KRISTEN M. BORCHERT, MICHELE S. DOWLESS, PALANIAPPAN KULANTHAIVEL, YOUYAN ZHANG, THOMAS P. BEYER, ROBERT SCHMIDT, JEFFREY S. THOMAS, LAURA F. MICHAEL, ROBERT BARR, CHAHRZAD MONTROSE, PATRICK I. EACHO, GUOQING CAO, and THOMAS P. BURRIS Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana Received April 8, 2003; accepted June 12, 2003 Downloaded from

ABSTRACT Natural products have been identified as ligands for a number well as glucose metabolism. We show that paxilline, a fungal of members of the nuclear hormone receptor (NHR) superfam- metabolite, is an efficacious agonist of both LXR␣ and LXR␤ in ily. Often these natural products are used as dietary supple- biochemical and in vitro cell-based assays. Paxilline binds di- ments to treat myriad ailments ranging from perimenopausal rectly to both receptors and is an activator of LXR-dependent hot flashes to hypercholesterolemia and reduced cognitive transcription in cell-based reporter assays. We also demon- jpet.aspetjournals.org function. Examples of some natural product ligands for NHRs strate that paxilline binding to the receptors results in efficient include genestein (estrogen receptors NR3A1 and NR3A2), activation of transcription of two physiological LXR target guggulsterone (farnesoid X receptor NR1H4), and St. John’s genes, ABCA1 and SREBP. The discovery of paxilline, the first wort (pregnane X receptor, NR1I2). In this study, we identified reported nonoxysterol natural product ligand of the LXRs, may the first nonoxysterol natural product that functions as a ligand provide insight into the mechanism of ligand recognition by for the liver X receptor (LXR␣ and LXR␤; NR1H3, NR1H2), a these receptors and reaffirms the utility of examining natural NHR that acts as the receptor for oxysterols and plays a key product libraries for identifying novel NHR ligands. role in regulation of cholesterol metabolism and transport as at ASPET Journals on March 9, 2015

Natural products are proven sources of biologically active edy for depression, is a potent agonist of the xenobiotic nu- molecules that have played a critical role in pharmacology. clear receptor, pregnane X receptor (PXR, NR1I2) (Moore et Many of these natural products have been demonstrated to al., 2000; Watkins et al., 2003). Although the antidepressent be useful for medicinal purposes. A limited number of natu- activity of St. John’s wort does not appear to be mediated by ral products appear to act as ligands of nuclear hormone its PXR activity, the induction of cytochrome P450 (CYP) 3A4 receptors. One of the most well characterized natural product is mediated by activation of this receptor potentially leading nuclear receptor ligands is genestein, a product of soy, which to a significant increase in the metabolism of a variety of binds directly to estrogen receptors (NR3A1 and NR3A2) and drugs taken by individuals using this supplement (Moore et is used as a dietary supplement to alleviate menopausal al., 2000). The most recent report of a natural product ligand symptoms in women (Kuiper et al., 1998; Tham et al., 1998). of a nuclear receptor is the discovery that guggulsterone, Interestingly, genestein has been recently shown to also found in the resin of the guggul tree, is an efficacious antag- function as a low-affinity ligand for yet another nuclear re- onist of the farnesoid X receptor (FXR, NR1H4), the bile acid ␥ ceptor, peroxisome proliferator-activated receptor (NR1C3) receptor (Urizar et al., 2002; Wu et al., 2002). Guggul tree (Dang et al., 2003). A related isoflavone soy product also used extract has been suggested to lower low-density lipoprotein in dietary supplements, daidzein, is another estrogen recep- levels in animal models, has been successfully used in tor agonist, albeit weaker than genestein (Wiseman, 2000). Ayurveda medicine to treat obesity and lipid disorders, and a The active compound within St. John’s wort, an herbal rem- modern antihyperlipoproteinemic drug based on the actions of guggulsterone is marketed in India (Satyavati, 1988; Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. Singh et al., 1990; Dev, 1997). Beyond the use of natural DOI: 10.1124/jpet.103.052852. products directly as dietary supplements, identification of

ABBREVIATIONS: PXR, pregnane X receptor; LXR, liver X receptor; FXR, farnesoid X receptor; LBD, ligand binding domain; SRC-1, steroid receptor coactivator-1; TIF-2, transcription intermediary factor-2; GST, glutathione S-transferase; OHC, hydroxycholesterol; ER, estrogen receptor; bDNA, branched DNA; RXR, retinoid X receptor; DMEM, Dulbecco’s modified Eagle’s medium; SRE, sterol regulatory element; SREBP, SRE binding protein; 22(R)-OHC, 22(R)-hydroxycholesterol. 291 292 Bramlett et al.

novel natural product ligands for a given receptor often pro- 1 s per well in a PerkinElmer fusion microplate analyzer using the vides pharmacological tools and unique insight into drug manufacturers standard AlphaScreen detection protocol. design. Cell Culture and Transfections. HEK293 cells were cultured in The liver X receptors [LXR␣ (NR1H3) and LXR␤ (NR1H2)] 3:1 DMEM/F-12 containing 10% fetal bovine serum and supplemented are additional members of the nuclear receptor superfamily with 1% penicillin and streptomycin, 1% L-glutamine, and 20 mM HEPES. Cells were seeded 48 h before transfection at 6 ϫ 106 cells/T225 and were originally identified as orphan receptors (Shinar et flask in 30 ml of growth medium. Cells were transfected with Fugene al., 1994; Song et al., 1994; Seol et al., 1995; Teboul et al., transfection reagent (Roche Diagnostics, Indianapolis, IN) according to 1995; Willy et al., 1995). Subsequently, oxysterol cholesterol the Fugene protocol with 330 ng of pcDNA3-hRXR-␣, 33 ng of pCMV6 metabolites were demonstrated to be physiological ligands LXR␣ or LXR␤, 660 ng of pGL3B-E1b-3XLXRE luc (Thomas et al., for LXR (Janowski et al., 1996). These two receptors play a 2003), and 10 ␮l of Fugene per 106 cells. Growth medium was replaced key role in the regulation of cholesterol metabolism and during transfection with 3:1 DMEM/F-12 containing 10% charcoal/ transport as well as glucose metabolism and inflammation dextran-treated, heat-inactivated fetal bovine serum and supplemented (Repa and Mangelsdorf, 2002; Cao et al., 2003; Joseph et al., with 1% penicillin and streptomycin, 1% L-glutamine, and 20 mM 2003). Modulation of the activity of these receptors may be HEPES. After 24 h, cells were harvested and plated into 96-well white ␮ useful in the treatment of a number of pathophysiological plates at 50,000 cells per well in 90 l of complete transfection medium, allowed to attach for 2 h, then treated with 10 ␮lof10ϫ compound and states including dyslipidemia, atherosclerosis, and diabetes dimethyl sulfoxide controls. After 24 h, cells were lysed and assayed for (Joseph et al., 2002; Tangirala et al., 2002; Cao et al., 2003). luciferase activity. In this study, we describe the discovery of the first nonox- ABCA1 mRNA Quantitation. ABCA1 mRNA expression was Downloaded from ysterol natural product ligand of LXR. The indole alkoid measured in THP-1 macrophage cells using a bDNA assay (Quanti- fungal metabolite from Penicillium paxilli, paxilline, func- Gene high volume kit; Bayer Corp.-Diagnostics Div., Tarrytown,

tions as a ligand for both LXR␣ and LXR␤. Paxilline binds NY). THP-1 cells were grown in suspension at 37°C 5%/95% CO2/air directly to both receptors leading to recruitment of coactiva- incubator in growth medium (RPMI 1640 medium containing 0.05 tors and activates the receptors in a cell-based context. In mM 2-mercaptoethanol and 10% fetal bovine serum at a density of addition, paxilline efficaciously induces the expression of 250,000 cells per milliliter and allowed to reach a density of 1 million LXR target genes, ABCA1 and SREBP. Our data confirm cells per milliliter. Growth medium was then changed to growth jpet.aspetjournals.org medium containing 10 nM phorphol 12-myristate 13-acetate and that natural product libraries are a rich source of ligands for cells were plated in 96-well dishes at a density of 100,000 cells per nuclear receptors and may provide pharmacological agents well. After an overnight incubation, medium was changed to a for investigation of the function of these receptors as well as growth medium containing 10% lipoprotein depleted fetal bovine potential drugs. serum. Cells were treated with various concentrations of compounds serially diluted to obtain a 10-point concentration curve from a final concentration of 20 to 0.001 ␮M. After a 24-h incubation with com- at ASPET Journals on March 9, 2015 Materials and Methods pound, cells were lysed using 50 ␮l/well bDNA assay kit lysis re- agent. The kit reagents as well as ABCA1-specific primer sets were LXR Ligands. 22(R)-Hydroxycholesterol and paxilline were ob- used to process the samples for the bDNA assay as previously de- tained from Sigma-Aldrich (St. Louis, MO). scribed (Zhang et al., 2002). After a 15-min incubation at 37°C, 100 Radioligand Binding Assay. The LXR radioligand binding as- ␮l of the lysis buffer from each well were transferred to the corre- say was performed using scintillation proximity technology as pre- sponding wells of the capture plate. The capture plate was incubated viously described (Thomas et al., 2003). We used 800 ng of baculov- overnight at 53°C. The capture plate was then washed twice with irus-expressed, His-tagged LXR␣-LBD protein (amino acids 162– QuantiGene wash buffer followed by the addition of 100 ␮l/well 447) or 600 ng of LXR␤-LBD protein (amino acids 202–461), 25 nM QuantiGene amplifier working reagent. The plate was incubated for 3H-labeled 25-hydroxycholesterol (Amersham Biosciences, Inc., Pis- 60 min at 46°C followed by two washes. The mRNA to be measured cataway, NJ), 0.05 mg of yttrium silicate polylysine-coated SPA was then labeled by the addition of 100 ␮l of QuantiGene label probe beads (Amersham Biosciences, Inc.), and varying concentrations of working buffer followed by a 60-min incubation at 46°C. The capture competitor per well of a 96-well OptiPlate (PerkinElmer Life Sci- plate was then washed twice followed by the addition of 100 ␮l/well ences, Boston, MA). Protein, radioligand, and competitor were added QuantiGene substrate plus QuantiGene enhancer reagent. The to the plate. SPA beads were then added to the assay plate followed plates were incubated at 37°C for up to 30 min and then read on a by 10 min of gentle shaking at room temperature and protected from luminometer to detect the luminescent signal. The induction of light. The plates were incubated in the dark at room temperature for ABCA1 mRNA expression was calculated as a ratio of compound- 2 h before reading in a TopCount plate reader (PerkinElmer Life treated luminescent levels compared with untreated control levels. Sciences). SRE Assay and SREBP mRNA Quantitation. As previously Coactivator Interaction Assay. Interaction between LXR␣/ described (Thomas et al., 2003), HepG2 cells stably transfected with LXR␤ and the coactivators SRC-1 or TIF-2 were assayed using a 3XSRE thymidine kinase luciferase reporter construct were AlphaScreen (amplified luminescent proximity homogenous assay) treated for 24 h with an LXR ligand to assess SREBP activity. technology (PerkinElmer Life Sciences). The assay was performed in SREBP mRNA was quantitated by Taqman real-time polymerase white, low volume, 384-well plates using a final volume of 15 ␮l chain reaction as previously described (Thomas et al., 2003). containing final concentrations of 20 nM His-tagged baculovirus Data Analysis. Dose responses and displacement curves were expressed LXR␣-LBD or LXR␤-LBD protein, 5 nM GST-TIF-2 or analyzed in GraphPad Prism (GraphPad Software Inc., San Diego,

GST-SRC-1 protein that contained the entire nuclear receptor inter- CA) allowing calculation of both EC50 and Ki values. Each point of acting domain of the coactivator protein fused to GST and 10 ␮g/ml data represents minimal triplicate wells, and the results shown are of both Ni2ϩ chelate donor beads and anti-GST acceptor beads representative of at least three independent experiments. (PerkinElmer Life Sciences). The assay buffer contained 25 mM HEPES (pH 7.0), 100 mM NaCl, 0.1% bovine serum albumin, and 2 Results mM dithiothreitol. All manipulations involving assay beads were done in ambient light. Assay plates were covered with a clear seal A screen of a natural product library for compounds with and incubated in the dark for 2 h after which the plates were read for the ability to modulate the activity of LXR yielded the iden- A Natural Product Ligand of LXR 293 ϭ tification of the first nonoxysterol natural product ligand for nM [22(R)-OHC 1,400 nM]. The EC50 for paxilline-medi- this receptor, paxilline. Paxilline is an indole alkoid metab- ated LXR␤ recruitment of SRC-1 was 930 nM [22(R)-OHC ϭ olite from the fungus, P. paxilli. The structure of paxilline is 300 nM] whereas TIF-2 was 1,200 nM [22(R)-OHC ϭ 780 shown in Fig. 1 and compared with several LXR ligands nM]. The ability of paxilline to induce coactivator recruit- including a natural ligand, 22(R)-hydroxycholesterol, as well ment by LXR suggested that paxilline might function as an as synthetic ligands GW3965, T0901317, and APD (Repa et agonist. This was confirmed in a transfection experiment in al., 2000; Schultz et al., 2000; Collins et al., 2002; Sparrow et which HEK293 cells were cotransfected with either LXR␣ or al., 2002). LXR␤ and a reporter containing three copies of a DR4 ele- We examined the ability of paxilline to bind directly to both ment derived from the ABCA1 promoter (Fig. 4). Paxilline LXR␣ and LXR␤ using a radioligand binding assay. A scin- activated transcription of both LXR␣ and LXR␤ with equiv- ϳ tillation proximity assay format was employed using triti- alent potency and efficacy as 22(R)-OHC (EC50 4,000 nM ated 25-hydroxycholesterol as the radioligand. As illustrated for both receptors and ligands). The apparent discrepancy in Fig. 2, paxilline displaced 25-hydroxycholesterol from both between the affinity of 22(R)-OHC for LXR in the binding ␣ ϭ ␤ ϭ receptors (LXR Ki 660 nM; LXR Ki 1,100 nM). In assay and the potency in a cotransfection assay has been contrast a natural oxysterol ligand, 22(R)-hydroxycholesterol previously described and is apparently a function of the ␣ [22(R)-OHC] displayed higher affinity in this assay (LXR Ki physiochemical properties of this compound (Janowski et al., ϭ ␤ ϭ 250 nM; LXR Ki 490 nM) (Fig. 2). Paxilline did not bind 1999). Interestingly, paxilline functions as a partial agonist and/or activate any other nuclear receptor examined (IC50 in the coactivator interaction assay [60–90% 22(R)-OHC] but Ͼ ␮ ␣ ␤ Downloaded from and/or EC50 10 M for ER ,ER , TR, RXR, FXR) (data not as a full agonist in the cotransfection assays. This suggests shown). Paxilline also induced LXR recruitment of both co- that either additional coactivators are recruited in the cell- activators SRC-1 or TIF-2 in a cell-free AlphaScreen assay based assay that allow for retention of full efficacy or that system (Fig. 3). Using purified recombinant LXR␣ or LXR␤ only a threshold of coactivator recruitment is required to and purified GST-SRC-1 or GST-TIF-2, we demonstrated reach full agonism. Consistent with its function as an agonist that increasing amounts of paxilline resulted in dose-depen- in both the coactivator recruitment and cotransfection assay, dent recruitment of these coactivators to both LXRs. The paxilline induced the expression of ABCA1 in THP-1 cells jpet.aspetjournals.org ␣ EC50 for paxilline-mediated LXR recruitment of SRC-1 was very efficaciously with a maximal induction of approximately ϭ ϭ 1,800 nM [22(R)-OHC 2,600 nM] whereas TIF-2 was 660 7-fold (EC50 1,300 nM) (Fig. 5). In this paradigm, the dose at ASPET Journals on March 9, 2015

Fig. 1. Chemical structures of LXR ligands. Paxilline is compared with previously described ligands including the natural ligand, 22(R)-hydroxycho- lesterol. The structures of three LXR ligands, T0901317 (T1317), GW 3965, and APD, identified by Tularik, GlaxoSmithKline, and Merck are also shown. 294 Bramlett et al. Downloaded from jpet.aspetjournals.org

Fig. 2. Paxilline binds to both LXR␣ and LXR␤. A scintillation proximity radioligand binding assay was performed utilizing either recombinant LXR␣ or LXR␤ and a selective radioligand 3H-labeled 25-OHC. Paxilline Fig. 4. Paxilline activates LXR-mediated transcription from a reporter ␣ gene. Full-length LXR␣ or LXR␤ (along with RXR␣) were transfected into displaced the radiolabeled 25-OHC with a Ki of 660 nM for LXR and 1,100 nM for LXR␤. The natural ligand 22(R)-OHC displayed higher HEK293 cells along with a luciferase reporter containing three copies of ␣ LXRE derived from the DR4 element of the ABCA1 promoter. Paxilline affinity for both receptors with a Ki of 250 nM for LXR and 490 nM for LXR␤. and 22(R)-OHC had similar potency and efficacy for both LXR␣ and ␤ at ASPET Journals on March 9, 2015 LXR .EC50 values were approximately 4,000 nM for each ligand for both receptors.

Fig. 5. Paxilline induces the expression of a natural target gene, ABCA1, in THP-1 cells. THP-1 cells were differentiated as described under Ma- Fig. 3. Binding of paxilline to LXR results in coactivator recruitment. terials and Methods followed by treatment with either paxilline or 22(R)- Utilizing Alpha-Screen technology, we evaluated whether paxilline would OHC for 24 h. After this treatment, the cells were harvested and ABCA1 recruit coactivators (SRC-1 or TIF-2) to either LXR subtype in a biochem- mRNA levels were assessed by bDNA measurement. Paxilline stimulated ical model. Recombinant LXRs were attached to the donor bead via ABCA1 mRNA expression approximately 7.5-fold with an EC50 of 1,300 2ϩ His-tagged Ni ion interaction while GST-fusion coactivators were at- nM. 22(R)-OHC was less potent with an EC50 of greater than 5,000 nM, tached to acceptor beads via a GST-Ab. Paxilline treatment resulted in and maximal efficacy was not measured due to limiting toxicity. dose-dependent recruitment of coactivators to either receptor subtype. ␣ For paxilline, the EC50 for recruitment of SRC-1 and TIF-2 to LXR was response for 22(R)-OHC was limited by toxicity above 10 ␮M; 1,800 and 660 nM, respectively. The EC50 for recruitment of SRC-1 and TIF-2 to LXR␤ was 930 and 1,200 nM, respectively. For the natural LXR however, the potency is greater than 5,000 nM and the max- ␣ imal efficacy at 10 ␮M is approximately 6-fold. Paxilline also ligand, 22(R)-OHC, the EC50 for recruitment of SRC-1 and TIF-2 to LXR was 2,600 and 1,400 nM, respectively. The EC for recruitment of SRC-1 increased the expression of a second LXR target gene, ␤ and TIF-1 to LXR was 300 and 780 nM, respectively. In all cases, SREBP, in HepG2 cells (Fig. 6A). We used a SREBP-respon- paxilline appears to have partial agonist activity compared with 22(R)- OHC with maximal efficacies ranging from 60 to 90% of 22(R)-OHC sive luciferase reporter stably transfected into HepG2 cells levels. (Thomas et al., 2003) to investigate the dose-responsiveness A Natural Product Ligand of LXR 295

lesterol to bile acids (Lehmann et al., 1997), which was later confirmed in the LXR null mouse (Peet et al., 1998). In addition to its function in regulation of CYP7A, which ap- pears to be rodent-specific, an array of additional target genes have been identified that establish LXR as a key reg- ulator of cholesterol homeostasis. These genes include sterol transporters such as ABCA1, ABCG1, ABCG5, and ABCG8, as well as other genes demonstrated to be critical in lipid metabolism including apoE, apoCII, lipoprotein lipase, phos- pholipid transfer protein, and cholesterol ester transfer pro- tein (Luo and Tall, 2000; Repa et al., 2000, 2002; Ven- kateswaran et al., 2000; Laffitte et al., 2001; Zhang et al., 2001; Cao et al., 2002; Mak et al., 2002). The manner in which LXR regulates these genes suggests that activation of this receptor may be antiatherogenic, which has been con- firmed in mouse models (Joseph et al., 2002; Tangirala et al., 2002). The therapeutic potential of a LXR agonist has re- cently expanded by the demonstration that activation of this Downloaded from receptor results in both anti-inflammatory and antidiabetic activity by regulating an array of genes involved in either inflammatory processes or gluconeogenesis, respectively (Cao et al., 2003; Fowler et al., 2003; Joseph et al., 2003). Development of selective, high-affinity ligands for LXR such as T1317 and GW3965 has proven to provide essential tools in characterization of the physiological and pathophys- jpet.aspetjournals.org iological roles of LXR. In this study, we identified and char- acterized an additional ligand of LXR, which represents the first nonoxysterol natural product ligand for this receptor. Natural products have proven to be an abundant source of agents for pharmacological characterization of biomolecules as well as for medicinal purposes. Although natural product libraries have not been as profitable for identification of at ASPET Journals on March 9, 2015 Fig. 6. Paxilline induces the expression of a natural target gene, SREBP, ligands for nuclear hormone receptors as they have been for in HepG2 cells. A, HepG2 cells were treated for 24 h with either 100 nM other fields, such as ion channel pharmacology, key ligands T1317 or various concentrations of paxilline for 24 h before lysis of the have been identified in the past that target receptors such as cells, preparation of total RNA, and quantitation of SREBP mRNA by real-time polymerase chain reaction as previously described (Thomas et ER, FXR, PXR, and peroxisome proliferator-activated recep- al., 2003). Paxilline dose dependently increased SREBP expression. B, tor ␥. We identified paxilline, an indole alkoid fungal metab- HepG2 cells stably transfected with a SRE reporter construct were olite from P. paxilli, as an efficacious LXR agonist. Paxilline treated with various concentrations of paxilline to assess the ability of ␣ ␤ this ligand to induce SREBP. Paxilline increased reporter expression binds directly to both LXR and LXR resulting in coactiva- dose dependently displaying an EC50 of 2,800 nM. tor recruitment and activation of LXR-dependent gene tran- scription. Paxilline exhibits similar potency and efficacy as of paxilline induction of SREBP expression. As illustrated in the natural ligand, 22(R)-OHC, in cotransfection assays and Fig. 6B, paxilline efficiently increased transcription from the in terms of induction of expression of a natural target gene,

SRE reporter with an EC50 of 2,800 nM. These data demon- ABCA1. Additional pharmacological activities of paxilline strate that paxilline binds directly to both LXR␣ and LXR␤, precluded examination of modulation of LXR activity in vivo functions as an agonist with the ability to mediate recruit- due to toxicity. Paxilline is a tremorgenic mycotoxin that is a ment of coactivators to the receptors, and activates transcrip- well characterized antagonist of high conductance calcium- tion of a reporter gene as well as two natural LXR target activated K channels (BK channel) with potencies in the genes. range of 100 nM, which is clearly greater than the potencies we detected for the LXRs in the low single digit micromolar Discussion range (Knaus et al., 1994). Given the limitations for evaluation of paxilline in vivo, Since the identification of LXRs as orphan members of the this compound still provides an additional tool for pharma- nuclear hormone receptor superfamily in the mid-1990s, sev- cological characterization of LXR. In addition, the novel eral ligands both natural and synthetic have been identified. chemical structure provides insight into the diversity of Endogenous oxysterols were the first ligands identified for chemical structures that can recognize the ligand binding LXR, which suggested a role for this receptor in regulation of pockets of both LXR␣ and LXR␤ leading to coactivator re- cholesterol homeostasis via action as a “cholesterol sensor” cruitment and transcriptional activation. Furthermore, iden- (Janowski et al., 1996). The first target gene identified for tification of an additional natural product ligand for a nu- LXR, cholesterol 7␣-hydroxylase (CYP7A), was consistent clear hormone receptor indicates that natural product with this suggestion, illustrating a role for this receptor in libraries may be a rich source for ligands of additional nu- regulation of the rate-limiting step in the conversion of cho- clear hormone receptors including the orphans. 296 Bramlett et al.

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