Primary in vivo screen for MAPK/ERK pathway inhibitors among 433 annotated oncology related compounds

<p>The aim was to test if a C. elegans model could identify known MEK inhibitors in a blinded screen of 433 annotated oncology related compounds. Primary hits were further validated for pathway specificity. Please see the paper cross-reference for more details.</p> <p>We bought a chemical library (SciLife lab, Stockholm, Sweden) with 433 oncology-related compounds (see table S1 for the full list), where compounds had been acoustically dispensed from 10 mM stocks into 96-well plates. DMSO concentrations were adjusted to 0.5 % in all wells, including negative controls. Trametinib was used as a positive control at 7 muM. All chemicals were kept at -20 degrees C until use. The C. elegans strain ST65 was used. We dispensed 50 muL of worm culture into each well and incubated animals for 4-5 days at 20 degrees C until DMSO control animals reached early adulthood. Animals were then sedated for 30 minutes by adding 4 muL 20 mM levamisole per well before scoring.<br><br>Primary hit selection: 1) Wells were excluded if they contained progeny or fluorescent specles from drugs or bacteria, indicated by too many objects classified as vulvae. The cutoff was set to vulvae &gt;2x the number of adults in the positive control. 2) Wells were excluded they contained &lt; 30 % adults compared to the positive control, indicative of severely reduced larval growth. 3) Remaining compounds with vulvae/adult scores &lt; 0.4 were considered primary hits. Scoring of the Muv phenotype in adult lin-1(e1777) mutants was done manually from ImageXpress images. See the paper cross-reference for more details.</p>