Extended Data Fig. 3: In vitro characterization of potent and selective KIF18A inhibitors.
a, KIF18A compounds (1 µM) were assessed across a panel of kinases (n = 96) by a competitive binding assay. Graph presented as percentage of control (POC); dashed line indicates POC value of 30 (n = 1 experiment). b, Tubulin polymerization analysis. Profile of KIF18A compounds (10 µM) relative to DMSO, paclitaxel (5 µM, tubulin stabilizer), or nocodazole (5 µM, tubulin destabilizer). Graphs presented as tubulin fluorescence signal at 440 nm expressed as arbitrary units (a.u.) versus time (min) with area-under-the-curve (AUC) values (n = 1 or 3 independent experiments). c, Mitotic spindle image analysis. MDA-MB-157 cells were treated for 24 h with DMSO, AM-1882 (0.2 µM), or AM-5308 (0.5 µM) and stained to detect DNA, α-tubulin, and pericentrin. Representative images were captured with x40 objective (scale bar = 10 µm) (n = 1 experiment). d-f, KIF18A compound effects on HeLa cells. d, Cells were treated for 96 h with DMSO or KIF18A compounds at the indicated concentrations. KIF18A compound concentration-response profiles presented as count relative to the percentage of DMSO control (POC) with count EC50 values (n = 2 independent experiments in duplicate). e, KIF18A protein localization in cells after treatment for 6 hours with DMSO or AM-1882 (0.05 µM). Cells stained to detect DNA, KIF18A, and centrin-3. Representative images of mitotic cells (n = 3 per group) were captured with x60 objective (scale bar = 5 µm) and graphs of line scan measurements were obtained for DNA, KIF18A, and centrin-3 channels expressed as fluorescence intensity (a.u.) versus distance (µm) (n = 1 experiment). f, Mitotic cell fate imaging analysis. HeLa Kyoto cells co-expressing α-tubulin-EGFP and H2B-mCherry proteins released from a G1S block in media containing DMSO or AM-1882 (0.2 µM). Analysis was performed on cells entering the first mitosis (n = 40 cells per group). Graph presented as time in mitosis (h) versus mitotic cell fate, either complete cell division (CCD) or death in mitosis (DiM). Classified cells exiting mitosis by the number of daughter cells and cell death in early interphase (n = 1 experiment). Images were captured every 15 min for 48 h with x20 objective (see Supplementary Videos 1, 2).
