NSC-632839 (Ubiquitin Isopeptidase Inhibitor II) is a nonselective isopeptidase inhibitor, which inhibits USP2 (EC50: 45±4 μM), USP7 (EC50: 37±1 μM), and SENP2 (EC50: 9.8±1.8 μM).

SENP2:9.8±1.8 μM(EC50), USP2:45±4 μM(EC50), USP7:37±1 μM(EC50)

NSC 632839 selectively inhibits ubiquitin isopeptidases, evidenced by its mid-micromolar inhibition of z-LRGG-AMC cleavage in crude lysates, without affecting PLA2 activity at concentrations of 1.2-150 μM.

In a 96-well-plate, 40 nM USP2, 40 nM USP7, or 20 nM SENP2 is preincubated with a concentration range of NSC 632839 (NCI/NIH developmental therapeutics program) or control for 30 min before supplementation with an equal volume of 60 nM Ub-PLA2/40 μM NBD C6-HPC (USP2 or 7) or 20 nM SUMO3-PLA2/40 μM NBD C6-HPC (SENP2). Relative activity of the enzymes is determined by measuring the RFU values at single time points within the initial linear range (USP, 50 min; USP7, 50 min; and SENP2, 30 min). The RFU values within the initial linear range are normalized such that isopeptidase+vehicle=0% inhibition and isopeptidase+NEM=100% inhibition. The EC50 values are determined as above. The inhibitory activity of the test compound against the reporter enzyme PLA2 is performed as described above except there is no preincubation step and the data are normalized such that free PLA2+vehicle=0% inhibition and free PLA2+EDTA=100% inhibition. PLA2 activity is determined 8 min after the addition of the reagents[1].