The design, synthesis and pharmacological characterization of novel β₂-adrenoceptor antagonists

Abstract

Background and purpose: Selective and potent antagonists for the β(2) -adrenoceptor are potentially interesting as experimental and clinical tools, and we sought to identify novel ligands with this pharmacology.

Experimental approach: A range of pharmacological assays was used to assess potency, affinity, selectivity (β(2) -adrenoceptor vs. β(1) -adrenoceptor) and efficacy.

Key results: Ten novel compounds were identified but none had as high affinity as the prototypical β(2) -adrenoceptor blocker ICI-118,551, although one of the novel compounds was more selective for β(2) -adrenoceptors. Most of the ligands were inverse agonists for β(2) -adrenoceptor-cAMP signalling, although one (5217377) was a partial agonist and another a neutral antagonist (7929193). None of the ligands were efficacious with regard to β(2) -adrenoceptor-β-arrestin signalling. The (2S,3S) enantiomers were identified as the most active, although unusually the racemates were the most selective for the β(2) -adrenoceptors. This was taken as evidence for some unusual enantiospecific behaviour.

Conclusions and implications: In terms of improving on the pharmacology of the ligand ICI-118,551, one of the compounds was more selective (racemic JB-175), while one was a neutral antagonist (7929193), although none had as high an affinity. The results substantiate the notion that β-blockers do more than simply inhibit receptor activation, and differences between the ligands could provide useful tools to investigate receptor biology.

Figure 1

The chemical structures of active compounds isolated using a virtual field screen.

Figure 2

[³H]-CGP-12177 competition binding curves. These curves show the ability of ICI-118,551 (A), 5731869 (B), JB-143 (C) and JB-176 (D) to concentration-dependently displace [³H]-CGP-12177 binding to β₁- and β₂-adrenoceptors. The curves are an average ± SEM of three experiments performed in triplicate.

Figure 3

Efficacy of ICI-118,551 and the Cresset compounds for β₂-adrenoceptor-mediated cAMP signalling. HEK239 cells stably expressing β₂-adrenoceptors were co-incubated with 30 µM forskolin and 100 µM of each compound, and the resulting cAMP response is expressed as a percentage of the response to forskolin alone. Responses above and below 100% (dotted line) signified partial and inverse agonism respectively. Antagonism was signified by no deviation from this response. **P < 0.01 compared with forskolin alone. Data are an average ± SEM of three experiments performed in triplicate.

Figure 4

Antagonism of inverse and partial agonists for the β₂-adrenoceptor-mediated cAMP pathway. β₂-adrenoceptor-expressing HEK293were incubated with 30 µM forskolin and stimulated with 1 µM ICI-118,551, JB-143 or 5217377 with or without 100 µM 7929193. The resulting cAMP responses from the treatments identified 7929193-mediated antagonism of both inverse and partial agonism. *P < 0.05 comparing the effect of antagonist on each ligand response. Data are an average ± SEM of three experiments performed in triplicate.

Figure 5

Ligand-mediated regulation of β₂-adrenoceptor-β-arrestin interactions. HEK293 cells were transiently transfected with β₂-adrenoceptors and GFP-βarr2(1–380), and subcellular localization of fluorescence was imaged by confocal microscopy. One image before (0 min) and two after (15 min) incubation with ligand are shown. Positive responses (membrane translocation) are highlighted with arrows. Images are a representation of at least 10 groups of cells from at least two separate experiments.

Figure 6

The chemical structures of ICI-118,551 and its derivatives. The compounds JB-163, JB-175 and JB-176 were synthesized and pharmacologically analysed as 2S,3S enantiopure preparations. The remaining compounds are racemates.

Figure 7

JB-175-mediated antagonism of adrenaline responses in the guinea-pig trachea. (A) Increasing concentrations of JB-175 produce increasing rightward shifts in the concentration–response curve to adrenaline. (B) The Schild-plot of these responses constructed by plotting log (dose ratio – 1) by concentration of JB-175. Results of each individual experiment are plotted together with their respective linear regression lines. This illustrates the low slope of these curves.

Figure 8

Efficacy of the structural derivatives of ICI-118,551 for β₂-adrenoceptor-mediated cAMP signalling. HEK239 cells stably expressing β₂-adrenoceptors were co-incubated with 30 µM forskolin and 100 µM of each compound, and the resulting cAMP response is expressed as a percentage of the response to forskolin alone. Responses above and below 100% (dotted line) signify partial and inverse agonism respectively. Antagonism was signified by no deviation from this response. **P < 0.01 compared with forskolin alone. Data are an average ± SEM of three experiments performed in triplicate.

Figure 9

The effect of pertussis toxin (PTX) on the negative cAMP responses of ICI-118,551 and JB-143. HEK239 cells stably expressing β₂-adrenoceptors were pretreated with or without 10 ng·mL⁻¹ PTX for 15 h. They were then co-incubated with forskolin and ICI-118,551 or JB-143, and cAMP responses measured. As PTX had an effect on forskolin responses alone, each ligand-mediated response is expressed as a percentage of the respective forskolin response with or without PTX. Data are an average ± SEM of three experiments performed in triplicate.

Figure 10

Conformation changes in the β-adrenoceptor on ligand binding. (A) Carazolol was replaced by JB-175 in the β₂-adrenoceptor X-ray structure (2RH1.PDB) (B) Cyanopindolol was replaced by JB-175 in the β₁-adrenoceptor X-ray structure (2VT4.PDB). In each case, the whole ensemble was fully optimized using the eXtended Electron Distribution molecular mechanics force field. The least squares Cα backbone root mean square difference between the fully optimized empty β₂-receptor and the receptor containing JB-175 was 0.49 and that for the β₁-adrenoceptor was 0.23 (C) Superimposition of the two optimized receptors indicates the relatively small change in ligand alignment (red; β₁-adrenoceptor with magenta ligand, black; β₂-adrenoceptor with grey ligand).