A B In-house kinase profiling Invitrogen SelectScreen Profiling % % % Kinase IC50 ( M) Kinase IC50 ( M) inhibition inhibition inhibition AKT3 >5 IKK-epsilon >5 Kinase at 500 nM Kinase at 500 nM Kinase at 500 nM ALK 0.26 INSR 0.38 ABL1 7 GRK6 -7 PAK1 4 AUR-A >5 JAK1 >5 GRK2 4 HCK 7 PASK 6 AUR-B >5 JNK1 0.11 AKT1 2 HIPK2 9 PHKG1 8 AUR-C >5 MEK1 >5 AXL 6 HIPK4 -6 PRKCB1 -2 B-RAF 3.2 MST4 >5 BMX 14 IGF1R 37 PRKCE 0 CDK1 >5 NEK2 >5 BRSK1 -9 IRAK4 0 PRKCG 3 CDK2 >5 PDK1 >5 CAMK2D -1 ITK 9 PRKCI -2 CDK6 >5 PKA >5 CDC42 BPA 0 LCK 29 PRKCN 6 CHK1 >5 PKC-delta >5 CDK5/p25 -1 LIMK1 17 PRKCZ -5 CHK2 >5 PLK1 >5 CSF1R 82 MAP2K1 11 PRKG2 2 C-MET >5 PLK4 3.3 CSK 1 MAP2K6 2 PTK6 15 C-SRC >5 ROCK2 >5 CSNK1D 10 MAP3K3 17 RET 11 DYRK2 >5 RSK2 >5 CSNK1G1 -1 MAP3K5 -1 RPS6KA2 23 ERK2 3.9 STK33 1.1 CSNK2A2 33 MAP3K8 12 RPS6KA5 2 FAK1 2.7 TAK1 >5 DCAMKL2 2 MAPK1 0 SGK2 4 FER 0.59 TAO1 >5 DYRK1B 1 MAPK11 2 SNF1LK2 3 FLT3 0.080 TBK1 >5 EEF2K 2 MAPK12 5 SRPK2 -3 HGK >5 TRKB >5 EGFR 14 MAPK3 -2 STK22B 11 IKK-alpha >5 YES1 >5 EPHA1 6 MAPK9 61 STK23 -1 IKK-beta >5 EPHA4 -10 MAPKAPK2 9 STK24 -2 EPHB1 -33 MARK4 3 STK25 0 ERBB2 5 MELK 6 TEK 6 FGFR1 15 MERTK 7 TYK2 10 FGR 17 MKNK1 2 TYRO3 -2 FLT4 15 MST1R 3 ZAP70 5 FRAP1 7 NEK1 1 GRK4 3 NEK4 -6 Supplementary Table S1. Kinase selectivity of MPI-0479605. (A). The effect of MPI- 0479605 on the activity of 42 protein kinases was determined. IC50 less than 1 M is highlighted in grey. (B) The inhibitory effect of 500 nM MPI-0479605 against a panel of kinases was determined using Z’-Lyte kinase assays (Invitrogen SelectScreen™ kinase profiling). Inhibition greater than 50% is highlighted in grey. MPI-0479605 Doubling GI50 (M) time (hrs) 3-day 7-day Cell Line A549 > 10 0.053 22.9 Colo205 0.105 0.044 23.8 DU-4475 > 10 0.042 45.0 DU-145 > 10 0.115 32.3 HCC827 > 10 0.054 28.0 HCT116 0.055 0.051 17.4 HT29 > 10 0.104 19.5 MDA MB 231 > 10 0.037 41.9 MiaPaCa2 > 10 0.055 40.0 NCI-H69 > 10 0.090 40.0 NCI-H460 0.178 0.091 17.8 NCI-N87 > 10 0.694 47.0 OPM2 > 10 0.0921 33.0 OVCAR-3 > 10 0.266 34.7 Supplementary Table S2. Effect of MPI-0479605 on cell viability. Cells were treated for 3 or 7 days with various concentrations of MPI-0479605 and the GI50 was determined. The reported time required for cells to double is also included. A B Mps1 WT Mps1 T676A MOCK Mps1 KD PPase untreated Mps1-pT676 Mps1-pT676 GFP-Mps1 Mps1 Endogenous Mps1 Mps1 GAPDH Supplementary Figure S1. Characterization of Mps1-pT676 antibody. (A) 50 ng recombinant Mps1 purified from E. Coli was pre-treated with or without lambda phosphatase. It was subsequently analyzed by Western blot with antibodies for Mps1 or phosphorylated Mps1 (Mps1-pT676). The Mps1-pT676 antibody only recognizes the phosphorylated form of recombinant Mps1. (B) HEK-293T cells were transfected with empty vector or plasmid encoding GFP-tagged Mps1 (wild-type (-WT), kinase-inactive (-KD), or phospho-acceptor mutant (-T676A)). After 48 hours, cell lysates were analyzed by Western blot with antibodies for Mps1, Mps1-pT676 or GAPDH. Loss of Mps1 kinase activity or the T676 phospho-acceptor site abolishes recognition of Mps1 by the Mps1-pT676 antibody. 40 30 EC50 = 71.3 nM 20 positive cells positive 10 % phospho-histone H3 0 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 [MPI-0479605], (M) Supplementary Figure S2. MPI-0479605 abrogates nocodazole-induced mitotic arrest. Nocodazole-arrested HeLa cells were treated with DMSO or increasing concentrations of MPI- 0479605 for 4 hours and subsequently stained for phosphohistone H3 (pHH3). Data are plotted as the percentage of cells that stained positive for pHH3 relative to the DMSO control sample. O O S HN N CH 3 N N N N N N H H O H C 3 Ispinesib 5-fluorouracil MPI-0485812 Supplementary Figure S3. Compound structures. HCT116 (p53WT) Colo-205 (p53Mut) 3 8 Vehicle Vehicle Z-VAD-FMK Z-VAD-FMK 6 2 4 1 (Fold Increase) (Fold Increase) 2 Caspase 3/7 Caspase 3/7 0 0 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 MPI-0479605, M MPI-0479605, M HCT116 (p53WT) Colo-205 (p53Mut) 150 150 Vehicle Vehicle GI50 = 84 nM GI50 = 100 nM Z-VAD-FMK Z-VAD-FMK 100 100 GI50 = 67 nM GI50 = 95 nM Viability 50 Viability 50 (% Vehicle Control) (% Vehicle Control) 0 0 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 MPI-0479605, M MPI-0479605, M Supplementary Figure S4. Inhibition of caspase activity does not affect MPI-0479605- induced cell death. HCT-116 and Colo-205 cells were treated with MPI-0479605 in the absence or presence of 100 M Z-VAD-fmk. Caspase-3/7 activity (A, B) was determined after 48 hours while cell viability (C, D) was measured after 72 hours. 1000000 100000 30 mg/kg 150 mg/kg 10000 1000 (ng/mL) 100 concentration median plasma median 10 1 0 2 4 6 8 10 12 14 16 18 20 22 24 time (hr) Dose Cmax AUC (0-last) (mg/kg) (ng/L) (hr*ng/L) 30 18.3 30.1 150 76.5 786.4 Supplementary Figure S5. Pharmacokinetic analysis of MPI-0479605. Mice were dosed intraperitoneally with 30 or 150 mg/kg of MPI-0479605 and plasma concentrations of MPI- 0479605 were determined at the indicated times by mass spectrometry analysis. The dotted line represents the dose that is predicted to inhibit Mps1 in vivo based on cellular activity of MPI- 0479605. Colo-205 800 10 Vehicle MPI-0479605 (30mg/kg, QD) MPI-0479605 (150mg/kg,Q4D) 600 Ispinesib (10 mg/kg,Q4D) 0 400 % Change in % -10 Body Weight Body Tumor Volume 200 0 -20 0 5 10 15 20 25 0 5 10 15 20 25 Day Day Supplementary Figure S6. Effect of MPI-0479605 on xenograft tumor growth. Nude mice bearing Colo-205 tumors were treated (n=10 per group) intraperitoneally with MPI-0479605 at 30 mg/kg daily (QD) or at 150 mg/kg every fourth day (Q4D) for a period of 21 days. Ispinesib dosed Q4D at 10 mg/kg was used as a reference treatment. Median tumor volume (left) and body weight (right) were measured. There were two deaths at day 15 in the 150 mg/kg group. H2AX p53-S15 p53 GAPDH 9605 NOC 9605 NOC DMSO DMSO - Z-VAD-fmk Supplementary Figure S7. Effect of caspase inhibitor on MPI-0479605-induced phosphorylation of H2AX and p53. HCT-116 cells were pretreated with 100 M Z-VAD-fmk for 1 hour followed by treatment with 1 M MPI-0479605 for 48 hours. Cell lysates were analyzed by Western blot with antibodies for H2AX, phospho-p53(S15), p53 and GAPDH. Supplementary Materials and Methods In Vitro Kinase Assays and Selectivity Screening To measure Mps1 activity, 25 ng of recombinant, full-length enzyme was incubated in reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.01% Triton X-100 and 5 M Myelin basic protein (MBP)) containing vehicle (DMSO alone) or inhibitors. Forty M ATP (2xKm) was added with 1 Ci [-33P]ATP and the reaction was incubated at room temperature for 45 minutes. Reactions were terminated with 3% phosphoric acid and transferred to P81 filter plates. Samples were washed in 1% phosphoric acid and 33P radioactivity was measured on a TopCount scintillation reader (PerkinElmer). In-house kinase assays were all carried out at 2xKm ATP concentrations. MPI-0479605 (500 nM) was also screened against a larger kinase panel (SelectScreenTM Kinase Profiling; Invitrogen). Immunofluorescence Microscopy To measure phosphohistone H3 (pHH3), nocodazole-arrested cells were treated for 4 hours with MPI-0479605. Cells were fixed with 4% paraformaldehyde for 15 minutes at 37°C, permeabilized in 0.2% Triton-X 100 for 5 minutes, incubated with primary antibodies (rabbit polyclonal anti-phosphohistone H3 (diluted 1:200; Upstate) and mouse monoclonal anti- - tubulin (diluted 1:2000; Sigma)) for 45 min at 37oC, followed by a 45 min incubation with Alexa Fluor-conjugated secondary antibodies (Invitrogen). Cells were subsequently stained with Hoechst 33342 dye. The percentage of pHH3-positive cells was quantified by image analysis (BD Pathway High Content Imaging system). .