RyR1-targeted drug discovery pipeline integrating FRET-based high-throughput screening and human myofiber dynamic Ca2+ assays

Figures

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  Figure 1. Reproducibility of the FLT-FRET HTS readout. RyR1-specific FRET (E) was measured in the presence of LOPAC compounds (10 μM test-compounds) in 1536-well format. Normalization was relative to DMSO-only controls (E0). Hits were those compounds that altered FRET by > 4 SD in at least two of three repeats of the screen. Each data point is shown as a solid black square, and means ± SE are shown in green, n = 3.

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  Figure 2. FRET profiles for RyR1 and RyR2 over a range of Hit concentrations. The same FLT-detected FRET readout used in the primary HTS was measured at several Hit concentrations, using RyR1 and RyR2 samples. FRET dose-response of (a) suramin, (b) temsirolimus, (c) chloroxine, and (d) myricetin, measured at 30 nM Ca2+ using skeletal (green tones) and cardiac (red tones) SR membranes, in the presence of 5 mM GSSG (dark tones) or GSH (light tones). FRET efficiency in the presence of compound (E) was normalized to FRET efficiency in the presence of DMSO-only (E0). Data are presented as means ± SEM, n = 4. *P < 0.05 vs. DMSO by 2-sided Student’s unpaired T-test.

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  Figure 3. [3 H]ryanodine binding profiles for RyR1 and RyR2 in the presence of a range of myricetin concentrations. Dose-dependent (0–20 μM) effect of LOPAC HTS Hit, myricetin, on [3 H]ryanodine binding to skeletal SR (a) and cardiac SR (b) in the absence of CaM (closed symbol) or in the presence of 300 nM CaM (open symbol), at 30 nM (blue) or 30 μM (red) free Ca2+. Data are shown normalized relative to the values for no-drug DMSO control (gray line), means ± SEM, n = 4–6. *P < 0.05 for no-CaM samples vs. DMSO control using 2-sided Student’s unpaired t-test; #P < 0.05 for 0.3 μM CaM samples vs. DMSO control using 2-sided Student’s unpaired t-test.

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  Figure 4. Effects of HTS Hits on t-tubule Ca2+ measurements of RyR1 leak in human skinned skeletal muscle fibers. (a) Diagram showing how rhod-5N trapped in the sealed t-system can be used to detect RyR Ca2+ leak. At rest, Ca2+ leaks through the RyR into the junctional space, where it is taken up by the NCX and PMCA, thus resulting in a net increase of [Ca2+]t-sys (left). Blockage of the RyR inhibits Ca2+ leak into the JS (right). The difference in [Ca2+]t-sys under these conditions represents total Ca2+ leak31. (b) Representative traces of t-tubule Ca2+ in response to RyR modulators. Solution exchanges are indicated by the blue, vertical bars. The blue horizontal line indicates the presence of 200 nM [Ca2+]cyto (standard solution) in the fiber bathing solution, which loads the SR and t-system with Ca2+ in the presence of functional RyRs. The functional RyR leaks Ca2+ into the junctional space to increase local [Ca2+] at the t-system and increase t-system Ca2+-dependent fluorescence. The addition of RyR modulators to the standard solution are indicated by the horizontal lines. Each RyR modulator causes a reduction in rhod-5N fluorescence, indicating an effect on RyR Ca2+-leak. The red…

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  Figure 5. Effect of micromolar myricetin and chloroxine on electrically evoked Ca2+ transients. In rat skinned fibers, Ca2+ transients were obtained by confocal line scans parallel to the fiber long axis, with corresponding line averaged and normalized rhod-2 fluorescence signals (F/F0). Representative recordings of Ca2+ transients are shown in Supplementary Fig. 7. All solutions contained 1 mM EGTA and 100 nM free Ca2+. Cytosolic Ca2+ transients were elicited by electrical field stimulation at 1 Hz in the presence of DMSO (control), myricetin or chloroxine. Dotted line represents 0.1% DMSO control (which causes no detectable reduction in EC-coupling). Data presented as means ± SEM, *P < 0.05, n = 6.

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  Figure 6. RyR1 Drug Discovery Pipeline. Green boxes denote early-stage steps (assays) in the RyR1 drug discovery pipeline that are covered in this report. In red, we summarize additional steps that will be necessary in a full-scale drug discovery campaign.