The estrogen receptors (ERs) are members of the steroid hormone receptor family, which taxonomically are within the nuclear receptor (NR) superfamily (Figure 1). The steroid hormone receptor family (a.k.a. NR3) has many members, including the mineralocorticoid, glucocorticoid, progesterone, androgen, and estrogen-related receptors (MRs, GRs, PRs, ARs and ERRs, respectively) [1,2]. These receptors are targets of the lipophilic steroid hormones sharing cholesterol as a common building block and are synthesized in the adrenal cortex (e.g., glucocorticoids, mineralocorticoids and adrenal androgens), testes (e.g., testosterone and testicular androgens), ovary and placenta (estrogens and progesterone) [3].

The role of the ERs far transcends the most commonly ascribed purposes of regulating development of female characteristics and the female reproductive system. ERs are also indispensable in a diversity of physiological processes that include regulation of lipid profile, bone integrity, hemostasis, endothelial functions, inflammatory markers, the growth of different tissues, and both prenatal and postnatal development [4,5,6]. This extensive involvement may be attributable to the status of an ER as the common ancestor of members of the steroid hormone receptor family [1,7]. Estrogen mediates both male and female health, having both adverse and beneficial effects in both sexes. For example, the ERs and their hormone ligands are implicated in stroke [8], cancers (e.g., endometrial [7] and breast [9]), as well as postmenopausal symptoms resulting from depleted circulating estrogen (e.g., osteoporosis, hot flushes and vaginal atrophy).

The ERs are divided into two major subtypes, ERα and ERβ, which possess distinctly different identities [5,10,11]. A third subtype, ERγ, found only in non-human species e.g., fish, has also been reported [12]. The ERα and ERβ subtypes are coded by different genes, ESR1 and ESR2 respectively, and are distributed and play multiple roles in a tissue-dependent manner. For example, the ERα is more prevalent in the gonads, mammary glands, kidney and lung bronchi, while ERβ predominates in bone, lung alveoli and prostate tissues [5,13,14]; furthermore, ERα has been found to promote cell proliferation while ERβ possesses an anti-proliferative effect in the mammary tissues [15,16,17]. The ERα and ERβ appear to share a modest 47% [10,18] and 56% [5], respectively, overall and ligand binding domain (LBD) sequence identity; yet, only two residues among those that line the binding pocket are found to be different: Leu384 (ERα) vs. Met336 (ERβ) and Met421(ERα) vs. Ile373 (ERβ) [5] (Figure 2).

The ERs share a common architecture with other members of the NR superfamily that are composed of six evolutionarily conserved domains A, B, C, D, E and F (Figure 3). The solvent-exposed A/B region located at the N-terminus contains the ligand-independent activation function 1 (AF1) and is responsible for protein-protein interactions [2,5]. The highly conserved C region is the DNA binding domain, and is responsible for DNA dependent and independent receptor dimerization [21], as well as for binding to specific regions of the DNA (e.g., the estrogen response elements, EREs) [5]. The little-known and poorly conserved D region is the linker region that contains the nuclear localization sequence as well as sites for co-modulator recruitment and post-translational modifications. The E and F regions located at the C terminus contain the LBD and the ligand-dependent activation function 2 domain (AF2), as well as sections for nuclear localization and homo/hetero dimerization [5,22]. It is worth noting that several splice variants exist for both ERα (over twenty) and ERβ (at least five) subtypes [23,24,25]. While the wild-type ERα and ERβ (66 and 59kDa, respectively) contain complete A-F regions and are considered as full-length, the alternative splice variants either lack a portion of the protein or contain duplicate regions compared to the wild-type ERs [23]. Examples of splice variants lacking a portion of the protein are the ERα36 and ERβ2 [2,23]. The former, devoid of the AF1 region, has been found to oppose ERα AF1-dependent transcription [26]; the latter, shorter around the AF2 region due to shorter helix 11, has an altered AF2 conformation that results in limited ligand access [27]. The ERα80 isoform is an example of a splice variant which contains duplicate region as demonstrated by its extended E domain; so far, its function remains unclear [28]. More comprehensive discussions of the ER splice variants are provided by Sotoca et al. [23], Lewandowski et al. [25] and Poola et al. [24].

The LBDs of the ERs have been of intense interest to the scientific community and pharmaceutical industry for obvious reasons: the conformational changes that occur in the LBD following ligand and co-regulator binding determine and often initiate a spectrum of important and different downstream estrogen responses. Concomitantly, the binding site is the target for designing ligands causing a specific and large response of a certain type, called subtype selectivity. This is also precisely the reason why a great number of crystal structures of ERα and ERβ LBDs in complex with a multitude of ligands have been elucidated over the years (See Table 1 and Table 2 for lists of ERα and ERβ LBD crystal structures available to date in the Protein Data Bank (PDB)).

Commonly described as a “three-layered anti-parallel α helical sandwich”, the LBD consists of 12 helices (H1 to H12) and a beta hairpin, with a core region made up of H5, H6, H9 and H10, as well as two flanking outer layers comprising the remaining helices [5,30] (Figure 4a). The H12 that sits at the end of the LBD forms the AF2 with H3, H4 and H5, and plays a key role as a molecular switch [55]. Influenced by ligand binding (although without direct ligand contact [29]), H12 regulates receptor activities and the recruitment of co-regulators to AF2 by adopting distinct conformations, i.e., active vs. inactive conformations [82] (Figure 4b,c, respectively). In the active conformation, H12 rests across H3 and H11, forming a groove to accommodate co-regulator binding; in the inactive conformation, such as when bound to an antagonist, H12 is displaced from this position, which distorts the co-regulator binding groove [59]. Flanked by the beta hairpin and H12, the ligand binding cavity (made up of H3, H4, H10 and H11) is a compact and enclosed ellipsoid cavity situated deep in the core of the LBD [29,82].

The ERs are the target of the natural estrogens, namely estradiol (E2, most potent) [83], estrone (E1) and estriol (E3). E2 is also commonly referred to as 17β-estradiol as it possesses the hydroxyl group at the carbon 17 position above the steroid plane (α and β indicate below and above the steroid plane, respectively) (Figure 5).

Apart from these endogenous ligands, the ERs, quite unlike the other members of the steroid receptor family, are also found to bind to a remarkably diverse range of exogenous substances [84,85,86], earning them a notorious reputation as the promiscuous receptors. Molecules that are able to bind to the ERs span from industrial byproducts (polychlorinated biphenyls, dioxins, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin), plasticizer (phthalates), plastics (bisphenol A), naturally occurring phytoestrogens (genistein), pharmaceuticals products (diethylstilbesterol) to pesticides (dichlorodiphenyltrichloroethane, popularly known as DDT [87,88]). Despite the structural diversity (Figure 6), these molecules appear to possess at least an aromatic ring structure, which is believed to be crucial for having affinity to bind [84,89,90,91,92]. Now frequently referred to as endocrine disruptors, these compounds and a large number more have been implicated in numerous and diverse adverse health effects. Worldwide, authorities regulating drugs, foods, food packaging, veterinary products and medical devices now consider endocrine activity in their regulatory duties, as do authorities responsible for regulating chemicals in the environment [93]. Some examples of the adverse health outcomes include infertility, precocious puberty, various cancers (e.g., breast [94,95], cervical and vaginal cancers [96,97,98]), obesity, diabetes, cardiovascular [87,99] and immune disorders [100].

Ligand recognition and receptor-ligand binding are generally deemed as biological processes that take place with considerably high specificity; this is pivotal in allowing the body to “switch on” only the correct receptors to produce responses appropriate to various in vivo/environmental stimuli amidst the highly complex labyrinth of signaling pathways. The conventional “lock-and-key” model as well as the refined “induced fit” model has been widely accepted to underlie the modus operandi of protein-ligand recognition. These models suggest that the binding site of a receptor only recognizes specific ligands (or those that closely resemble them), bearing complementary size, shape and molecular properties, to the binding site. The fairly “rigid” rules that govern protein-ligand recognition extend further to the ligand binding modes whereby it is widely assumed that a ligand binds to the receptor binding site in a single conformation. This is understandably so as a great number of X-ray structures had supported this point.

The ERs have appeared to be an exception to many aspects of the core concept of lock and key that implies high specificity. In fact, ER ligands have been found to bind to the ER binding pocket in more than one binding orientation which, in turn, affect the final conformations (active/inactive) of the LBD [66]. The apparent lack of specificity, or rather, the high flexibility as demonstrated by the ER in ligand binding can perhaps be justified by the versatility and multi-functional roles of this group of receptors (see review by Lathe and Kotelevtsev [101]). Broadly speaking, the observed flexibility is required for: (1) binding to different endogenous ligands that vary according to tissue type, (2) multifunctional biological roles played by the ERs, and (3) graded activity i.e., partial agonism and antagonism. For the first instance, while E2 is commonly seen as the default endogenous ligand for the ERs, this is not always the case: the 5α-androstene-3β,17β-diol (adiol) is found to replace the E2 as the physiological ligand for ERβ in prostate tissue, microglia and astrocytes; 27-hydroxycholesterol (27OHC) has been found to be the first endogenous selective estrogen receptor modulator (SERM) that acts as the modulator of both ERα and ERβ [101,102,103,104] (Figure 7). The existence of these additional endogenous ligands demonstrates the degree of binding flexibility required for ERs to fulfill their multiple functions. As for multifunctional biological roles of ERs, contrary to the oversimplified concept of a ligand (i.e., an agonist or an antagonist) binding to the receptor with a specific conformation thus leading to a response (i.e., activation or inhibition), the ERs paint a more multifaceted picture of receptor signaling. In the case of ERs, the binding of different ligands, depending on their nature and pharmacological classes, will lead to different LBD conformations and subsequent recruitment of co-regulator proteins. These different conformations and co-regulators, in turn, lead to different signaling through specific secondary pathways [46,82,101,105], eventually producing distinctly different biological responses. For graded activity, Bruning et al. [59] have shown that the ligand binding orientations in the binding pocket affect signaling outcomes which lead to a gradation of activities such as those expected of the partial agonists or antagonists.

The ability of the ERs to flexibly bind to various ligands can be attributed to a number of unique features, as displayed in both ligand association as well as the binding cavity. In the classic example, E2 binding to ERs requires little contact between the receptor and the ligand in that only a portion of the molecule tightly fits within the binding pocket (Figure 8). The E2, as shown by X-ray crystal structures [32,34,63,64,78,81], fits the length and breadth of the pocket perfectly but leaves empty spaces (i.e., sub-pockets) above and below the steroid core, particularly in the C and D ring regions [89]. The phenolic moiety (A ring) of the steroid core binds securely to the pocket by forming hydrophobic contacts with the residues situated above and below the ring, forming a network of hydrogen bonds with E353/305 and R394/346 (ERα and ERβ respectively) [5]. The C and D rings, on the other hand, are anchored to the pocket only by a single hydrogen bond between the D ring hydroxyl group and H524. Given the presence of sub-pockets, there exists adequate space to accommodate conformational changes [5] as well as a variety of substituents (e.g., 7α, 11β, 17α) with little to no steric clash in the vicinity [89].

Finally, the ER’s binding pockets have been shown to exhibit considerable structural plasticity. Larger ligands that would not have been expected to fit into the ER binding pocket have been reported to bind to the ER pocket as a result of the formation of novel binding grooves [30,55].

Clearly, the ERs belie common lock and key dogma, instead showing considerable non-discriminatory nature in the ligands they will bind. ERs evolved this way to fulfill diverse and tissue-dependent biological roles, a characteristic highly conserved from ancestral receptor forms to present. The complexity of ER interaction grows further when subtype selectivity between the ERα and ERβ is considered.

In spite of the enormous structural diversity of compounds that bind to the ERs, the receptors exhibit high sensitivity to ligand structure as measured by differential binding affinity or downstream activation and signaling response. Minor modifications to ER-binding compounds will normally cause pronounced change in the binding affinity or even complete loss of activity [82,89]. For example, ketosteroids (e.g., androstenedione), although structurally similar to E2, do not bind to the ERs due to the switch from hydroxyl to ketone functional group in the A ring, which results in the disruption of the crucial hydrogen bond network.

Given that receptor promiscuity is by Nature’s design, it is not surprising that nature has also evolved alternate means to achieve specificity and, indeed, subtype selectivity. Some of the alternate ways of managing proper hormone signaling include elevating the concentration of circulating and tissue-specific hormone levels to the μM or mM range (e.g., 27OHC), and the use of “gating” enzymes (e.g., CYP7B1) to limit the access of certain hormones to a specific tissues [101]. Apart from these, the differences between the residues that lie within and beyond the binding pocket are widely known to play a major role in conferring ER subtype selectivity [106]. Nettles et al. [106] have used three compounds (THC, HPTE and PPT) to illustrate how the amino acid differences within and beyond the binding cavity can impact subtype selectivity as well as actions of these ligands. Impacts occur through the direct effects of different molecular interactions and through the indirect effects of long range interactions (arising from the differences in primary sequence beyond the binding pocket), both altering the selectivity of compounds. They have also identified structural differences in the surface, hydrophobic core, H12, β sheets and H1-H3 coils between the ERα and ERβ subtypes that caused differences in the cavity shape between the two ER subtypes (narrowing of back pocket of ERβ), which subsequently determine the different actions of ligands [106]. Indeed, unravelling the subtleties that lie between the ERα and ERβ has become the main impetus for many research efforts in the pursuit of designing more subtype selective ligands.

A fair number of ERα and ERβ selective compounds and SERMs have been synthesized over the past decade, and a number of past reviews have provided summaries: a brief overview was provided by Redden [107] in 2004, followed by Zhao et al. [108], Henke and Heyer [109] and Veneeman [110] in 2005. Blizzard [111] in 2008 published a high level review of the SERMs investigated by Merck; and Minutolo et al. [112] provided an excellent review on ERβ ligands with specific focus on the ones published in 2005 to 2008.

Here a (non-exhaustive) summary that includes some of the most recent subtype selective ligands is provided. The intent is to delve into how subtype selectivity is achieved in these compounds, with particular focus given to how selectivity is mainly determined, as opposed to a full discussion on how the desired activity/therapeutic profile is attained.

Like the great majority of the ER subtype selective compounds, the selectivity of ERα and ERβ selective compounds discussed in the following sections, mostly arises from the interactions between certain moiety/ies in the small molecules with the two critical amino acids in the ERα and ERβ.

The ERs are an inherently versatile group of receptors, demonstrating a remarkable ability to recognize and interact with a wide range of small molecule ligands and consequently produce an impressive diversity of downstream responses. The disadvantage of this flexibility is manifested in an apparent non-discriminatory nature of the ERs in ligand binding that, unfortunately, often leads to a multitude of adverse health effects due to exogenous chemicals including those in the diet and environment. Though often co-expressed in certain tissue types, the ER subtypes are also differentially expressed in others [130]. In view of this, having ER subtype selective ligands is highly desirable to achieve selective and fine-tuned ER modulated responses in the course of disease treatments. A range of different subtype selective ER ligands have been discussed in this review. These selective ligands have been obtained through different strategies but mostly by the exploitation of the subtle differences in the ligand binding sites of ERα and ERβ. Typically, selectivity is achieved through the introduction of different substituents as well as through non-planarity to the small molecules – strategies that have been successful so far. All that said, the road to translate observed ER selective ligands to eventual clinical outcomes is not straightforward. It bears emphasizing that the lack of correlation between the binding affinity and efficacy should also be taken into consideration, at point illustrated by comparing E2 and genistein. While E2 binds with similar affinity to ERα and ERβ, it has been found to display higher transcriptional potency in ERα [106]. In contrast, despite binding with high affinity to the ERβ subtype (25 fold higher than ERα), genistein only acts as a partial agonist for the ERβ subtype [121]. Notwithstanding, the availability of more ER selective ligands will undeniably help to improve the clinical outcomes when treating ER mediated diseases by offering clinicians a wider selection of compounds to tailor to individual needs.