Design of pathway preferential estrogens that provide...

R E S E A R C H A R T I C L E

D R U G D E S I G N

Design of pathway preferential estrogens thatprovide beneficial metabolic and vascular effectswithout stimulating reproductive tissuesZeynep Madak-Erdogan,1 Sung Hoon Kim,2 Ping Gong,1 Yiru C. Zhao,1 Hui Zhang,3

Ken L. Chambliss,3 Kathryn E. Carlson,2 Christopher G. Mayne,4 Philip W. Shaul,3

Kenneth S. Korach,5 John A. Katzenellenbogen,2 Benita S. Katzenellenbogen1*

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There is great medical need for estrogens with favorable pharmacological profiles that support desir-able activities for menopausal women, such as metabolic and vascular protection, but that lack stim-ulatory activities on the breast and uterus. We report the development of structurally novel estrogensthat preferentially activate a subset of estrogen receptor (ER) signaling pathways and result in favor-able target tissue–selective activity. Through a process of structural alteration of estrogenic ligandsthat was designed to preserve their essential chemical and physical features but greatly reduced theirbinding affinity for ERs, we obtained “pathway preferential estrogens” (PaPEs), which interacted withERs to activate the extranuclear-initiated signaling pathway preferentially over the nuclear-initiatedpathway. PaPEs elicited a pattern of gene regulation and cellular and biological processes that didnot stimulate reproductive and mammary tissues or breast cancer cells. However, in ovariectomizedmice, PaPEs triggered beneficial responses both in metabolic tissues (adipose tissue and liver) thatreduced body weight gain and fat accumulation and in the vasculature that accelerated repair of en-dothelial damage. This process of designed ligand structure alteration represents a novel approach todevelop ligands that shift the balance in ER-mediated extranuclear and nuclear pathways to obtaintissue-selective, non-nuclear PaPEs, which may be beneficial for postmenopausal hormone replace-ment. The approach may also have broad applicability for other members of the nuclear hormone re-ceptor superfamily.

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INTRODUCTION

Estrogens regulate many essential physiological processes and are neededfor the functional maintenance of many adult target tissues within andoutside of the reproductive system. However, they can have deleteriousactions in promoting breast and uterine cancers (1–4). This balance betweendesirable and undesirable activities in diverse target tissues offers anintriguing opportunity for the development of tissue-selective estrogens thatprovide a net benefit with minimal risk for menopausal hormone replace-ment, such as ones affording metabolic and vascular protection withoutstimulation of the breast or uterus. Estrogens act through estrogen receptors(ERs) by using two distinct signaling pathways, the nuclear-initiated (“ge-nomic”) pathway, wherein ER functions as a chromatin-binding ligand-regulated transcription factor, and the extranuclear-initiated (“nongenomic”)pathway, which involves kinase cascades initiated by ER action fromoutside the nucleus (5, 6). Here, we used a process involving structural al-teration of steroidal and nonsteroidal ER ligands in ways that enhanced theirselectivity for the extranuclear-initiated pathway over the nuclear-initiatedER pathway, resulting in ER ligands having effective metabolic and vascularprotective activity but lacking stimulatory activity on reproductive tissues.

1Department of Molecular and Integrative Physiology, University of Illinois atUrbana-Champaign, Urbana, IL 61801, USA. 2Department of Chemistry, Uni-versity of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. 3Center forPulmonary and Vascular Biology, Department of Pediatrics, University of TexasSouthwestern Medical Center, Dallas, TX 75390–9063, USA. 4Beckman Insti-tute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. 5National Institute of EnvironmentalHealth Sciences, Research Triangle Park, NC 27709, USA.*Corresponding author. Email: [email protected]

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The activation of specific kinases by the action of estrogens throughextranuclear ER action is generally rapid and often transient (7–12),and its initiation likely requires only the input of a triggering signalby the ER-hormone complex to initiate a kinase cascade and cellularactivities through the extranuclear-initiated ER signaling pathway thatcould collectively have important downstream cellular effects. By con-trast, the activation of genes through the nuclear ER signaling pathwayappears to require a sustained action of ER-hormone complexes, suf-ficient to effect dissociation of heat shock proteins, recruit co-regulatorproteins, stimulate ER binding to chromatin, alter chromatin architectureand modify histones, and activate RNA polymerase (pol) II to initiate genetranscription. ER ligands with potent nuclear ER activity form kineticallystable receptor-cofactor complexes, and coactivator binding can slow lig-and dissociation rates by orders of magnitude (13). Thus, it seemed pos-sible that ER ligands preferential for extranuclear over nuclear ERsignaling might be obtained by redesigning the structures of certain estro-gens in ways that would preserve their essential chemical features, a phe-nol and often a secondary alcohol, as well as their overall composition andgeometry, but would reduce considerably their high-affinity ER binding.Our aim was to preserve the ability of such a modified estrogen to have aneffective interaction with ER that would be appropriate and sufficient toactivate the extranuclear-initiated ER pathway, but insufficient to sustainactivity through the nuclear pathway.

Here, we describe the unique activities of such pathway preferentialestrogens (PaPEs) that alter the balance of utilization of extranuclear-and nuclear receptor–initiated signaling pathways. By selectively trigger-ing extranuclear-initiated ER signaling pathway activities, PaPEs regulatethe transcription of a subset of ER target genes and evoke biological

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outcomes both in cells in culture and in animals in vivo in ways that pro-vide a favorable balance of desired versus undesired effects. Thus, PaPEsrepresent novel tissue-selective estrogens that might have potential asclinically useful pharmaceuticals. The structural alteration approach we
have used to design PaPEs could be ap-plied to other nuclear hormone receptorsas well.
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RESULTS

PaPE-1 has impeded ER bindingand coactivator interactionsTo create PaPE-1, we rearranged key ele-ments of the steroid structure of estradiol(E2) that, while preserving its key functionalgroups, reduced considerably its ER bindingaffinity (Fig. 1A). First, we deleted the B-ringof the steroid, a change that we knew fromprevious work on A-CD and other B-secoestrogens would cause a drop in ER bindingaffinity (14). Second, we added the twocarbon atoms lost from the B-ring deletionto the A-ring, flanking both sides of thephenol; this type of ortho substitution sterical-ly impedes the important hydrogen bondsbetween the phenol and the glutamate and ar-ginine residues (Glu353 and Arg394) in theER ligand-binding pocket and also reducesreceptor binding (15). Finally, we convertedthe C-ring to a benzene, simplifying the struc-ture by eliminating the C-18 methyl groupand at the same time creating a biphenyl sys-tem, a ligand core element that is present invarious nonsteroidal estrogens (16, 17). Froma comparison of their properties (Fig. 1A),it is evident that both E2 and the structural-ly permuted estrogen, PaPE-1, have closelymatched estrogenic functional groups andphysical properties (composition, shape,size, volume, lipophilicity, and polar sur-face area) but very different binding affi-nities for ERa and ERb.

The relative binding affinity (RBA) val-ues of E2 and PaPE-1 (Fig. 1A) were ob-tained by a competitive radiometric bindingassay using purified full-length human ERaand ERb (18). The RBAs for E2 are definedas 100, and relative to E2, PaPE-1 binds50,000-fold less well to ERa and ERb (fig.S1A). The equivalent dissociation constant(Kd) values are 10 and 25 mM for PaPE-1on ERa and ERb, respectively, comparedto the subnanomolar Kd values for E2. Thus,our design greatly lowered the ER bindingaffinities of PaPE-1 for both ERs and alsogreatly increased its dissociation rate fromERa (fig. S1B) while preserving as muchas possible the physical and functional groupattributes of E2.

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A computational model showing the structural details of how theligand-binding pocket of ERa accommodates PaPE-1 in comparison toE2 is presented in Fig. 1B. Helices 3 and 6 constrict the A-ring end ofthe binding pocket, and E2 (silver gray) forms hydrogen bonds to both

A

Property E2 PaPE-1 PaPE-2 PaPE-3 PaPE-4

Molecular formula C18H24O2 C17H18O2 C16H18O2 C18H20O2 C21H26N2O4

MW 272.4 254.3 242.1 268.4 370.5

cLogP 3.78 3.64 3.53 4.20 1.54

Volume 269 Å3 244 Å3 238 Å3 261 Å3 351 Å3

Polar surface area 40.5 Å2 40.5 Å2 40.5 Å2 40.5 Å2 98.7 Å2

ER Ki [RBA] 0.2 nM [100] 10 µM [0.002] 10 µM [0.002] 10 µM [0.002] 20 µM [0.001]

ERβα

Ki [RBA] 0.5 nM [100] 25 µM [0.002] 13.µM [0.004] 17 µM [0.003] 17 µM [0.003]

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Arg

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394His524

B

Fig. 1. Structures and molecular and binding properties of E2 and four PaPEs, and a model of E2 and
PaPE-1 binding to ERa-LBD. (A) MW is molecular weight; cLogP is log10 of the calculated octanol-water partition coefficient; volume is molecular volume; and polar surface area is a measure of com-pound polarity. All values were obtained using ChemBioDraw Ultra (version 13.0.0.3015). RBA valueswere determined by competitive radiometric binding assays (18). E2 is set at 100 on both ERs. Inhi-bition constant (Ki) values were calculated as Ki = Kd (for E2) × (100/RBA), where Kd of E2 is 0.2 nM(ERa) and 0.5 nM (ERb). Values are average of three to four determinations with coefficient of variation<0.3. (B) Computational model comparing PaPE-1 and E2 in the ligand-binding pocket of ERa. Themodel of ERa + E2, based on a crystal structure [Protein Data Bank (PDB) ID: 1GWR], has E2 andhelical elements shown in silver gray and the pocket volume contour in slate blue. The model forPaPE-1 was generated from the ERa + E2 structure by progressive transformation of the ligand struc-ture from E2 to PaPE-1, partnered with progressive minimization of the ligand and the LBD. The result-ing positions of the PaPE-1 ligand and hydrogen-bonding residues are shown in yellow.



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Glu353 and Arg394, further stabilized by a crystallographic water (red dot)bridging these two residues. By contrast, the ortho-methyl groups ofPaPE-1 (in yellow) introduce steric clashes with helices 3 and 6, inducinga substantial shift in ligand positioning. Although the A-ring OH of PaPE-1 maintains a hydrogen bond to Glu353 and the crystallographic water stillbridges between Glu353 and Arg394, direct ligand contact to Arg394 isbroken (ER structure in yellow). At the D-ring end of the pocket, thereis also a subtle shift in the positioning of His524. Overall, it appears thatthe reduced volume of the PaPE-1 ligand and the increased planarity dueto the aromatic C-ring mimetic result in fewer van der Waals contactsthroughout the central region of the pocket. All of these changes withrespect to the ER-E2 complex are consistent with the greatly lowerbinding affinity of PaPE-1. Nevertheless, the overall shape of the ligand-binding pocket is not altered in a major way by the binding of PaPE-1,suggesting that PaPE-1 can still form a structurally competent complexwith ER.

Using a time-resolved Förster resonance energy transfer (FRET) as-say we have previously developed, we monitored the binding of thesteroid receptor coactivator 3 (SRC3) to ERa (19, 20). In coactivatortitration assays (fig. S1C), SRC3 bound with high affinity to ERa com-plexes with E2 but showed no binding to ERa complexes with PaPE-1.Notably, however, the antagonist trans-hydroxytamoxifen (OH-Tam)reversed the ER-SRC3 interaction promoted by E2; PaPE-1 also reversedthe ER-SRC3 interaction, but only at much higher concentrations, com-mensurate with its lower ERa binding affinity (fig. S1D). The E2-elicitedinteraction of ERa with SRC3 was also observed by coimmunoprecipi-tation of the complexes from MCF-7 cells, whereas no coimmunopreci-pitated complexes were observed for ERa and SRC3 after exposure toPaPE-1 (fig. S1E).

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PaPE-1 regulates a subset of estrogen-modulated genesand activates kinases, but does not stimulate breastcancer cell proliferationIn earlier studies of an estrogen-dendrimer conjugate (EDC) that acti-vates ER nongenomic signaling with high selectivity (8, 10, 21, 22), weidentified a set of genes in MCF-7 human breast cancer cells whose ex-pression was increased effectively by either extranuclear or nuclear ERaction, as well as ones that required only nuclear action by ER (10). Wefound that PaPE-1 was selective in activating extranuclear-initiated ER-regulated genes, as shown by LRRC54 stimulation, but was essentialwithout activity on the nuclear-initiated ER gene target PgR (Fig.2A). Activation of LRRC54 gene expression by PaPE-1 was blockedby treatment with the antiestrogen fulvestrant (ICI 182,780) (Fig. 2B)and by knockdown of ERa (Fig. 2C and fig. S2). By contrast, knockdownof GPR30 did not affect gene stimulation (Fig. 2C and fig. S2). PaPE-1 also

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Fig. 2. Comparison of regulation of gene expression, cell proliferation, andpathway signaling by PaPE-1 and E2. (A) PaPE-1 preferentially activates
extranuclear-initiated genes (LRRC54) over nuclear genes (PgR) comparedto E2 in MCF-7 cells. Cells were treated with control vehicle (Veh), E2, orPaPE-1, and gene expression was monitored by quantitative polymerasechain reaction (qPCR) (n = 3 biological replicates). (B) MCF-7 cells werepretreated with ICI 182,780 (ICI) and then treated with vehicle, E2, orPaPE-1 in the presence or absence of ICI 182,780. RNA was isolatedand subjected to qPCR analysis for the indicated genes (n = 3 biologicalreplicates). (C) MCF-7 cells transfected with siControl (siCtrl), siERa, orsiGPR30 were treated with vehicle, E2, or PaPE-1. RNA was isolated andsubjected to qPCR analysis (n = 3 biological replicates). (D) MCF-7 cellswere treated with vehicle, E2, or the indicated concentrations of PaPE-1 for6 days, and proliferation was monitored by WST assay (n = 4 biologicalreplicates). OD, optical density. (E) PaPE-1 selectively activated mTORand MAPK signaling in MCF-7 cells. Left: Cells were treated with controlvehicle (V) or the indicated concentrations of E2 or PaPE-1 for 15 min(upper panel) and 45 min (lower panel), and Western blots were doneto assess the activation of signaling proteins and phosphorylation (p) ofSer118 in ERa. Total ERa was monitored, and total ERK2 was used as aloading control (n = 3 biological replicates). Right: Total amount of thesefactors in cells receiving the indicated treatments (n = 3 biological repli-cates). (F) PaPE-1 induces interaction between ERa and RAPTOR. MCF-7cells were treated with E2 or PaPE-1. Cells were crosslinked and incubatedwith ERa and RAPTOR antibodies overnight, and PLA was performed.Quantitation of signal intensities is shown in the graph (n = 3 biologicalreplicates). Two-way analysis of variance (ANOVA), Bonferroni posttest,*P < 0.05, **P < 0.01, ***P < 0.001. Scale bar, 50 mm.



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did not stimulate proliferation of MCF-7 cells, whereas E2 potently stimu-lated proliferation (Fig. 2D).

When we monitored activation of major signaling pathways in MCF-7cells, we observed that PaPE-1 efficiently activated mammalian target ofrapamycin (mTOR) and mitogen-activated protein kinase (MAPK)signaling (Fig. 2E), as seen by increased phosphorylation of P70S6Kand 4EBP1, which are associated with mTOR complex 1 (mTORC1) ac-tivation, increased phosphorylation of SGK1, which is associated withmTORC2 activation, and increased phosphorylation of MAPK. PaPE-1increased phosphorylation of AKT and SREBP1 (sterol regulatory element–binding protein 1) to a greater extent than E2. However, PaPE-1 did notdetectably induce phosphorylation of Ser118 in ERa, which was ob-served with E2 (Fig. 2E) and is associated with the mitogenic activityof ERa (23).

The mTOR pathway is the major signaling system that senses thenutrient state of the environment and modulates metabolic functions inthe cell. mTOR kinase is present in two distinct complexes, mTORC1and mTORC2, which are distinguished by the presence of RAPTORand RICTOR scaffolding proteins, respectively. It is thought that mTORC1primarily modulates cell metabolism, whereas mTORC2 is principallyinvolved in regulating the cytoskeleton and cell proliferation (24, 25).To further characterize the mTOR activation by PaPE-1, we performedproximity ligation assays (PLAs) in MCF-7 cells and found that ERainteracted with RAPTOR to a greater extent in the presence of PaPE-1 thanE2 (Fig. 2F). We did not detect any interaction between ERa and mTOR, orERa and proline-rich AKT substrate of 40 kD (PRAS40), which is im-portant in AKT and mTOR signaling, suggesting that ERa modulated themTOR pathway through direct interactions with RAPTOR (fig. S3).

PaPE-1 regulates metabolism-related genes in an mTORand MAPK activity–dependent manner, but does notcause recruitment of ERa to chromatinNext, we performed RNA sequencing (RNA-seq) analysis to compare genesthat were changed in their expression by PaPE-1 and/or E2. We treatedMCF-7 cells with 10 nM E2 or 1 mM PaPE-1 for 4 and 24 hours and com-pared genes regulated by each compound at these two time points (Fig. 3A).At both times, E2 regulated the expression of more genes than did PaPE-1,and the magnitude of gene regulation by E2 was generally higher than thatby PaPE-1. At 4 hours, E2 regulated nearly 1500 genes, whereas PaPE-1modulated expression of only 500 genes (Fig. 3A, upper Venn diagram).At 24 hours, about 3000 genes were regulated by E2, whereas PaPE-1regulated about 2200 genes (Fig. 3A, lower Venn diagram). At both 4and 24 hours, about three quarters of the genes regulated by PaPE-1 werealso targets of E2. Notably, only E2 increased the expression of mitosisgenes and decreased the expression of apoptosis genes at 24 hours (tableS1), consistent with our observation that PaPE-1 did not stimulate MCF-7cell proliferation.

Next, we used pathway-selective inhibitors to assess the effect of mTORor MAPK pathway activation on PaPE- and E2-mediated gene regulation.The mTOR pathway inhibitor PP242 blocked the regulation of ~60% ofPaPE-1–regulated genes, but only 40% of E2-regulated genes. Similarly,the MAPK inhibitor AZD6244 blocked almost 50% of PaPE-1–regulatedgenes, but only ~23% of E2-regulated genes (Fig. 3B). Examination ofenriched gene ontology (GO) functions revealed that these inhibitors blockedE2 regulation of cell migration–, immune-, cell cycle–, and angiogenesis-related genes, whereas they blocked PaPE-1 regulation of genes involvedin nucleotide metabolism, inflammatory response, noncoding RNA pro-cessing, amino acid transport, and glycoprotein metabolism (table S1).

To investigate the roles of ERa, extracellular signal–regulated kinase 2(ERK2), and active RNA pol II recruitment in PaPE-1–mediated transcrip-

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tional events, we performed chromatin immunoprecipitation sequencing(ChIP-seq) analysis, which revealed that ERa was recruited to chromatinupon treatment of cells with E2, but not with PaPE-1 (Fig. 3C and tableS2). PaPE-1 induced recruitment of ERK2 to the proximal promoter re-gions of genes, whereas E2 caused recruitment of ERK2 to enhancer re-gions together with ERa. We observed distinct RNA pol II recruitmentsites with PaPE-1, further suggesting that PaPE-1 induces transcriptionalevents through recruitment of RNA pol II without affecting ERa or ERK2recruitment to enhancer regions of target genes. These distinct patterns ofERa, ERK2, and phosphorylation of Ser5 in RNA pol II binding after celltreatment with vehicle, E2, and PaPE-1 are shown for the TFF1/pS2 andLRRC54/TSKU genes in Fig. 3C (right). The lack of ERa and ERK2 re-cruitment by PaPE-1 may be associated with the lack of effect of PaPE-1on the phosphorylation of Ser118 in ERa (Fig. 2E).

PaPE-1 is a tissue-specific modulator of ER and mTORsignaling, with preferential estrogen-like activity innonreproductive (metabolic and vascular) tissuesWe next characterized the in vivo biological activities of PaPE-1 in ovar-iectomized female mice. Similar to E2, PaPE-1 was cleared rapidly aftersubcutaneous injection [half-time (t1/2) ~1 hour; fig. S4A]; so, to providemore prolonged exposure for in vivo studies, we used either administrationby ALZET minipump or by a pellet containing PaPE-1, both implantedsubcutaneously (fig. S4B). Mice at 8 weeks of age were ovariectomized,and after 2 weeks, they were treated with control vehicle, E2, or PaPE-1 for 3 weeks. Although E2 was effective in increasing growth of the uter-us, PaPE-1 did not exhibit any stimulatory effects on uterine weight (Fig.4A). As has been previously demonstrated (26), we observed that E2 in-duced marked involution of the thymus, but PaPE-1, even at a high dose,had no effect on thymus size (Fig. 4A). Likewise, PaPE-1 did not changemammary gland ductal morphology from that of vehicle control mice,whereas E2 elicited marked ductal growth (elongation and branching) (Fig.4B). Of interest, however, both E2 and PaPE-1 reduced the mammary glandadiposity that develops in mice after ovariectomy. This effect was associatedwith reduced adipocyte area after E2 or PaPE-1 treatment (Fig. 4C).

Body weights of the mice increased after ovariectomy, as has beenpreviously reported, and this increase in weight was suppressed byPaPE-1 and by E2 over the 3-week period monitored (Fig. 5A). Bodyweights were statistically lower in E2- and PaPE-1–treated mice than invehicle control–treated animals, and the PaPE-1 and E2 groups werenot statistically different from one another. The reduction in bodyweight gain that we observed with PaPE-1 was not due to a changein food consumption (Fig. 5B). Because of the anorexigenic effect ofE2, there was an early drop in food intake in E2-treated animals, aneffect that was not observed in PaPE-1–treated animals, suggesting thatthis effect did not trigger the differences in body weights and changesin the fat depots.

A major impact of PaPE-1 was on fat depots of the animals, as ob-served in EchoMRI measurements. Both E2 and PaPE-1 greatly reducedfat mass, but total lean mass and water mass were increased only by E2and were not affected by PaPE-1, further indicating that PaPE-1 workedby changing overall adiposity rather than lean mass of the animals (Fig.5C). Hematoxylin and eosin (H&E) and Oil Red O staining of perigonadaladipose tissue (Fig. 5D) highlighted the similar and marked estrogeniceffects of both PaPE-1 and E2 on this tissue. PaPE-1 and E2 decreasedthe weight of all of the fat depots examined (Fig. 5E). The weights ofthe perigonadal, perirenal, and subcutaneous adipose tissues were reducedby both ligands, but to a larger extent by E2 at the dosages tested. Mesen-teric adipose tissue weight was similarly reduced by both ligands, and bothligands also decreased triglyceride concentrations in blood (Fig. 5F).




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n = 1478 PaPE-1 4h

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Fig. 3. PaPE-1 and E2 regulate common as well as distinct groups of genes before treatment with E2 or PaPE-1 in the presence or absence of inhi-
in MCF-7 cells. (A) Cells were treated with E2 or PaPE-1 for 4 and 24 hours.RNA was isolated, and RNA-seq was performed. Regulated genes areconsidered to be those with P < 0.05 and expression fold change >2(n = 2 biological replicates). (B) PaPE-1–mediated gene expressionchanges are sensitive to mTOR and MAPK pathway inhibitors. Effectof mTOR and MAPK inhibitors on gene regulation by E2 and PaPE-1 inMCF-7 cells is shown. Cells were pretreated with PP242 or AZD6244
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bitors. RNA was isolated, and RNA-seq was performed. (P < 0.05, foldchange >2; n = 2 biological replicates). (C) PaPE-1 does not induce recruit-ment of ERa or ERK2 to chromatin but stimulates recruitment of RNA pol II.MCF-7 cells were treated with E2 or PaPE-1. ChIP-seq was performed forthe indicated proteins. UCSC (University of California, Santa Cruz) genometracks of cistromes in the presence of E2 or PaPE-1 are shown (right panel)(n = 3 biological replicates that were pooled). bp, base pairs.




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Ovariectomy resulted in increased lipid droplet accumulation in theliver, and E2 or PaPE-1 decreased hepatocyte lipid content, as observedin Oil Red O staining of liver sections (Fig. 5G). We verified changesin fatty acid synthesis pathways by monitoring two biomarkers of hepaticsteatosis, FASN (27, 28) and SREBP1c (29, 30), and found that expressionof these genes was reduced after 3 weeks of E2 or PaPE-1 treatment (Fig.

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5H). Expression of SREBP1 transcriptionaltargets, FASN, and the gene encoding theenzyme acetyl–coenzyme A (CoA) car-boxylase a (ACACA), which catalyzes thecarboxylation of acetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acidsynthesis, was decreased by 24 hours ofPaPE-1 or E2 treatment and remained lowover the 2-week monitoring period (Fig.5H).

PaPE-1 and E2 show similaritiesand differences in geneexpression and signalingpathway activations in multipletissues in vivoPaPE-1 induced tissue-specific molecularchanges in gene expression and signalingpathway activation patterns. To character-ize these changes in the various tissues har-vested from animals in long-term studies,we performed analysis of the expression ofgenes reported in the literature to bealtered in liver, skeletal muscle, perigona-dal fat, pancreas, and uterus. PaPE-1 andE2 generated quite similar profiles in meta-bolic tissues, namely, in liver and skeletalmuscle; in contrast, only E2 induced geneexpression changes in uterus (Fig. 6A),consistent with the selective stimulatoryactions of PaPE-1 in nonreproductive tissues.

To gain more mechanistic insight, weprofiled the signaling pathways activatedby E2 and PaPE-1 in different tissues(Fig. 6B). E2 and PaPE-1 had similar path-way effects in nonreproductive tissues,whereas E2, but not PaPE-1, stimulatedreproductive tissues (uterus and mamma-ry gland). Furthermore, in metabolic tis-sues such as liver and skeletal muscle,both E2 and PaPE-1 were effective in in-ducing mTOR signaling, as observed byS6 phosphorylation.

PaPE-1 activity is lost in ERaknockout miceA select number of effects of E2 and PaPE-1on physiologic processes and gene expres-sion were compared in wild-type and ERaknockout (ERKO) mice, in which thecomplete ERa gene was deleted (fig. S5).Ovariectomized mice were treated with E2or PaPE-1 for 3 weeks, and the ability ofE2 or PaPE-1 to diminish the body weight

gain after ovariectomy was lost in the ERKO mice (fig. S5A). Asexpected, E2 did not stimulate uterine growth in ERKO mice (fig.S5B), and E2 or PaPE-1 did not decrease blood triglycerides in thesemice (fig. S5C). Furthermore, ERa mediated the suppression of FASNand SREBP1c gene expression by PaPE-1 or E2 in the liver of wild-typemice, an effect that was lost in ERKO mice (fig. S5D).

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Fig. 4. Unlike E2, PaPE-1 does not change uterus or thymus weight and does not induce mammary glandductal branching, but like E2, PaPE-1 reduces mammary gland adipocyte area. (A) PaPE-1 does not affect
uterus or thymus weight. C57BL/6 mice were ovariectomized and then were given daily subcutaneousinjections of E2 or were implanted with PaPE-1 pellets for 4 days. Weights of uterus and thymus weremonitored (n = 8 mice per treatment). Scale bars, 5 mm. (B) PaPE-1 stimulates minimal mammary ductalelongation but it greatly reduces adipocyte size in mammary gland. Ovariectomized C57BL/6 mice were im-planted with E2 or PaPE-1 pellets. Whole-mount stain (scale bars, 1 mm) and H&E stain (scale bars, 200 mm)of mammary gland are shown. (C) Mammary gland adipocyte area was calculated from the H&Eimages (n = 4 mice per treatment). A one-way ANOVA model was fitted to assess the contribution of ligandtreatment on uterine weight, thymus weight, or mammary gland adipocyte area. When the main effects werestatistically significant at a = 0.05, pairwise t tests with a Bonferroni correction were used to identify the treat-ments that were significantly different from each other. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
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PaPE-1 and E2 show beneficial vascular effectsEstrogens have potential beneficial actions on vascular cells, as exem-plified by in vivo studies of carotid artery reendothelialization afterperivascular electric injury in female mice. After ovariectomy, micewere treated with vehicle, E2, or PaPE-1 for 18 days, at which timeE2- and PaPE-1–treated mice were also administered a single dose ofvehicle or the antiestrogen ICI 182,780. Three days later, carotid arterydenudation was performed, and the mice received a second dose of ve-hicle or ICI 182,780, whereas E2 or PaPE-1 was continued, and 72 hoursafter denudation, Evans blue dye was administered systemically to assess

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the remaining area of denudation in the intimal surface of the carotidartery (Fig. 7A, upper panel). E2 and PaPE-1 caused similar markedendothelial repair, as indicated by the minimal area of remaining denu-dation, and the responses elicited by E2 and PaPE-1 were fully pre-vented by the antiestrogen fulvestrant (ICI 182,780) (Fig. 7A, lowerpanel). In the same animals, uterine weight, which was greatly elevatedby E2, was unaffected by PaPE-1, and as expected, the antiestrogen ICI182,780 blocked uterine stimulation by E2 (Fig. 7B).

To evaluate direct effects on endothelium, endothelial nitric oxide syn-thase (eNOS) activation was assessed by measuring the conversion of

Fig. 5. Like E2, PaPE-1 reduces the increasein body weight after ovariectomy and re-duces adipose stores and blood triglycerideconcentrations. (A) PaPE-1 is effective innormalizing body weight after ovariectomy.Ovariectomized C57BL/6 mice were im-planted with pellets containing E2 or PaPE-1or vehicle control for 3 weeks (n = 8 miceper group). Animals were on a normal chowdiet. Two-way ANOVA, Bonferroni post-test, *P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001, comparing treatments tovehicle. (B) Food consumption for eachmouse from (A) was monitored weekly.(C) Body composition for each mouse from(A) was monitored using EchoMRI at the endof 3 weeks. One-way ANOVA, Newman-Keuls posttest, *P < 0.05, **P < 0.01, ***P <0.001, ****P < 0.0001. (D) H&E staining ofperigonadal adipose tissue (AT). Imagesare representative of eight mice per group.Scale bar, 500 mm. (E) Weights of variousadipose tissue depots after 3 weeks ofcontrol vehicle or ligand exposure. (F) Tri-glycerides were measured in the blood ofanimals (n = 6 mice per group) at theend of 3 weeks of vehicle, E2, or PaPE-1treatment. (G) H&E staining (upper tworows; scale bars, 100 mm) and Oil Red Ostaining (lower panel; scale bar, 20 mm) ofthe liver after 3 weeks of treatment. Imagesare representative of eight mice per group.(H) Gene expression analysis of SREBP1cand FASN in the liver at 3 weeks (upper pan-el) (n = 12 mice per group), and time courseof FASN and ACACA expression in the liversof E2- and PaPE-1–treated mice (n = 3 miceper group). A two-way ANOVA model wasfitted to assess the contribution of ligandtreatment and time of ligand treatment onbody weight, food consumption, or gene ex-pression. A one-way ANOVA model wasfitted to assess the contribution of ligandtreatment on fat mass, lean mass, watermass, triglyceride concentrations, weightof different fat depots, or gene expression.
When the main effects were statistically significant at a = 0.05, pairwise t tests with a Bonferroni correction were used to identify the treatments that weresignificantly different from each other. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
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[14C]L-arginine to [14C]L-citrulline by intact primary bovine endothelialcells in culture. eNOS was activated by E2, as previously observed (22),and there was a comparable response to PaPE-1; the effects of both E2 andPaPE-1 were fully attenuated by ICI 182,780 (Fig. 7C).

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Additional PaPEs show pathway- and tissue-selectiveactivities similar to those of PaPE-1To further exemplify compounds that preferentially activate ER nonge-nomic signaling, we present results from PaPE-2 and PaPE-3, two ligands

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Fig. 6. Gene regulation and signaling pathway activations by PaPE-1 and phorylation of S6) in liver and skeletal muscle. Ovariectomized C57BL/6
E2 in tissues in vivo. (A) Ovariectomized C57BL/6 mice were implantedwith pellets containing E2 and PaPE-1. Liver, skeletal muscle, perigona-dal fat, pancreas, and uterus were harvested. RNA was isolated, andqPCR was performed for the indicated genes (n = 8 mice per treatment).(B) PaPE-1 activates mTOR signaling (as monitored by increased phos-
mice were injected with E2 (E) or PaPE-1 (P) for 2 hours. The indicatedtissues were collected and subjected to Western blot analysis for phos-phorylated S6 and MAPK1/2. b-Actin and total ERK2 were used asloading controls. Total MAPK1/2 and total P70S6K are also shown (n = 3mice per treatment).




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that are structurally related to PaPE-1 by being altered forms of thesteroidal ligand E2, but having variations in the structure of whatwas originally the E2 D-ring, namely, ring cleavage in PaPE-2 and ringenlargement in PaPE-3 (Fig. 1A). To further diversify the structures ofPaPEs, we also studied an additional ligand, PaPE-4; this PaPE is derivedfrom a nonsteroidal estrogen, bisphenol A (BPA), and it retains the corebisphenol structure of BPA but has been modified to have reduced ERbinding affinity by replacing one of the methyl groups with a polar bis-amide substituent. The binding and physical properties of PaPE-2, PaPE-3,and PaPE-4 are given in Fig. 1A.

All of the PaPEs showed similar biological activities in vitro and in vivo.They all caused preferential stimulation of extranuclear-initiated over nu-clear gene activity (LRRC54 > PgR) compared to E2 (10), which efficient-ly stimulated expression of both genes (Fig. 8A). In contrast to E2, none ofthe PaPEs stimulated proliferation of MCF-7 cells over a broad concen-tration range tested (Fig. 8B). All of the PaPEs increased activation ofMAPK, mTOR, and AKT signaling pathways, as monitored by the phos-phorylation of MAPK, P70S6, SREBP1, and AKT in these cells (Fig. 8, Cand D). In endothelial cells, PaPE-2, PaPE-3, and PaPE-4 also increasedNOS activity (Fig. 8E), similar to E2 and PaPE-1 (Fig. 7C), and the NOSstimulation was fully blocked with the antiestrogen ICI 182,780. In vivo, thefour PaPEs and E2 reduced body weight gain after ovariectomy (Fig. 8F),without altering food consumption (Fig. 8G). In contrast, unlike E2, the fourPaPEs did not elicit increases in uterine weight (Fig. 8H). The reduction inbody weight with the four PaPEs was largely due to a change in body fatmass, with little or no change in lean mass or water mass, as monitored byEchoMRI. E2 reduced fat mass more markedly and increased lean mass andwatermass (Fig. 8I). These differential effectsmay account for the fact that thebody weight of E2-treated animals matched that of the PaPE-treated animals.

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DISCUSSION

The preferential extranuclear-initiated pathway activityof PaPEs results in a distinctive pharmacologicaloutcome, favoring beneficial metabolic and vasculareffects over reproductive tissue stimulationHere, we present a new approach to develop small-molecule estrogenswith a favorable profile of tissue-selective activity through preferentialactivation of the extranuclear-initiated signaling pathway over the nuclear-initiated pathway. This type of estrogen molecule was derived through astructural alteration process designed to preserve essential physical andfunctional features of E2 or BPA, but purposefully lessen the high-affinitybinding required for nuclear ER signaling.

Our analyses of cellular pathways and gene expression changes inresponse to PaPE-1 and E2 treatments indicated that E2 and the PaPE-1ligand regulated certain common as well as various different groups ofgenes and biological processes. In MCF-7 breast cancer cells, PaPE-1regulated genes involved in nucleotide, amino acid, and lipid metabo-lism, and induced RNA pol II recruitment to chromatin, but PaPE-1 didnot induce ERa or ERK2 recruitment to gene enhancers or stimulateexpression of proliferation-associated genes, as seen with E2. However,like E2, PaPE-1 strongly activated MAPK and mTOR pathways, andPaPE-1 relied on these pathways for a considerable fraction of its generegulation on the basis of the effect of MAPK and mTOR inhibitors.These results suggest a role of kinase pathways for some of the tran-scriptional responses we observed, and that these kinases might beworking through other transcription factors that are downstream ofMAPK or mTOR signaling [such as cyclic adenosine monophosphateresponse element–binding protein (CREB)] to affect gene regulation.

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Fig. 7. Like E2, PaPE-1 elicits repair of the vascular endothelium afterinjury, an effect that is prevented by the antiestrogen ICI 182,780. (A)Carotid artery reendothelialization after an injury that denudes the endo-thelial layer in ovariectomized mice treated with PaPE-1 or E2 in the ab-sence or presence of the antiestrogen ICI 182,780 (n = 6 to 9 mice pergroup). *P < 0.05 compared to basal control. Scale bar, 400 mm. (B) E2,but not PaPE-1, increases uterine weight, an effect that is blocked by ICI182,780 (n = 7 mice per treatment). (C) eNOS stimulation by E2 andPaPE-1 in the presence and absence of the antiestrogen ICI 182,780in bovine aortic endothelial cells (BAECs) (n = 4 biological replicates;*P < 0.05 compared to control).




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Fig. 8. Assessment othe activities of PaPE-2
PaPE-3, and PaPE-4 inMCF-7 cells, in BAECsand in mice. (A) Assessment of extranuclearinitiated LRRC54 geneexpression compared todirect nuclear PgR geneexpression inMCF-7 cellstreated for 4 hours withcontrol vehicle, E2, or theindicated PaPE (n = 4biological replicates). (BProliferation of MCF-7cells after treatment withdifferent concentrationsof E2 or the PaPE for 6days (n = 4 biologicareplicates). (C) Stimulation of various celsignaling pathways bydifferent concentrationsof E2, PaPE-1, or PaPE4 after 15 min in MCF-7cells. ERa abundanceis also shown (n = 3 biological replicates). (D) Timecourse of cell signalingpathway activations byPaPE-2 or PaPE-3, monitored at the indicatedtimes. ERa abundanceis shown, and total ERK2is used as a loadingcontrol (n = 2 biological replicates). (E) Stimulation oNOS activity during 15min treatment of BAECswith ligand either aloneor with ICI 182,780 (n =4 biological replicates)(F) The PaPEs and E2
reduce weight gain afteovariectomy in C57BL/6mice. Ovariectomizedanimals received pelletsof E2, the PaPE, or vehicle, and body weighwas monitored over thenext 3 weeks. A group
of intact non-ovariectomized mice were included for comparison (n = 4mice per group). A two-way ANOVA model was fitted to assess the con-tribution of ligand treatment and time of ligand treatment on body weight orfood consumption. When the effects were statistically significant at a =0.05, pairwise t tests with a Bonferroni correction were used to identifythe treatments that were significantly different from each other. *P < 0.05,**P < 0.01, ***P < 0.001, ****P < 0.0001. (G) Food consumption of the micein (F) was monitored over time. (H) Assessment of uterine weight gain in theovariectomized C57BL/6 mice in (F) after 3 weeks of receiving E2 or PaPE-1,
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PaPE-2,PaPE-3, andPaPE-4.One-wayANOVA,Newman-Keulsposttest, *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001. (I) Fat mass, leanmass, andwater mass were measured by EchoMRI at the end of the 3-week treatmentperiod in the mice in (F). A one-way ANOVA model was fitted to assess thecontribution of ligand treatment on gene expression, fat mass, lean mass,and water mass. When the main effects were statistically significant at a =0.05, pairwise t tests with a Newman-Keuls correction were used to identifythe treatments that were significantly different from each other. *P < 0.05,**P < 0.01, ***P < 0.001, ****P < 0.0001.




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The preferential regulation of cellular responses through the extra-nuclear signaling pathway by PaPE-1 and the three other PaPEs resultedin distinctive biological outcomes, favoring metabolic actions in liver andadipose tissues and actions on the vasculature, with negligible if any stim-ulatory activity in reproductive tissues. Thus, PaPE-1 lacked estrogen-likeactivity on uterus, thymus, and mammary gland but closely mimicked theactions of E2 in metabolic tissues and on the vasculature. Regarding theimpact on adipose sites, it is notable that both PaPE-1 and E2 reducedcentral fat depots (perigonadal and perirenal) that are associated withthe metabolic syndrome and obesity in postmenopausal women (31).All four PaPEs suppressed body weight gain after ovariectomy, althoughsuppression by E2 was more marked and was accompanied by an increasein lean mass and water mass not seen with PaPEs. PaPE-1 greatly reducedexpression of liver genes associated with lipid synthesis (such as FASNand ACACA) and decreased blood triglyceride concentrations. Like E2,PaPE-1 also decreased the expression of the mRNA encoding the tran-scription factor SREBP1c, which plays a key role in lipogenesis. Theseeffects required ERa and were lost in ERKO mice.

The design of PaPEs represents a new approachfor developing estrogens with pathway- andtissue-selective activityPrevious efforts to prepare agents having tissue-selective activity have usedthree different mechanisms: differential interaction with co-regulators,differing activity on the two ER subtypes, and restricted tissue, cellular,or subcellular distribution. SERMs (selective ER modulators; such as ta-moxifen, raloxifene, and bazedoxifene) have different amounts of partialagonist–antagonist activity in different target tissues, a result of differentialengagement of co-regulator proteins in a target cell– and gene-specific man-ner (32). ER subtype–selective ligands were developed to exploit the dis-tinctly different tissue distributions of ERa and ERb (33) so as to regulatethe different sets of genes and physiological outcomes controlled by thesetwo ERs (9, 34, 35), a major focus being on ERb-selective compounds toavoid the proliferative drive of ERa in reproductive tissues (36–38).

To activate the extranuclear-initiated pathways of ER action in pref-erence to nuclear-initiated actions, we originally developed a synthetichormone-polymer conjugate, denoted EDC, that bound well to ER buthad little genomic activity because it was excluded from the nucleus dueto its charge and polymeric size (8, 10). Although EDC provided robustER-mediated protection against vascular injury and cortical bone losswithout stimulation of the uterus and breast cancer (21, 22), its polymericnature complicates its development as a pharmaceutical agent. Otherapproaches have been taken to obtain selective estrogen action by restrict-ing the sites of hormone action (39). Recent studies using transgenicanimals with ER engineered to have only nuclear effects [NOER mouse(40) or C451A-ERa mouse (41)] or extranuclear, membrane-initiatedeffects [MOER mouse (42)] have further highlighted that preferential ac-tivation of one or the other of these pathways can result in distinctive,often beneficial patterns of selectivity (40, 43–45); genetic engineering,however, is not a pharmaceutical approach. Hence, as described in thisstudy, we were prompted to undertake a different approach to discoversmall molecules that would have preferential activity on the extranuclearsignaling pathway and might be used ultimately as novel pharmaceuticalagents in women for hormone replacement therapy without detrimentaleffects on reproductive tissues.

Little is known in any system about the intimate molecular details ofhow a ligand binds to and dissociates from a receptor, and how the dy-namics of these physical interactions are coupled to the dynamics ofdownstream signal transduction events. In general, as binding affinitydrops, the rate of ligand dissociation increases, whereas the rate of ligand

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association changes comparatively little. This has been characterized insome detail with a number of ER ligands having a spectrum of affinities(46). Despite wide differences in their binding affinities, ER agonist as-sociation rate constants all fell within the relatively narrow, 5-fold range(1.3 × 106 to 6.0 × 106 M−1 s−1), whereas their dissociation rates covereda >5000-fold range (from 5 × 10−5 to 2.7 × 10−1 s−1), with rates beinginversely related to ligand-binding affinities. These dissociation ratescorresponded to a half-life of the ER-ligand complex that ranged fromseveral hours to under 1 min for ligands with progressively decreasingaffinities (46).

To investigate how ER ligand-binding dynamics were coupled withsignal transduction pathways, we altered the structure of the steroidal es-trogen E2 to produce PaPE-1, PaPE-2, and PaPE-3, and modified thestructure of the nonsteroidal estrogen BPA to produce PaPE-4, with a par-ticular aim in mind. We wanted to retain, as much as possible, the keyphysical, functional, and structural features of active estrogens, so that theywould still be recognized by the ER ligand-binding domain (LBD), but togreatly lower the affinity of their interaction with ER.

We anticipated that the short lifetime of these ER-PaPE complexesmight be sufficient to activate extranuclear signaling pathways but wouldbe less effective in activating nuclear action, the former requiring the trig-gering of kinase cascades, but the latter requiring an ER-ligand complex ofsufficient lifetime to alter chromatin architecture and promote transcription.By our measurements, the dissociation rates of PaPE-1 and E2 for ERadiffered by nearly 2000-fold; the half-life of the ER-E2 complex was nearly30 hours, whereas that of the PaPE-1 complex was less than 1 min (fig.S1B). These measurements were done with purified ER proteins in acell-free system, and one should consider that, in a cellular context, the in-teraction of ER-ligand complexes with other proteins, such as co-regulators,or signaling or scaffold proteins, such as seen with RAPTOR in thesestudies, might modulate the ligand-binding properties of PaPEs.

There have been other attempts to produce estrogens havingpathway-selective activity (47, 48), but these compounds had eithermixed endocrine effects or had limited selectivity and in vivo efficacy(49–51). Thus, the structural permutation and modification approacheswe used to generate PaPEs appear to provide a robust way to producesmall molecules having distinct pathway preferential activity that ismatched by a pattern of favorable biological actions. All four of thePaPEs described in this report provide favorable actions in metabolicand vascular tissues by selective activation of signaling pathways crit-ical for ERa action in these tissues, yet they fail to activate these path-ways and increase growth of the uterus or proliferation of mammarytissue. The tissue-selective actions of the PaPEs appear to result fromthe greater retention of their activity through the extranuclear-initiatedpathway than through the nuclear pathway.

Other features of the PaPEs are of note: Although the affinity of PaPE-1and the other PaPEs for ER is about 50,000-fold less than that of E2, wewere able to stimulate extranuclear ER effects using only ~500-fold excessof PaPE-1 over E2, an observation suggesting that the extranuclear signal-ing pathway might depend to a lesser extent on the affinity of ligand for thereceptor than the nuclear pathway. Also, whereas PaPE-1, PaPE-2, andPaPE-3, which were patterned after E2, all have physical characteristics(lipophilicity, polar surface area, volume, and so on) similar to that ofE2, PaPE-4 is considerably larger and more polar than E2 and the otherPaPEs, yet it has biological activities similar to those of the other threePaPEs, suggesting that the class of PaPE-like compounds can cover arather broad range of physical and structural characteristics.

Our studies with PaPEs establish a new conceptual framework for thedevelopment of novel PaPEs that might have promise as clinically usefulpharmaceuticals for estrogen replacement therapy to improve the metabolic




and vascular health of postmenopausal women without risk to the breast oruterus. Furthermore, our ligand structural permutation process, whichdefined a distinct class of tissue- and pathway-selective estrogens, mightalso have broad applicability to other nuclear hormone receptors such asthe glucocorticoid and vitamin D receptors, and liver X receptor b, forexample, which are similar to ER in mediating both nuclear andextranuclear processes (52). Changes that reduce affinity but preserve es-sential bioactive structural features of drugs for other classes of drug re-ceptors that operate through more than one signaling pathway mightenable the development of drug analogs with an altered balance of cellularsignaling pathway utilization and possibly favorable selective activity.

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MATERIALS AND METHODS

Cell culture and siRNA treatmentsMCF-7 cells were obtained from and grown as recommended by theAmerican Type Culture Collection. Receptor abundance was measuredby qPCR and Western blotting, and gene expression and proliferative re-sponse to E2 were monitored regularly. For experiments with E2 and PaPEtreatment, cells were maintained in phenol red–free tissue culture mediumfor at least 5 days before use. ERa knockdown experiments used theSMARTpool of four small interfering RNAs (siRNAs) from Dharmaconand siRNA from Santa Cruz Biotechnology and were performed as de-scribed (10) with 30 nM siControl or siERa or siGPR30 for 72 hoursand resulted in knockdown of the corresponding mRNA and protein bygreater than 80%. Primary bovine aortic endothelial cells were harvested,maintained, and used as previously described (22).

Animals and ligand treatmentsStudies used wild-type and ERKO mice in C57BL/6 background. Allexperiments involving animals were conducted in accordance with theNational Institutes of Health standards for the use and care of animals, withprotocols approved by the University of Illinois at Urbana-Champaign andthe University of Texas Southwestern Medical Center. Wild-type C57BL/6mice were purchased from The Jackson Laboratory/National Cancer Insti-tute. ERKO mice with complete excision of ERa and wild-type littermateswere obtained from Taconic and were used as described previously (53, 54).

In studies of metabolic parameters and gene expression in vivo, ligandswere administered to ovariectomized recipient mice by subcutaneous im-plantation of pellets containing compound mixed with cholesterol to a totalweight of 20 mg. Animals were single-housed during the study. For 3-weekstudies, E2 (Sigma-Aldrich) dosage (0.125mg per pellet) was chosen on thebasis of our previous findings (38). In carotid artery reendothelializationexperiments, compounds were delivered at a dose of 6 mg/day by a 4-weekALZETminipump (model 2006), as described (22). Total body weight andfood intakeweremonitored everyweek after ovariectomy.Body fat and leanmass composition were monitored at the end of 3 weeks using EchoMRI-700 Body Composition Analyzer (Echo Medical Systems), which enablesone to quantify longitudinal body composition in the live animal.

Western blotting and ChIP assaysWestern blot analysis used specific antibodies for ERa (HC-20; Santa CruzBiotechnology); ERK2 (D-2; Santa Cruz Biotechnology); and pS6, pS6K,pmTOR, pRAPTOR, pRICTOR, and pMAPK (Cell Signaling). Coim-munoprecipitation assays used antibodies for SRC3 (C-20; Santa CruzBiotechnology) andERa (F10; SantaCruzBiotechnology).ChIPassayswerecarried out as described (9, 11). Antibodies used were for ERa (HC-20),ERK2 (D2 and C14; Santa Cruz Biotechnology), and pSer5 RNA pol II(sc-47701; Santa Cruz Biotechnology). ChIP DNAwas isolated using QIA-

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quickPCRPurificationKit (Qiagen) andused forChIP-seq analysis andqPCR.qPCRwas used to calculate recruitment to the regions studied, as described (9).

ChIP-seq analysis and clusteringFor genome-wide ChIP-seq, the ChIP DNA was prepared from three bi-ological replicates and pooled. Libraries were prepared according toIllumina Solexa ChIP-seq sample processing methods, and single-read se-quencing was performed using the Illumina HiSeq 2000. Sequences gen-erated were mapped uniquely onto the human genome (hg18) by Bowtie2(9, 11). MACS (model-based analysis of ChIP-seq) algorithm was used toidentify enriched peak regions (default settings) with a P value cutoff of6.0 × 10−7 and false discovery rate of 0.01, as we have described (9). TheseqMINER density array method with a 500-bp window in both directionswas used for the generation of clusters, or groups of loci having similarcompositional features (9). BED (Browser Extensible Data) files for eachcluster were used for further analysis with Galaxy Cistrome integrativeanalysis tools (Venn diagram, conservation, CEAS).

RNA-seq transcriptional profilingFor gene expression analysis, total RNAwas extracted from three biolog-ical replicates for each ligand treatment using TRIzol reagent and furthercleaned using the RNeasy kit (Qiagen). For time course studies, MCF-7cells were treated with vehicle [0.1% ethanol (EtOH)], 10 nM E2, or 1 mMPaPE for 4 and 24 hours. For inhibitor studies, MCF-7 cells were pre-treated with control [0.1% dimethyl sulfoxide (DMSO)], 1 mM PP242,or 1 mM AZD6244 for 30 min and then treated with vehicle, 10 nME2, or 1 mM PaPE-1 in the presence or absence of inhibitors for 4 hours.Once the sample quality and replicate reproducibility were verified, twosamples from each group were subjected to sequencing. RNA at a concen-tration of 100 ng/ml in nuclease-free water was used for library construc-tion. Complementary DNA (cDNA) libraries were prepared with themRNA TruSeq Kit (Illumina Inc.). Briefly, the polyadenylate-containingmRNA was purified from total RNA, RNA was fragmented, double-stranded cDNA was generated from fragmented RNA, and adapters wereligated to the ends.

The paired-end read data from the HiSeq 2000 were processed and ana-lyzed through a series of steps. Base calling and demultiplexing of sampleswithin each lane were done with Casava 1.8.2. FASTQ files were trimmedusing FASTQTrimmer (version 1.0.0). TopHat2 (version 0.5) (55) was usedto map paired RNA-seq reads to version hg19 of the Homo sapiensreference genome in the UCSC genome browser (56) in conjunction withthe RefSeq genome reference annotation (57). Gene expression values(raw read counts) from BAM files were calculated using Strand NGS(version 2.1) quantification tool. Partial reads were considered, and optionof detecting novel genes and exons was selected. Default parameters forfinding novel exons and genes were specified. DESeq normalizationalgorithm using default values was selected. Differentially expressed geneswere then determined by fold change and P value with Benjamini andHochberg multiple test correction for each gene for each treatment relativeto the vehicle control (9). We considered genes with fold change >2 andBenjamini-Hochberg–adjusted P value less than or equal to 0.05 as statis-tically significant, differentially expressed.All RNA-seq data sets have beendeposited with the National Center for Biotechnology Information (NCBI)under Gene Expression Omnibus (GEO) accession no. GSE73663.

Motif and GO category analysisOverrepresented GO biological processes were determined by the Web-based DAVID bioinformatics resource database (58), ClueGO, andWeb-based GREAT software (59). Motif enrichment analysis was doneusing SeqPos (60).




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ImmunohistochemistryH&E staining and whole-mount staining were performed on paraffin-embedded tissue sections (61). Images were quantified by monitoringaverage cell size from three randomly chosen fields in Fiji software(http://fiji.sc/wiki/index.php/Fiji).

Immunofluorescence microscopy, PLAs in cells, anddata analysisCells treated with vehicle (0.1% EtOH), 10 nM E2, or 1 mM PaPE-1 for15 min were washed in phosphate-buffered saline (PBS), fixed on glasscoverslips, and incubated with antibodies against ERa (F10; Santa CruzBiotechnology), pSer5 RNA pol II (47701; Santa Cruz Biotechnology), orRAPTOR (Cell Signaling). The next day, a PLAwas performed using theDuolink in situ kit (Olink Bioscience) according to the manufacturer’s in-structions, as described (62). Briefly, overnight incubation with primaryantibodies was followed by hybridization with two PLA probes at 37°Cfor 1 hour, and then by ligation for 15 min and amplification for 90 min at37°C. A coverslip was mounted on each slide, and image acquisition andanalysis were conducted. Samples were imaged using a 63×/1.4 oil differ-ential interference contrast M27 objective in a Zeiss LSM 700 or 710 laserscanning confocal microscope. Images were obtained in a sequentialmanner using a 488-nm Ar (10 mW) laser line for PLA signal. Theindividual channels for 4′,6-diamidino-2-phenylindole (DAPI) andPLA signal were obtained using a sequential scanning mode to preventbleed-through of the excitation signal. Laser power, gain, and offset werekept constant across the samples and scanned in a high-resolution format of512 × 512 or 1024 × 1024 pixels with 2/4 frame averaging. Further quanti-fication of the images used Fiji software (http://fiji.sc/wiki/index.php/Fiji)(63). Briefly, images were converted to 8 bit for segmentation for eachchannel, and images were background-subtracted using a rolling ball method,with a pixel size of 100 and segmented using the DAPI channel.

Cell proliferation assaysCells were seeded at 1000 cells per well in 96-well plates. On the secondday, the cells were treated with 0.1% ethanol vehicle, 10 nM E2, or PaPEat the concentrations indicated and proliferation was assessed usingWST-1 reagent (Roche) as described (9).

eNOS activationeNOS activation was assessed in intact primary endothelial cells by mea-suring [14C]L-arginine conversion to [14C]L-citrulline over 15 min usingpreviously reported methods (22). Cells were treated with vehicle (yield-ing basal activity), E2, or PaPE alone or with the antiestrogen ICI 182,780at the concentrations indicated.

Pharmacokinetic analysesFor short-term studies, ovariectomized C57BL/6 mice were injected sub-cutaneously with 100 mg of PaPE-1 in 100-ml DMSO. Three mice weresacrificed at each time point, and 400 ml of blood was obtained from theabdominal aorta. Samples were centrifuged at 2000g for 10 min, and serumwas collected. A 50-ml portion of each sample was submitted to the Meta-bolomics Center at the University of Illinois for analysis. For longer-termstudies, ovariectomized C57BL/6 mice were implanted subcutaneouslywith a pellet fabricated with 8 mg of PaPE-1 and 12 mg of cholesterol.Blood samples (30 ml) were collected by tail snipping every week until thethird week of treatment. Samples were centrifuged at 2000g for 10 min,and serum was collected. Serum (10 ml) was mixed with 40 ml of PBS andsubmitted to the Metabolomics Center for analysis. For analysis, a massstandard of PaPE-1 (labeled with three deuterium atoms) was added toeach sample before analysis by liquid chromatography–mass spectrom-

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etry using the 5500 QTrap with Agilent 1200 high-performance liquidchromatography.

Carotid artery reendothelializationCarotid artery reendothelialization was studied after perivascular electricinjury in mice by assessing Evans blue dye uptake 72 hours after injury.Endothelial denudation and recovery after injury in this model have beenconfirmed by immunohistochemistry for von Willebrand factor (64). Atthe time of ovariectomy at 8 to 9 weeks of age, female mice received in-traperitoneal osmotic minipumps prepared to deliver E2 (6 mg/day) orPaPE-1. Carotid artery denudation was performed 21 days later. In selectstudies, additional treatments included subcutaneous injections of vehicleor ICI 182,780 (360 mg per mouse) administered 3 days before carotid in-jury and on the day of injury. At the end of the study, uteri were alsoharvested and weighed.

Ligand dissociation assaysThe fluorescence polarization or anisotropy characteristics of fluores-cein attached to C530 in ERa, which is sensitive to the nature of thebound ligand, can be exploited to detect the dissociation of one ligandand the association of a second ligand, with the rate of ligand exchangebeing limited by the rate of dissociation of the initially bound ligand(65). PaPE-1 gives an anisotropy value of about 20% lower than that ofE2 when bound to ER, and OH-Tam gives an 80% lower anisotropyvalue than E2 when bound to the ER; so, these differences can be usedto monitor the rate of dissociation of the initially bound ligand.

ERa-LBD, mutated to have one active cysteine at Cys530, was site-specifically labeled with 5-iodoacetamidofluorescein (65) and then dilutedinto t/g [50 mM tris, 10% glycerol (pH 8)] buffer with 0.01 M mercapto-ethanol and ovalbumin (0.03 mg/ml) added as a carrier protein to give 2 nMER. To minimize homoFRET, a fivefold excess (10 nM) of unlabeled ERa-LBD (10 nM) was added, and the fluorescein-labeled and unlabeled ERdimers were allowed to exchange at room temperature in the dark for 1 hour,thereby producing dimers in which essentially only one monomer is fluorescein-labeled. The ER was then bound with 100 nM E2, or 100 nM/RBA ofPaPE-1; the RBA of PaPE-1 is 0.002%; therefore, 100 nM/RBA = 50 mMPaPE-1 was used. These samples were allowed to complete ligand binding atroom temperature in the dark for 2.5 hours.

The anisotropy was measured on a Spex Fluorolog II cuvette-basedfluorometer under constant wavelength conditions. The excitation wasset at 488 nm and emission at 520 nm, under magic angle conditions,and three to five time points were taken for a zero time. To initiate thedissociation of PaPE-1, 300 nM E2 was added to the PaPE-1 sample,and the time course of dissociation was subsequently followed by changesin anisotropy. Because of glycerol viscosity changes, there is a change inthe protein anisotropy between room temperature and 5°C; therefore, carewas taken to prechill the protein as well as the cuvette chamber to 5°C.

To measure the dissociation of E2, 300 nM E2 was added to the pre-exchanged sample of fluorescein-labeled and unlabeled apo-ER dimerand allowed to bind for 2.5 hours, as above. The cuvette and chamberwere chilled to 5°C, and after taking the zero time points, E2 dissoci-ation was initiated by adding 5 mM OH-Tam, and change in anisotropywas followed with time. The data for both dissociation experiments werefitted to an exponential decay function by linear regression using Prism 4.

Computational modeling of the complex of ERa withPaPE-1 or E2Starting from the ERa + E2 crystal structure (PDB code: 1GWR), thestructure preparation routine in MOE (Molecular Operating Environment;Chemical Computing Group) was used to fill missing loops and side


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chains and add explicit hydrogen atoms. A custom volume visualizationcode was used to create the binding volume for the ERa + E2 structure,shown in slate blue in Fig. 1B; the red dot is a structural water. The modelof the binding of PaPE-1 was built by progressive generation of the PaPE-1 ligand structure from that of E2, coupled with progressive minimizationof the LBD: Atoms were first deleted from E2 to open the B-ring andconvert the C-ring into an aromatic ring, but the two ortho-methyl groupson the A-ring were not yet added. At this stage, the positions of the ligandoxygen atoms and all protein atoms were fixed, and energy minimizationwas performed using the MMFF94× force field with a termination gradi-ent cutoff of 0.1 kcal/(mol·Å) to obtain a low-energy conformation of thePaPE-1 ligand core. All atoms were then unfixed, and energy minimiza-tion was further performed while constraining protein backbones to opti-mize interactions with hydrogen-bonding side chains. After the two A-ringortho-methyl groups were added, another energy minimization was per-formed with constrained backbone atoms, and then a final unconstrainedenergy minimization was performed, all to the same gradient cutoff. Theresulting positions of the ligand and hydrogen-bonding residues are shownin yellow in Fig. 1B.

Statistical analysesData from in vivo animal metabolism studies were analyzed using either aone-way ANOVA model to compare different ligand effects or a two-wayANOVA model to compare time-dependent changes. For every main ef-fect that was statistically significant at a = 0.05, pairwise t tests were con-ducted to determine which treatment levels were significantly differentfrom each other. For these t tests, the Bonferroni correction was used tocontrol the experiment-wise type I error rate at a = 0.05 followed by Bon-ferroni post hoc test using GraphPad Prism 6 for Windows (GraphPadSoftware). Differences were considered statistically significant if *P <0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/9/429/ra53/DC1Fig. S1. ER and coactivator binding and interaction assays with ligands and ligand disso-ciation rates.Fig. S2. Verification of siRNA results in Fig. 2C with another siRNA.Fig. S3. PLAs with E2 and PaPE-1.Fig. S4. Pharmacokinetic studies for analysis of blood concentrations of PaPE-1 afterinjection or pellet implantation.Fig. S5. Effects of PaPE-1 require ERa.Table S1. List of genes differentially expressed by cell treatment with E2 and PaPE-1.Table S2. BED files for ERa, ERK2, and pSer5 RNA pol II ChIP-seq data from experimentswith MCF-7 cells.
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Acknowledgments: We thank A. Hernandez of the University of Illinois Keck Bio-technology Center for assistance with RNA-seq analyses; R. Ventrella, L. Petry, F. Lee,and L. Xu for technical assistance. Funding: This work was supported by a grant from theBreast Cancer Research Foundation (to B.S.K.); NIH grants R37 DK015556 (to J.A.K.)and P50AT006268 (to B.S.K.) from the National Center for Complementary and Inte-grative Health (NCCIH), the Office of Dietary Supplements (ODS), and the NationalCancer Institute (NCI); National Institute of Food and Agriculture, U.S. Department of Ag-riculture award ILLU-698-909 (to Z.M.-E.); NIH HL087564 (to P.W.S.); the Associates FirstCapital Corporation Distinguished Chair in Pediatrics at University of Texas (UT) South-western (to P.W.S.); a fellowship from Sun Yat-sen University, China (to H.Z.); and Division ofIntramural Research, National Institute of Environmental Health Sciences 1ZIAES070065(to K.S.K.). Its contents are solely the responsibility of the authors and do not necessarilyrepresent the official views of the NIH, NCCIH, ODS, NCI, or the U.S. Department ofAgriculture. Author contributions: Z.M.-E., S.H.K., P.W.S., J.A.K., and B.S.K. con-ceived and designed the project; Z.M.-E., S.H.K., P.G., Y.C.Z., H.Z., K.L.C., K.E.C., andC.G.M. performed the experiments; K.S.K. provided key reagents and advice; all authors




discussed and analyzed data. Z.M.-E., P.W.S., J.A.K., and B.S.K. wrote the manuscript; allauthors added parts and comments. All authors read and approved the manuscript.Competing interests: The authors declare that they have no competing interests. Dataand materials availability: All RNA-seq data sets have been deposited with the NCBI andare available under GEO accession no. GSE73663. ChIP-seq data for ERa, ERK2, and pSer5

RNA pol II from experiments with MCF-7 cells treated with vehicle, E2, or PaPE-1 can befound as BED files in table S2. A U.S. Provisional Patent Application (serial no. 62/275,416)entitled as “Estrogen-derived compositions and methods of using the same” was filed on6 January 2016 by the University of Illinois and the UT Southwestern Medical Center.

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Submitted 5 November 2015Accepted 3 May 2016Final Publication 24 May 201610.1126/scisignal.aad8170Citation: Z. Madak-Erdogan, S. H. Kim, P. Gong, Y. C. Zhao, H. Zhang, K. L. Chambliss,K. E. Carlson, C. G. Mayne, P. W. Shaul, K. S. Korach, J. A. Katzenellenbogen,B. S. Katzenellenbogen, Design of pathway preferential estrogens that provide beneficialmetabolic and vascular effects without stimulating reproductive tissues. Sci. Signal. 9, ra53(2016).


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effects without stimulating reproductive tissuesDesign of pathway preferential estrogens that provide beneficial metabolic and vascular

Christopher G. Mayne, Philip W. Shaul, Kenneth S. Korach, John A. Katzenellenbogen and Benita S. KatzenellenbogenZeynep Madak-Erdogan, Sung Hoon Kim, Ping Gong, Yiru C. Zhao, Hui Zhang, Ken L. Chambliss, Kathryn E. Carlson,

DOI: 10.1126/scisignal.aad8170 (429), ra53.9Sci. Signal.

postmenopausal hormone replacement therapies.ovariectomized mice. These estrogens provided vascular and metabolic benefits and thus could be further developed asthat did not enhance ductal mammary gland branching (a sign of mammary gland growth) or increase uterine weight in

designed estrogen-like molecules that had reduced receptor affinity andet al.these reproductive tissues. Madak-Erdogan liver and adipose tissue, but they can also cause breast or uterine cancer, because they stimulate cell proliferation in

Estrogen and synthetic versions can enhance the repair of blood vessels after injury or improve metabolism in theDesigning better estrogens

ARTICLE TOOLS http://stke.sciencemag.org/content/9/429/ra53

MATERIALSSUPPLEMENTARY http://stke.sciencemag.org/content/suppl/2016/05/20/9.429.ra53.DC1

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