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

Abstract

Selective and potent antagonists for the b2-adrenoceptor are potentially interesting as experimental and clinical tools, and we sought to identify novel ligands with this pharmacology. EXPERIMENTAL APPROACHA range of pharmacological assays was used to assess potency, affinity, selectivity (b2-adrenoceptor vs. b1-adrenoceptor) and efficacy. KEY RESULTSTen novel compounds were identified but none had as high affinity as the prototypical b2-adrenoceptor blocker ICI-118,551, although one of the novel compounds was more selective for b2-adrenoceptors. Most of the ligands were inverse agonists for b2-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 b2-adrenoceptor-b-arrestin signalling. The (2S,3S) enantiomers were identified as the most active, although unusually the racemates were the most selective for the b2-adrenoceptors. This was taken as evidence for some unusual enantiospecific behaviour. CONCLUSIONS AND IMPLICATIONSIn 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 b-blockers do more than simply inhibit receptor activation, and differences between the ligands could provide useful tools to investigate receptor biology.

A Systematic Review of Inverse Agonism at Adrenoceptor Subtypes

“…Frequently applied assays include GTPγS binding as a very proximal assay of receptor activity [ 16 , 17 , 18 ]; more distally, early second messengers, such as the formation of inositol phosphates (IP) [ 19 ], formation of cAMP [ 15 , 20 ], or inhibition of the formation of cAMP [ 21 , 22 ]; or, even more distally, up-regulation of the receptor number [ 23 , 24 , 25 , 26 , 27 ]. Less frequently applied readouts involve AR phosphorylation [ 26 , 28 ], FRET signals for receptor/G-protein interactions [ 29 , 30 , 31 ], GTPase activity [ 32 ], β-arrestin recruitment [ 33 ], the activity of phospholipase D [ 23 ] or extracellular signal-regulated kinase [ 14 , 34 ], free intracellular Ca 2+ concentrations [ 35 ], the modulation of ion channels such as L-type Ca 2+ channels [ 36 ] or the iK Ca 1 K + channel [ 37 ], or the modulation of cAMP gene transcription by acting on cAMP response elements [ 15 , 38 ]. However, it needs to be considered that the more distant an observed response occurs in the signal transduction cascade, the greater the biological complexity of the assay and the possibility that it becomes affected by factors other than IA [ 39 ].…”

“…IA has been studied more often at β 2 -AR than any other AR subtype. Accordingly, this has been conducted in a wide range of models, including transfected mammalian COS-7 [ 47 , 53 ], BC3H1 [ 42 ], CHW [ 49 , 54 ], CHO [ 15 , 28 , 48 , 82 , 83 ], HEK [ 26 , 27 , 31 , 33 , 84 , 85 ], NG 108-15 cells [ 25 , 51 , 53 , 86 ], Burkitt lymphoma [ 38 ], H9C2 cells [ 87 ], fibroblasts [ 84 ], and insect Sf9 cells [ 32 , 48 , 52 , 88 ]. Models with an endogenous expression of β 2 -AR have also been used, including A431 [ 20 ] cells and cardiomyocytes [ 36 , 89 , 90 ].…”

“…Models with an endogenous expression of β 2 -AR have also been used, including A431 [ 20 ] cells and cardiomyocytes [ 36 , 89 , 90 ]. While the most frequently used readout was cAMP formation, others included FRET assays for the receptor/G protein interaction [ 31 , 91 ], G protein activity [ 32 ], arrestin recruitment [ 33 ], receptor phosphorylation [ 26 ], intracellular Ca 2+ levels [ 90 ], Ca 2+ channel activity [ 36 ], the activation of extracellular signal-regulated kinase [ 15 ], reporter gene assays [ 15 , 38 ], and receptor up-regulation [ 25 , 26 , 27 , 85 , 86 ].…”

“…Extending these observations, acute exposure to nadolol (5 min) reduced cAMP formation and forskolin-stimulated phosphorylation of the β 2 -AR at Ser 355 and Ser 356 , whereas longer exposure (24 h) increased cAMP formation, presumably by up-regulation of the receptor, and did not change receptor phosphorylation [ 26 ]. In contrast to the agonist adrenaline, ICI 118,551 did not affect arrestin recruitment [ 33 ].…”

“…More than 20 studies have demonstrated the inhibition of basal cAMP formation via WT β 2 -AR: These studies demonstrated IA for ICI 118,551 as the most frequently studied compound [ 15 , 20 , 28 , 31 , 32 , 33 , 42 , 49 , 51 , 52 , 53 , 54 , 83 , 84 , 85 , 88 , 89 , 90 ], but also for alprenolol [ 42 , 48 , 82 ], atenolol [ 15 , 42 ], betaxolol [ 28 , 51 , 82 , 85 , 86 ], bisoprolol [ 15 ], cyanopindolol [ 88 ], dichloroisoproterenol [ 48 ], labetolol [ 48 , 82 ], metoprolol [ 15 , 31 ], nadolol [ 26 ], pindolol [ 48 , 82 ], propranolol [ 15 , 42 , 48 , 82 , 88 ], sotalol [ 15 , 86 ], and timolol [ 15 , 53 ] [ 48 , 51 , 82 , 84 ,…”