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 1

Discovery of C11 through DNA-encoded small-molecule library screening.

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Discovery of C11 through DNA-encoded small-molecule library screening. (...
(A) Schematic of screening conditions: empty nanodisc and un-liganded (apo)-β1AR nanodisc controls; β1AR nanodiscs bound to high-affinity orthosteric agonist, BI-167107 (BI); and BI-bound β1AR or β1V2Rpp in complex with transducers (heterotrimeric Gs or β-arrestin 1, respectively). Transducer complexes were further reinforced using conformation-stabilizing nanobodies Nb35 (included in β1AR/G protein complex), Nb25, and Fab30 (included in β1V2Rpp/β-arrestin complex). (B) Purified β1ARs reconstituted in biotinylated lipid nanodiscs were immobilized using neutravidin beads and incubated with HitGen’s OpenDEL small-molecule library. After washing, bound compounds were eluted (elution 1) and applied as input for a second round of affinity selection with fresh β1AR nanodiscs. Molecules from the final elution (elution 2) were identified by high-throughput DNA sequencing. (C and D) DNA copy number in eluted samples (E1–E2) was determined by qPCR after each round of affinity selection. Compared with library input (~1015 molecules), copy number was reduced to approximately 107 after 2 rounds of screening in each condition (C). Approximately 104–5 molecules were lost in round 1 and approximately 102–3 molecules were lost in round 2 (D). (E and F) 3D plots of each screening condition (F) and all 5 conditions merged (E) depict an enriched chemical line feature — where C11 was identified — which was enriched in Apo-β1AR and BI-β1AR samples, minimally present in transducer complex samples, and completely absent in the empty nanodisc condition. Axes enumerate the chemical building blocks utilized in each round of chemical synthesis (R1–R3). Data point size corresponds to sequence count for a particular compound; the copy number of C11 in each condition is indicated in red text. Compounds outside of the feature that contains C11 were filtered out to facilitate visualization of the enriched chemotype. (G) Schematic of chemical structure of C11 generated through 3 rounds of chemical synthesis: R1, red; R2, green; R3, blue.