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Pharmacoproteomic profiling directed by C. elegans-driven phenotypic quantitative HTS

ABSTRACT: Quantitative high-throughput screening (qHTS) pharmacologically evaluates libraries of drugs and investigational agents 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 for rapid signal acquisition with concurrent quantification of the fluorescent protein-expressing organism to select phenotype-modifying compounds for subsequent life-stage and proteomic analysis. E. coli bacterial ghosts, a non-replicating nutrient, allow compound exposures over 7-days spanning two life cycles to mitigate complications from bacterial overgrowth. We demonstrate the method using 643 anti-infective biased agents tested in 7-pt titration to assess feasibility of nematode-based in vivo qHTS. A pharmacological profile from the primary screen confirmed the efficacy of known anti-parasitic molecules, such as ivermectin and levamisole as well as illuminating anthelmintic properties of general chemical classes, including MEK, -secretase, bromodomain and proteasome inhibitors. Subsequent life-stage and proteomics analysis helped elucidate the physiologic consequence of specific compounds on C. elegans viability. For example, bortezomib induced a parallel upregulation of core proteasome subunits and the transcription factor skn-1-activating DDI aspartyl protease, while the kinase inhibitor trametinib upregulated the C. elegans MEK-1 ortholog, Mnk-1, downstream aminoacyl tRNA synthetases and ribosomal survival pathway proteins. 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Caenorhabditis Elegans

TISSUE(S): Cell Culture

SUBMITTER: Dingyin Tao

LAB HEAD: Dingyin Tao

PROVIDER: PXD033552 | Pride | 2023-02-21

REPOSITORIES: Pride

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Project description:Metformin is a type 2 diabetes medication which extends life span across species when given from young adulthood onwards; late life effects of metformin are not well understood. Here we used C. elegans to investigate the outcome of metformin treatment initiated late in life. We found that, contrary to young age administration, old age metformin treatment interferes with energy homeostasis and shortens life span by aggravating age-associated mitochondrial dysfunction. Nematode mutants defective in mitochondrial respiration, mitochondrial biogenesis and mitochondrial quality control were highly susceptible to metformin killing already at young age while insulin receptor deficient nematodes carrying healthier mitochondria were protected from late life metformin toxicity. In the mammalian cell culture model of replicative senescence metformin killing correlated with loss of mitochondrial membrane potential and strong reduction of systemic ATP levels. Reduced ATP levels and depletion of lipid stores consistent with energy deficit were observed also in nematodes following late life metformin treatment. At the molecular level young animals responded to metformin by inducing adaptive stress responses and longevity-assurance pathways while old nematodes demonstrated a protein expression signature consistent with lipid turnover and energy deficit. Ectopic ATP supplementation was sufficient to alleviate metformin toxicity in human skin fibroblasts highlighting the key contribution of energy deficit to the detrimental effect of metformin. Also, co-exposure with rapamycin, known to stabilize cellular ATP levels under conditions of mitochondrial failure, significantly reduced metformin-inflicted lethality in both cells and old animals. In summary we uncovered a novel negative synergism between metformin treatment and mitochondrial dysfunction which gains importance late in life and may limit therapeutic benefits of metformin for older patients; these side effects can partially be overcome by co-treatment with agents stabilizing cellular ATP levels such as rapamycin.

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	Ce_7groups_Jan2022msf.mzML	Mzml
	Ce_7groups_Jan2022msf.mzid.gz	Mzid
	Ce_DMSO1ugB01.raw	Raw
	Ce_DMSO1ugB02.raw	Raw
	Ce_DMSO1ugB03.raw	Raw

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Pharmacoproteomic profiling directed by C. elegans-driven phenotypic quantitative HTS

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