In vitro activity: Altiratinib (also known as DCC-270, DP-5164) is an orally active, highly potent and selective multi-kinase inhibitor with IC50 values of 2.7, 8, 9.2, 9.3, 0.85, 4.6, 0.83 nM for MET, TIE2, VEGFR2, FLT3, Trk1, Trk2, and Trk3. It was designed based on the rationale of engineering a single therapeutic agent able to address multiple hallmarks of cancer. Specifically, altiratinib inhibits not only mechanisms of tumor initiation and progression, but also drug resistance mechanisms in the tumor and microenvironment through balanced inhibition of MET, TIE2 (TEK), and VEGFR2 (KDR) kinases. Altiratinib potently inhibits cellular proliferation in MET-amplified EBC-1 and MKN-45 cells, as well as TPM3-TRKA fusion KM-12 cells. Altiratinib exhibits properties amenable to oral administration and exhibits substantial blood-brain barrier penetration, an attribute of significance for eventual treatment of brain cancers and brain metastases.

Kinase Assay: Kinase activity was determined using the pyruvate kinase/lactate dehydrogenase (PK/LDH) system. Kinases are described in Supplementary Table S2. Assay mixtures were mixed with test compound. ATP was added to start the reaction immediately or following preincubation. The absorbance at 340 nm was measured at 30°C. Reaction rates were compared with controls and IC50 values were calculated using Prism software (GraphPad). Kinetic determination of off-rate from MET was performed as previously published. Michaelis–Menten analysis using the PK/LDH assay evaluated competitive or noncompetitive inhibition versus ATP. Kinome-wide profiling was performed at Reaction Biology. Altiratinib also inhibits MET isoforms METD1228H, MET D1228N, METY1230C, METY1230D, METY1230H, METM1250T with IC50s of 3.6, 1.3, 1.2, 0.37, 1.5 and 6 nM, respectively. Altiratinib inhibits MET phosphorylation with IC50 values of 0.85 and 2.2 nM, respectively. In the U-87 glioblastoma cell line, MET and HGF are both expressed. Altiratinib blocks autocrine activation of MET phosphorylation in these cells (IC50=6.2 nM). Altiratinib potently inhibits cellular proliferation in MET-amplified EBC-1 and MKN-45 cells, as well as TPM3-TRKA fusion KM-12 cells. Activation of MET is known to increase the motility and invasiveness of cancer cells: Altiratinib inhibits HGF-induced A549 cell migration, with an IC50 of 13 nM. Altiratinib also inhibits FLT3-ITD mutant MV-4-11 cell proliferation with an IC50 of 12 nM.

Cell Assay: Cells are added to 96-well (EBC-1, M-NFS-60, and SK-MEL-28: 2,500 cells/well; MKN-45: 5,000 cells/well; MV-4-11: 10,000 cells/well) or 384-well plates (A375 and HCT-116: 625 cells/well; BT-474, KM-12, PC-3, and U-87-MG: 1,250 cells/well). Plates are incubated for 72 hours. Viable cells are quantified using resazurin using a plate reader with excitation at 540 nm and emission at 600 nm.