To screen for compounds that can inhibit TRABID DUB activity in vitro, we established an assay to measure the DUB activity of TRABID. Consistent with the previous report by Tran et al. [11
], we observed that recombinant TRABID proteins purified from an E coli expression system or pulled down from HEK293 cells overexpressing Flag-TRABID by an Flag antibody were able to specifically cleave hexa-K63 (Figure B,C), but not penta-K48 (data not shown), ubiquitin chains. We also tested a TRABID DUB-deficient mutant containing a substitution mutation (C443A), a residue located in the OTU domain and critical for its catalytic activity [11
]. The mutation abrogated the ability of TRABID to cleave the Hexa-K63 ubiquitin substrate (Figure C), confirming the importance of this residue for the DUB activity. Homology modeling of TRABID OTU domain based on the crystal structure of A20 OTU domain [17
] revealed that the common cysteine catalytic site was well conserved in TRABID. This site is characterized by a cysteine residue that forms an electrostatic network with a histidine and a residue with an acidic side chain. In TRABID, these 3 residues are C443, H596 and D410, respectively, based on the homology model (Figure D).
Figure 1 TRABID DUB activity assays and structural modeling of TRABID OTU domain. A) Sequence alignment of TRABID OTU domain and A20 OTU domain, the identical (yellow) and homologous (green) pairs are highlighted. B,C) Cleavage of K63 Chains by Recombinant TRABID. (more ...)
Searching on the surface of the equilibrated structure of TRABID OTU domain modeled from the crystal structure of A20 OTU revealed that there was a potential pocket adjacent to the catalytic center of OTU (Figure E). This pocket is part of a distal ubiquitin binding site at the catalytic site in A20 [17
]. Because of its spatial proximity to the catalytic site and its role in ubiquitin binding, it is reasonable to postulate that targeting this site may block the entrance of the substrates and hence inhibit the OTU DUB activity. Of note, during the molecular dynamics refinement, considerable conformational changes occurred in this site, making it more expanded comparing with the pocket size in the A20 crystal structure (Figure F).
We performed hierarchical virtual screening towards this pocket as described in the “Materials and Methods”, and requested 200 compounds that were ranked highest in the virtual screen and were provided with 125 by NCI. These 125 compounds were screened for their abilities to inhibit the cleavage of Hexa-K63 ubiquitin by TRABID. Seventeen of these compounds showed more than 50 percent (denote with ++ in Additional file 1
. Table S1) and five showed between 25–50 percent (denote with
in Additional file 1
. Table S1) inhibition of TRABID DUB activity. Figure A shows a representative Western blot used in the TRABID DUB activity assay. All of the positive compounds were retested in a dose-dependent experiment. Figure B shows the data for the two of the strongest inhibitors, which showed the IC50 values of about 3 μM. Neither compound showed significant inhibition of A20 up to 30 μM, though both inhibited A20 at 100 μM (Figure C-D). Compound NSC60650 as well as a number of other compounds shown in Additional file 1
. Table S1, which share similar chemical structures to NSC112200 and NSC267309 (Figure E), did not inhibit TRABID DUB activity (Figure B), suggesting that the two hydroxyl groups and their locations on the benzene ring may be important for the inhibitory activity. The molecular modeling of the bindings of NSC112200, NSC267309 and NSC60650 to TRABID OTU domain suggests that while all three compounds can form electrostatic interactions with the side chain of E522 and the backbone NH of T556, both NSC112200 and NSC267309 can form an additional hydrogen bond with the side chain of S491 (Figure F-H). Moreover, NSC112200 can form another hydrogen bond with the side chain of S520, and its one of methyl groups provides extra hydrophobic interaction with the carbon atoms located at the side chain of R557 (Figure F). The rescoring of binding free energies by ICM shows that the binding free energies of NSC112200 and NSC267309 with TRABID OTU are quite similar (−20.28 kcal/mol and −20.03 kcal/mol, respectively), while the binding free energy of NSC60650 with TRABID OTU is −16.89 kcal/mol, which is 3.39 kcal/mol higher than NSC112200. On the other hand, the calculated binding free energies of NSC112200 and NSC267309 with A20 OTU are −15.23 kcal/mol and −13.43 kcal/mol, respectively. As the high binding free energy means a lower binding affinity, our modeling predictions are consistent with our experimental data. TRABID was shown to play a positive role in canonical Wnt signaling, and its DUB activity appeared to be essential for this function [11
]. We hence tested the TRABID DUB inhibiting compounds for their effects on canonical Wnt signaling. SW480 or HCT116 colorectal cancer cell lines, which were used in the previous study for TRABID’s role in Wnt signaling [11
], were transfected with a Wnt reporter gene construct TOPFLASH and incubated with compounds for 24 hrs. Because these two cell lines contain mutations in the APC and β-catenin genes, respectively, there was high reporter gene activity in these cells (Figure A-B). However, neither NSC112200 nor NSC267309 inhibited the reporter gene activity (Figure A-B). As controls knockdown β-catenin resulted in significant reduction in Wnt reporter gene activity (Figure A,B). We also tested other TRABID inhibitors in Additional file 1
. Table S1, and none of them inhibited the reporter gene activity (data not shown). We then examined two endogenous Wnt target gene expression. Neither Axin2 nor c-Myc expression in SW480 cells was affected by NSC112200 or NSC267309 treatment (Figure C). These results together suggest that TRABID might not be involved in Wnt$β-catenin-mediated gene transcription regulation. To further evaluate the role of TRABID in the regulation of Wnt/β-catenin-mediated gene transcription, we generated 6 constructs that produce six independent shRNAs for TRABID. When these shRNAs were coexpressed with TRABID cDNA in HEK293 cells, they showed varying efficiencies in suppressing TRABID expression with shTrbd5 and 1 being the most efficiency and shTrbd2 and 4 showing little effects (Figure A). We also evaluated their knockdown efficiencies using quantitative RT-PCR analysis of RNAs from YFP-positive HCT116 transfectants (shRNA and YFP were in the same transcript) (Figure B). However, expression of these shRNAs had no inhibitory effect on Wnt reporter gene expression in either SW480 or HCT-116 cells (Figure C,D). These shRNAs also did not show inhibitory effect on the expression of endogenous Wnt target gene Axin2 in SW480 cells (Figure E). Furthermore, overexpression of wildtype TRABID or its DUB-deficient mutant had little effects on Wnt reporter gene expression in either SW480 or HCT-116 cells (Figure F,G) or in HEK293 cells (Figure H). Thus, we failed to demonstrate that TRABID has an important role in Wnt target gene expression regulation in these two cancer cells.
Figure 2 Identification of TRABID DUB inhibitors. A,B) Effects of compounds on TRABID DUB activity. Compounds (30 μM in A) were incubated with Hexa-K63 ubiquitin and immunoprecipitated FLAG-TRABID for 3 hrs. Samples were then analyzed by Western under (more ...)
Figure 3 Effect of TRABID inhibitors on Wnt/β-catenin-mediated transcriptional activity. A,B) TRABID inhibitors fail to inhibit Wnt reporter gene activity. HCT-116 or SW480 cells were transfected with FOPFLASH or TOPFLASH and a plasmid expressing Renilla (more ...)
Figure 4 Effects of TRABID shRNAs or overexpression on Wnt reporter gene activity and Axin2 expression. A) Effects of TRABID shRNAs on TRABID expression. HEK293T cells were cotransfected with GFP (an internal control), FLAG-TRABID, and one of TRABID shRNA. Western (more ...)
Nevertheless, both TRABID inhibitors NSC112200 and NSC267309, but not the non-active analog NSC60650, inhibited the growth of not only the colorectal tumor cell lines HCT-116 and SW480, but also two para-normal cell lines NIH3T3 and HEK293 at 20 μM (Figure ). These compounds showed no effects on the growth of these cells at 5 μM (data not shown). Because of the lack of effect of Compound NSC60650 on cell growth, we believe that the effects of Compounds NSC112200 and NSC267309 are likely due to their inhibition of the deubiquitinase activity.
Figure 5 Effect of TRABID inhibitors on cell growth. Compounds (20 μM) were added to cells at seeding and cell growth was determined by counting live cells at indicated times. Experiments are performed in triplicates, and data are presented as means±SD. (more ...)
Wnt signaling plays a crucial role in cancer cell growth and cancer stem cell maintenance, which are believe to be responsible for cancer metastasis and poor prognosis of chemotherapy. Thereby, regulation of Wnt signaling holds promise in chemotherapy of cancer. TRABID, one of K63-specific DUB enzymes, became an attractive candidate to achieve this task since it has been reported to regulate Wnt signal positively [11
], as inhibition of TRABID presumably decreases Wnt signaling activity. However, in this study, we failed to confirm the notion that TRABID positively regulates canonical Wnt signaling based on several lines of evidence. Firstly, the compounds, which blocked TRABID DUB activity in vitro, failed to inhibit Wnt transcription activity in two tumor cell lines whereβcatenin-mediated transcription are known to be elevated. Secondly, the TRABID shRNAs failed to downregulate Wnt/β-catenin-mediated transcription activity or endogenous Wnt target gene Axin2 mRNA levels in these tumor cells. Thirdly, overexpression of WT and C443S mutant TRABID failed to show any effect on Wnt target transcription activity. We did observe that the siRNA described in the previous report [11
] was able to inhibit Wnt reporter gene activity (Figure 4C-E). One possibility is that the effect of the siRNA reported in [11
] is off target. A less likely possibility is that a minute amount of TRABID is sufficient for its regulation of Wnt signaling in these cells, and our shRNAs or inhibitors were not efficient enough to alter Wnt activity. Nevertheless, the fly data [11
] have already suggested that TRABID might not be a core component of Wnt signaling. The observation that the TRABID inhibitors were able to inhibit cell growth suggests that TRABID may have a role in cell growth regulation. However, the mechanisms for this role of TRABID need to be further investigated.