A positive allosteric modulator of the β1AR with antagonist activity for catecholaminergic polymorphic ventricular tachycardia
Alyssa Grogan, Robin M. Perelli, Seungkirl Ahn, Haoran Jiang, Arun Jyothidasan, Damini Sood, Chongzhao You, David I. Israel, Alex Shaginian, Qiuxia Chen, Jian Liu, Jialu Wang, Jan Steyaert, Alem W. Kahsai, Andrew P. Landstrom, Robert J. Lefkowitz, Howard A. Rockman
Alyssa Grogan, Robin M. Perelli, Seungkirl Ahn, Haoran Jiang, Arun Jyothidasan, Damini Sood, Chongzhao You, David I. Israel, Alex Shaginian, Qiuxia Chen, Jian Liu, Jialu Wang, Jan Steyaert, Alem W. Kahsai, Andrew P. Landstrom, Robert J. Lefkowitz, Howard A. Rockman
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Research Article Cardiology

A positive allosteric modulator of the β1AR with antagonist activity for catecholaminergic polymorphic ventricular tachycardia

Abstract

Orthosteric beta blockers represent the leading pharmacological intervention for managing heart diseases owing to their ability to competitively antagonize β-adrenergic receptors (βARs). However, their use is often limited by adverse effects such as fatigue, hypotension, and reduced exercise capacity, due in part to nonselective inhibition of multiple βAR subtypes. These challenges are particularly problematic in treating catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease characterized by lethal tachyarrhythmias directly triggered by cardiac β1AR activation. To identify small-molecule allosteric modulators of the β1AR with enhanced subtype specificity and robust functional antagonism of β1AR-mediated signaling, we conducted a DNA-encoded small-molecule library screen and discovered Compound 11 (C11). C11 selectively potentiates the binding affinity of orthosteric agonists to the β1AR while potently inhibiting downstream signaling after β1AR activation. C11 prevents agonist-induced spontaneous contractile activity, Ca2+ release events, and exercise-induced ventricular tachycardia in the CSQ2–/– murine model of CPVT. Our studies demonstrate that C11 belongs to an emerging class of allosteric modulators termed positive allosteric modulator antagonists that positively modulate agonist binding but block downstream function. Its pharmacological properties and selective functional antagonism of β1AR-mediated signaling make C11 a promising therapeutic candidate for the treatment of CPVT and other forms of cardiac disease associated with excessive β1AR activation.

Authors

Alyssa Grogan, Robin M. Perelli, Seungkirl Ahn, Haoran Jiang, Arun Jyothidasan, Damini Sood, Chongzhao You, David I. Israel, Alex Shaginian, Qiuxia Chen, Jian Liu, Jialu Wang, Jan Steyaert, Alem W. Kahsai, Andrew P. Landstrom, Robert J. Lefkowitz, Howard A. Rockman

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Figure 5

C11 does not suppress cellular signaling mediated by alternative receptors β2AR, AT1R, or M3R.

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C11 does not suppress cellular signaling mediated by alternative recepto...
(A–C) β2AR nanodiscs were incubated with a fixed amount of radiolabeled orthosteric antagonist, I125-CYP; serial doses of unlabeled orthosteric ligand; and either vehicle or 30 μM C11. Competition binding curves (A and B) and corresponding IC50 shifts (C) revealed that C11 enhanced the binding affinity of carvedilol (Carv) to the β2AR but not isoproterenol (Iso). Dose-response curves are presented as percentage of maximum I125-CYP binding. IC50 values were calculated from the nonlinear fit (1-site binding; GraphPad Prism) and plotted as the difference between IC50 (DMSO) and IC50 (C11). F tests were performed on the nonlinear fit; ***P < 0.001. Data points represent mean ± SEM of at least 3 independent experiments performed in duplicate. (D–K) HEK293T cells transiently overexpressing receptor (β2AR, β2V2R, or AT1R) and BRET fusion proteins (Figure 3, A and J) were pretreated with vehicle or 30 μM C11 and stimulated with either isoproterenol (for β2AR or β2V2R) or angiotensin II (Ang II, for AT1R). C11 treatment had no significant effect on G-protein dissociation or β-arrestin internalization downstream of agonist-activated β2AR/β2V2R (D–G) or AT1R (H–K). (L–N) HEK293T cells pretreated with vehicle or 30 μM C11 were stimulated with carbachol to activate the endogenous Gq-coupled muscarinic M3 receptor (M3R). C11 treatment had no significant effect on the resulting Ca2+ response (L–M). Representative time-course plots of the baseline-subtracted raw fluorescence at each carbachol dose depicted comparable Ca2+ responses between vehicle- and C11- treated cells (N); data points represent mean ± SEM of at least 3 independent experiments performed in duplicate or triplicate; curve fits were plotted using a log(agonist) 3-parameter model in GraphPad Prism; net BRET ratios (emission of RLuc8/GFP) are baseline-subtracted according to the nonlinear fit of each treatment condition. Ca2+ responses are presented as the baseline-subtracted AUC and normalized to the percentage of DMSO maximum.