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
View: Text | PDF
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

×

Figure 3

C11 suppresses G protein signaling and β-arrestin function downstream of agonist-activated β1AR in HEK293T cells.

Options: View larger image (or click on image) Download as PowerPoint
C11 suppresses G protein signaling and β-arrestin function downstream of...
(A–L) HEK293T cells transiently transfected with β1AR (or β1V2R) and BRET or luciferase biosensor plasmids were pretreated with DMSO (0.19%) or 30 μM C11 and then stimulated with serial doses of the agonist isoproterenol (Iso). Schematic representation of the BRET-based TRUPATH G protein dissociation assay where Gαs-RLuc8 dissociates from Gβ/Gγ-GFP upon β1AR activation, resulting in BRET signal decay (A). C11 treatment significantly reduced maximal G protein dissociation compared with vehicle (B and C). Schematic representation of the GloSensor luciferase-based biosensor that emits light in response to binding intracellular cAMP (D). C11 treatment significantly reduced maximal cAMP accumulation compared with vehicle (E and F). Schematic representation of the BRET-based β-arrestin recruitment assay wherein β-arrestin–GFP is recruited to β1V2R-RLucII upon receptor activation and generates a BRET signal (G). C11 treatment significantly reduced maximal β-arrestin recruitment to β1V2R-RLucII compared with vehicle (H and I). Schematic representation of BRET-based β-arrestin–mediated receptor internalization assay (J). Upon β1V2R activation, the receptor/β-arrestin–RLucII complex is internalized into endosomes and a BRET signal is generated when internalized β-arrestin–RLucII and endosomal marker FYVE-GFP are in proximity. C11 treatment significantly reduced maximal β-arrestin internalization compared with vehicle (K and L); F test, *P < 0.05, ***P < 0.001; data points represent mean ± SEM of at least 3 independent experiments performed in duplicate or quadruplicate; 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; luminescence values and Emax quantifications (derived from the nonlinear fit) are presented as the percentage of maximal signal in the vehicle condition.