Fig. 5: G-1749 modulation of IRE1 RNase is dependent on the kinase phosphorylation state.

a IRE1 RNase activity in the presence of AMG-18, G-9807, or G-1749. RNase activity was measured by the same assay as Fig. 1, but using an IRE1 KR construct (G547-L977) unphosphorylated (0P, left), or fully phosphorylated (3P, right) on S724, S726, S729. Source data are provided as a Source Data file. Data are presented as the mean for measurements from two independent experiments (n = 2). b (SV-AUC) experiments for IRE1 LKR autophosphorylated alone or in the presence of slight excess G-1749 or AMG-18. Source data are provided as a Source Data file. c Co-crystal structure of 2 (purple sticks) in complex with IRE1-3P (purple cartoon) aligned with co-crystal structure of G-1749 (gold sticks) in complex with IRE1-0P (gray cartoon). d Details of the IRE1 kinase activation loop from the crystal structure of apo IRE1-3P (green cartoon). Phosphorylated serines and interacting residues are highlighted in sticks. e RNase activity for IRE1 KR phosphoserine S/A mutants in the presence of increasing concentration of G-1749. 1P or 2P refers to the phosphorylation state of each mutant, as verified by intact mass spectrometry and phosphorylation mapping after protein digestion (see “Methods” and SI). Background from the no-protein control was subtracted from signal before calculating fold change. Source data are provided as a Source Data file. Data are presented as the mean for measurements from two independent experiments (n = 2). f RNase activity in the presence of 10 µM G-1749 for fully phosphorylated IRE1 KR mutants of residues that would interact with phosphoserines, as in (d). Background from the no-protein control was subtracted from signal before calculating fold change. Source data are provided as a Source Data file. Data are presented as the mean and S.D. for measurements from three independent experiments (n = 3).