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Identification Of Small Molecule TRABID Deubiquitinase Inhibitors By Computation-Based Virtual Screen.

Shi T, Bao J, Wang NX, Zheng J, Wu D - BMC Chem Biol (2012)

Bottom Line: However, these inhibitors failed to show inhibitory effects on β-catenin-mediated gene transcription.In addition, expression of TRABID shRNAs, wildtype TRABID, or the DUB activity-deficient mutant showed little effects on β-catenin-mediated gene transcription.TRABID may not be a critical component in canonical Wnt/β-catenin signal transduction or that a minute amount of this protein is sufficient for its role in regulating Wnt activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, USA. jie.zheng@stjude.org.

ABSTRACT

Background: Wnt/β-catenin-mediated gene transcription plays important roles in a wide range of biological and pathophysiological processes including tumorigenesis where β-catenin-mediated transcription activity frequently elevates. TRABID, a deubiquitinase, was shown to have a positive Wnt/β-catenin-mediated gene transcription and hence holds a promise as a putative anti-cancer target.

Results: In this study, we used a combination of structure based virtual screening and an in vitro deubiquitinase (DUB) assay to identify several small molecules that inhibit TRABID DUB activity. However, these inhibitors failed to show inhibitory effects on β-catenin-mediated gene transcription. In addition, expression of TRABID shRNAs, wildtype TRABID, or the DUB activity-deficient mutant showed little effects on β-catenin-mediated gene transcription.

Conclusions: TRABID may not be a critical component in canonical Wnt/β-catenin signal transduction or that a minute amount of this protein is sufficient for its role in regulating Wnt activity.

No MeSH data available.


Related in: MedlinePlus

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 analyzed was performed 48 hours after transfection. B) TRABID shRNA knockdown efficiency. HCT116 cells were transfected with shRNAs and sorted for YFP positive cells. As a control, HCT116 cells were transfected with the TRABID siRNA (siTrbd). Relative TRABID mRNA levels were determined by q-RT-PCR. C,D) Effects of TRABID shRNAs on Wnt reporter gene activity. HCT116 (B) or SW480 (C) cells were cotransfected with FOPFLASH or TOPFLASH, Renilla luciferase, and one of the TRABID shRNAs. TOPFLASH activity was detected 72 hrs after transfection. E) Effects of TRABID shRNAs on Axin2 expression in SW480 cells. Cells were transfected with one of the TRABID shRNAs for 72 hours, and total RNA was extracted. The levels of Axin2 mRNA were determined by qRT-PCR using beta-actin as control. F,G) Effect of overexpression of TRABID WT or C443S mutant on Wnt reporter gene activity. SW480 (E) or HCT116 (F) cells were cotransfected with WT TRABID or C443S TRABID and TOPFLASH or FOPFLASH. After 24 hrs, luciferase activity was determined. Experiments are performed in triplicates, and data are presented as means ± SD. H) Effect of expression of TRABID WT or C443S mutant on Wnt reporter gene activity in HEK293 cells. Cells were transfected as indicated, and luciferase activity was determined next day.
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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 analyzed was performed 48 hours after transfection. B) TRABID shRNA knockdown efficiency. HCT116 cells were transfected with shRNAs and sorted for YFP positive cells. As a control, HCT116 cells were transfected with the TRABID siRNA (siTrbd). Relative TRABID mRNA levels were determined by q-RT-PCR. C,D) Effects of TRABID shRNAs on Wnt reporter gene activity. HCT116 (B) or SW480 (C) cells were cotransfected with FOPFLASH or TOPFLASH, Renilla luciferase, and one of the TRABID shRNAs. TOPFLASH activity was detected 72 hrs after transfection. E) Effects of TRABID shRNAs on Axin2 expression in SW480 cells. Cells were transfected with one of the TRABID shRNAs for 72 hours, and total RNA was extracted. The levels of Axin2 mRNA were determined by qRT-PCR using beta-actin as control. F,G) Effect of overexpression of TRABID WT or C443S mutant on Wnt reporter gene activity. SW480 (E) or HCT116 (F) cells were cotransfected with WT TRABID or C443S TRABID and TOPFLASH or FOPFLASH. After 24 hrs, luciferase activity was determined. Experiments are performed in triplicates, and data are presented as means ± SD. H) Effect of expression of TRABID WT or C443S mutant on Wnt reporter gene activity in HEK293 cells. Cells were transfected as indicated, and luciferase activity was determined next day.

Mentions: 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 2A 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 2B 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 2C-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 2E), did not inhibit TRABID DUB activity (Figure 2B), 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 2F-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 2F). 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 3A-B). However, neither NSC112200 nor NSC267309 inhibited the reporter gene activity (Figure 3A-B). As controls knockdown β-catenin resulted in significant reduction in Wnt reporter gene activity (Figure 3A,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 3C). 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 4A). 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 4B). However, expression of these shRNAs had no inhibitory effect on Wnt reporter gene expression in either SW480 or HCT-116 cells (Figure 4C,D). These shRNAs also did not show inhibitory effect on the expression of endogenous Wnt target gene Axin2 in SW480 cells (Figure 4E). 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 4F,G) or in HEK293 cells (Figure 4H). Thus, we failed to demonstrate that TRABID has an important role in Wnt target gene expression regulation in these two cancer cells.


Identification Of Small Molecule TRABID Deubiquitinase Inhibitors By Computation-Based Virtual Screen.

Shi T, Bao J, Wang NX, Zheng J, Wu D - BMC Chem Biol (2012)

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 analyzed was performed 48 hours after transfection. B) TRABID shRNA knockdown efficiency. HCT116 cells were transfected with shRNAs and sorted for YFP positive cells. As a control, HCT116 cells were transfected with the TRABID siRNA (siTrbd). Relative TRABID mRNA levels were determined by q-RT-PCR. C,D) Effects of TRABID shRNAs on Wnt reporter gene activity. HCT116 (B) or SW480 (C) cells were cotransfected with FOPFLASH or TOPFLASH, Renilla luciferase, and one of the TRABID shRNAs. TOPFLASH activity was detected 72 hrs after transfection. E) Effects of TRABID shRNAs on Axin2 expression in SW480 cells. Cells were transfected with one of the TRABID shRNAs for 72 hours, and total RNA was extracted. The levels of Axin2 mRNA were determined by qRT-PCR using beta-actin as control. F,G) Effect of overexpression of TRABID WT or C443S mutant on Wnt reporter gene activity. SW480 (E) or HCT116 (F) cells were cotransfected with WT TRABID or C443S TRABID and TOPFLASH or FOPFLASH. After 24 hrs, luciferase activity was determined. Experiments are performed in triplicates, and data are presented as means ± SD. H) Effect of expression of TRABID WT or C443S mutant on Wnt reporter gene activity in HEK293 cells. Cells were transfected as indicated, and luciferase activity was determined next day.
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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 analyzed was performed 48 hours after transfection. B) TRABID shRNA knockdown efficiency. HCT116 cells were transfected with shRNAs and sorted for YFP positive cells. As a control, HCT116 cells were transfected with the TRABID siRNA (siTrbd). Relative TRABID mRNA levels were determined by q-RT-PCR. C,D) Effects of TRABID shRNAs on Wnt reporter gene activity. HCT116 (B) or SW480 (C) cells were cotransfected with FOPFLASH or TOPFLASH, Renilla luciferase, and one of the TRABID shRNAs. TOPFLASH activity was detected 72 hrs after transfection. E) Effects of TRABID shRNAs on Axin2 expression in SW480 cells. Cells were transfected with one of the TRABID shRNAs for 72 hours, and total RNA was extracted. The levels of Axin2 mRNA were determined by qRT-PCR using beta-actin as control. F,G) Effect of overexpression of TRABID WT or C443S mutant on Wnt reporter gene activity. SW480 (E) or HCT116 (F) cells were cotransfected with WT TRABID or C443S TRABID and TOPFLASH or FOPFLASH. After 24 hrs, luciferase activity was determined. Experiments are performed in triplicates, and data are presented as means ± SD. H) Effect of expression of TRABID WT or C443S mutant on Wnt reporter gene activity in HEK293 cells. Cells were transfected as indicated, and luciferase activity was determined next day.
Mentions: 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 2A 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 2B 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 2C-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 2E), did not inhibit TRABID DUB activity (Figure 2B), 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 2F-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 2F). 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 3A-B). However, neither NSC112200 nor NSC267309 inhibited the reporter gene activity (Figure 3A-B). As controls knockdown β-catenin resulted in significant reduction in Wnt reporter gene activity (Figure 3A,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 3C). 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 4A). 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 4B). However, expression of these shRNAs had no inhibitory effect on Wnt reporter gene expression in either SW480 or HCT-116 cells (Figure 4C,D). These shRNAs also did not show inhibitory effect on the expression of endogenous Wnt target gene Axin2 in SW480 cells (Figure 4E). 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 4F,G) or in HEK293 cells (Figure 4H). Thus, we failed to demonstrate that TRABID has an important role in Wnt target gene expression regulation in these two cancer cells.

Bottom Line: However, these inhibitors failed to show inhibitory effects on β-catenin-mediated gene transcription.In addition, expression of TRABID shRNAs, wildtype TRABID, or the DUB activity-deficient mutant showed little effects on β-catenin-mediated gene transcription.TRABID may not be a critical component in canonical Wnt/β-catenin signal transduction or that a minute amount of this protein is sufficient for its role in regulating Wnt activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, USA. jie.zheng@stjude.org.

ABSTRACT

Background: Wnt/β-catenin-mediated gene transcription plays important roles in a wide range of biological and pathophysiological processes including tumorigenesis where β-catenin-mediated transcription activity frequently elevates. TRABID, a deubiquitinase, was shown to have a positive Wnt/β-catenin-mediated gene transcription and hence holds a promise as a putative anti-cancer target.

Results: In this study, we used a combination of structure based virtual screening and an in vitro deubiquitinase (DUB) assay to identify several small molecules that inhibit TRABID DUB activity. However, these inhibitors failed to show inhibitory effects on β-catenin-mediated gene transcription. In addition, expression of TRABID shRNAs, wildtype TRABID, or the DUB activity-deficient mutant showed little effects on β-catenin-mediated gene transcription.

Conclusions: TRABID may not be a critical component in canonical Wnt/β-catenin signal transduction or that a minute amount of this protein is sufficient for its role in regulating Wnt activity.

No MeSH data available.


Related in: MedlinePlus