AR Ligands Research Articles

Abstract A44: Carnosol, a dietary diterpene functions as a dual AR and ER-alpha antagonist in prostate cancer.

Abstract A44 Emerging evidence suggest that androgens may not be the only hormone responsible for the etiology of prostate cancer (PCa). Recent data has suggested that estrogens may be another important piece to the PCa puzzle. For example the use of the Selective Estrogen Receptor Modulator (SERM) toremifene in HG-PIN patients for one year resulted in a 21% reduction in the conversion of HG-PIN to PCa after one year of treatment. It is well established that race correlates with the levels of androgens with African Americans having the highest, followed by Caucasians and lowest in Japanese males. A similar trend has been observed in regard to serum estrogen levels and it is noteworthy that with age estrogen levels remain constant while androgens levels decrease. Given that experimental animal models suggest that both estrogens and androgens are necessary for PCa development it may be a reasonable approach to simultaneously disrupt both androgen and estrogen signaling in PCa. Several SERMs that include tamoxifen, toremifine, and fulvestrant have been evaluated for their ability to also target the AR and function as a dual AR and ER antagonist, however, experiments have suggested these effects are saturable and in some instances SERMs exert AR agonist properties. In our quest to find a dual AR and ER antagonist present in dietary substances that humans consume we found that the dietary diterpene Carnosol displays growth inhibitory properties in the hormone sensitive prostate (LNCaP and CWR22Rv1) and breast (MCF7 and AU565) cancer cell lines. Given the structural similarity between Carnosol to dihydrotestosterone and estradiol we hypothesized that it may interact with both the androgen and estrogen receptor alpha (ER-α). Using a TR-FRET cell free biochemical assay we found that Carnosol can interact with both AR and ER-alpha with similar potencies. Next, we evaluated the antagonist and agonist properties of Carnosol using a functional assay where the AR and ER-alpha ligand binding domains are expressed as a fusion protein in HEK293 cells. We observed that Carnosol exhibits antagonist properties that are dose responsive and even more interestingly, regardless of dose, did not exert agonist properties. In LNCaP, CWR22Rv1, and MCF7 cells Carnosol was found to disrupt both androgen and estrogen signaling in a dose and time dependent manner. Our finding that Carnosol displays the ability to disrupt both androgen and estrogen signaling simultaneously suggesting that Carnosol may be developed or chemically modified through more rigorous structure activity relationship studies of other such chemical entities for a new class of investigational agents - a dual AR/ER modulator. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A44.

Argon cluster-mediated isolation and vibrational spectra of peroxy and nominally D3h isomers of CO3− and NO3−

Vibrational predissociation spectra are reported for two isomeric forms of the gas-phase ions, CO(3)(-) and NO(3)(-). The peroxy forms, (OOCO(-) and OONO(-)) were isolated using an Ar-mediated synthetic scheme involving exchange of CO and NO for the more weakly bound Ar ligands in O(2)(-)Ar(m) clusters, while the forms based on a central heteroatom (CO(3)(-) and NO(3)(-)) were generated by electron impact on CO(2) and HNO(3) vapor. The simple two-band spectrum of OOCO(-) indicates that it is best described as the O(2)(-) x CO ion-molecule complex, whereas the covalently bound CO(3)(-) form yields a much more complicated vibrational spectrum with bands extending out to 4000 cm(-1). In contrast, the NO(3)(-) ion yields a simple spectrum with only one transition as expected for the antisymmetric NO stretching fundamental of a species with D(3h) structure. The spectrum of the peroxynitrite isomer, OONO(-), displays intermediate complexity that can be largely understood in the context of fundamentals associated with its cis and trans structures previously characterized in an Ar matrix.

Promising core structure for nuclear receptor ligands: Design and synthesis of novel estrogen receptor ligands based on diphenylamine skeleton

Novel diphenylamine-type estrogen receptor ligands were designed and synthesized, and their biological activities were evaluated by means of binding assays for estrogen receptor-α and -β and cell proliferation assay using MCF-7 cells. Compounds 4f, 11b, 12c, and 8 showed moderate estrogenic activities. We propose that the diphenylamine skeleton may be a privileged structure for various nuclear receptor ligands, including RAR, RXR, and AR ligands.

Androgen Receptor-Mediated Apoptosis Is Regulated by Photoactivatable Androgen Receptor Ligands

We have studied nonsteroidal ligands of the human androgen receptor (hAR) and have shown elsewhere that when photoactivated by visible light they collide with O2 to yield singlet oxygens (1O2) in vitro. Here we report cell killing after brief light activation (405 nm) of 1,2,3,4-tetrahydro-2,2-dimethyl-6-(trifluoromethyl)-8-pyridono[5,6-g]quinoline (TDPQ) in human prostate tumor cells. TDPQ/AR complexes were required for the death response because AR-positive LNCaP cells were killed, whereas AR-negative PC-3 cells were resistant. Excess dihydrotestosterone (DHT) blocked the TDPQ effect when the two were added together; irradiation of cells containing DHT alone had no effect. When LNCaP AR expression was suppressed using small interfering oligonucleotides targeting AR, photocytotoxicity was diminished. Conversely, stable transfection of hAR into PC-3 cells made the cells photosensitive to TDPQ. Similar results were obtained using a structural isomer of TDPQ, and also the synthetic steroidal AR ligand R1881. Cell death occurred via apoptosis as demonstrated by annexin V immunostaining, nuclear condensation, and caspase inhibition. Death involved oxidative stress, because it was prevented by addition of the antioxidant ascorbic acid during photoactivation. Detection of elevated levels of 8-hydroxy-2'-deoxyguanosine in nuclei of irradiated cells indicated oxidative DNA damage. Apoptosis spread into adjacent nonirradiated cells by direct cell-cell contacts, indicative of a bystander effect. Other photoactivatable ligands are described, implying a general method for ablation of cells bearing specific nuclear hormone receptors.

Pharmacological characterization of AC-262536, a novel selective androgen receptor modulator

Because of the limitations and liabilities of current testosterone therapies, non-steroidal tissue-selective androgen receptor modulators may provide a clinically meaningful advance in therapy. Using a functional cell-based assay AC-262536 was identified as a potent and selective AR ligand, with partial agonist activity relative to the natural androgen testosterone. A 2-week chronic study in castrated male rats indicated that AC-262536 significantly improves anabolic parameters in these animals, especially in stimulating the growth of the levator ani and in suppressing elevated LH levels. In sharp contrast to testosterone, AC-262536 has weak androgenic effects, as measured by prostate and seminal vesicle weights. Thus, AC-262536 represents a novel class of selective androgen receptor modulators (SARMs) with beneficial anabolic effects.

Ockham's Razor and Selective Androgen Receptor Modulators (SARMs): Are We Overlooking the Role of 5 -Reductase?

Selective Androgen Receptor Modulators (SARMs) are a novel class of AR ligands that possess tissue-selective pharmacological activities. SARMs of various chemical structures have been discovered and characterized, and lead compounds with much improved specificity for AR, in vivo pharmacokinetic profiles, and higher degree of tissue selectivity have entered clinical development, and are expected to dramatically expand the clinical applications of androgens. With the rapid progress in SARM discovery and increasing demand for mechanism-based drug design, more and more research efforts have been devoted to the mechanisms of action of the observed tissue selectivity of SARMs. There is increasing enthusiasm in adapting the molecular mechanisms of action from SERM research to the SARM field; however, is the SARM story really so complicated? The tissue-specific expression of 5alpha-reductase might provide a simple explanation for this puzzle.

Potent Adenosine A1 and A2A Receptors Antagonists: Recent Developments

This review summarizes the current tendencies observed in the past 5 years in the development of A(1) and A(2A) adenosine receptor antagonists performed in various academia and industry. A(1) and A(2A) AR antagonists are as well xanthines as heteroaromatic derivatives and are most commonly 6:5 fused heteroatomic compounds. Among xanthine-based compounds, some common features could be pointed out. The recent A(1) AR ligands which show good biological profile, possess long alkyl chains in position 1 and 3 as well as bulky C(8)-substituent, while A(2A) AR antagonists with a high A(2A) AR affinity are C(8)-styryl substituted with N(1)-alkyl/alkynyl moiety or fused tricyclic xanthines possessing heteroatom(s) in the third cycle. The research in the field of heteroaromatic A(1) and A(2A) ARs antagonists impressively has a wide range. Ligands are as well non-fused monocyclic substituted compounds as fused bi- and tricyclic derivatives with the nitrogen, oxygen and sulfur heteroatoms. Most often, adenosine A(1) receptor non-xanthine antagonists are adenine-based, having substituted amino group and variable nitrogen atoms positions in the molecules. A(2A) AR ligands show good affinity when furanyl function, which is crucial for binding, is present in the fused bicyclic and tricyclic analogs. Moreover, tricyclic nitrogen containing antagonists in order to be active, frequently possess long-alkylphenyl moiety.

Arylisothiocyanato selective androgen receptor modulators (SARMs) for prostate cancer

A new series of androgen receptor targeted agents (ARTA) was prepared and tested in androgen-dependent and -independent prostate cancer cell lines. These agents were bicalutamide analogs with isothiocyanato substituted B-rings. Also, the linker sulfone of R-bicalutamide was maintained or replaced with several alternative linkages including ether, amine, N-methylamine, thioether, and methylene (in this case the product was a racemic mixture) functional groups at the X-position. To expand the structure–activity relationship (SAR) of these arylisothiocyanato AR ligands, B-ring halogenated arylisothiocyanato ligands were also prepared and tested. The arylisothiocyanato AR ligands showed strong binding affinities to AR ranging from 0.6 to 54nM. Among them, thioether and ether linkages demonstrated high binding affinities (0.6 and 4.6nM, respectively) and selective cell growth inhibition (approximately 3- to 6-fold) for LNCaP, an androgen-dependent prostate cancer cell line, when compared to the androgen independent prostate cell lines (DU145, PC-3, and PPC-1) and a bladder cell line (TSU-Pr1). However, the ligands were inactive (IC50>100mM) in a normal monkey kidney cell line (CV-1) that was used as the control for non-specific toxicity.

Aggregation and proteasome: The case of elongated polyglutamine aggregation in spinal and bulbar muscular atrophy

Aggregates, a hallmark of most neurodegenerative diseases, may have different properties, and possibly different roles in neurodegeneration. We analysed ubiquitin-proteasome pathway functions during cytoplasmic aggregation in polyglutamine (polyQ) diseases, using a unique model of motor neuron disease, the SpinoBulbar Muscular Atrophy. The disease, which is linked to a polyQ tract elongation in the androgen receptor (ARpolyQ), has the interesting feature that ARpolyQ aggregation is triggered by the AR ligand, testosterone. Using immortalized motor neurons expressing ARpolyQ, we found that a proteasome reporter, YFPu, accumulated in absence of aggregates; testosterone treatment, which induced ARpolyQ aggregation, allowed the normal clearance of YFPu, suggesting that aggregation contributed to proteasome de-saturation, an effect not related to AR nuclear translocation. Using AR antagonists to modulate the kinetic of ARpolyQ aggregation, we demonstrated that aggregation, by removing the neurotoxic protein from the soluble compartment, protected the proteasome from an excess of misfolded protein to be processed.

α2C-Adrenergic Receptors Exhibit Enhanced Surface Expression and Signaling upon Association with β2-Adrenergic Receptors

The alpha(2C)-adrenergic receptor (alpha(2C)AR) is known to be poorly trafficked to the cell surface when expressed in a variety of cell types. We tested the hypothesis that the surface expression and signaling of alpha(2C)AR might be enhanced by heterodimerization with other G protein-coupled receptors (GPCRs). Cotransfection of alpha(2C)AR with more than 25 related GPCRs revealed that only coexpression with the beta(2)-adrenergic receptor (beta(2)AR) increased the surface localization of alpha(2C)AR in human embryonic kidney-293 cells. Coimmunoprecipitation of alpha(2C)AR with beta(2)AR confirmed a physical interaction between the two receptors. Confocal microscopy studies demonstrated that alpha(2C)AR expressed alone was mainly intracellular, whereas alpha(2C)AR coexpressed with beta(2)AR was predominantly localized to the plasma membrane. Ligand binding studies revealed a significant increase in alpha(2C)AR binding sites upon coexpression with beta(2)AR, with no apparent change in affinity for alpha(2)AR ligands. Functional assays with the alpha(2)AR-specific agonist brimonidine (UK 14,304) revealed that coexpression of beta(2)AR with alpha(2C)AR enhanced alpha(2C)AR-mediated activation of extracellular signal-regulated kinase 1/2. Furthermore, analyses of agonist-promoted receptor endocytosis demonstrated enhanced alpha(2C)AR internalization in response to alpha(2)AR agonists when alpha(2C)AR and beta(2)AR were coexpressed. In addition, substantial cointernalization of alpha(2C)AR in response to betaAR agonists was observed when alpha(2C)AR was coexpressed with beta(2)AR. These data reveal that alpha(2C)AR can interact with beta(2)AR in cells in a manner that regulates alpha(2C)AR surface expression, internalization, and functionality.

Studies of C−S Bond Cleavage Reactions of Re(V) Dithiolates: Synthesis, Reactivity, and Mechanism

A series of rhenium(V) complexes, [(X)(ReO)(dt)(PPh(3))] and [(o-SC(6)H(4)PPh(2))(ReO)(mtp)], were prepared to explore electronic effects on the C-S cleavage reaction that occurs upon reaction with PAr(3) at ambient temperature [where X = S(C(6)H(4)-p-Z) (Z = OMe, Me, H, F, Cl), OPh, Cl, and SC(2)H(5), and dt is the chelating dithiolate ligand derived from 2-(mercaptomethyl)thiophenol, 1,2-ethanedithiol, 1,3-propanedithiol, 1,3-butanedithiol, and 2,4-pentanedithiol]. The scope and selectivity of the C-S activation were examined. The C-S bond cleavage to form metallacyclic Re(V) complexes with a ReS core occurs only for the complexes with mtp and pdt frameworks and X = SAr and SC(2)H(5). The difference in reactivity is due to the different donating abilities of ancillary and dithiolate ligands, especially their pi-donating ability, which plays a critical role in C-S activation. The kinetics of the C-S activation process was determined; nucleophilic attack of PPh(3) on the oxo group of the Re(V)O core appears to be the rate-controlling step. The reaction is accelerated by electron-poor ArS ligands, but is unaffected by the substituents on phosphines. A detailed mechanistic study is presented. The results represent a rare example of migration of alkanethiolate leading to the formation of alkylthiolato complexes.

Synthesis and first in vivo evaluation of new selective high affinity β 1-adrenoceptor radioligands for SPECT based on ICI 89,406

The results of cardiac biopsies suggest that myocardial β 1-adrenoceptor (AR) density is reduced in patients with chronic heart failure, while changes in cardiac β 2-ARs vary. A technique for visualization and quantification of β 1-AR populations rather than total β-AR densities in the human heart would be of great clinical interest. Molecular imaging techniques, either single photon emission computed tomography (SPECT) or positron emission tomography (PET), with appropriate radiopharmaceuticals offer the possibility to assess β-AR density noninvasively in humans, but to date, neither a SPECT nor a PET-radioligand is clinically established for the selective imaging of cardiac β 1-ARs. The aim of this study was to design a high affinity selective β 1-AR radioligand for the noninvasive in vivo imaging of cardiac β 1-AR density in man using SPECT. Based on the well-known selective β 1-AR antagonist, ICI 89,406, both the racemic iodinated target compound 11a and the ( S)-enantiomer 15a were synthesized. Competition studies using the nonselective AR ligand, [ 125I]iodocyanopindolol ([ 125I]ICYP), and ventricular membrane preparations from mice showed that 11a and 15a possess higher β 1-AR affinities (up to 265-fold) and β 1-AR selectivities (up to 245-fold) than ICI 89,406. Encouraged by these results, the radioiodinated counterparts of racemic 11a ( 11b: 125I, 11c: 123I) and ( S)-configurated 15a ( 15b: 125I, 15c: 123I) were synthesized. The target compounds were evaluated in rats. Biodistribution and metabolism studies in rats indicated that there is a specific heart uptake of 11b– c and especially 15b– c accompanied by rapid metabolism of the radioligands. Therefore, radioiodinated 11c and 15c appeared to be unpromising SPECT-radioligands for assessing β 1-ARs in vivo in the rat. However, the rat may metabolize β-AR ligands more rapidly than other species as demonstrated for ( S)-[ 11C]CGP 12177, a radioligand structurally related to 11a– c and 15a– c. Therefore further studies in a different animal model will be carried out.

Effect of organochlorine pesticides on human androgen receptor activation in vitro

Many persistent organochlorine pesticides (OCs) have been implicated in adverse effects, that is, reproductive and developmental effects, in man and in wildlife alike. It has been hypothesized that these so-called xeno-hormones could be responsible for the increased incidence in various male sexual differentiation disorders such as hypospadias, cryptorchidism, low sperm counts and quality. In this report, OCs, called endocrine disrupters, were tested for their interaction with the androgen receptor. The stable prostatic cell line PALM, which contains a human androgen receptor (hAR) expression vector and the reporter MMTV-luciferase, was used to characterize the response of hAR to OC and was compared with synthetic androgen compound R1881. We found that all the OC pesticides tested were able to shift the agonist [ 3H]-R1881 from its binding site to the AR in competitive binding assays. In addition, these compounds antagonize—in a dose-dependent manner—the AR-mediated transcription by synthetic AR ligand R1881. None of the pesticides reacted as agonists. These results demonstrate that OC endocrine activities in vivo probably result from direct and specific binding to the AR ligand-binding domain. Although the antagonistic potential of OC pesticides is lower than that of hydroxyflutamide, they are capable of disrupting the male hormone signaling pathway. Because these chemicals are extremely persistent and tend to bioaccumulate, these results support the hypothesis that the recent increase in the incidence of male sexual disorders could be due to long exposure to ubiquitous OC pesticides found in the environment.

N–H⋯O hydrogen bonding interactions in tetrahedral [ZnS 4] complexes of relevance to zinc enzymes: the synthesis, structures and reactivity of tris(2-mercapto-1-arylimidazolyl)hydroborato zinc(2-mercapto-1-arylimidazole) complexes, {[Tm Ar]Zn(mim Ar)}[ClO 4] (Ar=Ph, p-Tol)

The tris(2-mercapto-1-arylimidazolyl)hydroborato complexes {[Tm Ar]Zn(mim Ar)}[ClO 4], which feature tetrahedrally coordinated zinc centers in a sulfur rich environment, may be prepared by the reaction of [Tm Ar]Li with Zn(ClO 4) 2 in the presence of 2-mercapto-1-arylimidazole (mim Ar; Ar=Ph, p-Tol). X-ray diffraction studies demonstrate that the mim Ar ligands are almost orthogonal to the Zn–S–C plane, in contrast to the almost planar arrangement that is calculated for {[Tm H]Zn(mim H)} + and {[Tm H]Zn(mim Me)} +. The orthogonal arrangement observed experimentally for both {[Tm Ph]Zn(mim Ph)} + and {[Tm p-Tol ]Zn(mim p-Tol )} + is a consequence of the existence of a N–H⋯O hydrogen bond between the N–H group of the mim Ar ligand and a molecule of methanol. Evidently, the hydrogen bonding interactions in {[Tm Ar]Zn(mim Ar)⋯(HOMe)} + are sufficiently strong to counter the electronic destabilization resulting from rotation of the mim Ar ligand from the Zn–S–C plane. The mim Ph ligand of {[Tm Ph]Zn(mim Ph)} + may be immediately displaced by I − to give [Tm Ar]ZnI; however, {[Tm Ph]Zn(mim Ph)} + does not react rapidly with MeI, thereby demonstrating that the zinc coordinated mim Ph ligand is less susceptible to electrophilic attack than is a zinc thiolate ligand.

Design of new β 1-selective adrenoceptor ligands as potential radioligands for in vivo imaging

In general, the failing human heart is characterized by a selective reduction in β 1-adrenoceptors (β 1-ARs) without change in β 2-AR density. Medical imaging techniques, either single photon emission computed tomography (SPECT) or positron emission tomography (PET) with appropriate radioligands, offer the possibility of assessing β-adrenoceptor density non-invasively in humans. To date, neither a SPECT nor a PET radioligand is available for the selective imaging of cardiac β 1-ARs. The aim of this study was to develop potential high affinity β 1-selective AR radioligands for the non-invasive in vivo imaging of the β 1-AR density in the human heart using SPECT or PET. A variety of racemic N-aryl- N′-[2-[3-aryloxy-2-hydroxy-propylamino]-ethyl]-urea derivatives and chain-elongated analogues, related to the established β 1-AR antagonist, ICI 89,406 8i, were synthesized. Competition studies using the non-selective AR ligand, [ 125I]iodocyanopindolol ([ 125I]ICYP), and ventricular membrane preparations of wild-type mice revealed nine ligands with higher β 1-AR affinities (up to 76-fold) and β 1-AR selectivities (up to 139-fold) than 8i. Mostly, these ligands possess a 2-substituted phenoxy group and a 4-substituted phenyl residue in contrast to the lead compound 8i. The non-radioactive counterparts of the desired SPECT- and PET-radiotracers were synthesized as reference compounds [e.g., 8f, 8g, 8h and 8l as the non-radioactive analogues of the radioiodinated SPECT radioligands, 8e and 8h as the non-radioactive compounds of C-11 labelled PET-tracers (C-11 in the methoxy group)]. The established library of high affinity β 1-selective AR antagonists was screened for chemical precursors for the radiosynthesis of the mentioned radioligands. Furthermore, the library consists of some comparison compounds that are unsubstituted, allyl- and alkyl-substituted or chain-elongated (e.g., 8a, 8j, 8o and 8r– t). Future steps will include radiolabelling and pharmacokinetic evaluation of the β 1-selective target compounds, which could be applied as sympathetic innervation agents for in vivo investigations and diagnostics in patients suffering from cardiac diseases like heart failure and ventricular arrhythmias.

New pyrimido[5,4-b]indoles as ligands for alpha(1)-adrenoceptor subtypes.

A new series of compounds were designed as structural analogues of the alpha(1)-AR ligand RN5 (4), characterized by a tricyclic 5H-pyrimido[5,4-b]indole-(1H,3H)2,4-dione system connected through an alkyl chain to a phenylpiperazine (PP) moiety. These compounds were synthesized and tested in binding assays on human alpha(1A)-AR, alpha(1B)-AR, and alpha(1D)-AR subtypes expressed in HEK293 cells. Several structural modifications were performed on the PP moiety, the tricyclic system, and the connecting alkyl chain. Many of the new molecules showed a preferential affinity for the alpha(1D)-AR subtype. Some compounds, including 39 and 40, displayed substantial alpha(1D)-AR selectivity with respect to alpha(1A)-AR, alpha(1B)-AR, serotonergic 5-HT(1A), 5-HT(1B), 5-HT(2A), and dopaminergic D(1) and D(2) receptors. Two conformationally rigid analogues of 4, useful for studying the architecture of the receptor/ligand complex, were also prepared and tested. A subset of the new compounds was then used to evolve a preliminary pharmacophore model for alpha(1D)-AR antagonists, based on a more generalized model we had developed for alpha(1)-AR antagonists. This new model rationalized the relationships between structural properties and biological data of the pyrimido[5,4-b]indole compounds, as well as other compounds.

The Presence of [Ar2Cu]- and [(RC⋮C)2Cu]- Homocuprate Moieties in the Neutral Mixed Cuprate [Cu2Li2(C⋮CR)2Ar2] (Ar = C6H4{CH2N(Me)CH2CH2NMe2}-2; R = C6H4Me-4 or C6H4SiMe3-4)

The neutral, mixed 2:2 aryl-alkynyl-cuprates [Cu2Li2(C⋮CR)2Ar2] (Ar = [C6H4{CH2N(Me)CH2CH2NMe2}-2]-; R = C6H4Me-4 (3a) or C6H4SiMe3-4 (3b)) are the first examples of cuprates that contain two different organic homocuprate parts ([Ar−Cu−Ar]- and [(RC⋮C)−Cu−(C⋮CR)]-) in one assembled structure. Each part has a two-coordinate Cu atom in linear geometry. They are held together by Li−Cipso interactions to two Li atoms. Tetrahedral four-coordination at the Li centers is completed by N,N‘-chelation of the ortho-diamine substituent of the Ar ligand. Compound 3 is formed as the only reaction product in almost quantitative yield when LiAr and Cu(C⋮CR) aggregates are mixed together in equimolar amounts. This cuprate is an example of ion-pairing between two different homocuprate anions and two Li cations.

Elucidation of the Solution Structures of Transition Metal Complex Ion Pairs by NOE NMR Experiments

AbstractFor Abstract see ChemInform Abstract in Full Text.

Dissimilar pharmacological responses by a new series of imidazoline derivatives at precoupled and ligand-activated alpha 2A-adrenoceptor states: evidence for effector pathway-dependent differential antagonism.

Whereas agonist-directed differential signaling at a single receptor subtype has become an accepted pharmacological concept, distinct behaviors by ligands that are assumed to be antagonists is less documented. The intrinsic activity and capacity of antagonism for a new series of imidazoline-derived adrenergic ligands analogous to dexefaroxan were investigated by measuring two distinct signaling pathways at the recombinant human alpha 2A-adrenoceptor (alpha 2A AR): 1) pertussis toxin-resistant guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding responses mediated by either a recombinant G alpha oCys351Ile or G alpha i2Cys352Ile protein in CHO-K1 cells, and 2) inhibition of cAMP formation in a stably transfected C6-glial cell line. Ligands could be differentiated as inverse agonists [i.e., 2-(4-methoxy-2-ethyl-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 851062], neutral antagonists [i.e., 2-(4-hydroxy-2-ethyl-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 851057], partial [i.e., 2-(4-chloro-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 821008], and high-efficacy [i.e., 2-(6,7-dichloro-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 821010] agonists at a precoupled alpha 2A AR state in the copresence of a G alpha oCys351Ile protein but not G alpha i2Cys352Ile protein by monitoring [35S]GTP gamma S binding responses. Neither positive nor negative efficacy was observed for these compounds by monitoring the adenylate cyclase pathway at a presumably low-affinity alpha 2A AR state. The capacity of the dexefaroxan analogs to antagonize the (-)-epinephrine-mediated [35S]GTP gamma S binding response at a G alpha oCys351Ile protein was inversely correlated with their magnitude of intrinsic activity and unrelated to their ligand binding affinity for the alpha 2A AR. On the other hand, their capacity to antagonize either (-)-epinephrine or 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline tartrate (UK 14304)-mediated inhibition of forskolin-stimulated cAMP formation was not related with the rank order of antagonist capacity for the (-)-epinephrine-mediated [35S]GTP gamma S binding response. In conclusion, these data demonstrate that certain alpha2 AR ligands that are assumed to be antagonists, may yield dissimilar pharmacological responses, dependent on the investigated agonist-stimulated effector pathway.

Selective modulation of genomic and nongenomic androgen responses by androgen receptor ligands.

Steroids can induce both transcription-dependent (genomic) and independent (nongenomic) signaling. Here, several classical androgen receptor ligands were tested for their ability to modulate genomic and nongenomic responses, focusing on the role of the oocyte-expressed Xenopus classical androgen receptor (XeAR) in mediating these processes. Cellular fractionation and immunohistochemistry revealed that the XeAR was located throughout oocytes, including within the plasma membrane. RNA interference and oocyte maturation studies suggested that androgen-induced maturation was mediated in part by the XeAR in a transcription-independent fashion, perhaps by altering G protein-mediated signaling. While inducing minimal transcription in oocytes, all AR ligands promoted significant XeAR-mediated transcription in CV1 cells. In contrast, only testosterone and androstenedione potently induced oocyte maturation, whereas dihydrotestosterone and R1881 actually inhibited testosterone and human chorionic gonadotropin-induced maturation and signaling. These results suggest that the nature of a steroid-induced signal (genomic vs. nongenomic) may depend on the type of target cell, the receptor location within cells, as well as the ligand itself. The identification of molecules capable of selectively altering genomic vs. nongenomic signaling may be useful in delineating the roles of these pathways in mediating androgen responses and might lead to the development of novel compounds that specifically modulate these signals in vivo.