Activation of ß2-adrenergic receptor by (R,R')-4'-methoxy-1-naphthylfenoterol inhibits proliferation and motility of melanoma cells.

(R,R')-4'-methoxy-1-naphthylfenoterol [(R,R')-MNF] is a highly-selective ß2 adrenergic receptor (ß2-AR) agonist. Incubation of a panel of human-derived melanoma cell lines with (R,R')-MNF resulted in a dose- and time-dependent inhibition of motility as assessed by in vitro wound healing and xCELLigence migration and invasion assays. Activity of (R,R')-MNF positively correlated with the ß2-AR expression levels across tested cell lines. The anti-motility activity of (R,R')-MNF was inhibited by the ß2-AR antagonist ICI-118,551 and the protein kinase A inhibitor H-89. The adenylyl cyclase activator forskolin and the phosphodiesterase 4 inhibitor Ro 20-1724 mimicked the ability of (R,R')-MNF to inhibit migration of melanoma cell lines in culture, highlighting the importance of cAMP for this phenomenon. (R,R')-MNF caused significant inhibition of cell growth in ß2-AR-expressing cells as monitored by radiolabeled thymidine incorporation and xCELLigence system. The MEK/ERK cascade functions in cellular proliferation, and constitutive phosphorylation of MEK and ERK at their active sites was significantly reduced upon ß2-AR activation with (R,R')-MNF. Protein synthesis was inhibited concomitantly both with increased eEF2 phosphorylation and lower expression of tumor cell regulators, EGF receptors, cyclin A and MMP-9. Taken together, these results identified ß2-AR as a novel potential target for melanoma management, and (R,R')-MNF as an efficient trigger of anti-tumorigenic cAMP/PKA-dependent signaling in ß2-AR-expressing lesions.

Tyrosine 308 is necessary for ligand-directed Gs protein-biased signaling of ß2-adrenoceptor.

Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, ß2-adrenoceptor (ß2-AR) couples dually to Gs and Gi proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of ß2-AR causes a switch in receptor coupling from Gs to Gi. More recent studies have demonstrated that phosphorylation of ß2-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the Gi coupling. We have previously shown that although most ß2-AR agonists cause both Gs and Gi activation, (R,R')-fenoterol preferentially activates ß2-AR-Gs signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on ß2-AR essential for Gs-biased signaling. Following stimulation with a ß2-AR-Gs-biased agonist (R,R')-4'-aminofenoterol, the Gi disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type ß2-AR. Interestingly, Y308F substitution on ß2-AR enabled (R,R')-4'-aminofenoterol to activate Gi and to produce these responses in a pertussis toxin-sensitive manner without altering ß2-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of ß2-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of ß2-AR stabilize receptor conformations favoring the receptor-Gs protein coupling and subsequently result in Gs-biased agonism.

Comparative molecular field analysis of fenoterol derivatives interacting with an agonist-stabilized form of the ß2-adrenergic receptor.

The ß2-adrenergic receptor (ß2-AR) agonist [(3)H]-(R,R')-methoxyfenoterol was employed as the marker ligand in displacement studies measuring the binding affinities (Ki values) of the stereoisomers of a series of 4'-methoxyfenoterol analogs in which the length of the alkyl substituent at α' position was varied from 0 to 3 carbon atoms. The binding affinities of the compounds were additionally determined using the inverse agonist [(3)H]-CGP-12177 as the marker ligand and the ability of the compounds to stimulate cAMP accumulation, measured as EC50 values, were determined in HEK293 cells expressing the ß2-AR. The data indicate that the highest binding affinities and functional activities were produced by methyl and ethyl substituents at the α' position. The results also indicate that the Ki values obtained using [(3)H]-(R,R')-methoxyfenoterol as the marker ligand modeled the EC50 values obtained from cAMP stimulation better than the data obtained using [(3)H]-CGP-12177 as the marker ligand. The data from this study was combined with data from previous studies and processed using the Comparative Molecular Field Analysis approach to produce a CoMFA model reflecting the binding to the ß2-AR conformation probed by [(3)H]-(R,R')-4'-methoxyfenoterol. The CoMFA model of the agonist-stabilized ß2-AR suggests that the binding of the fenoterol analogs to an agonist-stabilized conformation of the ß2-AR is governed to a greater extend by steric effects than binding to the [(3)H]-CGP-12177-stabilized conformation(s) in which electrostatic interactions play a more predominate role.

Sub-anesthetic concentrations of (R,S)-ketamine metabolites inhibit acetylcholine-evoked currents in α7 nicotinic acetylcholine receptors.

The effect of the (R,S)-ketamine metabolites (R,S)-norketamine, (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine on the activity of α7 and α3ß4 neuronal nicotinic acetylcholine receptors was investigated using patch-clamp techniques. The data indicated that (R,S)-dehydronorketamine inhibited acetylcholine-evoked currents in α7-nicotinic acetylcholine receptor, IC(50) = 55 ± 6 nM, and that (2S,6S)-hydroxynorketamine, (2R,6R)-hydroxynorketamine and (R,S)-norketamine also inhibited α7-nicotinic acetylcholine receptor function at concentrations ≤ 1 µM, while (R,S)-ketamine was inactive at these concentrations. The inhibitory effect of (R,S)-dehydronorketamine was voltage-independent and the compound did not competitively displace selective α7-nicotinic acetylcholine receptor ligands [(125)I]-α-bungarotoxin and [(3)H]-epibatidine indicating that (R,S)-dehydronorketamine is a negative allosteric modulator of the α7-nicotinic acetylcholine receptor. (R,S)-Ketamine and (R,S)-norketamine inhibited (S)-nicotine-induced whole-cell currents in cells expressing α3ß4-nicotinic acetylcholine receptor, IC(50) 3.1 and 9.1 µM, respectively, while (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine were weak inhibitors, IC(50) >100 µM. The binding affinities of (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine at the NMDA receptor were also determined using rat brain membranes and the selective NMDA receptor antagonist [(3)H]-MK-801. The calculated K(i) values were 38.95 µM for (S)-dehydronorketamine, 21.19 µM for (2S,6S)-hydroxynorketamine and>100 µM for (2R,6R)-hydroxynorketamine. The results suggest that the inhibitory activity of ketamine metabolites at the α7-nicotinic acetylcholine receptor may contribute to the clinical effect of the drug.

Cannabinoid receptor activation correlates with the proapoptotic action of the ß2-adrenergic agonist (R,R')-4-methoxy-1-naphthylfenoterol in HepG2 hepatocarcinoma cells.

Inhibition of cell proliferation by fenoterol and fenoterol derivatives in 1321N1 astrocytoma cells is consistent with ß(2)-adrenergic receptor (ß(2)-AR) stimulation. However, the events that result in fenoterol-mediated control of cell proliferation in other cell types are not clear. Here, we compare the effect of the ß(2)-AR agonists (R,R')-fenoterol (Fen) and (R,R')-4-methoxy-1-naphthylfenoterol (MNF) on signaling and cell proliferation in HepG2 hepatocarcinoma cells by using Western blotting and [(3)H]thymidine incorporation assays. Despite the expression of ß(2)-AR, no cAMP accumulation was observed when cells were stimulated with isoproterenol or Fen, although the treatment elicited both mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt activation. Unexpectedly, isoproterenol and Fen promoted HepG2 cell growth, but MNF reduced proliferation together with increased apoptosis. The mitogenic responses of Fen were attenuated by 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118,551), a ß(2)-AR antagonist, whereas those of MNF were unaffected. Because of the coexpression of ß(2)-AR and cannabinoid receptors (CBRs) and their impact on HepG2 cell proliferation, these Gα(i)/Gα(o)-linked receptors may be implicated in MNF signaling. Cell treatment with (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55,212-2), a synthetic agonist of CB(1)R and CB(2)R, led to growth inhibition, whereas inverse agonists of these receptors blocked MNF mitogenic responses without affecting Fen signaling. MNF responses were sensitive to pertussis toxin. The ß(2)-AR-deficient U87MG cells were refractory to Fen, but responsive to the antiproliferative actions of MNF and WIN 55,212-2. The data indicate that the presence of the naphthyl moiety in MNF results in functional coupling to the CBR pathway, providing one of the first examples of a dually acting ß(2)-AR-CBR ligand.

Thermodynamics and docking of agonists to the ß(2)-adrenoceptor determined using [(3)H](R,R')-4-methoxyfenoterol as the marker ligand.

G protein-coupled receptors (GPCRs) are integral membrane proteins that change conformation after ligand binding so that they can transduce signals from an extracellular ligand to a variety of intracellular components. The detailed interaction of a molecule with a G protein-coupled receptor is a complicated process that is influenced by the receptor conformation, thermodynamics, and ligand conformation and stereoisomeric configuration. To better understand the molecular interactions of fenoterol analogs with the ß(2)-adrenergic receptor, we developed a new agonist radioligand for binding assays. [(3)H](R,R')-methoxyfenoterol was used to probe the binding affinity for a series of fenoterol stereoisomers and derivatives. The results suggest that the radioligand binds with high affinity to an agonist conformation of the receptor, which represents approximately 25% of the total ß(2)-adrenoceptor (AR) population as determined with the antagonist [(3)H]CGP-12177. The ß(2)-AR agonists tested in this study have considerably higher affinity for the agonist conformation of the receptor, and K(i) values determined for fenoterol analogs model much better the cAMP activity of the ß(2)-AR elicited by these ligands. The thermodynamics of binding are also different when interacting with an agonist conformation, being purely entropy-driven for each fenoterol isomer, rather than a mixture of entropy and enthalpy when the fenoterol isomers binding was determined using [(3)H]CGP-12177. Finally, computational modeling identified the molecular interactions involved in agonist binding and allow for the prediction of additional novel ß(2)-AR agonists. The study underlines the possibility of using defined radioligand structure to probe a specific conformation of such shape-shifting system as the ß(2)-adrenoceptor.

Effect of fenoterol stereochemistry on the ß2 adrenergic receptor system: ligand-directed chiral recognition.

The ß(2) adrenergic receptor (ß(2)-AR) is a model system for studying the ligand recognition process in G protein-coupled receptors. Fenoterol (FEN) is a ß(2)-AR selective agonist that has two centers of chirality and exists as four stereoisomers. Radioligand binding studies determined that stereochemistry greatly influences the binding affinity. Subsequent Van't Hoff analysis shows very different thermodynamics of binding depending on the stereoconfiguration of the molecule. The binding of (S,x')-isomers is almost entirely enthalpy controlled whereas binding of (R,x')-isomers is purely entropy driven. Stereochemistry of FEN molecule also affects the coupling of the receptor to different G proteins. In a rat cardiomyocyte contractility model, (R,R')-FEN was shown to selectively activate G(s) protein signaling while the (S,R')-isomer activated both G(i) and G(s) protein. The overall data demonstrate that the chirality at the two chiral centers of the FEN molecule influences the magnitude of binding affinity, thermodynamics of local interactions within the binding site, and the global mechanism of ß(2)-AR activation. Differences in thermodynamic parameters and nonuniform G-protein coupling suggest a mechanism of chiral recognition in which observed enantioselectivities arise from the interaction of the (R,x')-FEN stereoisomers with a different receptor conformation than the one with which the (S,x')-isomer interacts.

The effect of stereochemistry on the thermodynamic characteristics of the binding of fenoterol stereoisomers to the beta(2)-adrenoceptor.

The binding thermodynamics of the stereoisomers of fenoterol, (R,R')-, (S,S')-, (R,S')-, and (S,R')-fenoterol, to the beta(2)-adrenergic receptor (beta(2)-AR) have been determined. The experiments utilized membranes obtained from HEK cells stably transfected with cDNA encoding human beta(2)-AR. Competitive displacement studies using [(3)H]CGP-12177 as the marker ligand were conducted at 4, 15, 25, 30 and 37 degrees C, the binding affinities calculated and the standard enthalpic (DeltaH degrees ) and standard entropic (DeltaS degrees ) contribution to the standard free energy change (DeltaG degrees ) associated with the binding process determined through the construction of van't Hoff plots. The results indicate that the binding of (S,S')- and (S,R')-fenoterol were predominately enthalpy-driven processes while the binding of (R,R')- and (R,S')-fenoterol were entropy-driven. All of the fenoterol stereoisomers are full agonists of the beta(2)-AR, and, therefore, the results of this study are inconsistent with the previously described "thermodynamic agonist-antagonist discrimination", in which the binding of an agonist to the beta-AR is entropy-driven and the binding of an antagonist is enthalpy-driven. In addition, the data demonstrate that the chirality of the carbon atom containing the beta-hydroxyl group of the fenoterol molecule (the beta-OH carbon) is a key factor in the determination of whether the binding process will be enthalpy-driven or entropy-driven. When the configuration at the beta-OH carbon is S the binding process is enthalpy-driven while the R configuration produces an entropy-driven process.

Comparative molecular field analysis of fenoterol derivatives: A platform towards highly selective and effective beta(2)-adrenergic receptor agonists.

PURPOSE: To use a previously developed CoMFA model to design a series of new structures of high selectivity and efficacy towards the beta(2)-adrenergic receptor. RESULTS: Out of 21 computationally designed structures 6 compounds were synthesized and characterized for beta(2)-AR binding affinities, subtype selectivities and functional activities. CONCLUSION: the best compound is (R,R)-4-methoxy-1-naphthylfelnoterol with K(i)beta(2)-AR=0.28microm, K(i)beta(1)-AR/K(i)beta(2)-AR=573, EC(50cAMP)=3.9nm, EC(50cardio)=16nm. The CoMFA model appears to be an effective predictor of the cardiomocyte contractility of the studied compounds which are targeted for use in congestive heart failure.

Comparative molecular field analysis of the binding of the stereoisomers of fenoterol and fenoterol derivatives to the beta2 adrenergic receptor.

Stereoisomers of fenoterol and six fenoterol derivatives have been synthesized and their binding affinities for the beta2 adrenergic receptor (Kibeta2-AR), the subtype selectivity relative to the beta1-AR (Kibeta1-AR/Kibeta2-AR) and their functional activities were determined. Of the 26 compounds synthesized in the study, submicromolar binding affinities were observed for (R,R)-fenoterol, the (R,R)-isomer of the p-methoxy, and (R,R)- and (R,S)-isomers of 1-naphthyl derivatives and all of these compounds were active at submicromolar concentrations in cardiomyocyte contractility tests. The Kibeta1-AR/Kibeta2-AR ratios were >40 for (R,R)-fenoterol and the (R,R)-p-methoxy and (R,S)-1-naphthyl derivatives and 14 for the (R,R)-1-napthyl derivative. The binding data was analyzed using comparative molecular field analysis (CoMFA), and the resulting model indicated that the fenoterol derivatives interacted with two separate binding sites and one steric restricted site on the pseudo-receptor and that the chirality of the second stereogenic center affected Kibeta2 and subtype selectivity.

In vitro detection of differential and cell-specific hepatobiliary toxicity induced by geldanamycin and 17-allylaminogeldanamycin using dog liver slices.

Experiments on rat liver slices demonstrated the differential hepatobiliary toxic potency of two anticancer agents, geldanamycin (GEL) and 17-allylaminogeldanamycin (17-AAG), over a 5-day period. This report describes the pattern of toxicity of these agents in dog liver tissue, using the in vitro liver slice culture model. Liver slices (200 microm thick) from male beagle dogs were cultured for 5 days in chemically defined culture medium containing a range of GEL and 17-AAG concentrations (0.1-5 microM). Tissues were evaluated using a panel of clinically relevant biomarkers and histological endpoints. GEL-induced reduction of alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) slice levels, indicators of biliary epithelial cell (BEC) viability, was supported by histological findings showing an increasing loss of BEC as higher concentrations were applied. At the highest concentrations studied, GEL caused both hepatocellular necrosis and BEC loss. Biomarker pattern results in the medium concurred with those from slice biochemistry measurements and histology. 17-AAG, a less potent compound in vivo, elicited more biomarker retention at higher concentrations than did GEL. Histological analysis revealed higher BEC viability and significant retention of BEC proliferation as compared with GEL. However, at the highest concentration, the toxic insult caused a marked decrease in BEC viability and proliferation. Comparison of responses with both compounds indicated that slices exposed to the same concentrations were more sensitive to GEL than to 17-AAG. Dog liver slices can thus be used to evaluate species-, compound-, and concentration-dependent differences in toxicity.