Screening for Ligands of Human Retinoid XReceptor-r Using Ultrafiltration MassSpectrometry

Dongting Liu,† Jian Guo,† Yan Luo,† David J. Broderick,‡ Michael I. Schimerlik,‡ John M. Pezzuto,§ andRichard B. van Breemen*,†

Department of Medicinal Chemistry and Pharmacognosy, University of Illinois College of Pharmacy, 833 South Wood Street,Chicago, Illinois 60612, Department of Biochemistry and Biophysics, Oregon State University, ALS 2011, Corvallis, Oregon97331, and College of Pharmacy, University of Hawaii at Hilo, 60 Nowelo Street, Suite 101, Hilo, Hawaii 96720

Retinoid X receptors (RXRs) function as ligand-activatedtranscription factors and are obligatory components of alarge number of nuclear receptor heterodimers. RXRshelp regulate diverse physiological responses includingthe cancer prevention responses of cell proliferation,inflammation, cell differentiation, and apoptosis. SinceRXRs represent important targets for cancer chemopre-vention, an ultrafiltration mass spectrometry-based assaywas developed to facilitate the discovery of potentialchemoprevention agents that bind to human RXRr. Natu-ral and synthetic ligands for RXRr including 9-cis-retinoicacid, docosahexaenoic acid, and LG100268 could bedetected and identified in DMSO (dimethyl sulfoxide) oreven complex matrixes such as extracts of marine bacte-ria. Specific binding of ligands to RXRr was demonstratedthrough competitive binding using ultrafiltration LC-MS/MS (liquid chromatography-tandem mass spectrometry),and ligands could be ranked in order of affinity for RXRr.Therefore, ultrafiltration LC-MS/MS is suitable for thescreening of complex mixtures such as natural productextracts for the discovery of new ligands to RXRr.

The retinoic acid receptors (RARs) and the retinoid X receptors(RXRs) are two families of nuclear receptors, which are thereceptors for retinoic acids. RARs bind all trans-retinoic acid (alltrans-RA) and 9-cis-retinoic acid (9-cis-RA), whereas RXRs bindonly the 9-cis-RA stereoisomer (see structures in Figure 1).1 RXRsinclude the forms RXRR, RXRâ, and RXRγ and are obligatorycomponents of a large number of nuclear receptor heterodimersthat include the RARs, the vitamin D receptor, thyroid hormonereceptors, peroxisome proliferator-activated receptors, and manyorphan receptors.2-5 RXRs also form heterodimers with NGFIB

and NURR1, which are two members of the small nerve growthfactor-induced clone B subfamily that can interact with DNA asmonomers and homodimers.1 The interaction of receptors withRXR increases their DNA-binding efficiency, and then theseheterodimers act as ligand-dependent transcriptional regulatorsby binding to specific DNA-response elements found in thepromoter region of target genes.1 It has been reported that theRXR partner does not exhibit a significant preference for any ofthe three subtypes in most of the cases.1

Because ligand activation of RXRs have potentially pleiotropiceffects on numerous biological pathways, RXR-selective ligands(rexinoids) have potential use as therapeutic agents for thetreatment or chemoprevention of cancer and metabolic diseases.6,7

Rexinoids might elicit the beneficial pharmacological activities ofretinoic acids without toxic side effects such as teratogenicity andhypervitaminosis.1 The synthetic rexinoid LGD1069 (bexarotene,Targretin) was recently approved for treating refractory advanced-stage cutaneous T-cell lymphoma. However, adverse side effectssuch as induction of hyperglyceridemia, hypercholesterolemia,central hypothyroidism, and headache were observed.8 As animportant new class of therapeutic agents, an efficient screeningassay is needed to identify ligands of RXR. Therefore, wedeveloped a new screening assay based on ultrafiltration massspectrometry.

Electrospray mass spectrometry has been used for the directcharacterization of RXR-ligand complexes.9-11 These studiesdemonstrated the principle that ligands with high affinity for RXRmay be distinguished from compounds that bind nonspecifically

* Corresponding author. Phone: 312-996-9353. Fax: 312-996-7107. E-mail:breemen@uic.edu.

† University of Illinois College of Pharmacy.‡ Oregon State University.§ University of Hawaii at Hilo.

(1) Germain, P.; Chambon, P.; Eichele, G.; Evans, R. M.; Lazar, M. A.; Leid,M.; De Lera, A. R.; Lotan, R.; Mangelsdorf, D. J.; Gronemeyer, H. Pharmacol.Rev. 2006, 58, 760-772.

(2) Yu, V. C.; Delsert, C.; Andersen, B.; Holloway, J. M.; Devary, O. V.; Naar,A. M.; Kim, S. Y.; Boutin, J. M.; Glass, C. K.; Rosenfeld, M. G. Cell 1991,67, 1251-1266.

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(4) Zhang, X. K.; Hoffmann, B.; Tran, P. B.; Graupner, G.; Pfahl, M. Nature1992, 355, 441-446.

(5) Sporn, M. B.; Suh, N. Nat. Rev. Cancer 2002, 2, 537-543.(6) Dragnev, K. H.; Petty, W. J.; Shah, S.; Biddle, A.; Desai, N. B.; Memoli, V.;

Rigas, J. R.; Dmitrovsky, E. J. Clin. Oncol. 2005, 23, 8757-8764.(7) Shulman, A. I.; Mangelsdorf, D. J. N. Engl. J. Med. 2005, 353, 604-615.(8) Lowe, M. N.; Plosker, G. L. Am. J. Clin. Dermatol. 2000, 1, 245-250;

discussion 251-252.(9) Lengqvist, J.; Mata De Urquiza, A.; Bergman, A. C.; Willson, T. M.; Sjovall,

J.; Perlmann, T.; Griffiths, W. J. Mol. Cell. Proteomics 2004, 3, 692-703.(10) Lengqvist, J.; Alvelius, G.; Jornvall, H.; Sjovall, J.; Perlmann, T.; Griffiths,

W. J. J. Am. Soc. Mass Spectrom. 2005, 16, 1631-1640.(11) Lengqvist, J.; Mata de Urquiza, A.; Perlmann, T.; Sjovall, J.; Griffiths, W. J.

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9398 Analytical Chemistry, Vol. 79, No. 24, December 15, 2007 10.1021/ac701701k CCC: $37.00 © 2007 American Chemical SocietyPublished on Web 11/13/2007

and with low affinity. However, this approach has not beenextended to include the screening of mixtures of compounds suchas natural product extracts or combinatorial libraries for thediscovery of RXR ligands or for ranking ligands according to theiraffinity for RXR.

Ultrafiltration mass spectrometry was invented and developedfor the screening of combinatorial library mixtures and naturalproduct extracts in order to identify ligands to macromoleculartargets, such as adenosine deaminase,12,13 dihydrofolate reduc-tase,14 cyclooxygenase-2,15 serum albumin,16 and estrogen recep-tors.17 During ultrafiltration mass spectrometric screening, ligandsin a mixture are allowed to bind to a receptor such as a proteintherapeutic target, and then ultrafiltration is used to separate thereceptor-ligand complexes from unbound low-mass compounds.Finally, the ligands are released by disrupting the ligand-receptorcomplexes using organic solvent, a pH change, or other destabiliz-ing conditions and then analyzed using mass spectrometry or

liquid chromatography-mass spectrometry. Control assays arecarried out using inactive receptor or no receptor to test fornonspecific binding to the macromolecule or the ultrafiltrationmembrane. This process is summarized in Figure 2.

In this study, an ultrafiltration mass spectrometry assay wasdeveloped for the screening of natural product extracts andcombinatorial libraries for the discovery of ligands to humanRXRR. With the use of this assay, drugs or drug leads may bediscovered that might be useful for chemoprevention or cancertherapy. In addition, the utility of this assay for ranking RXRligands according to their binding affinity was investigated.

EXPERIMENTAL SECTIONChemicals and Reagents. All organic solvents were HPLC

grade or better and were purchased from Fisher (Hanover Park,IL). The RXR agonist LG100268 was purchased from CVChem(Cary, NC), and 9-cis-RA, 13-cis-retinoic acid (13-cis-RA), all trans-RA, docosahexaenoic acid (DHA), and 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (TT-NPB) (see structures in Figure 1) were purchased from Sigma-Aldrich (St. Louis, MO). The human RXRR ligand binding domainwas expressed and purified as described previously and confirmedto be a homodimer by size exclusion chromatography and massspectrometry.18 A dichloromethane extract of a culture of a

(12) van Breemen, R. B.; Huang, C. R.; Nikolic, D.; Woodbury, C. P.; Zhao, Y.Z.; Venton, D. L. Anal. Chem. 1997, 69, 2159-2164.

(13) Zhao, Y. Z.; van Breemen, R. B.; Nikolic, D.; Huang, C. R.; Woodbury, C.P.; Schilling, A.; Venton, D. L. J. Med. Chem. 1997, 40, 4006-4012.

(14) Nikolic, D.; van Breemen, R. B. Comb. Chem. High Throughput Screening1998, 1, 47-55.

(15) Nikolic, D.; Habibi-Goudarzi, S.; Corley, D. G.; Gafner, S.; Pezzuto, J. M.;van Breemen, R. B. Anal. Chem. 2000, 72, 3853-3859.

(16) Gu, C.; Nikolic, D.; Lai, J.; Xu, X.; van Breemen, R. B. Comb. Chem. HighThroughput Screening 1999, 2, 353-359.

(17) Sun, Y.; Gu, C.; Liu, X.; Liang, W.; Yao, P.; Bolton, J. L.; van Breemen, R. B.J. Am. Soc. Mass Spectrom. 2005, 16, 271-279.

(18) Yan, X.; Broderick, D.; Leid, M. E.; Schimerlik, M. I.; Deinzer, M. L.Biochemistry 2004, 43, 909-917.

Figure 1. Structures of the RXRR ligands and other test compounds used during this investigation.

Figure 2. Experimental design of ultrafiltration LC-MS/MS screening of solutions for ligands to human RXRR.

Analytical Chemistry, Vol. 79, No. 24, December 15, 2007 9399

Streptomyces species from marine sediment was used as a naturalproduct matrix for screening.19

Binding to RXRr and Ultrafiltration. In preparation forultrafiltration screening, 85 µL of binding buffer consisting of 50mM Tris-HCl (pH 7.5), 10% glycerol, 50 mM KCl, and 1 mMEDTA, 5 µL of a sample solution in dimethyl sulfoxide (DMSO),and 10 µL of RXRR (10 µM in binding buffer) were mixed andincubated for 2 h at room temperature. In some incubations,sample solutions consisted of an extract of a marine sedimentbacterial culture (4 mg/mL) reconstituted in DMSO and spikedwith ligands for RXRR or binding buffer spiked with ligands forRXRR. For the competitive binding assay, the samples consistedof equimolor mixtures (1.0 or 0.2 µM) of LG100268, 9-cis-RA, andDHA in DMSO. After incubation, each mixture was filteredthrough a Microcon (Millipore, Bedford, MA) YM-10 centrifugalfilter containing a regenerated cellulose ultrafiltration membranewith a 10 000 MW cutoff by centrifugation at 13 000g for 10 minat 4 °C. The RXRR-ligand complexes were washed three timeswith 200 µL aliquots of 30 mM ammonium acetate (pH 7.5)followed by centrifugation again at 13 000g for 15 min at 4 °C toremove the unbound compounds. For the two-filter method, thewashed RXRR-ligand solution (∼10 µL) was transferred to asecond filter where it was treated with 200 µL of 90% methanol indeionized water to disrupt the receptor-ligand complexes. Forthe one-filter method, the washed RXRR-ligand solution was nottransferred. The released ligands were then isolated from thedenatured protein by centrifugation at 13 000g for 15 min. Thesolvent in the ultrafiltrate was evaporated under vacuum, and theligands were reconstituted in 50 µL of methanol/water (80:20, v/v)containing 200 nM TTNPB as an internal standard for analysisusing liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described below. For comparison, control analyseswere carried out that were identical except for the use ofdenatured RXRR.

LC-MS/MS. Aliquots (10 µL each) of each reconstitutedultrafiltrate were analyzed using an Applied Biosystems (FosterCity, CA) API 4000 triple-quadrupole mass spectrometer equippedwith Shimadzu (Columbia, MD) LC-10A pumps and a Leap(Carrboro, NC) HTS PAL autosampler. HPLC separations werecarried out using a Thermo Quest (San Jose, CA) column (3 µm,2.1 mm × 30 mm) with a mobile phase consisting of isocratic80:20 (v/v) methanol/10 mM aqueous ammonium acetate at aflow rate of 400 µL/min. Negative ion electrospray at an ion sourcetemperature of 350 °C was used with tandem mass spectrometry(MS/MS) for the detection of each compound. During MS/MS,collision-induced dissociation was carried out followed by multiplereaction monitoring (MRM) with a dwell time of 200 ms/ion assummarized in Table 1. Data were acquired and analyzed usingAnalyst software version 1.2 (Applied Biosystems).

RESULTS AND DISCUSSIONThe RXRR ligands, LG100268, 9-cis-RA, and DHA,1 and the

structurally similar nonligands, 13-cis-RA and all trans-RA, wereused for the development of the ultrafiltration screening assay.Figure 3 shows the LC-MS/MS analysis of an ultrafiltrateobtained following the incubation of LG100268, 9-cis-RA, DHA, 13-

cis-RA, and all trans-RA with 1 µM recombinant human RXRR. Asa control for nonspecific binding, the LC-MS/MS analysis ofanother ultrafiltrate is shown in Figure 3 that was prepared in anidentical manner except for the use of denatured RXRR. In bothLC-MS/MS analyses, TTNPB was used as an internal standard.

When specific binding to RXRR occurred, the peak area of theligand was greater in the chromatogram corresponding to incuba-tion with active RXRR than in the chromatogram of sampleincubated with denatured RXRR. As shown in Figure 3, LG100268,9-cis-RA, and DHA showed enhanced peak areas relative to thecontrol chromatograms. The peaks for 9-cis-RA and DHA wereenhanced approximately 10-fold compared to the correspondingcontrol peaks representing nonspecific binding, and the signal forLG100268 was enhanced to an even greater extent. Since thepeaks corresponding to 13-cis-RA and all trans-RA could not bedistinguished from the control incubations using denatured RXRR,the ultrafiltration screening assay confirmed that these compoundswere not ligands. These results demonstrate the specificity of thisultrafiltration screening method for the detection of RXRR ligands

(19) Soria-Mercado, I. E.; Prieto-Davo, A.; Jensen, P. R.; Fenical, W. J. Nat. Prod.2005, 68, 904-910.

Table 1. Precursor/Product Ion Pairs and Parametersfor Multiple Reaction Monitoring LC-MS/MS ofCompounds Used in This Investigation

compdsprecursor

ion f (m/z)declustering

potentialentrancepotential

collisionenergy

collisioncell exitpotential

LG100268 362.2 f 317.9 -85 -10 -32 -179-cis-RA 299.2 f 255.0 -85 -10 -22 -13DHA 327.3 f 283.2 -68 -10 -15 -1713-cis-RA 299.2 f 255.0 -85 -10 -22 -13all trans-RA 299.2 f 255.0 -85 -10 -22 -13TTNPB 347.3 f 303.2 -95 -10 -26 -19

Figure 3. Ultrafiltration LC-MS/MS screening of LG100268, 9-cis-RA, DHA, 13-cis-RA, and all trans-RA for binding to RXRR. Theconcentration of RXRR and each compound was 1 µM, and theincubations were carried out for 2 h. TTNPB was added to thesamples as an internal standard immediately before analysis usingLC-MS/MS with negative ion electrospray. The control incubations(dashed lines) containing denatured RXRR were used to test fornonspecific binding and adsorption of sample to the ultrafiltrationapparatus. Enhancement of HPLC peak areas in the experimentalincubations (solid lines) indicates specific binding of ligands to RXRR.Note that the known ligands, LG100268, 9-cis-RA, and DHA, but not13-cis-RA and all trans-RA, which are not known to bind to RXRR,were shown to bind.

9400 Analytical Chemistry, Vol. 79, No. 24, December 15, 2007

such as 9-cis-RA but not its nonligand isomers 13-cis-RA and alltrans-RA.

Since RXRR ligands such as LG100268 and 9-cis-RA tend to bevery hydrophobic, the use of ultrafiltration for screening can resultin high nonspecific binding that might interfere with the detectionof specifically bound ligands. By replacing the ultrafiltration filterbefore the ligand dissociation and elution step (see Figure 2) witha clean filter uncontaminated by ligands, the level of nonspecificbinding was decreased more than 5-fold as shown in Figure 4,which compares ultrafiltration RXRR screening results for LG100268and 9-cis-RA obtained using one or two filters. By adding a secondfilter to the screening process, the specific binding (expressedas a ratio of the ligand peak area for the experiment to that of thecontrol) increased from 3.8 ( 0.4 to 26.9 ( 5.2 (N ) 3) forLG100268 and from 2.1 ( 0.4 to 10.6 ( 1.1 (N ) 3) for 9-cis-RA.

Ultrafiltration LC-MS/MS screening data may be expressedin terms of the percentage of ligand specifically bound to areceptor relative to the total amount of the ligand in the incubation.Defined by Nikolic et al.15 as an “enrichment factor”, this value isan indicator of the relative affinity of ligands for a receptor. Sunet al.17 showed that enrichment factors may be used to rankligands in order of their affinities for a receptor. In order tocalculate enrichment factors for RXRR, a quantitative LC-MS/MS assay was developed for the measurement of ligands LG100268,9-cis-RA, and DHA. The calibration curves for all three compoundswere linear (r2 > 0.99) from the limit of quantitation to 1 µM.The limits of detection (LODs) of LG100268, 9-cis-RA, and DHAwere 0.036 pg (10 pM, 10 µL injection), 0.090 pg (30 pM, 10 µLinjection), and 0.082 pg (25 pM, 10 µL injection), respectively,based on a signal-to-noise ratio of 3:1. The limits of quantitationfor these compounds were 0.108 pg (30 pM, 10 µL injection), 0.300

pg (100 pM, 10 µL injection), and 0.262 pg (80 pM, 10 µLinjection), respectively, for a signal-to-noise ratio of 10:1.

The enrichment factors for the ultrafiltration LC-MS/MSscreening assay are shown in Figure 3 and others are shown inTable 2. At test concentrations of 1 µM for RXRR and each ligand(assayed individually), the highest and lowest enrichment factorswere 14.9% ( 2.6% and 0.9% ( 0.2% for LG100268 and DHA,respectively (N ) 3). The enrichment factor for 9-cis-RA wasintermediate at 11.7% ( 1.8%. These factors are consistent withthe reported affinities of these compounds, which are 3 ( 1 nM20

for LG100268 and 11.7 nM21 for 9-cis-RA. DHA is a much loweraffinity compound compared to 9-cis-RA with an EC50 of 50-100µM.22 Compounds with no measurable affinity for RXRR, such as13-cis-RA and all trans-RA (Figure 3), have enrichment factors of0%. At a test concentration of 0.2 µM for each ligand and 1 µMfor RXRR, the enrichment factors for LG100268, 9-cis-RA, and DHAwere 41.9% ( 4.6%, 37.4% ( 7.4%, and 1.6% ( 0.7%, which are alsoconsistent with the reported affinities for these ligands. Theseresults also show that enrichment factors are dependent upon therelative concentrations of ligand and receptor.

When LG100268, 9-cis-RA, and DHA were assayed with 1 µMRXRR as equimolar mixtures of 1 µM or 0.2 µM, only LG100268and 9-cis-RA were identified as ligands. The enrichment factorsfor LG100268 and 9-cis-RA were 7.3% ( 2.5% and 5.5% ( 1.0% in 1µM mixture, 27.4% ( 5.4% and 26.1% ( 2.0% in 0.2 µM mixture,respectively. Because the amount of RXRR available for bindingwas the same as assayed individually, the higher affinity ligandsLG100268 and 9-cis-RA excluded DHA through competition forthe binding site. This experiment demonstrated that binding ofthese ligands to RXRR was specific to the active site.

To test for possible interference from common solvents usedin high-throughput screening such as DMSO or complex naturalproduct matrixes such as a bacterial culture extract, LG100268and 9-cis-RA were screened using ultrafiltration LC-MS/MS eitherin DMSO or in a matrix consisting of an extract of a marinebacterial culture (4 mg/mL). The enrichment factors for LG100268

(20) Boehm, M. F.; Zhang, L.; Zhi, L.; McClurg, M. R.; Berger, E.; Wagoner,M.; Mais, D. E.; Suto, C. M.; Davies, J. A.; Heyman, R. A. J. Med. Chem.1995, 38, 3146-3155.

(21) Heyman, R. A.; Mangelsdorf, D. J.; Dyck, J. A.; Stein, R. B.; Eichele, G.;Evans, R. M.; Thaller, C. Cell 1992, 68, 397-406.

(22) de Urquiza, A. M.; Liu, S.; Sjoberg, M.; Zetterstrom, R. H.; Griffiths, W.;Sjovall, J.; Perlmann, T. Science 2000, 290, 2140-2144.

Figure 4. Comparison of specific and nonspecific binding ofLG100268 and 9-cis-RA to recombinant human RXRR during ultra-filtration LC-MS/MS screening using the one-filter and two-filterprocedures. In the MRM chromatograms, the solid lines representincubations with active RXRR, and the dashed lines represent theuse of denatured RXRR as a control for nonspecific binding. Theconcentrations of ligand and protein were each 1 µM, and eachincubation was carried out for 2 h. (A) When the RXRR-ligandcomplexes were washed with ammonium acetate to remove unboundcompounds and then dissociated using methanol/water (90:10; v/v)using the same ultrafiltration filter, nonspecific binding to the mem-brane produced a strong signal for LG100268 and 9-cis-RA. (B)Transferring the RXRR-ligand complexes after the washing step toa clean ultrafiltration filter for dissociation resulted in much lessnonspecific binding.

Table 2. Ultrafiltration LC-MS/MS Signal EnrichmentFactors for Ligands to RXRr (1 µM)a

RXRRligand

ligand(1 µM)

ligand(0.2 µM)

mixture(three

ligands,1 µM each)

mixture(three

ligands, 0.2µM each) Kd

LG100268 14.9 ( 2.6b 41.9 ( 4.6 7.3 ( 2.5 27.4 ( 5.4 3 ( 1 nMc

9-cis-RA 11.7 ( 1.8 37.4 ( 7.4 6.2 ( 1.0 26.1 ( 2.0 11.7 nMd

DHA 0.9 ( 0.2 1.6 ( 0.7 LODe LOD lower affinitycomparedto 9-cis-RAf

a Enrichment factor ) (amount of compound specifically bound)/(total amount of compound in incubation) × 100%. b Mean ( SD (N )3). c Ref 20. d Ref 21. e The percent DHA bound to RXRR could not becalculated for the mixture because the signal was reduced to near thelimit of detection by competition with the higher affinity ligandsLG100268 and 9-cis-RA. f Ref 22.

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in binding buffer, DMSO, and the bacterial culture extract were13.1%, 14.6%, and 16.6%, respectively; and for 9-cis-RA, the enrich-ment factors were 10.9%, 11.8%, and 9.0%, respectively. Theseresults indicate that both LG100268 and 9-cis-RA could be detectedas ligands in the presence of DMSO or an extract of a bacterialculture.

CONCLUSIONSAn ultrafiltration LC-MS/MS screening assay has been

developed for the discovery of ligands to human RXRR. Themethod was optimized to minimize potential interference due tononspecific binding of hydrophobic compounds to the ultrafiltra-tion membrane. The use of DMSO to dissolve samples, which iscommon practice for combinatorial libraries used for high-throughput screening, did not interfere with the assay. Becauseno interference was observed from an extract of a bacterial cellculture, this ultrafiltration method should be suitable for screening

samples in complex matrixes such as natural product extracts.Finally, this assay shows high sensitivity and only requires 100pmol of RXRR and ligand per assay. Overall, ultrafiltration LC-MS/MS is a powerful new method for the discovery andcharacterization of RXRR ligands.

ACKNOWLEDGMENTThe work was supported by Grant 5 P01 CA48112 from the

National Cancer Institute. We thank Drs. Michael B. Sporn,Guowen Liu, and Dejan Nikolic for helpful suggestions anddiscussions.

Received for review August 10, 2007. Accepted October 1,2007.

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