Target engagement assay

To analyze the interaction between small-molecule ligands and their target proteins in intact cells, a cellular thermal shift assay (CETSA) was performed as described previously with minor modifications^9,34. Briefly, HeLa cells cultured in a 100-mm dish to 80% confluency, were treated with NPD7155 (300 μM), NPD9948 (300 μM), or (S)-crizotinib (30 μM) for 1 h. After treatment, cells were collected with trypsin and resuspended in Tris-buffered saline (TBS). The cell suspension was aliquoted into four polymerase chain reaction (PCR) tubes and heated at 45, 50, 55, or 60 °C for 5 min. Subsequently, cells were lysed by three repeated freeze-thaw cycles using liquid nitrogen. Precipitated proteins were separated from the soluble fraction by centrifugation at 17,000 × g for 20 min. Soluble proteins collected in the supernatant were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) for western blot analysis. The membrane was incubated with a primary antibody against MTH1 (1:500, sc-67291; Santa Cruz Biotechnology) and a horseradish peroxidase–labeled secondary antibody (Vector Laboratories) in sequence. They were then visualized with Fusion Solo S (Vilber Lourmat) using a Super Signal West Pico Chemiluminescence Substrate (Pierce). After detection of MTH1 by western blot, proteins on the membrane were visualized by staining with Coomassie brilliant blue (CBB).