The β₂ adrenergic receptor (β2-AR) has emerged as a model system for studying the ligand recognition process and the mechanism of GPCR activation (c.f. [1]). Fenoterol, a β2-AR selective agonist is used in therapy as a racemic mixture of (R,R)- and (S,S)- isomers (Fig.1.). In the current project we studied the four stereoisomers of fenoterol and several derivatives modified on the aminoalkyl tail. Radioligand binding studies determined that stereochemistry greatly influences the binding affinity with relative order uniform for all studied derivatives: (R,R)>(R,S)>(S,R)>(S,S) [2]. Control experiments on binding affinity towards the β1-AR show that β2-AR vs. β1-AR selectivity is the greatest for (R,R)- configuration of the ligand, and in case of some new derivatives this selectivity reaches exceptionally high value (e.g., (R,R)-4-methoxy-1-naphtylfenoterol K_i(β1-AR)/K_i(β1-AR)=573) [3]. Functional activities of the compounds were determined using induced cAMP accumulation and cardiomyocyte contractility and the results confirmed that the (R,R)-configuration of fenoterol based derivatives is the most effective.

Figure 1. Stereochemistry of fenoterol.

Subsequent Van’t Hoff analysis of fenoterol isomers shows very different thermodynamic characteristic of binding depending on the stereoconfiguration of the molecule. The binding of (S,S)- and (S,R)- isomers is almost entirely enthalpy controlled whereas the binding of (R,R)- and (R,S)- fenoterol is purely entropy driven [4]. The latter observation contradicts the “enthalpy-entropy discrimination” paradigm commonly accepted for β-adrenergic receptors [5] according to which the binding of full agonist is controlled by the combination of enthalpy and entropy while binding of antagonists is mainly entropy controlled.

The stereochemistry of the fenoterol molecule also affects the coupling of the AR to the Gs and Gi proteins.  In cardiomyocyte studies, the addition of pertussis toxin had no effect on the activity of (R,R)- fenoterol indicating that the compound selectively activates Gs protein signaling upon binding to the receptor [6].  When (S,R)-fenoterol was used as the agonist, the addition of pertussis toxin significantly reduced cardiomyocyte contractility indicating that the compound activates both Gi and Gs protein [6]. The same pattern of stereo-discrimination was observed with the 4-methoxyfenoterol derivatives [6].

The data demonstrate that the stereochemistry of the fenoterol molecule influences the magnitude of binding affinity, the thermodynamics of local interaction of ligand within the binding site and the global mechanism of activation of the β2-receptor as evidenced by the Gs/Gi selectivity observation. It opens new perspectives for developing potent and highly selective β2-AR agonists. For example (R,R)-methoxyfenoterol is currently clinically tested for treatment of congestive heart failure, some other derivatives appears as very effective inhibitors of tumor cells mitogenesis in in vitro studies.

References

[1] Bokoch MP, Zou Y, Rasmussen SG, et. al., Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor Nature. 2010;463(7277):108-12.

[2] Jozwiak K, Khalid C, et al., Comparative molecular field analysis of the binding of the stereoisomers of fenoterol and fenoterol derivatives to the β2 adrenergic receptor. J Med Chem. 2007;50(12):2903-15.

[3] Jozwiak K, Woo YH, et al., Comparative molecular field analysis of fenoterol derivatives: A platform towards highly selective and effective β2-adrenergic receptor agonists. Bioorg Med Chem. 2010;18(2):728-736.

[4] Jozwiak K, Toll L, et al., The effect of stereochemistry on the thermodynamic characteristics of the binding of fenoterol stereoisomers to the β2-adrenoceptor. Biochem. Pharmacol. 2010; in press.

[5] Borea PA, Dalpiaz A et al., Can Thermodynamic Measurements of Receptor Binding Yield Information on Drug Affinity and Efficacy?  Biochem Pharm 2000; 60: 1549-1556.

[6] Woo YH, Wang TB, et al., Stereochemistry of an Agonist Determines Coupling Preference of β2-Adrenoceptor to Different G Proteins in Cardiomyocytes. Mol Pharmacol. 2009; 75: 158-165.

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