Fig. 1: ETI41 and ETI60 potently and selectively inhibit endosomal TLRs.

a Chemical structures of the main scaffold, TAC (red), and the potent inhibitors, ETI41 and ETI60. b Cell survival curve according to ETI concentration (1.6–200 μM) was measured by MTT assay in murine RAW 264.7 cell line and water-soluble tetrazolium assay in a human Daudi cell line. c Inhibitory effects of ETI15, ETI41 and ETI60 (ranging from 3.9 nM to 10 μM) on TLR7 and TLR9 were assessed by quantifying TNF-α secretion in murine RAW 264.7 cells and human Daudi cells, respectively. d ETI41 and ETI60 inhibited TLR3, TLR7 and TLR8 in a concentration-dependent manner (31.2 nM to 10 μM), as indicated by the reduction in TNF-α secretion in mouse RAW 264.7 cells. e Specificities of ETI41 and ETI60 were confirmed by measuring TNF-α secretion in surface TLRs (TLR1, TLR2, TLR4, TLR5 and TLR6. The cells were activated with agonistic ligands: TLR1/2 (FSL-1, 100 ng/ml, 4 h), TLR2/6 (Pam3CSK4, 100 nM, 4 h), TLR3 (poly I:C, 2 μg/ml, 24 h), TLR4 (LPS, 10 or 100 ng/ml, 4 h), TLR5 (FLA-ST, 500 ng/ml, 4 h), TLR7 (ORN06/LyoVec, 2 μg/ml, 24 h; and IMQ, 1 μg/ml, 4 h), TLR8 (TL8-506, 2 μg/ml, 24 h) and TLR9 (ODN2395, 1 μM, 4 h) at various concentrations in RAW 264.7 cells, human Daudi cells and THP-1 cells. Data are from at least three independent experiments (n = 3) and statistical differences between the induced case and other cases were analyzed and verified using a one-tailed Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001).