Fig. 5: WEE1 PROTAC elicits less ISR toxicity compared to AZD1775.
From: WEE1 inhibitors synergise with mRNA translation defects via activation of the kinase GCN2
a Chemical structure of AZD1775 and PROTACs utilising AZD1775 as a warhead. The chemical structures were adapted on ChemDraw 25.0.2 from a previous publication53. b Resazurin cell viability summary synergy scores (combined Loewe, Bliss and HSA synergy scores) of CC-90009 in combination with AZD1775 or ZNL-02-096 in the RPE TP53−/− cell line in a 96 well plate format (independant biological replicates n = 3). Heatmaps of the drug combinations can be found in Supplementary Fig. 16a. c Resazurin cell viability assay with varying concentrations of AZD1775 or ZNL-02-096 treated for 72 h in a 96 well plate format in the RPE-1 TP53−/− dCas9-KRAB cell line expressing sgRNAs that target GCN2 or the AAVS1 locus (independant biological replicates n = 3). Graphs are depicted with means ± SD. d Western blot of an in vitro experiment probing the total and phosphorylated flag-tagged GCN2 in the presence of DMSO, AZD1775 and ZNL-02-096. p-GCN2 and total GCN2 were run in parallel on separate blots. Data are representative of n = 3 independant biological replicates. An additional independant biological experiment can be found in Supplementary Fig. 16b. e Western blot comparing AZD1775 and ZNL-02-096 18 h treatments in the RPE TP53−/− cell line. Data are representative of n = 3 independant biological replicates. f Quantifications of western blots of RPE TP53−/− treated with either DMSO, 650 nM AZD1775 or 650 nM ZNL-02-096 for 18 h (independant biological replicates n = 3). ATF4, γH2AX, and WEE1 were normalised to vinculin loading control. AZD1775 and ZNL-02-096 treatments were normalised to the vehicle to calculate the fold change of each condition. Graphs are depicted with means ± SD. Statistical analysis was performed using unpaired two-tailed t-tests comparing AZD1775 to ZNL-02-096: ATF4, p = 0.01246; γH2AX, p = 0.01331; WEE1, p = 0.00006. g Schematic showing the balance between the two independent toxicities of DNA damage and ISR activation for WEE1 inhibitor treatments. Source data are provided as a Source data file.
