a hydrophobic groove on Ubc13 [6,7,8,9]. A critical contributor to the affinity and specificity of this interaction is Phe13 in Uev1, which fits into a deep pocket formed by residues Glu55, Leu56, Phe57 and Arg70 of Ubc13 [6,7,8]. Although other residues contribute to heterodimerization, the above configuration ac- counts for most of the specificity and affinity of the interaction between Uev1 and Ubc13 [8,9,10]. In the yeast S. cerevisiae, DNA damage induces K63 poly- ubiquitylation of the polymerase auxiliary factor PCNA, promot- ing its function in the error-free DNA damage response pathway, a process dependent on Ubc13 and Mms2 [11,12,13], which is conserved in mammalian cells [14,15]. Of the two Uev proteins in mammals, Uev2/Mms2, but not Uev1, appears to be specifically involved in DNA damage repair [16]. The Ubc13-Uev2 heterodimer, recruited by the ubiquitin ligases RNF8 and RNF168, also promotes the recruitment of the BRCA1 A DNA damage repair complex, and K63 polyubiquitylation of histones H2A and H2AX are critical in this process [12]. One of the best studied processes regulated by K63 polyubiquitylation in mam- mals are signaling pathways that activate the transcriptional factor NF-kB [17]. Upon binding of TNF-a to its receptor, the RING finger E3 ubiquitin ligase cIAP is recruited to the receptor complex and ubiquitylates RIP1 possibly through Ubc13-dependent K63 polyubiquitination, resulting in the recruitment of LUBAC, and the complexes TAK1/TAB2/TAB2 and NEMO/IKKa/IKKb [18]. LUBAC drives linear polyubiquitylation of several compo- nents of the TNF-R1 complex [19], which promotes the stabilization of the complex [20] and is essential for the recruitment of NEMO and activation of NF-kB [21]. Binding of IL-1b to IL-1R recruits TRAF6 which oligomerizes, self- polyubiquitylates in a reaction catalyzed by Ubc13-Uev1 and recruits the TAK1/TAB2/TAB2 and NEMO/IKKa/IKKb complexes. Both cytokines eventually activate a kinase cascade that leads to the phosphorylation-dependent ubiquitylation and degradation of IkB, allowing the nuclear translocation and activation of NF-kB [17]. Recent evidence indicates that, in addition to Ubc13, UbcH5 can also mediate K63 polyubiquityla- tion of RIP, and that NF-kB activation by TNF-a may not be as dependent on K63 polyubiquitylation as previously thought [22]. K63 polyubiquitylation is also critical for the activation of additional signaling pathways [23,24], and has been shown to regulate receptor endocytosis and processing [25,26], protein sorting in the multivesicular body pathway [27], cell motility [28], cell-cycle checkpoints [29], and autophagy [30]. Therefore, Ubc13-dependent K63 polyubiquitylation critically regulates processes that generally enhance the survival of cells and organisms in response to certain forms of stress, such as DNA damage or exposure to infectious agents. A pharmacological inhibitor of this modification would be useful not only to study the proteins and biochemical and cellular processes that are modified by this signal, but also to modulate these pathways for therapeutic purposes: for instance, to sensitize cancer cells to DNA damaging agents, or to blunt excessive immune activation. Results Development of antagonists of the Ubc13-Uev1 interaction The hydrophobic grooves and pocket on Ubc13 that serve as its interface with Uev1 present features of a candidate site for specific and high-affinity occupancy by small molecules [31]: a relatively small and deep surface, well-delimited by residues that are not highly polar on the average. Preliminary docking analyses using a comprehensive peptide-based structural library had suggested us Novel Ubc13 Inhibitors that this pocket could be effectively occupied by peptides with the preferred consensus sequence NH3-X – Pro – b-strand aa (Val/ Leu/Ile/Arg/Cys) – hydrophobic/aromatic – X – Pro-COOH (M. Murcia & A.R.O., unpublished observations). We reasoned that peptidomimetic molecules capable of occupying this pocket with sufficient affinity could compete with Uev1 for its interaction with Ubc13, thereby inhibiting the enzymatic activity of the heterodimer. We used a combinatorial chemical library based on trimers of N-alkylglycines (peptoids) [32,33] as an initial source of peptidomimetic structures. Peptoids are characterized by a peptide scaffold with side chains attached to the backbone nitrogen atoms [32], which confers them with several structural properties of peptides [34,35], together with the desirable pharmacological property of being more resistant to proteolytic enzymes [36]. The combinatorial arrangement of N-substituted free amines provided the necessary chemical diversity, and the positional scanning format adopted for this particular library afforded a convenient screening scheme with 52 pools, each containing 320 (pools 1–20) or 256 individual peptoids (pools 21–52), for a total of 5,120 compounds [33]. Conformational flexibility greatly multiplies the structural diversity provided by this library [37,38], a feature that we deliberately chose in spite of the predicted detriment to high- affinity interactions with specific targets of such flexible structures. The general structure of the peptoid library used in this screening is shown in Fig. 1A. As a system to test the capacity of peptoids to inhibit the interaction of Ubc13 with Uev1, we used the yeast-two hybrid assay, since it permits to readily determine the specificity of the inhibitory activities on a fully defined protein-protein interaction and, simultaneously, it constitutes a stringent filter for the bioavailability of active molecules. Yeast cells bearing interacting human Ubc13 and Uev1 were used to screen the combinatorial peptoid library pools for consistent and significant inhibition of this interaction. As a control, an unrelated protein-protein interaction was tested, that of p53 with SV40 large T. Of the 52 pools, those numbered 12, 36 and 46 produced the greatest inhibitory activity on the Ubc13-Uev1 interaction, without significant inhibition on the control p53-large T interaction (Fig. 1B). Deconvolution of the selected pools (i.e., 12 and 16 from the sublibrary where R1 was known, 36 from the sublibrary where R2 was known, and 42 and 46 from the sublibrary where R3 was known) indicated that the inhibitory activity corresponded to four preferred structures (Fig. 1C). The selected amines in these peptoids are: at position R1, either 49-fluorophenylethyl (peptoid N37-37-9C) or 29-49- dichlorophenylethyl (N15-37-13C); at position R2, 49-fluorophe- nylethyl; at position R3, either 49-methoxyphenylethyl (N37-37- 13C and N15-37-13C) or 2-(29-pyridyl)ethyl (N37-37-9C and N15-37-9C). Our hypothesis is that these peptoids inhibit the Ubc13-Uev1 interaction by occupying the Ubc13 dimerization interface with Uev1. Therefore, we analyzed whether these molecules fit onto the Ubc13 surface by means of molecular docking. Because of the inherent flexibility of the carbon framework and N-substituted bonds in alkylglycines, the selected peptoids present a high degree of conformational heterogeneity. To reduce this conformational flexibility, we evaluated 8 families of cyclic structures that are relatively more rigid than the planar equivalents of the selected peptoids, with the added feature that they are readily suitable for synthesis following standard chemical procedures. All energetically favourable spatial arrangements for these structures were com- puted. Of the 8 families of cyclic structures, six had 4 enantiomers and two had 2 enantiomers, and each molecule had between 10 and 14 torsional angles. Thus, the total number of ligands generated was <56106. This small structural library was subjected PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e11403