Click synthesis of estradiol–cyclodextrin conjugates as cell compartment selective estrogens

Cyclodextrin (CD) is a well known drug carrier and excipient for enhancing aqueous solubility. CDs themselves are anticipated to have low membrane permeability because of relatively high hydrophilicity and molecular weight. CD derivatization with 17-beta estradiol (E₂) was explored extensively using a number of different click chemistries and the cell membrane permeability of synthetic CD–E₂ conjugate was explored by cell reporter assays and confocal fluorescence microscopy. In simile with reported dendrimer–E₂ conjugates, CD–E₂ was found to be a stable, extranuclear receptor selective estrogen that penetrated into the cytoplasm.     

A critical review of fundamental controversies in the field of GPR30 research

The female sex hormone estradiol plays an important role in reproduction, mammary gland development, bone turnover, metabolism, and cardiovascular function. The effects of estradiol are mediated by two classical nuclear receptors, estrogen receptor α (ERα) and estrogen receptor β (ERβ).In 2005, G-protein-coupled receptor 30 (GPR30) was claimed to act as a non-classical estrogen receptor that was also activated by the ERα and ERβ antagonists tamoxifen and fulvestrant (ICI 182780). Despite many conflicting results regarding the potential role of GPR30 as an estrogen receptor, the official nomenclature was changed to GPER (G-protein-coupled estrogen receptor).This review revisits the inconsistencies that still exist in the literature and focuses on selected publications that basically address the following two questions: what is the evidence for and against the hypothesis that GPR30 acts as an estrogen receptor? What is the potential in vivo role of GPR30?Thus, in the first part we focus on conflicting results from in vitro studies analysing the subcellular localization of GPR30, its ability to bind (or not to bind) estradiol and to signal (or not to signal) in response to estradiol. In the second part, we discuss the strengths and limitations of four available GPR30 mouse models. We elucidate the potential impact of different targeting strategies on phenotypic diversity.     

Enhanced A3 adenosine receptor selectivity of multivalent nucleoside-dendrimer conjugates

Background

An approach to use multivalent dendrimer carriers for delivery of nucleoside signaling molecules to their cell surface G protein-coupled receptors (GPCRs) was recently introduced.

Results

A known adenosine receptor (AR) agonist was conjugated to polyamidoamine (PAMAM) dendrimer carriers for delivery of the intact covalent conjugate to on the cell surface. Depending on the linking moiety, multivalent conjugates of the N ⁶-chain elongated functionalized congener ADAC (N ⁶-[4-[[[4-[[[(2-aminoethyl)amino]carbonyl]methyl]anilino]carbonyl]methyl]phenyl]-adenosine) achieved unanticipated high selectivity in binding to the cytoprotective human A₃ AR, a class A GPCR. The key to this selectivity of > 100-fold in both radioreceptor binding (K_{i app} = 2.4 nM) and functional assays (EC₅₀ = 1.6 nM in inhibition of adenylate cyclase) was maintaining a free amino group (secondary) in an amide-linked chain. Attachment of neutral amide-linked chains or thiourea-containing chains preserved the moderate affinity and efficacy at the A₁ AR subtype, but there was no selectivity for the A₃ AR. Since residual amino groups on dendrimers are associated with cytotoxicity, the unreacted terminal positions of this A₃ AR-selective G2.5 dendrimer were present as carboxylate groups, which had the further benefit of increasing water-solubility. The A₃ AR selective G2.5 dendrimer was also visualized binding the membrane of cells expressing the A₃ receptor but did not bind cells that did not express the receptor.

Conclusion

This is the first example showing that it is feasible to modulate and even enhance the pharmacological profile of a ligand of a GPCR based on conjugation to a nanocarrier and the precise structure of the linking group, which was designed to interact with distal extracellular regions of the 7 transmembrane-spanning receptor. This ligand tool can now be used in pharmacological models of tissue rescue from ischemia and to probe the existence of A₃ AR dimers.     

Negative feedback loop of BRCA1–BARD1 ubiquitin ligase on estrogen receptor alpha stability and activity antagonized by cancer-associated isoform of BARD1

Estrogen is involved in breast cancer risk, which is increased for BRCA1 mutation carriers, suggesting a role for BRCA1 in estrogen signaling. BRCA1 exerts its function through forming an E3 ubiquitin ligase with BARD1. We report that the estrogen receptor alpha is a target of the BRCA1–BARD1 ubiquitin ligase in vivo. BRCA1 and BARD1 are required for estrogen receptor alpha ubiquitination and degradation, and repression of either one leads to ERα accumulation, suggesting a feedback loop between BRCA1–BARD1 and estrogen receptor alpha, since BRCA1 and BARD1 are induced by estrogen receptor alpha. While the ubiquitin ligase activity maps to the N-terminal RING finger domains of BRCA1 and BARD1, we demonstrate that the BARD1 C-terminus is important for target recognition. Furthermore, a BARD1 isoform lacking the RING domain binds and stabilizes estrogen receptor alpha. Thus deficiencies of BRCA1 or BARD1 and/or upregulation of BARD1 isoforms lead to estrogen receptor alpha upregulation, providing a functional link between BRCA1 deficiency, estrogen signaling, and tumorigenesis.     

Cyclopia Extracts Act as ERα Antagonists and ERβ Agonists,In Vitro and In Vivo

Hormone replacement therapy associated risks, and the concomitant reluctance of usage, has instigated the search for new generations of estrogen analogues that would maintain estrogen benefits without associated risks. Furthermore, if these analogues display chemo-preventative properties in breast and endometrial tissues it would be of great value. Both the selective estrogen receptor modulators as well as the selective estrogen receptor subtype modulators have been proposed as estrogen analogues with improved risk profiles. Phytoestrogen containing extracts of Cyclopia, an indigenous South African fynbos plant used to prepare Honeybush tea may serve as a source of new estrogen analogues. In this study three extracts, P104, SM6Met, and cup-of-tea, from two species of Cyclopia, C. genistoides and C. subternata, were evaluated for ER subtype specific agonism and antagonism both in transactivation and transrepression. For transactivation, the Cyclopia extracts displayed ERα antagonism and ERβ agonism when ER subtypes were expressed separately, however, when co-expressed only agonism was uniformly observed. In contrast, for transrepression, this uniform behavior was lost, with some extracts (P104) displaying uniform agonism, while others (SM6Met) displayed antagonism when subtypes were expressed separately and agonism when co-expressed. In addition, breast cancer cell proliferation assays indicate that extracts antagonize cell proliferation in the presence of estrogen at lower concentrations than that required for proliferation. Furthermore, lack of uterine growth and delayed vaginal opening in an immature rat uterotrophic model validates the ERα antagonism of extracts observed in vitro and supports the potential of the Cyclopia extracts as a source of estrogen analogues with a reduced risk profile.     

Molecular modeling of a PAMAM-CGS21680 dendrimer bound to an A2A adenosine receptor homodimer

The theoretical possibility of bivalent binding of a dendrimer, covalently appended with multiple copies of a small ligand, to a homodimer of a G protein-coupled receptor was investigated with a molecular modeling approach. A molecular model was constructed of a third generation (G3) poly(amidoamine) (PAMAM) dendrimer condensed with multiple copies of the potent A_2A adenosine receptor agonist CGS21680. The dendrimer was bound to an A_2A adenosine receptor homodimer. Two units of the nucleoside CGS21680 could occupy the A_2A receptor homodimer simultaneously. The binding mode of CGS21680 moieties linked to the PAMAM dendrimer and docked to the A_2A receptor was found to be similar to the binding mode of a monomeric CGS21680 ligand.     

Design, synthesis, and biological functionality of a dendrimer-based modular drug delivery platform

A modular dendrimer-based drug delivery platform was designed to improve upon existing limitations in single dendrimer systems. Using this modular strategy, a biologically active platform containing receptor mediated targeting and fluorescence imaging modules was synthesized by coupling a folic acid (FA) conjugated dendrimer with a fluorescein isothiocyanate (FITC) conjugated dendrimer. The two different dendrimer modules were coupled via the 1,3-dipolar cycloaddition reaction ("click" chemistry) between an alkyne moiety on the surface of the first dendrimer and an azide moiety on the second dendrimer. Two simplified model systems were also synthesized to develop appropriate "click" reaction conditions and aid in spectroscopic assignments. Conjugates were characterized by (1)H NMR spectroscopy and NOESY. The FA-FITC modular platform was evaluated in vitro with a human epithelial cancer cell line (KB) and found to specifically target the overexpressed folic acid receptor.     

Follicle-stimulating hormone promotes the transformation of cholesterol to estrogen in mouse adipose tissue

Follicle-stimulating hormone (FSH) levels increase estrogen biosynthesis in obese menopausal women. Ovariectomized mice and 3T3-L1 cells were used to explore estrogen biosynthesis in the decline of ovarian function. After ovariectomy, lipid deposition, and FSH and estrogen levels changed, and feed intake increased significantly. In mouse adipose tissue, FSH was found to have a role in accelerating lipid deposition via the peroxisome proliferators-activated receptor pathway, and in inducing estrogen biosynthesis via the steroid hormone metabolism pathway. Furthermore, FSH bound to the FSH receptor promoted CREB phosphorylation, which was activated by cAMP-PKA. Moreover, pCREB could up-regulate PPARγ and SREBP2 mRNA levels, resulting in an increased transformation of cholesterol to estrogen. Overall, this study shows that FSH induces fat deposition and promotes the transformation of cholesterol to estrogen through CREB activation by cAMP-PKA in mouse adipose tissue. Our findings provide a new understanding of menopause treatment.     

Novel effects of diosgenin on skin aging

Extracts of Dioscorea coomposita or Dioscorea villosa are consumed as supplemental health foods at the time of climacteric. The extracts contain large amounts of the plant steroid, diosgenin. Here, we studied the safety and efficacy of diosgenin against skin aging at the time of climacteric. In vitro, diosgenin enhanced DNA synthesis in a human 3D skin equivalent model, and increased bromodeoxyuridine uptake and intracellular cAMP level in adult human keratinocytes. The increase of bromodeoxyuridine uptake by diosgenin was blocked by an adenylate cyclase inhibitor, but not by antisense oligonucleotides against estrogen receptor α, estrogen receptor β or an orphan G-protein-coupled receptor, GPR30, indicating the involvement of cAMP but not estrogen receptor α, estrogen receptor β or GPR30. In vivo, administration of diosgenin improved the epidermal thickness in the ovariectomized mice, a climacteric model, without altering the degree of fat accumulation. In order to examine the safety of diosgenin, diosgenin and 17β-estradiol were administered to breast cancer-burdened mice. The results revealed that while 17β-estradiol accelerated the tumor growth, diosgenin did not show this effect. Our finding, a restoration of keratinocyte proliferation in aged skin, suggests that diosgenin may have potential as a safe health food for climacteric.     

A middle-affinity estrogen-specific binding protein in livers of vitellogenic and nonvitellogenic Xenopus laevis

Administration of estradiol-17β to male Xenopus laevis evokes massive liver synthesis of the egg yolk precursor protein, vitellogenin, and its cognate mRNA. Since previous experiments had implicated only a nuclear estrogen receptor in vitellogenesis, we examined Xenopus liver cytosol for other estrogen-binding proteins. Cytoplasmic extracts from unstimulated Xenopus liver contain high levels (approximately 500,000 sites/cell) of an estrogen-specific binding protein. This protein exhibits a K_d of approximately 4 × 10^?8M for estradiol-17β, binds estrogenic steroids only, and has a sedimentation coefficient in the range 2–3 S. It is not a classical estrogen receptor, as it does not translocate into the nucleus following estrogen administration. We discuss possible functions of this protein, which include a role in the ontogeny of the vitellogenic response, and in the cytoplasmic transport and storage of estrogen.     

Kaempferol is an estrogen-related receptor α and γ inverse agonist

Kaempferol is a dietary flavonoid that is thought to function as a selective estrogen receptor modulator. In this study, we established that kaempferol also functions as an inverse agonist for estrogen-related receptors alpha and gamma (ERRα and ERRγ). We demonstrated that kaempferol binds to ERRα and ERRγ and blocks their interaction with coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). Kaempferol also suppressed the expressions of ERR-target genes pyruvate dehydrogenase kinase 2 and 4 (PDK2 and PDK4). This evidence suggests that kaempferol may exert some of its biological effect through both estrogen receptors and estrogen-related receptors.

Structured summary:

MINT-6824653:PGC-1 alpha (uniprotkb:Q9UBK2) and ERR gamma (uniprotkb: P62508) bind (MI:0407) by surface plasmon resonance (MI:0107)     

Effects of Combination of Estradiol with Selective Progesterone Receptor Modulators (SPRMs) on Human Breast Cancer Cells In Vitro and In Vivo

Use of estrogen or estrogen / progestin combination was an approved regimen for menopausal hormonal therapy (MHT). However, more recent patient-centered studies revealed an increase in the incidence of breast cancer in women receiving menopausal hormone therapy with estrogen plus progestin rather than estrogen alone. Tissue selective estrogen complex (TSEC) has been proposed to eliminate the progesterone component of MHT with supporting evidences. Based on our previous studies it is evident that SPRMs have a safer profile on endometrium in preventing unopposed estrogenicity. We hypothesized that a combination of estradiol (E2) with selective progesterone receptor modulator (SPRM) to exert a safer profile on endometrium will also reduce mammary gland proliferation and could be used to prevent breast cancer when used in MHT. In order to test our hypothesis, we compared the estradiol alone or in combination with our novel SPRMs, EC312 and EC313. The compounds were effectively controlled E2 mediated cell proliferation and induced apoptosis in T47D breast cancer cells. The observed effects were found comparable that of BZD in vitro. The effects of SPRMs were confirmed by receptor binding studies as well as gene and protein expression studies. Proliferation markers were found downregulated with EC312/313 treatment in vitro and reduced E2 induced mammary gland proliferation, evidenced as reduced ductal branching and terminal end bud growth in vivo. These data supporting our hypothesis that E2+EC312/EC313 blocked the estrogen action may provide basic rationale to further test the clinical efficacy of SPRMs to prevent breast cancer incidence in postmenopausal women undergoing MHT.     

Neuroprotective effects of genistein in VSC4.1 motoneurons exposed to activated microglial cytokines

Pro-inflammatory cytokines released from activated microglia may be responsible for neuronal damage and resulting motor deficits associated with CNS disorders such as spinal cord injury, Parkinson’s disease, and multiple sclerosis. Estrogen (17β-estradiol) is capable of ameliorating motoneuron death following spinal cord injury, but has a number of deleterious side effects. Genistein (GEN), an estrogen receptor beta agonist and potent antioxidant, may represent an alternative to estrogen in treating neurodegenerative disorders. However, little is known about the neuroprotective effects of GEN. We therefore tested whether GEN would prevent apoptosis in cultured motoneurons following exposure to pro-inflammatory cytokines released from IFN-γ activated microglia. Exposure of ventral spinal cord 4.1 motoneurons to microglial cytokine supernatant in vitro caused significant apoptosis and reduced mitochondrial membrane potential. An increase in reactive oxygen species, intracellular Ca²⁺, calpain, caspases, cytochrome c, and the bax:bcl-2 ratio were also noted. GEN treatment reversed apoptotic death and cellular changes following cytokine exposure and was associated with increased expression of estrogen receptor β suggesting that GEN may promote neuroprotection via receptor-mediated pathways. The addition of ICI 182, 780, an estrogen receptor antagonist following GEN treatment attenuated neuroprotection, suggesting that GEN may act mainly via estrogen receptor β to protect VSC4.1 motoneurons. We conclude that GEN protects cultured ventral spinal cord 4.1 cells from inflammatory insult and thus may represent a potential beneficial therapy in the treatment of neurodegenerative disorders.     

Uncoupling the BMP receptor antagonist function from the WNT agonist function of R-spondin 2 using the inhibitory peptide dendrimer RWd

Signaling by bone morphogenetic proteins (BMPs) plays pivotal roles in embryogenesis, adult tissue homeostasis, and disease. Recent studies revealed that the well-established WNT agonist R-spondin 2 (RSPO2) is also a BMP receptor (BMP receptor type 1A) antagonist, with roles in early Xenopus embryogenesis and human acute myeloid leukemia (AML). To uncouple the BMP antagonist function from the WNT agonist function and to promote development of AML therapeutics, here we identified a 10-mer peptide (RW) derived from the thrombospondin 1 domain of RSPO2, which specifically prevents binding between RSPO2 and BMP receptor type 1A without altering WNT signaling. We also show that a corresponding RW dendrimer (RW^d) exhibiting improved half-life relieves inhibition of BMP receptor signaling by RSPO2 in human AML cells, reduces cell growth, and induces differentiation. Moreover, microinjection of RW^d in Xenopus embryos ventralizes the dorsoventral embryonic patterning by upregulating BMP signaling without affecting WNT signaling. Our study corroborates the function of RSPO2 as a BMP receptor antagonist and provides a proof of concept for pharmacologically uncoupling BMP antagonist from WNT agonist functions of RSPO2 using the inhibitor peptide RW^d with enhanced target selectivity and limited side effects.     

Genetic and pharmacologic strategies to determine the function of estrogen receptor α and estrogen receptor β in cardiovascular system

Background: The biological functions of estrogens extend beyond the female and male reproductive tract, affecting the cardiovascular and renal systems. Traditional views on the role of postmenopausal hormone therapy (HT) in protecting against heart disease, which were challenged by clinical end point studies that found adverse effects of combined HT, are now being replaced by more differentiated concepts suggesting a beneficial role of early and unopposed HT that does not include a progestin.Objective: We reviewed recent insights, concepts, and research results on the biology of both estrogen receptor (ER) subtypes, ERα and ERβ, in cardiac and vascular tissues. Knowledge of these ER subtypes is crucial to understanding gender and estrogen effects and to developing novel, exciting strategies that may have a profound clinical impact.Methods: This review focuses on in vivo studies and includes data presented at the August 2007 meeting of the American Physiological Society as well as data from a search of the MEDLINE and Ovid databases from January 1986 to November 2007. Search results were restricted to English-language publications, using the following search terms: estrogen, estrogen receptor α, estrogen receptor β, estrogen receptor α agonist, estrogen receptor α antagonist, estrogen receptor β agonist, estrogen receptor β antagonist, PPT, DPN, heart, vasculature, ERKO mice, BERKO mice, transgenic mice, and knockout mice.Results: Genetic mouse models and pharmacologic studies that employed selective as well as nonselective ER agonists support the concept that both ER subtypes confer protective effects in experimental models of human heart disease, including hypertension, cardiac hypertrophy, and chronic heart failure.Conclusions: Genetic models and novel ligands hold the promise of further improving our understanding of estrogen action in multiple tissues and organs. These efforts will ultimately enhance the safety and efficacy of HT and may also result in new applications for synthetic female sex hormone analogues.     

Modulation of pro-inflammatory activation of monocytes and dendritic cells by aza-bis-phosphonate dendrimer as an experimental therapeutic agent

Introduction

Our objective was to assess the capacity of dendrimer aza-bis-phosphonate (ABP) to modulate phenotype of monocytes (Mo) and monocytes derived dendritic cells (MoDC) activated in response to toll-like receptor 4 (TLR4) and interferon γ (IFN- γ) stimulation.

Methods

Mo (n = 12) and MoDC (n = 11) from peripheral blood of healthy donors were prepared. Cells were preincubated or not for 1 hour with dendrimer ABP, then incubated with lipopolysaccharide (LPS; as a TLR4 ligand) and (IFN-γ) for 38 hours. Secretion of tumor necrosis factor α (TNFα), interleukin (IL) -1, IL-6, IL-12, IL-10 and IL-23 in the culture medium was measured by enzyme-linked immunosorbent assay (ELISA) and Cytokine Bead Array. Differentiation and subsequent maturation of MoDC from nine donors in the presence of LPS were analyzed by flow cytometry using CD80, CD86, CD83 and CD1a surface expression as markers.

Results

Mo and MoDC were orientated to a pro-inflammatory state. In activated Mo, TNFα, IL-1β and IL-23 levels were significantly lower after prior incubation with dendrimer ABP. In activated MoDC, dendrimer ABP promoted IL-10 secretion while decreasing dramatically the level of IL-12. TNFα and IL-6 secretion were significantly lower in the presence of dendrimer ABP. LPS driven maturation of MoDC was impaired by dendrimer ABP treatment, as attested by the significantly lower expression of CD80 and CD86.

Conclusion

Our data indicate that dendrimer ABP possesses immunomodulatory properties on human Mo and MoDC, in TLR4 + IFN-γ stimulation model, by inducing M2 alternative activation of Mo and promoting tolerogenic MoDC.