Assay Development and Screening
James Inglese, PhD
National Center for Advancing Translational Sciences (NCATS), NIH, Maryland, United States
Phenotypic high-throughput screening (HTS) assays aim to examine pathways and networks, primarily through cell culture systems, to reveal novel compound pharmacology and implicate associated druggable targets. Caenorhabditis elegans have served as a powerful eukaryotic model organism for human biology and disease by virtue of genetic conservation and experimental tractability, as well as a surrogate host for infectious microbes and free-living substitute for parasitic nematodes. Here C. elegans was employed to develop a quantitative HTS (qHTS)-based forward chemical physiology platform utilizing non-replicating E. coli as a nutrient source and 384-well microtiter plate laser scanning cytometry for rapid quantification of the fluorescent protein-encoded phenotype, used here to score worm viability. In a proof-of-concept study, the pharmacological qHTS profile obtained from a library of 643 anti-infective compounds confirmed the efficacy and potency of known anti-parasitic molecules, such as ivermectin and levamisole. In addition, anthelmintic properties of general chemical classes, including g-secretase, bromodomain and proteasome inhibitors were observed. Each of these inhibitor classes consisted of species-selective chemotypes, for example bortezomib potently inhibited worm viability, while MG132 had no effect. Moreover, these classes differentially affected the organism’s life-stage progression. Finally, quantitative mass-spectrometry proteomic analysis of bortezomib-treated C. elegans showed significant increase in the 20S and 19S subunit proteins of the 26S proteasome, demonstrating how pharmacological activity may be correlated with mechanism of action from this platform. We anticipate broader applications for this approach will include the use of C. elegans orthologous transgenic phenotypes based on the genetics underlying human pathologies to facilitate phenotypic drug discovery for a range of therapeutic indications.