Figure 6. Effect of compound Ia on the growth and tumorige- nicity of PC-3 prostate cancer cells. (A) Inhibition by compound Ia of the invasiveness through Matrigel of PC-3/luc cells (left panel) and of the clonogenicity in soft agar of PC-3/M cells (right panel). Error bars correspond to standard deviations for values obtained in triplicate experiments. (B) Activity of compound Ia in a mouse tumor xenograft model. Balb/c nu/nu mice were grafted with luminescent PC-3/luc cells on both rear extremities. After tumors had developed at the grafted sites, mice were placed in 4 groups. A control group received only intratumoural injections of PBS. A group was treated intravenously with doxorubicin (5 mg/Kg/week). A third group was treated with compound Ia, (100 mM intratumoral, twice a week). A fourth group was treated both with doxorubicin (5 mg/Kg/week) and compound Ia (0.15 mg/Kg intratumoral, twice a week). (C) Quantitative data analysis (relative light units) corresponding to 6 tumors (3 mice) for each treatment group. RLUs were normalized in each group relative to values for day 0. doi:10.1371/journal.pone.0011403.g006 Novel Ubc13 Inhibitors was not directly antiproliferative in vitro for a variety of cell lines tested, but it inhibited the invasiveness of PC-3 cells through extracellular matrix in Boyden chamber experiments, and also inhibited the formation of colonies in 3-dimensional soft-agar cultures. The NF-kB pathway is known to play a prominent role in promoting invasiveness [57,58], being constitutively active in PC-3 cells [59], and thus the observed inhibition of in vitro invasiveness by compound Ia could be one of the consequences of the inhibition of NF-kB activation by this compound. Clonogenicity in soft agar is associated with the capacity of cells for self-renewal, and tends to correlate well with tumorigenicity in vivo. This property, exhibited by distinct cellular subpopulations in some tumors, is not necessarily positively correlated with NF-kB activity [60], and thus the inhibition by compound Ia of the clonogenicity of PC-3 cells could reflect a requirement for Ubc13 activity in other pathways regulating the self-renewal capacity of these cells. In any case, the sum of both activities of compound Ia could explain at least part of the observed direct antitumoral effect. In summary, we have developed specific and potent small molecule antagonists of the Ubc13-Uev1 interaction that inhibit the enzymatic activity of this heterodimer, K63 polyubiquitylation, and we have shown that one of these molecules produces significant effects in the activation of NF-kB by TNF-a, and in invasiveness and clonogenicity in vitro and tumorigenicity of cancer cells in vivo. Based on these activities, we anticipate that tese compounds should be useful to probe other biochemical pathways and cellular processes regulated by K63 polyubiquitylation and to test their effects in relevant models of human pathologies in which these processes are dysregulated. Materials and Methods Synthesis of trialkylglycine-based combinatorial mixtures and of individual compounds An optimized library of 5,120 peptoids in 52 controlled mixtures was synthesized by using the positional scanning format on solid phase [33] (Supporting Methods S1). Yeast two-hybrid Plasmids pBD-Ubc13 and pACT2-Uev1 were co-transfected into the S. cerevisiae strain AH109 in quadruple selection medium. After 3 days, colonies positive for the Ubc13-Uev1 interaction were subjected to liquid culture assays of b-galactosi- dase activity using ONPG as the substrate, in the presence of 0.1 mM of peptoid-mix from each peptoid-pool. One unit of b- galactosidase was defined as the amount which hydrolyzed 1 mmol O-nitrophenol D-galactose per min per cell. Docking Docking was performed using the crystal structure of the Ubc13-Mms2 complex (entry 1JAT in the Brookhaven Protein Data Bank). From the heterodimeric complex, chain A (Ubc13) was selected as the receptor (Supporting Methods S1). Protein-protein binding assays Purified GST-Ubc13 (1 mg) was bound to glutathione-Sephar- ose, washed and preincubated for 1 h with varying concentra- tions of compounds Ia IIa, or a control cyclic compound, and then allowed to bind Uev1 (1 mg) for 1 h in a buffer containing 50 mM Tris-HCl pH 7.6, 0.1 mM EDTA, 150 mM NaCl and 0.2 mM DTT. Unbound fractions were collected, the beads washed 5 times with the same buffer, and finally incubated with glutathione elution buffer. Fractions were resolved by PLoS ONE | www.plosone.org 8 June 2010 | Volume 5 | Issue 6 | e11403