File S1 - The Potent Cdc7-Dbf4 (DDK) Kinase Inhibitor XL413 Has Limited Activity in Many Cancer Cell Lines and Discovery of Potential New DDK Inhibitor Scaffolds

Supporting files. Figure S1, Two DDK inhibitors, PHA-767491 and XL413, exhibit differential activity against cultured HCT116 colon cancer cells. To determine the IC50, HCT116 (p53+/+) cells were treated with increasing concentrations of PHA-767491 or XL413 (A) and the cell viability was measured 72 hrs post drug addition. HCT116 (p53-/-) cells were treated with increasing concentrations of PHA-767491 or XL413 (B) and the cell viability was measured 72 hrs post drug addition. All data represent the mean of at least three separate measurements +/− SD. Figure S2, DDK thermal stability shift assays (TSA) screen using 400 known kinase inhibitors. Figure S3, Top hits identified by TSA screen inhibit DDK in vitro. (γ)-32P ATP DDK kinase assays in presence of increasing concentrations of SB 218078 (A), PKR Inhibitor (B), UCN-01 (C), JAK3 Inhibitor VI (D), Rho Kinase Inhibitor III (E), and PI3-Kα Inhibitor VIII (F). Kinase activities represent the mean of two independent measurements +/− SD on separate days. Figure S4, The Chk1 inhibitor (SB 218078) inhibits DDK activity and induces low levels of apoptosis in breast cancer cells. IC50 values for SB 218078 (structure shown in Figure 7) were determined against purified DDK (A) and HCC1954 cells (B). Caspase 3/7 assays show that low levels of apoptosis were induced at 24 hours following SB 218078 addition and this was eliminated using the pan-caspase inhibitor z-VAD (C). SB 218078 induces cell growth arrest in HCC1954 cells over time (D) and also modestly inhibits Mcm2 phosphorylation in cells, a known DDK target (E). The measurements in panels A-D represent the averages of at least two measurements +/− SD and were highly reproducible. Figure S5, XL413 acquired from the commercial supplier (MedKoo Biosciences, North Carolina) behaved similar to the chemically synthesized compound. To determine the IC50 values, HCC1954 and Colo-205 cells were treated with increasing concentrations of XL413 (MedKoo) and the cell viability was measured 72 hrs post drug addition. All data represent the mean of at least three separate measurements +/− SD and were highly reproducible on separate days. Table S1, Characteristics of top kinase inhibitors discovered in TSA screen.
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