Table 1.
Production of TSLP in NHBE cells from the 2 donors used in this study.
Table 2.
Summary of final conditions used for the phenotypic assay.
Fig 1.
Activity of reference compound BX795 in the TSLP production assay.
Activity in donors #1 (left panel) and #2 (right panel) is expressed as IC50 (M). Two representative curves are shown (n = 4 in each case).
Fig 2.
Quality control of primary phenotypic screen.
(A) Z’ factor versus plate number. (B) Global representation of total and basal activity values for all plates.
Fig 3.
Overview of the screening and confirmation process.
Left panel shows % inhibition of TSLP production (vertical axis) for all screened compounds distributed by plates (horizontal axis). Upper horizontal blue line in the left panel indicates cut off value for hit selection (45%). Right panel is a summary of the hit selection process (DR = dose response evaluation, I = inhibition).
Fig 4.
Upregulation of TSLP protein production and mRNA levels by compound 1.
Results were obtained using NHBE cells from donor #1 and are shown as average % upregulation ± SD (n = number of replicates).
Fig 5.
List of selected hit compounds.
For TSLP protein and mRNA determinations results were obtained using NHBE cells from donor #1. Results are shown as geometric mean IC50 (μM) (95% confidence interval) or average % inhibition ± SD at the indicated concentration [μM]. Specific activities evaluated are indicated in each case and shown in bold. Activities reported in a [44], b [45], c [46], d [47] and e [32].
Fig 6.
Correlation between inhibition of TSLP production and biochemical and cellular kinase activities of reference compound 7 (p38 inhibitor) and tofacitinib (JAK inhibitor).
For TSLP protein determinations, results were obtained using NHBE cells from donor #1. Results are shown as geometric mean IC50 (μM) (95% confidence interval) or average % inhibition ± SD at the indicated concentration [μM]. Specific activities evaluated are indicated in each case and shown in bold.
Fig 7.
BioMAP activity profile of compound 4.
Black thin arrow indicates cytotoxicity at 1 μM in the 3C system. The 3C system was excluded from further analysis at this concentration (indicated by horizontal bracket). Grey arrows indicate anti-proliferative activity. Abbreviations (full details in Materials & methods and S2 Table): 3C and 4H (HuVEC, venular endothelial cells), LPS and SAg (PBMC, peripheral blood mononuclear cells + HuVEC), BT (B cells + PBMC), BE3C (NHBE, normal human bronchial epithelial cells), BF4T (NHBE + NHDF, normal human dermal fibroblasts), HDF3CGF (NHDF), KF3CT (NHK, normal human keratinocytes + NHDF), CASM3C (CASMC, coronary artery smooth muscle cells), MyoF (HLF, human lung fibroblasts) and /Mphg (HuVEC, M1 macrophages).
Table 3.
Inhibition of TSLP production by mTOR inhibitors everolimus and AZD8055.
Table 4.
Main pharmacological activities identified in compound 4.
Fig 8.
Active compounds identified in the hit expansion exercise.
Results obtained using NHBE cells from donor #1 in the TSLP assay. Results shown as geometric mean IC50, μM (95% confidence interval) or average % inhibition ± SD at the indicated concentration. Biochemical and cell based specific activities evaluated are indicated in each case and shown in bold.
Fig 9.
(A) Phenotypic and target based drug discovery strategies are complementary. (B) Phenotypic based optimization relies on biological profiling to identify both potential liabilities (represented in red) and targets responsible for activity (represented in green). SAR: structure—activity relationship.