Synergistic FRET assays for drug discovery targeting RyR2 channels

https://doi.org/10.1016/j.yjmcc.2022.04.002Get rights and content
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Highlights

  • RyR2- targeted FRET assays are compatible with high-throughput screening.

  • Calmodulin and DPc10 binding to RyR2 are indicative of its pathologically leaky state.

  • Ro 90–7501 reduces sarcoplasmic reticulum Ca2+ leak in adult cardiomyocytes.

  • Ro 90–7501 has minimal effects on excitation-contraction coupling in cardiomyocytes.

Abstract

A key therapeutic target for heart failure and arrhythmia is the deleterious leak through sarcoplasmic reticulum (SR) ryanodine receptor 2 (RyR2) calcium release channels. We have previously developed methods to detect the pathologically leaky state of RyR2 in adult cardiomyocytes by monitoring RyR2 binding to either calmodulin (CaM) or a biosensor peptide (DPc10). Here, we test whether these complementary binding measurements are effective as high-throughput screening (HTS) assays to discover small molecules that target leaky RyR2. Using FRET, we developed and validated HTS procedures under conditions that mimic a pathological state, to screen the library of 1280 pharmaceutically active compounds (LOPAC) for modulators of RyR2 in cardiac SR membrane preparations. Complementary FRET assays with acceptor-labeled CaM and DPc10 were used for Hit prioritization based on the opposing binding properties of CaM vs. DPc10. This approach narrowed the Hit list to one compound, Ro 90–7501, which altered FRET to suggest increased RyR2-CaM binding and decreased DPc10 binding. Follow-up studies revealed that Ro 90–7501 does not detrimentally affect myocyte Ca2+ transients. Moreover, Ro 90–7501 partially inhibits overall Ca2+ leak, as assessed by Ca2+ sparks in permeabilized rat cardiomyocytes. Together, these results demonstrate (1) the effectiveness of our HTS approach where two complementary assays synergize for Hit ranking and (2) a drug discovery process that combines high-throughput, high-precision in vitro structural assays with in situ myocyte assays of the pathologic RyR2 leak. These provide a drug discovery platform compatible with large-scale HTS campaigns, to identify agents that inhibit RyR2 for therapeutic development.

Keywords

Calcium channel
RyR
Sarco/endoplasmic reticulum
Fluorescence lifetime
Calcium leak

Abbreviations

7,8-DHF
7,8-Dihydroxyflavone hydrate
A-CaM
acceptor labeled calmodulin
A-DPc10
acceptor labeled DPc10
AET
2-(2-Aminoethyl)isothiourea dihydrobromide
APDC
Ammonium pyrrolidinedithiocarbamate
BSA
bovine serum albumin
CaMKII
Calcium/calmodulin-dependent protein kinase II
CaT
calcium transient
cDPCP
cis-diammine(pyridine)chloroplatinum(II) chloride
CPVT
catecholaminergic polymorphic ventricular tachycardia
Ctrl
control
D-FKBP
donor-labeled FK506 binding protein 12.6
DMPP
1,1-dimethyl-4-phenylpiperazinium iodide
E
FRET efficiency
E
east
EGTA
(ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetra-acetic acid)
FLT
fluorescence lifetime
HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
HTS
high-throughput screening
LOPAC
library of pharmacologically active compounds
N
North
NOBP
6-nitroso-1,2-benzopyrone
Pice
piceatannol
PIPES
piperazine-N,N′-bis(2-ethanesulfonic acid)
PKA
protein kinase A
R(−)-2-OH-Npa HBr
R(−)-2,10,11-trihydroxy-N-propylnoraporphine hydrobromide
R(−)-NPA
N-propylnorapomorphine
Ro90
Ro 90–7501
RyR
ryanodine receptor
S
south
SR
sarcoplasmic reticulum
SERCA
sarco/endoplasmic reticulum Ca-ATPase
W
west

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