The effect of stereochemistry on the thermodynamic characteristics of the binding of fenoterol stereoisomers to the β2-adrenoceptor
Graphical abstract
Introduction
The thermodynamics of the binding of agonists and antagonists to β-adrenergic receptors (β-ARs) have been described as fundamentally different processes in which the binding of an agonist is enthalpy-driven while the binding of an antagonist is entropy-driven [1], [2], [3]. This observation has been generalized as the principle of “thermodynamic agonist–antagonist discrimination”, which has been defined as a relationship that when the binding of an agonist is entropy-driven, the binding of an antagonist is enthalpy-driven, or vice versa [4]. This principle has been recently reviewed and appears to hold for the β-AR, adenosine A1 and A2A, A2B and A3, cannabinoid CB1 and CB2, histamine H3, glycine, GABAA, serotonin 5-HT3, nicotinic acetylcholine and purinergic P2X3 receptors [4], [5]. However, “thermodynamic agonist–antagonist discrimination” does not appear to apply to cholecystokinin CCK2, dopamine D2, histamine H1, δ- and μ-opiod, purinergic P2X1 and serotonin 5-HT1A receptors [4], [5].
To our knowledge, the data used to establish “thermodynamic agonist–antagonist discrimination” at the β-AR were obtained using racemic mixtures or only one enantiomer of a chiral compound. One of these compounds was the agonist fenoterol. Fenoterol, is an asymmetric molecule that contains two chiral centers and, therefore, exists as 4 stereoisomers, (R,R′)-, (S,S′)-, (R,S′)-, (S,R′)-fenoterol, Fig. 1 , where the configuration at the carbon containing the β-hydroxyl moiety, β-hydroxy carbon, is denoted as R or S and the configuration at the chiral center on the aminoalkyl portion of the molecule is denoted as R′ or S′. The compound identified as “fenoterol” in the initial study by Wieland et al. [3] was the racemic (50:50) mixture of (R,R′)- and (S,S′)-fenoterol, rac-fenoterol.
We have recently synthesized all of the stereoisomers of fenoterol and demonstrated that the chirality at the two chiral centers affected the magnitude of the interactions with the β2-AR. In these studies, the β2-AR binding affinities (K i ) and activities (EC50) were determined using a stably expressed cell line [6], [7]. The calculated K i values differed for each stereoisomer and ranged from 7158 nM (S,S′)-fenoterol to 164 nM (R,R′)-fenoterol (25 °C), see Table 1 . The data also demonstrated that all of the fenoterol stereoisomers were full agonists, defined as ≥100% induced stimulation of cAMP accumulation relative to isoproterenol, with EC50cAMP values of 0.3 nM (R,R′), 4.70 nM (R,S′), 8.50 nM (S,R′) and 580 nM (S,S′) [7] and Supplemental Data, Figs. S1–4. The activity of the fenoterol stereoisomers also differed in a rat cardiomyocyte contractility model with EC50cardio values of 73 nM (R,R′) [8], 575 nM (R,S′) [7], 2340 nM (S,R′) [8], and 55,000 nM (S,S′) [unpublished data]. In addition, we have also demonstrated that the stereochemistry of the fenoterol molecule determines the β2-AR coupling preference for G-proteins in cardiomyocytes, as (R,R′)-fenoterol preferentially activates Gs signaling while (S,R′)-fenoterol activates both Gs and Gi proteins [8].
The objective of the current study was to further explore the role that stereochemistry plays in the interactions of fenoterol with the β2-AR through the determination of the binding thermodynamics of the four fenoterol stereoisomers. The results indicate that the binding of (S,S′)-fenoterol and (S,R′)-fenoterol were enthalpy-driven, while the binding of (R,R′)-fenoterol and (R,S′)-fenoterol was entropy-driven. Thus, the binding of these compounds to the β2-AR does not conform to the previously described “thermodynamic agonist–antagonist discrimination” and instead is similar to the processes described for the cholecystokinin CCK2, dopamine D2, histamine H1, δ- and μ-opiod, purinergic P2X1 and serotonin 5-HT1A receptors, in which agonists bind to the receptors with both enthalpy-driven and entropy-driven mechanisms. In addition, the data demonstrate that the chirality of the β-OH carbon is a key factor in the determination of whether the binding process will be enthalpy-driven or entropy-driven, with the S configuration resulting in an enthalpy-driven and the R configuration producing an entropy-driven process. To our knowledge this is the first report in which the binding thermodynamics of enantiomers differ not only quantitatively but also qualitatively, i.e. the binding of one enantiomer is enthalpy-driven (ΔH° < 0) while the binding of the other is purely entropy-driven (ΔH° ≥ 0).
Section snippets
Materials
(R,R′)-Fenoterol, (R,S′)-fenoterol, (S,R′)-fenoterol and (S,S′)-fenoterol were synthesized as previously described [6]. [5,7-3H]-(−)-CGP-12177 was purchased from PerkinElmer (Shelton, CT), DMEM was purchased from Lonza Walkersville, Inc. (Walkersville, MD), fetal bovine serum was purchased from Atlas Biologicals (Fort Collins, CO), penicillin–streptomycin and geneticin (G418) were purchased from Invitrogen (Carlsbad, CA), sodium chloride and calcium chloride were purchased from Mallinckrodt
Results
The effect of a decrease in temperature, from 37 to 4 °C, on the K i values of the fenoterol stereoisomers is reported in Table 1. The agonist (R)-isoproterenol and the antagonist rac-propranolol were utilized as a prototypical agonist and antagonist as the thermodynamics of the β2-AR binding of these compounds have been previously examined [1], [2], [3], [4]. The results obtained for these two compounds are also presented in Table 1. The binding affinity, K d value, of the marker ligand, [3
Discussion
All of the fenoterol stereoisomers used in this study are full agonists of the β2-AR, and, therefore, the results of this study are inconsistent with the hypothesis that agonist binding to the β-AR [1] and β2-AR [2] is an enthalpy-driven process. Instead, the data indicate that agonist binding to the β2-AR is similar to the processes described for the cholecystokinin CCK2, dopamine D2, histamine H1, δ- and μ-opiod, purinergic P2X1 and serotonin 5-HT1A receptors, in which agonists bind to the
Acknowledgements
This work was supported in part by funds from the National Institute on Aging Intramural Research Program, by the National Institutes on Aging under contract number N01AG-3-1009 and the Foundation for Polish Science (FOCUS 4/2006 Programme).
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