A Nonsteroidal Glucocorticoid Receptor Antagonist

JEFFREY N. MINER, CURTIS TYREE, JUNLIAN HU, ELAINE BERGER, KEITH MARSCHKE,MASAKI NAKANE, MICHAEL J. COGHLAN, DAVE CLEMM, BEN LANE, AND JON ROSEN

Department of Molecular and Cell Biology (J.N.M., J.H., D.C., J.R.), New Leads Discovery (C.T., E.B.,K.M.), Ligand Pharmaceuticals, Inc., San Diego, California 92121; and Abbott Laboratories (M.N.,M.J.C., B.L.) D-4NB J35 Pharmaceutical Discovery, Abbott Park, Illinois 60064-3535

Selective intracellular receptor antagonists areused clinically to ameliorate hormone-dependentdisease states. Patients with Cushing’s syndromehave high levels of the glucocorticoid, cortisol, andsuffer significant consequences from this overex-posure. High levels of this hormone are also impli-cated in exacerbating diabetes and the stress re-sponse. Selectively inhibiting this hormone mayhave clinical benefit in these disease states. To thisend, we have identified the first selective, nonste-roidal glucocorticoid receptor (GR) antagonist.This compound is characterized by a tri-aryl meth-ane core chemical structure. This GR-specific an-

tagonist binds with nanomolar affinity to the GRand has no detectable binding affinity for the highlyrelated receptors for mineralocorticoids, andro-gens, estrogens, and progestins. We demonstratethat this antagonist inhibits glucocorticoid-medi-ated transcriptional regulation. This compoundbinds competitively with steroids, likely occupyinga similar site within the ligand-binding domain.Once bound, however, the compound fails to in-duce critical conformational changes in the recep-tor necessary for agonist activity. (Molecular Endo-crinology 17: 117–127, 2003)

THE INTRACELLULAR RECEPTOR family is a largegroup of transcription factors defined by homol-

ogy within their DNA-binding domains. Modulation ofintracellular receptor activity in vitro and in vivo usingsynthetic ligands has yielded remarkable insights intothe molecular mechanisms of receptor function (1, 2)and enormous benefit in disease treatment (3–7). Spe-cific chemical structural classes that bind with highaffinity to intracellular receptors and either mimic (ago-nists) or suppress (antagonists) the activity of the en-dogenous ligand have been identified (8). Ligandsused clinically target the receptors for estrogen (ER)(9), progesterone (PR) (10), glucocorticoid (GR) (11–13), androgen (AR) (10), and mineralocorticoid (MR)(14). Typically, these ligands are steroidal, consistingof the classic tetracyclic structure with various sub-stituents decorating this core. Nonsteroidal com-pounds that interact with members of the steroid re-ceptor subclass have been identified. Nonsteroidalcompounds have been derived that have primarily an-tagonist properties; these include the partial ER an-tagonists, tamoxifen and raloxifene-related com-pounds (15, 16), and the AR antagonists, casodex andflutamide (17–19), as well as several more recentlyidentified AR antagonists (20, 21). Additionally, selec-tive nonsteroidal modulators for the androgen recep-tor have been identified, which also act as partialagonists and may represent a new class of agonist

with a beneficial therapeutic profile (22–26). Similarly,the progesterone receptor ligands have been devel-oped with both antagonist and partial agonist activity(27, 28). However, functional nonsteroidal antagoniststhat interact in cells with the remaining steroid recep-tors, GR or MR, have not yet been identified. Severalnonsteroidal compounds have been identified that ap-pear to bind directly to the GR, including 5,5-diphe-nylhydantoin (29) and �-Lapachone (30), although it isnot yet clear whether these compounds bind the re-ceptor in cells.

Selective antagonists of GRs could be useful intreating hypercortisolemia associated with Cushing’ssyndrome and other conditions in which the endoge-nous GR is hyperactivated either through higher glu-cocorticoid levels or increased receptor sensitivity (5).Other possible uses include a reduction of the immu-nosuppression associated with ongoing HIV infection,depression (31), and other stress-associated phenom-ena (32–34). Finally, a selective GR antagonist may beuseful in treating diabetic patients. Glucocorticoidsappear to play a pathological role in high serum glu-cose levels in diabetic patients by up-regulating therate-limiting enzyme in gluconeogenesis, phospho-enol pyruvate carboxykinase. Regardless of their po-tential for therapeutic use, selective antagonists willlikely prove useful in furthering our understanding ofthe GR signaling pathway itself.

During the course of screening compound librariesfor GR modulators, we discovered an antagonist, des-ignated “AL082D06” (D06) that bound specifically toGR with nanomolar affinity. This antagonist is unlikethe other frequently used steroidal antagonists for GR,RU-38486 (RU-486) and ZK-98299 (ZK-299), in that it

Abbreviations: AR, Androgen receptor; Dex, dexametha-sone; ER, estrogen receptor; GR, glucocorticoid receptor;GRE, glucocorticoid response element; MMTV, mouse mam-mary tumor virus; MMTV:Luc, MMTV promoter driving a lu-ciferase reporter; MR, mineralocorticoid receptor; PR, pro-gesterone receptor; TAT, tyrosine amino transferase.

0888-8809/03/$15.00/0 Molecular Endocrinology 17(1):117–127Printed in U.S.A. Copyright © 2003 by The Endocrine Society

doi: 10.1210/me.2002-0010

117

has no measurable binding affinity for the progester-one receptor. As has been described previously, thethree-dimensional structure of the ligand defines notonly its affinity for the receptor, but also the confor-mation of that receptor once it has associated withligand (35–37). This new compound appears to binddirectly to receptor without inducing the same confor-mational changes associated with steroidal ligands.These ligands prevent the occurrence of some of theearliest steps in receptor activation. We report herethe molecular and cellular characterization of thisantagonist.

RESULTS

We conducted a high-throughput screen of a definedcompound library using a GR-based assay. Thisscreen revealed a nonsteroidal compound that exhib-ited strong antagonist activity against GR. We under-took the molecular and cellular characterization of thisligand.

Transient transfection of GR into CV1 cells in thepresence of the synthetic glucocorticoid, dexametha-sone (Dex), and a glucocorticoid-responsive mousemammary tumor virus (MMTV) promoter driving a lu-ciferase reporter (MMTV:Luc) plasmid results in anincrease in luciferase activity (�2000-fold) (Fig. 1). Thisactivity can be inhibited with known antagonists likeRU-486 and ZK-299 (Fig. 1), which compete with Dexfor the ligand-binding pocket of the receptor but fail, inmost cases, to induce an active conformation (1, 35,36, 38). Addition of D06 causes a dose-dependentdecrease in transcriptional activation from the MMTV:Luc reporter stimulated with half-maximal DEX con-centrations. D06 acts to antagonize reporter activityusing several glucocorticoid-responsive promoter-reporter systems including the 3-kb tyrosine aminotransferase (TAT) promoter and less complex promot-ers comprised of isolated glucocorticoid response el-ement (GRE) sequences (data not shown) (37).

This compound, bis(4-N,N-dimethylaminophenyl)(2-chloro-5-nitrophenyl) methane, is related to a wellknown series of dyes such as Malachite green, a fun-gicide used in aquariums (Fig. 2). This general class ofmolecule has been described previously as ligands forthe estrogen receptor (39).

We used a competitive binding assay to determinethe affinity of D06 for GR as well as the other steroidreceptors. D06 competes with 3H-Dex for baculovirus-expressed GR with nanomolar affinity. Other intracel-lular receptors (AR, ER, PR, and MR) have no affinityfor D06 in a similarly structured binding assay with theappropriate receptor and tritiated ligand (�2500 nM).This selectivity for binding is in contrast to the signif-icant PR cross-reactivity of other known GR antago-nists, RU-486 and ZK-299 (Fig. 2).

We tested the functional specificity of D06 by as-sessing its agonist and antagonist activity against abattery of related intracellular receptors (Fig. 3). Theseresults confirm the in vitro binding data and clearlyindicate that D06 has no activation efficacy on theprogesterone, androgen, mineralocorticoid, retinoid,glucocorticoid, or estrogen receptors (Fig. 3A). Fur-thermore, these data indicate that D06 is very effica-cious at antagonizing GR activity but exhibits muchweaker efficacy when tested against the other steroidreceptors in contrast to the reference antagonistsused as controls (Fig. 3B).

D06 was tested for antagonist effects in cell-basedmodels of transcriptional activation. We demonstratethat D06 can antagonize steroid-mediated induction ofglutamine synthetase RNA in MG63 cells (data notshown) and TAT enzyme in human skin fibroblasts(Fig. 4A). We also tested D06 for effects on genesnormally repressed by glucocorticoids. We measuredrepression using an E-Selectin promoter:luciferaseconstruct. This plasmid contains the E-Selectin genepromoter upstream from the luciferase reporter. TNFand IL-1 strongly induce expression from this plasmid,and glucocorticoids are effective repressors of thisinduction (Fig. 4B), In contrast, D06 is unable to re-

Fig. 1. D06 Antagonist Activity on GRThis shows the results of a cotransfection experiment with a concentration response of D06 (0.01 nM to 10 �M in log steps).

The MMTV:Luc reporter and hGR expression vector are cotransfected into CV-1 cells and compound is added. Curves for Dexand D06 are shown in agonist mode (F, Dex; f, D06). In addition, D06 and ZK-299 are tested in antagonist mode (Œ, ZK-299 andF, D06) in the presence of an EC50 of Dex (3 � 10�10 M). Symbols for the antagonists experiment are as follows (Œ, ZK299; andF, D06). The experimental data for the experiment shown are the average of three independent replicates. This is a representativeexperiment from more than three similar experiments with the same result.

118 Mol Endocrinol, January 2003, 17(1):117–127 Miner et al. • Nonsteroidal GR Antagonist

press transcription when added alone at any concen-tration (black squares). However, D06 is able to fullyreverse the repression mediated by Dex at the E-selectin promoter. D06 is also capable of partially in-hibiting Dex-mediated repression of IL-6 and collage-nase protein production from untransfected humanskin fibroblast cells using endogenous receptors (datanot shown). In summary, D06 can act to inhibit bothtranscriptional activation and repression by receptor ina variety of cell types on a variety of genes.

Antagonists like RU-486 can cause receptor to bindto GRE sequences and exhibit agonist activity whentested in certain cells at certain promoters (37). Incontrast, ZK-299-bound GR exhibits no detectableDNA binding activity in vitro and does not activatetranscription under any circumstances although this iscontroversial (40). We tested D06 for its effect on DNAbinding and determined that much like ZK-299, D06did not induce DNA binding by GR in vitro (Fig. 5A) andwill inhibit both Dex- and RU-486-induced GR DNAbinding activity (Fig. 5A and data not shown).

To confirm the in vitro DNA binding results in cells,we used an in vivo competition assay to monitor DNAbinding activity of antagonist-bound GR (40). The as-say utilizes coexpression of a constitutively active C-terminal deletion of GR with wild-type GR. The con-stitutively active receptor binds to and activatestranscription from the MMTV reporter. The wild-typeprotein will compete with this activity if it is bound toan antagonist like RU-486 that allows DNA binding,but not transcriptional activation. The results of thistype of assay with D06 reveal that unlike either ZK-299or RU-486, D06-bound GR does not compete with theconstitutively active GR (Fig. 5B). This result demon-

strates a clear difference between the known GR an-tagonists and this nonsteroidal compound.

The structure and function of the receptor are in partdetermined by the structure of the bound ligand (41,42). Agonists as well as antagonists such as RU-486induce a particular structural conformation in the re-ceptor that can be detected by limited protease diges-tion (35, 36, 43, 44). In our hands, compounds such asZK-299 induce a different, more protease-sensitivestructure that is similar to the unbound state (37, 45).As shown in Fig. 6A, D06 produces a pattern that, likeZK-299, is highly sensitive to protease digestion. Weare convinced that D06 is bound to the receptor underthese conditions because much like ZK299, D06 in-hibits the formation of a Dex-protected band at thesame concentrations (Fig. 6B).

Early in the GR signal transduction pathway, thereceptor translocates to the nucleus (46). We com-pared the nuclear translocation activity of RU-486 andZK-299 to D06 using an immunofluorescence assay intransfected COS cells (40). In agreement with previ-ously published data for progesterone receptor (40)and for GR (47), Fig. 7 shows that both RU-486 andZK-299 induced significant nuclear translocation inthis immunofluoresence assay. In contrast, D06 exhib-ited only weak nuclear translocation, even whenadded at 10 �M.

DISCUSSION

A nonsteroidal GR ligand, D06, has been identified.We have tested this compound in a variety of assays

Fig. 2. Tri-aryl Methane, D06, Exhibits Selective GR Binding ActivityThe structure of D06 is shown [bis(4-N,N-dimethylaminophenyl)(2-chloro-5-nitrophenyl) methane] together with the binding

data for D06 covering a number of related intracellular receptors (GR, PR, AR, MR, ER). Reference steroidal agonists (Dex,hydrocortisone, and prednisolone) and antagonists (RU-486 and ZK-299) are shown for comparison. The data are shown as Ki

(nM). NT, Not tested. K, 1000.

Miner et al. • Nonsteroidal GR Antagonist Mol Endocrinol, January 2003, 17(1):117–127 119

to assess its agonist and antagonist activities. Thiscompound is characterized by a tri-aryl methane corechemical structure and has strong (�250 nM) affinityfor the GR and binds competitively with other knownGR ligands. The most likely explanation for our com-petitive binding profiles is that D06 occupies the samehydrophobic pocket in the ligand-binding domain assteroids, although it is conceivable that binding couldoccur elsewhere on the receptor and this in turn altersthe steroidal ligands’ affinity. Once bound, the com-pound is a very efficacious antagonist of receptor ac-tivity. It is capable of antagonizing both gluco-corticoid-mediated transcriptional activation andrepression in multiple cellular contexts. D06 is an an-tagonist in these assays with a potency of 200 nM. Thiscompound does not have any effect in these assays in

the absence of steroid, classifying it as a pureantagonist.

One of the problems with current attempts at glu-cocorticoid antagonist therapy is cross-reactivity withother steroid receptors. The currently available glu-cocorticoid antagonists mifepristone (RU-486) andonapristone (ZK-299) are also potent progesterone an-tagonists, making their clinical use problematic. Wehave demonstrated that D06 is selective for GR, withno measurable affinity for, and little or no activity on,the progesterone, mineralocorticoid, androgen, estro-gen, and retinoid receptors (Fig. 2 and data notshown). Thus, this antagonist appears to be useful forselectively inhibiting GR in different contexts.

The mechanism of antagonist ligands has been ex-tensively reviewed (11, 48–50). Current literature sug-

Fig. 3. D06 Activity Is GR SelectiveA, Agonist assay: This graph represents the data from a 96-well cotransfection experiment into CV1 cells with the specified

transfected receptors and the MMTV:Luc (or MMTV:3�ERE:Luc for ER) promoter in the presence of D06 or appropriate referencesteroidal agonists [GR-Dex (D, 100 nM); PR-progesterone (P, 100 nM], MR-aldosterone [100 nM (A); ER-estrogen (E, 100 nM);AR-DHT (Dht,100 nM)]. The results are shown as a percent agonist activity. This experiment has been repeated more than threetimes with similar results. The data shown are an average of three replicates within a single experiment. B, Antagonist: Identicalto panel A except that the assays are done in the presence of an EC50 of each reference steroidal agonist [GR-Dex (3 � 10�10

M); PR-progesterone (1 � 10�9); MR-aldosterone (1 � 10�9); ER-estrogen (1 � 10�9); AR-DHT (3 � 10�10 M)]. Referenceantagonists are shown to validate the assays [GR-RU486 (1 �M), PR-RU-486 (1 �M), MR-RU28318 (RU318) (1 �M), ER-Tamoxifen(Tam.) (1 �M), AR-flutamide (Flut.) (1 �M)]. D06 and the reference antagonists are used at two different concentrations designatedby 7 for D06 (1 �M and 10 �M). The results are expressed such that maximal antagonist activity by the reference antagonist isset to 100%. These experiments were repeated three times with similar results.

120 Mol Endocrinol, January 2003, 17(1):117–127 Miner et al. • Nonsteroidal GR Antagonist

Fig. 4. Antagonism of GR-Mediated Activation and RepressionA, TAT antagonist activity of D06. TAT activity was measured as described (41) in human skin fibroblasts in the presence of

vehicle (F), an EC50 of Dex (F) (10 nM), or hydrocortisone (F) (HC 100 nM). Antagonists ZK299, RU-486, or D06 were added inincreasing concentration. These experiments were repeated three times each with similar results and a representative experimentis shown. B, D06 inhibits DEX-mediated repression of E-selectin promoter activity. Luciferase activity is shown upon inductionwith TNF and IL-1� (open vertical bar). D06 in dose response in the absence of DEX is shown in the black squares. Thisdemonstrates no effect of D06 on TNF-IL-1-induced luciferase activity. In the presence of half-maximal concentrations of DEXthe luciferase activity is suppressed. Addition of D06 relieves repression by DEX (open squares). This is a representativeexperiment showing the average of three replicates within the experiment. This experiment has been repeated in this format atleast three times.

Miner et al. • Nonsteroidal GR Antagonist Mol Endocrinol, January 2003, 17(1):117–127 121

gests that they induce a conformation in the receptorthat differs from that induced by agonists. Thesechanges affect the interaction of the receptor withcritical components of the cell, be it other proteins orDNA. Many antagonists do, in fact, exhibit some ag-onist activity when bound to the receptor. Tamoxifen,an antagonist in breast tissue, has partial agonist ac-tivity in the uterus that may increase the risk of signif-icant proliferative side effects.

We established the point in the glucocorticoid signaltransduction pathway at which D06 inhibits GR func-tion. The known GR/PR antagonist RU-486 inducesDNA binding activity of the receptor. In contrast, insimple gel shift assays, ZK-299 and D06 antagonizeDNA binding by GR (Fig. 5A). We also examined theDNA binding activity in a cell-based assay used tomeasure GR DNA binding activity; (37). We have pre-viously shown that under certain conditions, even ZK-299-receptor complexes can occupy GREs in vivo(37). Using similar in vivo competition assays, D06 failsto induce the formation of receptor-GRE complexes,whereas ZK-299 and RU-486 are fully capable of in-hibiting activation from a constitutively activated re-porter (Fig. 5B). We interpret the results from the cel-lular competition assay with the view that this assaymeasures competition at the level of DNA, although itis possible that the competition we are seeing is com-petition for some type of limiting coactivator requiredfor the constitutive activator to function. At present, wecannot distinguish between these models. This ques-tion is especially relevant for ZK-299 because it fails toinduce DNA binding in vitro, but competes nicely invivo. The interpretation of the D06 results is morestraightforward because it fails to induce DNA bindingand is extremely weak in the in vivo competition assay.In contrast to other known antagonists, D06 has littleor no agonist activity in any assay we have devised;therefore, we consider it a pure, type 1 GR antagonistas defined by McDonnell et al. (51, 52).

To roughly assess the conformation of the receptorwhen bound to D06, we used a protease digestionassay (Fig. 6). This assay demonstrated that D06 andZK-299 generate a conformation different from bothpartial antagonists (RU-486) and full agonists (Dex)(37, 45, 53). Furthermore, this conformation is moresensitive to proteases than agonist-like conforma-tions. Thus the receptor is in an antagonist conforma-tion when bound to D06. After binding ligand andchanging conformation, the receptor undergoes nu-clear translocation. We tested the impact of D06 onthis process. Confirming the results of others (54, 55),we show that the known steroidal agonists and antag-onists all induce significant nuclear translocation un-der our assay conditions. This result is controversialbecause the nuclear translocation activity of RU-486may be cell type specific (56, 57). In our assay, incontrast to RU-486 and the other steroids, D06 exhib-ited reduced nuclear translocation (Fig. 7). These re-sults indicate that at least part of the antagonist ac-tivity of this class occurs by reducing the amount ofGR transported to the nucleus (Fig. 7). Furthermore,

Fig. 5. D06 Is an Antagonist to GR DNA Binding ActivityA, In vitro DNA binding activity was measured using an

EMSA. PA is radiolabeled GRE probe alone. In vitro trans-lated GR was incubated with either solvent (�), Dex (10 nM),or D06 (1 and 10 �M). This assay produces strong hormone-dependent DNA binding by GR in response to glucocorti-coids, but not by D06. The antagonist experiment is similarexcept that GR was incubated with 10 nM Dex and increasingconcentrations of D06 (0.01, 0.1, 1, and 10 �M) or ZK-299(0.01 and 1 �M) as a positive antagonist control. B, Cellularcompetition assay of D06. This assay uses cotransfection ofthe MMTV:Luc reporter, together with a constitutively activetruncation of GR (I550) and the wild-type receptor (RSVhGR).The first lane shows the basal level of transcription from theMMTV promoter without transfected constitutive activator.The second lane shows the signal generated in response tothe constitutive activator in the presence of the solvent eth-anol. Known antagonists such as RU-486 (10 �M) and ZK-299(10 �M) will inhibit the constitutive activator by binding towild-type GR and inducing competitive DNA binding signifi-cantly (P � 0.05). In contrast, shown in the last lane, D06 (10�M) exhibits only weak, inconsistent inhibition that is notsignificantly different from vehicle plus constitutive GR (P �0.05) (Fisher’s least significant differences). This experimenthas been conducted four times with the same conclusion,although some differences between absolute levels of lucif-erase produced by the constitutive GR were found.

122 Mol Endocrinol, January 2003, 17(1):117–127 Miner et al. • Nonsteroidal GR Antagonist

the portion of GR bound to the D06 that does reachthe nucleus is unable to bind to DNA (Fig. 5, A and B).

In summary, we have characterized a compound,D06, that is a full antagonist for GR, but not for othersteroid receptors. D06 partially blocks GR transloca-tion to the nucleus, and completely blocks DNA bind-ing by the receptor. This molecule may be a usefultemplate for the development of clinically useful oralantagonists of the GR.

The search for selective antagonists of GR activityhas been driven by both clinical and research needs.The selective inhibition of GR action may allow thedifferentiation of its activities from those of MR and PRwhen studying the physiology and cellular biology ofglucocorticoid action. In addition, compounds of thistype may provide better treatment for patients with avariety of cortisol-related endocrine disorders.

MATERIALS AND METHODS

Cellular Assays

Transfection. CV-1 cells (African green monkey kidneyfibroblasts, American Type Culture Collection, Manassas, VA)

were grown in DMEM (BioWhittaker, Inc., Walkersville, MD)containing 10% (vol/vol) fetal calf serum (HyClone Laborato-ries, Inc., Logan, UT), 2 mM L-glutamine, and 55 �g/ml gen-tamycin. Cells were transiently transfected using the calciumphosphate coprecipitation method (58). Unless otherwisenoted, 5 �g/ml of a human GR-expression plasmid vector(RSV:hGR), 5 �g/ml MMTV:Luc reporter plasmid, 2.5 �g/mlof pRSV-�-Gal (�-galactosidase) as a control for transfectionefficiency, and 7.5 �g of filler DNA (pGEM4) at a final con-centration of 20 �g/ml were precipitated and then added tothe cells. The medium was changed 16 h later to contain 5%charcoal-stripped fetal calf serum and steroid ligands with orwithout test compounds (10 �M) for 24 h. Cells were thenlysed and assayed as described previously (43, 59). TheE-selectin transfection assay is similar except that 5 �g/mlE-sel/luc reporter plasmid was added instead of MMTV:Luc.The medium was changed 16 h after transfection to contain10% charcoal-stripped fetal calf serum, TNF� (10 ng/ml),IL-1� (1 ng/ml), and test compounds (10 nM to 10 �M) with orwithout 0.32 nM Dex for 24 h. Cells were then lysed andassayed as described above.TAT Assay. TAT activity in H4IIE cells was measured asdescribed previously (60). Preconfluent H4IIE cells in 96-wellplates were incubated for 24 h with compound, washed withPBS, and lysed. Extracts were subjected to enzymatic assayas described (60).

IL-6 was measured in confluent human skin fibroblasts ininduction media (1.75% BSA/antibiotics/DMEM) after incu-bation with induction media for 4–6 h. Media were changed

Fig. 6. Protease Digestion Assay of D06-Bound GRA, The conformation induced by D06 was assayed by incubation of radiolabeled GR together with increasing concentrations

of trypsin in the presence or absence of solvent, the reference agonist (Dex 1 �M), reference antagonists ZK299 (10 �M), RU-486(10 �M), and with D06 (10 �M). Protected bands correspond to segments of the ligand-binding domain that are more resistant toprotease in the presence of hormone. B, Protease digestion assay in antagonist mode. This assay uses 10 nM Dex to producea small amount of the protected species denoted by an arrow. Addition of either antagonist D06 or ZK299 effectively competeswith Dex for the receptor, which creates a less protease-resistant structure resulting in the disappearance of the band.

Miner et al. • Nonsteroidal GR Antagonist Mol Endocrinol, January 2003, 17(1):117–127 123

and cells incubated a further 1 h in induction media; com-pound. IL-1� was then added to a final concentration of 1ng/ml in induction media (Roche Molecular Biochemicals,Indianapolis, IN), and cells were cultured for 24 h. Media wereremoved and added to Maxisorp Plate (Nunc) with captureantibody (IL-6-monoclonal mouse antihuman IL-6)-coatedwells (M-620, Endogen, Inc., Boston, MA) and incubated atroom temperature (RT) overnight. Plate was washed twice inPBS, blocked with 4% BSA/PBS, and incubated 1 h at RT.Secondary antibody-biotinylated monoclonal antihuman IL-6(M-621-B, Endogen, Inc.), 500 �g/ml in 4% BSA/PBS, wasadded and incubated for 2 h at RT, and washed three timesin PBS. A 1:5000 diluted ExtrAvidin-horseradish peroxidasesolution (E-2886, Sigma, St. Louis, MO) in 4% BSA/PBS wasadded and incubated for 30 min at RT. Plates were washedthree times in PBS, and substrate solution (One hundredmicroliters of 3,3�,5,5� tetramethyl benzidine-hydrogen per-oxide (Sigma) was added and incubated 15 min at RT. Re-action was stopped with 50 �l per well of 2 N H2SO4 and ODwas read at 450 nm/540 nm. Collagenase was measured inconfluent human skin fibroblasts induced in 1.75% BSA-DMEM with compounds for 1 h. IL-1� was added (RocheMolecular Biochemicals) in induction medium (final 1 ng/ml)and the cells were cultured for 24 h.Collagenase Assay. Culture supernatants were added to0.1% BSA/PBS and incubated for 2 h at RT; after washing,polyclonal rabbit antihuman MMP-1 in assay buffer wasadded and incubated for 2 h at RT. After washing, horserad-ish peroxidase-donkey antirabbit Ig in 0.1% BSA/0.1%Tween 20/PBS was added and incubated for 1 h at RT. Onehundred microliters of 3,3�,5,5� tetramethyl benzidine-hydro-gen peroxide were added and incubated for approximately5–30 min at RT, after which 100 �l per well of stop solution (1N H2SO4) were added and the OD read at 450/540 nm.

Plasmids

The constitutive activator I550, pRSV:hGRnx, was obtainedfrom Ron Evans (Salk Institute, La Jolla, CA) (61). T7hGRnxgwas constructed by first inserting the Glu-Glu tag epitopesequence (62, 63) into pRSVhGRnx at the KpnI and SalI sites;hGRnxg was then inserted into the pT7-link expression vec-tor at the NcoI and BamHI sites (61, 64). MMTV:Luc wasobtained from Ron Evans (Salk Institute, La Jolla, CA). MMTV:ERE3x:Luc contains three tandem estrogen response ele-

ments cloned into a version of the MMTV promoter in whichthe GREs have been deleted (59).

A reporter construct containing 600 bp of the E-selectinpromoter region fused to the luciferase gene (E-sel/luc) wasused in the E-selectin repression assays.

Protease Digestion Assay

The protease digestion was performed essentially as de-scribed by Allan and colleagues (35, 36, 43) with minor mod-ifications. The plasmid pGR107 containing the GR-wt cDNAwas used to produce 35S-radiolabeled GR using the TNTsystem (Promega Corp., Madison, WI). After the translationreaction, an aliquot (25 �l) of the lysate was incubated for 20min at room temperature in the presence or absence of testcompound at a final concentration of 1 �M. Aliquots (5 �l) ofligand-treated receptor mixture were subsequently incubatedfor 10 min with 0.6 �l of a trypsin solution (WorthingtonBiochemical Corp., Freehold, NJ) yielding a final enzymeconcentration of 5, 10, 25, and 50 �g/ml. After terminationand electrophoresis, the gels were fixed with a 30% (vol/vol)methanol, 10% (vol/vol) acetic acid solution for 30 min, andthen immersed in Amplify (Amersham Pharmacia Biotech,Arlington Heights, IL) for 30 min.

EMSAs

Human GR was prepared by in vitro translation using the T7expression vector pT7hGRnxg in a TNT T7-coupled reticulo-cyte lysate system (Promega Corp.). Test compounds wereadded at the beginning of the translation reaction at theindicated concentration. The specific probe is based on apalindromic GRE and was formed by annealing oligonucleo-tides with the sequences 5�-TCGACAGAACATCATGTTCT-GAGCTAC-3� and 5�-TCGAGTAGCTCAGAACATGATGT-TCTG-3�. The annealed oligonucleotide was labeled by fillingin the overhanging ends with the Klenow fragment of DNApolymerase in the presence of [�32P]dATP and dGTP. Bind-ing reactions were performed as described (65). Reactionswere incubated on ice for 5–10 min and then resolved on 4%polyacrylamide gels containing 0.25� TBE [1� TBE is 89 mM

Tris borate, 1 mM EDTA (pH 8.0)] at 4 C and 20 V/cm, whichwere then dried and autoradiographed.

Fig. 7. Nuclear Translocation Induced by HormoneCos cells transfected with a Glu-tagged GR expression vector (RSVhGRnxg) were monitored for receptor localization by

immunofluoresence using anti-Glu antibodies in response to various treatments. Cells were assessed for receptor localization bya blinded analysis; a minimum of 60 cells were analyzed per experiment. Comparisons were made between a nuclear DNA stainand the anti-GR immunofluoresence to ensure proper localization of the nuclei and cytoplasm. Cells were scored 0–3 (0 �cytoplasmic entirely; 1� C � N; 2 � C � N; 3 � nuclear entirely). The scores of all the cells counted for a given treatment wereaveraged, and the SEM was calculated. The results were graphically expressed as a percentage of nuclear translocation where100% is complete translocation (a perfect score of 3). RU-486 and ZK-299 are used as antagonist controls. All compounds wereused at saturating concentrations (D06, 10 �M; Dex, 1 �M; RU-486, 1 �M; ZK299 10 �M).

124 Mol Endocrinol, January 2003, 17(1):117–127 Miner et al. • Nonsteroidal GR Antagonist

Competitive Binding Assay

Growth and purification of recombinant hGR baculovirus fol-lowed the protocol outlined by Summers and Smith (66).The extract and binding assay buffer consisted of 25 mM

sodium phosphate, 10 mM potassium fluoride, 10 mM sodiummolybdate, 10% glycerol, 1.5 mM EDTA, 2 mM dithiothreitol,2 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), and 1 mM phenylmethylsulfonyl fluoride(pH 7.4), at room temperature. Intracellular receptors pro-duced in this fashion exhibit reproducible interaction withknown ligands at the published affinity. These preparationswere subjected to extensive quality control experiments be-fore the assays, covering receptor response, specificity, size,and reference ligand affinity. Receptor assays were per-formed with a final volume of 250 �l containing from 50–75�g of extract protein, plus 1–2 nM [3H]Dex at 84 Ci/mmol andvarying concentrations of competing ligand (0 to 10�5 M).Assays were set up using a 96-well minitube system, andincubations were carried out at 4 C for 18 h. Equilibrium underthese conditions of buffer and temperature was achieved by6–8 h. Nonspecific binding was defined as that binding re-maining in the presence of 1000 nM unlabeled Dex. At the endof the incubation period, 200 �l of 6.25% hydroxyapatitewere added in wash buffer (binding buffer in the absence ofdithiothreitol and phenylmethylsulfonyl fluoride). Specificligand binding to receptor was determined by a hydroxyap-atite-binding assay according to the protocol of Weckslerand Norman (67). Hydroxyapatite absorbs the receptor-ligand complex, allowing for the separation of bound fromfree radiolabeled ligand. The mixture was vortexed and incu-bated for 10 min at 4 C and centrifuged, and the supernatantwas removed. The hydroxyapatite pellet was washed twotimes in wash buffer. The amount of receptor-ligand complexwas determined by liquid scintillation counting of the hy-droxyapatite pellet after the addition of 0.5 mM EcoScint Ascintillation cocktail from National Diagnostics (Atlanta, GA).

After correcting for nonspecific binding, IC50 values weredetermined. The IC50 value is defined as the concentration ofcompeting ligand required to reduce specific binding by50%; the IC50 values were determined graphically from alog-logit plot of the data. Kd values for the analogs werecalculated by application of the Cheng-Prussof equation (68,69). Steroid standards are included in each assay, and re-sulting Kd values are determined by use of a modified Cheng-Prussoff equation (49, 50).

MR, AR, PR, and ER� expression in the baculovirus sys-tem and binding assays was conducted similarly except thatlabeled ligands were aldosterone [1–2 nM 3H-aldosteronefrom Amersham Pharmacia Biotech (TRK 434), specific ac-tivity 60 Ci/mmol], DHT (1–2 nM 3H-DHT at 130 Ci/mmol),progesterone [2–3 nM 3H -progesterone (Sigma, 93 Ci/mmol],and estradiol [2–3 nM 3H-estradiol (NEN Life Science Prod-ucts), 114 Ci/mmol], respectively. Each binding assay point isdone in duplicate, and each full experiment is repeated threeor more times.

Nuclear Translocation

Cos cells were transfected as above, and the immunofluores-ence assay was done as described (70). Glu tag antibody wasused to detect GLU-tagged receptor (BABCO, Berkeley, CA).The results were quantified by a blinded analysis of views ofat least 60 cells defined as follows: N, entirely nuclear (3points); C, entirely cytoplasmic (0 points); N � C, 2 points;and C � N, 1 point. Nuclei were localized by Hoechst stain33342, which binds to DNA.

Acknowledgments

We thank Emily Guido for her expert technical assistance.We appreciate the plasmids and helpful discussions provided

by Marc Elgort, Ron Evans, Keith Yamamoto, and DavidPearce.

Received January 8, 2002. Accepted September 20, 2002.Address all correspondence and requests for reprints to:

Jeffrey N. Miner, Ligand Pharmaceuticals, Molecular and Cel-lular Biology, 10275 Science Center Drive, San Diego, Cali-fornia 92121. E-mail: jminer@ligand.com.

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