Review

10.1517/17460441.1.2.137 © 2006 Informa UK Ltd ISSN 1746-0441 137

Steroid-binding G-protein-coupled receptors: new drug discovery targets for old ligandsEric R Prossnitz†, Jeffrey B Arterburn, Bruce S Edwards, Larry A Sklar & Tudor I Oprea†Department of Cell Biology & Physiology, MSC08-4750, University of New Mexico, Albuquerque, NM 87131, USA

Steroid-binding receptors have long been a successful target class for thepharmaceutical industry. Clinical applications for steroids range from contra-ception and hormone replacement therapy to immune regulation and can-cer therapy. With the recent demonstration that the orphan GPCR, GPR30binds and is activated by estrogen, as well as the identification of aGPR30-selective agonist, it is likely that GPR30 represents a novel drug targetwith many potential clinical applications. This review discusses the role ofGPR30 in mediating the effects of estrogen, as well as recent efforts to iso-late GPR30-specific ligands using a combination of virtual and biomolecularscreening. Finally, comments are made on the future directions regardingGPCRs, steroids and drug discovery.

Keywords: biomolecular screening, drug discovery, estrogen, G-protein-coupled receptors, GPR30, steroids, virtual screening

Expert Opin. Drug Discov. (2006) 1(2):137-150

1. Introduction

Drugs that alter the actions of steroids by altering their synthesis, replacing them orblocking their effects represent an important treatment for a wide range of diseases.In the case of estrogen, indications include i) hormone replacement therapy (HRT)(to alleviate the symptoms of menopause, including hot flashes and osteoporosis);ii) contraception when combined with progestins; and iii) breast cancer, where thetumour-promoting effects of estrogen are blocked by antiestrogens or selective estro-gen receptor modulators (SERMs; such as tamoxifen or raloxifene) or aromataseinhibitors, which prevent the synthesis of estrogen [1]. In all cases, undesirable sideeffects limit the ideality of the treatment. In the case of HRT, estrogen increases theincidence of breast and uterine cancers, venous thromboembolisms, heart diseaseand stroke [2]. In the case of adjuvant tamoxifen treatment for breast cancer, resist-ance to the drug and an increase in the incidence of endometrial hyperplasia andcancer represent serious problems [3]. Due to the complexity of estrogen-mediatedactivity, significant efforts continue to be dedicated towards the identification ofsmall molecules that maintain the beneficial effects of estrogen as well as preventingthe harmful effects of the hormone and side effects of the drugs [4,5]. In addition, theexistence of two isoforms of the classical estrogen receptor (ER)-α and -β, has led toefforts to develop subtype-specific agonists and antagonists [4]. The discovery of anew transmembrane G-protein-coupled ER, GPR30 [6,7], that binds estrogen andcan mediate the effects of estrogen in ER-deficient cells, adds further complexity tothe development of small molecules targeting estrogen function and suggests thatmolecules that exhibit selectivity among these three estrogen-binding receptors willbe required in the therapeutic arsenal.

The search for novel receptor ligands displaying nanomolar binding affinity is anontrivial task, given the size and diversity of chemical space. The high-throughput

1. Introduction

2. Estrogen and its receptors

3. Virtual screening for

estrogen-like ligands

4. Biomolecular screening for

GPR30-specific ligands

5. Molecular and cellular

characterisation of a

GPR30-specific ligand

6. Conclusion

7. Expert opinion

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screening (HTS) paradigm seeks novel lead compounds byphysically screening relatively large numbers (104 – 106) ofcompounds. The screening collection, sometimes termedscreening deck or screening library, is often subject to rigorousprocedures prior to assembly [8]. However, the HTS processhas been found to yield disappointing hit rates [9]. Virtualscreening (VS) technologies have emerged as an adaptiveresponse to the massive throughput screening and synthesis(e.g., combinatorial chemistry) paradigm [10]. Unlike ran-domly selected libraries, VS attempts to incorporate knowl-edge about the receptor and/or ligands into the deck; theresulting subset of the screening deck is sometimes sufficientto identify interesting candidates, although the twoapproaches are widely acknowledged to complement eachother [11-13].

2. Estrogen and its receptors

2.1 EstrogenEstrogen is a critical hormone in the human body, regulatingfunctionally dissimilar processes in diverse tissues. Estrogen isa member of the steroid hormone family, which also includesprogesterone, testosterone, cortisol (glucocorticoids) andaldosterone (mineralocorticoids) that together control manyaspects of mammalian physiology. Steroid hormones are syn-thesised in tissues throughout the body, including the ovaries(estrogen and progesterone), testes (androgens or testoster-one) and adrenal glands (cortisol, androgens and aldosterone).Additional physiologically relevant estrogen-based steroids,such as estrone and estriol, are also known to mediate biologi-cal functions in the body. Among estrogen’s physiologicaleffects are the regulation of growth, development and home-ostasis in tissues and organs. The best understood of thesefunctions are mammalian female reproduction and breastdevelopment [14]. In addition, estrogen regulates skeletal phys-iology [15], (cardio)vascular function [16], the CNS [17] andimmune system [18]. In the clinical arena, estrogen is perhapsmost appreciated for its role in stimulating the proliferation ofapproximately two-thirds of breast cancers [19,20].

Estrogen-like activity can also be found in a large variety ofsources, both natural and man made. These includephytoestrogens/isoflavonoids, from plants and fungi [21], aswell as xenoestrogens, which include a variety of pesticides,polychlorinated biphenyls and plasticisers [22,23]; for example,diethylstilbestrol (DES) was used during 1938 – 1971 to pre-vent miscarriages or premature deliveries [24]. In uteroexposure to DES has been shown to have carcinogenic, tera-togenic and reproductive effects on both the original patientas well as the children of treated individuals [25]. Most of thesecompounds are thought to exert their effects through theinappropriate activation of ERs [26].

2.2 Classical estrogen receptorsThe effects of estrogen are mediated by specific receptors thatbind to the hormone and traditionally regulate transcription,

resulting in the modulation of gene expression. The first ERto be characterised (termed ER and later ERα) was discoveredin 1973 as a consequence of specific estrogen activity in ratuterus extracts [27]. In 1995, the discovery of a related ER,ERβ [28] that was highly homologous to ERα, created newcomplexities in the understanding of the mechanisms of estro-gen action. Steroid receptors display a modular organisationconsisting of a ligand-binding domain, DNA-binding domainand two transcriptional activation domains. Estrogen bindingto ERs results in dimerisation and association with coregula-tory transcriptional factors, resulting in transcriptional activa-tion or repression, depending on the target gene. TheDNA- and ligand-binding domains of ERα and -β are highlyhomologous; therefore, both receptors bind compounds withsimilar affinities and recognise identical DNA sequences,leading to the conclusion that they may exhibit overlappingfunctions. However, the lack of homology in the transcrip-tional activation domain and differences in the tissue distribu-tion of the two receptors [29,30] suggest that there arefunctional physiological differences between the two receptorsubtypes. The unique phenotypic properties of ERα and -βknockout mice lend support to the unique roles of the indi-vidual ERs in vivo [31] and have led to the search forsubtype-specific ERα and -β modulators [4].

2.3 A novel intracellular transmembrane estrogen receptorOver the years, reports have described estrogen-mediatedresponses (initiated by receptors that are either similar to or dis-tinct from the classical ERs [32-36]) that result in both traditionalgenomic (transcriptional) activity as well as nongenomic (rapid)signal transduction events [37]. Nongenomic signalling path-ways are those that are traditionally thought of as arising fromclassical growth factor receptors and GPCRs and include theproduction of second messengers such as Ca2+, cAMP andnitrogen oxide. Kinase activation (e.g., PI3K, MAPK familymembers, Src family members and PKA/PKC) has also beenshown to result from estrogen stimulation of cells [36,38-42]. Thedescription of G-protein-dependent estrogen-mediated signal-ling and membrane localisation of estrogen-binding sites led tospeculation of the existence of a transmembrane ER, possibly ofthe seven-transmembrane GPCR family. Between 1996 and1998, a GPCR homologue was cloned by four different groups[43-46] and shown to be expressed in many tissues throughoutthe body [44-46]. In 2000, Filardo et al. demonstrated MAPK(Erk1/2) activation by estrogen in breast cancer cell lines thatexpress GPR30, but not in cell lines lacking the receptor [47]. Inaddition to estrogen, ER antagonists or SERMs (ICI 182,780and tamoxifen) were also capable of activating Erk inGPR30-expressing cells. Cell activation was characterised tooccur through the transactivation of EGFRs via release of cellsurface heparin-bound EGF [47] as well as through cAMP gen-eration [48]. It was subsequently reported that GPR30 couldpromote Bcl-2 [49], cyclin D [50] and c-fos expression [51],suggesting that GPR30 may play a role in the regulation of

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cellular proliferation and apoptosis, which are natural targetsfor drug discovery.

The involvement of GPR30 in rapid estrogen signallingwas first suggested from the correlation of receptor expressionwith estrogen-mediated signal transduction [47,49-52]. To deter-mine whether the expression of GPR30 results in the genera-tion of estrogen-binding sites, Prossnitz et al. synthesisedfluorescent Alexa-based derivatives of ethynyl estradiol (ahigh-affinity ER agonist), permitting subcellular localisationand quantitative measurements of estrogen-binding sites to beassessed by confocal fluorescence microscopy and flowcytometry, respectively. The fluorescent estrogen exhibitedstoichiometric colocalisation with both classical ERs in thenucleus or GPR30 in the endoplasmic reticulum, consistentwith anti-GPR30 staining patterns [7]. Binding of the fluores-cent estrogen derivative is highly specific for both ER andGPR30 as demonstrated by competition with 17β-estradiol(inhibition constant [Ki] 5 – 10 nM), but not 17α-estradiol.At about the same time, binding of tritiated 17β-estradiol tomembrane preparations from GPR30-expressing cellswas reported to exhibit a Ki value of 3 nM, consistent with thelatter results [53].

Using both the fluorescent estrogen derivative andantibodies directed against GPR30, a correlation was deter-mined between GPR30 protein expression and estrogenbinding in a number of cell lines [7]. For endometrial cancerand choriocarcinoma cell lines, an ‘aggressive’ line was com-pared with a ‘normal’ cell line. The aggressive and normalcell lines were HEC50 and Ishikawa H cells for endometrialcancer, respectively, and JEG and HTR8 cells for choriocar-cinoma, respectively. In each case, the aggressive cell linestained strongly for GPR30 using the antibody, whereas thenormal cell line expressed low levels of GPR30. This patternwas reproduced using the fluorescent estrogen, which fur-thermore confirmed the intracellular expression of endog-enously expressed GPR30. In addition, three breast cancercell lines (MCF7, SKBr3 and MDA-MB-231) were exam-ined. Although the MDA-MB-231 cells expressed low ormoderate levels of GPR30, both MCF7 and SKBr3 cells dis-played high expression of GPR30. Interestingly, SKBr3 celllines are often described as estrogen unresponsive becausethey lack classical ERs, despite reports of cellular effects ofestrogen treatment [54]. However, results demonstrate thatSKBr3 show PI3K activation in response to estrogen stimu-lation and that depletion of GPR30 (with antisense) elimi-nates this response [7]. Similarly, MCF7 cells also expresshigh levels of GPR30. This cell line has been used exten-sively since its development in 1973 [55] in thousands ofpublications (based on a search of PubMed) as anERα/β-expressing cell line with the assumption that all ofthe effects of estrogen are mediated solely by these receptors.Having demonstrated that these cells also express GPR30,the question arises as to whether this assumption is valid andwhether some of the effects of estrogen in MCF7 cells are(in fact) mediated by GPR30. With the introduction of

GPR30 as a validated estrogen-binding and -responsivereceptor, the need for receptor (ERα, ERβ and GPR30)-specific ligands becomes paramount to resolve the propertiesof these receptors in a complex environment.

3. Virtual screening for estrogen-like ligands

3.1 Ligand versus target-based virtual screening for estrogen receptorsThere are two distinct, although not mutually exclusive,approaches to VS: ligand-based VS (LBVS), which uses noinput from the presumed receptor target; and target-based VS(TBVS), which is predominantly based on information gath-ered from the receptor and, sometimes, the known ligands.Steroid receptors as a class are particularly interesting as theendogenous ligands (e.g., estradiol, testosterone, progesteroneand cortisol) are rigid in structure. This leaves no room forspeculation regarding the bioactive conformation and favoursLBVS in particular if using three-dimensional information [5].The ligand-binding domain of ERα has been solved in bothits agonist (17β-estradiol) and antagonist (4-hydroxy-tamoxifen)-bound conformations (pdbId = 1ERE [56] and3ERT [57], respectively), and several TBVS reports have identi-fied active ligands for the nuclear ER; for example, selectiveERα antagonists [58] and selective ERβ ligands [59]. However,the authors of this review recommend LBVS as the VS tech-nology of choice for steroid receptors in general as it can(in the proper bioassay context) also capture structures ofinterest for example; membrane-bound steroid receptors suchas GPR30 (which do not at present have an availablethree-dimensional structure).

3.2 Virtual screening methodologyThe LBVS strategy is rooted in the chemical similarity princi-ple; that is, ligands with similar features are expected to havesimilar biological activity [60]. Thus LBVS does not work inthe absence of at least one known, preferably potent and rigidligand. It is ideally suited when using 17βE2 as a query for allof the targets that bind estradiol with high affinity.

There are three elements that define a LBVS query: the refer-ence point (the query itself ) against which all other ligands inthe virtual library are compared; the chemical similarity index(the numerical value that is assigned to the similarity value);and the chemical space metric (chemical descriptor space). Byusing different queries, similarity indices and chemical descrip-tors, one is very likely to obtain different results, even when per-forming a LBVS for the same end point and using the samevirtual library. However, this is no different than the variedresults that could be obtained running physical or biomolecular(HTS) screens based on different assays (e.g., ligand binding,signal transduction and gene transcription).

3.2.1 Similarity indicesChemical similarity is expressed as a number (or set ofnumbers) that quantifies the extent to which two

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compounds are related, according to a given index. In thisstudy, Tanimoto’s symmetric distance between patterns [61]

and Tversky’s asymmetric contrast model [62] were usedwhen evaluating the overall similarity. In addition to thesetwo metrics, the Euclidian distance was used across the firstsix different principal components extracted fromALMOND descriptors [63] using principal componentanalysis (PCA) [64].

3.2.2 Chemical descriptorsMolecular (or chemical) fingerprints are binaryrepresentations for chemical structure characterisation thatcapture different molecular descriptors such as atomic dis-tances, pharmacophore patterns or unique structural paths.Two basic types of fingerprints are distinguished: structuralkeys and hashed fingerprints. In the 17βE2 LBVS study, itwas decided to use prototypes of both. With structural keys,

Figure 1. The top five ranked compounds according to the composite similarity scores (Tanimoto and Tversky) for the MDLand Daylight keys are shown. Compounds are depicted in the appropriate ranking order. See also Figure 2.

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Figure 2. The top five ranked compounds according to the composite similarity scores for ROCS (Tanimoto and Tversky) andfor ALMOND are shown. The ALMOND scores are based on the Euclidian distance in principal component analysis space. Compoundsare depicted in the appropriate ranking order. Note how two-dimensional-based methods (see Figure 1) are biased by the estrogenscaffold, whereas three-dimensional methods reflect a significant departure from it. Because ALMOND does not include a shape-basedcontrol (pharmacophore only), its top ranking compounds reflect the hydrogen bonding pattern (donor/acceptor) of 17β-estradiol.

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individual bits are set to 1 or 0 according to the presence orthe absence of a particular (predefined) chemical fragmentidentified from a dictionary of such fragments. The MDL320 keys [65] were used, generated with the Mesa Analyticsand Computing Fingerprint module [101]. Alternatively, withhashed fingerprints, there is no direct relationship betweenbits and features as the software does not use a predefined setof fragments. Therefore the 2048 bits fingerprints were usedfrom Daylight Chemical Information Systems [102], with thepath length set to 8 (default).

As 17βE2 is a rigid ligand, one should take advantage ofthe information provided when searching for three-dimen-sional similarity as well. The authors used ROCS(Rapid Overlay of Chemical Structures) [66], a Gaus-sian-shape volume overlap filter that rapidly identifiesshapes matching the 17βE2 shape. Multiple conformers perlibrary molecule were explored, as ROCS is a computation-ally efficient algorithm; the multiple conformers wereobtained using OMEGA (OpenEye Scientific Software,New Mexico [103]) a conformer generator that has the abilityto retrieve the bioactive conformation [67].

However, the use of three-dimensional similarity withROCS strictly provides a shape-based comparison, as noinformation concerning the electrostatics of the 17βE2 andlibrary molecule is considered during the similarity compu-tation. Therefore, pharmacophore perception methods canbe used to evaluate those molecules that have significantshape similarity with the query; for example, usingALMOND [63]. This method processes GRID interactionfields [68] starting with the hydrophobic, H-bond donor andH-bond acceptor GRID probes, and encodes them to a fixednumber of auto- and crosscorrelograms.

3.3 Virtual screening resultsThe top five LBVS hits according to the composite index(Tversky and Tanimoto) are shown for both the chemicalfingerprints (Daylight and MDL, Figure 1) as well as forROCS (Figure 2). Only the top 10% of compounds werescored with ALMOND; of these, the final top five moleculesare also presented in Figure 2. ALMOND was used to ‘fine-tune’ the three-dimensional-based similarity selection, whichis done by adding pharmacophore features to an alreadymatching shape. However, different similarity metrics arelikely to yield very different answers [8]. Because one cannotknow a priori which combination of similarity metric andchemical descriptors provides the ‘best’ answer (that is, ahigh-activity molecule), a combined similarity score was usedas follows: 40% weight was attributed to two-dimensional fin-gerprints, 40% to the ROCS-based similarities and, finally,20% to ALMOND-based similarity (Figure 3). Thus, Tanim-oto and Tversky (substructure and superstructure) coefficientswere given a 6.66% contribution each for both Daylight andMDL fingerprints and a 13.33% contribution each for shape.The Euclidian distance in PCA was given a 20% weight to thefinal score.

Given this composite score, the top 100 ranked moleculeswere selected for biomolecular screening. This cutoff (100)proved to be quite arbitrary as the G-1 structure (vide infra)was ranked 97 and could have been missed. However, twoother true hits that were active on the classical ERs (manu-script in preparation) were ranked higher in the list, whichseems to imply that different factors (perceived by the chemi-cal descriptors) contribute to binding affinity in a differentmanner for different ERs.

4. Biomolecular screening for GPR30-specific ligands

4.1 Design of fluorescent estrogen ligandsIn general, fluorescent probes offer greater convenience andversatility for investigating receptor binding in vitro and cellculture than traditional radiolabelled probes. Fluorescencemicroscopy enables characterisation of the spatial distributionand intracellular localisation of steroid receptors, whereasrapid quantification of receptor content and high-throughputbinding assays are possible using flow cytometry. Solvato-chromic- and pH-dependent shifts in absorption/emissionproperties and the dependence of fluorescence quantum yieldon the environment are important practical considerations forcertain fluorometric assays. Efficient fluorescent emissionfrom the receptor-bound probe and the ability to distinguishfree ligand, nonspecific binding and cell autofluorescence arenecessary for direct detection. Emission quenching by reso-nance energy transfer can occur following binding or throughselfassociation of hydrophobic dyes; examples includenitrobenzoxadiazole labelled estrogens and progestins (thatwere nonemissive when receptor bound [69]) and BODIPYestradiol derivatives that display high relative binding affinity,but fluoresce more intensely when bound nonspecifically [70].Careful optimisation of structural and spectroscopic charac-teristics can provide steroid probes that exhibit high recep-tor-binding affinity, low levels of nonspecific binding, anddesirable absorption and emission properties. Cell membranepermeability can be affected by the size and electronic chargeof the probe and this provides an additional parameter forprobe development.

Two strategies have been used for the construction of fluo-rescent steroid probes: pendant and integrated. In the pen-dant approach, fluorescent moieties are connected to thesteroid ligand. Understanding of rational design and detailedstructures are required to accommodate the large steric vol-ume and multiple charges of most dyes without compromis-ing receptor binding. The commercial availability offluorescent dyes covering a wide range of excitation/emissionspectra maximises the versatility of this approach. Thelength, conformational flexibility, and polarity of the linkageconnecting the steroid and fluorophore are important designconsiderations that must be optimised to minimise unfa-vourable steric interferences and balance the loss in entropywith favourable enthalpies of receptor binding. Estradiol

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derivatives incorporating rigid alkyne linkages in the17α-position have provided high-affinity probes in combina-tion with AlexaFluors, fluorescein (Figure 4) andCy dyes [7,44,71].

Integrated probes consist of a high-affinity receptor ligandpossessing inherent fluorescent properties. This approachoffers an obvious steric advantage over the pendant design,

although compatible photophysical properties typicallyrequire rigid, planar molecules with extended conjugation.

Examples include naturally occurring fluorescentphytoestrogens, such as coumestrol [72] and synthetic estro-gens such as 12-oxo-9(11)-dehydrestradiol [73]. High-affinitystilbene-like tetrahydrochrysene derivatives have been used forvisualisation of ER in transfected COS7 cells expressing ele-vated levels of ER [74]. Detailed structural information fromX-ray crystallography of ER-bound agonists and antagonistscombined with computational docking studies have providedinsight into the observed kinetics of estradiol binding to ERαand -β measured using fluorescence spectrometry [75].

4.2 Assay design and implementationBased on the competitive binding of the fluorescent estrogenin GPR30-expressing cells by 17β-estradiol, a flowcytometric approach was used to screen the 100 compoundsselected by VS [76]. COS7 cells were transfected with aGPR30 green fluorescent protein construct and serumstarved overnight. Cells were incubated with 10 µM testcompound for 20 min in a final volume of 10 µl in a 96-wellplate. The fluorescent estrogen derivative (an Alexa633 con-jugate of 17α-[4-aminomethyl- phenylethnyl]-estra-1,3,5(10)-triene-3,17β-diol; Figure 4) diluted insaponin-based permeabilisation buffer was added to a finalconcentration of ∼ 4 nM. After 10 min incubation at 37°C,the cells were washed once and re-suspended to 20 µl. Sam-ples consisting of 2 µl volumes were analysed from the96-well plate using HyperCyt (see Section 4.3) and analysedon a FACS Calibur. To maximise the binding signal, cellswere gated for high GFP (GPR30) expression in FL1. Bind-ing of the Alexa633 estrogen derivative was detected in FL4with nonspecific binding determined in the presence of 100nM 17β-estradiol. Z′ scores (assessing robustness of theassay) were in the range of 0.5 – 0.7 [77]. The results of thisscreening approach yielded one compound referred to asG-1 (GPR30-specific compound 1; Figure 5), a substituteddihydroquinoline that reproducibly competed for binding ofthe fluorescent estrogen. The structure of the compoundwas reconfirmed using 1H NMR and mass spectrometry.

Figure 3. The top five ranked compounds according to theweighted similarity score based on the four descriptorsystems. The weighting scheme was: 40% two dimensional(MDL and Daylight), 40% ROCS and 20% ALMOND.

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Figure 4. Structure of fluorescent estrogen derivative (anAlexa633 conjugate of 17α-[4-aminomethyl-phenylethynyl]-estra-1,3,5(10)-triene-3, 17β-diol) usedfor characterising and screening GPR30.

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In addition, resynthesis of the compound yieldedactive material.

4.3 Biomolecular screening with the HyperCyt platformThe HyperCyt platform interfaces an auto sampler with a flowcytometer [78-80]. As the sampling probe of the autosamplermoves from one well to the next of a multiwell microplate, aperistaltic pump sequentially aspirates sample particle suspen-sions from each well. Between wells, the continuously runningpump draws a bubble of air into the sample line. This resultsin the generation of a tandem series of bubble-separated sam-ples for delivery to the flow cytometer. Sample and bubble vol-umes are determined by the time the auto sampler probe is ina microplate well aspirating liquid or above a well aspiratingair; ∼ 2 µl is aspirated from each well during a single round ofsampling. A 96-well plate is processed in < 2.5 min and a384-well plate in < 10 min. Between 1000 and 3000 cells aretypically analysed from each well. Due to the relatively smallvolume and time requirements for a single round of sampling,a second round is typically performed immediately after thefirst and the results averaged.

The platform currently used is a custom instrument builtand patented by the authors at the University of New Mexico.It is the key biological screening platform used by the NewMexico Molecular Libraries Screening Centre, a foundingmember of the NIH-sponsored Molecular Libraries ScreeningCentre Network (see Section 7.4).

5. Molecular and cellular characterisation of a GPR30-specific ligand

5.1 Binding propertiesDetailed analyses of the binding properties of G-1 involvedcompetition dose responses for GPR30, ERα and -β, per-formed both manually and using HyperCyt. Using the com-petition binding assay 17β-estradiol yields Ki values of 0.3and 0.4 for ERα and -β, respectively, in comparison withGPR30, which yields a Ki value of 6 nM [7,76]. Surprisingly,the Ki value exhibited by G-1 for GPR30 was only two timeslower (at 11 nM) than that observed for estradiol. Perhapsequally surprising, G-1 displayed no significant binding toeither ERα or -β at concentrations of ≤ 10 µM. Thus, G-1exhibits both high affinity and high selectivity for GPR30. It should be emphasised that GPR30 also displays highspecificity for 17β-estradiol over other steroids. Theauthors [7] and others [53] have shown that 17α-estradiol (aswell as progesterone, testosterone and a collection of otherphysiological steroids) is inactive in terms of binding oractivation of GPR30. This is consistent with a well-defined ligand-binding pocket that exhibits highstereoselectivity in the recognition of the estrogenmolecule. Overlay of the structure of G-1 and17β-estradiol reveals that the two oxygen atoms of17β-estradiol and two of the three oxygen atoms of G-1can occupy similar regions in space [76]. However, the G-1molecule is slightly longer than estrogen, thus providing apossible explanation for its lack of binding to classical ERs.Docking of G-1 to both the estrogen- andtamoxifen-bound conformations of ERα suggests that evenin the best-docked pose, G-1 would exhibit a bindingaffinity of at least two to three orders of magnitude worsethan that of estrogen or tamoxifen in their respective ERαconformers [76].

5.2 Functional propertiesEstrogen is known to mediate multiple rapid, nongenomiccellular signalling pathways following its administration.The authors initially chose to use two distinct signallingpathways to characterise the effects of GPR30 activation.The first was calcium mobilisation because of its high tem-poral resolution, where responses can be convenientlydetected on the second time scale using fluorescent calciumindicators such as Indo1. The second was PI3K activation,an important regulator of cell function. Although ofteninterrogated via the phosphorylation of Akt using westernblots, it was decided to use a reporter capable of yieldingsubcellular spatial resolution. This reporter consists of a flu-orescent protein (either GFP or RFP) fused to the pleckstrinhomology domain of Akt that is responsible for localisingAkt to membrane sites rich in PIP3 resulting from PI3Kactivity. If transiently expressed in cells, this reporter is usu-ally found in the cytoplasm, where it translocates to theplasma membrane following stimulation through a plasma

Figure 5. Structure of the GPR30-specific agonist G-1 (top)compared with the structure of 17β-estradiol (bottom).

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membrane-localised receptor (e.g., EGFR), the site ofPI3-kinase-mediated synthesis of PIP3. The exception is cer-tain cancer cell lines with constitutively activated PI3K,resulting (for example) from ErbB2 overexpression(e.g., SKBr3 cells), where the reporter is found associatedwith the plasma membrane even in ‘unstimulated’ cells.

An important aspect to the characterisation of recep-tor-specific estrogen-mediated effects is the use of definedreceptor expression patterns in cells. The use of multiplehighly divergent cells lines (e.g., breast cancer cell lines suchas MCF7 and MB-MDA-231) that are only defined withrespect to a small collection of receptors (e.g., ERα/β,EGFR, and so on) can lead to misleading results due to themyriad of additional differences between the cell lines thatmay not be recognised. For this reason, many of the signaltransduction studies were carried out in COS7 cells (whichlack ERα/β and GPR30, and do not respond to estrogenstimulation) transfected to specifically express one receptortype. The results obtained with COS7 cells were thenextended to and confirmed in cancer cell lines expressing theendogenous receptors.

The results produced have demonstrated that estrogencan activate calcium and PI3K pathways through both clas-sical ERs as well as through GPR30. However, the signaltransduction pathways are distinct for the two receptortypes, with one major difference being the transactivationof EGFRs by GPR30, but not ERs [7]. However, it shouldbe noted that ER-mediated transactivation of EGFR hasalso been reported [36], although the GPR30 status of thecells used is unknown. Characterisation of G-1 revealedthat this compound is capable of mediating calcium mobi-lisation and PI3K activation through GPR30, but not ERsexpressed in COS7 cells. The EC50 values correspondedclosely to the binding affinity determined for G-1 fromcompetition assays. Interestingly, the kinetics of calciummobilisation for G-1 were slower than that observed forestrogen, suggesting either that G-1 traverses the mem-brane more slowly, or binds/activates GPR30 more slowlythan estrogen. To determine whether G-1 also activatesGPR30 when expressed endogenously, the effect of G-1on the activation of PI3K in both SKBr3 cells (ERα/βneg-ative and GPR30-positive) and MCF7 cells (ERα/β-posi-tive GPR30-positive) was tested. In both cell types, G-1was capable of activating PI3K. These results are importantfor two reasons. First, SKBr3 cells, which lack classicalERs, express GPR30 and respond to both estrogen andG-1 stimulation with PI3K activation. Second, MCF7cells, are routinely used as ‘ER positive’ cells to examinesignalling, both genomic and non genomic, through classi-cal ERs. As these cells also express GPR30, effects of estro-gen cannot be definitively assigned to ERs. The activationof PI3K in these cells by G-1 establishes thatGPR30-mediated effects must also be considered in thisand other cell lines.

6. Conclusion

It has become apparent that the actions of steroids (andestrogen specifically) may be mediated by receptors otherthan the classical nuclear steroid receptors. To investigate theunique physiological functions of the individual receptors,specific ligands are required much in the same way as classi-cal pharmacology revealed the roles of GPCR subtypes.Because of the rigid nature of steroids, a LBVS approach wasemployed to identify a subset of library compounds for sub-sequent biomolecular screening. The importance of VS tech-nologies is, by now, widely recognised in public andindustrial sectors. Its application as a ‘preselection tool’ priorto biomolecular screening allows the users to enhance theinformation extracted by querying the receptor binding siteusing chemical probes, thus ensuring that maximum knowl-edge is gained from the experiment. Coupled with flow cyto-metric-based biomolecular screening (employing a novelfluorescent estrogen derivative) a ligand capable of bindingto GPR30 was rapidly identified [76]. Given that bothclassical ERs and GPR30 are likely to be coexpressed inmany cells and tissues, it becomes even more imperativeto develop receptor-specific ligands capable of probing thespecific functions of these receptors.

The GPR30-binding compound displayed high selectiv-ity for GPR30 compared with the classical ERα and -β.This was demonstrated not only in ligand binding assays,but also in functional assays, where it was revealed that thecompound behaved as an agonist for GPR30 with no activ-ity towards the classical ERs. Most importantly perhaps, isthe fact that in cells expressing both classical ERs andGPR30, it could be demonstrated that there was activityspecifically through GPR30, leading to the conclusion thatin cells and tissues expressing both receptor types, GPR30 isprobably mediating some of the effects of estrogen. Clearly,the work described in this review represents merely thebeginning of a new understanding for the mechanismsinvolved in the actions of estrogen and potentially othersteroids. Future work will focus on deciphering the individ-ual roles of specific estrogen-binding receptors, through thedevelopment of specific probes to target these receptors,with the eventual goal of developing improved therapeuticapproaches for hormone-dependent diseases.

7. Expert opinion

7.1 Future of virtual screeningVS technologies evolved initially by sacrificing accuracy forthe speed of computation, to match the throughput ofexperimental methods [10]; however, algorithmic and hard-ware advances in the past decade have enabled the use ofmore sophisticated methods, thus it can be considered thataccuracy is no longer an issue except for the limited abilityto appropriately model complex biophysical phenomena as

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parameterised by computational methods in general. Forsteroid-binding targets, the VS technique of choice appearsto remain rooted in ligand-based methods. A recent queryin WOMBAT 2006.1, an annotated database of bioactivemolecules [81], indicates that there are 298 potent (≥ 10 nMaffinity or better) and rigid (≤ 1 flexible bonds) moleculesactive on class 3 estrogen-like (estrogen receptors, estro-gen-related receptors, glucocorticoid receptors, mineraloco-rticoid receptors, progesterone receptors, androgenreceptors) and class 1 thyroid-hormone-like nuclear hor-mone receptors (oxysterol receptors and farnesoid recep-tors). This wide array of molecules, or a subset thereof, canbe used to seek novel active ligands using ligand-basedmethods. As discussed in this review, in the right biomo-lecular screening context, such queries are also likely toretrieve active molecules for non-nuclear receptors. This ismore likely to occur using LBVS compared with TBVS, asthe ligand-binding domains for non-nuclear steroidreceptors (e.g., GPCRs) are unknown. Conversely, there are99 potent steroid-based ligands in WOMBAT 2006.1 thatare reported as being active on non-nuclear hormone tar-gets. These targets are as follows: the σ-type 1 receptor,cholestenol δ-isomerase, δ-24-sterol-reductase,17α-hydroxylase, 5α-steroid dehydrogenase, steroid sulfa-tase, inducible nitric-oxide synthase, aromatase and thedigitalis receptor site of the Na+/K+ ATPase. Thus, there iswide applicability for steroid-based ligand screening out-side the nuclear receptor and GPCR target classes. Theauthors anticipate that steroid-binding targets will turn outto be more amenable to LBVS technologies, particularly ifone is equipped with the necessary tools to run multipletarget profiling, and thus be able to discriminate differentbinding classes of ligands.

7.2 Opportunities for membrane-localised estrogen receptorsIn addition to the classical ERs and GPR30, many reportshave been published over the years characterising the existenceof ‘novel’ ERs and binding sites. Some of these appear to rep-resent truncated forms or splice variants of the classical recep-tors. Other reports describe the presence of estrogen bindingsites or activity at the plasma membrane. These include locali-sation of classical receptors or their variants to the inner leafletof the plasma membrane (e.g., caveolae [33,82]), as well asreceptor proteins and estrogen-mediated effects via receptorson the exterior of the cell [83]. Whether GPR30 (under certaincircumstances or in certain cells/tissues) is expressed on thecell surface instead of the endoplasmic reticulum also remainsto be determined, although so far no convincing reports ofplasma membrane-localised GPR30 exist [53]. To the authors’knowledge, no other GPCRs have been shown tofunction from an intracellular location, although a putativepigment cell-specific orphan GPCR, OA1, is localised tomelanosomes [84] and data suggest that lipid mediators mayinitiate long-term transcriptional effects through GPCRs

localised to the cell nucleus [85]. Defining the plethora ofdescribed estrogen-binding receptors will be an importanttask for researchers in the foreseeable future. Only once thespectrum of physiologically relevant estrogen-bindingreceptors is well defined and specific reagents developed foreach member will a thorough understanding of the mecha-nisms through which estrogen mediates its effects be gained.This understanding is requisite for the development oftarget-specific drugs exhibiting minimal side effects.

7.3 Other GPCR steroid receptorsA significant question to consider is whether estrogen isunique in its ability to bind and signal through both classicalnuclear as well as seven-transmembrane GPCRs. This possi-bility seems to be unlikely particularly given the description ofa family of transmembrane G-protein-coupled progesteronereceptors first characterised in fish [86] with homologues inhumans [87]. Although these receptors do not exhibit the con-served amino acids that define classical GPCRs, progesteronestimulation leads to cAMP modulation and ERK activationthrough transfected receptors [86]. Recent descriptions of pro-gesterone receptor membrane component-1, a singletransmembrane-spanning protein expressed in the ovary,further support the existence of additional steroid receptortypes [88]. Nongenomic effects and membrane binding ofandrogens have also been reported, leading to the proposalthat the effects of this hormone may be mediated through sexhormone-binding globulin as well as GPCRs [89]. In addition,membrane active glucocorticoid and mineralocorticoid recep-tors, as well as rapid signalling events in response to these ster-oids, have been described [90-93]. Thus the effects of all steroidsmay be mediated by multiple classes of receptors leading tothe existence of highly complex networks of signalling eventsthat are ultimately integrated to produce the appropriatephysiological response.

7.4 Academic screeningFinally, HTS, traditionally the realm of large pharmaceuticalcompanies, is now being carried out by academic researchers.At present, 10 academic institutions (funded by the NIHRoadmap initiative) are performing HTS based on assays sub-mitted by individual academic researchers. The results of all ofthe screens will be posted to PubChem and will be freely avail-able to the public. This is in contrast to the screening resultsobtained by pharmaceutical companies, which are naturallyconsidered trade secrets.

There are multiple benefits anticipated from this effort.First, the overall initiative is predicated on the notion ofchemical genomics to complement traditional genomics andthe human genome project. The idea is that a library of sub-stantial size (> 100,000 small molecules) will be screenedagainst 500 biological targets over the next few years. Theproject itself will contribute to determining the specificityand selectivity of novel families of molecules across a diverseselection of biological target space. Second, the data from the

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500 high-throughput screens and follow-up optimisationchemistry will be publicly disclosed, leading to unprece-dented rapid experimentation characterising the activity ofthe molecules in many laboratories in many more experimen-tal systems than would have ever been possible before. Third,the screening centres are organised within a network thatencourages extensive collaboration. Although targets areassigned to individual centres, it is expected that complemen-tary technologies in screening and VS will emerge to pro-mote the overall success of the discovery process. Fourth, inthe case of the authors’ centre and team of investigators, the

work looks forward in a bench to bedside trajectory. Thus theauthors are engaged not only in the discovery of smallmolecules, but in the elucidation of signalling pathways andpharmacological mechanisms. The authors are also activelyengaged in target validation to define the role of targets, suchas GPR30, in disease outcome and as biomarkers and alsoexpect to use discovered probes for the generation of radiola-belled agents to be used in diagnostics and therapeutics.Thus academic screening is poised to advance our under-standing of complex biological systems through the discoveryof novel molecular probes.

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Websites

101. http://www.mesaac.com This is the company website for Mesa Analytics and Computing LLC. One of their products, the Fingerprint Module, encodes the MDL 320 keys.

102. http://www.daylight.com/ This is the company website for Daylight Chemical Information Systems. One of their products, the Daylight Toolkit package, encodes the Daylight 2048 fingerprints.

103. http://www.eyesopen.com/products/applications/omega.htmlThis is a direct link to OMEGA, one of the products from OpenEye Scientific Software. OMEGA provides multi-conformer (3D) structures from two-dimensional input chemical structures.

AffiliationEric R Prossnitz†1,2, Jeffrey B Arterburn5, Bruce S Edwards2,3, Larry A Sklar2,3 & Tudor I Oprea2,4

†Author for correspondence1Department of Cell Biology & Physiology, MSC08-4750 University of New Mexico, Albuquerque, NM 87131, USATel: +1 (505) 272 5647; Fax: +1 (505) 272 1421; E-mail: eprossnitz@salud.unm.edu2Cancer Research and Treatment Center University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA3Department of Pathology, University of New Mexico, Albuquerque, NM 87131, USA4Division of Biocomputing, MSC11-6145, University of New Mexico, Albuquerque, NM 87131, USA5Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico, 88003, USA

Author Proof