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bioRxiv · 10.1101/2023.06.22.546083

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

Seflova, J.·Schwarz, J. A.·Smith, A. N.·Svensson, B.·Blackwell, D. J.·Phillips, T. A.·Nikolaienko, R.·Bovo, E.·Rebbeck, R. T.·Zima, A. V.·Thomas, D. D.·Van Petegem, F.·Knollmann, B. C.·Johnston, J. N.·Robia, S. L.·Cornea, R. L.
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Abstract

Hyperactivity of cardiac sarcoplasmic reticulum (SR) ryanodine receptor (RyR2) Ca2+-release channels contributes to heart failure and arrhythmias. Reducing 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) Ca2+ 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). Multi-exponential analysis of FLT-FRET measurements between four donor-labeled FKBP12.6 variants and acceptor-labeled 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.

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Seflova, J., Schwarz, J. A., Smith, A. N., Svensson, B., Blackwell, D. J., Phillips, T. A., Nikolaienko, R., Bovo, E., Rebbeck, R. T., Zima, A. V., Thomas, D. D., Van Petegem, F., Knollmann, B. C., Johnston, J. N., Robia, S. L., Cornea, R. L.. 2023-06-22. RyR2 Binding of an Antiarrhythmic Cyclic Depsipeptide Mapped Using Confocal Fluorescence Lifetime Detection of FRET. https://doi.org/10.1101/2023.06.22.546083

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