Synergistic FRET assays for drug discovery targeting RyR2 channels

Fig. 1.. Modulators of RyR2 that promote normal (closed) resting channel or pathologically leaky channel, and FRET-based biosensor systems for monitoring modulator binding to RyR2. A) Schematic of the interplay between RyR modulator binding and the healthy vs. pathologically leaky states of the RyR2 in cardiomyocytes. Post-translational modifications that promote RyR2 leak state associated with HF and arrhythmias may reduce binding of RyR stabilizers, FKBP and CaM, and enhance binding of domain peptide, DPc10 [14,15]. Conversely, dantrolene reduces the RyR2 leaky state, increases CaM-RyR2 binding and reduces DPc10-RyR2 binding in cardiomyocytes [14–16]. B) Schematic of FKBP, CaM and DPc10 bound to RyR2 for our FRET assays that use fluorescence lifetime (FLT) measurements of donor (Alexa Fluor 488) attached to FKBP (D-FKBP) in the presence of acceptor (Alexa Fluor 568 or HiLyte Fluor 647) attached to CaM and DPc10, respectively, to gauge shifts in RyR-modulator binding. These assays, previously using fluorescence intensity measurements of FRET in SR membranes and adult cardiomyocytes [14,15,17–20], have been adapted for HTS by exploiting rapid, high-precision FLT measurements of FRET in a…

Fig. 2.. Representative orthogonal display of changes in FRET between D-FKBP and A-CaM FRET or A-DPc10 in a screen of the 1280-compound LOPAC collection. Representative sets of fluorescence waveforms (primary readout) are shown in Supplementary Fig. 1. FRET efficiency (E) corresponding to each compound was normalized to the DMSO control (Econtrol). Hits were selected outside a 4SD threshold (pink lines) from the mean. For reference, 3 SD (blue lines) and 5 SD (green lines) are shown. The Hits localize to eight sectors (each indicated within a purple box) depending on the direction of their effects on FRET to A-CaM or A-DPc10. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.. Reproducibility of FLT-FRET HTS Hits. Normalized plot of the effects of 10 μM Hit on FRET to A-CaM (A) or A-DPc10 (B) exceeding 4SD in 2 of 3 screens. SR was exposed to no H2O2 (black for individual values and green for mean values±SD) and H2O2 treatment (purple for individual values and pink for mean values±SD). The first 18 Hits altered both A-CaM and A-DPc10 FRET, Hits 19–37 only altered A-CaM FRET, and Hits 38–42 altered A-DPc10 FRET. These groups were assigned based on the Fig. 2 representation, sectors being indicated in parentheses for each compound. A-CaM FRETcontrol values of SR exposed to no H2O2 and plus H2O2 treatment were 0.11 ± 0.01 and 0.09 ± 0.01, respectively. A-DPc10 FRETcontrol values of SR exposed to no H2O2 and plus H2O2 treatment were 0.1 ± 0.02 and 0.11 ± 0.04, respectively. Chemical structures are shown in Supplementary Figs. 2 and 3. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4.. Dose-dependent effects of Ro 90–7501. (A) Normalized FLT-FRET response to the indicated Ro 90–7501 concentrations using cardiac SR and A-CaM (purple) or A-DPc10 (green), at 30 nM Ca2+ following pre-treatment with no H2O2 (light colors), and 100 μM H2O2 (dark colors). (B) Response of RyR2 [3H]ryanodine binding to the indicated concentrations of Ro 90–7501, in 100 nM (blue) or 30 μM (red) free Ca2+. Data are normalized relative to the values for no-drug DMSO control (grey line), means ± SEM, n = 3. *P < 0.05 vs. DMSO by 2-sided Student’s unpaired t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5.. Ro 90–7501 does not affect Ca2+ handling in normal rabbit myocytes. (A) Time series of a representative experiment with Ro 90–7501 (blue underbar), decimated from >1 × 106 data points to a more practical 1 × 104 points. Red dots indicate 30-s rests, showing that the resting fluorescence was stable, thus justifying free [Ca2+]i pseudo-ratio measurements with resting [Ca2+]i at baseline assumed to be 100 nM. (B) Detail of the same experiment before, during and after drug contact, each showing the final 10 s of steady state Ca2+ transients, followed by 10 mM caffeine (duration 8 s, green underbars) to assay SR Ca2+ loading. (C) Peak transient [Ca2+]i, (D) transient [Ca2+]i decay time constant and (E) residual (diastolic) [Ca2+]i at the end of each transient, predicted by exponential fitting. (C-E) #cells/#animal (n/N) = 24/10, 24/10, and 26/10 for ctrl, Ro90 and wash, respectively. (F) SR Ca2+ loading, represented as total [Ca2+] released to cytosol by caffeine (calculated as in text). (G) Ca2+ transient decay time constant measured with caffeine present, representing non-SR Ca2+ removal, mainly via Na/Ca exchange. (F and G) n/N = 18/10, 24/10, and 15/10, respectively. [Ca2+] in…

Fig. 6.. Ro 90–7501 effect on Ca2+ spark rate in permeabilized rat myocytes. (A) Representative paired line-scan recordings (6 s) of Ca2+ sparks response to acute exposure (5 min) to 10 μM Ro 90–7501 or 500 μM tetracaine treatment, as indicated, under either control (Ctrl) conditions or following 50 μM H2O2 pretreatment (1 h). Colour display scales span the fluorescence range of Ca2+ sparks. (B) Time course of average (±SEM, n/N (# of cells/# of animals) = 7/4 Ctrl, 9/4 Ro 90–7501) Ca2+ spark frequencies (CaSpF) with and without 10 μM Ro 90–7501 treatment over 5 min (repeated measures two-way ANOVA with Geisser-Greenhouse correction; Dunnett multiple comparison post-hoc test). (C) Explicit paired comparisons of CaSpF before or after 5 min exposures to either Ro 90 or tetracaine, with or without H2O2 pretreatment (Wilcoxon matched-pairs rank tests, n/N = 13/7, 8/3 and 7/3, respectively). (D) CaSpF for individual groups, including unpaired measures, analyzed via one-way ANOVA vs. Ctrl and Dunnett’s multiple comparison post-hoc test (n/N = 28/11, 15/7, 8/3, 9/3, 7/3, respectively). (E) Percent change in average Ca2+ spark frequency and amplitude relative to respective control averages, and…

Fig. 7.. RyR2 Drug Discovery Process. Green boxes denote early-stage steps (assays) that are covered in this report. In red, we summarize some of the additional steps that will be necessary to advance compounds in a fully developed drug discovery campaign. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)