Figure 2. Selection of cyclic structures with optimal fitting on Ubc13 by large-scale molecular docking. (A, B) Connolly surface representation of Ubc13 along with the conformers of cyclic structures with highest ranked energy and fitting values for docking on the surface of Ubc13 (A, family I cyclicization mode, enantiomer 2; B, family II cyclicization mode, enantiomer 1). C, Structure of cyclic compound Ia, synthesized after the structure for family I cyclic molecules, derived from peptoid N37-37-9C. D, Structure of cyclic compound IIa, synthesized after the structure for family II cyclic molecules, derived from peptoid N37-37-9C. doi:10.1371/journal.pone.0011403.g002 Next, the ability of compound Ia to affect the enzymatic activity of Ubc13-Uev1 was tested in polyubiquitin chain extension reactions with defined components. In these reactions, the substrate was either wild-type ubiquitin or a variant ubiquitin in which all Lys residues, except at positions 63 or 48, were mutated to Arg (UbK63 or UbK48). The Ubc13-Uev1 dimer, but not either protein alone, supported the formation of free polyubiquitin chains of increasing lengths with either form of ubiquitin as a substrate (Fig. 3D) with robust kinetics (Fig. 3E). Addition of compound Ia strongly inhibited this reaction (Fig. 3D and E). In control experiments, the UbK48 mutant was not a substrate for free polyubiquitin chain extension by Ubc13-Uev1 (Fig. 3F), which supports the specificity of the Ubc13-Uev1 heterodimer for K63- only ubiquitin. Neither compound affected the in vitro Ubc4/5- dependent polyubiquitylation of proteasomal components (Fig. S3), supporting that they specifically target Ubc13 and not other E2 enzymes with canonical (K48) polyubiquitylation activities. Therefore, compound Ia is a potent disruptor of the interaction between Ubc13 and Uev1, and an effective inhibitor of K63 polyubiquitylation catalyzed by Ubc13-Uev1. Effects of compound Ia on cellular pathways regulated by K63 polyubiquitylation Both compound Ia and compound IIa were shown to efficiently enter cultured human cells, as determined by HPLC of cytosolic lysates (not shown) and by microscopic localization of fluorescei- nated derivatives of the compounds (Fig. S4 and S5). To test whether compound Ia could disrupt the Ubc13-Uev1 interaction in vivo, HeLa cells were incubated with compound Ia, lysed and subjected to co-immunoprecipitation. It can be seen (Fig. 4A) that incubation with compound Ia significantly inhibited the abun- dance of the Ubc13-Uev1 heterodimer in HeLa cells. The activation of NF-kB by extracellular signals is regulated by Ubc13-dependent K63 polyubiquitylation in HEK293T and other Novel Ubc13 Inhibitors epithelial cells [41], but not in immune cells [24]. Preincubation of HeLa cells with compound Ia prevented the downregulation of IkB by TNF-a in a dose-dependent manner, with strong effects at concentrations as low as 10 nM (Fig. 4B), an evidence of its activity as an antagonist of NF-kB activation induced by TNF-a in these cells. Compound Ia also inhibited the phosphorylation of IKKa/b induced by TNF-a in HeLa cells (Fig. 4B), consistent with the known role of Ubc13 in TNF-a signaling upstream from the IkB kinases [41]. In addition, compound Ia also strongly inhibited in a dose-dependent manner the nuclear translocation of a GFP form of p65 (a subunit of NF-kB) by TNF-a in HeLa cells (Fig. 4C). We tested the effect of compound Ia on a second pathway regulated by K63-linked polyubiquitylation. Thus, compound Ia inhibited the UV-induced K63 polyubiquitylation of PCNA in vivo (Fig. 5A), although the effects were evident only when cells were exposed to relatively high concentrations of the compound (100 mM). In control experiments, the UV-induced K63-depen- dent polyubiquitylation of PCNA was inhibited by transfection with a dominant-negative form of Ubc13 (Ubc13C87A), indicating that this modification of PCNA indeed requires enzymatically active Ubc13 (Fig. S6). Chemosensitization of mammalian cells by a compound Ia Given the role of K63 polyubiquitylation in DNA damage recognition and repair and NF-kB signaling, we reasoned that mammalian cells exposed to compound Ia might show increased sensitivities (decreased survival) to DNA damaging agents. PC-3 prostate cancer cells and HeLa cells were exposed to increasing doses of the topoisomerase II inhibitors etoposide or doxorubicin, respectively, which induce double-strand breaks in DNA, in the presence of compound Ia. In PC-3 cells, treatment with 100 mM compound Ia decreased the LD50 for doxorubicin from 0.55 mM to 0.1 mM (5.5-fold sensitization, Fig. 5B), and in HeLa cells, compound Ia decreased the LD50 for etoposide from 2.5 mM to 0.2 mM (12.5-fold sensitization, Fig. 5C). Effects of compound Ia on the growth and invasiveness of cultured cells Compound Ia did not significantly inhibit the growth on plastic substrate of HeLa cells under standard growth conditions (Fig. S7), suggesting a low direct cytotoxicity on a range of cell types. In contrast, it significantly inhibited colony formation in soft agar of highly metastatic and clonogenic PC-3/M cells at low micromolar concentrations (Fig. 6A, left panel), and it inhibited the in vitro invasiveness through extracellular matrix of parental PC-3 prostate cancer cells also at low micromolar concentrations (Fig. 6A, right panel). Neither compound Ia, nor the less active compound IIa, significantly affected the growth or differentiation of human endothelial cells in vitro (data not shown). Antitumoral activity of compound Ia in a mouse xenograft model The chemosensitizing effect of compound Ia could be caused either by an inhibitory activity on DNA repair, a diminished activation of NF-kB, or a combination of both activities. Compound Ia also inhibits the clonogenicity and invasiveness of PC-3 cells. We next tested the in vivo antitumoral activity of this compound. PC-3 prostate cancer cells, bearing a stably integrated luciferase gene under the control of a constitutive promoter [42], were grafted into the rear extremities of Balb/c nude mice. Tumor-bearing mice were injected intravenously with doxorubicin (5 mg/Kg, weekly), combined, or not, with intratumoral injections PLoS ONE | www.plosone.org 4 June 2010 | Volume 5 | Issue 6 | e11403