Figure 4. Inhibition of NF-kB signalling by compound Ia. (A) Compound Ia inhibits the formation of Ubc13-Uev1 heterodimers in vivo. HeLa cells were co-transfected with FLAG-Ubc13 and HA-Uev1, treated with different concentrations of compound Ia, lysed and subjected to co-immunoprecipitation. It can be seen that incubation of cells with increasing concentrations of compound Ia leads to decreasing amounts of Uev1 that can be co-immunoprecipitated with Ubc13. (B) Dose-dependent inhibition by compound Ia of NF-kB activation by TNF- a. HeLa cells were stimulated for 15 min with TNF-a (20 ng/mL) without (0) or with overnight preincubation with the indicated final concentra- tions of compound Ia, and analyzed by Western blotting for IkB protein levels or for phosphorylation of IKKa/b. (C) Dose-dependent inhibition by compound Ia of the nuclear translocation of p65-GFP induced by TNF-a. HeLa cells were transfected with p65-GFP were incubated overnight with varying concentrations of compound Ia, treated with TNF-a (20 ng/mL) for 30 min, fixed and cells counted for nuclear localization of p65-GFP. Background (proportion of cells with nuclear p65-GFP in untreated samples) was subtracted, and values normalized against the proportion of cells with nuclear p65-GFP in cells stimulated with TNF-a only. doi:10.1371/journal.pone.0011403.g004 Novel Ubc13 Inhibitors of compound Ia (0.15 mg/Kg in phosphate-buffered saline, twice a week). A third group of mice received intratumoral compound Ia and no doxorubicin. Control mice were sham injected with phosphate-buffered saline by both routes. After 8 weeks of treatment, control mice had grown extensive tumors at the site of grafting (Fig. 6B, C). In contrast, mice receiving doxorubicin alone, compound Ia alone, or a combined treatment of systemic doxorubicin and local (intratumoral) compound Ia had signifi- cantly reduced tumors as compared to control mice, by the end of the treatment (Fig. 6B, C). Analysis of the corresponding quantitative data (normalized photon counts) showed that the average sizes of the tumors at the end of the experiment were 24.7% of control tumors for mice treated with compound Ia alone, 22.0% for mice treated with doxorubicin alone, and 15.5% for mice with the combined treatment (Fig. 6C), the differences between the three treatment regimes not being statistically significant. There were no evidences of acute or chronic toxicity either at the site of injection or in other organs and tissues associated with the administration of compound Ia (unpublished observations). Therefore, administration of compound Ia alone significantly limits the growth of PC-3 prostate cancer xenografts, to a degree comparable to treatment with the chemotherapeutic agent doxorubicin. Discussion In terms of drug design, the specific contribution of our approach has been to use in vivo screenings to find hit structures from a combinatorial chemical library, and to further select the best ligands by virtual screening. Our drug development scheme should be applicable to the design of small molecules capable of specifically interfering with many other well-characterized inter- or intra-molecular interactions with amenable surfaces [31]. Other non-peptide small molecules that disrupt specific protein-protein interactions have been successfully developed in recent times [43,44,45,46,47], and they are beginning to show great promise for the therapy of human cancer. In practical terms, we have developed small molecules that can effectively and selectively antagonize the Ubc13-Uev1 interaction and inhibit K63 poly- ubiquitylation in both yeast and mammalian cells, and we have shown that these compounds can be used in combination therapy schemes as antitumoral agents in cultured and animal models of cancer. Specifically, compound Ia sensitizes PC-3 prostate cancer cells to the antiproliferative activity of doxorubicin in cultured cells and it shows direct antitumoral activity in mouse tumor xenografts. A number of mechanisms can be at play to cause increased sensitivities of tumor cells to chemotherapy or radiotherapy, including inhibition of NF-kB [48], downregulation of transporters of the MDR family [49] or the Akt-mTOR pathway [50,51,52]. The evidence provided here suggests that at least two mechanisms may be relevant for the increased sensitivity to doxorubicin caused by compound Ia, namely inhibition of NFk-B activity and compromise of DNA repair. The demonstration that this compound disrupts the interaction between Uev1 and Ubc13 provides a mechanistic explanation for its inhibitory activity on the NF-kB signaling pathway [17]. Recently, it has been shown that another ubiquitin conjugating enzyme, UbcH5, can promote K63 polyubiquitylation, and that NF-kB activation by IL-1b is much more strongly dependent on Ubc13-dependent K63 polyubiqui- tylation than activation by TNF-a [22]. However, a large body of literature strongly suggests a critical role of Ubc13 and K63 polyubiquitylation in the activation of NF-kB not only by IL-1b but also by TNF-a. In this regard, the chain type of ligand-induced PLoS ONE | www.plosone.org 6 June 2010 | Volume 5 | Issue 6 | e11403