Protein-Protein Interaction Antagonists as Novel Inhibitors of Non-Canonical Polyubiquitylation Johanna Scheper1., Marta Guerra-Rebollo1., Glo` ria Sanclimens2, Alejandra Moure2, Isabel Masip2, Domingo Gonza´ lez-Ruiz3, Nuria Rubio4, Bernat Crosas1, O´ scar Meca-Corte´ s1, Noureddine Loukili1, Vanessa Plans1, Antonio Morreale3, Jero´ nimo Blanco4, Angel R. Ortiz3, A` ngel Messeguer2, Timothy M. 1 Thomson * 1 Department of Cell Biology, Institute for Molecular Biology (IBMB-CSIC), Barcelona, Spain, 2 Department of Biological Organic Chemistry, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain, 3 Bioinformatics Unit, Centro de Biolog´ıa Molecular Severo Ochoa (CBM-UAM-CSIC), Madrid, Spain, 4 Catalan Center for Cardiovascular Research (CIC-CSIC), and CIBER de Bioingenier´ıa, Biomateriales y Nanomedicina, Barcelona, Spain Abstract Background: Several pathways that control cell survival under stress, namely RNF8-dependent DNA damage recognition and repair, PCNA-dependent DNA damage tolerance and activation of NF-kB by extrinsic signals, are regulated by the tagging of key proteins with lysine 63-based polyubiquitylated chains, catalyzed by the conserved ubiquitin conjugating heterodimeric enzyme Ubc13-Uev. Methodology/Principal Findings: By applying a selection based on in vivo protein-protein interaction assays of compounds from a combinatorial chemical library followed by virtual screening, we have developed small molecules that efficiently antagonize the Ubc13-Uev1 protein-protein interaction, inhibiting the enzymatic activity of the heterodimer. In mammalian cells, they inhibit lysine 63-type polyubiquitylation of PCNA, inhibit activation of NF-kB by TNF-a and sensitize tumor cells to chemotherapeutic agents. One of these compounds significantly inhibited invasiveness, clonogenicity and tumor growth of prostate cancer cells. Conclusions/Significance: This is the first development of pharmacological inhibitors of non-canonical polyubiquitylation that show that these compounds produce selective biological effects with potential therapeutic applications. Citation: Scheper J, Guerra-Rebollo M, Sanclimens G, Moure A, Masip I, et al. (2010) Protein-Protein Interaction Antagonists as Novel Inhibitors of Non-Canonical Polyubiquitylation. PLoS ONE 5(6): e11403. doi:10.1371/journal.pone.0011403 Editor: Rafael Linden, Universidade Federal do Rio de Janeiro (UFRJ), Brazil Received September 30, 2009; Accepted June 8, 2010; Published June 30, 2010 Copyright: 2010 Scheper et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by grants from the Consejo Superior de Investigaciones Cientificas (PIF200580 to T.M.T., A.R.O. and A. Messeguer), the Ministerio de Educacion y Ciencia (SAF2005-05109-CO2-01 to T.M.T.; CTQ2005-00995 and GEN2003-20642-C09-09 to A. Messeguer; BIO2005-0576, and GEN2003-206420- C09-08 to A.R.O.), the Comunidad de Madrid (GR/SAL/0306/2004 and 200520M157 to A.R.O.), and an institutional grant from the Fundacion Ramon Areces. J.S. and M.G.R. are receipients of fellowships from the Ministerio de Educacion y Ciencia, and A. Moure of a I3P fellowship from the Consejo Superior de Investigaciones Cientificas. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: J.S., G.S., A. Moure, I.M., D.G-R., A. Morreale, A.R.O., A. Messeguer and T.M.T. are co-inventors in, and the Consejo Superior de Investigaciones Cient´ıficas the holder of, a patent application related to the results described in this manuscript. * E-mail: titbmc@cid.csic.es . These authors contributed equally to this work. Introduction Modifications by ubiquitin (ubiquitylation) control the fate and participation of proteins in fundamental biological processes [1]. The ubiquitylation of a protein involves the formation of a isopeptide bond between a substrate lysine residue and the carboxy terminal Gly76 on ubiquitin. Ubiquitin is activated by an ATP-hydrolyzing ubiquitin-activating enzyme (Uba or E1), that forms a high energy thioester bond between a Cys of its active site and the carboxy terminus of ubiquitin. Activated ubiquitin is transferred to a ubiquitin-conjugating enzyme (Ubc or E2) and a thioester-linked E2-ubiquitin complex is formed. Finally, E2 interacts with a ubiquitin-protein ligase (E3), which conjugates ubiquitin to the substrate protein and confers substrate specificity to the pathway. Ubiquitin has several lysine residues that may be substrates themselves of ubiquitylation, leading to the formation of polyubiquitin chains. The signaling properties of ubiquitylation vary according to the topology of polyubiquitin chains, which depends on the particular lysine residue on the ubiquitin molecule used to form these chains [2]. Thus, polyubiquitin chains linked through K48 (often dubbed as ‘‘canonical’’) are recognized by specific subunits of the 26S proteasome regulatory particle, leading to the degradation of the modified protein [1,2]. Polyubiquitin chains based on K63 are not as efficiently recognized by the proteasome, and rather modify substrate proteins for interactions with other proteins that participate in signaling and other nonproteolytic processes [2,3]. The formation of this class of ‘‘non-canonical’’ polyubiquitin chains is mostly catalyzed by the heterodimeric ubiquitin conjugating enzyme formed by Ubc13 and a Uev protein, Uev1 or Uev2/Mms2 in higher eukaryotes, or Mms2 in the yeast S. cerevisiae [2,4,5]. The N-terminal alpha helix of Uev1 (or Mms2) engages in high affinity interactions with PLoS ONE | www.plosone.org 1 June 2010 | Volume 5 | Issue 6 | e11403