Figure 5. Chemosensitizing activity of compound Ia. (A) Inhibition by compound Ia of UV-induced K63-dependent polyubiqui- tylation of PCNA. HEK-293T cells, transfected with a construct expressing HA-tagged K63-only mutant ubiquitin, were pretreated (Ia) or not (Control) overnight with 100 mM of compound Ia, irradiated or not with UV light (60 J/m2), lysates immunoprecipitated with anti-PCNA and blotted with anti-HA. The immunoprecipitates were blotted in parallel with anti-PCNA for loading control (PCNA, lower panel). Novel Ubc13 Inhibitors Arrowhead, position cooresponding to monoubiquitylated PCNA. (B and C) Sensitization by compound Ia to doxorubicin-induced cytotoxicity in mammalian cells. PC-3 prostate cancer cells (B) and HeLa cervical cancer cells (C) were exposed to varying concentrations of doxorubicin or etoposide, with or withoud compound Ia (100 mM). Viable cell numbers were determined with the CyQuant assay after 72 h (PC-3 cells) or 48 h (HeLa cells) of treatment. doi:10.1371/journal.pone.0011403.g005 ubiquitylation by cIAP of TNF-R1 complex components has not been determined, and, given the recruitment of Ubc13 by cIAP, it is quite possible that such chains are of the K63 type. Furthermore, mice haploinsuficient for Ubc13 display cell-type- specific defects in chemokine and NF-kB signaling [53], supporting a critical role of Ubc13 and K63 polyubiquitylation in the activation of NF-kB by different stimuli in vivo, including TNF-a and LPS. Our observations showing that the small molecule antagonist of Ubc13-Uev interactions compound Ia inhibits NF-kB activation by TNF-a would also support a role for Ubc13 in this pathway. Alternative explanations would include the possibility that our compounds inhibit other ubiquitin conjugating enzymes or additional components of the TNF-a signaling cascade, which has not been formally ruled out in the present study. On the other hand, it has also been shown that unanchored K63-linked polyubiquitin chains are essential for the activation of the RIG-I pathway in response to viral infection, and that both Ubc13 and Ubc5 are required in this pathway [54,55]. Therefore, the inhibition of Ubc13 by small compounds could limit the response to viral infections mediated through this pathway. Regarding the role of Ubc13 and K63 polyubiquitylation in DNA damage response, the very high similarity of Uev2 (Mms2) to Uev1, and the computed interaction of compound Ia on the hydrophobic pocket of Ubc13, allows to predict with sufficient confidence that this compound should disrupt also the interaction of Uev2 (Mms2) with Ubc13. Indeed, we have shown that compound Ia inhibits the UV-induced K63 polyubiquitylation of PCNA, a modification that requires Ubc13-Uev2 [11,15,16]. Therefore, the predicted disruption of the Ubc13-Uev2 heterodi- mer should be associated with a compromise in tolerance to DNA damage by radiation or radiomimetic drugs in mammalian cells [16]. Additional mechanisms, not explored here but possibly also involved in the chemosensitization caused by compound Ia, could be related to the regulation by Ubc13 of double-strand DNA damage recognition and repair through its interaction with the ubiquitin ligase RNF8 [12]. The fact that we have observed inhibition by compound Ia of K63 polyubiquitylation of PCNA only at high concentrations of the compound may suggest either that the compound, although it enters the cells, does not reach the nucleus efficiently, or that K63 polyubiquitylation of PCNA can be catalyzed in mammalian cells by other ubiquitin conjugating enzymes in addition to Ubc13. This may also be the case for K63 polyubiquitylation associated with damage foci in response to DNA double-strand breaks [56]. Indeed, in immunofluorescent c- H2AX focus assays, the same batches of compound Ia that inhibited NF-kB activation at low micromolar concentrations only modestly inhibited the maintenance of c-H2AX in ionizing radiation-induced foci (data not shown). Given the limited effects of compound Ia on both PCNA K63-linked polyubiquitylation and on DNA damage focus formation and resolution, it is possible that the chemosensitization to doxorubicin and etoposide observed in PC-3 and HeLa cells may be better explained by its inhibitory effects on NF-kB signaling. We have observed that compound Ia exerts a direct antitumoral activity in a PC-3 mouse xenograft tumor model. This compound PLoS ONE | www.plosone.org 7 June 2010 | Volume 5 | Issue 6 | e11403