Fig. 2. Target discovery for oxfendazole identifies IRAK1.
(a) Similarity Ensemble Approach (SEA) predicted targets of oxfendazole and analogs. E-value color-coded white to red. Black bars, predicted targets for oxfendazole. (b) DTC assay of p53MK/MK embryos (120 hpf) treated with inhibitors of SEA-predicted oxfendazole targets (see a) with or without 15 Gy γIR (red and blue bars, respectively). Dotted line, penetrance cutoff at DMSO+IR levels. Data represented as means +/− SD of n = 4 independent experiments (DMSO, IRAK1/4i), n = 3 independent experiments (oxfendazole, buparvaquone, rebastinib) or n = 2 independent experiments (remaining drugs) with >12 embryos per experiment. Relative to DMSO-treated irradiated embryos (bar 2), **P < 0.005, ***P < 0.0005, ns, not significant, two-tailed Student’s t-test. (c) Chemical structures of indicated drugs. (d) Dose-response to IRAK1/4i of p53MK/MK embryos scored in DTC assay. Data represented as means +/− SD of n = 3 independent experiments, *P < 0.05, **P < 0.005, ***P < 0.0005, n.s., not significant, relative to DMSO-treated irradiated embryos (bar 2), two-tailed Student’s t-test. (e) Sequence alignment of human (Hs) and zebrafish (Dr) IRAK1 kinase domains, ATP binding domain boxed in blue. Id, identity, sim., similarity. (f,g) Induced-fit docking of oxfendazole to ATP-binding site of zebrafish (f) and human (g) IRAK1 models. Gate-keeper residue: Y255 (Y288 in human). 3 residue changes near proposed docking pose within binding site highlighted in cyan in zebrafish model: M239, Y257, and M258 (V272, F290 and L291 in human). (h-i) KINOMEScan in vitro kinase capture assay for indicated kinases (h) and in vitro kinase assay vs. IRAK1 (i), curved-fit from 2 and 3 replicates, respectively. (j) IRAK1 inhibitor R406 (40 μg/ml) phenocopies oxfendazole (see 1d) in AO assay at 48 hpf, with images representative of 2 independent experiments. See Supplementary Table 4 for statistics source data including precise P values. Scale bar, 0.2 mm.