Oncogene. compounds, we identified Mc as novel SENP1 inhibitor that inhibited proliferation of prostate cancer cells and deSUMOylation assay (Figure ?(Figure1B).1B). The IC50 of AMD 3465 Hexahydrobromide Mc-induced SENP1C inhibition was 15.37 M (Figure ?(Figure1C).1C). As only SENP1C contained the appropriate catalytic domain, we next examined whether Mc inhibited the activity of full-length SENP1 in cells. To this end, HEK293T cells were transfected with full-length SENP1 and Flag-tagged SUMO2 and then treated with Mc. As shown in Figure ?Figure1D,1D, the accumulation of SUMO-modified proteins increased as the Mc treatment concentration increased, indicating that Mc inhibits the isopeptidase activity of full-length SENP1 in cells. Open in a separate window Figure 1 Mc is a SENP1 inhibitor(A). The chemical structure of Mc. (B). In an gel-based SENP1 activity assay, various concentrations of Mc were preincubated with 20 nM SENP1C before SUMO2-RanGAP1 was added. After incubation, the reactions were stopped and the products were separated by 12% SDS-PAGE AMD 3465 Hexahydrobromide and visualized with coomassie brilliant blue (G250). NEM stands for N-Ethylmaleimide, an irreversible inhibitor of all cysteine peptidases. (C). After the gel-based SENP activity assay, gray scanning analysis was carried out using ImageJ software, and a curve was fitted using GraphPad Prism 5.0 after three independent experiments. The IC50 of Mc was 15.37 M. (D). HEK293T cells were transiently transfected with Flag-SUMO2 and empty vector or RGS-SENP1 for 24 h and then treated with DMSO or 6.25, 12.5, or 25 M Mc for 2 h; the indicated proteins were detected by Western blotting. Mc interacts with SENP1 in cells Because Mc inhibited the activity of SENP1 (Supplementary Figure S1). Next, we used CETSA to evaluate the interaction of SENP1 with Mc in androgen receptor-negative prostate cancer PC3 cells. As the commercially available SENP1 antibody did not reliably detect endogenous SENP1, we transfected Flag-tagged SENP1 into PC3 cells (PC3Flag-SENP1). As shown in Figure ?Figure2C,2C, compared to DMSO, Mc markedly increased the accumulation of Flag-SENP1 in the soluble fraction at the temperatures examined. We also tested whether Flag-SENP1 stability during heating depended on the dose of Mc. As shown in Figure ?Figure2D,2D, Flag-SENP1 accumulation markedly increased as Mc concentration increased. As a negative control, we demonstrated that Mc did not increase the stability of vinculin in cells. These data suggest that Mc directly interacts with SENP1 in cells. Open in a separate window Figure 2 Mc alters SENP1 thermal stabilization(ACB). Four g of purified SENP1C was incubated with 50 M Mc at the indicated temperatures (A), and 4 g purified SENP1C was incubated with indicated concentrations of Mc at 45C for 3 min (B). After centrifugation, supernatant was analyzed by western blot with anti-SENP1 antibody and bands were scanned for densitometric analysis. The thermal melt curve (A) and the isothermal dose-response fingerprint (B) are shown. (CCD). Lysate from PC3 cells stably transfected with pBabe-Flag-SENP1 was treated with 100 M Mc at the indicated temperatures (C) or with the indicated concentrations of Mc at 60C for 3 min (D), then analyzed by western blot with anti-flag antibody. The bands were scanned for densitometric analysis, and the thermal melt curve (C) and the isothermal dose-response fingerprint (D) are shown. Mc increases SUMOylated protein levels in prostate cancer cells Given that Mc inhibits SENP1 activity and interacts with SENP1 in cells, Mc likely inhibits SENP1 activity in PC3 cells. Because the intracellular concentration of SUMO1 is low and less dynamic in PC3 cells, and because there are no specific antibodies to distinguish endogenous SUMO2 from SUMO3, we stably transfected PC3 cells with pBabe-Flag-SUMO1/2/3 plasmids (PC3Flag-SUMO1/2/3) to increase the pool of free SUMO1 and to distinguish between proteins modified with SUMO2 or SUMO3. 25 M Mc treatment induced IL-20R2 a large increase in SUMOylated protein levels in SUMO2-transfected PC3 cells (PC3Flag-SUMO2) (Figure ?(Figure3B)3B) and a moderate increase in SUMO1/3-transfected PC3 cells (PC3Flag-SUMO1/3) (Figure ?(Figure3A3A and ?and3C),3C), as indicated by the appearance of smeared high molecular weight bands. These results suggest that Mc inhibits the isopeptidase activity of endogenous SENP1 and subsequently leads to the accumulation of SUMOylated proteins. To further confirm that Mc inhibits SENP1 activity, we examined whether Mc altered the SUMOylation of the known SUMO substrates HIF-1 and nucleus accumbens-associated protein 1 (NAC1). HIF-1 is a well-known AMD 3465 Hexahydrobromide and important oncogene in PCa . NAC1 is associated with pathogenesis in several types of cancer cells [29C31], and we previously identified NAC1 as a possible SUMO substrate in PCa cells . PC3 cells were transiently transfected with Flag-HIF-1 and HA-SUMO2 and then treated with Mc for 2 hours. Flag-HIF-1 was immunoprecipitated from cell lysate and SUMOylation status was detected by western blot. As.