Figure 1.
Development and optimization of an ARE induction assay in AREc32 Cells.
(A) The luciferase assay signal stability in the presence of 0, 10, 20, or 80 µM tBHQ.(B) The effect of AREc32 cells seeding density on the luciferase assay sensitivity in the presence of 0, 10, 20, or 80 µM. (C) The effect of DMSO concentration on the luciferase assay sensitivity using a range of 0%–10% DMSO in the presence or absence of 0 or 10 µM tBHQ.
Figure 2.
Validation of the ARE induction assay using known Nrf2 activators and through pilot screening.
(A) Concentration-response curves of tBHQ and CDDO-Im to increase the luminescent signal in AREc32 cells. (B) Activity spread of compounds in Microsource, Prestwick, and CMLD libraries together with positive controls. Compounds in Microsource and CMLD libraries were tested at 10 µM, and compounds in Prestwick library were tested at 8 µM. (C) Z′ scores for 16 pilot screening plates.
Figure 3.
Full library screening for Nrf2 activators through ARE induction assay in AREc32 cells.
(A) The scatterplot distribution of the screening actives from the ARE library screen was calculated using data from the Chembridge, Prestwick, Microsource, and CMLD library compounds. The fold increase in luminescence, indicative of ARE activation by compounds in AREc32 cells, is plotted against each individual well of the libraries. (B) Frequency distribution of the Chembridge, Prestwick, Microsource, and CMLD library compounds to increase the luminescent signal in AREc32 cells.
Figure 4.
Concentration-response curves of the most effective and potent compounds.
(A) Concentration-response curves and (B) chemical structures of top four compounds with greatest maximum fold-increase in luminescent signal. (C) Concentration-response curves and (D) chemical structures of top four compounds with lowest EC50. AREc32 cells were treated with compound (0.01–30 µM) or DMSO vehicle. 24 hours after treatment, luciferase activity was assessed using the Steady-Glo luciferase assay with luminescence readout. The luminescence of each well was divided by the median luminescence of the DMSO vehicle control wells to generate the fold ARE activation.
Figure 5.
Chemical scaffolds clustered in the top 247 validated hits from the primary screening.
Preliminary hit clustering was based on the EC50 value from the concentration-response curves of the top 240 compounds, and accomplished via the Selector program from Tripos via the Jarvis Patrick routine, using default parameters.
Figure 6.
Concentration-response curves of known Nrf2 activators and most active compounds from the secondary screening.
Concentration-response curves of (A) tBHQ and CDDO-Im, and (B) top 4 compounds from the secondary screening. Hepa1c1c7 cells were treated with compound (0.01–3 µM) or DMSO vehicle. 24 hours after treatment, mRNA of Nqo1 was quantified using reverse transcription q-PCR analysis. Fold-increase in Nqo1 mRNA was normalized by cells treated with DMSO vehicle.
Figure 7.
Hypothetical modes of action of actives compounds.
Four potential mechanisms of action for hit compounds to activate Keap1-Nrf2-ARE pathway. Hit compounds can disrupt binding of Keap1 to Nrf2, facilitate Keap1 ubiquitination, inactivate Nrf2 export signaling, or activate protein kinase cascades for Nrf2 phosphorylation.