SUMOylation is one of the posttranslational modifications that mediate cellular activities such as transcription,DNA repair,and signal transduction and is involved in the cell cycle.However,only a limited number of small molecule inhibitors have been identified to study its role in cellular processes.Here,we report a F(o)rster resonance energy transfer (FRET) high-throughput screening assay based on the interaction between E2 Ubc9 and E3 PIAS1.Of the 3200 compounds screened,34 (1.1％) showed higher than 50％ inhibition and 4 displayed dose-response inhibitory effects.By combining this method with a label-free surface plasmon resonance (SPR) assay,false positives were excluded leading to discovering WNN060S-F008 and WNN1062-D002 that bound to Ubc9 with Ko values of 1.93 ± 0.62 and 524 ± 3.73 μM,respectively.We examined the effect of the two compounds on SUMO2-mediated SUMOylation of RanGAP1,only WNN0605-F008 significantly inhibited RanGAP1 SUMOylation,whereas WNN1062-D002 did not show any inhibition.These compounds,with novel chemical scaffolds,may serve as the initial material for developing new SUMOylation inhibitors.