Fig. 4: TMA inhibits IRAK4 and rescues LPS-induced TLR4-mediated pro-inflammatory response.
a, Kinome screen for a single dose of TMA (0.1 mM) against 456 kinases. The kinases covered by the kinome scan assay are visualized using a phylogenetic layout. Preliminary positive hits with potential binding >35% vs control (DMSO) are represented by a red dot; non-binding kinases are represented by a green dot. Kinase group names: TK, tyrosine kinases; TKL, tyrosine kinase-like; STE, STE kinase group; CK1, cell linase 1; AGC, protein kinase A, G and C families; CAMK, calcium and calmodulin-regulated kinases; CMGC, CMGC kinase group. b, Functional characterization of the inhibition of IRAK4 by TMA. IRAK4 phosphorylation activity was determined in the presence of various concentrations of TMA and resulted in an IC50 of 3.4 µM. c,d, TMA preincubation (100 µM for 30 min) suppresses LPS-induced (1 µg ml−1, 4 h) IL-6 (c) and TNF (d) release by human PBMCs. The values of the unstimulated controls were arbitrarily set to 1. For c and d, statistical significance (P < 0.05) was assessed with a two-sided unpaired Student’s t-test (vs LPS-stimulated control). e, TMA (100 μM) or the IRAK4-specific inhibitor PF06650833 (50 nM) do not inhibit TNF secretion from human PBMCs after 4 h stimulation with PMA (50 ng ml−1) and ionomycin (1 μg ml−1). f, Impact of TMA pre-treatment (100 µM, 30 min) on human PBMCs’ Thr209IRAK1/IRAK1 phosphorylation upon LPS (1 µg ml−1) stimulation for the indicated times. g, Human PBMCs were pre-treated (for 30 min) with the indicated concentrations of TMA, and Thr209IRAK1/IRAK1 phosporylation was assessed after 10 min of LPS (1 μg ml−1) challenge. The time-dependent (h) and dose-dependent (i) effect of TMA on phosphorylation of Ser536NF-κBp65/NF-κBp65 of human PBMCs stimulated with LPS (1 μg ml−1) was assessed as in f and g. For f–i, the control value was arbitrarily set to 1, and statistical significance (P < 0.05) was assessed with a one-sided unpaired Student’s t-test. For PBMC experiments in c–i, each data point represents a separate experiment. j, A 24 h Kaplan–Meier survival curve of mice challenged with a lethal dose of LPS (30 mg kg−1) that received a single dose of TMA (59 mg kg−1, red line), or vehicle (black line); *P = 0.0047 determined by two-sided log-rank Mantel–Cox test. Data are means; error bars, s.e.m.
