In vivoquantitative 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 HTS assays interrogate molecular pathways, often relying on cell culture systems, historically less focused on multicellular organisms. C. 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. GFP-expressing organisms revealing phenotype-modifying structure-activity relationships guide subsequent life-stage and proteomic analyses. E. 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 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 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.

Nonspecific membrane bilayer perturbations by ivermectin underlie SARS-CoV-2 in vitro activity

“…In contrast to the antiviral assay, IVM and analogs displayed SAR spanning approximately three orders of magnitude in a quantitative HTS C. elegans assay for nematode viability ( Figures 1C, 1D ). 20 Like IVM, the antiviral activity of analogs 2 - 15 occurred at the same concentrations where A549-ACE2 cellular viability was reduced in the CTG counter-screen, again with a relatively flat SAR ( Figure 1E ). Of note, there is extensive SAR spanning multiple orders of magnitude for the antiparasitic activity of IVM ( Supplementary Note 1 ).…”

“…The activity of the primary ivermectin sample ( 1a ) was assessed using a previously described C. elegans quantitative high-throughput screening assay for nematode viability. 20 Briefly, test compounds were arrayed in 11-pt, 1:3 or 22-pt, 1:2 intra-plate titrations. Compounds were tested at a high concentration of 0.1-25 μM for final assay titrations spanning 0.20 pM – 333 nM to 39.7 pM – 83.3 μM.…”

Semi‐automated, high‐content imaging of drug transporter knockout sea urchin (Lytechinus pictus) embryos

“…A major use case for genetically modified embryo models is in high‐throughput drug and toxicant screening (Dranchak et al, 2023; MacRae & Peterson, 2023). To this end, we developed an approach in which we describe transporter activity and differential xenobiotic sensitivity using canonical substrates and inhibitors of ABCB1 using our drug transporter knockout sea urchin line.…”

“…This has mirrored a broader effort in the field to generate stable genetic models in this group of organisms (Oulhen et al, 2023;Yaguchi & Yaguchi, 2022;Yaguchi et al, 2020), yet the question has remained open how these advances may best be applied. A major use case for genetically modified embryo models is in high-throughput drug and toxicant screening (Dranchak et al, 2023;MacRae & Peterson, 2023). To this end, we developed an approach in which we describe transporter activity and differential xenobiotic sensitivity using canonical substrates and inhibitors of ABCB1 using our drug transporter knockout sea urchin line.…”