Allosteric modulator potentiates β2AR agonist–promoted bronchoprotection in asthma models
Seungkirl Ahn, Harm Maarsingh, Julia K.L. Walker, Samuel Liu, Akhil Hegde, Hyeje C. Sumajit, Alem W. Kahsai, Robert J. Lefkowitz
Seungkirl Ahn, Harm Maarsingh, Julia K.L. Walker, Samuel Liu, Akhil Hegde, Hyeje C. Sumajit, Alem W. Kahsai, Robert J. Lefkowitz
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Research Article

Allosteric modulator potentiates β2AR agonist–promoted bronchoprotection in asthma models

Abstract

Asthma is a chronic inflammatory disease associated with episodic airway narrowing. Inhaled β2-adrenergic receptor (β2AR) agonists (β2-agonists) promote — with limited efficacy — bronchodilation in asthma. All β2-agonists are canonical orthosteric ligands that bind the same site as endogenous epinephrine. We recently isolated a β2AR-selective positive allosteric modulator (PAM), compound-6 (Cmpd-6), which binds outside of the orthosteric site and modulates orthosteric ligand functions. With the emerging therapeutic potential of G-protein coupled receptor allosteric ligands, we investigated the impact of Cmpd-6 on β2AR-mediated bronchoprotection. Consistent with our findings using human β2ARs, Cmpd-6 allosterically potentiated β2-agonist binding to guinea pig β2ARs and downstream signaling of β2ARs. In contrast, Cmpd-6 had no such effect on murine β2ARs, which lack a crucial amino acid in the Cmpd-6 allosteric binding site. Importantly, Cmpd-6 enhanced β2 agonist–mediated bronchoprotection against methacholine-induced bronchoconstriction in guinea pig lung slices, but — in line with the binding studies — not in mice. Moreover, Cmpd-6 robustly potentiated β2 agonist–mediated bronchoprotection against allergen-induced airway constriction in lung slices obtained from a guinea pig model of allergic asthma. Cmpd-6 similarly enhanced β2 agonist–mediated bronchoprotection against methacholine-induced bronchoconstriction in human lung slices. Our results highlight the potential of β2AR-selective PAMs in the treatment of airway narrowing in asthma and other obstructive respiratory diseases.

Authors

Seungkirl Ahn, Harm Maarsingh, Julia K.L. Walker, Samuel Liu, Akhil Hegde, Hyeje C. Sumajit, Alem W. Kahsai, Robert J. Lefkowitz

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Figure 1

Positive cooperativity of Cmpd-6 with albuterol at the human β2AR in functional assays.

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Positive cooperativity of Cmpd-6 with albuterol at the human β2AR in fun...
(A) HEK293 cells stably expressing the GloSensor reporter monitoring the cAMP level were pretreated with either 25 μM Cmpd-6 or the control vehicle (DMSO) and stimulated with a serial dilution of either isoproterenol (ISO) or albuterol (Alb). The level of cAMP production induced by the endogenously expressed β2AR was determined as described in Methods. (B) HEK293T cells expressing all components for the Tango assays monitoring β-arrestin recruitment to the stably expressed β2V2R as described in Methods were pretreated with Cmpd-6 or DMSO and stimulated with the agonists as described for (A). The extent of β-arrestin recruitment was determined as described in Methods. The values in (A and B) were expressed as percentage of the ISO-stimulated maximal response in the DMSO-treated condition. (C and D) Isolated membranes from Expi293F cells transiently expressing the human β2AR were incubated together with either Cmpd-6 at indicated concentrations or DMSO, a serial dilution of the indicated agonist competitor, Alb (C) and ISO (D), and 60 pM [125I]-cyanopindolol (125I-CYP). The reaction was terminated, and 125I-CYP bound to the receptor was read as described in Methods. Values were normalized to the percentage of the maximal 125I-CYP binding level obtained in each of the Cmpd-6- and DMSO-treated conditions, with data points representing mean ± SEM, obtained from 4 (A and B) or 5 (C and D) independent experiments performed in duplicate. The shift of curves was expressed as fold changes in either EC50 and Bmax (A and B) or IC50 (C, D) values between Cmpd-6– and DMSO-treated conditions. Statistical analyses for these shifts in each of the directions were performed using paired 2-tailed Student’s t tests. P values shown were *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control DMSO-treated condition.