(A) The core scaffold of APY29 (aminopyrazole pyrimidine-base indicated in beige). (B) Structure-activity analysis of activating compounds. Compounds were assayed at 1 μM in a RNA cleavage assay containing IRE1α-KR43 (200 nM) and 5′ [32P]-labeled RNA substrate HP21 (see ‘Materials and methods’). IPAx is methylated at the N position in the pyrazole ring indicated as shown in R1. (C) The effects of activating compounds (20 μM) as a function of IRE1α-KR43 concentration. 1/2kmax, obs and Hill coefficients (‘n’) were calculated (IPA: 1/2kmax, obs = 0.43 μM, n = 4.2; cmp1: 1/2kmax, obs = 3 μM, n = 3.8; IPAx 1/2kmax, obs = 8 μM, n = 3.3, DMSO control: 1/2kmax, obs = 8 μM, n = 3.3). (D) Compounds and FDA-approved drugs (all at 1 µM) were screened against a panel of 266 recombinant human kinases (‘S/T’: Ser/Thr protein kinases; ‘Y’: Tyr protein kinases, ‘PI’: phosphatidylinositide lipid kinases). APY24 contains the aminopyrozole pyrimidine-base, which is similar to APY29 (Figure 1—figure supplement 2).