摘要Dear Editor,An increasing body of evidence suggests that USP28 could be a target for cancer treatment1-4.To identify its inhibitors,we screened a 100-thousand synthetic compound library and found 3 lead compounds,CT1001-1003,that showed significant inhibi-tory activity(Supplementary Fig.1a).The optimization of these compounds led to CT1018,a much stronger inhibitor(Supple-mentary Fig.1 b).CT1018 is almost the same as CT1002 except that it is semi-methylated at the 3-amino group.Interestingly,fully methylating the 3-amino group(CT1038,Supplementary Fig.1c)almost killed the inhibitory activity,and replacing the methyl group with an ethyl group(CT1047,Supplementary Fig.1c)also led to a weaker inhibitor.These results indicate that the semi-methylation of the 3-amino group is critical for the inhibitory activity.However,CT1018 was inactive in cell-based assays.Continued optimization resulted in CT1073 and CT1113(Fig.1 a).These two compounds are potent against USP28 as well as the closely-related USP25(Supplementary Fig.1f).The importance of the semi-methylation at the 3-amino group is also true for CT1073,as its unmethylated version,CT1008,is a much weaker inhibitor(Supplementary Fig.1d).CT1113 contains a chiral center and one enantiomer is much more potent than another(Supplementary Fig.1e).The specificity of these compounds was demonstrated by the lack of activities of as much as 10μM CT1073 or CT1113 against other deubiquitinases and SENP1(Supplementary Fig.1g).Furthermore,we measured the interaction kinetics between the inhibitors and USP25/28 with an SPR instrument.As shown in Supplementary Fig.1h,i,CT1073 and CT1113 have similar KDs for USP25 and USP28.