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Optimization of a High-Throughput Cell-Based Screening Strategy to Identify Small-Molecule Inhibitors of IL-23 Signaling

Varghese, Teena M. ; Dudas, Paul L. ; Allen, Samantha J. ; Schneeweis, Jonathan E. ; Finley, Michael F.A.

SLAS discovery, 2021-01, Vol.26 (1), p.122-129 [Periódico revisado por pares]

Los Angeles, CA: Elsevier Inc

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  • Título:
    Optimization of a High-Throughput Cell-Based Screening Strategy to Identify Small-Molecule Inhibitors of IL-23 Signaling
  • Autor: Varghese, Teena M. ; Dudas, Paul L. ; Allen, Samantha J. ; Schneeweis, Jonathan E. ; Finley, Michael F.A.
  • Assuntos: 1536-well ; Drug Discovery - methods ; DT40 ; Gene Expression ; Gene Expression Regulation - drug effects ; Genes, Reporter ; High-Throughput Screening Assays - methods ; Humans ; IL-23 ; Interleukin-23 Subunit p19 - metabolism ; luciferase ; Signal Transduction - drug effects ; STAT5
  • É parte de: SLAS discovery, 2021-01, Vol.26 (1), p.122-129
  • Notas: ObjectType-Article-1
    SourceType-Scholarly Journals-1
    ObjectType-Feature-2
    content type line 23
  • Descrição: Interleukin-23 (IL-23) is a key cytokine implicated in the pathogenesis of autoimmune disorders, including psoriasis and ulcerative colitis. Although targeted IL-23 antibody therapeutics are used clinically, there are no small-molecule therapeutics that selectively inhibit IL-23 signaling. To address this gap, we developed a high-throughput screening strategy employing an IL-23-responsive cell-based luciferase reporter gene assay as the primary screen, with cellular cytotoxicity and off-target counter screening assays to identify IL-23 pathway-specific inhibitors. The primary screening assay utilized avian DT40 cells, genetically engineered to overexpress IL-23R, IL-12Rβ1, STAT5, and firefly luciferase, in a 1536-well format. Treatment of these cells with IL-23 resulted in the phosphorylation and activation of STAT5, which was completely inhibited by the pan-JAK inhibitor tofacitinib. Assay performance was robust, with signal-to-background >7-fold and Z′ > 0.5 over 40 screening plates (approximately 24,000 compounds), with a hit rate of 5% (>66.9% activity cutoff). Of these 1288 hits, 66% were identified as cytotoxic by incubating the IL-23 reporter cells with compound overnight and measuring cell viability. Further assessment of specificity via examination of impact on off-target IFN-γ signaling eliminated an additional 230 compounds, leaving 209 that were evaluated for dose–response activity. Of these compounds, 24 exhibited IC50 values of <7 µM and ≥80% inhibition of IL-23 activity, with >3-fold selectivity over IFN-γ inhibition, thus representing promising starting points for prospective IL-23 pathway small-molecule inhibitors.
  • Editor: Los Angeles, CA: Elsevier Inc
  • Idioma: Inglês
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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