1521-0111/91/4/264–276$25.00 http://dx.doi.org/10.1124/mol.116.105213MOLECULAR PHARMACOLOGY Mol Pharmacol 91:264–276, April 2017Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics

Identification of a Novel Liver X Receptor Agonist that Regulatesthe Expression of Key Cholesterol Homeostasis Genes withDistinct Pharmacological Characteristics

Ni Li, Xiao Wang, Yanni Xu, Yuan Lin, Ningyu Zhu, Peng Liu, Duo Lu, and Shuyi SiState Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academyof Medical Sciences, Beijing, People’s Republic of China (N.L., Y.L., D.L.); and Institute of Medicinal Biotechnology, ChineseAcademy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China (N.L., X.W., Y.X., N.Z.,P.L., S.S.)

Received May 21, 2016; accepted January 6, 2017

ABSTRACTActivation of liver X receptor (LXR) is associated with cholesterolmetabolism and anti-inflammatory processes, which makes itbeneficial to antiatherosclerosis therapy. Nevertheless, existingagonists that target LXR, for example TO901317, are related tounwanted side effects. In the present study, using a screeningmethod we identified IMB-808, which displayed potent dualLXRa/b agonistic activity. In vitro, IMB-808 effectively increasedthe expressing quantity of genes related to reverse cholesteroltransport process as well as those associated with cholesterolmetabolism pathway in multiple cell lines. Additionally, IMB-808remarkably promoted cholesterol efflux from RAW264.7 as wellas THP-1 macrophage cells and reduced cellular lipid accumu-lation accordingly. Interestingly, comparedwith TO901317, IMB-

808 almost did not increase the expressing quantity of genesrelated to lipogenesis in HepG2 cells, which indicated that IMB-808 could exhibit fewer internal lipogenic side effects with acharacteristic of selective LXR agonist. Furthermore, in compar-ison with the full LXR agonist TO901317, IMB-808 recruitscoregulators differently and possesses a distinct predictivebinding pattern for the LXR ligand-binding domain. In summary,our study demonstrated that IMB-808 could act as an innovativepartial LXR agonist that avoids common lipogenic side effects,providing insight for the design of novel LXR modulators. Ourdata indicate that this compound might be used as a promisingtherapeutic agent for the prospective treatment of atherosclero-sis in the future.

IntroductionAtherosclerosis, as the dominant cause of mortality in

developed nations, is gradually becoming a health issuearound world (Roger et al., 2012). The liver X receptor (LXR)was capable of inhibiting inflammatory reactions driven bymacrophages and promoting the process of reverse cholesteroltransport (RCT) which made it a prospective target fortreating atherosclerosis (Joseph et al., 2003; Naik et al.,2006; Zhang et al., 2012). LXRs, including LXRa and LXRb,bind to response elements of their target genes to modulategene expression (Edwards et al., 2002). Although the express-ing range of LXRb is ubiquitous, LXRa is expressed

exclusively in kidney, lung, intestine, adipose tissue, liver,as well as certain kinds of immunocytes (Auboeuf et al., 1997;Heine et al., 2009). Activation or repression of LXR depends onthe presence or absence of its ligands. In the absence ofligands, LXRs are in a nonactive state, combining to corepres-sors, for example, the nuclear receptor corepressor (NcoR)(Chen and Evans, 1995). The binding of ligands results in achange of the conformation of LXRs that enables corepressorsto be released, coactivators to be recruited, and the targetgenes to be transactivated (Wiebel et al., 1999).LXRs serve as a sensor of cholesterol that protects cells from

the adverse effect of overloaded cholesterol by inducing theexpression of target genes. RCT is a primary mechanism forremoving cholesterol from cells and transferring it into theliver, which can be stimulated by LXRs (Bełtowski, 2008).Several proteins of the ATP-binding cassette (ABC) trans-porter family contribute to cholesterol metabolism regulationand are regarded as target genes of LXRs. ABCA1 and ABCG1play important roles in cholesterol efflux, and ABCG5 and

This work was supported by the National Natural Science Foundation ofChina [Grants 81273515, 81321004, and 81503065], the Key New DrugCreation and Manufacturing Program [Grants 2012ZX09301002-003 and2012ZX09301002-001]; and the Basic Scientific Research Program of MateriaMedica, CAMS [Grant 2014ZD03].

N.L. and X.W. contributed equally to this work.dx.doi.org/10.1124/mol.116.105213.

ABBREVIATIONS: ABC, ATP-binding cassette; ApoA-I, apolipoprotein A-I; ApoE, apolipoprotein E; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; FAS, fatty acid synthase; FL, fluorescein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;GST, glutathione S-transferase; HDL, high-density lipoprotein; LBD, ligand-binding domain; LXR, liver X receptor; NcoR, nuclear receptorcorepressor; NPC1L1, Niemann-Pick C1 like 1; ox-LDL, oxidized low-density lipoprotein; PBS, Phosphate-buffered saline; PCR, polymerase chainreaction; PDB, Protein Data Bank; RCT, reverse cholesterol transport; SCD-1, stearoyl-coenzyme A desaturase 1; SMRT, silencing mediator ofretinoic acid and thyroid hormone receptor; SREBP-1c, sterol response element binding protein 1c; Tb, terbium; TR-FRET, time-resolvedfluorescence resonance energy transfer.

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ABCG8 influence hepatic cholesterol excretion and intestinalabsorption (Yu et al., 2003; Cavelier et al., 2006; Wang, 2007).LXR can also increase apolipoprotein E (ApoE) expression,which serves as a critical regulator for atherogenesis tomaintain cholesterol homeostasis (Laffitte et al., 2001;Parikh et al., 2014). Moreover, Niemann-Pick C1 like1 (NPC1L1) is indispensable for absorbing cholesterol inintestine. According to reports, the expressing of NPC1L1can be downregulated by LXR activators in human intestinaland mouse cells (Duval et al., 2006).A previous report (Schuster et al., 2002) demonstrated that

either LXRa or LXRb could exert an effort on the antiathero-sclerosis role of macrophage cells while lacking LXRa andLXRb led to accumulating lipids in the foam cells of lesions inarteries. Several synthesized LXR ligands, for example fullagonists TO901317 and GW3965, were widely focused on andstudied substantially for many years (Janowski et al., 1996;Houck et al., 2004; Geyeregger et al., 2006). Nevertheless, theseligands have not yet been developed as drugs because of theirundesirable side effects. Through the activation of hepaticsterol regulatory element–binding protein-1c (SREBP-1c), theligands could induce lipogenesis and hypertriglyceridemia(Peet et al., 1998; Schultz et al., 2000). Consequently, particularLXR activators that induced no hepatic synthesis of fatty acidsinterested us. It was reported that such ligands, for example N,N-dimethyl-3b-hydroxy-cholenamide and WAY-252623, couldreduce atherosclerosis without lipogenesis increasing andSREBP-1c activating (Kratzer et al., 2009; Quinet et al.,2009). This finding increased the probability that some LXRagonists with the effect of treating atherosclerosis mightenhance RCT while not causing significant lipid accumulationin the liver. Consequently, it was our target to identify LXRagonists, which exhibited satisfactory selectivity.In our current study, we identified IMB-808, which was an

innovative analog of benzo-dioxepine-carboxamide exhibitingimpressive activity of LXR agonist using a cell-based lucifer-ase reporter assay. We found that IMB-808 had an effecton LXR target genes and influenced some cholesterolmetabolism–related pathways in multiple cells. Moreover,the molecular docking result provided us with a theoreticalbasis to study the interaction site between this compound andthe construction of both LXRa and LXRb ligand-bindingdomains (LBDs). Furthermore, based on the coregulatorrecruitment and site mutation activation assays, the possiblemechanism of LXRa/b interaction with IMB-808 was clarified.

Materials and MethodsReagents. TO901317, which was also called T1317 in the current

study, accompanied by phorbol-12- myristate-13-acetate and Oil RedO stain were obtained from Sigma-Aldrich (St. Louis, MO). IMB-808was obtained from the compound library of the National Laboratoryfor Screening New Microbial Drugs, Institute of Medicinal Biotech-nology, Peking Union Medical College (Beijing, People’s Republic ofChina). Opti-MEM Reduced Serum Medium as well as fetal bovineserum were obtained from Invitrogen (ThermoFisher Scientific,Carlsbad, CA). RPMI 1640 medium, Dulbecco’s modified Eagle’smedium as well as modified Eagle’s medium were obtained fromHyclone (Thermo Scientific, Rockford, IL). 22-NBD-cholesterol andLipofectamine 2000 and were obtained from Invitrogen. Oxidized low-density lipoprotein (ox-LDL), apolipoprotein A-I (ApoA-I) as well ashigh-density lipoprotein (HDL) were bought from Union BiologyCompany (Beijing, People’s Republic of China).

Plasmids. The wild-type genes of human LXRa-LBD and LXRb-LBD were obtained by polymerase chain reaction (PCR) from HepG2cells and cloned into pBIND vector (Promega, Madison, WI), in whichGAL4 DNA binding domain existed. GAL4-pGL4-luc plasmids wereprepared as similarly described before (Li et al., 2013).

The method of mutagenesis directed by sites was used to createmutations in pBIND-LXRa-LBD or pBIND-LXRb-LBD using the FastMutagenesis System (TransGen Biotech, Beijing, People’s Republic ofChina). A few important amino acids of LXRa-LBD were convertedinto other molecules. The plasmids were mutated according to thefollowing pattern: F257Y (Phe257 to Tyr), T302I (Thr302 to Ile),R305G (Arg305 to Gly), H421D (His421 to Asp), and W443G (Trp443to Gly). Meanwhile, key amino acids in LXRb-LBD were changed toalanines accordingly: F271A (Phe271 to Ala), M312A (Met312 to Ala),T316A (Thr316 to Ala), H435A (His435 to Ala), andW457A (Trp457 toAla). Sequencing was used to verify the successful pBIND-LXRa-LBDand pBIND-LXRb-LBD mutated plasmids.

Human LXRa-LBD (amino acids 182–447) and LXRb-LBD cDNA(amino acids 196–461) were cloned into pET30a vector separately. Toconstruct the mutation expression plasmids, Arg305 was subsequentlymutated to Gly in pET30a-LXRa-LBD (named pET30a-LXRa-R305G),whereas Phe271 was mutated to Ala in pET30a-LXRb-LBD (namedpET30a-LXRb- F271A).

The Reporter Assay of LXR -GAL4 Chimera and CellCulture. In brief, RAW264.7 macrophages, HepG2, HEK293T,Caco-2 cells as well as the human monocyte cell line THP-1 werecultured in different media separately, as described previously (Liet al., 2014). Phorbol-12- myristate-13-acetate 100 nM was added toTHP-1 cells and incubated for 24 hours to produce fully differentiatedmacrophages, after which serum-free mediumwas used to replace theformer medium.

IMB-808 was identified through the screening of a synthesizedcompound library that contained 20,000 drug-like constructions, asdescribed previously (Li et al., 2016). In the activity assay of IMB-808,HEK293T cells were transfected by pBIND-LXRa-LBD (or pBIND-LXRb-LBD) expression plasmid as well as GAL4-pGL4-luc reporterplasmid by Lipofectamine 2000 and incubated for 6 hours before beingtreated by compounds for 18 hours. The Luciferase Assay System(Promega) was used to determine luciferase activity using a micro-plate reader (PerkinElmer, Waltham, MA).

Real-Time Quantitative Reverse-Transcription PCR Analysis.HepG2andCaco-2 cellswere inoculated in sixwell plateswith thedensityof 4� 105 cells/ml.RAW264.7macrophagesandhumanTHP-1monocyteswere cultured in six-well plates with densities of 6 � 105 and 1.5 � 106

cells/ml, respectively. TRIzol reagent (ThermoFisher Scientific) was usedto extract total RNA of the cells, whereas reverse transcriptional kits(TransGen Biotech) were used to reverse transcribe it. The real-timequantitative PCR assay was performed on a CFX96 Real-Time PCRDetection System (Bio-Rad, Hercules, CA) by using SYBR Green (RocheDiagnostics, Lewes, UK) detecting reagents. Table 1 shows the primersequences used in this study. Glyceraldehyde-3-phosphate dehydroge-nase (GAPDH) level was used to normalize all mRNA expressionquantities, and the DDCt method was used to conduct quantitativemeasurement.

Western Blotting. RAW264.7 macrophages, HepG2 cells, THP-1humanmonocytes, and Caco-2 cells were cultured as described above.Varied concentrations of IMB-808 were added into cells after whichthe cells had established attachment (24 hours). Cells were collectedafter 18 hours of incubation, and protein samples were extractedaccording to the above-mentioned protocol. The protein samples weredetected using the corresponding primary antibodies and then in-cubated with secondary anti-rabbit and anti-mouse IgG antibodies (1:5000; Novus, Littleton, CO). An Enhanced Chemiluminescence re-action kit (EMD Millipore, Billerica, MA) was used for blot detection.The following primary antibodies were used: anti-GAPDH (1:2000;Abmart; Shanghai, People’s Republic of China), anti-ApoE (1:1000;Abcam, Cambridge, UK), anti-ABCG1 (1:500; Novus), anti-ABCA1 (1:1000; Novus), anti-ABCG5 (1:1000; Abcam), anti-ABCG8 (1:500;

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Novus), anti-NPC1L1 (1:1000; Abcam), and anti-SREBP-1c (1:1000;Novus). GAPDH was used to normalize entire proteins.

Oil Red O Staining. Oil Red O staining was used to evaluateaccumulated cellular lipids in RAW264.7macrophages. The cells werecultured in 96-well plates and supplemented with 60 mg/ml ox-LDLafter attachment. After 12 hours, they were stimulated with variedconcentrations of IMB-808 for 18 hours then fixed and stainedwithOilRed O according to previous methods, and observed by light micros-copy (Li et al., 2013). To extract Oil Red O, isopropanol was added toeach well. The samples were measured at 510 nm by a microplatereader 10 minutes after that (Zou and Shen, 2007).

Cholesterol Efflux Experiment. 22-NBD-cholesterol was usedto conduct a cell cholesterol efflux experiment on RAW264.7 macro-phages and THP-1 monocytes separately (Li et al., 2014). Briefly, thecells were seeded into 96-well clear-bottom black plates (Costar;Corning, Corning, NY) and marked using 22-NBD-cholesterol with afinal concentration of 2.0 mM in serum-free medium that contained0.2% (w/v) bovine serum albumin (medium A; Sigma-Aldrich) for24 hours. Phosphate-buffered saline (PBS) was used to wash cellstwice, after which the cells were incubated with IMB-808 or vacantcontrol for an additional 18 hours. Subsequently, the receptor proteinof ApoA-I or HDL with final concentrations of 10 or 50 mg/ml wasadded into cells and incubated for 6 hours. A microplate reader wasused to test the quantities of cholesterol in cells as well as of themedium separately (PerkinElmer; excitation 485 nm, emission535 nm). The calculating mode of the 22-NBD-cholesterol effluxpercentage was (medium)/(medium 1 cell) � 100%. Each efflux testwas conducted in triplicate.

Expression and Purification of LXRa-R305G and LXRb-F271A. The pET30a-LXRa-R305G and pET30a-LXRb-F271A plas-mids were transformed into Rosetta (DE3) Escherichia coli cellsseparately. The expression of the two proteins was induced by theaddition of 0.2 mM isopropyl b-D-1-thiogalactopyranoside at 20°Covernight. The supernatants of LXRa-R305G and LXRb-F271A wereharvested by centrifugation then filtered through a 0.45 mM filter andloaded onto a Ni21 His Trap chelating column (GE Healthcare, LittleChalfont, UK). The binding buffer contained 20mMTris-HCl (pH 7.4),500mMNaCl, and 20mM imidazole. LXRa-R305G proteinwas elutedby 20 mM Tris-HCl (pH 7.4), 500 mM NaCl, 200 mM imidazole,whereas LXRb-F271A proteinwas eluted by 20mMTris-HCl (pH 7.4),500 mM NaCl, and 250 mM imidazole. The purified proteins wereconcentrated to 1 mg/ml by ultrafiltration and subsequently stored inthe buffer of 50 mM potassium phosphate (pH 8.0), 150 mM KCl,0.5 mM EDTA, 0.5% CHAPS, 5 mM dithiothreitol, and 20% glycerolat 280°C.

LanthaScreen Time-Resolved Fluorescence Energy TransferLXR-Coregulator Peptide Interaction assays. LanthaScreen time-resolved fluorescence energy transfer (TR-FRET) LXRa-CoactivatorAssay Kit (PV4655; ThermoFisher Scientific) and LXRb-CoactivatorAssay Kit (PV4658; ThermoFisher Scientific) were used to performTR-FRET LXR-coregulator peptide interaction assays according to themanufacturer instructions separately. Human glutathione S-transferase

(GST)-LXRa-LBD, GST-LXRb-LBD, terbium (Tb)-labeled anti-GST tag antibody, fluorescein (FL)-labeled peptides, includingFL-TRAP220/DRIP2 (PV4549), FL-D22 (PV4387), FL-NcoR (NcoR ID2,PV4624), and FL-silencingmediator of retinoic acid and thyroid hormonereceptor (SMRT) (SMRTID2, PV4423), as well as all of the buffers wereincluded in the current study. TO901317 or IMB-808was diluted and firstadded to 384-well black plates (Costar) according to the kit instructions.LXRa-LBD or LXRb-LBD protein was then added, followed by mixedcoregulator and FL-peptide/Tb-anti-GST, which was added last.

Human LXRa-R305G and LXRb-F271A mutation proteins wereobtained as described before. Tb-labeled anti-His tag antibody(PV5863, Invitrogen) was purchased from Invitrogen. TR-FRET assayofmutation LXR and coregulator interaction was performed under thesimilar condition. TO901317 or IMB-808 was diluted and added to theplate, LXRa-R305G (final concentration 30 nM) or LXRb-F271A (finalconcentration 100 nM) was then added, followed by coregulator andFL-peptide/Tb-anti-His mixed together.

The plates were shaken in darkness under ambient temperature for2 hours. A PerkinElmer EnVision plate reader was used to measurethe TR-FRET ratio (520/495 nm) of all assay wells, and the emissionsignal at 520 nm was divided by the emission signal at 495 nm toobtain the data. Each assay for each FL peptide was performed fourtimes separately (n 5 4).

Virtual Molecular Docking. The ligand action of IMB-808 wasevaluated by the docking programDiscovery Studio 4.1 (Accelrys, SanDiego, CA) with the crystal structure of LXRa [Protein Data Bank(PDB) code: 1UHL, LXRa with TO901317] and LXRb (PDB code:1PQC, LXRb with TO901317) separately. After removing all crystal-lized H2O molecules from the former construction, hydrogen wasadded into the DS CDOCKER module. An optimized start conforma-tion was obtained by minimizing the compound to achieve the lowestenergy level ahead of docking in silico.

Statistical Analysis. The software of GraphPad Prism 5.0(GraphPad, San Diego, CA) was used to calculate statistics as wellas best-fit curves. The data were represented as the mean 6 SEM.One-way analysis of variance and Student’s t test were used to analyzeresults with SPSS version 11.0 (SPSS Inc., Chicago, IL). A P valueof ,0.05 was regarded as statistically significant (*P , 0.05; **P ,0.01; and ***P , 0.001).

ResultsIMB-808 Displays LXRa/b Dual-Agonist Activity. In

this study, IMB-808, an LXRb agonist with an analogousstructure of benzodioxepine-carboxamide (Fig. 1A) wasverified using LXRb-GAL4 luciferase reporter screening accord-ing to the above-mentioned protocol. The chemical name ofIMB-808 is N-methyl-N-(2-oxo-2-((2,3,4-trifluorophenyl)amino)ethyl) -3,4-dihydro-2H-benzo[b][1,4] dioxepine-7-carboxamide,and this compound has not been reported to display any activitypreviously. IMB-808 significantly dose-dependently induced

TABLE 1Primers for real-time quantitative PCR

Gene Forward Primers Reverse Primers

hABCA1 59-TTCCCGCATTATCTGGAAAGC-39 59-CAAGGTCCATTTCTTGGCTGT-39hABCG1 59-ATTCAGGGACCTTTCCTATTCGG-39 59-CTCACCACTATTGAACTTCCCG-39hApoE 59-GTTGCTGGTCACATTCCTGG-39 59-GCAGGTAATCCCAAAAGCGAC-39hFAS 59-TGGAAGTCACCTATGAAGCCA-39 59-ACGAGTGTCTCGGGGTCTC-39hGAPDH 59-AGCCACATCGCTCAGACAC-39 59-GCCCAATACGACCAAATCC-39hNPC1L1 59-TCACTCGAGGTGTTGTGCTGC-39 59-CAAGCAGGTACGAGTCCTTGGGCA-39hSCD-1 59-TGGGTGGCTGCTTGTG-39 59-GCGTGGGCAGGATGAAG-39hSREBP-1c 59-CGGAGCCATGGATTGCACTTTC-39 59-GATGCTCAGTGGCACTGACTCTTC-39mABCA1 59-AAAACCGCAGACATCCTTCAG-39 59-CATACCGAAACTCGTTCACCC-39mABCG1 59-GCTCCATCGTCTGTACCATCC-39 59-ACGCATTGTCCTTGACTTAGG-39mGAPDH 59-AGGTCGGTGTGAACGGATTTG-39 59-GGGGTCGTTGATGGCAACA-39

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LXRb activation under concentrations ranging from 0.001 to30 mM, with an EC50 of 0.53 mM, and displayed a maximizedactivity of nearly 2-fold (Fig. 1D). In this model, TO901317

showed approximately 3-fold LXRb activation (Fig. 1E). Sub-sequently, we examined the activity of IMB-808 using LXRa-GAL4 luciferase reporter assays. It was revealed that IMB-808

Fig. 1. IMB-808 regulated LXRb. (A) Structure of IMB-808. (B) LXRa activation by IMB-808. HEK293T cells were transfected with the GAL4-pGL4-lucreporter plasmid and the pBIND-LXRa expression plasmid. IMB-808 displayed significant LXRa agonistic activity in the luciferase activity assay. (C)LXRa activation by T1317. (D) LXRb activation by IMB-808. HEK293T cells were transfected with the GAL4-pGL4-luc reporter plasmid and the pBIND-LXRb expression plasmid. (E) LXRb activation byT1317. Similar results were obtained in three independent experiments. Data are reported as themean6SEM (n = 3).

Fig. 2. Effect of IMB-808 on ABCA1, ABCG1, and ApoE protein expression. (A) RAW264.7 macrophages were incubated with IMB-808 at variousconcentrations or with T1317 for 18 hours, and ABCA1 and ABCG1 protein levels were determined by Western blotting. (B and C) THP-1–derivedmacrophages were treated with IMB-808 (1 or 10 mM) or T1317 (1 mM) for 18 hours, and ABCA1, ABCG1, and ApoE protein levels were determined byWestern blotting. (D) The mRNA levels of ABCA1 and ABCG1 were measured by real-time quantitative PCR in RAW264.7 macrophages. (E and F)ABCA1, ABCG1, and ApoE mRNA levels were determined by real-time quantitative PCR separately in THP-1–derived macrophages. Induction factorswere normalized against GAPDH, and the control groups were treated with dimethylsulfoxide (0.1%). Similar results were obtained in four independentexperiments. Data are reported as the mean 6 SEM (n = 4, *P , 0.05 versus control, **P , 0.01 versus control, ***P , 0.01 versus control).

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also could dose-dependently activate LXRawith a lower EC50 of0.15 mM (Fig. 1B), and TO901317 showed 3.3-fold LXRaactivation with an EC50 of 0.05 mM (Fig. 1C).IMB-808 Could Induce the Expression of ABCG1,

ABCA1, and ApoE In Vitro. ABCA1 and ABCG1 areimportant target genes of LXR that were associated with theRCT pathway in macrophages (Geyeregger et al., 2006). Theinfluence of IMB-808 on the expressing profiles of ABCA1 andABCG1 in murine and humanmacrophages was first detectedby Western blot and real-time quantitative PCR assays. IMB-808 significantly increased both protein and mRNA levels ofABCG1 as well as ABCA1 in RAW264.7 macrophages (Fig. 2,A and D) and THP-1–derived macrophages (Fig. 2, B and E).ApoE is another crucial target gene of LXR, which is involved

in cholesterol homeostasis and is beneficial for the protection ofatherosclerosis (Laffitte et al., 2001). In the present study, theexpressing quantities of mRNA and the protein of ApoE wereslightly increased after treatment with IMB-808 in THP-1–derived macrophages. Moreover, these genes were induced toa greater extent while treating the cells with full agonistTO901317 (Fig. 2, C and F).IMB-808 Promotes Cholesterol Efflux from Macro-

phages. ABCG1 and ABCA1 were crucial transporter pro-teins for facilitating cholesterol efflux out of macrophages toplasma HDL and ApoA-I (Repa and Mangelsdorf, 2000).

Subsequently, the influence on the cholesterol efflux ofmacrophages derived fromTHP-1 andRAW264.7was studied.HDL (50 mg/ml) or ApoA-I (10 mg/ml) was added with the aimof promoting cholesterol efflux separately. It was also discov-ered that IMB-808 could dose-dependently promote choles-terol efflux toward ApoA-I and HDL and reduce the cellularcholesterol concentration in these two cell lines (Fig. 3).IMB-808 Reduces Cellular Lipid Accumulation. To

determine the potential effect of IMB-808 on the inhibition oflipid accumulation and foam cell formation, assays of foamcells were carried in RAW264.7 cells. According to Fig. 4, C–G,IMB-808 could effectively reduce the quantity of accumulatedlipid in comparison with that of a single ox-LDL (Fig. 4B).Furthermore, foam cell formation was significantly inhibitedafter stimulation with 3 mM IMB-808 (Fig. 4F), resulting inlevels comparable to those of the control group (Fig. 4A) andinferior to that seen after treatment with TO901317 (Fig. 4H).Then, the content of lipid in cells was quantitated. The resultshowed that IMB-808 was capable of significantly reducingaccumulated lipid in cells from 0.1 to 10 mM (Fig. 4I).IMB-808 Regulates the Expressing Profiles of

ABCG5, ABCG8, and NPC1L1, Which Are Related tothe Absorbing and Secreting Process of Cholesterol. Itwas proposed by previous studies that LXRs influencedcholesterol level by reducing the quantity of cholesterol

Fig. 3. IMB-808 promoted cholesterol efflux. (A and B) RAW264.7 macrophages were preincubated with 22-NBD-cholesterol for 24 hours. The cells werethen washed with PBS and incubated with IMB-808 (0, 0.01, 0.1, 1, or 10 mM). After 18 hours, 10 mg/ml ApoA-I or 50 mg/ml HDL (final concentration) wasadded and incubated for 6 hours at 37°C. The amount of cholesterol in themediumand cells wasmeasured separately. Relative 22-NBD-cholesterol effluxto ApoA-I or HDL induced by IMB-808 was calculated as described in the Materials and Methods. (C and D) Similar cholesterol efflux assays wereperformed in THP-1–derived macrophages. Similar results were obtained in three independent experiments. Data are reported as the mean6 SEM (n =3, *P , 0.05 versus control, **P , 0.01 versus control).

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absorbed in the intestine through the regulation of NPC1L1,ABCG8, as well as ABCG5. Moreover, LXRs upregulate theexpression of ABCG8 as well as ABCG5 to promote cholesterolefflux into bile (Yu et al., 2003). In this study, IMB-808increased the expression of ABCG5 and ABCG8 protein inHepG2 cells by 1.70-fold and 1.82-fold, respectively (Fig. 5, Aand B). We also found that IMB-808 significantly induced theexpression of ABCG5 and ABCG8 proteins of Caco-2 cells dosedependently (Fig. 5, C and D). Furthermore, in Caco-2 cellsboth protein and mRNA expression quantities of NPC1L1decreased after treatment with IMB-808 (Fig. 5, E and F).IMB-808 Almost Does Not Induce Lipogenic Gene

Expression. Another crucial target gene of LXR, SREBP-1c,which can induce hepatic fatty acid synthesis, was analyzed(Wagner et al., 2003). Interestingly, our results demonstratedthat IMB-808 almost did not increase the protein expression ofSREBP-1c in comparison with that after TO901317 treatment(approximately 4-fold for 1 mM TO901317) (Fig. 6A). Moreover,the mRNA levels of stearoyl-coenzyme A desaturase-1 (SCD-1),

fatty acid synthase (FAS) as well as SREBP-1c did not increaseobviously after treatmentwith0.01–10mMIMB-808, in contrastwith treatment with TO901317 (Fig. 6B).IMB-808 Shows Distinct Recruitment of Coregulators

in Comparison with TO901317. The expression of geneswas regulated by LXR, which was transcriptionally activatedin a ligand-dependent manner through dissociating core-pressors and subsequently recruiting coactivators (Leo andChen, 2000). The specific conformation alteration of LXRinduced by IMB-808 might be due to coregulator interac-tions, which probably explains the different regulatingpatterns on genes that were discovered during lipogenesis.It was probably caused by distinct patterns of ligand bindingwith LXR-LBD, which consequently led to an alternatingcapacity of dissociating corepressors and/or recruiting coac-tivators. To further analyze the molecular mechanism ofcompound binding with LXR-LBD, the TR-FRET assay wasconducted with the aim of comparing the capacity ofTO901317 and IMB-808 to adjust the interacting activityof LXRa and LXRb with corepressors or coactivators,respectively.For LXRa, IMB-808 displayed a weak displacement with

the corepressor NcoR dose dependently compared with that ofTO901317 (approximately 42%) (Fig. 6C) and showed a moremoderate ability to recruit coactivator TRAP220 (approxi-mately 38%) compared with that of TO901317 (Fig. 6D). At thesame time, IMB-808 also exhibited an inferior influencecompared with that of TO901317 to displace the corepressorSMRT (approximately 40%) (Fig. 6E) and recruit coactivatorD22 (approximately 44%) (Fig. 6F) for LXRb.IMB-808 Docks to the LXR-LBD in Silico. To investi-

gate the supposed binding pattern and possible interactionbetween the ligand and pocket of IMB-808, the structure ofIMB-808 was virtually docked to the crystal structures of LBDof LXRa (PDB code: 1UHL) and LXRb (PDB code: 1PQC) usingthe docking program DS CDOCKER separately. The virtualbinding result suggested that IMB-808 can fit well in eitherLXRa LBD (Fig. 7, A and B) or LXRb LBD (Fig. 8, A and B).First, for the LXRa LBD model, the fluorine atom in thetrifluorophenyl ring formed a hydrogen bondwith Arg305, andthe phenyl ring of the benzodioxepine system formed ahydrogen bond with Trp443. IMB-808 forms a p–p bond withthe imidazole ring of His421 and an amide–Pi bond withThr302. In addition, IMB-808 is surrounded by Phe257,Ala261, Met298, and Glu301 through the Pi-alkyl interaction,the hydrophobic interaction, and van der Waals forces.Second, for the LXRb LBD model IMB-808 virtually docked

into the pocket including two hydrogen bonds, two p–pstacking interactions, some van der Waals force, and hydro-phobic interactions with amino acids around. Specifically, theoxygen atom of IMB-808 separately forms two hydrogen bondswith Met312 and Thr316 in a conjugated structure, while thebenzene ring of IMB-808 is close to Phe271 and Phe329 of theLXRb LBD and forms pp–p stacking interactions with theseamino acids. In addition, IMB808 is also surrounded byThr272, Ala275, Phe340, Leu345, and Phe349 of the LXRbLBD through van der Waals forces or hydrophobic interac-tions to form a complete agonistic conformation.IMB-808 Displays Distinct Interacting Sites from

Those of TO901317. With the aim of investigating theinteraction sites of IMB-808 with LXRa LBD or LXRbLBD compared with those of TO901317, we performed the

Fig. 4. IMB-808 reduced ox-LDL–induced lipid accumulation in RAW264.7macrophages. RAW264.7 macrophages were preincubated with PBS (vehicle)(A) or ox-LDL (60 mg/ml) (B–H). After 24 hours, these cells were treated with0.1% dimethylsulfoxide (B), IMB-808 (0.1, 0.3, 1, 3, or 10 mM) (C–G), or T1317(1 mM) (H) for 18 hours. Cells were fixed with 4% paraformaldehyde andstained with 0.5% Oil Red O to detect lipid accumulation. Representativeimages of the eight study groups are displayed (original magnification, 400�).(I) After Oil Red O staining, bound dye was solubilized and quantifiedspectrophotometrically at 510 nm. Similar results were obtained in threeindependent experiments. Data are reported as themean6SEM (n = 3, *P,0.05 versus control, ***P , 0.001 versus control).

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site-directed mutagenesis and then examined the luciferaseactivity of mutation plasmids on the LXRa or LXRb agonistscreening model, respectively.Five critical amino acid residues of LXRa LBD, which

played an essential role in binding IMB-808 or TO901317,were replaced with others respectively, and the obtainedmutant proteins were tested to figure out whether they couldbe activated by IMB-808 or TO901317 (Fig. 7, C and D,separately). The activation levels of W443G, H421D, andT302I mutants conferred were low and the increasing extentwas slight, whereas the R305G mutant was barely capable ofactivating LXRa. Interestingly, the mutant and the wild-typeLXRa plasmids had nearly the same activity when Phe257was replaced with Tyr (Fig. 7C). In addition, T302I and F257Ymutants were slightly activated whereas W443G and H421Dwere barely activated by TO901317 (Fig. 7D).Furthermore, five different amino acids of LXRb that were

determined to be crucial residues for IMB-808 binding wereindividually replaced with alanine residue, and the obtained

mutant proteins were tested to determine whether they couldbe activated by IMB-808 or TO901317 (Fig. 8, C and D,respectively). F271A, M312A, as well as T316A mutantsdisplayed significant decreases in agonistic activation byIMB-808, which indicated that these residues played animportant role in transcriptional activation. Interestingly,when H435 and W457 were transformed into alanine, themutants that were obtained displayed activity similar to thatof the wild-type LXRb plasmids (Fig. 8C). On the contrary,T316, H435, and W457 mutants barely displayed any activa-tion, whereas M312 mutants were only mildly activated byTO901317 (Fig. 8D). Conforming to these findings, variousmutants displayed different agonist activity compared withthe wild-type group after stimulation by 10 mM IMB-808 (Fig.7E; Fig. 8E) or 1 mM TO901317 (Fig. 7F; Fig. 8F).IMB-808 Shows Distinct Effect of the LXR-LBD

Mutation on Coregulator Recruitment. To investigatethe differential effect of IMB-808 and T1317 on LXRmediatedby the interaction of LXR-LBD and cofactors, experiments to

Fig. 5. IMB-808 regulated ABCG5, ABCG8, and NPC1L1 expression. (A and B) HepG2 cells were treated with IMB-808 at various concentrations orT1317 (1 mM) for 18 hours, and ABCG5 and ABCG8 protein levels were determined by Western blot assays. (C and D) Caco-2 cells were incubated withvarious concentrations of IMB-808 or T1317 for 18 hours. ABCG5 andABCG8 protein levels were thenmeasured.Western blotting (E) and real-time PCR(F) were performed on Caco-2 cells that were incubated with IMB-808 or T1317 for 18 hours. NPC1L1 protein and mRNA levels were then determined.Induction factors were normalized against GAPDH, and the control groups were treated with dimethylsulfoxide (0.1%). Similar results were obtained infour independent experiments. Data are reported as the mean 6 SEM (n = 4, *P , 0.05 versus control, **P , 0.01 versus control).

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determine the effect of the mutation on cofactor recruitmentwas performed by TR-FRET. According to the effects of theluciferase activity of mutation plasmids on LXRa or LXRb,LXRa-R305G and LXRb-F271A were chosen as representa-tive mutation proteins for the TR-FRET assay.For LXRa-R305G, IMB-808 exhibited moderate displace-

ment with the corepressor NcoR compared with that ofTO901317 (approximately 31%) (Fig. 9A) and showed a weakability to recruit coactivator TRAP220 (approximately 18%)compared with that of TO901317 (Fig. 9B). Moreover, IMB-

808 also displayed an influence that was inferior to that ofTO901317 to displace the corepressor SMRT (approximately23%) (Fig. 9C) and recruit coactivator D22 (approximately13%) (Fig. 9D) for LXRb-F271A.

DiscussionLXRs were known as nuclear receptors with key functions

on the regulation of lipid and cholesterol homeostasis intissues and recently have attracted attention because they

Fig. 6. (A) Effect of IMB-808 on SREBP-1c expression. HepG2 cells were incubated with IMB-808 or T1317 for 18 hours, and SREBP-1c protein levelswere determined by Western blotting. (B) Effects of IMB-808 on SREBP-1c, FAS, and SCD-1 mRNA expression. HepG2 cells were treated with IMB-808at various concentrations, and T1317 was used as a positive control. After 18 hours, the SREBP-1c, FAS, and SCD-1 mRNA expression levels weredetermined by real-time PCR. Four independent experiments were performed, and representative graphs are shown. Data are reported as the mean 6SEM (n = 4, *P, 0.05 versus control, **P, 0.01 versus control). (C) The TR-FRET assaywas used to examine corepressor peptideNcoR ID2 displacementfrom human LXRa-LBD treated with IMB-808 or TO901317. (D) The TR-FRET assay was performed to examine coactivator TRAP220/DRIP2recruitment to human LXRa-LBD treated with IMB-808 or TO901317. (E) The TR-FRET assay was used to examine corepressor peptide SMRT ID2displacement from human LXRb-LBD treated with IMB-808 or TO901317. (F) The TR-FRET assay was performed to examine coactivator D22recruitment to human LXRb-LBD treated with IMB-808 or TO901317. The data are expressed as themean ratio of the emission signal at 520 nm and thesignal at 495 nm. All assays were repeated four times independently (n = 4). Max, maximum.

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also display anti-inflammatory activities (Willy et al., 1995;Miyata et al., 1996). Synthesized LXR agonists have drawnmuch attention in the development of new drugs for treatingatherosclerosis (Hong and Tontonoz, 2014). However, full LXRagonism commonly leads to lipid accumulation in the liverbecause it activates the LXRa subtype by increasing theexpressing quantity of SREBP-1c genes associated with thelipogenesis pathway (Baranowski, 2008). Therefore, our cur-rent study aimed to find an innovative partial LXR agonistthat had antiatherosclerotic activity and fewer lipogenic sideeffects as determined by screening.Here, we found that IMB-808 is an activator of LXRb using

a cell-based screening method that revealed a novel struc-tural agonist with an EC50 of 0.53 mM. Subsequently, wefound that IMB-808 was not only a partial modulator forLXRb, it could also dose-dependently activate LXRa with alower EC50 of 0.15 mM. LXRs were important transcriptionfactors while regulating RCT. In fact, it was proposed by the

following studies that LXRs could influence almost everypart of this pathway. The first step of RCT was cholesterolefflux out of cells that were mainly regulated by ABCG1 andABCA1 transporters. Here, we found that the levels of bothprotein andmRNA expression of ABCG1 as well as of ABCA1were improved by IMB-808 dose dependently in two macro-phage cell lines. Furthermore, IMB-808 could reduce cellularlipid accumulation and inhibit foam cell formation inRAW264.7 macrophages. ABCA1 and ABCG1 are responsi-ble for transferring both phospholipid and/or cholesterolmolecules through plasma membranes toward ApoA-I with-out lipids or HDL (Dean et al., 2001; Kennedy et al., 2005). Atthe same time, we determined that IMB-808 could signifi-cantly increase cholesterol efflux toward HDL or ApoA-I andreduce cholesterol levels inside cells dose dependently inboth macrophage cell lines. Moreover, as one of otherimportant LXR target genes, the transcription of ApoE,which maintains cholesterol homeostasis in plasma, is

Fig. 7. (A and B) The result of IMB-808 virtually docking into the active site of the LXRa LBD based on the X-ray co–crystal structure of TO901317. (Cand D) Activation of various LXRa mutants by IMB-808 or T1317 using the LXRa-GAL4 reporter assay. Data are displayed as mean values. IMB-808(10 mM) (E) or T1317 (1 mM) (F) displayed different LXRa agonist activities between the wild-type group (WT) and the different mutants in the LXRa-GAL4 chimera reporter assays. Similar results were obtained in four independent experiments. Data are reported as the mean 6 SEM (n = 4).

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promoted through loading cholesterol (Laffitte et al., 2001).Therefore, we concluded that the improvement in ABCA1,ABCG1, and ApoE protein expression was conducive topreventing atherosclerosis via the activation of both LXRaand LXRb by IMB-808.LXR could regulate systematic cholesterol homeostasis by

reducing cholesterol absorbed in the intestine and promotingthe excretion of bile cholesterol through regulating trans-porters on the membrane, which included ABCG5, ABCG8, aswell as NPC1L1 (Repa et al., 2002). It was revealed in thisarticle that IMB-808 efficaciously upregulated ABCG8 andABCG5 protein expression of HepG2 and Caco-2 cells dosedependently. Additionally, after stimulation with IMB-808,both NPC1L1 mRNA and protein were downregulated. Con-sequently, we suggest that IMB-808 could increase theexpression level of LXR-related target genes in vitro and

regulate the entire cholesterol metabolism pathway by acti-vating LXRa/b.To date, several LXR agonists with various structures from

different pharmaceutical companies have been reported andinvestigated, such as GW3965 (GlaxoSmithKline, Brentford,UK) and TO901317 (Tularik, South San Francisco, CA), whichare regarded as classic potent full LXRa/b agonists (Collinset al., 2002). In response to natural or synthetic ligands, LXRsstimulate SREBP-1c, acetyl-CoA carboxylase, SCD-1, andFAS expression in the liver, leading to increased fatty acidsynthesis and plasma triglyceride levels. However, manystudies have suggested that LXRa is the dominant isoformin this pathway (Repa et al., 2000). In this study, it wasdiscovered that IMB-808 neither induced the expression oflipogenesis genes nor exhibited any toxicity at 200mM inRAW264.7 macrophages and HepG2 cells (data not shown). The

Fig. 8. (A andB) The result of IMB-808 virtually docking into the active site of the LXRbLBD based on the X-ray co–crystal structure of T1317. (C andD)Activation of various LXRb mutants by IMB-808 or T1317 using the LXRb-GAL4 chimera reporter assay. Data are displayed as mean values. IMB-808(10 mM) (E) or T1317 (1 mM) (F) displayed different LXRb agonist activities between the wild-type group (WT) and the different mutants in the LXRb-GAL4 chimera reporter assays. Similar results were obtained in four independent experiments. Data are reported as the mean 6 SEM (n = 4).

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crystal structures of the two LXR subtypes revealed that mostof the crucial residues in the LBD are conserved, suggestingthat the discovery of subtype-selective LXR ligands may bechallenging (Williams et al., 2003; Fradera et al., 2010; Liet al., 2010). Surprisingly, our study indicated that IMB-808has a number of advantages with good selectivity for geneexpression regulation, and it may have few lipogenic sideeffects in vivo. Furthermore, we speculate that the selectiveactivity of IMB-808 is due to a distinct mode of interactingwith LXRa and LXRb compared with TO901317.Coregulator recruiting capacity was typically analyzed to

study the ligand qualitatively as an agonist. We wanted toelucidate how IMB-808 displayed specificity for LXRa andLXRb compared with TO901317. First, in the TR-FRETanalysis, IMB-808 weakly displaced the corepressors SMRTand NcoR, and recruited coactivator D22 and TRAP220 atapproximately 40% of that of TO901317. Thus, IMB-808action was characteristic of a partial agonist of LXR ratherthan of the full agonist, like TO901317. Recently, somegroups demonstrated that one of the coactivators thatinteracted with LXRa was specifically recruited to SREBP-1c, which was the responding element of LXR rather thanABCA1 (Kim et al., 2015). This theory indicated that themechanism of coactivator specificity was probably associ-ated with modulating the specified expression level ofother critical regulating genes. Therefore, this led to ourconclusion that IMB-808 regulated cholesterol metabolism

without significant lipogenic side effects compared withTO901317.Moreover, virtual docking was performed to analyze IMB-

808 ligand characteristics. Key amino acid residues werepredicted according to the result of docking and were sub-sequently replaced with other different residues by site-directed mutagenesis. Interestingly, we determined that theamino acids in LXRa-LBD or LXRb-LBD interacting withIMB-808 differed from those of TO901317. In LXRa-LBD,Phe257 formed a Pi-alkyl interaction with IMB-808, but ahydrophobic interaction with TO901317. Moreover, Arg305significantly influenced the activity of IMB-808 but not ofTO901317. Three amino acids, Phe271, Met312, and Thr316,in LXRb form critical interactions with IMB-808. In contrast,H421 and W443 in LXRa (H435 and W457 in LXRb), whichare important for binding TO901317, interact with IMB-808 ina moderate level. Partial agonists may form interaction with aportion of the crucial amino acids in the LBD-active pocketof the nuclear receptor, such as LXR and peroxisomeproliferator–activated receptor, resulting in diminished sta-bilization of LBD AF-2 surface (Bruning et al., 2007). In thestudy by Liu et al. (2015), they also found that alterations inthe conformation of peroxisome proliferator–activated recep-tor g could lead to the process of recruiting differentiated setsof cofactors and subsequently reduce the side effects of thecompound, which were possibly associated with such a specificinteraction.

Fig. 9. (A) The TR-FRET assay was used to examine corepressor peptide NcoR ID2 displacement from human LXRa-R305G treated with IMB-808 orTO901317. (B) TheTR-FRET assaywas performed to examine coactivator TRAP220/DRIP2 recruitment to humanLXRa-R305G treatedwith IMB-808 orTO901317. (C) The TR-FRET assay was used to examine corepressor peptide SMRT ID2 displacement from human LXRb-F271A treated with IMB-808or TO901317. (D) The TR-FRET assay was performed to examine coactivator D22 recruitment to human LXRb-F271A treated with IMB-808 orTO901317. The data are expressed as the mean ratio of the emission signal at 520 nm and the signal at 495 nm. All assays were repeated four timesindependently (n = 4). Max, maximum.

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According to the virtual docking result and the luciferaseactivity assay of mutation plasmids on LXRa or LXRb, weadopted LXRa-R305G and LXRb-F271A as being representa-tive to further discuss the differential effect of the LXR-LBDmutation on coregulator recruitment between IMB-808 andTO901317 by TR-FRET. In the TR-FRET analysis, IMB-808weakly displaced the corepressors SMRT and NcoR, andrecruited coactivator D22 and TRAP220 at approximately10–30% that of TO901317. The mutations LXRa-R305G andLXRb-F271A significantly influenced the activity of IMB-808on coregulator recruitment but not of TO901317. Conse-quently, it was suggested that IMB-808 had a unique mech-anism as an innovative partial dual agonist of LXRa/b withunique regulatory pattern for different target genes, and itsinteraction mode differs from that of the traditional LXRagonist TO901317.Overall, IMB-808 is a novel potent LXR agonist that could

regulate the gene expression involved in the pathway ofmetabolizing cholesterol by relying on the activation ofLXRa/b. IMB-808 remarkably promoted cholesterol effluxout of macrophages and reduced the accumulated lipids infoam cells. Moreover, in comparison with TO901317, our datashowed that IMB-808 had an obvious advantage because italmost did not increase lipogenesis gene expression, whichsuggested that IMB-808 may have lower lipogenic side effectsin vivo. The findings in this study provide us with directionsfor the design of innovative drugs targeting LXR for thetreatment of atherosclerosis in the future.

Acknowledgments

The authors thank GuohuaDu for the TR-FRET assay and analysisin our research.

Authorship Contributions

Participated in research design: Li and SiConducted experiments: Li, Wang, Xu, and LiuContributed new reagents or analytic tools: Lin, Zhu, and LuPerformed data analysis: Li and WangWrote or contributed to the writing of the manuscript: Li and Si

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Address correspondence to: Shuyi Si, Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences and Peking Union Medical College,Tiantan Xili #1, Beijing 100050, People’s Republic of China. E-mail: sisyimb@hotmail.com or Duo Lu, Institute of Materia Medica, Chinese Academy ofMedical Sciences and Peking Union Medical College, Xiannongtan Street #1,Beijing 100050, China. E-mail: luduo@imm.ac.cn

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