Beschreibung:
Myriad Pharmaceuticals has developed a chemical proteomics process to identify cellular proteins that bind immobilized small molecules. This process was used to identify off‐targets of early stage TTK inhibitors and to guide development of TTK inhibitors for our oncology program. It is an unbiased method and reduces the need to generate and test a large number of kinases in vitro. MPI‐0471537 inhibits TTK kinase in vitro with an IC50 of 8 nM and is cytotoxic to HCT116 cells (IC50 = 1.2 μM). MPI‐0471537 coupled to beads was used to affinity purify proteins from cellular lysates, with subsequent LC‐MS/MS‐based protein identification. As expected, TTK was identified as a high affinity binder of the MPI‐0471537‐beads. Additional kinases identified included AurA, FER, JNK1 and JAK1. In vitro assay of these kinases revealed IC50 values of 420, 15, 57 and 320 nM, respectively. MPI‐0478574, a derivative of MPI‐0471537 with modifications designed to increase the selectivity for TTK kinase (IC50 = 3 nM) was subsequently analyzed. MPI‐0478574‐beads failed to bind AurA and showed reduced affinity for FER, JNK1 and JAK1, but maintained similar affinity for TTK. Biochemical assays confirmed the reduced activity of MPI‐0478574 against AurA, FER, JNK and JAK1 (IC50 values >5,000, 380, 120 and >5,000 nM, respectively). These results demonstrate the efficacy of chemical proteomics to assess and advance kinase inhibitor selectivity.