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

Abstract

Quantitative high-throughput screening (qHTS) evaluates the pharmacology of drug and investigational agent libraries for potential therapeutic uses, toxicological risk assessment, and increasingly for academic chemical tool discovery. Phenotypic HTS assays aim to interrogate molecular pathways and networks, often relying on cell culture systems, historically with less emphasis on multicellular organisms. C. elegans has served as a powerful eukaryotic model organism for human biology and disease by virtue of genetic conservation and experimental tractability. Here we describe a paradigm to enable C. elegans in qHTS using 384-well microtiter plate laser scanning cytometry. GFP-expressing organisms are used to reveal phenotype-modifying structure-activity relationships to guide subsequent life stages and proteomic analysis. E. coli bacterial ghosts, a non-replicating nutrient source, allow compound exposures over 7-days spanning two life cycles to mitigate complications from bacterial overgrowth. We demonstrate the method with a library composed of anti-infective agents, or molecules of general toxicological concern. Each was tested in 7-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 having a physiological impact. We anticipate a broader application of this qHTS-coupled proteomics approach will enable the analysis of C. elegans orthologous transgenic phenotypes of human pathologies to facilitate drug and probe profiling from high-impact chemical libraries for a range of therapeutic indications and study of potential toxicological signatures.Graphic Abstract

Use of Viscous medium to study anthelmintic drug action in Caenorhabditis elegans

“…The use of the V medium could also facilitate analysis of other drug delivery systems since C. elegans have been used in high throughput drug screens to understand drug mechanics, neurodegenerative diseases and test antimicrobials and antifungals [43][44][45][46] . We propose that instead of using S medium for many of these assays that only span one generation to use instead V medium to build multigenerational experiments.…”