RyR2 Binding of an Antiarrhythmic Cyclic Depsipeptide Mapped Using Confocal Fluorescence Lifetime Detection of FRET

Abstract

Hyperactivity of cardiac sarcoplasmic reticulum (SR) ryanodine receptor (RyR2) Ca 2+ -release channels contributes to heart failure and arrhythmias. Reducing the RyR2 activity, particularly during cardiac relaxation (diastole), is a desirable therapeutic goal. We previously reported that the unnatural enantiomer (ent) of an insect-RyR activator, verticilide, inhibits porcine and mouse RyR2 at diastolic (nanomolar) Ca 2+ and has in vivo efficacy against atrial and ventricular arrhythmia. To determine the ent-verticilide structural mode of action on RyR2 and guide its further development via medicinal chemistry structure−activity relationship studies, here, we used fluorescence lifetime (FLT)measurements of Forster resonance energy transfer (FRET) in HEK293 cells expressing human RyR2. For these studies, we used an RyR-specific FRET molecular-toolkit and computational methods for trilateration (i.e., using distances to locate a point of interest). Multiexponential analysis of FLT-FRET measurements between four donor-labeled FKBP12.6 variants and acceptorlabeled ent-verticilide yielded distance relationships placing the acceptor probe at two candidate loci within the RyR2 cryo-EM map. One locus is within the Ry12 domain (at the corner periphery of the RyR2 tetrameric complex). The other locus is sandwiched at the interface between helical domain 1 and the SPRY3 domain. These findings document RyR2-target engagement by ent-verticilide, reveal new insight into the mechanism of action of this new class of RyR2-targeting drug candidate, and can serve as input in future computational determinations of the ent-verticilide binding site on RyR2 that will inform structure−activity studies for lead optimization.

ent-Verticilide B1 Inhibits Type 2 Ryanodine Receptor Channels and is Antiarrhythmic inCasq2−/−Mice

“…Our in vitro assays suggest that, like ent-1, ent-B1 directly binds to RyR2 and produces incomplete inhibition, unlike recently identified RyR2 inhibitory compounds (chloroxylenol, methyl orsellinate, and riluzole) which produce complete RyR2 block (Takenaka et al, 2023). ent-B1 likely acts as a negative allosteric modulator of channel function, similar to ent-1, for which we recently identified a putative binding site (Seflova et al, 2023). ent-B1's combination of sub-micromolar potency and partial channel inhibition is ideal for our target, as a stronger inhibition of RyR2 could be fatal given its central role in physiologic excitation-contraction coupling.…”

Backbone-Determined Antiarrhythmic Structure–Activity Relationships for a Mirror Image, Oligomeric Depsipeptide Natural Product

“…In 2019, we reported that its non-natural enantiomer ( 1 ) exhibits activity at mammalian ryanodine receptor 2 (RyR2), while nat -verticilide was inactive, recording an evidently unprecedented case where all detectable activity against a specific target is harbored by the non-natural enantiomer of a natural product. 3 , 4 RyR2 is a target for antiarrhythmic development, and selective inhibitors promise to develop a more complete picture of its role in heart disease. 5 − 7 We have since discovered a second example of activity harbored entirely by the non-natural enantiomer in the antiarrhythmic ent -verticilide B1—the 18-membered oligomer of 1 .…”