Article

Development of bivalent triarylalkene- and cyclofenil-derived dual estrogen receptor antagonists and downregulators

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Abstract

Up to 80% of mammary carcinoma initially exhibit estrogen-dependent growth, which can be treated by aromatase inhibitors or SERMs/SERDs. To increase the options after failure of the hormonal therapy with these drugs, the search for alternatives with a different mode of action to prevent estrogen action is of high relevance. Therefore, this study focused on the inhibition of coactivator recruitment at the estrogen receptor (ER) by targeted attachment of bivalent compounds at the coactivator binding site besides the primary binding at the ligand binding domain. Eight homodimeric 4-[1-(4-hydroxyphenyl)-2-phenyl-1-butenyl]cinnamic acid (GW7604)- or cyclofenilacrylic acid-based ER ligands with diaminoalkane linkers (C2–C5) were synthesized and their effects on the ER subtypes were assessed in vitro. All compounds possessed full antagonistic potency at ERα/β as determined in a transactivation assay. Furthermore, they exerted medium downregulatory effects dependent on the spacer length and did not stimulate the ER expression as observed for 4-hydroxytamoxifen. The cyclofenil-derived dimer with C4 spacer (15b) showed the highest binding affinity to ERα (RBA = 79.2%) and downregulated the ER content in MCF-7 cells with an efficiency of 38% at 1 μM.

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... Alkyl spacers of different lengths between the molecules should guarantee sufficient flexibility to reach two different pockets within this exposed surface, which emerge as potential binding areas. 24 As proposed, it was possible to increase the binding affinity and to inhibit ER transactivation. ...
... It is based on the crystal structure of the ERβ-LBD (PDB entry 2FSZ) 26 cocrystallized with two 4-OHT molecules. 24 The first one is attached at the LBS and the second one at the CABS. Both can formally be connected by an alkyl spacer, enabling a view on possible binding modes of homodimeric compounds. ...
... 39 Such a reaction was already observed in the case of GW7604-based homodimers. 24 For the interpretation of the biological results, it is necessary to obtain information about the isomerization by simulating physiological conditions. Therefore, we incubated 17 and 36 as examples in a mixture of methanol (MeOH) and 2× phosphate-buffered saline (PBS) (75:25, v/v) and analyzed the E/Z isomerization by HPLC using an RP18 column and acetonitrile (ACN)/water (TFA, 0.1% or Na 2 SO 4 , 20 mM (pH 3), respectively) gradients. ...
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(E/Z)-3-(4-((E)-1-(4-Hydroxyphenyl)-2-phenylbut-1-enyl)phenyl)acrylic acid (GW7604) as a derivative of (Z)-4-hydroxytamoxifen (4-OHT) was linked by diaminoalkane spacers to molecules that are known binders to the coactivator binding site (benzimidazole or thioxo-quinazolinone scaffolds). With this modification, an optimization of the pharmacological profile was achieved. The most active thioxo-quinazolinone derivative 16 showed extraordinarily high affinity to the estrogen receptor (ER) β (RBA = 110%), inhibited effectively the coactivator recruitment (IC50 = 20.88 nM (ERα) and 28.34 nM (ERβ)), acted as a pure estradiol (E2) antagonist in a transactivation assay (IC50 = 18.5 nM (ERα) and 7.5 nM (ERβ)), and downregulated the ERα content in MCF-7 cells with an efficacy of 60% at 1 μM. The cytotoxicity was restricted to hormone-dependent MCF-7 (IC50 = 4.2 nM) and tamoxifen-resistant MCF-7TamR cells (IC50 = 476.6 nM). The compounds bearing a thioxo-quinazolinone moiety can therefore be assigned as pure E2-antagonistic selective ER degraders/downregulators. By contrast, the benzimidazole derivatives acted solely as pure antagonists without degradation of the ER.
... Knox, A.K. et al used another approach and developed compounds in such a way that besides the LBS, the coactivator binding site (CABS) is targeted simultaneously. They evaluated the consequences on the receptor binding affinity and the intracellular responses [13]. In their first study, homodimers of GW7604 and of the related cyclofenil acrylic acid were designed, because an X-ray crystal structure revealed a hydrophobic groove at the CABS suitable to bind 1,1-diaryl-or 1,1,2-triarylalkenes. Alkyl spacers of different lengths between the molecules should guarantee sufficient flexibility to reach two different pockets within this exposed surface, which emerge as potential areas [13]. ...
... They evaluated the consequences on the receptor binding affinity and the intracellular responses [13]. In their first study, homodimers of GW7604 and of the related cyclofenil acrylic acid were designed, because an X-ray crystal structure revealed a hydrophobic groove at the CABS suitable to bind 1,1-diaryl-or 1,1,2-triarylalkenes. Alkyl spacers of different lengths between the molecules should guarantee sufficient flexibility to reach two different pockets within this exposed surface, which emerge as potential areas [13]. As proposed by them, it was possible to increase binding affinity and to inhibit ER transactivation [13]. ...
... In their first study, homodimers of GW7604 and of the related cyclofenil acrylic acid were designed, because an X-ray crystal structure revealed a hydrophobic groove at the CABS suitable to bind 1,1-diaryl-or 1,1,2-triarylalkenes. Alkyl spacers of different lengths between the molecules should guarantee sufficient flexibility to reach two different pockets within this exposed surface, which emerge as potential areas [13]. As proposed by them, it was possible to increase binding affinity and to inhibit ER transactivation [13]. In continuation of this structure−activity relationship (SAR) study, they tried to optimize the CABS-binding properties. ...
Article
Breast cancer is still the most common cancer in women worldwide, affecting one in eight women in high-income countries, and the incidence is further increasing. Endocrine therapy, including aromatase inhibitors or selective estrogen receptor modulators (SERMs)/selective estrogen receptor down-regulators (SERDs), consequently represents an indispensable treatment opportunity. Unfortunately, acquired endocrine resistance is an inevitable issue, which manifests after prolonged therapy. Consequently, developing a novel drug for the treatment of breast cancer is need of the hour. But it is an established fact that designing or repurposing a drug using ‘trial and error’ approach is a tricky, long, expensive and could be a failure in clinical stage. Hence, there is a need to employ alternative approaches like computer aided drug design (CADD) to overcome these shortcomings of conventional approach. Recently, CADD has gained a high popularity among drug designers and medicinal chemists due to several advances associated with it. Pharmacophore modeling is an efficient and useful approach to identify important patterns in a series of molecules for optimizations. Hence, in this analysis, an attempt is made to develop consensus pharmacophore model of heterodimeric GW7604 derivatives using alignment approach. The dataset consists of fourteen heterodimeric GW7604 derivatives exhibiting the binding activity in a transactivation assay ERα and ERβ to the coactivator binding site. The heterodimeric GW7604 derivatives possess good variation in substation pattern like the presence of different diaminoalkane spacer and CABS binder. The consensus pharmacophore model revealed the importance of structural features and their correlation with the biological activity.
... With the aim of modulating the activity of the estrogen receptor (ER), a recent approach consisted of designing C 2 -symmetric dimers to bridge both ligand binding sites of a dimeric ER [20]. In order to exploit this strategy, Knox et al. developed a series of cyclophenylacrylic acid dimers, which can downregulate the activity of ER [21]. The choice of structure for those dimers was based on crystallographic and theoretical studies [22,23]. ...
... With the aim of modulating the activity of the estrogen receptor (ER), a recent approach consisted of designing C2-symmetric dimers to bridge both ligand binding sites of a dimeric ER [20]. In order to exploit this strategy, Knox et al. developed a series of cyclophenylacrylic acid dimers, which can downregulate the activity of ER [21]. The choice of structure for those dimers was based on crystallographic and theoretical studies [22,23]. ...
... All compounds were tested in vitro for biological activity [21]. A transactivation assay showed that all compounds possess full antagonistic potency against ERα/β. ...
Article
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Abstract: The design of C2-symmetric biologically active molecules is a subject of interest to the scientific community. It provides the possibility of discovering medicine with higher biological potential than the parent drugs. Such molecules are generally produced by classic chemistry, considering the shortness of reaction sequence and the efficacy for each step. This review describes and analyzes recent advances in the field and emphasizes selected C2-symmetric molecules (or axial symmetric molecules) made during the last 10 years. However, the description of the dimers is contextualized by prior work allowing its development, and they are categorized by their structure and/or by their properties. Hence, this review presents dimers composed of steroids, sugars, and nucleosides; known and synthetic anticancer agents; polyphenol compounds; terpenes, known and synthetic antibacterial agents; and natural products. A special focus on the anticancer potential of the dimers transpires throughout the review, notwithstanding their structure and/or primary biological properties.
... The success of this strategy is corroborated by results from in vitro assays showing that the cyclofenil-based bivalent compound AK-15b ( Fig. 2a) with a C4 spacer exhibited the highest binding affinity to ERa, with an ER downregulatory potency of 38% at 1 mM in MCF-7 cells. Furthermore, AK-15b completely blocked the recruitment of peroxisome proliferator-activated receptor-g coactivator 1 (PGC1) and abolished the estradiol-induced transactivation effect in U2OS cells [30]. This study furnished ER modulators with a novel mode of action to prevent estrogen action. ...
... It has been documented that the length of the spacer can have a profound influence on the binding affinity and functional activity of bivalent compounds. This optimal length has been determined for a number of different GPCR dimers, including the examples in this review [24,30,[43][44][45][46][47][48][49][50]. ...
... Hydrophobic spacers are likely to bridge the pharmacophores directly through the hydrophobic TM domains, whereas hydrophilic spacers might span the extracellular compartments of receptors. We have found that the PEG repeat (e.g., [44,47,51,52]) and peptidic chains (e.g., [24,30,48,53]) seem to be the most popular spacers, because these allow for a gradual increase in spacer length with suitable intrinsic physiochemical properties [18]. In addition, spacers containing the 1,2,3-triazole moiety have been increasingly used in recent years, because the 1,2,3-triazole motif can be rapidly constructed through the efficient and prevalent CuAAC (copper(I)-catalyzed alkyne-azide cycloaddition) click reaction. ...
Article
G protein-coupled receptors (GPCRs) have been exploited as primary targets for drug discovery, and GPCR dimerization offers opportunities for drug design and disease treatment. An important strategy for targeting putative GPCR dimers is the use of bivalent ligands, which are single molecules that contain two pharmacophores connected through a spacer. Here, we discuss the selection of pharmacophores, the optimal length and chemical composition of the spacer, and the choice of spacer attachment points to the pharmacophores. Furthermore, we review the most recent advances (from 2018 to the present) in the design, discovery and development of bivalent ligands. We aim to reveal the state-of-the-art design strategy for bivalent ligands and provide insights into future opportunities in this promising field of drug discovery.
... Tamoxifen (TMX) was the first-licensed selective estrogen receptor modulator (SERM) whereas, the 4-hydroxytamoxifen (4-OH-TMX) ( Fig. 1) is the active metabolite that performs antagonist activity on the ER located in the breast endocrine system and moderate agonist activity on the ER located in the endometrium [42][43][44][45]. This variation in the drug performance leads to endometrial carcinoma [46] as a concomitant side effect of the drug upon the long-term therapy of estrogen-dependent breast cancer. ...
... Moreover, the moderate agonist activity of Tamoxifen is the reason which justified the resistance of ER+ breast cancer patients to the drug [47,48]. It is worth also mentioning that Tamoxifen was a good starting point for the triphenyl-ethylene class of antiestrogens in which the trans-biphenyl is a conserved motif to maintain the ER affinity [44]. We lean to attribute the ER-partial agonist activity of 4-OH-TMX to the lacking of the second p-OH-phenolic group. ...
Article
Nuclear Estrogen receptors (ER) are cytoplasmic proteins; translocated to the nucleus to induce transcriptional signals after getting bound to the estrogen hormone. ER activation implicated in cancer cell proliferation of female reproductive organs. Thus, the discovery of ER antagonists is a reliable strategy to combat estrogen-dependent breast cancer. Endometrial carcinoma is one of the complications encountered upon long-term therapy by selective estrogen receptor modulators (SERMs) like Tamoxifen (TMX) and methyl piperidinopyrazole (MPP). Thus, the ER-full antagonist is a solution to improve the safety of this class of therapeutics during the treatment of breast cancer. We selected MPP as a lead structure to design conformationally constrained analogs. Structural rigidification is a proven strategy to transform the SERMs into full antagonists. Accordingly, we synthesized 7-methoxy-3-(4-methoxyphenyl)-4,5-dihydro-2H-benzo[g]indazoles (4), (6a-c), (8-12) along with the biphenolic counterparts (13-19) that are the anticipated active metabolites. The 4-nitrophenyl derivative (4) is with the most balanced profile regarding the in vivo anti-uterotrophic potential (EC50 = 4.160 μM); and the cytotoxicity assay of the corresponding active metabolite (13) against ER+ breast cancer cell lines (MCF-7 IC50 = 7.200 μM, T-47D IC50 = 11.710 μM). The inconsiderable uterotrophic activities of the elaborated ER-antagonists and weak antiproliferative activity of the compound (13) against ovarian cancer (SKOV-3 IC50 = 29.800 μM) highlighted it as a good start point to elaborate potential ER-full antagonists devoid of endometrial carcinoma. Extending the pendant chain that protrudes from the 2-(4-(substituted)-phenyl) ring of the new benzo-indazoles is recommended for enhancing the potency based on the binding mode of compound (13) in the ligand-binding domain (LBD) of ER.
... Previous literature focused on the effect of substitution on position 4 of ring B [13]; additionally, recent studies even suggested different substituents on ring B to design a homodimeric ER ligand that can act as ER antagonist and SERD (selective estrogen receptor Previous literature focused on the effect of substitution on position 4 of ring B [13]; additionally, recent studies even suggested different substituents on ring B to design a homodimeric ER ligand that can act as ER antagonist and SERD (selective estrogen receptor degrader) [14]. In our work, the effect of the length of the alkoxy chain, size, and bulkiness of N-substituents and cyclization are thoroughly studied. ...
... Compounds (10)(11)(12)(13)(14) bear a methoxy substituent on ring A. Both compounds 10 and 13 were the most potent congeners. They bear a dimethylaminopropoxy side chain and a morpholinylethoxy side chain, respectively, on ring B (relative β-galactosidase activity = 11.61 and 12.41, respectively). ...
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Abstract: Selective estrogen receptor modulators (SERMs) act as estrogen receptor (ERα) agonists or antagonists depending on the target issue. Tamoxifen (TAM) (a non-steroidal triphenylethylene derivative) was the first SERM approved as anti-estrogen for the treatment of metastatic breast cancer. On the hunt for novel SERMs with potential growth inhibitory activity on breast cancer cell lines yet no potential to induce endometrial carcinoma, we designed and synthesized 28 novel TAM analogs. The novel analogs bear a triphenylethylene scaffold. Modifications on rings A, B, and C aim to attenuate estrogenic/anti-estrogenic activities of the novel compounds so they can potentially inhibit breast cancer and provide positive, beneficial estrogenic effects on other tissues with no risk of developing endometrial hyperplasia. Compound 12 (E/Z-1-(2-{4-[1-(4-Chloro-phenyl)-2-(4-methoxy- phenyl)-propenyl]-phenoxy}-ethyl)-piperidine) showed an appreciable relative ERα agonistic activity in a yeast estrogen screen (YES) assay. It successfully inhibited the growth of the MCF-7 cell line with GI50 = 0.6 µM, and it was approximately three times more potent than TAM. It showed no potential estrogenicity on Ishikawa endometrial adenocarcinoma cell line via assaying alkaline phosphatase (AlkP) activity. Compound 12 was tested in vivo to assess its estrogenic properties in an uterotrophic assay in an ovariectomized rat model. Compared to TAM, it induced less increase in wet uterine wet weight and showed no uterotrophic effect. Compound 12 is a promising candidate for further development due to its inhibition activity on MCF-7 proliferation with moderate AlkP activity and no potential uterotrophic effects. The in vitro estrogenic activity encourages further investigations toward potential beneficial properties in cardiovascular, bone, and brain tissues.
... This specificity is likely to be lost with the trimeric agent 21, where the potency is in double digit micromolar region as well. Our data are similar to what has been seen in investigations of linker lengths in dimeric agents targeting G protein-coupled receptors [39], estrogen receptors [40], proteolysis targeting chimeras [39], among others [41]. It is noteworthy that polygodial and ophiobolin A unsaturated esters displayed comparable potencies in cells both sensitive and resistant to apoptosis induction, indicating that this family of compounds is capable of overcoming apoptosis resistance in the clinic (Table 1) [18,22]. ...
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In a search of small molecules active against apoptosis-resistant cancer cells, including glioma, melanoma, and non-small cell lung cancer, we previously prepared α,β- and γ,δ-unsaturated ester analogues of polygodial and ophiobolin A, compounds capable of pyrrolylation of primary amines and demonstrating double-digit micromolar antiproliferative potencies in cancer cells. In the current work, we synthesized dimeric and trimeric variants of such compounds in an effort to discover compounds that could crosslink biological primary amine containing targets. We showed that such compounds retain the pyrrolylation ability and possess enhanced single-digit micromolar potencies toward apoptosis-resistant cancer cells. Target identification studies of these interesting compounds are underway.
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The signing of the National Cancer Act in 1971, was designed to take laboratory discoveries rapidly from the bench to the bedside. A “war on cancer” had been declared. Combination cytotoxic chemotherapy was predicted to cure all cancers based on the stunning success in treating childhood leukemia. Breast cancer treatments were primitive; radical mastectomy and radiation was standard of care for disease that had not spread. Ablative endocrine surgery (oophorectomy, hypophysectomy, and adrenalectomy) was a palliative last option for metastatic breast cancer. However, only 30% responded for a year or two: everybody died. The discovery of the estrogen receptor (ER), and translation to breast cancer treatment triggered a revolution in women’s health. Two important, but interconnected events occurred at the Worcester Foundation for Experimental Biology (WFEB), which would exploit the breast tumor ER as the first target to save lives and prevent breast cancer development. Two new groups of medicines: Selective Estrogen Receptor Modulators (SERMs) and aromatase inhibitors (AIs) would continue the momentum of research at the WFEB to improve women’s health. Here we recount the important progress made in women’s health based upon knowledge of the endocrinology of breast cancer. We propose future opportunities in SERM therapeutics to “refresh” the current standards of care for breast cancer treatment. The opportunity is based upon emerging knowledge about acquired resistance to long term adjuvant AI therapy used to treat breast cancer.
Article
Estrogen receptor α (ERα) is a crucial target for ERα positive breast cancer treatment. Previous drug discovery efforts were focused on developing inhibitors that targeted the canonical ligand binding pockets of the ligand binding domain (LBD) of ERα. However, significant percentage of patients developed cancer relapse with drug-resistance. ERα peptidomimetic modulators have been considered as promising treatments for drug resistant breast cancers as they are targeting ERα-coactivator interacting interface instead of the ligand binding pocket of ERα. Herein, we reviewed the recent development of ERα peptidomimetics antagonists.
Article
Chemical protein synthesis allows the generation of milligram quantities of correctly folded and previously inaccessible tyrosine phosphorylated estrogen receptor α (ERα) and β (ERβ) ligand binding domains. By using this synthetic strategy, the crystal structure of a post-translationally modified nuclear receptor (pY488 ERβ) could be obtained for the first time (see figure).
Article
To search for new antiestrogens more effective in treating breast cancers, we explored alternatives to the acrylic acid side chain used in many antiestrogens. To facilitate our search, we used a simple adamantyl ligand core that by avoiding stereochemical issues enabled rapid synthesis of acrylate ketone, ester, and amide analogs. All compounds were high affinity estrogen receptor-alpha (ERα) ligands, but displayed a range of efficacies and potencies as antiproliferative and ERα-downregulating agents. There were large differences in activity between compounds having minor structural changes, but antiproliferative and ERα-downregulating efficacies generally paralleled one another. Some compounds with side chain polar groups had particularly high affinities. The secondary carboxamides had the best cellular activities, and the 3-hydroxypropylamide was as efficacious as fulvestrant in suppressing cell proliferation and gene expression. This study has produced structurally novel antiestrogens based on a simple adamantyl core structure with acrylate side chains optimized for cellular antagonist activity.
Article
Long-term (five years) adjuvant tamoxifen treatment for oestrogen receptor-positive, or ER-positive, breast cancer post-surgery is recognised as a major advance in healthcare. Clinical trials comparing standard and extended tamoxifen therapy found a major decrease in mortality occurred in the ten years following a decade of adjuvant therapy. Studies in premenopausal breast cancer patients showed that ovarian function suppression (OFS) plus an aromatase inhibitor (AI), such as exemestane, which compared to tamoxifen has fewer serious side effects and decreases mortality rates further, is superior to OFS plus tamoxifen in reducing breast cancer recurrence. Current studies using AIs in combination with the selective ER downregulator (SERD) fulvestrant to treat ER-positive metastatic breast cancer (MBC) may provide clinical benefit over AI alone. Preliminary studies suggest that high-dose fulvestrant is superior to anastrazole (an AI) as first-line treatment in MBC, and has prompted the search for new orally-active SERDs. The development of acquired resistance to AIs and fulvestrant led to the development of a treatment strategy with either an mTOR inhibitor or a cyclin-dependent kinase (CDK) 4/6 inhibitor. An mTOR inhibitor (e.g. everolimus) improves responsiveness to an AI over an AI alone and a CDK 4/6 inhibitor (e.g. palbociclib), either with an AI or fulvestrant versus the endocrine therapy alone, demonstrates significant improvements in progression-free survival. Unfortunately, both mTOR and CDK 4/6 inhibitors have considerable toxic adverse effects and are expensive. It is logical to predict that the novel targeted therapeutics used in combination with either an AI or tamoxifen (or indeed in the future with a new SERD) would dramatically improve response rates when used in the adjuvant setting. Nevertheless, the success of adjuvant therapy depends on compliance, and compliance depends upon the severity of side effects.
Article
The discovery of an orally bioavailable selective estrogen receptor downregulator (SERD) with equivalent potency and pre-clinical pharmacology to the intramuscular SERD, fulvestrant is described. A directed screen identified the 1-aryl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole motif as a novel, druglike ER ligand. Aided by crystal structures of novel ligands bound to an ER construct, medicinal chemistry iterations led to (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (30b, AZD9496), a clinical candidate with high oral bioavailability across pre-clinical species that is currently being evaluated in Phase I clinical trials for the treatment of advanced estrogen receptor (ER) positive breast cancer.
Article
Approximately eighty percent of breast cancers are estrogen receptor alpha (ER-) positive, and although women typically initially respond well to anti-hormonal therapies such as tamoxifen and aromatase inhibitors, resistance often emerges. Although a variety of resistance mechanism may be at play in this state, there is evidence that in many cases the ER still plays a central role, including mutations in the ER leading to constitutively active receptor. Fulvestrant is a steroid-based, selective estrogen receptor degrader (SERD) that both antagonizes and degrades ER-, and is active in patients who have progressed on anti-hormonal agents. However fulvestrant suffers from poor pharmaceutical properties and must be administered by intra-muscular injections that limit the total amount of drug that can be administered, and hence lead to the potential for incomplete receptor blockade. We describe the identification and characterization of a series of small-molecule, orally bioavailable SERDs which are potent antagonists and degraders of ER-, and in which the ER- degrading properties were prospectively optimized. The lead compound 11l (GDC-0810 or ARN-810) demonstrates robust activity in models of tamoxifen-sensitive and tamoxifen-resistant breast cancer, and is currently in clinical trials in women with locally advanced or metastatic estrogen receptor-positive breast cancer.
Article
Linear and branched compounds that contain two, three or five units of galloyl (3,4,5-trihydroxybenzoyl) or its isomer 2,3,4-trihydroxybenzoyl, as well as other mono- or dihydroxybenzoyl moieties have been synthesized. These molecules have been evaluated for their in vitro inhibitory effects against a wide panel of viruses showing preferential activity against HIV and HCV. Our structure-activity relationship studies demonstrated that the 2,3,4-trihydroxybenzoyl moiety provides better antiviral activities than the galloyl (3,4,5-trihydroxybenzoyl) moiety that is present in natural green tea catechins. This observation can be of interest for the further rational exploration of compounds with anti-HCV/HIV properties. The most notable finding with respect to HIV is that the tripodal compounds 43 and 45, with three 2,3,4-trihydroxybenzoyl moieties, showed higher activities than linear compounds with only one or two. With respect to HCV, the linear compounds, 52 and 41, containing a 12 polymethylene chain and two 2,3 di- or 2,3,4 tri-hydroxybenzoyl groups respectively at the ends of the molecule showed good antiviral efficiency. Furthermore, the anti-HCV activity of both compounds was observed at concentrations well below the cytotoxicity threshold. A representative member of these compounds, 41, showed that the anti-HCV activity was largely independent of the genetic make-up of the HCV subgenomic replicon and cell lines used. Copyright © 2015 Elsevier Masson SAS. All rights reserved.
Article
Estrogen receptors alpha (ERα) and beta (ERβ) are nuclear transcription factors that are involved in the regulation of many complex physiological processes in humans. Modulation of these receptors by prospective therapeutic agents is currently being considered for prevention and treatment of a wide variety of pathological conditions, such as, cancer, metabolic and cardiovascular diseases, neurodegeneration, inflammation, and osteoporosis. This review provides an overview and update of compounds that have been recently reported as modulators of ERs, with a particular focus on their potential clinical applications.
Article
Background: The selective estrogen receptor modulators (SERMs) raloxifene and tamoxifen are triphenylethylene derivatives that affect transcriptional regulation by the estrogen receptors (ERα and ERβ) but show different effects in different tissues. A third triphenylethylene derivative, GW-5638, displays tissue selectivity in rats identical to that of raloxifene, suggesting that GW-5638 and raloxifene share a mechanism of action that is different from that of tamoxifen.Results: Both GW-5638 and its hydroxylated analog GW-7604 were tested for their ability to bind to ERα and ERβ and their ability to affect transcription of ERα and ERβ at a consensus estrogen response element and an ER/AP-1 response element. The drugs were found to have the same affinity for ERα and ERβ, although they were also found to activate transcription from an AP-1 promoter element more potently with ERβ than with ERα. Derivatives of GW-5638 with alterations at the carboxylic acid still showed increased ERβ potency compared to ERα, but the magnitude of the activation with ERα was much higher than with ERβ.Conclusions: Despite similar binding affinities to isolated ERα and ERβ, GW-5638 and GW-7604 show markedly lower EC50 values with ERβ at an AP-1-driven promoter as compared to ERα. This suggests that the two compounds produce a more active ER/AP-1 conformation of the ER/AP-1 transcription factor complex when bound to ERβ than when bound to ERα.
Article
PyBOP® (benzotriazolyloxy-tris[pyrrolidino]-phosphonium hexafluorophosphate), an analog of BOP where dimethylamino groups are replaced with pyrrolidino, is the only analog exhibiting equivalent properties in peptide bond formation. It can be used instead of BOP for the sake of safety.
Article
The estrogen receptor (ER) is a hormone-regulated transcription factor that binds, as a dimer, to estrogens and to specific DNA sequences. To explore at a fundamental level the geometric and topological features of bivalent-ligand binding to the ER dimer, dimeric ER crystal structures were used to rationally design nonsteroidal bivalent estrogen ligands. Guided by this structure-based ligand design, we prepared two series of bivalent ligands (agonists and antagonists) tethered by flexible spacers of varying lengths (7-47Å) and evaluated their ER-binding affinities for the two ER subtypes and their biological activities in cell lines. Bivalent ligands based on the agonist diethylstilbestrol (DES) proved to be poor candidates, but bivalent ligands based on the antagonist hydroxytamoxifen (OHT) were well suited for intensive study. Binding affinities of the OHT-based bivalent ligands were related to spacer length in a distinctive fashion, reaching two maximum values at 14 and 29Å in both ER subtypes. These results demonstrate that the bivalent concept can operate in determining ER-ligand binding affinity and suggest that two distinct modes operate for the binding of bivalent estrogen ligands to the ER dimers, an intermolecular as well as an intramolecular mode. Our insights, particularly the possibility of intramolecular bivalent binding on a single ER monomer, may provide an alternative strategy to prepare more selective and active ER antagonists for endocrine therapy of breast cancer.
Article
In many estrogen responsive systems the isomers of tamoxifen are known to have different biological character-the trans isomer is generally an antagonist and the cis isomer an agonist. Attempts to similarly characterize the isomers of hydroxytamoxifen (which differ greatly in their affinity for the estrogen receptor) are shown to be complicated by their facile isomerization. This isomerization was studied in cultures of estrogen receptor positive MCF-7 human breast cancer cells and monitored by HPLC under reversed phase conditions. Hydroxytamoxifen isomers that are initially 99% pure, undergo a time and temperature dependent isomerization, so that after 2 days in tissue culture medium at 37°C they have isomerized to the extent of 20%. This isomerization occurs in the cell-free medium alone and cannot be attributed to a metabolic conversion by the cells. The isomerization occurs much more slowly at 4 than at 37° C and can be reduced considerably by various antioxidants (butylated hydroxytoluene, ascorbate, α-tocopherol, retinoic acid and retinal); however, at concentrations that block isomerization, these antioxidants are toxic to the cells. Although the medium contains both the cis and trans isomers of hydroxytamoxifen, the MCF-7 cells preferentially accumulate the trans isomer and the material associated with the nuclear estrogen receptor is, in all cases, mainly the higher affinity trans isomer. A similar preference of the estrogen receptor for the trans isomer is seen with diethylstilbestrol, resulting again in almost exclusive accumulation of the trans isomer in the receptor binding site.
Article
The estrogen receptor (ER) is a hormone-regulated transcription factor which is thought to bind to specific DNA sequences as a homodimer. In order to better understand structural requirements for dimerization and its functional role in ER action, we synthesized a series of bivalent ligands based on the non-steroidal estrogen hexestrol. These molecular probes join two hexestrol molecules of the erythro (E, active) configuration with either 4 or 8 carbon linkers (designated E-4-E and E-8-E series, respectively), or with longer linkers comprised of ethylene glycol units (E-eg-E series). Several other bi- and monovalent control compounds were prepared. The bivalent ligands binds to ER with a relative affinity 1–7% that of estradiol. While most of the ligands demonstrated normal monophasic displacement curves in competitive binding assays with [3H]estradiol, uncharacteristic biphasic competitive binding curves were seen for some of the ligands, indicating possible structure-specific, negative site-site interaction. In ER-deficient Chinese hamster ovary (CHO) cells transfected with an expression vector encoding ER, one series of bivalent ligands (E-4-E) had little stimulatory activity and inhibited transcription stimulated by hexestrol, as determined by a transient transfection assay using an estrogen-responsive reporter gene construct [(ERE)2-TATA-CAT, containing two estrogen response elements linked to a TATA promoter and the chloramphenicol acetyl transferase reporter gene]. Monovalent or control bivalent ligands failed to antagonize hexestrol-stimulated activity and were as fully active as hexestrol itself. Studies performed in MCF-7 human breast cancer cells, which contain endogenous ER, yielded similar bioactivity profiles for the E-4-E bivalent inhibitory ligands, showing them to be effective estrogen antagonists, when using either induction of progesterone receptor or (ERE)2-TATA-CAT transcriptional activation as the endpoint. The E-8-E ligand, however, acted as a partial agonist/antagonist of ERE-reporter gene transactivation and a full agonist of progesterone receptor induction in MCF-7 cells, thus showing cell- and response-specific differences in the effects of the bivalent ligand. The bivalent ligands for ER do not show enhanced potency or receptor binding affinity; however, some of them display binding properties that suggest the possibility of structure-specific negative site-site interaction, and some of them function as quite effective estrogen antagonists.
Article
The estrogen receptor binding affinities of bivalent raloxifene ligands tethered by flexible spacers of different lengths have been evaluated in vitro. Two bivalent binding modes, intra- and intermolecular, were hypothesized to explain their different binding properties. The binding affinities of these bivalent ligands in an aqueous environment are influenced by their conformations, which can be determined by 2D NMR and UV spectral methods. Moreover, computer modeling and simulations were performed to explain the binding modes of these bivalent ligands and to estimate the conformational entropy difference between their unbound and bound states. It was found that bivalent ligands tethered by long spacers had weaker binding affinities because of the shielding of the binding moieties that results from their folded conformations; those tethered by short spacers had stronger affinities because they exposed their ligands to the receptor.
Article
Selective estrogen receptor modulators, such as 17β-estradiol derivatives bound to metal complexes, have been synthesized as targeted probes for the diagnosis and treatment of breast cancer. Here, we report the detailed 3D structure of estrogen receptor α ligand-binding domain (ERα-LBD) bound with a novel estradiol-derived metal complex, estradiol-pyridine tetra acetate europium(III), at 2.6 Å resolution. This structure provides important information pertinent to the design of novel functional ERα targeted probes for clinical applications.
Article
Small molecules, namely coactivator binding inhibitors (CBIs), that block estrogen signaling by directly inhibiting the interaction of the estrogen receptor (ER) with coactivator proteins act in a fundamentally different way to traditional antagonists, which displace the endogenous ligand estradiol. To complement our prior efforts at CBI discovery by de novo design, we used high-throughput screening (HTS) to identify CBIs of novel structure and subsequently investigated two HTS hits by analogue synthesis, finding many compounds with low micromolar potencies in cell-based reporter gene assays. We examined structure-activity trends in both series, using induced-fit computational docking to propose binding poses for these molecules in the coactivator binding groove. Analysis of the structure of the ER-steroid receptor coactivator (SRC) complex suggests that all four hydrophobic residues within the SRC nuclear receptor box sequence are important binding elements. Thus, insufficient water displacement upon binding of the smaller CBIs in the expansive complexation site may be limiting the potency of the compounds in these series, which suggests that higher potency CBIs might be found by screening compound libraries enriched with larger molecules.
Article
Selective estrogen receptor (ER) down-regulators (SERDs) reduce ERalpha protein levels as well as block ER activity and therefore are promising therapeutic agents for the treatment of hormone refractory breast cancer. Starting with the triarylethylene acrylic acid SERD 4, we have investigated how alterations in both the ligand core structure and the appended acrylic acid substituent affect SERD activity. The new ligands were based on high affinity, symmetrical cyclofenil or bicyclo[3.3.1]nonane core systems, and in these, the position of the carboxyl group was extended from the ligand core, either retaining the vinylic linkage of the substituent or replacing it with an ether linkage. Although most structural variants showed binding affinities for ERalpha and ERbeta higher than that of 4, only the compounds preserving the acrylic acid side chain retained SERD activity, although they could possess varying core structures. Hence, the acrylic acid moiety of the ligand is crucial for SERD-like blockade of ER activities.
Article
Steroidal bivalent ligands for the estrogen receptor (ER) were designed using crystal structures of ERalpha dimers as a template. The syntheses of several 17alpha-ethynylestradiol-based bivalent ligands with varying linker compositions and lengths are described. The binding affinities of these bivalent ligands for ERalpha and ERbeta were determined. In the two series of bivalent ligands that we synthesized, there is a clear correlation between linker length and binding affinity, both of which reach a maximum at the same tether length. Further studies are underway to explore aspects of bivalent ligand and control compound binding to the ERs and their effects on ER dimer formation; these results will be reported in a subsequent publication.
Article
The oestrogenic and antioestrogenic properties of tamoxifen and its monohydroxylated (monohydroxytamoxifen) and dihydroxylated (dihydroxytamoxifen) metabolites have been investigated in the immature rat. Whether administered orally or subcutaneously, monohydroxytamoxifen was more active than tamoxifen as an antioestrogen. Dihydroxytamoxifen was less active than tamoxifen as an antioestrogen, but this derivative alone was unable to induce a uterotrophic response. Both metabolites of tamoxifen were potent inhibitors of the binding of [3H]oestradiol to oestrogen receptors in vitro. It is possible that the metabolites play a supportive role in the antioestrogenic activity of tamoxifen. The potent activity of monohydroxytamoxifen in vivo and in vitro suggests that this compound could be an important new tool for the subcellular investigation of oestrogenic and antioestrogenic events.
Article
Chronic administration of tamoxifen to female rats causes hepatocellular carcinomas. We have investigated damage to liver DNA caused by the administration of tamoxifen to female Fischer F344/N rats or C57B1/6 or DBA/2 mice using 32P-postlabelling. Following the administration of tamoxifen for 7 days (45 mg/kg/day) and extraction of hepatic DNA, up to 7 radiolabelled adduct spots could be detected after PEI-cellulose chromatography of the 32P-labelled DNA digests. Tamoxifen caused a time-dependent increase in the level of adduct detected up to a value of at least 1 adduct/10(6) nucleotides after 7 days dosing. A dose response relationship was demonstrated over the range of 5-45 mg/kg/day (0.013-0.12 mmol/kg/day). On cessation of dosing there was a loss of adducts from the liver DNA. These adducts were not detected in DNA from vehicle-dosed controls or in DNA from kidney, lung, spleen, uterus or peripheral lymphocytes. Pyrrolidinotamoxifen caused a similar level of adduct formation as tamoxifen. In contrast, no significant adduct formation could be detected in liver DNA from rats given droloxifene or toremifene. Mice given tamoxifen (45 mg/kg/day for 4 days) showed levels of adducts in the liver which were 30-40% of those present in rats. Exposure of rat hepatocytes to tamoxifen in vitro, resulted in induction of unscheduled DNA synthesis, when preparations from rats which had been pretreated with tamoxifen in vivo were used. No such increase could be detected in hepatocytes from control rats, suggesting tamoxifen may induce enzymes responsible for its own activation. Tamoxifen induced a significant increase in micronucleus formation in a dose dependent manner in cultures of MCL-5 cells, a human cell line that expresses 5 different human cytochrome P450 isoenzymes, as well as epoxide hydrolase.
Article
In this paper we describe the microcomputer-aided determination of cell proliferation kinetics and doubling times utilizing a crystal violet assay and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay in microtitration plates. The analysis of spectrophotometric data provides the doubling times at any time of incubation. Plots of doubling time versus time of incubation give reproducible information on the exact duration of the logarithmic growth phase. This method is applicable to anchorage-dependent as well as anchorage-independent cells when colorimetric or fluorometric data are accessible.
Article
Facile isomerization of molecular diethylstilbestrol (DES) occurs by a second order reaction on solution in organic solvents. The isomerization of the disodium salt of DES which occurs in basic methanolic solutions is a reversible first order reaction. Mechanisms of the isomerization processes of molecular and anionic DES are proposed. The estrogenic response of isolated disodium salt of cis-DES was determined to be approximately 45% of the activity observed for trans-DES using a mouse uterine weight assay. Since the trans isomer is believed to be considerably more active biologically because of its structural similarity with estradiol, it appears that the observed appreciable activity of the cis-DES test solution is a consequence of the isomerization in vivo to the trans isomer. These results may have significance in the use of DES as a ruminant growth promoter since estrogenic response appears not to be restricted solely to the amount of trans isomer content.
Article
1 The oestrogenic and antioestrogenic activities of tamoxifen and monohydroxytamoxifen have been compared with those of para-methoxy, -methyl, -fluoro, and -chloro tamoxifen in the 3 day immature rat uterine weight test.2 The oestrogenic activity of mestranol, a steroid with low oestrogen receptor binding affinity which is believed to be demethylated to ethinyl oestradiol before exerting its effects, was less potent than ethinyl oestradiol when assayed in the 3 day immature rat uterine weight test. Similarly, para-methoxytamoxifen was less active than monohydroxytamoxifen in oestrogenic and antioestrogenic tests.3 The introduction of a para-methoxy group into tamoxifen did not affect oestrogenic or antioestrogenic activity.4 All the derivatives of tamoxifen were partial oestrogen agonists when compared with oestradiol benzoate in the 3 d immature rat uterine weight test. All test compounds inhibited the uterotrophic activity of oestradiol benzoate (0.16 mug daily) in a dose-related manner. The order of potency was: monohydroxytamoxifen > tamoxifen identical with methoxytamoxifen > p-fluoro identical with p-chloro identical with p-methyltamoxifen.5 Tamoxifen was approximately equiactive with its p-methyl, p-fluoro and p-chloro derivatives in the ability to inhibit [(3)H]-oestradiol binding to rat uterine oestrogen receptors in vitro.6 Tamoxifen was approximately equiactive with its p-methyl and p-fluoro derivatives in the ability to inhibit vaginal cornification of ovariectomized rats upon intravaginal administration with oestradiol (3.2 ng total dose).7 Since tamoxifen in vivo was more active as a partial oestrogen agonist and antagonist than the para substituted fluoro, chloro and methyl derivatives that cannot undergo metabolic hydroxylation to monohydroxytamoxifen, whereas the antioestrogenic activity of the compounds upon local application in the vaginal cornification test was equivalent as was their ability to inhibit [(3)H]-oestradiol-17beta binding to the oestrogen receptor in vitro, it is suggested that at low doses; i.e. over the range of the partial agonist dose-response curve, the biological activity of tamoxifen is the net result of the activities of the parent compound and its metabolites.8 The results demonstrate that metabolic activation of non-steroidal antioestrogens is only an advantage and not a requirement for antioestrogenic activity.
Article
The mouse estrogen receptor was shown to be constantly shuttling between the nucleus and cytoplasm although under steady-state conditions it is detected predominantly in the cell nucleus in both the absence and presence of estradiol. Shuttling was demonstrated by monitoring the transfer of protein between nuclei in heterokaryons and by examining the subcellular distribution of mutant receptors. In the presence of the partial antiestrogen 4-hydroxytamoxifen the receptor was retained in the nucleus whereas it accumulated in the cytoplasm when cells were treated with the pure antiestrogen ICI 182780. The effect of the pure antiestrogen was to inhibit nucleocytoplasmic shuttling of the receptor by blocking its nuclear uptake. Thus although ligand binding is not required by the estrogen receptor to undergo nucleocytoplasmic shuttling, this process can be disrupted by the binding of a pure antiestrogen.
Article
Prediction of small molecule binding modes to macromolecules of known three-dimensional structure is a problem of paramount importance in rational drug design (the "docking" problem). We report the development and validation of the program GOLD (Genetic Optimisation for Ligand Docking). GOLD is an automated ligand docking program that uses a genetic algorithm to explore the full range of ligand conformational flexibility with partial flexibility of the protein, and satisfies the fundamental requirement that the ligand must displace loosely bound water on binding. Numerous enhancements and modifications have been applied to the original technique resulting in a substantial increase in the reliability and the applicability of the algorithm. The advanced algorithm has been tested on a dataset of 100 complexes extracted from the Brookhaven Protein DataBank. When used to dock the ligand back into the binding site, GOLD achieved a 71% success rate in identifying the experimental binding mode.
Article
Two experiments were conducted to determine the effect of tamoxifen (TAM) in mouse endometrium in comparison with that of 17beta-estradiol (E2). In a medium-term assay, TAM as well as E2 treatment semi-dose-dependently increased the levels of fos/jun mRNA and their oncoproteins (Fos/Jun). The long-term effect of TAM on mouse endometrial carcinogenesis was also examined in the following model. A total of 150 female ICR mice, 12-13 weeks of age, were used. Of these, 125 mice received an injection of N-methyl-N-nitosourea (MNU) solution (1 mg/100 g body weight) into their left uterine tube and saline into the right. One week later, they were divided into four groups: groups 1 (35 mice) and 2 (30 mice) were given 25 ppm and ppm E2-containing diet, respectively, while group 3 (30 mice) was fed 5 ppm TAM-containing diet. Group 5 (30 mice) was fed basal diet alone. The remaining 25 mice (group 4) received 5 ppm TAM-containing diet alone. At the termination of the experiment (30 weeks), endometrial carcinomas were confirmed to be present in the groups exposed to MNU. TAM increased the incidence of preneoplastic lesions of the endometrium, while E2 enhanced the occurrence of the carcinoma. No carcinomas were found in the group given TAM alone. In the ovaries, corpora lutea were lacking in most of the mice exposed to TAM, suggesting that the animals were not cycling. Such findings indicated that TAM has an enhancing effect on endometrial carcinogenesis in mice, probably via a mechanism involving overexpression of Fos/Jun proteins.
Article
In eukaryotic cells, the ubiquitin-proteasome pathway is the major mechanism for the targeted degradation of proteins with short half-lives. The covalent attachment of ubiquitin to lysine residues of targeted proteins is a signal for the recognition and rapid degradation by the proteasome, a large multi-subunit protease. In this report, we demonstrate that the human estrogen receptor (ER) protein is rapidly degraded in mammalian cells in an estradiol-dependent manner. The treatment of mammalian cells with the proteasome inhibitor MG132 inhibits activity of the proteasome and blocks ER degradation, suggesting that ER protein is turned over through the ubiquitin-proteasome pathway. In addition, we show that in vitro ER degradation depends on ubiquitin-activating E1 enzyme (UBA) and ubiquitin-conjugating E2 enzymes (UBCs), and the proteasome inhibitors MG132 and lactacystin block ER protein degradation in vitro. Furthermore, the UBA/UBCs and proteasome inhibitors promote the accumulation of higher molecular weight forms of ER. The UBA and UBCs, which promote ER degradation in vitro, have no significant effect on human progesterone receptor and human thyroid hormone receptor beta proteins.
Article
Tamoxifen is the endocrine treatment of choice for all stages of estrogen receptor (ER)-positive breast cancer, and it is the first drug approved to reduce the incidence of breast cancer in high-risk women. Unfortunately, tamoxifen also possesses some estrogen-like effects in the uterus that cause a modest increase in the risk of endometrial cancer. GW5638 is a tamoxifen derivative with a novel carboxylic acid side chain with no uterotropic activity in the rat (Willson et al., J Med Chem, 1994, 37:1550-1552). We have compared and contrasted the actions of 4-hydroxytamoxifen (4-OHT, the active metabolite of tamoxifen) with GW7604 [the presumed metabolite of GW5638 in breast (MCF-7) and endometrial (ECC-1) cell lines in vitro]. GW7604 did not cause the growth of ECC-1 cells at any concentration (10(-11)-10(-6) M), but 4-OHT was weakly estrogen-like at low concentrations (10(-11)-10(-10) M). Compounds (10(-7) M) blocked the growth promoting action of estradiol (10(-10) M) in both ECC-1 and MCF-7 cells. Western blotting was used to show that GW7604 and raloxifene did not affect ER levels significantly, compared with controls, in MCF-7 cells; whereas the pure antiestrogen ICI182,780 decreased ER levels (P < 0.05). An assay system was used that can classify compounds into tamoxifen-like, raloxifene-like, or pure antiestrogens. The assay depends on the activation of the transforming growth factor alpha (TGFalpha) gene in situ by wild-type or D351Y mutant ER stably transfected into MDA-MB-231 cells (MacGregor-Schafer et al., Cancer Res, 1999, 59:4308-4313). GW7604 inhibited both estradiol (10(-9) M) and 4-OHT (10(-8), 10(-7) M) induction of TGFalpha in a concentration related manner (10(-9)-10(-6) M). GW7604 and raloxifene stimulated TGFalpha with the D351Y ER. In contrast, ICI 182,780 (10(-6) M) did not initiate TGFalpha and blocked the induction of TGFalpha with GW7604, raloxifene, and 4-OHT in D351Y-transfected cells. Using computer-assisted molecular models of ER complexes, we found that the antiestrogenic side chain of 4-OHT weakly interacted with the surface amino acid 351 (aspartate), but the carboxylic acid of GW7604 caused a strong repulsion of aspartate 351. We propose that GW7604 is less estrogen-like than 4-OHT, because it disrupts the surface charge around aa351 required for coactivator docking in the 4-OHT:ER complex. This charge is restored in the D351Y ER, thus converting GW7604 from an antiestrogen to an estrogen-like molecule.
Article
Estrogens exert their effects on target tissues by binding to a nuclear transcription factor termed the estrogen receptor (ER). Previous structural studies have demonstrated that each class of ER ligand (agonist, partial agonist, and SERM antagonist) induces distinctive orientations in the receptor's carboxy-terminal transactivation helix. The conformation of this portion of the receptor determines whether ER can recruit and interact with the components of the transcriptional machinery, thereby facilitating target gene expression. We have determined the structure of rat ERbeta ligand binding domain (LBD) in complex with the pure antiestrogen ICI 164,384 at 2.3 A resolution. The binding of this compound to the receptor completely abolishes the association between the transactivation helix (H12) and the rest of the LBD. The structure reveals that the terminal portion of ICI's bulky side chain substituent protrudes from the hormone binding pocket, binds along the coactivator recruitment site, and physically prevents H12 from adopting either its characteristic agonist or AF2 antagonist orientation. The binding mode adopted by the pure antiestrogen is similar to that seen for other ER antagonists. However, the size and resultant positioning of the ligand's side chain substituent produces a receptor conformation that is distinct from that adopted in the presence of other classes of ER ligands. The novel observation that binding of ICI results in the complete destabilization of H12 provides some indications as to a possible mechanism for pure receptor antagonism.
Article
C2-Alkyl-substituted 1,1-bis(4-hydroxyphenyl)-2-phenylethenes were synthesized and assayed for estrogen receptor binding in a competition experiment with radiolabeled estradiol ([3H]-E2) using calf uterine cytosol. The relative binding affinity decreased with the length of the side chain R = H (3a: 35.2%) > Me (3b: 32.1%) > Et (3c: 6.20%) approximately CH2CF3 (3d: 5.95%) > n-Pr (3e: 2.09%) > Bu (3f: 0.62%). Agonistic and antagonistic effects were evaluated in the luciferase assay with MCF-7-2a cells stably transfected with the plasmid ERE(wtc)luc. All compounds showed high antiestrogenic activity without significant agonistic potency. The comparison of the IC(50) values for the inhibition of E2 (1 nM) documented the dependence of the antagonistic effects on the kind of the side chain: 3a (IC50 = 150 nM), 3b (IC50 = 30 nM), and 3f (IC50 = 500 nM) were weak antagonists, while 3c (IC50 = 15 nM), 3d (IC50 = 9 nM), and 3e (IC50 = 50 nM) were full antiestrogens and antagonized the effect of E2 completely. The most active compound 3d possessed the same antagonistic potency as 4-hydroxytamoxifen (4OHT: IC50= 7 nM) without bearing a basic side chain. 3d as well as all other 1,1-bis(4-hydroxyphenyl)-2-phenylalkenes were not able to influence the proliferation of hormone dependent MCF-7 cells despite the antagonistic mode of action. In this assay tamoxifen (TAM) and 4OHT reduced the cell growth concentration dependent up to T/C(corr) = 15% and 25%, respectively.
Article
In the search for a breast cancer prevention strategy which would avoid undesirable effects of orally administered tamoxifen, the percutaneous administration of the highly active metabolite 4OHTamoxifen (4OHTam) has been proposed. Percutaneous 4OHTam penetrates the skin to reach breast tissues. It, thus, avoids the hepatic first pass effect, and offers an optimal local/systemic effect. However, trans-4OHTamoxifen can spontaneously isomerize into the cis-isomer, which may have estrogen agonist action. The aim of this study was to examine the effect of cis-4OHTam on normal human breast epithelial (HBE) cells in culture. Spontaneous isomerization of trans- into cis-4OHTam occurred within 24-48h, but stabilized rapidly at a trans/cis ratio of 70/30, whether in stock solution, culture medium or cultured cells. The cis-4OHTam did not stimulate HBE cell growth according to histometric cell counts and scanning electron microscopy analysis, but inhibited E(2)-induced cell growth, albeit two to three times less than trans-4OHTam. In conclusion, spontaneous isomerization of trans- to cis-4-OHTam is limited and 4OHTam retains a marked antiestrogenic effect. It may prove to be a useful alternative to tamoxifen in breast cancer prevention, especially if administered percutaneously.
Article
Nuclear receptors (NRs) complexed with agonist ligands activate transcription by recruiting coactivator protein complexes. In principle, one should be able to inhibit the transcriptional activity of the NRs by blocking this transcriptionally critical receptor-coactivator interaction directly, using an appropriately designed coactivator binding inhibitor (CBI). To guide our design of various classes of CBIs, we have used the crystal structure of an agonist-bound estrogen receptor (ER) ligand binding domain (LBD) complexed with a coactivator peptide containing the LXXLL signature motif bound to a hydrophobic groove on the surface of the LBD. One set of CBIs, based on an outside-in design approach, has various heterocyclic cores (triazenes, pyrimidines, trithianes, cyclohexanes) that mimic the tether sites of the three leucines on the peptide helix, onto which are appended leucine residue-like substituents. The other set, based on an inside-out approach, has a naphthalene core that mimics the two most deeply buried leucines, with substituents extending outward to mimic other features of the coactivator helical peptide. A fluorescence anisotropy-based coactivator competition assay was developed to measure the specific binding of these CBIs to the groove site on the ER-agonist complex with which coactivators interact; control ligand-binding assays assured that their interaction was not with the ligand binding pocket. The most effective CBIs were those from the pyrimidine family, the best binding with K(i) values of ca. 30 microM. The trithiane- and cyclohexane-based CBIs appear to be poor structural mimics, because of equatorial vs axial conformational constraints, and the triazene-based CBIs are also conformationally constrained by amine-substituent-to-ring resonance overlap, which is not the case with the higher affinity alkyl-substituted pyrimidines. The pyrimidine-based CBIs appear to be the first small molecule inhibitors of NR coactivator binding.
Article
A new group of steviolbioside amide dimers 2a-g, derivatives 2h-i and their related steviol and isosteviol amide dimers 3a and 4a were prepared by reacting aliphatic alkylamine and alkyldiamines with PyBOP and DIEA. The synthesized compounds had cytotoxic effects on cancer and human embryonic lung cells. Compounds 3a, 4a, 2b and 2h were cytotoxic to cancer cells and to a lesser extent to human embryo lung cells. Compounds 2f, 2g and 4 of this series had favorable antibacterial effects, and were superior to penicillin G at inhibiting growth of Bacillus subtilis (BCRC 10029). The cytotoxicity and antibacterial effects may depend on the dimerization and derivative moieties in relation to the respective aglycons.
Article
From the historically grown archive of protein-ligand complexes in the Protein Data Bank small organic ligands are extracted and interpreted in terms of their chemical characteristics and features. Subsequently, pharmacophores representing ligand-receptor interaction are derived from each of these small molecules and its surrounding amino acids. Based on a defined set of only six types of chemical features and volume constraints, three-dimensional pharmacophore models are constructed, which are sufficiently selective to identify the described binding mode and are thus a useful tool for in-silico screening of large compound databases. The algorithms for ligand extraction and interpretation as well as the pharmacophore creation technique from the automatically interpreted data are presented and applied to a rhinovirus capsid complex as application example.
Article
Estrogen receptor alpha (ER) turnover in MCF-7 cells was assessed by pulse chase analysis and measurement of ER steady-state level. In untreated cells, degradation of (35)S-labeled ER was characterized by a slow phase followed by a more rapid decline. Without ligand, ER elimination was totally compensated by synthesis which maintained receptor homeostasis. Estradiol (E(2)) and the pure antiestrogen RU 58,668 abolished the slow phase of ER breakdown and enhanced the degradation of neosynthesized ER, producing a low ER steady-state level. By contrast, the partial antiestrogen OH-Tam was ineffective in this respect and caused ER accumulation. Regardless of the conditions, ER breakdown was abolished by proteasome inhibition (MG-132). ER ligands decreased cell capacity to bind [(3)H]E(2), even in the presence of MG-132, indicating that the regulation of ER level and E(2) binding capacity occurs through distinct mechanisms. MG-132 partially blocked the basal transcription of an ERE-dependent reporter gene and modified the ability of E(2) to induce the expression of the latter: the hormone was unable to restore the transactivation activity measured without MG-132. RU 58,668 and OH-Tam failed to enhance the inhibitory action of MG-132, suggesting that a loss of basal ER-mediated transactivation mainly affects the stimulatory effect of estrogens. Overall, our findings reveal that ER steady state level, ligand binding capacity and transactivation potency fit in a complex regulatory scheme involving distinct mechanisms, which may be dissociated from each other under various treatments.
Article
Tamoxifen is effective for the prevention and treatment of estrogen-dependent breast cancers, but is associated with an increased incidence of endometrial tumors. We report the crystal structure of the estrogen receptor alpha (ERalpha) ligand binding domain (LBD) bound to the structurally similar compound GW5638, which has therapeutic potential and does not stimulate the uterus. Like tamoxifen, GW5638 relocates the carboxy-terminal helix (H12) to the known coactivator-docking site in the ERalpha LBD. However, GW5638 repositions residues in H12 through specific contacts with the N terminus of this helix. In contrast to tamoxifen, the resulting increase in exposed hydrophobic surface of ERalpha LBD correlates with a significant destabilization of ERalpha in MCF-7 cells. Thus, the GW5638-ERalpha LBD structure reveals an unexpected mode of SERM-mediated ER antagonism, in which the stability of ERalpha is decreased through an altered position of H12. This dual mechanism of antagonism may explain why GW5638 can inhibit tamoxifen-resistant breast tumors.
Article
Antiestrogens used for breast cancer therapy can be categorized into two classes that differ in their effect on estrogen receptor (ER) alpha stability. The selective estrogen receptor modulators (SERMs) stabilize ER alpha and the selective estrogen receptor downregulators (SERDs) cause a decrease in cellular ER alpha levels. A clinically relevant antiestrogen, GW7604, appears to work through a SERD-like mechanism, despite sharing the same molecular scaffold as 4-hydroxytamoxifen, a SERM. In order to investigate potential structural features of GW7604 responsible for SERD activity, GW7604 and two analogs were synthesized using a new, improved synthetic route and tested for their effects on ER alpha function and cell proliferation. The two analogs, which have an acrylamide or a methyl vinyl ketone replacing the acrylic acid group of GW7604, display lower binding affinity for ER alpha than GW7604, but show similar antagonism of estradiol-induced activation of ER alpha-mediated transcription as GW7604 and inhibit estradiol-induced proliferation of the MCF-7 cell line with a similar potency as GW7604. Unlike GW7604, neither analog has a significant effect on cellular ER alpha levels, suggesting that the carboxylate is a key determinant in GW7604 action and, for the first time, showing that this group is responsible for inducing ER alpha degradation in breast cancer cells.