blocked. estrogenic compounds span a spectrum of activity ranging from: full agonists (agonistic in all tissues) such as the natural endogenous hormone estradiol mixed agonists/antagonistics (agonistic in some tissues while antagonistic in others) such as tamoxifen (a serm). pure antagonists (antagonistic in all tissues) such as fulvestrant. serms are known to stimulate estrogenic actions in tissues such as the liver, bone and cardiovascular system but known to block estrogen action where stimulation is not desirable, such as in the breast and the uterus. this agonistic or antagonistic activity causes varied structural changes of the receptors, which results in activation or repression of the estrogen target genes. serms interact with receptors by diffusing into cells and there binding to erα or erβ subunits, which results in dimerization and structural changes of the receptors. this makes it easier for the serms to interact with estrogen response elements which leads to the activation of estrogen-inducible genes and mediating the estrogen effects. serms unique feature is their tissue- and cell-selective activity. there is growing evidence to support that serm activity is mainly determined by selective recruitment of corepressors and coactivators to er target genes in specific types of tissues and cells. serms can impact coactivator protein stability and can also regulate coactivator activity through post-translational modifications such as phosphorylation. multiple growth signaling pathways, such as her2, pkc, pi3k and more, are downregulated in response to anti-estrogen treatment. steroid receptor coactivator 3 (src-3) is phosphorylated by activated kinases that also enhance its coactivator activity, affect cell growth and ultimately contribute to drug resistance. the ratio of erα and erβ at a target site may be another way serm activity is determined. high levels of cellular proliferation correlate well with a high erα:erβ ratio, but repression of cellular proliferation correlates to erβ being dominant over erα. the ratio of ers in neoplastic and normal breast tissue could be important when considering chemoprevention with serms. when looking at the differences between erα and erβ, activating function 1 (af-1) and af-2 are important. together they play an important part in the interaction with other co-regulatory proteins that control gene transcription. af-1 is located in the amino terminus of the er and is only 20% homologous in erα and erβ. on the other hand, af-2 is very similar in erα and erβ, and only one amino acid is different. studies have shown that by switching af-1 regions in erα and erβ, that there are specific differences in transcription activity. generally, serms can partially activate engineered genes through erα by an estrogen receptor element, but not through erβ. although, raloxifene and the active form of tamoxifen can stimulate af-1-regulated reporter genes in both erα and erβ. because of the discovery that there are two er subtypes, it has brought about the synthesis of a range of receptor specific ligands that can switch on or off a particular receptor. however, the external shape of the resulting complex is what becomes the catalyst for changing the response at a tissue target to a serm. x-ray crystallography of estrogens or antiestrogens has shown how ligands program the receptor complex to interact with other proteins. the ligand-binding domain of the er demonstrates how ligands promote and prevent coactivator binding based on the shape of the estrogen or antiestrogen complex. the broad range of ligands that bind to the er can create a spectrum of er complexes that are fully estrogenic or antiestrogenic at a specific target site. the main result of a ligand-binding to er is a structural rearrangement of the ligand-binding pocket, primarily in the af-2 of the c-terminal region. the binding of ligands to er leads to the formation of a hydrophobic pocket that regulates cofactors and receptor pharmacology. the correct folding of ligand-binding domain is required for activation of transcription and for er to interact with a number of coactivators. coactivators are not just protein partners that connect sites together in a complex. coactivators play an active role in modifying the activity of a complex. post-translation modification of coactivators can result in a dynamic model of steroid hormone action by way of multiple kinase pathways initiated by cell surface growth factor receptors. under the guidance of a multitude of protein remodelers to form a multiprotein coactivator complex that can interact with the phosphorylated er at a specific gene promoter site, the core coactivator first has to recruit a specific set of cocoactivators. the proteins that the core coactivator assembles as the core coactivated complex have individual enzymatic activities to methylate or acetylate adjacent proteins. the er substrates or coenzyme a can be polyubiquitinated by multiple cycles of the reaction or, depending on linkage proteins, they can either be activated further or degraded by the 26s proteasome. consequently, to have an effective gene transcription that is programmed and targeted by the structure and phosphorylation status of the er and coactivators, it is required to have a dynamic and cyclic process of remodeling capacity for transcriptional assembly, after which the transcription complex is then instantly routinely destroyed by the proteasome. structure and function structure–activity relationships the core structure of serms simulates the 17β-estradiol template. they have two aromatic rings separated by 1-3 atoms (often a stilbene-type of arrangement). between the two phenyls of the core, serms typically have a 4-substituted phenyl group that, when bound to er, projects from a position of an estratriene nucleus so that helix 12 moves from the receptor opening and blocks the space where coactivator proteins would normally bind and cause er agonist activity. there has been a lot of variations in the core portion of serms while there has been less flexibility with what is tolerated in the side chain. serms can be classified by their core structure. first-generation triphenylethylenes 4-hydroxytamoxifen (red) overlaid with 17β-estradiol (black) the first main structural class of serm-type molecules reported are the triphenylethylenes. the stilbene core (similar to the nonsteroidal estrogen, diethylstilbestrol) essentially mimics steroidal estrogens such as 17β-estradiol, while the side chain overlays with the 11th position of the steroid nucleus. triphenylethylene derivatives have an additional phenyl group attached to the ethylene bridge group. the 3-position h-bonding ability of phenols is a significant requirement for er binding. trans-form of clomifene with the triphenylethylene structure in red. the first drug, clomifene, has a chloro-substituent on the ethylene side chain which produces similar binding affinities as the later discovered drug tamoxifen. clomifene is a mixture of estrogenic (cis-form) and antiestrogenic isomers (trans-form). cis and trans are defined in terms of the geometric relationships of the two unsubstituted phenyl rings. the two isomers of clomifene have different profiles, where the trans-form has activity more similar to tamoxifen while the cis-form behaves more like 17β-estradiol. cis is approximately ten times more potent than trans. however, trans isomer is the most potent stimulator of epithelial cell hypertrophy since clomifene is antagonistic at low doses and agonistic at high doses. the antagonist isomers may cause inhibitory estrogenic effects in the uterus and mammary cancers, but the estrogenic isomer could combine with novel receptors to produce estrogen-like effects in bone. chemical structure of tamoxifen tamoxifen has become the treatment of choice for women diagnosed with all stages of hormone-responsive breast cancer, that is, breast cancer that is both er and/or progesterone positive. in the us, it is also administered for prophylactic chemoprevention in women identified as high risk for breast cancer. tamoxifen is a pure antiestrogenic trans-isomer and has differential actions at estrogen target tissues throughout the body. tamoxifen is selectively antiestrogenic in the breast but estrogen-like in bones and endometrial cancer. tamoxifen undergo phase i metabolism in the liver by microsomal cytochrome p450 (cyp) enzymes. the major metabolites of tamoxifen are n-desmethyltamoxifen and 4-hydroxytamoxifen. the crystallographic structure of 4-hydroxytamoxifen interacts with the amino acids of the er within the ligand-binding domain. the contact between the phenolic group, water molecule, and glutamate and arginine in the receptor (erα; glu 353/arg 394) resolves in high affinity binding so that 4-hydroxy tamoxifen, with a phenolic ring that resembles the a ring of 17β-estradiol, has more than 100 times higher relative binding affinity than tamoxifen, which has no phenol. if its oh group is eliminated or its position is changed the binding affinity is reduced. the triphenylethylene moiety and the side chain are required for tamoxifen binding to the er, whereas for 4-hydroxytamoxifen, the side chain, and the phenyl-propene do not appear as crucial structural elements for binding to the er. the basicity and length of the side chain do not seem to play a crucial role for tamoxifen binding affinity to the er nor the β-ring of tamoxifen, but the stilbene moiety of tamoxifen is necessary for binding to the er. the hydroxyl group is of particular importance for er binding of 4-hydroxytamoxifen, and the ethyl side chain of tamoxifen protrudes out of the ligand-binding domain of the er. few tamoxifen users have had increased rates of uterine cancer, hot flushes, and thromboembolisms. the drug can also cause hepatocarcinomas in rats. this is likely due to the ethyl group of the tamoxifen stilbene core that is subject to allylic oxidative activation causing dna alkylation and strand scission. this problem is later corrected in toremifene. tamoxifen is more promiscuous than raloxifene in target sites because of the relationship between er's amino acid in asp-351 and the antiestrogenic side chain of the serm. the side chain for tamoxifen cannot neutralize asp-351, so the site allosterically influences af-1 at the proximal end of the er. this issue is mended with the second-generation drug raloxifene. chemical structure of toremifene toremifene is a chlorinated derivative of the nonsteroidal triphenylethylene antiestrogen tamoxifen with a chloro substituent at the ethylene side chain producing similar binding affinities to that of tamoxifen. the structure and activity relationship of toremifene is similar to that of tamoxifen, but it has a substantial improvement from the older drug in regards to dna alkylation. the presence of the added chlorine atom reduces the stability of cations formed from activated allylic metabolites and thus decreases alkylation potential, and indeed toremifene does not display dna adduct formation in rodent hepatocytes. toremifene protects against bone loss in ovariectomized rat models and affects bone resorption markers clinically in a similar fashion to tamoxifen. toremifene undergoes phase i metabolism by microsomal cytochrome p450 enzymes, like tamoxifen, but primarily by the cyp3a4 isoform. toremifene forms its two major metabolites n-desmethyltoremifene and deaminohydroxy-toremifene (ospemifene) by undergoing n-demethylation and deamination-hydroxylation. n-desmethyltoremifene has similar efficacy as toremifene while 4-hydroxytoremifene has a higher binding affinity to the er than toremifene. 4-hydroxytoremifene has a role similar to that of 4-hydroxytamoxifen. second-generation benzothiophenes raloxifene has a benzothiophene group (red) and is connected with a flexible carbonyl hinge to a phenyl 4-piperidinoethoxy side chain (green). raloxifene belongs to the second-generation benzothiophene serm drugs. it has a high affinity for the er with potent antiestrogenic activity and tissue-specific effects distinct from estradiol. raloxifene is an er agonist in bone and the cardiovascular system, but in breast tissue and the endometrium it acts as an er antagonist. it is extensively metabolized by glucuronide conjugation in the gut and because of that has a low bioavailability of only 2% while that of tamoxifen and toremifene is approximately 100%. the advantage of raloxifene over the triphenylethylene tamoxifen is reduced effect on the uterus. the flexible hinge group, as well as the antiestrogenic phenyl 4-piperidinoethoxy side chain, are important for minimizing uterine effects. because of its flexibility the side chain can obtain an orthogonal disposition relative to the core so that the amine of raloxifens side chain is 1 å closer than tamoxifens to amino acid asp-351 in erα's ligand-binding domain. the critical role of the intimate relationship between the hydrophobic side chain of raloxifene and the hydrophobic residue of the receptor to change both the shape and charge of the external surface of a serm-er complex has been confirmed with raloxifene derivatives. when the interactive distance between raloxifene and asp-351 is increased from 2.7 å to 3.5-5 å it causes increased estrogen-like action of the raloxifene-erα complex. when the piperidine ring of raloxifene is replaced by cyclohexane, the ligand loses antiestrogenic properties and becomes a full agonist. the interaction between serm's antiestrogenic side chain and amino acid asp-351 is the important first step in silencing af-2. it relocates helix 12 away from the ligand-binding pocket thereby preventing coactivators