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.

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.

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.

Synthesis of Tritium Labeled (R,R)-4-Methoxyfenoterol.

The preparation of 2',4',6'-[(3)H(3)]-(R,R)-4-methoxyfenoterol, a tritium-labeled derivative of (R,R)-4-methoxyfenoterol was demonstrated on a 15 mCi scale providing material with a specific activity of 57 Ci/mmol.

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.

The combination of cobinamide and sulfanegen is highly effective in mouse models of cyanide poisoning.

CONTEXT: Cyanide is a component of smoke in residential and industrial fires, and accidental exposure to cyanide occurs in a variety of industries. Moreover, cyanide has the potential to be used by terrorists, particularly in a closed space such as an airport or train station. Current therapies for cyanide poisoning must be given by intravenous administration, limiting their use in treating mass casualties. OBJECTIVE: We are developing two new cyanide antidotes--cobinamide, a vitamin B(12) analog, and sulfanegen, a 3-mercaptopyruvate prodrug. Both drugs can be given by intramuscular administration, and therefore could be used to treat a large number of people quickly. We now asked if the two drugs would have an augmented effect when combined. MATERIALS AND METHODS: We used a non-lethal and two different lethal models of cyanide poisoning in mice. The non-lethal model assesses neurologic recovery by quantitatively evaluating the innate righting reflex time of a mouse. The two lethal models are a cyanide injection and a cyanide inhalation model. RESULTS: We found that the two drugs are at least additive when used together in both the non-lethal and lethal models: at doses where all animals died with either drug alone, the combination yielded 80 and 40% survival in the injection and inhalation models, respectively. Similarly, drug doses that yielded 40% survival with either drug alone, yielded 80 and 100% survival in the injection and inhalation models, respectively. As part of the inhalation model, we developed a new paradigm in which animals are exposed to cyanide gas, injected intramuscularly with an antidote, and then re-exposed to cyanide gas. This simulates cyanide exposure of a large number of people in a closed space, because people would remain exposed to cyanide, even after receiving an antidote. CONCLUSION: The combination of cobinamide and sulfanegen shows great promise as a new approach to treating cyanide poisoning.