Identification of drug modifiers for RYR1-related myopathy using a multi-species discovery pipeline
- Program for Genetics and Genome Biology, Hospital for Sick Children, Canada
- Department of Molecular Genetics, University of Toronto, Canada
- Department of Pharmacology and Toxicology, University of Toronto, Canada
- Department of Pharmacology, University of Rochester, United States
- UNC Eshelman School of Pharmacy, SGC Center for Chemical Biology, University of North Carolina, United States
Figures
Figure 1
Schematic of our multi-species translational pipeline aimed at identifying potential therapeutic targets for RYR1-RM.
The pipeline involved screening C. elegans and zebrafish with thousands of compounds for suppressors of RYR1 mutant phenotypes, followed by further characterization in zebrafish and evaluation in …
Figure 2 with 5 supplements
Chemical screen finds that p38 MAPK inhibition suppresses the nemadipine-A growth arrest of unc-68 mutants.
(A) Schematic of our screen methodology showing the expected growth arrest phenotype of unc-68 worms exposed to 25 μM nemadipine after 6 days of exposure and the expected phenotype of a chemical …
Figure 2—figure supplement 1
Phenotypes of unc-68 mutants considered for chemical screening.
(A) The ‘thrashing assay’ is performed by counting the waveforms propagated by individual C. elegans in one minute (Maryon et al., 1996). Consistent with previous reports (Maryon et al., 1996; Maryon…
Figure 2—figure supplement 2
Post-hoc statistical analysis showing that 142 chemicals out of the 278 chemicals identified from the primary screen produced developmental stage distributions that were significantly different from random escapee wells (red arrows).
It is important to note that this was performed with the assumption that 20 worms were in each well as a means of estimating the proportion of actual L4 and adult counts in each well. In other …
Figure 2—figure supplement 3
Post-hoc statistical analysis showing that 142 chemicals out of the 278 chemicals identified from the primary screen produced developmental stage distributions that were significantly different from random escapee wells (red arrows).
It is important to note that this was performed with the assumption that 20 worms were in each well as a means of estimating the proportion of actual L4 and adult counts in each well. In other …
Figure 2—figure supplement 4
Follow-up testing for compounds identified in the primary screen.
(A) Chemical dose-response re-testing of five compounds shows consistent ability to suppress nemadipine-A-induced growth arrest of unc-68 mutants. Note: wact-526 precipitated at 30 and 60 μM and …
Figure 2—figure supplement 5
Heat map visualization of Tanimoto scores from the ‘hit’ compounds (y-axis) screened from the 1280 compounds in the US Drug Collection library (x-axis).
Tanimoto scores were calculated for each pair of compounds as a measure of structural similarity and similar clusters were identified via hierarchical clustering of Tanimoto scores (legend indicates …
Figure 3 with 1 supplement
Schematic of our zebrafish screen methodology showing the expected motility of unaffected siblings (‘WT Siblings’) and double mutants (‘Dbl Mut’) and expected motility of immotile double mutants if a chemical suppressed the phenotype.
We did not identify any suppressors of the double mutant phenotype.
Figure 3—figure supplement 1
Phenotypes of zebrafish ryr1 mutants considered for chemical screening.
(A) ryr1a;ryr1b double mutants exhibit complete lack of movement as shown by optovin-6b8 induced swimming (n = 24 per genotype; Student’s T-test: ***p<0.001). They do not respond to touch stimuli …
Figure 4 with 3 supplements
Positive chemical-genetic interactions between p38 inhibitors and ryr1b mutants were observed using zebrafish larval movement speed as a readout.
Compared to the average speed of DMSO WT controls, treatment with (A) SB239063 and (B) PH-797804 reduced the average speed of WT siblings while the expected decrease in movement speed in treated ryr1…
Figure 4—figure supplement 1
Chemical-genetic interactions were tested using zebrafish larval movement speed as a readout.
Compared to the average speed of DMSO WT controls, treatment with (A) wact-405, (B) wact-488, (C) wact-526, (D) PI3K inhibitor PI-103, and (E) anthralin reduced the average speed of WT siblings …
Figure 4—figure supplement 2
Chemical-genetic interactions were tested using zebrafish larval movement speed as a readout.
Compared to the average speed of DMSO WT controls, chemical-genetic interactions were not observed for 18 hits from the C. elegans screen when tested in this assay (Figure 4—figure supplement 2–3F–W)…
Figure 4—figure supplement 3
Chemical-genetic interactions were tested using zebrafish larval movement speed as a readout.
Compared to the average speed of DMSO WT controls, chemical-genetic interactions were not observed for 18 hits from the C. elegans screen when tested in this assay (Figure 4—figure supplement 2–3F–W)…
Figure 5 with 4 supplements
Intracellular calcium measurement in C2C12 myotubes.
Ratiometric fura-2 imaging with 10 mM caffeine induction after treatment with p38 inhibitors (A) SB203580 (# of myotubes from left-right: n = 56, 30, 28, 43, 45, 57, 46, 79) and (B) SB202190 (n = 78,…
Figure 5—figure supplement 1
Characterization of Ryr1 knockout (KO) C2C12 cells.
(A) DNA sequence of Ryr1 KO showing frameshift mutation and premature stop codon. (B) Western blotting analysis of RyR1 in C2C12 myotubes at after 5 days of differentiation.
Figure 5—figure supplement 2
Quantitative PCR analysis was performed to validate the efficiency of siRNA knockdown of three p38 MAPK genes in both WT and Ryr1 KO myotubes.
As shown, the siRNAs reduce target gene expression by >50% in WT cells (A) while the degree of knockdown achieved in Ryr1 KO myotubes (B) was more variable. Data are presented as mean ± SEM fold …
Figure 5—figure supplement 3
Intracellular calcium measurement in C2C12 myotubes.
Ratiometric fura-2 imaging with 10 mM caffeine induction after treatment with A) SB203580, (B) SB202190, and C) p38 MAPK siRNAs. Black lines indicate individual myofibers while red lines present the …
Figure 5—figure supplement 4
Intracellular calcium measurement in C2C12 myotubes.
Ratiometric fura-2 imaging with 10 mM caffeine induction after treatment with D) SB203580, (E) SB202190, and F) p38 MAPK siRNAs. Panels on the left show the peak change in calcium concentration from …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (C. elegans) | unc-68(r1162) | C. elegans Genetics Center (University of Minnesota) | Strain TR2171 RRID:WB-STRAIN:WBStrain00034950 | |
| Genetic reagent (C. elegans) | unc-68(r1161) | C. elegans Genetics Center (University of Minnesota) | Strain TR2170 RRID:WB-STRAIN:WBStrain00034949 | |
| Strain, strain background (E. coli) | HB101 | C. elegans Genetics Center (University of Minnesota) | ||
| Strain, strain background (E. coli) | HT115 (DE3) | C. elegans RNAi Collection (Ahringer) from Source BioScience | Clones IV-9P08 (pmk-1), IV-4G23 (pmk-2), IV-4I01 (pmk-3), and I-1K04 (pop-1) | RNAi library |
| Genetic reagent (D. rerio) | ryr1a(z42) | Chagovetz et al., 2019 | ZFIN ID: ZDB-ALT-180925–10 | |
| Genetic reagent (D. rerio) | ryr1b(mi340) | Hirata et al. (2007) | ZFIN ID: ZDB-ALT-070928–1 | |
| Chemical compound, drug | The US Drug Collection | MicroSource Discovery Systems, Inc | Chemical library | |
| Chemical compound, drug | DiscoveryProbe Kinase Inhibitor Library | APExBIO | Catalog #L1024 | Chemical library |
| Chemical compound, drug | Nemadipine-A | ChemBridge | Catalog #5619779 | |
| Chemical compound, drug | Optovin analog 6b8 | ChemBridge | Catalog #5707191 | |
| Chemical compound, drug | SB203580 | Sigma | Catalog #S8307 | |
| Chemical compound, drug | SB202190 | Sigma | Catalog #S7067 | |
| Cell line (M. musculus) | C2C12 myoblasts | ATCC | Catalog #CRL1772 | |
| Cell line (M. musculus) | Ryr1 KO C2C12 myoblasts | This paper | 2 bp deletion in exon 6 of Ryr1: c.497_498del, p.Val166GlyfsX3 | |
| Antibody | anti-Ryanodine receptor (Mouse monoclonal) | Developmental Studies Hybridoma Bank (University of Iowa) | Catalog #34C, RRID:AB_528457 | WB (1:100) |
| Antibody | anti- beta actin (Mouse monoclonal) | Abcam | Catalog #ab8226 RRID:AB_306371 | WB (1:5000) |
| Sequence-based reagent | ON-TARGETplus Mouse Mapk11 siRNA | Horizon Discovery (Dharmacon) | Catalog # J-050928-05-0010 | Target sequence: 5’-AUGAGGAGAUGACCGGAUA-3’ |
| Sequence-based reagent | ON-TARGETplus Mouse Mapk12 siRNA | Horizon Discovery (Dharmacon) | Catalog # J-062913-05-0010 | Target sequence: 5’-AAUGGAAGCGUGUGACUUA-3’ |
| Sequence-based reagent | ON-TARGETplus Mouse Mapk14 siRNA | Horizon Discovery (Dharmacon) | Catalog # J-040125-06-0010 | Target sequence: 5’-GCAAGAAACUACAUUCAGU-3’ |
| Sequence-based reagent | ON-TARGETplus Non-targeting siRNA #1 | Horizon Discovery (Dharmacon) | Catalog # D-001810-01-05 | Target sequence: 5’-UGGUUUACAUGUCGACUAA-3’ |
| Sequence-based reagent | Tbp forward primer | This paper | Mouse Tbp gene: ENSMUSG00000014767 | 5’-TGCTGCAGTCATCATGAG-3’ |
| Sequence-based reagent | Tbp reverse primer | This paper | Mouse Tbp gene: ENSMUSG00000014767 | 5’-CTTGCTGCTAGTCTGGATTG-3’ |
| Sequence-based reagent | Mapk11 forward primer | This paper | Mouse Mapk11 gene: ENSMUSG00000053137 | 5’-CCAGCAATGTAGCGGTGAACGAG-3’ |
| Sequence-based reagent | Mapk11 reverse primer | This paper | Mouse Mapk11 gene: ENSMUSG00000053137 | 5’-GCATGATCTCTGGCGCCCGGTAC-3’ |
| Sequence-based reagent | Mapk12 forward primer | This paper | Mouse Mapk12 gene: ENSMUSG00000022610 | 5’-CACTGAGGATGAACCCAAGGCC-3’ |
| Sequence-based reagent | Mapk12 reverse primer | This paper | Mouse Mapk12 gene: ENSMUSG00000022610 | 5’-CTCCTAGCTGCCTAGGAGGCTTG-3’ |
| Sequence-based reagent | Mapk14 forward primer | This paper | Mouse Mapk14 gene: ENSMUSG00000053436 | 5’-CAGCAGATAATGCGTCTGACGGG-3’ |
| Sequence-based reagent | Mapk14 reverse primer | This paper | Mouse Mapk14 gene: ENSMUSG00000053436 | 5’-GCGAAGTTCATCTTCGGCATCTGG-3’ |
| Commercial assay or kit | RNeasy Mini Kit | Qiagen | Catalog #74104 |
Additional files
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Source data 1
Data files for the C. elegans chemical screen, zebrafish motility assays, and myotube calcium measurements.
- https://cdn.elifesciences.org/articles/52946/elife-52946-data1-v2.zip
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Supplementary file 1
List of 74 chemicals that suppressed the synthetic, nemadipine-A-induced unc-68 growth arrest.
- https://cdn.elifesciences.org/articles/52946/elife-52946-supp1-v2.xlsx
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Supplementary file 2
List of individual chemicals used in this study.
This list does not include all of the chemicals present in the chemical libraries screened.
- https://cdn.elifesciences.org/articles/52946/elife-52946-supp2-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/52946/elife-52946-transrepform-v2.pdf
