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Mol Biol Cell
Mol Biol Cell
molbiolcell
mboc
Molecular Biology of the Cell
1059-1524
1939-4586
The American Society for Cell Biology

38696259
E24-02-0073
10.1091/mbc.E24-02-0073
Special Issue on Cell Biology in China
new_hypothesis
Highlights from MBoC SelectionVerteporfin inhibits TGF-β signaling by disrupting the Smad2/3–Smad4 interaction
Nong Junxiu a †
Shen Shengqiang b †
Hong Fan c
Xiao Fan b
Meng Lingtian a
Li Pilong d
Lei Xiaoguang b *
Chen Ye-Guang a c e *
a The State Key Laboratory of Membrane Biology, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China
b Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China
c Guangzhou National Laboratory, Guangzhou International Bio Island, Guangzhou 510005, Guangdong Province, China
d Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China
e School of Basic Medicine, Jiangxi Medical College, Nanchang University, Nanchang 330031, China
Wu Congying Monitoring Editor
Peking University Health Science Center
†These authors contributed equally to this work.

Author contributions: J.N., P.L., X.L., and Y.C. conceived and designed the experiments; J.N., S.S., F.H., F.X., and L.M. performed the experiments; J.N., S.S., and F.H. analyzed the data; J.N., S.S., F.H., X.L., and Y.C. drafted the article; J.N. prepared the digital images; X.L. is also the corresponding author.

Conflicts of interests: The authors declare no financial conflict of interest.

National Natural Science Foundation of China (31988101 to Y.G.C)

National Key Research and Development Program of China (2022YFC3401500 and 2022YFC2502500 to X.L., 2023YFA1800603 to Y.G.C)

National Natural Science Foundation of China (22193073 and 92253305 to X.L.)

Beijing Outstanding Young Scientist Program (BJJWZYJH01201910001001 to X.L.)

ORCID ID: Fan Hong, 0009-0003-0318-4706

*Address correspondence to: Ye-Guang Chen (ygchen@tsinghua.edu.cn); Xiaoguang Lei (xglei@pku.edu.cn).
01 7 2024
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© 2024 Nong et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.
2024
https://creativecommons.org/licenses/by-nc-sa/4.0/ This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 4.0 Unported Creative Commons License.

Transforming growth factor-β (TGF-β) signaling plays a crucial role in pathogenesis, such as accelerating tissue fibrosis and promoting tumor development at the later stages of tumorigenesis by promoting epithelial–mesenchymal transition (EMT), cancer cell migration, and invasion. Targeting TGF-β signaling is a promising therapeutic approach, but nonspecific inhibition may result in adverse effects. In this study, we focus on the Smad2/3–Smad4 complex, a key component in TGF-β signaling transduction, as a potential target for cancer therapy. Through a phase-separated condensate-aided biomolecular interaction system, we identified verteporfin (VP) as a small-molecule inhibitor that specifically targets the Smad2/3–Smad4 interaction. VP effectively disrupted the interaction between Smad2/3 and Smad4 and thereby inhibited canonical TGF-β signaling, but not the interaction between Smad1 and Smad4 in bone morphogenetic protein (BMP) signaling. Furthermore, VP exhibited inhibitory effects on TGF-β–induced EMT and cell migration. Our findings indicate a novel approach to develop protein–protein interaction inhibitors of the canonical TGF-β signaling pathway for treatments of related diseases.

TGF-β signaling is involved in multiple pathophysiological events, and its signaling can be transduced via distinct Smad or non-Smad pathways. Therapeutic targeting of individual pathways may be important for achieving specific clinical effects.

Using a system based on phase-separated condensate-mediated biomolecular interactions, the authors discovered verteporfin as a small-molecule inhibitor that selectively targets the Smad2/3–Smad4 interaction.

Inhibitors targeting Smad complexes may exhibit reduced side effects and improved safety, making these inhibitors suitable for scientific and clinical applications such as against tissue fibrosis and metastasis of various cancers.
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pmcINTRODUCTION

Transforming growth factor-β (TGF-β) is a multifunctional growth factor that regulates cell proliferation, differentiation, migration, and death and plays a dual role in tumorigenesis (Hata and Chen, 2016). Initially, it acts as a tumor suppressor by inhibiting cell proliferation and promoting apoptosis. However, in later stages, it promotes tumor progression by inducing epithelial–mesenchymal transition (EMT), cancer cell migration, and invasion (Massague, 2008). Due to the pro-oncogenic role of TGF-β, cancer therapies targeting the TGF-β signaling pathway have been explored (Colak and Ten Dijke, 2017). Various approaches have been employed, including anti-ligand antisense oligonucleotides, ligand competitive peptides, antibodies against ligands, receptors, or associated proteins, and small-molecule inhibitors of TGF-β receptor kinases (Liu et al., 2021). As these strategies primarily target the TGF-β ligands or receptors, leading to nonspecific inhibitory outcomes and significant side effects in clinical treatment (Connolly et al., 2012; Huynh et al., 2019). Therefore, there is an urgent need for the strategies more specifically targeting TGF-β signaling.

The TGF-β superfamily consists of TGF-βs, activins, bone morphogenetic proteins (BMP) and others (Feng and Derynck, 2005). Smad proteins serve as central transducers in TGF-β superfamily signaling. Among the receptor-regulated Smads (R-Smad), Smad2/3 mediate TGF-β/activin signaling, while Smad1/5/8 are responsible for BMP signaling (Massague, 2012). Upon TGF-β stimulation, Smad2/3 undergo phosphorylation by the TGF-β type I receptor (TβRI), form a heterotrimeric complex with Smad4 and translocate into the nucleus to regulate target gene expression (Massagué et al., 2005). In addition to the Smad-mediated canonical signaling pathway, TGF-β can also activate AKT, MAPK, and other noncanonical signaling pathways (Moustakas and Heldin, 2005; Zhang, 2009). The Smad and non-Smad pathways act cooperatively or specifically to mediate TGF-β–induced pathophysiological functions.

Smad-mediated TGF-β signaling plays a crucial role in the EMT process during cancer development. Indeed, studies with murine models have demonstrated that targeting Smad3 can effectively reduce bone metastasis of breast cancer (Kang et al., 2005; Drabsch and ten Dijke, 2011). Blocking the intracellular Smad2/3–Smad4 complex may be a promising strategy to selectively inhibit the tumor-promoting function of TGF-β (Akhurst and Hata, 2012; Huang et al., 2021). Compared with conventional TGF-β inhibitors, small molecules targeting Smad complexes are expected to have a better safety profile, making them suitable for both scientific applications and clinical studies. However, to date, no small-molecule inhibitors have been reported to specifically target the Smad2/3–Smad4 complex.

In recent years, innovative methods to detect biomolecular interactions have been developed, such as condensate-aided enrichment of biomolecular interactions in test tubes (CEBIT) (Zhou et al., 2020) and compartmentalization of protein–protein interactions in cells (CoPIC) (Xu et al., 2021). The CEBIT-based assay consists of three parts: (1) the scaffold, which forms the phase-separated condensates driven by multivalent interactions and can be visualized with a fluorescent probe such as GFP; (2) the bridge, in which the interacting partner of interest is fused to a protein motif that can be recruited into the scaffold condensates; (3) the client, in which the test partner is fused with a fluorescent probe such as mCherry for visualization. As a result, the biomolecular interaction of interest is assessed by the enrichment of the mCherry-fused client within the GFP-labeled phase-separated condensates (Figure 1, A and B). These strategies use phase-separated condensates for efficient, high-throughput screening of biomolecular interactions and inhibitor discovery, offering a novel approach to study protein–protein interactions. In this study, we conducted a phase separation–based screening approach to identify small-molecule inhibitors of TGF-β signaling. Our results revealed that the compound verteporfin (VP) specifically disrupted the formation of the Smad2/3–Smad4 complex, thereby suppressing canonical TGF-β signaling. Moreover, VP inhibited the TGF-β–induced EMT and cell invasion. These findings provide a valuable insight into the development of novel therapeutic intervention against tissue fibrosis and metastasis of various cancers, including breast, lung, colon, and gastric cancers.

FIGURE 1: VP disrupts the Smad2/3–Smad4 interaction. (A) Schematic diagram of the CEBIT-based assay to assess the interaction between Smad3 and Smad4. This system comprises three parts, scaffold SmF-GFP-SH3-PDZ and SmF-GFP-PRM (shown in the green box), bridge PV-Smad3-MH2 (shown in the black box), and client mCherry-Smad4-MH2 (shown in the red box), in which four fusion proteins are included. The individual fusion proteins are shown in the box. The arrows indicate protein–protein interaction pairs by color. (B) CEBIT-based assay on the Smad3–Smad4 interaction. The partitioning of the client mCherry-fused Smad4-MH2 (5 μM) into the phase-separated condensates formed by 2 μM SmF-GFP-SH3-PDZ and 2 μM SmF-GFP-PRM, with or without PV-Smad3-MH2 (2.5 μM). (C) CEBIT-based assay showing the inhibitory effect of VP (10 μM) on Smad1/2/3–Smad4 interactions. The partitioning of the client mCherry-fused Smad4-MH2 (5 μM) into the phase-separated condensates formed by 2 μM SmF-GFP-SH3-PDZ and 2 μM SmF-GFP-PRM, was mediated by PV-Smad1/2/3-MH2 proteins (2.5 μM), PV (control) fused with mCherry without Smad4-MH2, and 5% DMSO were used as controls. All proteins were in KMEI buffer (150 mM KCl, 1 mM EGTA, 1 mM MgCl2, 10 mM imidazole, 1 mM Tris [2-carboxyethyl] phosphine and 10% glycerol, pH 7.4). Representative fluorescence images and quantification of mCherry signal in droplets are shown, with all mCherry signals normalized to DMSO groups. (D) Confocal images showing CoPIC analysis of VP inhibition on the Smad3–Smad4 interaction. HEK293T transfected with Nup98-GFP-Smad3-MH2 and Smad4-MH2-mCherry was treated with VP at the indicated concentrations for 30 min. Quantification of mCherry signal in the green puncta was shown. (E) GFP-trapped coimmunoprecipitation analysis of the Smad3–Smad4 interaction with indicated VP treatment as in D. (F) Purified MBP-Smad4 (60 μM) and 6His-Smad1/2/3 proteins (5 μM) were incubated with the indicated concentrations of VP, and Smad1/2/3 pulled down by MBP-Smad4 were revealed by immunoblotting. Quantification of Smad1/2/3 proteins was shown. (G) HEK293T cells treated with DMSO and VP for 30 min were harvested for anti-Smad2/3 immunoprecipitation. Total protein expression was confirmed using whole-cell lysates. The relative band intensity was quantified with ImageJ in E–G. Statistical analyses were performed with two-way ANOVA in C or one-way ANOVA in D, (**, P < 0.01; ***, P <0.001). Scale bar in A, B: 10 μm.

RESULTS

Identification of VP as an inhibitor of the Smad2/3–Smad4 interaction

To screen small molecules that can regulate the interaction between Smad3 and Smad4, we established a novel system based on the principles of CEBIT (Zhou et al., 2020) (Figure 1A). This system employed two multimeric scaffold proteins, SmF-GFP-SH3-PDZ and SmF-GFP-PRM, which promote phase separation and exhibit green fluorescence. The interaction between Smad3 and Smad4 resulted in the recruitment of mCherry-labeled Smad4-MH2 domain into the phase-separated droplets via the interaction of Smad3-MH2–linked PV and PDZ in the scaffold proteins (Figure 1B). Thus, the CEBIT system could be used for high-throughput screening of compounds targeting the Smad3–Smad4 interaction.

Using the CEBIT system, we screened and validated a total of 4773 known commercial compounds. Among them, VP (Supplemental Figure S1A) was identified as a potent inhibitor of the Smad3–Smad4 interaction (Figure 1C). Dose–response analysis further confirmed the inhibitory activity of VP (Supplemental Figure S1B). To investigate the selectivity of VP toward other R-Smads and Smad4, we employed different bridging proteins, including PV-Smad1-MH2 and PV-Smad2-MH2, with PV-mCherry protein as a control (Figure 1C; Supplemental Figure S1, C–E). The results demonstrated that VP specifically disrupted the interaction between Smad2/3 and Smad4, while having no effect on the interaction between Smad1 and Smad4. We have shown that both Axin1 and Dvl2 undergo LLPS (Nong et al., 2021; Kang et al., 2022), but VP did not affect LLPS of Axin1 and Dvl2 (Supplemental Figure S1, F and G). Therefore, the inhibitory effect of VP on the Smad2/3–Smad4 interaction is specific.

To further investigate the inhibitory effect of VP on the Smad2/3–Smad4 interaction, we conducted experiments in cellular systems. First, we confirmed that VP could rapidly enter the cytosol (Supplemental Figure S2A). In the CoPIC system (Xu et al., 2021), we utilized low complexity domains derived from nucleoporin 98 protein (Nup98) to form membraneless compartments with Smad3-MH2 labeled by GFP intracellularly. The recruitment of mCherry-Smad4-MH2 into these compartments served as a readout for the interaction. We observed that treatment with VP resulted in a dose-dependent decrease in the recruitment of mCherry-Smad4-MH2 into the Smad3-MH2 membraneless compartments (Figure 1D). These data were confirmed with coprecipitation assay, showing that VP decreased the Smad3–Smad4 interaction in a dose-dependent manner (Figure 1E). This observation strongly suggested that VP effectively inhibited the Smad3–Smad4 interaction in the cell.

We then performed pull-down assays to verify the inhibitory effect of VP on Smad1/2/3–Smad4 interaction. We examined the effect of VP on the interaction of the purified MBP-Smad4 protein and 6His-tagged Smad1/2/3 proteins and found that VP disrupted the Smad2/3–Smad4 interaction, but had no effect on the interaction between Smad1 and Smad4 (Figure 1F). The inhibitory effect of VP the binding between Smad2/3 and Smad4 was confirmed with endogenous coimmunoprecipitation experiment (Figure 1G). Collectively, these results demonstrate that VP possesses an inhibitory activity against the interaction between Smad2/3 and Smad4.

VP blocks the Smad2/3/4-mediated transcription

Upon TGF-β activation, Smad2/3 proteins form heterotrimeric complexes with Smad4, translocating to the nucleus to regulate signal transduction (Derynck and Zhang, 2003). We then examined the impact of VP on TGF-β–induced Smad-mediated transcription using CAGA and ARE-luciferase reporters. As shown in Figure 2, A and B, VP dose-dependently inhibited TGF-β–induced transcriptional activities of both Smad2 and Smad3. Additionally, VP significantly reduced the nuclear accumulation of Smad2 and Smad3 in response to TGF-β in human skin keratinocyte HaCaT and human mammary epithelial MCF10A cells (Figure 2, C–F).

FIGURE 2: VP blocks TGF-β/Smad signaling. (A, B) HEK293T cells transfected with Smad3-activated CAGA-luciferase (A) or Smad2-activated ARE-luciferase (B) reporters were treated with vehicle DMSO and indicated VP for 12 h before harvested for luciferase determination. (C–F) Immunofluorescence staining and quantification of Smad2/3 with or without TGF-β stimulation in HaCaT (C–D) and MCF 10A cells (E–F). Cells were treated with DMSO and VP or 100 pM TGF-β for 4 h, followed by anti-Smad3 and anti-Smad2 immunofluorescence, nuclei were counterstained with DAPI. Statistical analyses were performed with two-way ANOVA in A, B or one-way ANOVA in C–F (*, P < 0.05; **, P < 0.01; ***, P <0.001). Scale bar in C–F: 10 μm.

VP has been reported as a YAP (Yes-associated protein) inhibitor (Liu-Chittenden et al., 2012), and YAP/TAZ has been shown to mediate TGF-β signaling (Futakuchi et al., 2018). To explore whether the inhibitory effect of VP on Smad-mediated TGF-β signaling was dependent on YAP, we generated a YAP knockout in human embryonic kidney HEK293T cell line (Supplemental Figure S2B). We found that YAP ablation did not affect on the inhibitory effect of VP on TGF-β–induced CAGA-luciferase expression (Supplemental Figure S2C). As VP is a benzoporphyrin derivative used in photodynamic therapy (Parodi et al., 2016), we assess the possible effect of its photosensitivity by comparing its inhibitory effect under light protection and routine conditions. The results in Supplemental Figure S2D showed that VP’s inhibition on Smad3 transcriptional activity was independent of photoactivation.

SB431542, a specific TβRI kinase inhibitor (Inman et al., 2002), and SIS3, a specific inhibitor of Smad3 phosphorylation (Jinnin et al., 2006), have been previously reported to inhibit TGF-β signaling. To compare the effects of these two inhibitors with VP, we induced the CAGA-luciferase reporter expression by treating cells with TGF-β ligand (Supplemental Figure S3A) or by expressing a phosphorylation-mimicking mutant of Smad3 (Ser423/425 Asp mutant, Smad3-SD) (Supplemental Figure S3B). Both SB431542 and SIS3 exhibited inhibitory effects only in the TGF-β–treated group, but not in the Smad3-SD–expressing group. In contrast, VP had a significant inhibitory effect in both groups, supporting that VP acts downstream of Smad3 phosphorylation, consistent with the data of interfering with the interaction between Smad2/3 and Smad4. In agreement with it, VP did not affect TGF-β–induced Smad2/3 phosphorylation (Supplemental Figure S3C). Furthermore, TGF-β–induced phosphorylation of ERK, AKT, and p38 was not altered by VP either (Supplemental Figure S3D). These findings indicate that the inhibitory action of VP on TGF-β signaling does not involve the direct modulation of Smad2/3 phosphorylation or the activation of ERK, AKT, or p38.

Additionally, we tested the effect of VP on BMP signaling, and the results showed that VP had no effect on the transcriptional activity and nuclear accumulation of Smad1 induced by BMP4 (Supplemental Figure S4), in accordance with its lack of influence on the Smad1–Smad4 interaction. These findings indicate that VP selectively impairs the Smad2/3/4-mediated canonical TGF-β signaling.

VP inhibits TGF-β–induced EMT

EMT is a biological process characterized by the loss of intercellular adhesion and basal polarity in epithelial cells, accompanied by the acquisition of migratory and invasive characteristics resembling mesenchymal cells (Heldin et al., 2012; Hao et al., 2019). To investigate whether VP could block TGF-β–induced EMT, we treated HaCaT and MCF 10A cells with TGF-β in the presence or absence of VP. Upon TGF-β treatment, the cells became elongated and scattered, indicative of the morphologies similar to mesenchymal cells, while VP treatment prevented the TGF-β–induced EMT process (Figure 3, A and B). Furthermore, immunofluorescence staining revealed that the cytoskeleton of HaCaT and MCF 10A cells underwent noticeable deformation upon TGF-β treatment, characterized by decreased E-cadherin and F-actin–formed stress fibers, which are typical features of EMT (Figure 3, C and D). These alterations were effectively attenuated by VP treatment. Consistently, TGF-β stimulation upregulated the expression of EMT-related genes (fibronectin, N-cadherin, Snail1, and Snail2/Slug), while VP reduced the TGF-β effects in a dose-dependent manner (Figure 3, E–H). These results together indicate that VP has a potent inhibitory effect on the TGF-β–induced EMT process.

FIGURE 3: VP inhibits TGF-β–induced EMT. (A, B) HaCaT and MCF10A cells were treated with VP, with or without 100 pM TGF-β, for 24 h. The morphological changes of EMT induced by 100 pM TGF-β were observed. (C, D) HaCaT and MCF10A cells are treated with VP, with or without 100 pM TGF-β, for 24 h and followed by anti-E-cad and anti-F-actin immunofluorescence. And the nuclei were stained with DAPI. HaCaT and MCF10A cells were treated with 100 pM TGF-β and VP for 12 h. (E, F) The mRNA levels of Fibronectin (FN1), SNAI1, and CDH2 (N-cadherin) were examined by qPCR. (G, H) HaCaT and MCF10A cells were treated with 100 pM TGF-β and VP for 12 h. Cell lysates were harvested for immunoblotting. Protein expression levels of Fibronectin (FN1), N-cadherin (N-cad), Snail1, and Slug were examined. Relative band intensity was normalized to GAPDH. The relative band intensity was quantified with ImageJ in G–H. Statistical analyses were performed with two-way ANOVA in E, F, (*, P < 0.05; **, P < 0.01; ***, P <0.001). Scale bar in A, B: 50 μm; scale bar in C, D: 20 μm.

VP attenuates TGF-β–induced breast cancer cell migration

To investigate the inhibitory effect of VP on TGF-β–mediated tumor invasion, we examined the migration of human breast cancer MDA-MB-231 cells (Chiechi et al., 2013). VP demonstrated a dose-dependent attenuation of TGF-β–induced expression of mesenchymal markers, including Fibronectin, N-cadherin, Snail, and Slug (Figure 4A). Additionally, the transwell and wound-healing assays revealed that VP suppressed the migrative ability enhanced by TGF-β (Figure 4, B and C). These data collectively demonstrate that VP exerts a significant inhibitory effect on TGF-β–induced breast cancer cell migration.

FIGURE 4: VP attenuates TGF-β–induced breast cancer cell migration. (A) MDA-MB-231 cells were treated with TGF-β and VP for 12 h. Cell lysates were harvested for immunoblotting. Protein expression of mesenchymal markers N-cad, FN1, Snail1, and Slug was examined. Relative band intensity was normalized to GAPDH. (B) MDA-MB-231 cells were cultured in transwell plates treated with the indicated concentrations of VP and TGF-β for 12 h. The invading cells were stained with crystal violet and counted. (C) MDA-MB-231 cells were wounded and then treated VP and TGF-β for 12 h. The wound width was normalized to the baseline. The relative band intensity was quantified with ImageJ in A. Statistical analyses were performed with two-way ANOVA in B, C, (**, P < 0.01; ***, P <0.001). Scale bar in B, C: 100 μm.

Structure-activity relationship of VP derivatives

To improve the inhibitory activity of VP against the Smad2/3–Smad4 interaction, we analyzed the structure-activity relationship of VP. Considering that VP is an equal mixture of two regioisomers (Scott and Goa, 2000), VP-a and VP-b were separated to evaluate their influence on VP’s inhibitory activity. The CEBIT results showed that both VP-a and VP-b were equally active in inhibiting the Smad2/3–Smad4 interaction (Supplemental Figures S5A and S6A), indicating that interchanging the propionic acid and propionic acid methyl ester did not affect the activity.

In addition, we tested other porphyrin compounds including Protoporphyrin IX (PPIX), Temoporfin (TP), Vitamin B12 (VB12), and Methylcobalamin (MB) (Supplemental Figure S5B). However, none of these compounds showed any inhibition on the Smad3–Smad4 interaction (Supplemental Figure S5C), indicating that simple porphyrin rings alone do not contribute to the activity and emphasizing the importance of the parent ring structure of VP.

To investigate the role of side chains and double bond positions in VP’s inhibitory activity, we synthesized a series of VP derivatives, VP-A1-A15 (Figure 5A; Supplemental Figure S6, A and B) and subjected them to CEBIT screening. Among the derivatives, only VP-A6, which had a pentynyl group, inhibited Smad4 recruitment into droplets (Figure 5B), suggesting that the terminal alkene of VP is not primarily responsible for its activity to disrupt the Smad3–Smad4 interaction. Although VP-A6 exhibited lower cell permeability compared with VP (Figure 5C), it retained the inhibitory activity of VP on Smad3 and Smad2 transcriptional activity (Figure 5, D and E). These findings indicate that structural modifications at the terminal alkenes of VP could be a feasible strategy for optimization of its activity.

FIGURE 5: Effect of selected VP derivatives on Smad3–Smad4 interaction. (A) Chemical structure and basic information of VP derivatives (VP-A1-A15). VP-A1-A7 were synthesized to investigate the effects of the carboxyl group and the terminal alkenes of VP on its activity, while VP-A8-A15 to investigate the effects of different stereoisomers, double bond positions, and ester groups. (B) CEBIT-based assay showing the inhibitory effects of VP derivatives on the Smad3–Smad4 interaction. All proteins were in KMEI buffer (150 mM KCl, 1 mM EGTA, 1 mM MgCl2, 10 mM imidazole, 1 mM Tris (2-carboxyethyl) phosphine, and 10% glycerol, pH 7.4). (C) The uptake evaluation of VP and VP-A6 in HEK293T cells for 30 min. The control group was treated with the same amount of DMSO. (D–E) HEK293T cells transfected with Smad3-activated CAGA-luciferase (A) or Smad2-activated ARE-luciferase (B) reporters were treated with VP and VP-A6 for 12 h before harvested for luciferase determination. Statistical analyses were performed with one-way ANOVA in B, C or two-way ANOVA in D, E, (*, P < 0.05; **, P < 0.01; ***, P <0.001). Scale bar in B, C: 10 μm.

DISCUSSION

TGF-β signaling is involved in multiple pathophysiological events, and its signaling can be transduced via distinct Smad or non-Smad pathways (Massague, 2008). Current strategies to inhibit TGF-β signaling usually target either the ligand activity of TGF-β or the kinase activity of its receptors, leading to inhibition of downstream functions mediated by receptors (Huynh et al., 2019). It would be important to target one but not other pathways to achieve specific effect. For instance, Smad3 is critical for TGF-β–mediated EMT and fibrosis, specific target on Smad3-mediated TGF-β signaling could be used to treat tissue fibrosis (Heldin et al., 2012). Therefore, we chose the Smad2/3–Smad4 complex as the target and identified VP as a small-molecule inhibitor by a phase separation–based screening.

VP is a phenolic porphyrin derivative photosensitizer that has been approved for clinical use in the treatment of age-related macular degeneration with choroidal angiogenesis (Wei and Li, 2020). VP releases mono-oxygen when excited by light at a wavelength of 689 nm. This property can interfere with widely used high-throughput screening such as alpha-screen and FRET (Scott and Goa, 2000; Macarron et al., 2011). The discovery of VP in our study benefited from the advantages of phase separation–based screening, which allows the detection of VP’s activity without being affected by its photochemical properties (Zhou et al., 2020).

The YAP/TAZ pathway cooperates with the TGF-β pathway to exert profibrotic and tumorigenic effects (Noguchi et al., 2018). YAP/TAZ has been reported to induce EMT by regulating multiple EMT-related genes, such as Sox2, in cooperation with Oct4 (Bora-Singhal et al., 2015), or associating with transcription factors like ZEB1/2, Snail/Slug, and Twist (Lehmann et al., 2016; Tang et al., 2016). Furthermore, the interaction between YAP/TAZ, TEAD, and Smad2/3 regulates the transcription of metastatic and profibrotic genes (Fujii et al., 2012). The non-photoinduced molecular form of VP has been shown to inhibit YAP activity, by disrupting its interaction with TEAD and thus attenuating YAP-mediated tumor proliferation (Liu-Chittenden et al., 2012; Feng et al., 2016). In addition, VP has been reported as a potent inhibitor of TGF-β–mediated fibrosis, attributed to the cross-talk between YAP/TAZ and Smad2/3 signaling pathways (Szeto et al., 2016). VP reduced TGF-β–induced phosphorylation and the protein levels of Smad2/3, but YAP/TAZ depletion only decreased their phosphorylated Smad2/3 levels (Futakuchi et al., 2018), indicating that VP may also exert its effects through other non–YAP-dependent mechanisms. Here, our results demonstrate that VP blocked the interaction between Smad2/3 and Smad4 and impaired TGF-β/Smad signaling through a YAP-independent mechanism, highlighting the multifaceted role of VP. Furthermore, we did not observe the effect of VP on TGF-β–induced phosphorylation and the protein levels of Smad2/3 in HaCaT cells. SIS3 has been reported to act as Smad3-specific inhibitor by interfering its phosphorylation without impacting Smad2 (Jinnin et al., 2006). Although both SIS3 and VP act at the Smad level to block TGF-β signaling, VP specifically inhibits the interaction between Smad2 and Smad3 with Smad4, but has no effect on TGF-β–induced Smad2/3 phosphorylation or protein stability.

Targeting transcription factors like Smad proteins, which lack intrinsic enzymatic activity, poses challenges in developing small-molecule inhibitors due to limited ligand chemotypes interacting with their surfaces (Akhurst, 2017). Although VP disrupts the Smad2/3–Smad4 interaction, the structural mechanism remains unclear. Attempts at VP structure optimization for further insight into its modulatory activity on the TGF-β signaling pathway were made, but extensive optimization is needed. In conclusion, VP serves as a valuable lead compound for studying TGF-β signaling and designing more potent antagonists targeting the TGF-β/Smad pathway.

MATERIALS AND METHODS

Antibodies and reagents

Rabbit monoclonal anti-Smad4 (Cell Signaling Technology, 46535); Rabbit monoclonal anti-p-Smad2 (Cell Signaling Technology, 3104S); Rabbit monoclonal anti-Smad2 (Cell Signaling Technology, 5339); Rabbit monoclonal anti-p-Smad3 (Cell Signaling Technology, 9520S); Rabbit monoclonal anti-Smad3 (Cell Signaling Technology, 9523S); Mouse monoclonal anti-Smad1 (Cell Signaling Technology, 9743); Mouse monoclonal anti-N-cadherin (BD Biosciences, 610920); Mouse monoclonal anti-E-cadherin (BD Biosciences, 610181); Rabbit monoclonal anti-Snail (Cell Signaling Technology, 3879); Mouse monoclonal anti-Fibronectin (BD Biosciences, 610077); Rabbit monoclonal anti-Slug (Cell Signaling Technology, 9585); Rabbit monoclonal anti-p-ERK (Santa Cruz Biotechnology, SC-7383); Rabbit monoclonal anti-ERK (Santa Cruz Biotechnology, SC-93); Rabbit monoclonal anti-p-AKT S473 (Cell Signaling Technology, 4060S); Rabbit monoclonal anti-AKT (Cell Signaling Technology, 9272S); Rabbit monoclonal anti-p-p38 (Cell Signaling Technology, 9215L); Rabbit monoclonal anti-p38 (Santa Cruz Biotechnology, SC7972); Mouse monoclonal anti-GFP (Santa Cruz Biotechnology, SCC8334); Rabbit monoclonal anti-mCherry (Cell Signaling Technology, 43590); Mouse monoclonal anti-MBP (Sigma-Aldrich, M6295) were purchased from the indicated suppliers. VP, SIS3, and SB431542 from Selleck were dissolved in DMSO for 10 mM stock solution. Compounds utilized for high-throughput screening were sourced from commercial libraries, including Sigma-LOPAC-1280, Selleck-FDA Approved Drug Library-2148, Top Science-Natural Compound Library-409, and Pharmacodia-Natural Product Library-936.

Cell culture, plasmids, and transfection

All cell lines (HEK293T, HaCaT, MCF 10A, and MDA-MB-231) were obtained from American Type Culture Collection or China Infrastructure of Cell Line Resource. HEK293T and HaCaT were cultured in DMEM while MDA-MB-231 cells maintained in RPMI1640 medium, supplemented with 10% FBS (Hyclone) in a 37°C humidified incubator containing 5% CO2. MCF10A was supplemented with DMEM/F12 medium, 10% horse serum, 20 ng/mL EGF, 0.5 μg/ml hydrocortisone, 100 ng/ml cholera toxin, and 10 μg/ml insulin at 37°C in a humidified, 5% CO2 incubator. MCF 10A cells were used at less than four passages and other cells were used at less than 10 passages after thawing.

All genes were PCR amplified and cloned into the HA-pcDNA3.1 vector (for mammalian cell expression) to produce HA-tag fused recombinant proteins. To express EGFP- or mCherry-fused proteins, the EGFP or mCherry cDNA was fused to the 3′-end of target cDNAs, thus tagging the expressed proteins with HA at the N-terminus and EGFP or mCherry at the C-terminus.

Transfection of DNA plasmids into HEK293T, MCF-10A, and MDA-MB-231 cells was performed using Lipofectamine 2000 (Invitrogen) or Vigofect (Vigorous Biotechnology).

Gene editing with CRISPR/Cas9

HEK293T cells were transfected with plasmids containing Cas9 and two single-guide RNAs targeting exon 4 of YAP (5′-GCCCACAGGGAGGCGTCAT-3′ and 5′-ACCAGCAGCAACAGATGCG-3′). YAP knockout clones (KO1 and KO2) were identified by immunoblotting and confirmed by genomic DNA sequencing, and subsequently utilized in the study.

CEBIT high-throughput screening

The CEBIT system, as previously described (Zhou et al., 2020), comprises three components: scaffold (SmF-GFP-SH3-PDZ and SmF-GFP-PRM), bridge (KKETPV-Smad3-MH2), and client (mCherry-Smad4-MH2). SmF, derived from Saccharomyces cerevisiae, forms a stable tetradecamer and acts as the scaffold for biomolecular condensates. The scaffold components include SH3 (a domain of human NCK1) and PRM (the proline-rich motif of DLGAP2), along with PDZ (a domain of human PSD95) and KKETPV (a synthetic PDZ ligand, PV). The interaction between SmF-SmF and SH3-PRM promotes the formation of green fluorescent droplets. The bridge protein PV-Smad3-MH2 is then immobilized into droplets through the PDZ–PV interaction. Finally, the client protein mCherry-labeled Smad4-MH2 is recruited into droplets by the interaction between Smad3 and Smad4, resulting in enriched mCherry signals.

The assay system for screening compounds that disrupt the Smad3-Smad4 interaction consisted of all proteins in KMEI buffer (150 mM KCl, 1 mM EGTA, 1 mM MgCl2, 10 mM imidazole, 1 mM Tris (2-carboxyethyl) phosphine and 10% glycerol, pH 7.4) supplemented with 5% DMSO in a total volume of 20 μl in a 384-well microplate (Greiner Bio-One, 781090). Each compound was used at a concentration of 50 μM. After thoroughly mixing all the components (scaffolds, bridge, client proteins, and compounds), the microplates were incubated at 4°C for 30 min, images were taken by high content microscopy and data were collected.

Immunoblotting, coimmunoprecipitation, immunofluorescence, protein binding assay, and reporter assays

For immunoblotting, cells were lysed on ice with lysis solution (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% NP-40, 2 mM EDTA, 1 mM NaVO3, 10 mM NaF and protease inhibitors) and rotated at 4°C for 30 min. Lysates were subjected to SDS–PAGE and immunoblotting was performed with primary antibodies as indicated and secondary anti-rabbit or anti-mouse antibodies conjugated to horseradish peroxidase followed by enhanced chemiluminescence substrate (Pierce) detection according to the manufacturer’s instructions.

For coimmunoprecipitation, cells were lysed as described above. After taking an aliquot for total protein expression analysis, immunoprecipitation was performed by adding precleared GFP-nanoab-agarose (LABLEAD) to the remaining cell lysate, followed by incubation at 4°C overnight with gentle rotation. The immune complex was then isolated by centrifugation, washed with lysis solution and analyzed by SDS–PAGE and immunoblotting.

For immunofluorescence, cells transfected with the indicated plasmids were washed twice with PBS, fixed with 4% formaldehyde solution for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min and blocked with 5% BSA in PBS for 30 min at room temperature. After incubation overnight at 4°C with the primary antibody (anti-Smad2, 1:100; anti-Smad3, 1:100; anti-Smad1, 1:100; anti-E-cadherin [1:500]; anti-F-actin, 1:1000), the fluorescein-labeled secondary antibody and 4′, 6-diamidino-2-phenylindole (DAPI) were added for 1 h at room temperature. Imaging was with a Nikon A1 microscope. NIS-Elements AR Analysis was used to analyze these images.

For in vitro MBP pull-down assays, Smad1/2/3 and MBP-Smad4 proteins were incubated with the agarose in the buffer containing 20 mM HEPES pH7.4, 1 M NaCl and incubated at 4°C for 2 h. The beads were then collected, washed three times with buffer and then subjected to immunoblotting. For in vivo GFP pull-down assays, cells were lysed with lysis solution. Lysates were incubated with GFP-nanoab-agarose (LABLEAD) at 4°C for 2 h in binding buffer (0.5% NP-40, 150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA). The beads were then collected, washed three times with lysis buffer and subjected to immunoblotting.

For the reporter assay, HEK293T cells, cultured in 24-well plates, were transfected with 200 ng CAGA/ARE/BRE-luciferase plasmid and 30 ng Renilla plasmid for 3-well cells. At 12 h posttransfection, cells were treated with certain concentrations of VP with or without TGF-β for another 12 h. Luciferase activity was measured using the dual luciferase reporter assay system (Promega), experiments were repeated in triplicate and data are presented as means ± SD after normalization to Renilla activity.

Protein expression and purification

All genes were PCR amplified and cloned into the pET32M.3C vector to produce 6His tag-fused recombinant proteins or the pETMBP.3C vector to produce MBP tag-fused recombinant proteins. To express EGFP- or mCherry-fused proteins, EGFP or mCherry cDNA was fused to the 3′ end of target cDNAs. Point mutations and deletions were introduced by site-directed mutagenesis. All recombinant proteins used in this study were expressed in Escherichia coli (E. coli) BL21-Codon Plus (DE3) with induction by 1 mM IPTG for 16 h at 18°C.

For purification of 6His-SmF-GFP-SH3-PDZ, 6His-SmF-GFP-PRM, 6His-PV-Smad3-MH2, and 6His-mCherry-Smad4-MH2 proteins used in CEBIT, the bacteria were lysed by sonication in buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10% glycerol, and 1 mM phenylmethylsulfonyl fluoride), then proteins were purified using nickel-NTA-agarose beads (GE Healthcare) followed by ion exchange chromatography and finally by size exclusion chromatography in KMEI buffer (150 mM KCl, 1 mM EGTA, 1 mM MgCl2, 10 mM imidazole, pH 7.4, 1 mM Tris-HCl [2-carboxyethyl] phosphine, and 10% glycerol). All proteins were rapidly frozen with liquid nitrogen and stored at −80°C.

For purification of 6His-Smad1, 6His-Smad2, 6His-Smad3, MBP-6His-Smad4 proteins, E. coli cells were resuspended in binding buffer (50 mM Tris-HCl pH 7.9, 2 M NaCl and 10 mM imidazole), lysed with a high-pressure homogenizer and sedimented at 18000 rpm for 40 min. The supernatant lysates were purified on amylose resin (NEB). After extensive washing with binding buffer, proteins were eluted with 500 mM imidazole buffer or MBP elution buffer (50 mM Tris-HCl pH 7.9, 2M NaCl, and 10 mM imidazole, for MBP-labeled proteins), then purified on a HiPrep 26/60 Sephacryl S-200 HR column (GE Healthcare, 17-1195-01) on an AKTA purifier (GE Healthcare) and eluted with a buffer containing 20 mM HEPES, 1 M NaCl, pH 7.4. All purified proteins were concentrated by centrifugal filtration (Millipore) and stored in aliquots at −80°C.

Transwell and wound healing assays

For the transwell assay, cells were plated in medium containing 1% FBS and inhibitors in the upper chamber of a transwell plate (Corning), while the medium containing 10% FBS, VP, or TGF-β was placed in the lower well. After incubation for the indicated time, cells were fixed with methanol for 20 min, stained with crystal violet dye for 30 min and photographed. Experiments were performed at least three times.

For the wound healing assay, the confluent cell monolayer in a 12-well plate was wounded by scraping the cells with a 200 μl pipette tip and then treated with TGF-β or VP for the indicated time. Quantitation was performed by measuring the width of the migrating wound gap and comparing it to the initial wound from three independent experiments.

Quantitative RT-PCR

Total cell RNA was isolated using TRIzol (Invitrogen), and cDNA was synthesized using Revertra Ace (Toyobo). Real-time PCR was performed using a Roche 480 system (Roche). Expression levels were normalized to GAPDH expression. The PCR oligo sequences are shown. The following primers were used: FN1, 5′-GCAGCC­AACCAAGATGCAAA-3′ and 5′-ATTGCGGGCCAGACACTTAA-3′; SNAI1, 5′-CCAGTGCCTCGACCACTATG-3′ and 5′-CTGCTGG­AAGGTAAACTCTGGA-3′; CDH2, 5′-AGCCAACCTTAACTGAGGAGT-3′ and 5′-GGCAAGTTGATTGGAGGGATG-3′; GAPDH, 5′-AGGTCGGAGTCAACGGATTT-3′ and 5′-TATGAAGGGGTCATTGATGGCA-3′.

Synthesis of VP derivatives

VP was separated through silica gel column with DCM: Et2O = 50:1 and obtained VP-a and VP-b. Among them, isomer VP-a eluted before VP-b. The target compounds VP-A1-A7 are synthesized and outlined in Supplemental Figure S6A and Supplemental Schemes S1 and S2. Briefly, VP was selected as the starting material and reacted with different alcohols or amides in DCM to afford VP-A1-A5 (Supplemental Scheme S1). On the other hand, treatment of VP with excess Boc2O in the presence of t-BuOH and DMAP yielded the VP-OBut, and then stirred with alkene under the catalysis of Grubbs-II in THF to obtain Boc-protected precursors Boc-VP-A6-A7 (Liu et al., 2008). The deacetylation of Boc-VP-A6-A7 via TFA catalysis resulted in compounds VP-A6-A7 (Supplemental Scheme S2).

To further study the effects of different stereoisomers, double bond positions, and ester groups of VP on the inhibitory activity, VP derivatives VP-A8-A15 were synthesized (Supplemental Figure S6B). First, refer to procedures described in the literature (Brunner and Schellerer, 2002), compound PPIX was reacted with Boc2O to acquire PPIX-OBut, which on further reaction with R3OOCC≡CCOOR3 yielded key intermediates PPIX-DA-1-4. Direct removal of tert-butyl ester protecting group of PPIX-DA-1-4 resulted in compounds VP-A8-A9, VP-A12-A13 (Supplemental Scheme S3). In addition, treatment of PPIX-DA-1-4 with DBU and deprotection of tert-butyl ester obtained double bond migration products VP-A10-A11 and VP-A14-A15 (Supplemental Scheme S4).

Detailed synthetic procedures and characterization data for all of the synthesized compounds are given in the Supplemental Data.

Quantification and statistical analysis

Statistical analysis was carried out on GraphPad Prism 8 software. The one-way ANOVA and two-way ANOVA were employed to assess the difference between groups. The statistical significance was defined as follows: *, P < 0.05; **, P < 0.01; and ***, P < 0.001 were considered statistically significant. For bar graphs, data were presented as mean ± SD of at least three independent experiments. For immunoblot gel quantification, gels were scanned and band intensities quantified using Image J. The band intensity of total proteins was normalized to the loading control (GAPDH) and the band intensity of phosphorylated proteins was normalized to total proteins.

Supplementary Material

We thank Min Zhou for technical assistance. We thank the Protein Chemistry and Proteomics Facility at Technology Center for Protein Sciences of Tsinghua University for sample analysis. We are also grateful to SLSTU-Nikon Biological Imaging Center (Center of Pharmaceutical Technology, Tsinghua University, Beijing, China) for imaging support. This work was supported by grants from the National Natural Science Foundation of China (31988101 to Y.G.C), the National Key Research and Development Program of China (2022YFC3401500 and 2022YFC2502500 to X.L., 2023YFA1800603 to Y.G.C), the National Natural Science Foundation of China (22193073 and 92253305 to X.L.), and the Beijing Outstanding Young Scientist Program (BJJWZYJH01201910001001 to X.L.).

Abbreviations used:

BMP bone morphogenetic protein

CEBIT compartmentalization of enhanced biomolecular interactions in test tubes

CoPIC compartmentalization of protein-protein interactions in cells

EMT epithelial–mesenchymal transition

FRET fluorescence resonance energy transfer

TGF-β transforming growth factor-β.

This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E24-02-0073) on May 2, 2024.
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