Targeting FOXP4 by high-content screened small molecule. A, High-content small molecule screening was performed from 4,511 anticancer drugs to select the potent FOXP4 inhibitors. Four modeling tools were used to screen the most potent small molecules from the library. B, Four small molecules were deduced to potentially inhibit FOXP4 activity. C, The small molecule 42-(2-tetrazolyl) rapamycin displayed an inhibitory effect in the FOXP4 highly expressed gastric cancer lines MKN28 and NCI-N87. D and E, 42-(2-Tetrazolyl) rapamycin inhibited FOXP4 expression and cell colony formation ability in a dose-dependent manner. F, Dose–response combination assays of two drugs confirmed synergy among all the combinations of 5-FU and 42-(2-tetrazolyl) rapamycin, and the red peak of 3D plots represent the average highest single-agent model synergy scores. HSA, highest single agent. G, 42-(2-Tetrazolyl) rapamycin treatment enhanced the sensitivity towards 5-FU–induced apoptosis. H and I, Coadministration of 5-FU and 42-(2-tetrazolyl) rapamycin resulted in robust antitumor activity in gastric cancer xenografts and restrained gastric cancer peritoneal metastasis. The combination administration exhibited better survival in the mice model. *, P < 0.05; **, P < 0.01; ***, P < 0.001.