In vivo quantitative high-throughput screening for drug discovery and comparative toxicology

ABSTRACT Quantitative high-throughput screening (qHTS) pharmacologically evaluates chemical libraries for therapeutic uses, toxicological risk and, increasingly, for academic probe discovery. Phenotypic high-throughput screening assays interrogate molecular pathways, often relying on cell culture systems, historically less focused on multicellular organisms. Caenorhabditis elegans has served as a eukaryotic model organism for human biology by virtue of genetic conservation and experimental tractability. Here, a paradigm enabling C. elegans qHTS using 384-well microtiter plate laser-scanning cytometry is described, in which GFP-expressing organisms revealing phenotype-modifying structure–activity relationships guide subsequent life-stage and proteomic analyses, and Escherichia coli bacterial ghosts, a non-replicating nutrient source, allow compound exposures over two life cycles, mitigating bacterial overgrowth complications. We demonstrate the method with libraries of anti-infective agents, or substances of toxicological concern. Each was tested in seven-point titration to assess the feasibility of nematode-based in vivo qHTS, and examples of follow-up strategies were provided to study organism-based chemotype selectivity and subsequent network perturbations with a physiological impact. We anticipate that this qHTS approach will enable analysis of C. elegans orthologous phenotypes of human pathologies to facilitate drug library profiling for a range of therapeutic indications.

immediately after plating or (B) day 7. Plate images are auto contrasted based on total area of gated GFP objects where black is low total area and bright green is high total area. Worms were plated as L1 in columns 1-4, L2 in columns 5-8, L3 in columns 9-12, L4 in columns 13-16, and adults in columns 17-20 with the COPAS biosorter. Compound treatment is as described in Supplementary Methods. Representative well images from Acumen laser scanning cytometer for each day of time course for C. elegans grown on ghost capsules (C and D) or OP50 live bacteria (E and F) and treated with 0.5% DMSO (C and E) or 50 uM levamisole (D and F).

Fig. S6. Re-test 11-pt. dose response curves (DRCs) for compound classes displaying effects on C. elegans viability in the primary qHTS.
A. Antiparasitic agents with reported anti-nematodal properties (3/3); proteasome inhibitors (4/10); BRD inhibitors (4/6); the single antifungal agent; antimalarial agents (1/4); MEK 1/2 inhibitors (2/3); monoacylglycerol lipase (MAGL) inhibitors (2/2); and nuclear export inhibitors (2/2) in the anti-infectives library demonstrating reproducible effects on C. elegans viability. B. Compounds with anticancer/antioxidant properties (5/14); C-C chemokine receptor (CCR) antagonists (2/5); and DNA topoisomerase II inhibitors (3/5) in the anti-infectives library demonstrating reproducible effects on C. elegans viability. C. DRCs for compound classes with primary qHTS toxic effects on C. elegans that only partially reconfirmed activity in follow-up analysis. Eight antibacterial/antimicrobial compounds selected for follow-up from the 14 contained in the anti-infectives library, 5 reconfirmed with high quality inhibition curve classes; 13 antiviral compounds selected for follow-up from the 25 contained in the anti-infectives library, 8 demonstrated high quality inhibition curve classes with various potencies and efficacies; the γ-secretase inhibitor selected for follow-up from the six inhibitors contained in the collection reconfirmed similar activity (note that the low basal signal obtained at low concentrations of the compound was due to a plating artifact where the center of the plate demonstrated lower signal than the outer wells), and the β-secretase inhibitor selected for follow-up from the five inhibitors contained in the library did not reconfirm activity (the moderate increase in signal is also most likely due to the plating artifact above); two deubiquitinase inhibitors from the three inhibitors contained in the anti-infectives library, one reconfirmed good activity while the other demonstrated minimal to no activity in follow-up analysis; and two apoptosis activators of the five activators contained in the library, one reconfirmed moderate low potency activity, while the second was inactive in follow-up analysis. D. DRCs of compounds with individual annotated MOAs selected for follow-up based on primary qHTS toxic effects on C. elegans.
The two output parameters measured on the Acumen laser cytometer based on worm GFP intensity, GFP area (solid black circles) and worm number (open black circles), measured on day 7 of the screening time course are plotted as black lines where data was normalized to 41.7 µM levamisole screening control as -100% activity. Mammalian cellular toxicity response (red squares) are plotted for each compound where data is normalized to 83 μM digitonin cytotoxicity control as -100% activity. Fraction indicates number of active vs. total number of compounds in the anti-infective library annotated with a similar MOA. Curves were fit in GraphPad Prism.  Milbemycin lacks the α-l-oleandrosyl-α-l-oleandrosyl moiety found in the avermectins.
Dose-response curves for activity on C. elegans (black symbols) and HEK293 cells (red symbols) for days and times, respectively, indicated in the legend.

Figure S8
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