Chemical Biology‐Based Drug Discovery Workflow Highlights Target Identification as a Key Step in Developing a Novel, Potent, and Selective GLUT1 Inhibitor

Discovery of bioactive compounds with novel chemical scaffolds remains a cornerstone of drug discovery. We designed a spiro scaffold composed of a cyclic imide and a diazepane to construct a focused compound library with minimal diversity. Screening for HCT‐116 cell growth inhibition, supported by growth curve analysis and structural optimization, identified compounds 1a and 2a as a chemical probe and a lead candidate, respectively. Compound 2a displayed a distinctive gene expression profile and reduced the viability of U251 cells within the JFCR39 cancer cell panel. A fluorescent probe derived from 1a , localized to the cell membrane, provided crucial information for cell lysate preparation and the affinity‐based identification of facilitative glucose transporter type 1 (GLUT1) as a candidate target protein. Resistance to 2a was linked to a mutation at position N411 in GLUT1, and cells expressing the GLUT1 N411K mutant showed marked attenuation of both antiproliferative and glucose‐uptake inhibitory activities. In this study, we discuss the impact of an integrated approach from hit compound discovery to target identification, as well as 2a , a notable GLUT1 inhibitor that exhibits robust activity attributable solely to GLUT1 inhibition.