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Flavone and Isoflavone Phytoestrogens Are Agonistsof Estrogen-Related Receptors

Masatomo Suetsugi,1 Leila Su,2 Kimberly Karlsberg,2 Yate-Ching Yuan,2 and Shiuan Chen1

1Department of Surgical Research and 2Division of Information Sciences, Beckman Research Institute of theCity of Hope, Duarte, CA

AbstractWhile estrogen-related receptors (ERRA, ERRB, and

ERR;) share a high amino acid sequence homology with

estrogen receptors (ERs), estrogens are not ligands of

ERRs. Structure-function studies from this and other

laboratories have revealed that ERRs have small

ligand-binding pockets and have provided evidence

to show that these receptors can activate gene

transcription in a constitutive manner. To address the

question as to whether there is any agonist for ERRs,

our laboratory recently performed virtual ligand

screening on ERRA that predicted flavone and

isoflavone phytoestrogens to be ligands of this receptor.

Our mammalian cell transfection and mammalian

two-hybrid experiments revealed that three isoflavones

(genistein, daidzein, and biochanin A) and one flavone

(6,3V,4V-trihydroxyflavone) behaved as agonists of ERRs.

These phytoestrogens induced the activity of ERRA at

concentrations that are comparable to those for the

activation of ERA and ERB. In this study, we also used

the results of ERRA ligand-binding site mutant, F232A,

to verify our ERRA hypothetical computer model. Our

recent ERR research has determined for the first time

that flavone and isoflavone phytoestrogens are agonists

of ERRs. In addition, our studies have demonstrated that

an approach that combines structure-based virtual

screening and receptor functional assays can identify

novel ligands of orphan nuclear receptors.

IntroductionThere are three members in the estrogen-related receptor

(ERR) family, that is, ERRa, ERRh, and ERRg. The cDNAs

for ERRa and ERRh were first isolated by screening cDNA

libraries using probes corresponding to the DNA-binding

domain of the human estrogen receptor a (ERa; 1). ERRg

was identified during an analysis of the critical region of type

IIa Usher syndrome (2), and was also identified by yeast two-

hybrid screening, using the transcriptional coactivator gluco-

corticoid receptor interacting protein 1 (GRIP1) as bait (3).

While ERRs share a high amino acid sequence homology with

ERs, estrogens are not ligands of ERRs. In fact, ERRs are

transcriptionally active in the absence of exogenous ligand. In a

recent study, we generated results that lead us to propose that

Phe-2321 (4) in ERRa (analogous to Ala-350 in ERa) plays an

important role for the constitutive activity of ERRa (5). The

ERRa mutant F232A lost the transactivation activity and acted

as a dominant negative mutant. On the other hand, like wild-

type ERRa, the ERa mutant A350F was found to be

constitutively active (5). Our previous molecular model of

ERRa revealed that the side chain of Phe-232 might be able to

mimic bound ligand because it partially fills the binding pocket.

As a result, the receptor is constitutively active. The X-ray

structure of ERRg has been recently published (6). The

structure reveals that the ligand-binding pockets of ERRs are

very small and provide additional structural information to

support the conclusion that ERRs are constitutively active.

During the last several years, four compounds have been

shown to act as antagonists of ERRs. Using yeast-based assays

and mammalian transient transfection assays, we have

previously found that two organochlorine pesticides, toxa-

phene and chlordane, can act as antagonists of ERRa,

suppressing its constitutive activity (7). Diethylstilbestrol

(DES) and 4-hydroxytamoxifen (4-OHT) have also been

found to be antagonists of ERRs by two laboratories (8, 9).

Because ERRs are constitutively active, it is easier to search

for antagonists that suppress the basal activity than to identify

agonists, which have to be able to augment the basal activity.

To address the important question as to whether there are any

agonists of ERRs, we used an approach that combines

structure-based virtual screening (SVS) and receptor functional

assays to search for agonists. Our studies have revealed that

flavone and isoflavone phytoestrogens can act as agonists of

ERRs (Fig. 1). Results from these studies will be presented

and discussed.

ResultsComputer Modeling of the Human ERRa Ligand-Binding Domain

Virtual ligand screening by docking requires a good

representative three-dimensional structure of the considered

Received 8/25/03; revised 10/13/03; accepted 10/16/03.The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Grant support: National Institutes of Health Grants ES08258 and CA44735.Note: Masatomo Suetsugi and Leila Su contributed equally to this work.Requests for reprints: Shiuan Chen, Department of Surgical Research, BeckmanResearch Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA91010. Phone: (626) 359-8111, ext. 63454; Fax: (626) 301-8972.E-mail: [email protected] D 2003 American Association for Cancer Research.

1Please note that ERRa was numbered according to the protein sequence recentlyupdated in GenBank (NM_004451; 4). The ERRa numbering will be usedthroughout the text, if not otherwise mentioned.

Vol. 1, 981–991, November 2003 Molecular Cancer Research 981

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target. To screen for agonists of ERRa, it would be advantageous

to use an agonist-bound ERRa crystal structure. While an ERRa

ligand-bound structure has not yet been reported, a ligand-free

human ERRg structure (PDB code 1KV6) was reported by

Greschik et al. (6) that shares 57% amino acid sequence identity

with the human ERRa ligand-binding domain (LBD). We

prepared a homology model of ERRa based on the ERRg free

protein X-ray structure and found that it possessed a very small

‘‘ligand’’-binding pocket. More specifically, the side chain of

Phe-399 on helix 11 protruded into the ligand-binding pocket,

partially filled the cavity, and interfered with the formation of

hydrogen bonds between His-398 and the ligand. The position of

this Phe-399, together with the pull-in of Leu-305, Phe-286, as

well as helices 3, 5, 7, 11, and 12, led to a tightly packed pocket

that is only about half of the size of ERa as indicated by Greschik

et al. (6). Due to the small size of the pocket, we decided not to

use this model for ligand screening.

A homology model of ERRa using the DES-bound agonist

form of human ERa LBD as the template was previously

generated from our laboratory and was used to explain the

constitutive activity of ERRa (5). Since then, several high-

resolution X-ray crystal structures of the agonist-bound human

ERa have been published. Sequence homology between the

human ERRa LBD (235 residues) and the human ERa LBD is

34% amino acid identity and 55% similarity. The homology is

better conserved in the ligand-binding pocket formed by

22 residues (10), where the two receptors share amino acid

sequence identity of 45% and similarity of 74%. For the

purpose of generating an accurate, diversified, and unbiased

ligand-binding pocket for the screening of agonists of ERRa,

three human ERa crystal structures with different agonist

complexes [i.e. , DES, (R ,R)-5,11-cis-diethyl-5,6,11,12-tetrahy-

drochrysene-2,8-diol, and 17h-estradiol] were used as tem-

plates (PDB codes 3ERD, 1L2I, and 1GWR, respectively). This

second-generation structure model has the common classical

three-dimensional features of the nuclear receptor family. The

22 residues forming the ligand-binding cavity are mostly

hydrophobic (Fig. 2). The model was minimized as described in

‘‘Materials and Methods’’ and was used to screen for ERRa

agonists. Our previous study suggested that Phe-232 on helix 3,

one of the amino acids in the ligand-binding pocket of ERRa, is

responsible for the constitutive activity of ERRa (5). The

mutant F232A, noted as ERRaM, has a significantly lower

activity than the wild-type receptor. A homology model of

ERRaM was built by the same procedure as described for

ERRa and used to investigate the role of Phe-232 in the protein

activity and ligand binding. It is interesting to note that, like

ERa and ERh, ERRh and ERRg also have an alanine residue

in this site. Phe-232 is a unique structural feature in the ligand-

binding site of ERRa.

The quality of our ERRa and ERRaM models was

evaluated by checking the stereochemistry, local geometry,

solvent accessible surface areas, and side chain conformational

probabilities with the ProTable module from SYBYL (11).

Analysis of the Ramachandran plot showed that 97.5% of the

residues in the two models have the U/w dihedral angles in the

most favored or allowed regions. Only Thr-191 at the NH2

termini and Glu-343 in the loop region connecting H8 and H9,

which are far from the ligand-binding cavity, sit in disallowed

regions of the Ramachandran plot, and were corrected later by

energy minimization. The overall average energies of the

models calculated by MatchMaker energy plot (11) were below

zero, indicating that there is no major problem with the

structures and the models could be used for further analysis.

Structural Features of the Ligand-Binding Pocket of ERRaCompared with the ER family, members of the ERR family

have ligand-binding pockets that contain bulkier residues

(Fig. 2). As a result, human ERRg (ligand-binding pocket vol-

ume of 220 A3, 6) and human ERRa (295 A3 from our modeling

study, Fig. 3B) have smaller ligand-binding pockets than human

ERa (450 A3) and human ERh (390 A; 11). Our new ERRa

model based on ERa ligand-bound structures indicates that three

phenylalanine residues, Phe-232 from H3, Phe-399 from

H11, and Phe-414 from H12, line up on one side of the ERRa

FIGURE 1. Chemical structures of17h-estradiol (E2), 5,7,4V-trihydroxyi-soflavone (genistein), 7,4V-dihydroxyi-soflavone (daidzein), 5,7-dihydroxy-4V-methoxyisoflavone (biochanin A),and 6,3V,4V-trihydroxyflavone (fla-vone). Carbons of E2 and genisteinwere numbered.

Flavones and Isoflavones Are Agonists of ERRs982



ligand-binding cavity and stack with each other (Fig. 3, B and C).

These stacked aromatic side chains fill the upper portion of the

h-face of the cavity and change the pocket from the ‘‘wedge’’

shape in ER (Fig. 3A) to a more flattened shape (Fig. 3B).

A conserved water molecule has been indicated to be

important for ligand binding to human ERa and ERh(12–14), and is also structurally conserved among other

steroid receptors (15). The water molecule was manually

added to our ERRa model by superimposing the modeled

structure with the ERa template, followed by unrestrained

energy minimization. This water resides at the narrow end of

the h-face of the ligand-binding cavity of ERRa receptors and

could stabilize the ligand-receptor complex by forming

hydrogen bonds with hydroxyl groups of the ligand and

Glu-235 and Arg-276 of the receptor. The hydrogen bonding

between the water and the ligand could help the ligand dock

into the pocket (Fig. 3, B, D, E, and F).

Screening of a Virtual Database for Agonists of ERRaWhile our previous computer model of ERRa had revealed

that the side chain of Phe-232 might be able to mimic bound

ligand in the free protein, our new model suggests that in the

presence of ligand, Phe-232 took a different rotamer to stack

with Phe-414 and opened up the pocket for ligand binding. This

new model was used in the search for ERRa agonists. A virtual

screening of 603 compounds in the Indofine catalog was

performed (see ‘‘Materials and Methods’’). Each compound in

the Indofine database was flexibly docked into the modeled

ERRa agonist-binding pocket with the conserved water present.

Those ligands that failed to dock into the pocket were filtered

out using a TRIPOS SPL script. To verify our findings from the

virtual screening, 50 compounds in the hit list were purchased

and tested. Mammalian cell transfection and mammalian two-

hybrid functional assays were used to test these compounds

(discussed in the next section). Four ligands, ranked within the

top 30 in the hit list, including three isoflavones (genistein,

daidzein, and biochanin A) and a flavone (6,3V,4V-trihydroxy-flavone), demonstrated the ability to enhance ERRa activity

and were thus identified as ERRa agonists (Fig. 1). Two known

antagonists of ERRs, 4-OHT and DES, and the ERa agonist,

E2, failed to dock in our virtual screening. E2 is known not to

be a ligand of ERRs. 4-OHT and DES have been shown to be

antagonists of ERRh and ERRg, thus they should not bind to

the agonist-bound form of ERRa (16).

Confirmation of the Interaction of Phytoestrogens WithERRa by Mammalian Transfection and MammalianTwo-Hybrid Experiments

Our computer docking analyses have predicted that flavone

and isoflavone phytoestrogens are ligands of ERRa. Using

mammalian transfection and mammalian two-hybrid experi-

ments, we have found that three isoflavones (genistein,

daidzein, and biochanin A) and one flavone (6,3V,4V-trihydrox-yflavone; Fig. 1) can act as agonists of ERRa. The mammalian

transfection experiments demonstrated that these four com-

pounds also act as agonists of ERRh (Fig. 4). However, the

induction of the activation of ERRg by genistein and daidzein

were not statistically significant, suggesting that these two

isoflavones are relatively poor ligands of ERRg. Dose-response

studies to evaluate the binding of these compounds to ERRa

were performed, and the results were compared to those

generated with ERa and ERh (Fig. 5). Although ERRa has a

high constitutive activity, a dose-dependent increase of the

reporter activity in the presence of phytoestrogens was

observed. In addition, the maximal activity after phytoestrogen

treatment was similar among ERRa, ERa, and ERh (Fig. 5).

The interaction of phytoestrogens with ERRs was further

examined using mammalian two-hybrid analysis. The three

isoflavones were shown to enhance the interaction between

ERRs and the coactivator PNRC (Fig. 6). These results

FIGURE 2. Schematic comparison of amino acidresidues that form the ligand-binding pocket in hERRa

with the corresponding residues of hERa (underlined ).Genistein is shown in the binding pocket as in thecrystal structure 1KQM, where it takes the orientationas the phenyl ring sits in the narrow end of the bindingpocket and forms hydrogen bonds with the glutamatein helix 3, the arginine in helix 5, and a water molecule.Two distal hydroxyl groups (7-OH and 4V-OH ) arelabeled. The bulky phenylalanine residues couldcontribute to the constitutive activity of ERRa and areindicated by the box .

Molecular Cancer Research 983



confirmed that the compounds tested here are indeed agonists

of ERRs. It is recognized that the increase in the interaction

between ERRa and PNRC by genistein is not statistically

significant. Furthermore, while 6,3V,4V-trihydroxyflavone was

shown to be an agonist of ERRs using the mammalian

transfection assays, this compound was not able to enhance

the interaction between ERRs and PNRC (results not shown).

Several studies (17–19) have found that different ligands of a

FIGURE 3. The binding pocket of DES-bound ERa (A), the binding pocket of genistein-bound ERRa (B), and ribbon representation of the ligand-bindingsite (C). H3, H5, H7, and H12 are shown in yellow . F414 from H12 forms aromatic stacking interactions with F232 from H3 and F399 from H11. D. Thebinding pocket of ERRaM with genistein. E. The binding pocket of 6,3V,4V-trihydroxyflavone-bound ERRa. F. The binding pocket of ERRaM with 6,3V,4V-trihydroxyflavone. The pose of each ligand was chosen by CScore. Molecular surfaces of the ligand-binding cavity have been rendered with a translucentvan der Waals surface (1.4 A probe) and colored in green in A, B, D, E, and F. Proteins are shown as stick representations and colored by atom type(oxygen colored red , hydrogen in cyan , and carbon in white ). Side chains of key residues (E235, R276, H398, F/A232, F286, F399) in the active site aredepicted and labeled. The conserved water molecule is shown in ball-and-stick representation . Hydrogen bonds (colored in yellow ) are shown between theligands and the proteins, as well as the water.




FIGURE 5. Four phytoestrogens activate ERRa-, ERa-, and ERh-mediated transcriptional activities in a dose-dependent manner. HeLa cells weretransfected with (ERE)3SV40_LUC (0.25 Ag) and pSG5-hERRa (0.5 Ag), pCI-hERa (0.1 Ag), or pCI-hERh (0.1 Ag). The transfected cells were incubatedwith phytoestrogens for 24 h at the indicated concentrations. Each graph represents an average of three independent experiments. 0 AM, n 1 AM, 55 AM, 10 AM, 20 AM. The results between the DMSO groups and the treatment groups were subjected to statistical analyses [Student’s t test(unpaired)]. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIGURE 4. Four phytoestrogens areagonists of ERRs. HeLa cells weretransfected with (ERE)3SV40_LUC(0.25 Ag) and pSG5-hERRa, pSG5-ERRh, and pSG5-ERRg (0.5 Ag). Thetransfected cells were incubated withphytoestrogens for 24 h at 10 AM. Aftercells were washed twice with 1� PBS,the LUC activity was measured, and theactivities were shown by those taken ofthe solvent (DMSO) controls as 100%.Results are expressed as relative report-er activity averaged from three indepen-dent experiments. ERRa, n ERRh, 5ERRg. The results between the DMSOgroups and the treatment groups weresubjected to statistical analyses [Stu-dent’s t test (unpaired)]. *, P < 0.05;**, P < 0.01; ***, P < 0.001.




nuclear receptor can induce different conformational changes of

the receptor that lead to differential coactivator recruitment

capacities. It is possible that genistein and 6,3V,4V-trihydroxy-flavone induce conformational changes that reduce ERR

interaction with PNRC or enhance ERR interaction with endo-

genous coactivators in HeLa cells.

As discussed above, our laboratory has previously reported

that Phe-232 (analogous to Ala-350 in ERa) is responsible for

the constitutive activity of ERRa. In this study, we predicted

that Phe-232 is also playing a critical role in defining the

ligand-binding pocket of ERRa. Therefore, we examined the

interaction of the four phytoestrogens with the ERRa mutant

F232A using mammalian transfection experiments. As

reported by Chen et al. (5), the mutant F232A is a dominant

negative mutant. The LUC activity of the mutant F232A

(without ligand) was determined to be approximately 25% of

that of the wild-type ERRa. Our experiments revealed that

genistein and daidzein were more effective inducing agents for

F232A than for the wild-type ERRa (Fig. 7). However, the

mutation reduced the binding of biochanin A and 6,3V,4V-trihydroxyflavone. These results further confirmed that these

phytoestrogens indeed bind to the ligand-binding pocket of

ERRa and their interactions are modified by the mutation

F232A.

Interpretation of the Assay Results byComputer Modeling

The identified agonists, genistein, biochanin A, daidzein,

and 6,3V,4V-trihydroxyflavone, were carefully checked by

visual inspection in the ligand-binding pocket for their shape

complementarity, hydrogen bonding network, and van der

Waals clashes with the receptor. The ERRa agonists are

isoflavone and flavone analogues with extended aromatic

rings (Fig. 1). These four compounds are different from most

of the other flavones and isoflavones because they have two

hydroxyl or methoxyl groups lined up at the C7 and C4V end

positions with C2 symmetry. Our initial computer docking

analysis with ERRa also predicted several other phytochem-

icals as the ligands of this receptor, such as 4V-hydroxy-a-naphthoflavone, 5,7,3V,4V-tetrahydroxyflavone, phloretin, and

7-hydroxy-3(4V-methoxyphenyl)-4-methylcoumarin. But these

compounds failed to activate the receptor as indicated by

our bioassay. All of the failed compounds either do not

have hydroxyl groups at the two ends of the molecule or the

hydroxyl groups do not line up symmetrically. Those two

hydroxyl groups are predicted to provide key hydrogen

bonding interactions with the protein to stabilize the complex

formation. Ligands with bulky substituents, such as methyl or

ethyl, on the flavone rings, which likely cause steric hindrance

with the protein’s bulky phenylalanine residues, were also

found not to be ligands from our bioassay. As indicated

above, for the purpose of generating an unbiased ligand-

binding pocket for the screening of agonists of ERRa, three

human ERa crystal structures with different agonist com-

plexes were used as templates. Because ERa is known to have

a larger binding pocket than ERRa, that model could accept

ligands which were larger than the true ligands of this

receptor. The templates that were used to generate the model

had a direct impact on the results of virtual screening by

docking. The inclusion of a water molecule in the ligand-

binding site (see discussion below) helped us greatly by

placing the ligands in a more definitive orientation. We

believe that our ERRa model displays a significant degree of

accuracy because we have been able to identify the true

agonists (by bioassays) from the top 5% of our predicted list.

Our docking results showed that all of the four agonists were

tightly packed due to the small size of the cavity, and deeply

FIGURE 6. Demonstration of the in-teraction between ERRs and PNRCusing mammalian two-hybrid assays.HeLa cells were transfected with reporterplasmid Gal4-LUC (0.5 Ag) and PM-hERRa LBD (0.5 Ag), PM-hERRh LBD(0.5 Ag), or PM-hERRg LBD (0.5 Ag),along with pVP-PNRC coactivator frag-ment (aa 270 – 327, 0.5 Ag). Cells wereincubated with 10 AM phytoestrogens orthe same amount of DMSO for 24 h,washed twice with 1� PBS, and assayedfor LUC activities. Each graph representsan average of three independent experi-ments. The results between the DMSOgroups and the treatment groups weresubjected to statistical analyses [Stu-dent’s t test (unpaired)]. *, P < 0.05; **,P < 0.01; ***, P < 0.001.




docked into the ligand-binding site, partially due to the termini

water-bridged hydrogen bonds between hydroxyl group(s) of

the ligands and the conserved water molecule. The hydrogen

bonding groups on the two ends of the agonists form hydrogen

bonds with residues Glu-235, Arg-276, and His-398 at the

corresponding ends of the ligand-binding cavity (Fig. 3, B, D,

E, and F). Phe-286 on sheet 1 was fixed in an orientation by its

aromatic ring current interaction with the flat aromatic rings on

the agonists. The three phenylalanines, Phe-232, Phe-399, and

Phe-414, sit at the h face, while Phe-286 sits at the opposite a

face of the cavity to sandwich the agonists (Fig. 3, B, D, E, and

F). The hydrogen bonding, aromatic, and hydrophobic

interactions between the ligands and helices 3, 5, 7, 11, and

12 of the receptor stabilized the complexes of ERRa with the

phytoestrogens.

In summary, our modeling study indicated that the ERRa

agonist-binding cavity is rather flat due to the phenylalanines

in the ligand-binding site. This suggests that ligands with

extended aromatic structures make more effective agonists. Our

receptor functional analysis further indicates that an effective

agonist should be a molecule without bulky substituents in the

middle and with a pair of lined-up hydrogen bond forming

groups at the ends. The derived pharmacophore information

will enable us to identify additional agonists of ERRa by

screening larger commercial three-dimensional chemical data-

bases and verifying by mutagenesis and functional assays.

DiscussionVirtual Screening of the ERRa Agonists

SVS using a homology model has been proven to be a

valuable technique for nuclear hormone receptors, an important

therapeutic target family (20). Here, we identified for the first

time that flavone and isoflavone phytoestrogens are agonists of

all three isoforms of ERR through SVS and verified by

mammalian cell transfection and two-hybrid functional assays.

An important element of determining the success of the SVS is

the choice of the specific agonist conformation of nuclear

receptors used for screening. Our functional assay results

validate the effectiveness of using the homology model

generated from agonist-bound ERa X-ray complex structures

to automatically screen more than 600 structurally diverse

phytoestrogens and score using an effective consensus scoring

approach. Our studies have provided key structural information

FIGURE 7. Modification of the binding affinity of phytoestrogens by ERRa mutant F232A. HeLa cells were transfected with (ERE)3SV40_LUC (0.25 Ag)and pSG5-hERRa (0.5 Ag) or pSG5-hERRa F232A (0.5 Ag). Cells were incubated with 1 or 10 AM phytoestrogens, or the same amount of DMSO for 24 h. Allother experimental conditions are identical to those described in Fig. 5. The relative LUC activities were shown by those taken of the solvent (DMSO) controlsas 100%. n ERRa WT, 5 ERRa F232A. *, P < 0.05; **, P < 0.01; ***, P < 0.001.




to identify additional agonists of ERRa. We believe that our

study represents an excellent example of an approach that

combines SVS and receptor functional assays to identify novel

ligands of orphan nuclear receptors.

This study was aimed at the identification of ERRa novel

agonists and we presented a successful strategy to meet that goal.

An in-depth evaluation of the agonist binding mode and

structure-activity relationship requires extended work, such as

molecular dynamics and mutagenesis experiments, and that is

beyond the scope of this discussion. Enormous advances in

genomics have identified a significant number of orphan nuclear

receptors as potential therapeutic targets. We would like to place

our emphasis on aspects of combining the reliable and

inexpensive SVS technique with nuclear receptor functional

assays for lead discovery.

Functional Significance of Phe-232 of ERRaOur previous modeling study indicated that Phe-232 is re-

sponsible for the constitutive activity of ERRa (5). The current

modeling study suggested that the Phe-232 could contribute to the

protein activity by stacking with Phe-414 of H12 to help position

and stabilize H12 in the agonist position and uphold the

constitutive activity of ERRa. It was shown in the ERRa model

that Phe-414 was sandwiched by stacking to Phe-232 of H3 and

Phe-399 of H11 on each side (Fig. 3, B and C). A close inspection

of the free ERRg structure (1KV6, 6) indicates that the

phenylalanine on H12 (Phe-450 in ERRg) and the phenylalanine

on H11 (Phe-435 in ERRg), the two conserved phenylalanines in

all three ERR isoforms, sit very close to each other and fall in the

range of van der Waals interactions (about 2.5 A proton-proton

distance). A similar type of aromatic interaction has been observed

recently from the structural analysis of the constitutively active

orphan nuclear receptor Nurr1 (21). Together with results from

previous studies (5), we hypothesize that the constitutive activity

of ERRa results from (a) the side chain of Phe-232 mimicking

bound ligand, and (b) helix 12 being kept at the agonist-bound

conformation through an aromatic interaction among Phe-232

(H3), Phe-399 (H11), and Phe-414, which is in helix 12.

The receptor functional assays carried out in our laboratory

clearly indicated that the mutation of one of the three stacked

phenylalanines of ERRa, F232A, could significantly reduce

the constitutive activity of the receptor. Furthermore, genistein

and daidzein were found to be better agonists for the mutant

F232A, while biochanin A and 6,3V,4V-trihydroxyflavone were

more effective to activate wild-type ERRa (Fig. 7). This

raised the question of how the mutation, F232A, affected the

binding of each agonist and the protein activity in the

presence of the agonist. Unlike the ER-E2 complex, in the

ERRa-agonist system, ERRa has more bulky aromatic

residues at its ligand-binding site (Fig. 2), and the agonists

are molecules with extended aromatic ring structures (Fig. 1).

These new structure features imply that aromatic interactions

between the receptor and its agonists could play a very

important role in ligand binding and receptor activity.

Modeling of the mutant F232A (i.e. , ERRaM) indicated that

the side chain of Phe-414 took a different rotamer from the

stacking position in ERRa to fill part of the space left out by

Phe-232 in the mutant (Fig. 3, D and F). Phe-414 was then

stabilized by aromatic interactions with Phe-399 of H11

(Fig. 3, D and F). H12 could be further stabilized by

interacting with the agonists. The protein activity changes

induced by the mutation are the consequences of the adjusted

protein and ligand conformations. One of the many factors

accountable for the changes could be the orientation change of

the agonists in the ligand-binding site.

A detailed structure and activity analysis requires further

work considering the very similar structural features of the four

agonists. However, there are a few points that can be addressed

from this study. It was observed that the agonists of ERRa were

able to take two different docking orientations in the binding

pocket. One is the classical orientation of the nuclear receptor

ligand, where the phenolic ring sits at the A-ring end (Fig. 3, E

and F) as in the E2-ERa complex (22). The alternate orientation

is the flavone ring, instead, residing at this end (Fig. 3, B and

D). It was shown that each agonist had its favored orientation in

the binding site, depending on the specific structural character-

istics of the agonist. Genistein and daidzein, which have only

one functional group, the 4V-OH, on the phenolic ring, favor theflavone ring at the A-ring end in both the wild type and the

mutant (Fig. 3, B and D) and are better agonists for the mutant.

On the other hand, 6,3V,4V-trihydroxyflavone, which has an

extra hydroxyl group on the phenolic ring and is a better agonist

for the wild-type protein, tended to favor the phenolic ring at

the A-ring end in both the wild-type and the mutant receptors

(Fig. 3, E and F). Unlike 6,3V,4V-trihydroxyflavone, biochaninA, which acts as a better agonist for the wild-type protein and

has a bulkier methoxyl group lacking the favored hydrogen

donor for the A-ring end (23), kept its orientation with the

flavone ring at the A-ring end in both the mutated and wild-type

proteins. The preference of different orientations under specific

circumstances was not well understood.

Implication of Physiological Significance of Our Findingsof Phytoestrogens as Ligands of ERRa

As indicated in the introduction, it is not easy to identify

agonists for a receptor that is constitutively active, because they

have to be able to augment the basal activity. This work

suggested that ERRa stays at a stable agonist conformation

through the unique Phe-232 that mimics ligand and contributes

to aromatic interactions with Phe-399 in helix 11 and Phe-414

in helix 12. It is important to point out that the binding of

flavone and isoflavone ligands to ERRa increases its activity to

the levels of ERa and ERh in the presence of the same

compounds at similar concentrations. These results suggest that

ERRa has been stimulated to its maximal level in the presence

of the ligands.

ERRa is expressed in breast tissue and its expression has

recently been shown to associate with unfavorable bio-

markers in breast cancer (24). The action of ERRa and

enhancement of its activity by phytoestrogens in breast

cancer should not be overlooked. In addition, ERRa mRNA

has been found to be more highly expressed in rat calvaria

(RC) cell cultures than either ERa and ERh (25), and a

decrease in the synthesis of ERRa (through the use of

antisense oligonucleotides) led to an inhibition of RC

proliferation and bone nodule formation in vitro (26). The




interaction of phytoestrogens with ERRa can play important

roles in breast and bone, a newly identified action of

phytoestrogens. The effect of phytoestrogens on ERRa may

not be easily observed when functionally active ERa or ERhis present because these phytochemicals are also ligands of

ERs. The effect of phytoestrogens (through the interaction

with ERRa) in breast and bone in women could be detected

when the function of ERs is suppressed, such as through the

use of antiestrogens.

Our study has provided a novel approach to identifying

environmental chemicals that interact with ERRa as agonists

through the use of SVS and cell culture-based functional

analyses. We will further screen larger commercial chemical

databases for both agonists and antagonists of ERRa and verify

these results by mutagenesis and functional assays. This

research has a translational impact because the results generated

can be used for designing prevention strategies against breast

cancer. One should avoid exposure to those chemicals shown to

act as agonists of ERRs that may induce breast cancer

development. On the other hand, chemicals that act as

antagonists of these receptors may have value as preventative

agents against breast cancer. However, through the interaction

with ERRa in bone, agonists of ERRa may prevent bone loss

in women who use antiestrogens.

Materials and MethodsMaterials

5,7-Dihydroxy-4V-methoxyisoflavone (biochanin A), 7,4V-dihydroxyisoflavone (daidzein), 5,7,4V-trihydroxyisoflavone(genistein), and 6,3V,4V-trihydroxyflavone were purchased from

Indofine Chemical Co., Inc. (Somerville, NJ). The structures of

these compounds are shown in Fig. 1. hERRh cDNA and HeLa

cervix adenocarcinoma cells were purchased from American

Type Culture Collection (ATCC, Manassas, VA). HeLa cells

were maintained in Eagle’s MEMwith nonessential amino acids,

sodium pyruvate, and 10% fetal bovine serum at 37jC and 5%

CO2. Charcoal/dextran-treated serum was obtained from Gemini

Bio-Products (Woodland, CA). Lipofectin was purchased from

Invitrogen Life Technologies (Palo Alto, CA).

PlasmidAll recombinant DNA and plasmid construction experi-

ments were performed according to standard procedures. The

sequence and orientation of inserted DNA fragments in

plasmid constructs were verified by standard DNA sequenc-

ing. The expression plasmids, pSG5-ERRa and pSG5-ERRa

mutant F232A, were constructed by Chen et al. (5). The full-

length coding region of human ERRh was generated by PCR

using forward primer 5V-GCTGGAATTCATGTCGTCCGAAGACAGGCA-3V and reverse primer 5V-TGCGGAATTCT-CACACCTTGGCCTCCAGCA-3V, with the template cDNA,

hERR2 (ATCC). The PCR product was digested with EcoRI

and subcloned into vector pSG5 through the EcoRI sites. The

full-length coding region of human ERRg was generated by

PCR using forward primer 5V-CCGGGAATTCATG-

GATTCGGTAGAACTTTG-3V and reverse primer 5V-GAGC-GAATTCTCAGACCTTGGCCTCCAACA-3V, with the

template cDNA prepared from H295 cells (ATCC). The

PCR product was digested with EcoRI and subcloned into

vector pSG5 through the EcoRI sites. The luciferase reporter

plasmid, pGL3 (ERE)3-Luciferase, which contains three

copies of the ERE sequence, was constructed by Chen et al.

(5). The pSG5-GRIP1 plasmid was kindly provided by Dr.

Michael R. Stallcup (University of Southern California, Los

Angeles, CA), and pCI-ERa and pCI-ERh were kindly

provided by Dr. Y. Kinoshita (Beckman Research Institute

of the City of Hope, Duarte, CA).

Generation of the expression plasmids for mammalian two-

hybrid analysis, that is, PM-ERRa LBD, PM-ERRh LBD, PM-

ERRg LBD, and pVP-PNRC coactivator fragment (aa 270–

327), is briefly described below: The cDNA fragments of

human ERRa LBD were generated by PCR using forward

primer 5V-CCCCGAATTCACAGCAGCCCCCAGTGAATGC-3V and reverse primer 5V-ACCCGGATCCTCAGTCCAT-CATGGCCTCGA-3V, with the template DNA, pSG5 ERRa.

The cDNA fragment of human ERRh LBD was generated by

forward primer 5V-TCCCGACGCTAAAAAGCCATTGAC-TAA-3V and reverse primer 5V-TGCGGGATCCTCA-

CACCTTGGCCTCCAGCA-3V with the template DNA,

pSG5 ERRh. Finally, the cDNA fragment of human ERRg

LBD was generated by forward primer 5V-GGTTGAATTCGC-CAAAAAGCCATATAACAA-3V and reverse primer 5V-GAGCGGATCCTCAGACCTTGGCCTCCAACA-3V, with

the template DNA, pSG5 ERRg. The PCR products were

subcloned into vector pM through the EcoRI and BamHI sites.

For pVP-PNRC270–327, the cDNA fragment of PNRC270–327

(27) was generated by PCR using forward primer 5V-GCCGGATCCTAATGACTGAAGTGAGCCAAAAGGAA-3Vand reverse primer 5V-CGCTCGGATCCCTAAGTTT-

GAACTTTTGAGGAG-3V, and the PCR product was inserted

in proper reading frame into pVP16 activation domain vector

(Clontech Laboratories, Inc., Palo Alto, CA) at the BamHI site.

Mammalian Cell Transfection and Luciferase AssaysHeLa cells were cultured in MEM Earle’s salts medium

supplemented with 5% charcoal/dextran-treated fetal bovine

serum. Cells were divided and cultured in six-well plates until

80% confluent. The cells were transfected with 4 Ag Lipofectin,

and an equal amount of total DNAwas used in all transfections

by including appropriate amounts of the empty vector, pSG5, in

addition to specific amounts of the test plasmids indicated in each

experiment. After overnight incubation, medium containing

Lipofectin and DNAwas removed, and the cells were cultured in

growth medium containing 5% charcoal/dextran-treated fetal

bovine serumwith or without ligands. After a 24-h incubation, the

cells were harvested from the plates by scraping, and the luciferase

activities in the cell lysates (with the same amounts of protein)

were measured according to the manufacturer’s instructions

(Promega, Madison, WI). All experiments were performed in

triplicate.

Mammalian Two-Hybrid AssaysHeLa cells were transiently transfected with 0.5 Ag reporter

plasmid, Gal4-luciferase (Clontech), and 0.5 Ag PM-ERRa

LBD, PM-ERRh LBD, or PM-ERRg LBD along with 0.5 AgpVP-PNRC270–327.




Building the Models of ERRa LBDThree-dimensional homology modeling and SVS were

performed using the SYBYL program package (11), version

6.9 (Tripos, Inc., St. Louis, MO) on a Silicon Graphics O2+

workstation with the IRIX 6.5 operating system. The sequence

of hERRa was obtained from GenBank (NM_004451). The

FASTA search option available in the Protein Data Bank (28)

helped to identify several structural templates on which to base

the homology model. The homology models of hERRa LBD

and its mutant F232A were generated using the SYBYL

COMPOSER module (29). Three different agonist-bound X-ray

complex structures of the human ERa LBD templates (PDB

codes 3ERD, 1L2I, 1GWR) were chosen because of their high

resolution (2.03, 1.95, and 2.40 A). The structures were then

refined by torsional minimization and a series of energy

minimization steps first with ligand-binding pocket side chains,

followed by all protein side chains and finally the entire protein.

All the energy minimizations were performed using the Tripos

force field with the cutoff of nonbonded (NB) interactions at

8.0 A and the distance dielectric constant set at 4.0 following

the gradient termination of the Powell method with RMS of

0.005 kcal/mol A or the maximum 1000 iterations. The volume

of the ERRa ligand-binding pocket was estimated using

SYBYL MOLCAD separated surfaces with a grid width of

1.0 and a probe radius of 1.4 A.

Database PreparationThe Flavonoid and Coumarin Catalog was obtained from

Indofine Chemical Company. Three-dimensional structures of

the Indofine compounds were imported from the Available

Chemicals Directory (ACD; Molecular Design Limited, San

Leandro, CA) using ISIS/Base (MDL). For the known ligands of

ERs and ERRs not already included in the Indofine Catalog, such

as E2, DES, and 4-OHT, two-dimensional structures were

prepared by ISIS/Draw (MDL). Three-dimensional structures of

those molecules were generated using the Concord (Tripos)

conversion program. The new structures were then added to the

Indofine Catalog to create a final Indofine database of 603

molecules. Final coordinates were stored in a SYBYL database.

A subset of this database, containing 37 molecules (known ERa

agonists together with randomly chosen molecules), was created

as a test set to fine tune parameters for docking the Indofine

database and scoring the ligands for ERa (data not shown).

Receptor-Ligand DockingThe SYBYL FlexX program version 1.10 interfaced within

TRIPOS SYBYL 6.9 (11) was used to dock compounds to the

ligand-binding sites of ERRa and ERRaM. FlexX is a fast-

automated docking program that considers ligand conforma-

tional flexibility into a rigid protein structure by an

incremental fragment placing technique (30, 31). A structur-

ally conserved water molecule has been included in the

binding pocket for the docking. Standard parameters and

FlexX scores implemented in the program were used for

docking and scoring of FlexX poses. The ligand-protein

complex was relaxed by torsional minimization and a series of

constrained energy minimization steps. The ligand was first

minimized within the complex to an RMS of 0.001 kcal/mol

A to remove bad contacts. The side chains of amino acids in

the ligand-binding site and the entire complex were then

minimized respectively. During the minimization processes,

hydrogen bond constraints with 50 kcal/(mol A)2 force

constant were applied. Energy minimizations were carried

out using the Tripos force field with an NB cutoff of 8.0 A

and the distance dielectric constant set at 4.0 following

gradient termination using the Powell method to an RMS of

0.005 kcal/mol A or the maximum 1000 iterations.

Virtual Ligand Screening of the Indofine DatabaseEach flexible ligand of the Indofine databases composed of

flavonoids and coumarins was docked automatically into the

receptor. The FlexX score was used to guide the growing of the

ligand and was assigned to each successfully docked compound

to measure the goodness of its fit with the receptor. The FlexX

scoring function includes both polar (hydrogen bond and

charge-charge) and non-polar (hydrophobic) interactions that

are used to dock the ligand into the active site. The screening of

the Indofine database of 603 compounds took less than 5 h on a

Silicon Graphics O2+ workstation. Out of 603 screened

compounds, 426 were claimed to be ‘‘successfully docked’’

and up to 30 top scored poses of each docked compound were

saved. These 426 compounds were checked by an SPL (Sybyl

Programming Language) script to find out whether these

compounds were actually docked into the ligand-binding

pocket (flexx_pocket_mss.spl). Two hundred twenty-seven

out of these 426 ‘‘successful’’ compounds were found docked

into the pocket. ChemScore, Dock, FlexX, Gold, and PMF

scores were recalculated for the docked ligands using the

CScore module of SYBYL 6.9 for consensus scoring.

Consensus scoring has found to outperform any of the single

functions comparing the results from the test set. A hit list

containing the top scores from the three single scoring functions

(PMF, FlexX, and Gold) which were able to successfully

predict the non-binder such as E2 in the test set was generated.

The top 100 ligands in the hit list were visually inspected and

50 of them were selected, purchased, and experimentally tested.

AcknowledgmentsWe thank Dr. Dujin Zhou and TRIPOS Application Scientists for their commentsand suggestions to the research described here.

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2003;1:981-991. Mol Cancer Res Masatomo Suetsugi, Leila Su, Kimberly Karlsberg, et al. to this work.

Masatomo Suetsugi and Leila Su contributed equallyNote:Grants ES08258 and CA44735.

National Institutes of Health11Estrogen-Related ReceptorsFlavone and Isoflavone Phytoestrogens Are Agonists of

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