Chemical Biology-Based Drug Discovery Workflow Highlights Target Identification as a Key Step in Developing a Novel,...

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.