1521-0103/359/1/73–81$25.00 http://dx.doi.org/10.1124/jpet.116.232884 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 359:73–81, October 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Comparison of the b-Adrenergic Receptor Antagonists Landiolol and Esmolol: Receptor Selectivity, Partial Agonism, and Pharmacochaperoning Actions Shahrooz Nasrollahi-Shirazi, Sonja Sucic, Qiong Yang, Michael Freissmuth, and Christian Nanoff Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria Received February 15, 2016; accepted July 18, 2016 Downloaded from ABSTRACT Blockage of b1-adrenergic receptors is one of the most effective was very low. Both landiolol and esmolol caused a very modest treatments in cardiovascular medicine. Esmolol was introduced rise in cAMP levels but a robust increase in the phosphorylation some three decades ago as a short-acting b1-selective antag- of extracellular signal regulated kinases 1 and 2, indicating that the onist. Landiolol is a more recent addition. Here we compared the two drugs exerted partial agonist activity with a signaling bias. If two compounds for their selectivity for b1-adrenergic receptors cells were incubated for $24 hours in the presence of $1 mM jpet.aspetjournals.org over b2-adrenergic receptors, partial agonistic activity, signaling esmolol, the levels of b1-adrenergic—but not of b2-adrenergic— bias, and pharmacochaperoning action by using human embry- receptors increased. This effect was contingent on export of onic kidney (HEK)293 cell lines, which heterologously express the b1-receptor from endoplasmic reticulum and was not seen each human receptor subtype. The affinity of landiolol for b1- in the presence of landiolol. On the basis of these observa- adrenergic receptors and b2-adrenergic receptors was higher tions, we conclude that landiolol offers the advantage of: 1) and lower than that of esmolol, respectively, resulting in an improved selectivity and 2) the absence of pharmacochaperon- improved selectivity (216-fold versus 30-fold). The principal ing activity, which sensitizes cells to rebound effects upon drug metabolite of landiolol (M1) was also b -selective, but its affinity discontinuation. 1 at ASPET Journals on October 2, 2021 Introduction which the affinity is quoted in reviews, (e.g., Plosker, 2013), but for which the original data are inaccessible. b Landiolol is a short-acting -adrenergic receptor antagonist G protein-coupled receptor antagonists have been catego- – that has a half-life (3 4 minutes) shorter than the reference rized traditionally according to receptor selectivity, binding compound esmolol (9 minutes) (Plosker, 2013). Cleavage of an affinity, and the pharmacokinetic properties of the com- ester bond generates the major landiolol metabolite, M1, pounds. Additional possible discriminators that have been which has a half-life (1.8 hours) substantially longer than appreciated more recently are their intrinsic activity and the parent compound (Murakami et al., 2005). During short- signaling bias (Kenakin, 2005) and their ability to act as term infusion, M1 reaches peak concentrations in the range of pharmacochaperones (Tao and Conn, 2014): m 3.24 M (Murakami et al., 2005). Because of its long half-life, 1) G protein-coupled receptors elicit signals not only the concentrations of M1 are predicted to increase further by recruiting their cognate G protein. They can activate upon prolonged infusion and to reach their steady state, with additional signaling pathways in a manner independent of continuous intravenous infusion, after about 7 hours. In fact, heterotrimeric G proteins. Among all receptors known, the b2- the ratio of half-lives predicts that under steady-state condi- adrenergic receptor has been most extensively investigated tions, the concentration of M1 may exceed that of landiolol by with respect to its ability to generate a second wave of several fold. It was therefore of interest to document the intracellular signals: Agonist occupancy triggers phosphor- b b affinity and selectivity of M1 for human 1- and 2-adrenergic ylation of several serine and threonine residues in the receptors and to compare it to that of the parent compound, for C-terminus of the b2-adrenergic receptor by G protein- coupled receptor kinases (Lefkowitz and Shenoy, 2005). This supports binding of b-arrestins, which serve as versatile This work was supported by a grant from AOP Orphan Pharmaceuticals AG, adapters: They recruit the endocytotic machinery and thus Vienna, Austria, which markets esmolol and has a commercial interest in landiolol. support the clathrin-dependent endocytosis of receptors. dx.doi.org/10.1124/jpet.116.232884. In addition, b-arrestins scaffold via their C-terminus the ABBREVIATIONS: ANOVA, analysis of variance; BSA, bovine serum albumin; COPII, coatomer protein II; ER, endoplasmic reticulum; ERK, extracellular signal-regulated kinase (MAP kinase); FCS, fetal calf serum; GFP, green fluorescent protein; HEK, human embryonic kidney cells; M1, principal landiolol metabolite (3-{4-[(S)-2-hydroxy-3-(2-morpholinocarbonylamino) ethylamino]propoxy}phenylpropionic acid); MAP, mitogen- activated protein; PBS, phosphate-buffered saline; PMA, phorbol-12-myristat-13-acetate; PMSF, phenylmethylsulfonyl fluoride; siRNA, small- interfering RNA. 73 74 Nasrollahi-Shirazi et al. assembly of kinase cascades, most prominently the mitogen- 100, 31, and 33 mM, respectively. Isoproterenol (33 mM) was dissolved activated protein (MAP) kinase cascade (Lefkowitz and She- in 0.1 M HCl. noy, 2005; Shenoy and Lefkowitz, 2011). Receptor ligands Cell Transfections, Cell Cultures, and Cell Membrane b b differ in their ability to stabilize the active conformations of Preparations. Plasmids encoding human 1-and 2-adrenergic the receptor. Full agonists are thought to stabilize all active receptors,whichweretaggedontheirN-terminiwithaFLAG- epitope, were a generous gift of Dr. Mark von Zastrow (University conformations, pure antagonists are thought to trap the of California at San Francisco). Human embryonic kidney receptor in the inactive conformation. However, there is a (HEK)293 cells (a fibroblast cell line) were transfected using the continuum with respect to agonistic or antagonistic activity polycationic TurboFect reagent (Fermentas/Thermo Fisher Scien- and with respect to the conformations, which are achieved tific) as follows: The plasmid encoding the human b1-orthehuman upon binding of individual ligands (Kenakin, 2005). Accord- b2-adrenergic receptor plasmids (3 mg) were diluted with empty m m ingly, in the case of the b2-adrenergic receptor, antagonists carrier plasmid pcDNA3 (7 g) and 20 l of the TurboFect reagent have been found to act as b-arrestin-biased ligands, i.e., they in 1 ml Dulbecco’s modified Eagle’s medium (DMEM; Sigma- Aldrich). This mixture was incubated for 20 minutes at room block the canonical signaling pathway (i.e., Gs-dependent stimulation of adenylyl cyclase), but they support the re- temperature (22°C) to allow for complex formation between the cruitment of b-arrestin and the resulting MAP kinase stimu- DNA and the polycationic TurboFect reagent. Thereafter, it was pipetted dropwise onto the layer of HEK293 cells (80% confluent in lation (Azzi et al., 2003; Whalen et al., 2011). a 10-cm dish). After 24 hours the medium was exchanged and the 2) Continuous exposure to cell-permeable ligands can in- cells propagated in a humidified atmosphere (95% air/5% CO2)at crease the surface levels of a receptor and result in exagger- 37°C in DMEM containing 10% fetal calf serum (FCS) and Downloaded from ated responses to endogenous agonists, if the treatment with 0.7 mg/ml geneticin (G418) for selection of stable transfectants. an antagonist is suddenly stopped. In fact, this was first In some instances, a plasmid (3 mg/2 Â 106 cells) encoding a green observed with the b-adrenergic antagonist propranolol (“pro- fluorescent protein (GFP)-tagged bovine b-arrestin-2 (arrestin-3) pranolol withdrawal rebound”; Alderman et al., 1974; Miller or an empty control plasmid was subsequently introduced into et al., 1975) and linked to an increase in surface receptor levels stably transfected HEK293 cells. (Aarons et al., 1980). This effect is currently thought to reflect For membrane preparation, stably transfected cells were har- – jpet.aspetjournals.org pharmacochaperoning by cell-permeable antagonists, i.e., vested from 15-cm dishes (80 90% confluent) as follows: The dishes were first rinsed with phosphate buffered saline (PBS) and then specific ligands can assist receptor folding in the ER (endo- mechanically detached from the dish with a plastic scraper in 5 ml of plasmic reticulum) by binding to and stabilizing conforma- ice-cold PBS containing 0.1 mM phenylmethylsulfonyl fluoride tional intermediates on the trajectory to the stable low-energy (PMSF). Cells were recovered by centrifugation (400g for 10 minutes state of the mature receptor (Morello et al., 2000; Nanoff and at 4°C). The cell pellet was resuspended in 1 ml hypotonic HME Freissmuth, 2012). buffer (20 mM HEPES NaOH, pH 7.5, 1 mM EDTA, 2 mM MgCl2) The underlying chemistry behind the development of the containing 0.1 mM PMSF and the Complete Protease Inhibitor short-acting b-blocker landiolol resulted in a large molecular Cocktail (Roche Biochemical Reagents/Sigma-Aldrich) and subse- at ASPET Journals on October 2, 2021 structure. Given the chemical difference with conventional