Affinity For Estrogen Receptor Research Articles

Characterization of the hormone responsive element involved in the regulation of the progesterone receptor gene.

The transcription of the progesterone receptor gene is induced by estrogens and decreased by progestins. Studies were performed to define the regions of the gene and the molecular mechanisms involved. No hormonal regulation could be observed using 5' flanking regions of the gene up to -2762 in front of a heterologous gene. Estrogen and progestin regulation could be observed only when using fragments of the gene extending down to +788. Progressive deletions from the 5' and 3' ends, site-directed mutagenesis and DNase protection experiments with purified estrogen receptor suggested that the biologically active estrogen responsive element (ERE) is present at +698/+723, overlapping the initiation of translation. An oligonucleotide was synthesized bearing this ERE and shown to impart estrogen inducibility to a heterologous gene. Its regulation by anti-estrogens corresponded to that of the in situ progesterone receptor gene since tamoxifen was a partial agonist whereas ICI 164384 was a full antagonist. This ERE also mediated down-regulation by progestins in the presence of the progesterone receptor, even though it has no progesterone receptor binding ability. DNase footprinting showed that this effect was not due to a decrease of estrogen receptor affinity for the ERE in the presence of progesterone receptor. Finally, use of deletion mutants of the progesterone receptor showed that the steroid binding and the DNA binding domains were necessary for down-regulation whereas deletions of various parts of the N-terminal domain were without effect.

Efficient and selective photoaffinity labeling of the estrogen receptor using two nonsteroidal ligands that embody aryl azide or tetrafluoroaryl azide photoreactive functions

3-(4-Azido-2,3,5,6-tetrafluorobenzoyl)-6-hydroxy-2-(4- hydroxyphenyl)benzo[b]thiophene 1 (tetrafluoroaryl azide, TFAA) and its protio analogue 3-(4-azidobenzoyl)-6- hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene 2 (protioaryl azide, PAA), photoaffinity labeling (PAL) reagents for the estrogen receptor (ER), have been prepared in high specific activity tritium-labeled form (19 Ci/mmol) and shown to undergo selective and efficient photocovalent attachment to ER from rat uterus. Both azides 1 and 2 demonstrate high binding affinity for ER as determined by both a competitive binding assay (relative binding affinities: estradiol = 100; TFAA = 9.3; PAA = 66) and a direct binding assay (Kd: estradiol = 0.24 nM; TFAA = 2.64 nM; PAA = 0.37 nM). When unlabeled TFAA and PAA are irradiated at greater than 315 nm, they demonstrate site-specific photoinactivation of ER that reaches 43% and 55%, respectively, by 30 min. Specific photocovalent attachment to ER can be effected by irradiation of the tritium-labeled azides; the covalent attachment efficiency is good (1 = 20-30%, 2 = ca. 25%) and the selectivity of ER labeling is high. Characterization of the photolabeled proteins by SDS-polyacrylamide gel electrophoresis shows specific labeling of a major component at Mr 60,000 and a minor species at Mr 46,000, the same two species that are labeled by [3H]tamoxifen aziridine, a well-characterized affinity label for ER. The ER-specific antibodies H222Sp gamma and D547Sp gamma show a clean precipitation of only these two species. In the MCF-7 human breast cancer cell line, PAA is a full estrogen agonist in terms of stimulation of cell proliferation and induction of progesterone receptor. These two azides provide the first system in which the photocovalent attachment efficiency of an aryl azide can be compared to its tetrafluorosubstituted aryl azide analogue in a complex biological receptor system. Azides 1 and 2 are the most efficient and selective PAL reagents prepared to date for ER, and they should be useful in further studies of the hormone-binding domain of this protein.

Counteraction of estradiol-induced activation of tissue-type plasminogen activator in a human breast cancer cell line by an anti-estrogen, LY117018

LY117018 is a non-steroid anti-estrogen which exhibits about 100 times higher affinity for estrogen receptor than tamoxifen, another anti-estrogen. The cell line ES-1, which was isolated from human breast cancer MCF-7 cells, was highly sensitive to the cytocidal action of estradiol. Growth of ES-1 cells was inhibited by 10(-8)M 17 beta-estradiol, a concentration that stimulated the growth of parental MCF-7 cells. The estradiol-induced growth inhibition of ES-1 cells was almost completely reversed by treatment with LY117018, but not by treatment with tamoxifen. The relative binding affinity of LY117018 for estradiol receptor was equal to that of estradiol in both MCF-7 and ES-1 cells. Treatment of ES-1 cells with estradiol specifically induced tissue-type plasminogen activator (t-PA), whereas such estradiol-induced activation was not observed in parental MCF-7 cells. Quantitative immunoreactive assays and Northern blot analysis showed that estradiol-induced expression of t-PA was blocked by LY117018 in ES-1 cells. The inhibitory effect of tamoxifen was about 100 times lower than that of LY117018. The inhibition of t-PA gene expression by LY117018 might be due to competitive inhibition with estradiol in estradiol receptor binding.

Affinity and Conformation of the Estrogen Receptor When Bound to Single-Stranded vs. Double-Stranded Nonspecific DNA

The affinity of the hormone-bound estrogen receptor for single-stranded and double-stranded DNA was compared using isocratic elution chromatography. The receptor bound single-stranded DNA with a two-fold higher affinity than double-stranded DNA (17.9 x 10(4) M-1 vs. 9.1 x 10(4) M-1) at 0.2 M KCl. The same number of ions were released when the receptor bound either single-stranded or double-stranded DNA (11.8 vs. 10.6, respectively). These results indicate the hormone-bound estrogen receptor has no strong preference for single-stranded vs. double-stranded nonspecific DNA, and has a similar conformation when bound to either form of DNA at physiological salt concentrations.

Synthesis and receptor binding affinity of 7α- and 17α-substituted 2- and 4-chloroestradiol derivatives

Methods for the selective syntheses of 2- and 4-chloroestradiol derivatives substituted at the 17α- and 7α-positions are described. The relative binding affinities for estrogen receptors of these estrogens are also reported, and potential structure–activity relationships are put forth.

Multiple estrogen binding sites in the uterus: Stereochemistry of receptor and non-receptor binding of diethylstilbestrol and its metabolites

Indenestrol A (IA), an oxidative metabolite of the synthetic estrogen diethylstilbestrol (DES), has high binding affinity for estrogen receptor in mouse uterine cytosol but possesses weak biological activity. Racemic mixture of optically active [ 3H]indenestrol A (IA-Rac) was separated and purified into individual enantiomers on a semi-preparative scale by HPLC with a Chiralpak OP(+) column. The structure-activity relationship was investigated among the [ 3H]IA enantiomers (IA- R and IA- S) and [ 3H]DES through direct saturation binding assays using mouse uterine cytosol. Specific binding curves and Scatchard plots were obtained for each [ 3H]ligand; DES, IA-Rac, IA- R and IA- S. IA- S enantiomer ( K d =0.67) binds to the estrogen receptor with the same affinity as DES ( K d =0.71) and four times higher affinity than IA R( K d =2.56). The number of binding sites for IA- S is approximately the same as estradiol, DES and IA-Rac while IA- R binds far fewer sites than the other ligands. Saturation binding assays indicated that [ 3H]DES and [ 3H]IA enantiomers exhibited a higher level of non-specific binding to the cytosol receptor compared to estradiol which has a low level of non-specific binding. These binding studies led to the detection of an additional binding component for the stilbestrol compounds in estrogen target tissue cytosol preparations. Sucrose density gradient separation assays under low salt conditions showed that both [ 3H]DES and [ 3H]IA compounds bound to the 8S form of the receptor, the same as E 2. But, in addition both DES and IA bound to another binding component in 4S region. The binding to the 4S component were partially displayed by the addition of excess unlabeled E 2 and DES. Further characterization of the 4S component is described.

Structure-activity relationship of antiestrogens. Studies on 2,3-diaryl-1-benzopyrans

A series of 2,3-diaryl-1-benzopyran analogues substituted at position 4 of 2-phenyl with a hydroxy or pyrrolidinoethoxy residue were synthesized as models for (E)-triarylpropenones constrained in the s-trans conformation. The prototypes, belonging to five chemical series, were evaluated for their estrogen receptor affinity and for estrogen agonist-antagonist activities. The 4H-1-benzopyran-4-one, the 2,3-dihydro-4H-1-benzopyran-4-one, the 4H-1-benzopyran, and the 2,3-dihydro-1-benzopyran derivatives were found to be inactive or only marginally activate as receptor ligands or estrogen agonists-antagonists. In the 2H-1-benzopyran category the parent phenol was also inactive whereas the basic ethers 16 and 26 were modest receptor ligands while being quite active as antiestrogens. In a comparative study the benzopyran 16 was found to be more effective antiestrogen than tamoxifen while being as effective as LY-117018. The benzopyrans have thus emerged as a new class of potent antiestrogens.

Binding of oral contraceptive progestogens to serum proteins and cytoplasmic receptor

Some progestogens widely used in oral contraceptives are characterized at the level of high-affinity receptor binding as well as binding to sex hormone-binding globulin and corticosteroid-binding globulin. With regard to binding to sex hormone-binding globulin, gestodene, levonorgestrel, and to a lesser extent 3-ketodesogestrel (which is only formed from the prodrug desogestrel in the body), show a behavior that is manifested in the relatively high affinity to sex hormone-binding globulin, whereas desogestrel and norgestimate do not display any measurable affinity for this specific steroid-binding serum protein. Furthermore, levonorgestrel and gestodene dissociate very much more slowly from the binding sites of sex hormone-binding globulin than 3-ketodesogestrel. A natural affinity of all these synthetic progestogens tested for corticosteroid-binding globulin could not be established. Gestodene, levonorgestrel, and 3-ketodesogestrel bind to the progesterone, glucocorticoid, and androgen receptor with high affinity, apart from slight differences, whereas estrogen receptor affinity could not be demonstrated in any of the progestogens investigated. In relation to aldosterone, the relative binding affinity values of gestodene, levonorgestrel, and the natural progestogen progesterone are relatively high, whereas 3-ketodesogestrel does not display any measurable affinity for this receptor species.

4-aminoandrostenedione derivatives: A novel class of irreversible aromatase inhibitors. Comparison with FCE 24304 and 4-hydroxyandrostenedione

FCE 24928 (4-aminoandrosta-1,4,6-triene-3,17-dione) was selected among a series of 4-aminoandrostenedione derivatives as a novel irreversible aromatase inhibitor. Its in vitro and in vivo properties have been studied and compared to FCE 24304 (6-methylenandrosta-1, 4-diene-3,17-dione) and 4-OHA (4-hydroxyandrostenedione). FCE 24928 caused time-dependent inhibition of human placental aromatase with a T 1 2 of 4 min and K i of 59 nM. Enzyme inactivation by FCE 24928 was faster than by FCE 24304 ( t 1 2 13.9 min). In PMSG-treated rats, microsomal ovarian aromatase activity was reduced 24 h after FCE 24928 dosing by both the s.c. (ED 50 1.2 mg/kg) and the oral (ED 50 14.1 mg/kg) routes. The compound was more potent than FCE 24304 and 4-OHA (ED 50 1.8 and 3.1 mg/kg s.c.). FCE 24928 did not show any interference with 5α-reductase and desmolase activity nor any significant binding affinity for androgen and estrogen receptors. Slight binding affinity for androgen receptor was observed with FCE 24304 and 4-OHA (0.21 and 0.25% of DHT). In immature, castrated rats, FCE 24928 did not show any intrinsic androgenic activity, up to 100 mg/kg/day s.c., in contrast to a slight androgenic activity observed with FCE 24304 at 10 mg/kg s.c.

Over‐expression of the epidermal growth factor receptor in human breast cancer cells fails to induce an Estrogen‐independent phenotype

An association exists in primary human breast tumors between high epidermal growth factor receptor (EGFR) expression and a reduced number or even absence of estrogen receptors (ER). To determine whether an increase in EGFR expression might alter the estrogen responsiveness of an ER-positive human breast cancer cell line, ZR 75-1 cells were cotransfected with a plasmid containing the full-length cDNA for the human EGFR under the transcriptional control of the Harvey murine sarcoma virus (HaMSV) long terminal repeat (LTR) and with a pSV2neo plasmid. Two of the isolated G418-resistant clones were found to constitutively express EGFR levels 15- to 60-fold higher than those found on nontransfected ZR 75-1 cells. The EGFR in these clones were functionally normal since EGF could increase their autophosphorylation and since EGF could enhance the transphosphorylation of p185erbB-2. No change was seen in either the number or affinity of ER in these clones. In addition, the ability of estrogen to stimulate the anchorage-dependent and anchorage-independent growth of these clones was not significantly modified. These results suggest that an increase in EGFR expression alone is not sufficient to induce a hormone-independent phenotype in vitro in human breast cancer cells.

Estrogen receptor binding characteristics of 1,11β-ethanoestradiol: Effect of a 1,11β-bridge on steroidal estrogen

As part of an ongoing program to develop high affinity estrogenic ligands we have synthesized the 11β-vinyl, 11β-ethyl- and 1,11β-ethanoestradiols. Because the 1,11β-ethanoestradiol had not been previously reported in the literature, the investigation of its receptor binding characteristics would provide valuable insight into the effect of 1/11β-substitution. The data obtained in this study indicate that although significant estrogen receptor affinity is present for the 1,11β-ethano derivative, the RBA values, 5–22.4%, were far less than those observed (5–300-fold less) for the corresponding 11β-ethyl and 11β-vinyl estradiols and less than those for the 1-methyl and 11β-methyl estradiols. These results suggest that the orientation that the 11β-substituent must occupy is directed away from the A-ring and that substituents in the 1–11 pocket produce a detrimental effect on receptor interactions.

Platinum compounds with estrogen receptor affinity

The treatment of breast cancer, which is the leading cause of cancer deaths among West German women, includes both cytotoxic and hormonal therapy. Approximately one half of breast cancers possess estrogen receptors, and of these tumors about two-thirds are initially responsive to the better tolerated hormonal treatments (11). However, the first good results are usually followed by a regrowth of the patient’s tumor which is then resistant to further hormonal therapy. The clinician must turn to an aggressive regime of cytotoxic drugs, with toxic side effects feared by most patients. The idea behind the present drug-design approach was to begin treatment of estrogen receptor-positive (ER + ) tumors with well-tolerated cytotoxic therapy. This therapy would take advantage of the unique presence of estrogen receptors in the tumor cells. By coupling a DNA-reactive cytotoxin to a molecule with affinity for the estrogen receptor, it was thought that the cytotoxin would be selectively enriched in tumor cells positive for the estrogen receptor (7). A degree of sensitivity might also be gained if the estrogen receptor delivered the reactive cytotoxin to a critical region of DNA. Thus, with these added degrees of selectivity and sensitivity, cytotoxic chemotherapy might be achieved with fewer side effects. Such a drug might also be active against those tumors which are resistant to hormone therapy although the biopsies are positive for estrogen receptors. The strategy of linking the cytotoxic cisplatin (1) to either a nonsteroidal estrogen or antiestrogen appeared promising. The chemistry and pharmacology of cisplatin has been reviewed (2). Briefly, the reaction of cisplatin with DNA, specifically the intrastrand linking of adjacent guanines, is presumed to be the mechanism responsible for antineoplastic activity. Chemotherapy with cisplatin is well established in the cancer wards with a spectrum of activity that includes testicular, ovarian, endometrial, bladder, and head and neck cancer (14), but not breast cancer (5). One aim of the project was therefore to broaden the activity of cisplatin to include breast cancer. Considering the estrogen receptor-binding portion of the molecule, nonsteroidal estrogens and antiestrogens would appear to offer more synthetic possibilities for developing structure-activity relationships than steroidal estrogens.

1-(Aminoalkyl)-2-phenylindoles as novel pure estrogen antagonists

A number of 1-(omega-aminoalkyl)-5-hydroxy-2-(4-hydroxyphenyl)indoles were synthesized and studied for their binding affinities for the calf uterine estrogen receptor and estrogen antagonistic activities. Highest binding affinities were found with derivatives bearing a methyl group in position 3 and a hexamethylene chain between the indole and amino nitrogen atoms. Values for relative binding affinity (RBA) are between 20 and 30 for derivatives 5b, 5c, 5f, and 5h (17 beta-estradiol = 100). In the mouse uterine weight test, no significant agonistic (estrogenic) activity was observed up to a daily dose of 125 micrograms/animal, except for derivatives 5c, 5j, and 5l. 2-Phenylindoles with amino (5b), pyrrolidino (5f), piperidino (5h), and morpholino (5k) groups as the amino function completely suppressed estrone-stimulated uterine growth as a dose of 125 micrograms/animal (100% antagonism). Therefore, these derivatives can be considered as first examples of nonsteroidal pure antiestrogens.

Derivatives of ethynylestradiol with oxygenated 17α-alkyl side chain: Synthesis and biological activity

The coupling reaction of an acetylide ion with alkyl bromide or δ-valerolactone was used to synthesize twelve 1 17α-derivatives of ethynylestradiol having various 17α-side chain lengths and, except one, having mono- or di-oxygenated function on the side chain. All compounds had low (0.01–1.79%) relative binding affinity (RBA) for the rat uterine estrogen receptor. The highest RBA were obtained for compounds 26 (1.79%), 30 (1.55%) and 19 (0.42%). The length and polarity of the side chain decreases the affinity for the estrogen receptor. The in vivo estrogenic activity was measured on mouse uterine weight and was found to range from 0 to 35%, except for compound 30 (100%). The antiuterotrophic activity was measured by inhibition of estradiol-induced stimulation of uterine weight and was found to be 39% for compound 19, 25% for compound 26 and 0% for all other compounds. These two compounds ( 19 and 26) possess mixed agonist and antagonist activity.

1‐[4‐(N,N‐Bis‐β‐chloroethylcarbamoyloxy)‐phenyl]‐1,2‐bis‐(hydroxyphenyl)‐but‐1‐enes: Drugs Specifically Targeted against Estrogen Receptor Positive Mammary Tumors

1-[4-N,N-bis-2-chloroethylcarbamoyloxy)-phenyl]-1-(4-hydroxyphenyl )-2- phenylbut-1-ene (1) is a cytotoxic estrogen with a selective antitumor effect only on estrogen receptor (ER) containing tumors. In order to improve the ER affinity and thereby the antitumor activity of 1 a second OH-group in position 3 or 4 was introduced into the phenyl ring at C-atom 2 (8 and 9) and the respective acetates 10 and 11 were prepared. These modifications caused an enhanced ER affinity of 8-11 as compared to 1. The estrogenic properties were only slightly increased. In vitro, an enhanced antitumor effect on the ER positive MCF-7 mammary tumor cell line was determined for 8-11 as compared to 1 leading to inhibitions up to 90%. In vivo, 8-11 caused a pronounced growth inhibition of the hormone-dependent MXT M3.2 mammary tumor of the mouse though these effects were not better than those caused by 1 except the acetate 10, which inhibited tumor growth by 88%. These data show that the introduction of a second OH-group into 1 not only increased ER affinity, but also the antitumor activity in vitro and to a lesser extent in vivo in ER positive tumors. As none of these compounds had any significant effect on ER negative tumor models, 8-11 can be regarded as cytotoxic estrogens with a specific ER-mediated effect selectively on ER positive tumors.

Synthesis and structure-affinity of a series of 7.alpha.-undecylestradiol derivatives: a potential vector for therapy and imaging of estrogen-receptor-positive cancers

A series of 7 alpha-undecylestradiol derivatives, featuring various substituents at the end of the undecyl spacer chain, were synthesized and evaluated for their interaction with the estrogen receptor and nonreceptor sites. Their relative binding affinities (RBA) for calf uterine estrogen receptors were measured by competitive binding assays and varied between 0.5 and 8.4% of that of unlabeled 17 beta-estradiol. Enhanced lipophilicity and steric hindrance of the substituent on the end of the spacer chain resulted in decreased binding affinity for the estrogen receptor, while interactions with nonreceptor sites increased. RBA values were not affected by prolonged incubation times, suggesting a stable ligand-receptor complex. The potential to use the 7 alpha-undecylestradiol as a vector for site-selective delivery of diagnostic and therapeutic moieties to estrogen-receptor-positive human cancers is discussed.

Comparison of the effects of tamoxifen and of a tamoxifen analogue that does not bind the estrogen receptor on serum lipid profiles in the cockerel

Administration of the nonsteroidal antiestrogen tamoxifen to cockerels results in dose- and time-dependent decreases in the levels of free and esterified cholesterol, phospholipids, and triglycerides in serum and in very low density and low density lipoprotein fractions. Similar changes can be elicited using a tamoxifen analogue, N,N-diethyl-2-[(4-phenylmethyl)phenoxy]ethanamine.HCl (DPPE). Like tamoxifen, this compound is capable of binding antiestrogen binding sites and exhibits a relative binding affinity of 90% compared with tamoxifen (Ki approximately 4-5 nM). Unlike tamoxifen, DPPE shows no measureable affinity for the cockerel liver nuclear estrogen receptor. Further, DPPE exhibits no estrogen agonist or antagonist activity as measured at the level of synthesis of apolipoprotein II of very low density lipoprotein by liver, synthesis of ovalbumin by oviduct, or growth of the oviduct. Although it is possible that the lipid-lowering effects of tamoxifen result from the opposition of endogenous estrogen action in the cockerel, the similarity of the effects of tamoxifen and DPPE on the lipid profiles suggests common mechanisms that do not involve the estrogen receptor.

Cytotoxic Ester Derivatives of the Mammary Tumor Inhibiting Antiestrogen 2,3‐Bis(2‐fluoro‐4‐hydroxyphenyl)‐2,3‐dimethylbutane

The synthesis of the bis-acrylate 12, the bis-beta-chloropropionate 13 and the bis-beta-bromopropionate 14 of the "partial" antiestrogen 2,3-bis(2-fluoro-4-hydroxyphenyl)-2,3-dimethylbutane (7) is described. In the case of 13 and 14 the introduction of the beta-haloester-functions moderately reduces the estrogen receptor affinity of 7. However, it was quite strongly diminished in 12. The hydrolytic stability under in vitro-receptor-assay conditions decreases in the order 12 greater than 14 greater than 13. Compared with 7 the estrogenic potency of 12-14 is increased to a great extent. The title compounds cause a strong inhibition of the hormone-dependent MXT-M3.2 mouse mammary tumor.

Derivatives of tamoxifen. Dependence of antiestrogenicity on the 4-substituent.

A range of tamoxifen derivatives substituted in the 4-position of the 1-phenyl ring are described. The key steps in the synthesis of 4-iodo-, 4-bromo-, and 4-(methylthio)tamoxifen were reactions of 1,2-diarylbutanones with the (4-halogenophenyl)lithium or [4-(methylthio)phenyl]magnesium bromide. Oxidized precursors of 4-(methylthio)tamoxifen were used to prepare the methylsulfinyl and methylsulfonyl derivatives. Further derivatives (formyl, hydroxymethyl, oxirane, mercapto) were prepared from 4-bromotamoxifen via the 4-lithio derivative. Several of the derivatives (Br, I, SMe, SOMe, SO2Me, oxirane, CHO, CH2OH) displayed a higher affinity for estrogen receptors (ER) of calf uterine cytosol than did tamoxifen, but there was no relationship between affinity to ER and the ability to inhibit the growth of the MCF-7 breast cancer cell line in vitro.

Effects of the β-isomer of hexachlorocyclohexane on estrogen-sensitive human mammary tumor cells

The effects of the β-isomer of hexachlorocyclohexane (β-HCH) on the induction of the cytosolic progesterone receptor (PgRc), on the redistribution of the estrogen receptor (ER), and its affinity for ER were investigated in the estrogen-sensitive human mammary tumor cell line MCF-7. The effects of β-HCH were compared to those of estradiol-17β (E 2) and 2,4,6-trichlorophenol (TCP), a major urinary metabolite of β-HCH in rats. β-HCH in concentrations higher than 1 μ m caused induction of PgRc whereas TCP was essentially without effect up to a cytotoxic concentration of 100 μ m. Furthermore, β-HCH (10 μ m) caused redistribution of ER, i.e., decrease of cytosolic ER concentration and increase of nuclear ER concentration, in a way similar to E 2. In contrast to this, β-HCH up to a molar excess of 6 × 10 4 (concentration 30 μ m) caused no significant displacement of [ 3H]E 2 from ER indicating that β-HCH has no substantial affinity for ER. It is concluded that β-HCH has estrogenic properties which are at variance with the failure to demonstrate binding of β-HCH to ER.