Kerriamycin B inhibits protein SUMOylation

Post-translational conjugation of small ubiquitin-related modifier protein (SUMO) to protein substrates (SUMOylation) has been revealed as one of the major post-translational regulatory systems in animals and other eukaryotes. SUMO conjugation is catalyzed by a multi-step enzymatic reaction cascade similar to ubiquitinylation.1 In the first step, the SUMO precursor is cleaved near the C-terminus by SUMO-specific proteases to expose a C-terminal diglycine. The C-terminal glycine of mature SUMO then forms a thioester linkage to the cysteine residue of SUMO-activating enzyme (E1), the Aos1/Uba2 heterodimer, to generate the E1-SUMO intermediate in an ATP-dependent manner. Next, SUMO is transferred to the active site of the cysteine residue of the SUMO-conjugating enzyme (E2), Ubc9, through another thioester bond. In the last step, E2 and the SUMO ligase (E3) catalyze SUMOylation of substrate proteins at the ε-amino group of internal lysine residues. Although enzymatic reactions by E1 and E2 are sufficient for catalyzing in vitro SUMOylation in most cases, E3s facilitate both in vivo and in vitro conjugation and are important for substrate specificity.1 The structure of SUMO is similar to that of ubiquitin, but its functions are different. SUMOylation regulates protein subcellular localization, enzymatic activity and protein stability, which are associated with the cell cycle, transcription, DNA repair and innate immunity.2, 3 In addition, SUMOylation has been recently linked causally to diseases, such as Alzheimer's and Huntington's diseases,4 viral infection5 and cancer.6, 7 Notwithstanding the importance of SUMOylation in regulating diverse life phenomena and diseases, small molecule inhibitors of SUMOylation have been unexplored. Here, we report novel activity of kerriamycin B that inhibits protein SUMOylation, which will provide useful information about the role of SUMOylation in cells and drug development. Using an in situ cell-based SUMOylation assay method,9 we screened 1,839 samples of microbial cultured broth, and found extracts from three actinomycete strains showing activity to inhibit protein SUMOylation. We extracted the active substance with EtOAc from one of the extracts, and the active component was separated by using various adsorption column chromatographies. Finally, the pure compound was obtained by reverse phase HPLC. Physico-chemical properties and analysis of NMR and mass spectra showed that this compound was identical with a known antibiotic kerriamycin B (Figure 1a).10 To quantitatively analyze the SUMOylation inhibitory activity of kerriamycin B, we first characterized the effect of kerriamycin B on the in vitro protein SUMOylation using RanGAP1-C2 as a substrate. Kerrriamycin B completely inhibited SUMOylation of RanGAP1-C2 in vitro at 20 μM (Figure 1b) but not in vitro ubiquitinylation (data not shown). The IC50 value of kerriamycin B against SUMOylation of RanGAP1-C2 was determined to be 11.7 μM (Figure 1c). We then asked whether kerriamycin B also inhibits in vivo protein SUMOylation by analyzing the effect of the level of protein SUMOylation in 293T cells expressing Flag-tagged SUMO (Figure 1d). Immunoblotting using an anti-Flag antibody showed that kerriamycin B reduced the amount of high-molecular weight SUMO conjugates at 100 μM. Treatment with hydrogen peroxide also reduced the level of high-molecular weight SUMO conjugates (Figure 1d) as recently reported.11 Finally, we sought to determine the target of kerriamycin B. The complex of E1 with biotinylated SUMO-1 through the thioester bond can be detected in the presence of ATP under non-reducing conditions using a biotin–avidin detection system.8 The band corresponding to the E1-biotinylated SUMO-1 intermediate was detected after incubating E1 with biotinylated SUMO-1 in the presence of ATP, but this band disappeared after addition of the reducing agent DTT (Figure 2). The formation of the E1-biotinylated SUMO-1 intermediate was blocked by kerriamycin B at 20 μM (Figure 2). These results suggest that kerriamycin B inhibits protein SUMOylation by blocking the formation of the E1-SUMO-1 intermediate. Most recently, we identified ginkgolic acid and its related compound anacardic acid present in the plant extract as the first small molecule inhibitors of protein SUMOylation.12 Binding assays using a fluorescently labeled ginkgolic acid revealed that ginkgolic acid inhibited protein SUMOylation by directly binding to E1 to block the formation of the E1-SUMO intermediate. In this study, we rediscovered kerriamycin B as a novel inhibitor of protein SUMOylation from microbial metabolites, which also inhibited the formation of the E1-SUMO intermediate. These observations suggested that E1 is the common target for these structurally unrelated compounds. In addition to its antibacterial activity, kerriamycin B has been shown to possess antitumor activity against Ehrlich ascites carcinoma.10 However, the mechanism underlying the antitumor activity is totally unknown. Involvement of the aberrant SUMO system in tumorigenesis has recently been suggested. The increased expression of ubc9 encoding SUMO E2 was reported in several human ovarian cancer cell lines, such as PA-1 and OVCAR-8 as well as in ovarian tumor tissues,13, 14 human lung adenocarcinomas,15 and LNCaP metastastic prostate cancer cell line.16 These observations might reflect a possible role of Ubc9 in tumorigenesis by regulating SUMOylation of various cellular targets. Therefore, it seems possible that kerriamycin B activity to inhibit SUMOylation is responsible, at least in part, for its antitumor activity. Further analyses on the mechanisms of SUMOylation inhibition and structure–activity relationship of kerriamycin B are necessary for developing a novel anticancer agent targeting aberrant protein SUMOylation. We thank Dr Y Uchimura (Kumamoto University) for technical advice. Microbial cultured broth was supplied from the Broth Screening Network (BSN). This work was supported in part by the Chemical Genomics Research Project, RIKEN ASI, the CREST Research Project, the Japan Science and Technology Corporation, and a Grant-in-Aid for Scientific Research on Priority Area ‘Cancer’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan.