Molecular cloning of estrogen receptor and its function on vitellogenesis in pompano (Trachinotus ovatus).

Estrogen receptors (ERs) play a critical role in vitellogenesis (Vtgs). However, the contribution of each ER for the regulation of vtgs expression was not analyzed clearly in teleosts. In the present study, three ers isoforms (erα, erß1, and erß2) were cloned in pompano (Trachinotus ovatus). Real-time PCR and enzyme-linked immunosorbent assay (ELISA) was used to detect the effects of 17ß-estradiol (E2) on ERs and Vtgs in the liver of pompano. In vivo injection experiments showed that E2 significantly increased the expressions of ers and vtgs. ER broad spectrum antagonist Fulvestrant significantly attenuated the E2- induced up-regulation of ers and vtgs in a dose-dependent manner. ERα antagonist Methyl-piperidino pyrazole (MPP) significantly attenuated the up-regulation of erα, erß2, vtg-B and vtg-C, and promoted the expressions of erß1 and vtg-A. ERß antagonist Cyclofenil significantly inhibited the expressions of erß1, erß2, vtg-A and vtg-C, and promoted the expressions of erα and vtg-B. In addition, E2 significantly increased the protein level of Vtg, while Fulvestrant, MPP and Cyclofenil significantly inhibited the protein level of Vtg in a dose-dependent manner. Our results indicate that E2 may regulate the expression of each vtg with different subtypes of ERs, and shows a distinct compensatory expression effect on the regulation for ers and vtgs, which provides a theoretical basis for reproductive endocrinology study in pompano.

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

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/ß 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.

Structure-activity relationship study of estrogen receptor down-regulators with a diphenylmethane skeleton.

Selective estrogen receptor (ER) down-regulators (SERDs) are pure ER antagonists that also induce ER degradation upon binding to the receptor. Although SERDs have been developed for the treatment of ER-positive breast cancers for nearly a decade, their precise mechanism(s) of action and structure-activity relationship are still unclear. Generally, Western blotting is used to examine the effects of SERDs on ER protein levels, but the methodology is low-throughput and not quantitative. Here, we describe a quantitative, high-throughput, luciferase-based assay for the evaluation of SERDs activity. For this purpose, we established stable recombinant HEK-293 cell lines expressing ERα fused with emerald luciferase. We also designed and synthesized new diphenylmethane derivatives as candidate SERDs, and evaluated their SERDs activity using the developed system in order to examine their structure-activity relationship, taking EC50 as a measure of potency, and Emax as a measure of efficacy.

An estrogen antagonist, cyclofenil, has anti-dengue-virus activity.

Dengue virus (DENV) infections are a major cause of morbidity and mortality in tropical and subtropical areas. Several compounds that act against DENV have been studied in clinical trials to date; however, there have been no compounds identified that are effective in reducing the severity of the clinical manifestations. To explore anti-DENV drugs, we examined small molecules that interact with DENV NS1 and inhibit DENV replication. Cyclofenil, which is a selective estrogen receptor modulator (SERM) and has been used clinically as an ovulation-inducing drug, showed an inhibitory effect on DENV replication in mammalian cells but not in mosquito cells. Other SERMs also inhibited DENV replication in mammalian cells, but cyclofenil showed the weakest cytotoxicity among these SERMs. Cyclofenil also inhibited the replication of Zika virus. A time-of-addition assay suggested that cyclofenil may interfere with two stages of the DENV life cycle: the translation-RNA synthesis and assembly-maturation stages. However, the level of intracellular infectious particles decreased more drastically after treatment with cyclofenil than the viral RNA level did, indicating that the assembly-maturation stage might be the main target of cyclofenil. In electron microscopy analysis, many aggregated particles were detected in DENV-infected cells in the presence of cyclofenil, supporting the possibility that cyclofenil impedes the process of assembly and maturation of DENV.

Novel Selective Estrogen Receptor Ligand Conjugates Incorporating Endoxifen-Combretastatin and Cyclofenil-Combretastatin Hybrid Scaffolds: Synthesis and Biochemical Evaluation.

Nuclear receptors such as the estrogen receptors (ERα and ERß) modulate the effects of the estrogen hormones and are important targets for design of innovative chemotherapeutic agents for diseases such as breast cancer and osteoporosis. Conjugate and bifunctional compounds which incorporate an ER ligand offer a useful method of delivering cytotoxic drugs to tissue sites such as breast cancers which express ERs. A series of novel conjugate molecules incorporating both the ER ligands endoxifen and cyclofenil-endoxifen hybrids covalently linked to the antimitotic and tubulin targeting agent combretastatin A-4 were synthesised and evaluated as ER ligands. A number of these compounds demonstrated pro-apoptotic effects, with potent antiproliferative activity in ER-positive MCF-7 breast cancer cell lines and low cytotoxicity. These conjugates displayed binding affinity towards ERα and ERß isoforms at nanomolar concentrations e.g., the cyclofenil-amide compound 13e is a promising lead compound of a clinically relevant ER conjugate with IC50 in MCF-7 cells of 187 nM, and binding affinity to ERα (IC50 = 19 nM) and ERß (IC50 = 229 nM) while the endoxifen conjugate 16b demonstrates antiproliferative activity in MCF-7 cells (IC50 = 5.7 nM) and binding affinity to ERα (IC50 = 15 nM) and ERß (IC50 = 115 nM). The ER binding effects are rationalised in a molecular modelling study in which the disruption of the ER helix-12 in the presence of compounds 11e, 13e and 16b is presented These conjugate compounds have potential application for further development as antineoplastic agents in the treatment of ER positive breast cancers.

Effects of platelet-activating factor and its differential regulation by androgens and steroid hormones in prostate cancers.

BACKGROUND: Platelet-activating factor (PAF) is an arachidonic acid metabolite that plays an important role in cell proliferation, migration and neoangiogenesis, but whether it is involved in the progression of prostate cancer remains undiscovered. METHODS: Clinical prostate specimens were investigated with immunohistochemistry method and in vitro cell experiments referred to MTS cell proliferation assay, invasion and migration experiment, quantitative real-time RT-PCR assay, western blotting analysis and ELISA assay. RESULTS: Platelet-activating factor synthetase, lyso-PAF acetyl transferase (LPCAT1), increased significantly in castration-resistant prostate cancer (CRPC) specimens and CRPC PC-3 cells than that in controls. Intriguingly, PAF induced invasion and migration of PC-3 cells but not LNCaP cells. The PAF receptor antagonist inhibited proliferation of LNCaP and PC-3 cells. Dihydrotestosterone (DHT) treatment caused a decrease in LPCAT1 expression and PAF release in LNCaP cells, which could be blocked by androgen receptor antagonists. Finally, DHT increased LPCAT1 expression and PAF release in PC-3 cells in a Wnt/ß-catenin-dependent manner. CONCLUSION: For the first time, our data supported that PAF might play pivotal roles in the progression of prostate cancer, which might throw a new light on the treatment of prostate cancer and the prevention of the emergence of CRPC.

Stress induced hippocampal mineralocorticoid and estrogen receptor ß gene expression and long-term potentiation in male adult rats is sensitive to early-life stress experience.

Glucocorticoid hormones and their receptors have been identified to be involved in emotional and cognitive disorders in early stressed subjects during adulthood. However, the impact of other steroid hormones and receptors has been considered less. Especially, functional roles of estrogen and estrogen receptors in male subjects are largely unknown. Therefore, we measured hippocampal concentrations of 17ß-estradiol, corticosterone and testosterone, as well as the gene expression of estrogen receptor α and ß (ERα, ß), androgen receptor (AR), glucocorticoid (GR) and mineralocorticoid (MR) receptors after stress in adulthood in maternally separated (MS+; at postnatal days 14-16 for 6h each day) and control (MS-) male rats. In vivo hippocampal long-term potentiation (LTP) serves as a cellular model of learning and memory formation. Population spike- (PSA) and the fEPSP-LTP within the dentate gyrus (DG) were reinforced by elevated-platform-stress (EP-stress) in MS- but not in MS+ rats. MR- and ERß-mRNA were upregulated 1h after EP-stress in MS- but not in MS+ rats as compared to non-stressed littermates. Infusion of an MR antagonist before LTP induction blocked early- and late-PSA- and -fEPSP-LTP, whereas blockade of ERß impaired only the late PSA-LTP. Application of a DNA methyltransferase (DNMT) inhibitor partly restored the LTP-reinforcement in MS+ rats, accompanied by a retrieval of ERß- but not MR-mRNA upregulation. Basal ERß gene promoter methylation was similar between groups, whereas MS+ and MS- rats showed different methylation patterns across CpG sites after EP-stress. These findings indicate a key role of ERß in early-stress mediated emotionality and emotion-induced late-LTP in adult male rats via DNA methylation mechanisms.

Cyclofenil as a possible cause of non-arteritic anterior ischaemic optic neuropathy.

A 27-year-old woman receiving the steroid drug cyclofenil as a fertility adjunct, experienced blurred vision 24 h after missing a dose and taking a double dose to 'catch up' with her therapeutic protocol. She was found to have a non-arteritic anterior ischaemic optic neuropathy with a visual hemifield defect and impaired optic nerve function, which has not since shown any recovery. This case highlights the prothrombotic potential for the drug when used above normal dosing range, and is therefore of great guidance for those initiating it as a fertility treatment, or in unlicensed use.

Design, synthesis, and evaluation of cyclofenil derivatives for potential SPECT imaging agents.

To develop technetium- and rhenium-labeled nonsteroidal estrogen imaging agents for estrogen receptor (ER) positive breast tumors, two groups of rhenium and technetium cyclofenil derivatives were synthesized and characterized. The binding affinities of the rhenium complexes for ERs were determined. The tricarbonyl rhenium complex showed the highest binding affinity for ERs (81.2 for ERbeta, 16.5 for ERalpha). Tricarbonyl technetium cyclofenil complexes were obtained in high radiochemical purity and radiochemical yields. The results of studies of their octanol/water partition and in vitro stability are presented. These results demonstrate that these radiolabeled cyclofenil derivatives may be considered as potential breast cancer imaging agents.

Monitoring a coordinated exchange process in a four-component biological interaction system: development of a time-resolved terbium-based one-donor/three-acceptor multicolor FRET system.

Hormonal regulation of cellular function involves the binding of small molecules with receptors that then coordinate subsequent interactions with other signal transduction proteins. These dynamic, multicomponent processes are difficult to track in cells and even in reconstituted in vitro systems, and most methods can monitor only two-component interactions, often with limited capacity to follow dynamic changes. Through a judicious choice of three organic acceptor fluorophores paired with a terbium donor fluorophore, we have developed the first example of a one-donor/three-acceptor multicolor time-resolved fluorescence energy transfer (TR-FRET) system, and we have exemplified its use by monitoring a ligand-regulated protein-protein exchange process in a four-component biological system. By careful quantification of the emission from each of the three acceptors at the four channels for terbium donor emission, we demonstrate that any of these donor channels can be used to estimate the magnitude of the three FRET signals in this terbium-donor triple-acceptor system with minimal bleedthrough. Using this three-channel terbium-based, TR-FRET assay system, we show in one experiment that the addition of a fluorescein-labeled estrogen agonist displaces a SNAPFL-labeled antiestrogen from the ligand binding pocket of a terbium-labeled estrogen receptor, at the same time causing a Cy5-labeled coactivator to be recruited to the estrogen receptor. This experiment demonstrates the power of a four-color TR-FRET experiment, and it shows that the overall process of estrogen receptor ligand exchange and coactivator binding is a dynamic but precisely coordinated process.

Characterization of the pharmacophore properties of novel selective estrogen receptor downregulators (SERDs).

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.

Photocontrol of protein activity in cultured cells and zebrafish with one- and two-photon illumination.

We have implemented a noninvasive optical method for the fast control of protein activity in a live zebrafish embryo. It relies on releasing a protein fused to a modified estrogen receptor ligand binding domain from its complex with cytoplasmic chaperones, upon the local photoactivation of a nonendogenous caged inducer. Molecular dynamics simulations were used to design cyclofen-OH, a photochemically stable inducer of the receptor specific for 4-hydroxy-tamoxifen (ER(T2)). Cyclofen-OH was easily synthesized in two steps with good yields. At submicromolar concentrations, it activates proteins fused to the ER(T2) receptor. This was shown in cultured cells and in zebrafish embryos through emission properties and subcellular localization of properly engineered fluorescent proteins. Cyclofen-OH was successfully caged with various photolabile protecting groups. One particular caged compound was efficient in photoinducing the nuclear translocation of fluorescent proteins either globally (with 365 nm UV illumination) or locally (with a focused UV laser or with two-photon illumination at 750 nm). The present method for photocontrol of protein activity could be used more generally to investigate important physiological processes (e.g., in embryogenesis, organ regeneration and carcinogenesis) with high spatiotemporal resolution.

Nonclassical SNAPFL analogue as a Cy5 resonance energy transfer partner.

We have synthesized a new SNAPFL analogue (1) that exhibits a large Stokes shift (78 nm) (abs. 542 nm, em. 620 nm) and a good quantum yield. Because of the large overlap between the emission spectrum of 1 and the absorption spectrum of Cy5, 1 functions well as a fluorescence donor to Cy5 and has been used in FRET-based experiments using estrogen receptor site-specifically labeled with Cy5 and a receptor ligand conjugated to SNAPFL.

Synthesis and identification of hydroxylated metabolites of the anti-estrogenic agent cyclofenil.

The detection of metabolites of the anti-estrogenic substance cyclofenil, listed on the World Anti-Doping Agency (WADA) Prohibited List since 2004 is described. Target substances are hydroxylated metabolites, bearing an aliphatic hydroxyl group either in the 2-, 3- or 4-position of the aliphatic ring, in addition to the phenolic functions on the aromatic rings. Structural identification used NMR as well as high-resolution mass spectrometry after nano-electrospray ionisation (ESI). Unambiguous detection of all three synthesised cyclofenil metabolites M1-M3 was done using gas chromatography for separation and electron ionisation mass spectrometry for detection of the per-silylated compounds in comparison with a reference urine deriving from an excretion study within the WADA 2007 Educational Programme.

Fluorimetric determination of cyclofenil using photochemical derivatization.

In this work, a fluorimetric approach for the determination of cyclofenil, 4-4'-(cyclohexylidenemethylene)bis(phenylacetate), is presented. The method was based on the intense fluorescence (250/410 nm) observed after a UV photochemical treatment of cyclofenil. The influence of the pH and solvent system and UV exposure time on the fluorescence magnitude was studied. Optimization of parameters was made using experimental design (factorial design and central composite design). Limit of detection (3S(b)/m) were estimated to be 1.1 x 10(-8)mol L(-1) with the linear dynamic range extending up to 8 x 10(-5)mol L(-1). This analytical approach was tested through the analysis of a commercial pharmaceutical formulation. In this case, tests enabled an average recovery of 98.3+/-3.9% (for n=9) using the analytical curve. The identification of the fluorescent derivative is proposed based on results achieved from GC-MS.

Synthesis of new carbon-11 labeled cyclofenil derivatives for PET imaging of breast cancer estrogen receptors.

Carbon-11 labeled cyclofenil derivatives, [(11)C]methyl-2-{4-[bis(4-hydroxyphenyl)methylene]cyclohexyl}acetate ([(11)C]16a), [(11)C]methyl-4-[bis(4-hydroxyphenyl)methylene]cyclohexanecarboxylate ([(11)C]16b), [(11)C]methyl-2-{3-[bis(4-hydroxyphenyl)methylene]cyclohexyl}acetate ([(11)C]18a), and [(11)C]methyl-3-[bis(4-hydroxyphenyl)methylene]cyclohexanecarboxylate ([(11)C]18b), have been synthesized as new potential PET agents for imaging breast cancer estrogen receptors. The target tracers were prepared by O-[(11)C]methylation of their corresponding precursors using [(11)C]CH(3)OTf and isolated by a simplified SPE purification procedure in 35-50% radiochemical yields decay corrected to EOB, 15-20 min overall synthesis time, and 74-111 GBq/micromol specific activity at EOS.

Synthesis and biodistribution of fluorine-18-labeled fluorocyclofenils for imaging the estrogen receptor.

C4-[18F]Fluorocyclofenil ([18F]FCF, 6) and C3-[18F]fluoroethylcyclofenil ([18F]FECF, 9), two high-affinity nonsteroidal estrogens, were prepared and investigated as potential agents for imaging estrogen receptors (ERs) in breast tumors. Both of these compounds could be prepared conveniently from alkyl methanesulfonate precursors (5,8) by fluoride displacement reactions, and they were obtained in high radiochemical purity and radiochemical yields, with effective specific activities sufficient for in vivo biodistribution studies. While the biodistribution of [18F]FCF (6) in immature female rats showed no selective target tissue uptake, the biodistribution of [18F]FECF (9) showed selective uptake by the uterus, but this uptake could not be blocked by excess estradiol. The poor in vivo biodistribution of these otherwise high-affinity ligands arouses curiosity, and together with recent results on the biodistribution of other nonsteroidal ligands suggests that factors other than receptor binding affinity are important for in vivo imaging of estrogen target tissues and ER-positive breast tumors.

Fluorine-substituted cyclofenil derivatives as estrogen receptor ligands: synthesis and structure-affinity relationship study of potential positron emission tomography agents for imaging estrogen receptors in breast cancer.

In a search for estrogen receptor (ER) ligands to be radiolabeled with fluorine-18 for imaging of ER-positive breast tumors with positron emission tomography (PET), we investigated cyclofenil analogues substituted at the C3 or C4 position of the cyclohexyl group. McMurry coupling of 4,4'-dihydroxybenzophenone with various ketones produced key cyclofenil intermediates, from which C3 and C4 substituents containing alkyl and various oxygen or fluorine-substituted alkyl groups were elaborated. Binding assays to both ERalpha and ERbeta revealed that the C3 site is more tolerant of steric bulk and polar groups than the C4 site, consistent with a computational model of the ERalpha ligand binding pocket. Fluorine substitution is tolerated very well at some sites, giving some compounds having affinities comparable to or higher than that of estradiol. These fluoro and fluoroalkyl cyclofenils merit further consideration as fluorine-18 labeled ER ligands for PET imaging of ERs in breast tumors.

Exploration of the bicyclo[3.3.1]nonane system as a template for the development of new ligands for the estrogen receptor.

Three novel structural motifs based on a bicyclo [3.3.1]nonane template were examined as new ligands for estrogen receptor (ER). Type III compounds emerged as the most promising leads for developing high-affinity ER ligands, but they showed little selectivity for either ER subtype. Type II compounds, on the other hand, despite their lower affinity, exhibited significant ERbeta binding selectivity.