Comparative molecular field analysis of fenoterol derivatives interacting with an agonist-stabilized form of the ??-adrenergic receptor.
ABSTRACT: The ??-adrenergic receptor (??-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 ?' 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 EC?? values, were determined in HEK293 cells expressing the ??-AR. The data indicate that the highest binding affinities and functional activities were produced by methyl and ethyl substituents at the ?' position. The results also indicate that the Ki values obtained using [(3)H]-(R,R')-methoxyfenoterol as the marker ligand modeled the EC?? 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 ??-AR conformation probed by [(3)H]-(R,R')-4'-methoxyfenoterol. The CoMFA model of the agonist-stabilized ??-AR suggests that the binding of the fenoterol analogs to an agonist-stabilized conformation of the ??-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.
Project description: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 ?(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 ?(2)-adrenoceptor (AR) population as determined with the antagonist [(3)H]CGP-12177. The ?(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 ?(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 ?(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 ?(2)-adrenoceptor.
Project description:The effect of CGP-12177, originally developed as a radioligand with antagonist properties for binding studies of beta-adrenergic receptors, was investigated in brown adipose tissue. Contrary to expectations, CGP-12177 showed clear agonist properties in experiments with hamster brown-fat cells, with a maximal effect in stimulating oxygen consumption similar to that of the physiological stimulator noradrenaline, and also with a potency similar to that of noradrenaline [EC50 (50% effective concn.) approx. 70 nM]. This value could be contrasted with the very high affinity of CGP-12177 (KD about 1 nM) for ligand-binding sites on the cells. It is therefore suggested that the high-affinity binding site may not be the one that mediates the CGP-12177-stimulated thermogenesis in isolated cells. Also, when injected into cold-adapted rats, CGP-12177 stimulated non-shivering thermogenesis similarly to noradrenaline. This observation, in conjunction with the reported low general sympathomimetic effect of CGP-12177, may indicate that CGP-12177 could be of interest for the development of anti-obesity drugs.
Project description: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.
Project description:Recent interest has focused on antibodies that can discriminate between different receptor conformations. Here we have characterised the effect of a monoclonal antibody (mAb3), raised against a purified thermo-stabilised turkey ?1-adrenoceptor (?1AR-m23 StaR), on ?1-ARs expressed in CHO-K1 or HEK 293 cells. Immunohistochemical and radioligand-binding studies demonstrated that mAb3 was able to bind to ECL2 of the t?1-AR, but not its human homologue. Specific binding of mAb3 to t?1-AR was inhibited by a peptide based on the turkey, but not the human, ECL2 sequence. Studies with [3H]-CGP 12177 demonstrated that mAb3 prevented the binding of orthosteric ligands to a subset (circa 40%) of turkey ?1-receptors expressed in both CHO K1 and HEK 293 cells. MAb3 significantly reduced the maximum specific binding capacity of [3H]-CGP-12177 without influencing its binding affinity. Substitution of ECL2 of t?1-AR with its human equivalent, or mutation of residues D186S, P187D, Q188E prevented the inhibition of [3H]-CGP 12177 binding by mAb3. MAb3 also elicited a negative allosteric effect on agonist-stimulated cAMP responses. The identity of the subset of turkey ?1-adrenoceptors influenced by mAb3 remains to be established but mAb3 should become an important tool to investigate the nature of ?1-AR conformational states and oligomeric complexes.
Project description:1. Atypical beta-adrenoceptors resistant to propranolol, but blocked by bupranolol, increase contractile force and/or frequency of the heart in humans and rats. We compared the potencies of the enantiomers of bupranolol and examined the possible effects of seven bupranolol analogues including bevantolol (BEV) at this receptor in pithed and vagotomized rats. 2. CGP 12177, an agonist of the atypical beta-adrenoceptor, increased heart rate dose-dependently. Its dose-response curve was shifted to the right by S-(-)-bupranolol 10 micro mol kg(-1) by a factor of 8.4, but not affected by the same dose of R-(+)-bupranolol. 3. Desmethylbupranolol and compounds BK-21, BK-22, BK-23 and BK-25 also increased heart rate dose-dependently. The beta(1)-adrenoceptor antagonist CGP 20712 given in combination with the beta(2)-adrenoceptor antagonist ICI 118,551 (0.1 micro mol kg(-1) each) reduced the positive chronotropic action of the five bupranolol analogues without affecting that of CGP 12177. The potencies of the bupranolol analogues to increase heart rate were correlated (r=0.91, P<0.05) with their affinities for beta(1)-adrenoceptor binding sites in rat brain cortex membranes labelled with [(3)H]CGP 12177 (in the presence of ICI 118,551). 4. BK-26 and BEV, 10 micro mol kg(-1) each, had only minor effects on heart rate by themselves and did not antagonize the effect of CGP 12177. However, at 1 micro mol kg(-1), they antagonized the increase in heart rate elicited by the beta(1)-adrenoceptor agonist prenalterol. 5. In conclusion, bupranolol is a stereoselective antagonist at the atypical cardiostimulant beta-adrenoceptor. The effects of the bupranolol analogues are related to the activation or blockade of beta(1)-adrenoceptors, but not of atypical beta-adrenoceptors.
Project description:1 Fluorescence techniques offer a way to circumvent several problems associated with many radioligand binding and functional assays and the need for large numbers of cells. Fluorescent ligands also offer the advantage of allowing real time direct visualisation of ligand - receptors interactions. A fluorescent analogue of CGP 12177 (BODIPY-TMR-CGP) has thus been evaluated as a beta(2)-adrenoceptor ligand in CHO-K1 cells expressing the human beta(2)-adrenoceptor. 2 Studies of (3)H-cAMP accumulation showed that BODIPY-TMR-CGP stimulated an increase in cAMP accumulation and cyclic AMP response element (CRE)-mediated gene transcription with an EC(50) of 21-28 nM. Both of these responses were antagonised by the selective beta(2)-adrenoceptor antagonist ICI 118551. 3 Binding studies with (3)H-CGP 12177, and functional studies of CRE-regulated gene transcription showed that the BODIPY-TMR-CGP interaction with the human beta(2)-adrenoceptor is of very long duration. 4 Visualisation of the binding of BODIPY-TMR-CGP to single living mammalian cells was clearly demonstrated by confocal microscopy and showed that this ligand was able to selectively label cell surface beta(2)-adrenoceptors in living CHO-K1 cells transfected with the human beta(2)-adrenoceptor with an apparent K(D) of 27 nM. Studies with cells expressing a beta(2)-adrenoceptor-green fluorescent protein (GFP) fusion protein provided further strong evidence that BODIPY-TMR-CGP was binding to the beta(2)-adrenoceptor. 5 BODIPY-TMR-CGP is therefore a long-acting fluorescent beta(2)-adrenoceptor agonist that can be used to label beta(2)-adrenoceptors in the plasma membrane of living cells.
Project description:At the ?1-adrenoceptor, CGP 12177 potently antagonizes agonist responses at the primary high-affinity catecholamine conformation while also exerting agonist effects of its own through a secondary low-affinity conformation. A recent mutagenesis study identified transmembrane region (TM)4 of the ?1-adrenoceptor as key for this low-affinity conformation. Others suggested that TM4 has a role in ?1-adrenoceptor oligomerization. Here, assessment of the dissociation rate of a fluorescent analog of CGP 12177 [bordifluoropyrromethane-tetramethylrhodamine-(±)CGP 12177 (BODIPY-TMR-CGP)] at the human ?1-adrenoceptor expressed in Chinese hamster ovary cells revealed negative cooperative interactions between 2 distinct ?1-adrenoceptor conformations. The dissociation rate of 3 nM BODIPY-TMR-CGP was 0.09 ± 0.01 min(-1) in the absence of competitor ligands, and this was enhanced 2.2- and 2.1-fold in the presence of 1 µM CGP 12177 and 1 µM propranolol, respectively. These effects on the BODIPY-TMR-CGP dissociation rate were markedly enhanced in ?1-adrenoceptor homodimers constrained by bimolecular fluorescence complementation (9.8- and 9.9-fold for 1 µM CGP 12177 and 1 µM propranolol, respectively) and abolished in ?1-adrenoceptors containing TM4 mutations vital for the second conformation pharmacology. This study suggests that negative cooperativity across a ?1-adrenoceptor homodimer may be responsible for generating the low-affinity pharmacology of the secondary ?1-adrenoceptor conformation.
Project description:Recent advances in fluorescent ligand technology have enabled the study of G protein-coupled receptors in their native environment without the need for genetic modification such as addition of N-terminal fluorescent or bioluminescent tags. Here, we have used a non-imaging plate reader (PHERAstar FS) to monitor the binding of fluorescent ligands to the human adenosine-A<sub>3</sub> receptor (A<sub>3</sub>AR; CA200645 and AV039), stably expressed in CHO-K1 cells. To verify that this method was suitable for the study of other GPCRs, assays at the human adenosine-A<sub>1</sub> receptor, and ?<sub>1</sub> and ?<sub>2</sub> adrenoceptors (?<sub>1</sub>AR and ?<sub>2</sub>AR; BODIPY-TMR-CGP-12177) were also carried out. Affinity values determined for the binding of the fluorescent ligands CA200645 and AV039 to A<sub>3</sub>AR for a range of classical adenosine receptor antagonists were consistent with A<sub>3</sub>AR pharmacology and correlated well (<i>R</i><sup>2</sup> = 0.94) with equivalent data obtained using a confocal imaging plate reader (ImageXpress Ultra). The binding of BODIPY-TMR-CGP-12177 to the ?<sub>1</sub>AR was potently inhibited by low concentrations of the ?<sub>1</sub>-selective antagonist CGP 20712A (pK<sub>i</sub> 9.68) but not by the ?<sub>2</sub>-selective antagonist ICI 118551(pK<sub>i</sub> 7.40). Furthermore, in experiments conducted in CHO K1 cells expressing the ?<sub>2</sub>AR this affinity order was reversed with ICI 118551 showing the highest affinity (pK<sub>i</sub> 8.73) and CGP20712A (pK<sub>i</sub> 5.68) the lowest affinity. To determine whether the faster data acquisition of the non-imaging plate reader (~3 min per 96-well plate) was suitable for high throughput screening (HTS), we screened the LOPAC library for inhibitors of the binding of CA200645 to the A<sub>3</sub>AR. From the initial 1,263 compounds evaluated, 67 hits (defined as those that inhibited the total binding of 25 nM CA200645 by ?40%) were identified. All compounds within the library that had medium to high affinity for the A<sub>3</sub>AR (pK<sub>i</sub> ?6) were successfully identified. We found three novel compounds in the library that displayed unexpected sub-micromolar affinity for the A<sub>3</sub>AR. These were K114 (pK<sub>i</sub> 6.43), retinoic acid <i>p</i>-hydroxyanilide (pK<sub>i</sub> 6.13) and SU 6556 (pK<sub>i</sub> 6.17). Molecular docking of these latter three LOPAC library members provided a plausible set of binding poses within the vicinity of the established orthosteric A<sub>3</sub>AR binding pocket. A plate reader based library screening using an untagged receptor is therefore possible using fluorescent ligand opening the possibility of its use in compound screening at natively expressed receptors.
Project description:The smooth muscle relaxant responses to the mixed beta(3)-, putative beta(4)-adrenoceptor agonist, (-)-CGP 12177 in rat colon are partially resistant to blockade by the beta(3)-adrenoceptor antagonist SR59230A suggesting involvement of beta(3)- and putative beta(4)-adrenoceptors. We now investigated the function of the putative beta(4)-adrenoceptor and other beta-adrenoceptor subtypes in the colon, oesophagus and ureter of wildtype (WT) and beta(3)-adrenoceptor knockout (beta(3)KO) mice. (-)-Noradrenaline and (-)-adrenaline relaxed KCl (30 mM)-precontracted colon mostly through beta(1)-and beta(3)-adrenoceptors to a similar extent and to a minor extent through beta(2)-adrenoceptors. In colon from beta(3)KO mice, (-)-noradrenaline was as potent as in WT mice but the effects were mediated entirely through beta(1)-adrenoceptors. (-)-CGP 12177 relaxed colon from beta(3)KO mice with 2 fold greater potency than in WT mice. The maintenance of potency for (-)-noradrenaline and increase for (-)-CGP 12177 indicate compensatory increases in beta(1)- and putative beta(4)-adrenoceptor function in beta(3)KO mice. In oesophagi precontracted with 1 microM carbachol, (-)-noradrenaline caused relaxation mainly through beta(1)-and beta(3)-adrenoceptors. (-)-CGP 12177 (2 microM) relaxed oesophagi from WT by 61.4+/-5.1% and beta(3)KO by 67.3+/-10.1% of the (-)-isoprenaline-evoked relaxation, consistent with mediation through putative beta(4)-adrenoceptors. In ureter, (-)-CGP 12177 (2 microM) reduced pacemaker activity by 31.1+/-2.3% in WT and 31.3+/-7. 5% in beta(3)KO, consistent with mediation through putative beta(4)-adrenoceptors. Relaxation of mouse colon and oesophagus by catecholamines are mediated through beta(1)- and beta(3)-adrenoceptors in WT. The putative beta(4)-adrenoceptor, which presumably is an atypical state of the beta(1)-adrenoceptor, mediates the effects of (-)-CGP 12177 in colon, oesophagus and ureter.
Project description:Isoprenaline treatment of C6-glioma cells induced a fast decrease in the number of beta-adrenergic receptors as determined by binding of [3H]CGP-12177, which paralleled the decrease in the hormonally stimulated adenylate cyclase activity. The total number of receptors, as determined by binding of (-)-[3H]dihydroalprenolol, did not decrease. Separation of the beta-adrenergic receptors on a sucrose density gradient showed that the decrease in the number of receptors detectable with CGP-12177 was due to a movement of the receptors from the plasma membrane to a vesicular cell compartment. By using both (-)-[3H]dihydroalprenolol and [3H]CGP-12177 it is thus possible to differentiate between the total number of receptors and those present at the plasma membrane in an unfractionated cell lysate.