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. 2022 Sep 12;65(18):12014–12030. doi: 10.1021/acs.jmedchem.2c00671

Figure 2.

Figure 2

Nilotinib is a positive hit in a cellular MALDI-TOF MS drug discovery screen and inhibits inflammation in response to LPS. (A) Inhibition of the TLR2 and TLR4 signaling pathways. MRT68601 inhibits TBK1 (TANK-binding kinase-1), which blocks production of type I interferons (IFNs). NG-25 inhibits TAK1 (TGF-beta-activated kinase-1), which induces TNF-α and IL-6 production. BI2536 inhibits BET proteins (bromodomain and extra-terminal motif proteins), which are required for pro-inflammatory gene transcription. (B) TNF-α secretion of THP-1 cells treated in either vehicle control-treated (DMSO), 100 ng/mL LPS-treated, or pre-treated with 5 μM NG-25, BI2536 or 1 μM MRT68601 for 1 h before 100 ng/mL LPS treatment for up to 24 h measured by ELISA. (C) MALDI-TOF MS relative quantitation of the normalized ratio m/z 4964/4632 in THP-1 cells as treated in panel (B). (D) PCA of all features identified by the MALDI-TOF MS of experiment in panel (B) and 0.5 μM staurosporine-treated cells serving as control for apoptotic cells. (E) Compound hit map of the mean of three biological replicates with a 40% effectiveness cutoff showing nilotinib and NG-25 as positive hits and imatinib as the negative hit (arrows). (F) MALDI-TOF MS relative quantitation of the normalized ratio m/z 4964/4632 in THP-1 cells in vehicle control-treated (DMSO), 100 ng/mL LPS-treated, or pre-treated with 5 μM nilotinib, imatinib, 1 μM dasatinib, bosutinib, or ponatinib for 1 h before 100 ng/mL LPS-treatment for up to 24 h. (G) TNF-α secretion of cells treated as in panel (F) measured by ELISA after 24 h. Significant differences between two groups were determined by a Mann–Whitney U-test. The statistical significance of the comparisons with LPS is indicated as follows: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Error bars represent the standard deviation of four biological replicates.