Supplementary Materials and Methods, Supplementary Tables 1 through 4, and Supplementary Figures 1 through 9 from Altiratinib Inhibits Tumor Growth, Invasion, Angiogenesis, and Microenvironment-Mediated Drug Resistance via Balanced Inhibition of MET, TIE2, and VEGFR2

Supplementary Materials and Methods. Supplementary table 1: XRay crystallography parameters for DP-4157/MET complex. Supplementary table 2: Source and description of kinases. Supplementary table 3: Selectivity of profile of altiratinib inhibition from Reaction Biology 295 kinase panel (fold selectivity versus IC50 value of 2.7 nM MET inhibition). Supplementary table 4: A. Inhibition of MET phosphorylation in the MKN-45 xenograft model after a single oral dose of altiratinib (30 mg/kg); B. Inhibition of MET phosphorylation in the MKN-45 xenograft model after a single oral dose of altiratinib (10 mg/kg). Supplementary figure 1: Co-crystal structures of altiratinib analog DP-4157. Supplementary figure 2: Comparison of altiratinib with other clinical stage MET inhibitors regarding potency versus activation loop mutant forms D1228H, D1228N, Y1230D, Y1230C, and Y1230H. Supplementary figure 3: Determination of off-rate, residency time, and tight-binding inhibitor constant Kd for altiratinib binding to the MET kinase domain. Supplementary figure 4: Michaelis-Menton analysis of altiratinib inhibition of MET with respect to ATP. Supplementary figure 5: Kinome tree interaction profile for altiratinib vs 295 human kinases. Altiratinib was evaluated using Kinase HotspotSM platform from Reaction Biology (Malvern, PA). Supplementary figure 6: Inhibitory potencies for inhibiting HUVEC cell signaling activated by HGF/MET (black), VEGF-A/VEGFR2 (melon), and ANG2/TIE2 (tan). Comparative potencies are shown for altiratinib, E-7050, cabozantinib, and MGCD-265. Supplementary figure 7: Upper panel: inhibition of capillary tube formation by various concentrations of altiratinib. Tube formation was initiated by addition of 200 ng/mL of angiopoietin 2 (ANG2). Lower table: titrated IC50 values for inhibition of capillary tube formation induced by ANG2, HGF, and VEGF-A. Supplementary figure 8: Inhibition of proliferation in the mutant B-RAF melanoma SK-MEL-28 cell line in the absence or presence of HGF or MRC-5 fibroblast conditioned medium. Supplementary figure 9: Ratio of brain: plasma concentrations of altiratinib through 24 hr after administration of a single dose of 5 mg/kg IV to C57/Black mice.