1.Inhibition of proliferation and survival of diffuse large B-cell lymphoma cells by a small-molecule inhibitor of the ubiquitin-conjugating enzyme Ubc13-Uev1A.
Pulvino M1, Liang Y, Oleksyn D, DeRan M, Van Pelt E, Shapiro J, Sanz I, Chen L, Zhao J. Blood. 2012 Aug 23;120(8):1668-77. doi: 10.1182/blood-2012-02-406074. Epub 2012 Jul 12.
Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin lymphoma, remains a partially curable disease. Genetic alterations affecting components of NF-κB signaling pathways occur frequently in DLBCL. Almost all activated B cell-like (ABC) DLBCL, which is the least curable group among the 3 major subtypes of this malignancy, and a substantial fraction of germinal center B cell-like (GCB) DLBCL exhibit constitutive NF-κB pathway activity. It has been demonstrated that ABC-DLBCL cells require such activity for proliferation and survival. Therefore, inhibition of NF-κB activation in DLBCL may provide an efficient and targeted therapy. In screening for small-molecule compounds that may inhibit NF-κB activation in DLBCL cells, we identified a compound, NSC697923, which inhibits the activity of the ubiquitin-conjugating (E2) enzyme Ubc13-Uev1A. NSC697923 impedes the formation of the Ubc13 and ubiquitin thioester conjugate and suppresses constitutive NF-κB activity in ABC-DLBCL cells.
2.Screening of DUB activity and specificity by MALDI-TOF mass spectrometry.
Ritorto MS1, Ewan R2, Perez-Oliva AB2, Knebel A1, Buhrlage SJ3, Wightman M1, Kelly SM4, Wood NT1, Virdee S1, Gray NS3, Morrice NA5, Alessi DR1, Trost M1. Nat Commun. 2014 Aug 27;5:4763. doi: 10.1038/ncomms5763.
Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analysing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAB/MPN/Mov34 metalloenzyme DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs.
3.A small-molecule inhibitor of UBE2N induces neuroblastoma cell death via activation of p53 and JNK pathways.
Cheng J1, Fan YH2, Xu X2, Zhang H3, Dou J4, Tang Y5, Zhong X6, Rojas Y7, Yu Y3, Zhao Y3, Vasudevan SA7, Zhang H3, Nuchtern JG7, Kim ES7, Chen X8, Lu F8, Yang J2. Cell Death Dis. 2014 Feb 20;5:e1079. doi: 10.1038/cddis.2014.54.
Neuroblastoma (NB) is the most common extracranial neoplasm in children. In NB, loss of p53 function is largely due to cytoplasmic sequestration rather than mutation. Ubiquitin-conjugating enzyme E2 N (UBE2N), also known as Ubc13, is an E2 ubiquitin-conjugating enzyme that promotes formation of monomeric p53 that results in its cytoplasmic translocation and subsequent loss of function. Therefore, inhibition of UBE2N may reactivate p53 by promoting its nuclear accumulation. Here, we show that NSC697923, a novel UBE2N inhibitor, exhibits potent cytotoxicity in a panel of NB cell lines evidenced by its ability to induce apoptosis. In p53 wild-type NB cells, NSC697923 induced nuclear accumulation of p53, which led to its increased transcriptional activity and tumor suppressor function. Interestingly, in p53 mutant NB cells, NSC697923 induced cell death by activating JNK pathway. This effect was reversible by blocking JNK activity with its selective inhibitor, SP600125.
4.Covalent Inhibition of Ubc13 Affects Ubiquitin Signaling and Reveals Active Site Elements Important for Targeting.
Hodge CD1, Edwards RA1, Markin CJ1, McDonald D2, Pulvino M3, Huen MS4, Zhao J3, Spyracopoulos L1, Hendzel MJ2, Glover JN1. ACS Chem Biol. 2015 Jul 17;10(7):1718-28. doi: 10.1021/acschembio.5b00222. Epub 2015 May 1.
Ubc13 is an E2 ubiquitin conjugating enzyme that functions in nuclear DNA damage signaling and cytoplasmic NF-κB signaling. Here, we present the structures of complexes of Ubc13 with two inhibitors, NSC697923 and BAY 11-7082, which inhibit DNA damage and NF-κB signaling in human cells. NSC697923 and BAY 11-7082 both inhibit Ubc13 by covalent adduct formation through a Michael addition at the Ubc13 active site cysteine. The resulting adducts of both compounds exploit a binding groove unique to Ubc13. We developed a Ubc13 mutant which resists NSC697923 inhibition and, using this mutant, we show that the inhibition of cellular DNA damage and NF-κB signaling by NSC697923 is largely due to specific Ubc13 inhibition. We propose that unique structural features near the Ubc13 active site could provide a basis for the rational development and design of specific Ubc13 inhibitors.