Dvl PDZ Domain Research Articles

Disheveled-1 Interacts with Claudin-5 and Contributes to Norrin-Induced Endothelial Barrier Restoration.

Previous studies have revealed that norrin can reverse vascular endothelial-growth-factor (VEGF)-induced permeability in a β-catenin-dependent pathway. Here, we have explored the contribution of disheveled-1 (DVL1) in norrin-induced blood-retinal barrier (BRB) restoration. We provide evidence that in addition to canonical signaling, DVL1 promotes tight junction (TJ) stabilization through a novel, non-canonical signaling pathway involving direct claudin-5 (CLDN5) binding. Immunofluorescence staining of rat retinal cross-sections showed enriched expression of DVL1 and 3 at endothelial capillaries and co-localization with CLDN5 and ZO-1 at the TJ complex in primary bovine retinal endothelial cells (BRECs). Barrier properties of BRECs were determined via measurements of trans-endothelial electrical resistance (TEER) or permeability to 70 kDa RITC-dextran. These studies demonstrated that norrin restoration of barrier properties after VEGF treatment required DVL1 as an siRNA knockdown of Dvl1 but not Dvl2 or Dvl3, reduced basal barrier properties and ablated norrin-induced barrier restoration. However, loss of Dvl1 did not decrease β-catenin signaling activity as measured by Axin2 mRNA expression, suggesting the contribution of a non-canonical pathway. DVL and TJ protein interactions were analyzed via co-immunoprecipitation of endogenous protein in BRECs, which demonstrated that DVL1 interacts with both CLDN5 and ZO-1, while DVL3 interacts only with ZO-1. These interactions were most abundant after inducing BRB restoration by treating BRECs with VEGF and norrin. DVL has previously been shown to form intramolecular bindings between the C-terminal PDZ-binding motif (PDZ-BM) with an internal PDZ domain. Co-transfection of HEK293 cells with DVL1 and CLDN5 or relevant mutants revealed that DVL1 interacts with CLDN5 through the DVL PDZ domain binding, CLDN5 PDZ-BM, in competition with DVL1 PDZ-BM, since DVL/CLDN5 interaction increases with deletion of the DVL1 PDZ-BM and decreases by co-expressing the C-terminal fragment of DVL1 containing the PDZ-BM or through deletion of CLDN5 PDZ-BM. In BREC cells, transfection of the C-terminal fragment of DVL1 downregulates the expression of CLDN5 but does not affect the expression of other proteins of the TJs, including ZO-1, occludin, CLDN1 or VE-cadherin. Blocking DVL1/CLDN5 interaction increased basal permeability and prevented norrin induction of barrier properties after VEGF. Combined with previous data, these results demonstrate that norrin signals through both a canonical β-catenin pathway and a non-canonical signaling pathway by which DVL1 directly binds to CLDN5 to promote barrier properties.

Porcupine and CBP/β-catenin are the most suitable targets for the inhibition of canonical Wnt signaling in colorectal carcinoma cell lines*

Aim: The activation of canonical Wnt pathway is etiologically associated with the development of colorectal cancers. There are many possible molecular targets for the therapeutic abrogation of Wnt/β-catenin signaling. The aim of this study was to select the best molecular targets for the attenuation of β-catenin-dependent gene expression in colorectal cancer cell lines. Material/Methods: An siRNA screen was used for the selection of the best molecular targets for the down-regulation of TCF/LEF-dependent GFP expression in HCT116 cells. The level of the expression of β-catenin target genes was analyzed by qPCR. The effect of the tested chemicals on cell migration, cell cycle and apoptosis was assessed by the wound healing assay, flow cytometric analysis of propidium iodide stained cells and flow cytometric analysis of the activity of caspases-3/7, respectively. Results: Of the forty three genes which were tested in the screening stage, eight (KDM6A, KDM1A, PORCN, KDM4C, CARM1, DVL1, CBP, KMT2A) were selected as most promising. Small molecule inhibitors of these proteins (GSK-J4, GSK-LSD1, IWP-2, ML324, MS049, Dvl-PDZ Domain Inhibitor II, PRI-724, MM-102) were further used. The inhibitors of Porcupine (IWP-2) and CBP (PRI-724) were most effective in the down-regulation of the expression of β-catenin target genes and the induction of apoptosis in HCT116 cells, but showed weaker effects in DLD-1 cells. Conclusions: The inhibition of CBP and Porcupine is the most effective in attenuating canonical Wnt signaling in colon cancer cells. Future studies should determine which factors affect the sensitivity towards these promising anti-cancer agents.

Discovery of Potent Disheveled/Dvl Inhibitors Using Virtual Screening Optimized With NMR-Based Docking Performance Index.

Most solid tumors have their own cancer stem cells (CSCs), which are resistant to standard chemo-therapies. Recent reports have described that Wnt pathway plays a key role in self-renewal and tumorigenesis of CSCs. Regarding the Wnt/β-catenin pathway, Dvl (mammalian Disheveled) is an attractive target of drug discovery. After analyzing the PDZ domain of human Dvl1 (Dvl1-PDZ) using NMR, we subjected it to preliminary NMR titration studies with 17 potential PDZ-binding molecules including CalBioChem-322338, a commercially available Dvl PDZ domain inhibitor. Next, we performed virtual screening (VS) using the program GOLD with nine parameter sets. Results were evaluated using the NMR-derived docking performance index (NMR-DPI). One parameter set of GOLD docking showing the best NMR-DPI was selected and used for the second VS against 5,135 compounds. The second docking trial identified more than 1,700 compounds that exhibited higher scores than CalBioChem-322338. Subsequent NMR titration experiments with five new candidate molecules (NPL-4001, 4004, 4011, 4012, and 4013), Dvl1-PDZ revealed larger chemical shift changes than those of CalBioChem-322338. Finally, these compounds showed partial proliferation inhibition activity against BT-20, a triple negative breast cancer (TNBC) cell. These compounds are promising Wnt pathway inhibitors that are potentially useful for anti-TNBC therapy.

Discovery of a small-molecule inhibitor of Dvl-CXXC5 interaction by computational approaches.

The Wnt/β-catenin signaling pathway plays a significant role in the control of osteoblastogenesis and bone formation. CXXC finger protein 5 (CXXC5) has been recently identified as a negative feedback regulator of osteoblast differentiation through a specific interaction with Dishevelled (Dvl) protein. It was reported that targeting the Dvl-CXXC5 interaction could be a novel anabolic therapeutic target for osteoporosis. In this study, complex structure of Dvl PDZ domain and CXXC5 peptide was simulated with molecular dynamics (MD). Based on the structural analysis of binding modes of MD-simulated Dvl PDZ domain with CXXC5 peptide and crystal Dvl PDZ domain with synthetic peptide-ligands, we generated two different pharmacophore models and applied pharmacophore-based virtual screening to discover potent inhibitors of the Dvl-CXXC5 interaction for the anabolic therapy of osteoporosis. Analysis of 16 compounds selected by means of a virtual screening protocol yielded four compounds that effectively disrupted the Dvl-CXXC5 interaction in the fluorescence polarization assay. Potential compounds were validated by fluorescence spectroscopy and nuclear magnetic resonance. We successfully identified a highly potent inhibitor, BMD4722, which directly binds to the Dvl PDZ domain and disrupts the Dvl-CXXC5 interaction. Overall, CXXC5-Dvl PDZ domain complex based pharmacophore combined with various traditional and simple computational methods is a promising approach for the development of modulators targeting the Dvl-CXXC5 interaction, and the potent inhibitor BMD4722 could serve as a starting point to discover or design more potent and specific the Dvl-CXXC5 interaction disruptors.

Low-density lipoprotein receptor-related protein 6 is a novel coreceptor of protease-activated receptor-2 in the dynamics of cancer-associated β-catenin stabilization.

Protease-activated receptor-2 (PAR2) plays a central role in cancer; however, the molecular machinery of PAR2-instigated tumors remains to be elucidated. We show that PAR2 is a potent inducer of β-catenin stabilization, a core process in cancer biology, leading to its transcriptional activity. Novel association of low-density lipoprotein-related protein 6 (LRP6), a known coreceptor of Frizzleds (Fz), with PAR2 takes place following PAR2 activation. The association between PAR2 and LRP6 was demonstrated employing co-immunoprecipitation, bioluminescence resonance energy transfer (BRET), and confocal microscopy analysis. The association was further supported by ZDOCK protein-protein server. PAR2-LRP6 interaction promotes rapid phosphorylation of LRP6, which results in the recruitment of Axin. Confocal microscopy of PAR2-driven mammary gland tumors in vivo, as well as in vitro confirms the association between PAR2 and LRP6. Indeed, shRNA silencing of LRP6 potently inhibits PAR2-induced β-catenin stabilization, demonstrating its critical role in the induced path. We have previously shown a novel link between protease-activated receptor-1 (PAR1) and β-catenin stabilization, both in a transgenic (tg) mouse model with overexpression of human PAR1 (hPar1) in the mammary glands, and in cancer epithelial cell lines. Unlike in PAR1-Gα13 axis, both Gα12 and Gα13 are equally involved in PAR2-induced β-catenin stabilization. Disheveled (DVL) is translocated to the cell nucleus through the DVL-PDZ domain. Collectively, our data demonstrate a novel PAR2-LRP6-Axin interaction as a key axis of PAR2-induced β-catenin stabilization in cancer. This newly described axis enhances our understanding of cancer biology, and opens new avenues for future development of anti-cancer therapies.

Identification of small-molecule compounds targeting the dishevelled PDZ domain by virtual screening and binding studies

The Dishevelled (Dvl) protein, which conveys signals from receptors to the downstream effectors, is a critical constituent of the Wnt/β-catenin signaling pathway. Because the PDZ domain of Dvl protein functions through associations with a wide range of protein partners, Dvl protein involved in the Wnt signaling pathway has been considered to be therapeutic targets in cancers. In this study, we performed structure-based pharmacophore model of the Dvl PDZ domain to discover novel small-molecule binders and identified eight compounds with micromolar affinity. The most potent compound identified, BMD4702, efficiently bound to the Dvl PDZ domain with 11.2μM affinity and had a 0.186μM KD value according to surface plasmon resonance and fluorescence spectroscopy, respectively. Combining both structural–kinetic relationship analyses and docking studies, we fourmulated that the ligand-binding site is composed of three H-bonds and three hydrophobic features. Thus, our approach led to the identification of potent binders of the Dvl PDZ domain and the findings provide novel insights into structure-based approaches to design high-affinity binders for the Dvl PDZ domain.

Small molecule inhibitors of the Dishevelled-CXXC5 interaction are new drug candidates for bone anabolic osteoporosis therapy.

Bone anabolic agents promoting bone formation and rebuilding damaged bones would ideally overcome the limitations of anti‐resorptive therapy, the current standard prescription for osteoporosis. However, the currently prescribed parathyroid hormone (PTH)‐based anabolic drugs present limitations and adverse effects including osteosarcoma during long‐term use. Also, the antibody‐based anabolic drugs that are currently being developed present the potential limits in clinical application typical of macromolecule drugs. We previously identified that CXXC5 is a negative feedback regulator of the Wnt/β‐catenin pathway via its interaction with Dishevelled (Dvl) and suggested the Dvl–CXXC5 interaction as a potential target for anabolic therapy of osteoporosis. Here, we screened small‐molecule inhibitors of the Dvl–CXXC5 interaction via a newly established in vitro assay system. The screened compounds were found to activate the Wnt/β‐catenin pathway and enhance osteoblast differentiation in primary osteoblasts. The bone anabolic effects of the compounds were shown using ex vivo‐cultured calvaria. Nuclear magnetic resonance (NMR) titration analysis confirmed interaction between Dvl PDZ domain and KY‐02061, a representative of the screened compounds. Oral administration of KY‐02327, one of 55 newly synthesized KY‐02061 analogs, successfully rescued bone loss in the ovariectomized (OVX) mouse model. In conclusion, small‐molecule inhibitors of the Dvl–CXXC5 interaction that block negative feedback regulation of Wnt/β‐catenin signaling are potential candidates for the development of bone anabolic anti‐osteoporosis drugs.

Conformational change of Dishevelled plays a key regulatory role in the Wnt signaling pathways.

The intracellular signaling molecule Dishevelled (Dvl) mediates canonical and non-canonical Wnt signaling via its PDZ domain. Different pathways diverge at this point by a mechanism that remains unclear. Here we show that the peptide-binding pocket of the Dvl PDZ domain can be occupied by Dvl's own highly conserved C-terminus, inducing a closed conformation. In Xenopus, Wnt-regulated convergent extension (CE) is readily affected by Dvl mutants unable to form the closed conformation than by wild-type Dvl. We also demonstrate that while Dvl cooperates with other Wnt pathway elements to activate canonical Wnt signaling, the open conformation of Dvl more effectively activates Jun N-terminal kinase (JNK). These results suggest that together with other players in the Wnt signaling pathway, the conformational change of Dvl regulates Wnt stimulated JNK activity in the non-canonical Wnt signaling.

Abstract 322: The integrin α6A splice variant regulates Wnt/β-catenin and colorectal cancer cell proliferation through autophagic degradation of DVL2

Abstract Introduction: Colorectal cancer (CRC) is the second cause of death by cancer in North America. Recently, we found that both subunits of the integrin α6β4 are up-regulated in colon primary tumours. The α6 integrin subunit exists as two distinct splice isoforms, α6A and α6B, and only the α6A isoform is up-regulated in CRC. Moreover, we discovered that the integrin formed by the α6A isoform is a pro-proliferative integrin regulating the Wnt/β-catenin pathway and tumorigenesis of CRC cells. However, the exact mechanism by which the α6A splice variant controls the Wnt/β-catenin pathway is still unknown. Aim: In the present study, we investigated the mechanism underlying the regulation of Wnt/β-catenin by the integrin α6Aβ4 in human CRC, focusing on the degradation of DVL2 by autophagy. Methods: In order to investigate the mechanism involved in the regulation of the Wnt/β-catenin pathway by the integrin α6Aβ4, expression of the splice variant α6A was specifically abolished, using a small-hairpin RNA approach, in the colon adenocarcinoma cell line T84. Results: Knockdown of the integrin α6A subunit resulted in a significant repression of a number of Wnt/β-catenin pathway end points, including a decrease in DVL2 protein levels. Further analysis demonstrated that the Wnt/β-catenin pathway is restored by pharmacological inhibition of GSK3β, (SB216763) or by over-expression of a mutated β-catenin (S37A). To determine if DVL2 is degraded by autophagy following the abolition of the integrin α6A splice variant, autophagy was activated using an mTOR inhibitor (rapamycin) or inhibited using bafilomycin A1. Western blot analysis showed that subsequent activation of autophagy in T84 shα6A cells led to a significant decrease in DVL2 protein levels followed by a decrease in Wnt/β-catenin pathway end points, while inhibition of autophagy led to a significant increase in DVL2 protein levels. However, no significant change in DVL2 protein levels was observed by the modulation of autophagy in T84 shctl cells. Subsequent analysis revealed comparable autophagy flux between T84 shctl and T84 shα6A cells. The targeting of DVL2 to the autophagosome in T84 shα6A cells was confirmed by a strong co-localization of DVL with p62/SQSTM1. In order to confirm the significant relevance of the decrease of DVL2, both shctl and shα6A cells were treated with a DVL-PDZ domain inhibitor. Preliminary results demonstrated that inhibition of the DVL-PDZ domain led to a decrease in proliferation only in T84 shctl cells. Conclusion: Our study demonstrated for the first time a novel function for the α6A integrin subunit in the control of the degradation of DVL2 by autophagy to regulate the Wnt/β-catenin pathway and cell proliferation. Taken together, these results highlight the possibility for a new therapeutic treatment for CRC taking advantage of autophagy and the mechanism of regulation of the Wnt/β-catenin by the integrin α6A subunit. Citation Format: Jean-Francois Groulx, Jean-Francois Beaulieu. The integrin α6A splice variant regulates Wnt/β-catenin and colorectal cancer cell proliferation through autophagic degradation of DVL2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 322. doi:10.1158/1538-7445.AM2014-322

Cholesterol selectively activates canonical Wnt signalling over non-canonical Wnt signalling

Wnt proteins control diverse biological processes through β-catenin-dependent canonical signaling and β-catenin-independent non-canonical signaling. The mechanisms by which these signaling pathways are differentially triggered and controlled are not fully understood. Dishevelled (Dvl) is a scaffold protein that serves as the branch point of these pathways. Here, we show that cholesterol selectively activates canonical Wnt signaling over non-canonical signaling under physiological conditions by specifically facilitating the membrane recruitment of the PDZ domain of Dvl and its interaction with other proteins. Single molecule imaging analysis shows that cholesterol is enriched around the Wnt-activated Frizzled and low-density lipoprotein receptor-related protein 5/6 receptors and plays an essential role for Dvl-mediated formation and maintenance of the canonical Wnt signaling complex. Collectively, our results suggest a new regulatory role of cholesterol in Wnt signaling and a potential link between cellular cholesterol levels and the balance between canonical and non-canonical Wnt signaling activities.

Cholesterol binding is a determinant of canonical Wnt signaling activity of Dishevelled (598.3)

The Wnt cell‐cell signaling pathway plays a critical and evolutionarily conserved role in directing cell fates through the β‐catenin‐dependent canonical pathway and other non‐canonical pathways like planar cell polarity (PCP) pathway. Dishevelled (Dvl) determines the direction of Wnt and PCP signaling, however, it is still unknown how Dvl directs the two pathways. Here, we show that cholesterol in the plasma membrane serves as a regulatory lipid, selectively driving the progression of canonical Wnt signaling over PCP signaling by specifically interacting with the PDZ domain of Dvl. Single Molecule imaging shows that cholesterol is essential for sustained association of Dvl with Axin in response to a canonical Wnt ligand, but not for the formation of the PCP signaling complex component like Damm1. After the treatment with the cholesterol oxidase, it shows a tremendously decrease of Dvl and Axin interaction. Single molecule tracking also shows local enrichment of cholesterol near the Frizzle receptor upon its binding to a canonical Wnt ligand through the Frizzle co‐localization with the cholesterol binding domain, D4. Collectively, our results suggest a new regulatory role of cellular cholesterol and a potential link between cellular cholesterol levels and canonical Wnt signaling activities.

Synthesis of Potent Dishevelled PDZ Domain Inhibitors Guided by Virtual Screening and NMR Studies

Dishevelled (Dvl) PDZ domains transduce Wnt signals from the membrane-bound receptor Frizzled to the downstream. As abnormal Wnt signaling has been implicated in tumorigenesis, the Dvl PDZ domain is a potential target for small-molecule inhibitors that block Wnt signaling at the Dvl level. We expanded our in silico search to examine the chemical space near previously developed PDZ binders and identified nine additional compounds bind to the Dvl PDZ. We then performed a quantitative structure-activity relationship (QSAR) analysis of these compounds and combined these results with structural studies of the PDZ domain in complex with the compounds to design and synthesize a group of new, further optimized compounds. Two rounds of synthesis and testing yielded a total of six compounds that have greatly improved binding affinity to the Dvl PDZ domain and most potent ones competitively displace Dapper peptide from the PDZ domain. In addition to providing more potent Dvl PDZ domain inhibitors, this study demonstrates that virtual screening and structural studies can be powerful tools in guiding the chemical synthesis hit-to-lead optimization stage during the drug discovery process.

Identification of Transmembrane Protein 88 (TMEM88) as a Dishevelled-binding Protein

Wnt signaling pathways are involved in embryonic development and adult tissue maintenance and have been implicated in tumorigenesis. Dishevelled (Dvl/Dsh) protein is one of key components in Wnt signaling and plays essential roles in regulating these pathways through protein-protein interactions. Identifying and characterizing Dvl-binding proteins are key steps toward understanding biological functions. Given that the tripeptide VWV (Val-Trp-Val) binds to the PDZ domain of Dvl, we searched publically available databases to identify proteins containing the VWV motif at the C terminus that could be novel Dvl-binding partners. On the basis of the cellular localization and expression patterns of the candidates, we selected for further study the TMEM88 (target protein transmembrane 88), a two-transmembrane-type protein. The interaction between the PDZ domain of Dvl and the C-terminal tail of TMEM88 was confirmed by using NMR and fluorescence spectroscopy. Furthermore, in HEK293 cells, TMEM88 attenuated the Wnt/β-catenin signaling induced by Wnt-1 ligand in a dose-dependent manner, and TMEM88 knockdown by RNAi increased Wnt activity. In Xenopus, TMEM88 protein is sublocalized at the cell membrane and inhibits Wnt signaling induced by Xdsh but not β-catenin. In addition, TMEM88 protein inhibits the formation of a secondary axis normally induced by Xdsh. The findings suggest that TMEM88 plays a role in regulating Wnt signaling. Indeed, analysis of microarray data revealed that the expression of the Tmem88 gene was strongly correlated with that of Wnt signaling-related genes in embryonic mouse intestines. Together, we propose that TMEM88 associates with Dvl proteins and regulates Wnt signaling in a context-dependent manner.

Discovery and Characterization of a Small Molecule Inhibitor of the PDZ Domain of Dishevelled

Dishevelled (Dvl) is an essential protein in the Wnt signaling pathways; it uses its PDZ domain to transduce the Wnt signals from the membrane receptor Frizzled to downstream components. Here, we report identifying a drug-like small molecule compound through structure-based ligand screening and NMR spectroscopy and show the compound to interact at low micromolar affinity with the PDZ domain of Dvl. In a Xenopus testing system, the compound could permeate the cell membrane and block the Wnt signaling pathways. In addition, the compound inhibited Wnt signaling and reduced the levels of apoptosis in the hyaloid vessels of eye. Moreover, this compound also suppressed the growth of prostate cancer PC-3 cells. These biological effects suggest that by blocking the PDZ domain of Dvl, the compound identified in our studies effectively inhibits the Wnt signaling and thus provides a useful tool for studies dissecting the Wnt signaling pathways.

Sequence requirement and subtype specificity in the high‐affinity interaction between human frizzled and dishevelled proteins

Members of the Wnt family of lipoglycoproteins initiate signaling by binding to Frizzled (Fz) receptors, and the signal is then relayed by Disheveled (Dvl). The Dvl PDZ domain is known to interact directly with a peptide derived from the KTXXXW motif of Fz7, which is conserved in all known Fz subtypes. We found that an extended region spanning the KTXXXW motif on both its N-terminal and C-terminal sides dramatically influences the affinity of peptides derived from Fz7 for Dvl PDZ. An alanine scanning study identified the specific residues external to the KTXXXW motif that are important for high-affinity binding. In a circular dichroism analysis, mutation of some of these critical residues resulted in peptide conformational changes, suggesting that the secondary structure of the peptides contributes to Fz-Dvl PDZ binding. Of the 10 known Fz subtypes, peptides derived from only Fz1, Fz2, Fz3, Fz4, and Fz7 directly bound to Dvl PDZ domain in our study. Other Fz subtypes, including some known to be involved in Wnt/beta-catenin signaling (Fz5, Fz9), did not bind to Dvl, suggesting that direct interaction with Dvl PDZ does not determine the subtype-specific functionality of Fz. Molecular modeling and circular dichroism studies indicated that the Fz peptides that bind to Dvl PDZ domain form specific conformations that are different from those of nonbinding peptides.

Identification of tripeptides recognized by the PDZ domain of Dishevelled

The development of inhibitors of Dishevelled (Dvl) PDZ protein–protein interactions attracts attention due to a possible application in drug discovery and development. Using nuclear magnetic resonance (NMR) spectroscopy, we found that a tripeptide VVV binds to the PDZ domain of Dvl, which is a key component involved in Wnt signaling. Using a computational approach calculating the binding free energy of the complexes of the Dvl PDZ domain and each of the tripeptides VXV (X: any amino acid residue except Pro), we found that a tripeptide VWV had the highest binding affinity. Consistent with the computational result, experimental results showed that the binding of the tripeptide VWV to the Dvl PDZ domain was stronger than that of the tripeptide VVV. The binding affinity of the tripeptide VWV was comparable to that of the organic molecule NSC668036, which was the first identified Dvl PDZ inhibitor. The three-dimensional structure of the complex Dvl1 PDZ/VWV was determined to investigate the role of the energetically favorable W(−1) residue in binding. These interactions were also explored by using molecular dynamic simulation and the molecular mechanics Poisson–Boltzmann surface area method. Taken together, these two tripeptides may be used as modulators of Wnt signaling or as a scaffold to optimize an antagonist for targeting Dvl1 PDZ protein–protein interaction.

Simulations of a specific inhibitor of the dishevelled PDZ domain

The dishevelled (Dvl) PDZ domain is believed to play an essential role in the canonical and noncanonical Wnt signaling pathways, which are involved in embryo development as well as in tumorigenesis. Also, it binds directly to frizzled (Fz) receptors. An organic molecule (NSC668036) from the National Cancer Institute small-molecule library has been identified to be able to bind to the Dvl PDZ domain. Molecular dynamics simulation was used to provide detailed analyses of the binding between them.

Optimizing Dvl PDZ domain inhibitor by exploring chemical space

Because of advances in the high-throughput screening technology, identification of a hit that can bind to a target protein has become a relatively easy task; however, in the process of drug discovery, the following hit-to-lead and lead optimization still remain challenging. In a typical hit-to-lead and lead optimization process, the analogues of the most promising hits are synthesized for the development of structure-activity relationship (SAR) analysis, and in turn, in the effort of optimization of lead compounds, such analysis provides guidance for the further synthesis. The synthesis processes are usually long and labor-intensive. In silico searching has becoming an alternative approach to explore SAR especially with millions of compounds ready to be screened and most of them can be easily obtained. Here, we report our discovery of 15 new Dishevelled PDZ domain inhibitors by using such an approach. In our studies, we first developed a pharmacophore model based on NSC668036, an inhibitor previously identified in our laboratory; based on the model, we then screened the ChemDiv database by using an algorithm that combines similarity search and docking procedures; finally, we selected potent inhibitors based on docking analysis and examined them by using NMR spectroscopy. NMR experiments showed that all the 15 compounds we chose bound to the PDZ domain tighter than NSC668036.

Targeting Dishevelled, a mediator of Wnt/β-Catenin Signaling Pathway, in Human Non—Small-Cell Lung Cancer

Lung cancer is the leading cause among cancer-related deaths worldwide. The Wnt/β-catenin signaling pathway has been proven to be activated in non—small-cell lung cancer (NSCLC). We have previously reported that Dishevelled-3 (Dvl-3), interacting with the intracellular domain of Wnt signal receptor Frizzled (Fz) and functioning as a key mediator of the Wnt/β-catenin pathway, is over-expressed in NSCLC tissue samples. We also found that knocking down Dvl-3 by siRNA suppressed NSCLC cell growth through downregulating cytosolic β-catenin level and TCF-dependent transcriptional activity, hallmarks of activation of the Wnt/β-catenin signaling pathway. Moreover, knocking down Dvl-3 induced cell-cycle arrest and sensitized NSCLC cells to cisplatin treatment. More recently, we rationally designed a small-molecule inhibitor that interrupted the interaction between the intracellular domain of Fz receptor and the PDZ domain of Dvl. We found that this compound downregulated the Wnt/β-catenin signaling pathway induced apoptosis in NSCLC cells in vitro and suppressed tumor growth in vivo. Taken together, these data suggest that small-molecular inhibition of the oncogenic Dvl function might have potential therapeutic implications in the treatment of patients with NSCLC.

Indole-2-amide based biochemical antagonist of Dishevelled PDZ domain interaction down-regulates Dishevelled-driven Tcf transcriptional activity

We designed and synthesized a series of indole-2-amide-based compounds that antagonize interaction between the Dishevelled (Dvl) PDZ domain and a peptide derived from the natural PDZ ligand Frizzled-7 (Fz7). These compounds inhibit Tcf-mediated transcription activated by exogenous Dvl via the biochemical antagonism. We confirmed tumor cell-selective activation of caspases by these compounds.