The canonical Wingless-relatedintegration site signaling pathwayplays a critical role in human physiology, and its dysregulation canlead to an array of diseases. β-Catenin is a multifunctionalprotein within this pathway and an attractive yet challenging therapeutictarget, most notably in oncology. This has stimulated the search forpotent small-molecule inhibitors binding directly to the β-cateninsurface to inhibit its protein–protein interactions and downstreamsignaling. Here, we provide an account of the claimed (and some putative)small-molecule ligands of β-catenin from the literature. Throughin silico analysis, we show that most of these molecules contain promiscuouschemical substructures notorious for interfering with screening assays.Finally, and in line with this analysis, we demonstrate using orthogonalbiophysical techniques that none of the examined small molecules bindat the surface of β-catenin. While shedding doubts on theirreported mode of action, this study also reaffirms β-cateninas a prominent target in drug discovery. At the crux of the canonicalpathway, the protein β-catenin fulfills multiple roles underlyingsignal transduction and structural maintenance and is critical forembryonic development and adult tissue homeostasis.radation 1 left. Tradation 1, right.6 Overall, the degradative regulation mechanism of β-cateninoffers a fast cellular response mechanism at the post-translationallevel upon extracellular stimuli.7These oncogenes, and others, play key roles in the inception andprogression of many cancer types, giving rise to the various hallmarksof cancer.8 including osteoarthritis,9 neurodegenerative diseases,11 inflammatory bowel disease,12 type IIdiabetes,13 and multiple cancer types.22 In particular, an intimate relationship between the misregulationof Wnt signaling and numerous subtypes of colorectal cancer (CRC)is now well established.1 However, currenttherapies targeting CRC remain largely toxic and ineffective, makingCRC the second leading cause of cancer-related death in developedcountries.23 Crucially, the hyperactivityof the signal transducer protein β-catenin (encoded by the CTNNB1gene) is a hallmark of CRC, and the modulation of its activity hasbeen proposed as a promising strategy for developing novel classesof anticancer drugs.28 The small-molecule inhibition of diverse componentsof canonical Wnt signaling has recently been summarized in an excellentreview.29The importance of this regulatorymechanism is underscored by the observation that the dysregulationof Wnt/β-catenin signaling contributes to the development ofan array of diseases1, 30 XAV939 (2) stabilizes Axin through tankyrase inhibition, resulting in enhancedβ-catenin degradation.31 Pyrvinium(3) promotes β-catenin phosphorylation throughcasein kinase activation32 and ICG-001(4) binds to the cyclic adenosine monophosphate responseelement binding protein binding protein (CBP) and prevents its interactionwith β-catenin in the nucleus.33 Althoughall the abovementioned strategies aim to downregulate the activityof β-catenin, their indirect inhibitory effects on β-cateninactivity raise important concerns regarding their selectivity andmode of action as their molecular targets also interact with numerousother transcription factors from other signaling pathways. Additionally,oncogenic mutations leading to the inactivation of the DC  or the desensitization of β-catenin to the DC invariablyresult in β-catenin stabilization34 and will lead to limited response to molecular agents targetingthe DC or upstream components.1In its active form, thesignal transducer protein β-catenin acts as a transcriptionfactor but also plays a key role in cell adhesion through its interactionwith the cadherins. Despite the current paradigm, the relationshipslinking these complex and intricate protein–protein interaction(PPI) cascades and the resulting phenotypes are only partly understood.Thus, there is enormous interest in developing small molecules thattarget β-catenin-mediated signaling as chemical probes to dissectimportant molecular recognition events controlling these PPIs andtheir phenotypic effects, but also as proof-of-principle lead compoundstoward first-in-class therapeutics in disease, notably CRC. Importantly,the spatial and temporal controls that are afforded by small moleculescompared to more traditional gene knockouts or RNAi present significantadvantages for studying the complex molecular physiology of this system.To date, limited success has been achieved by indirectly targetingβ-catenin. For example, the porcupine inhibitor IWP-1 1, 2 prevent28 Full-length β-catenin is an 85 kDa protein, with residues127–682 forming the structured “armadillo repeat domain”, the cytoplasm, and the nucleus . Its distinctive structureresembling that of a super helix is composed of 12 repeat units, whichare themselves composed of three short α-helical segments .46 The α-helicalgroove in ARD-repeat 1  and the hydrophobic groove .49 These hotspot residues contribute disproportionatelyto the binding affinity of Tcf4/Lef1 and BCL9 and, ultimately, theoncogenicity of these PPIs.The direct targetingof β-catenin would conversely be highly desirable for the developmentof more specific chemical probes/drugs, as β-catenin is a specificcomponent of the Wnt/β-catenin signaling pathway. Notably, thetargeted/selective PPI disruption between β-catenin and itsnuclear co-regulators downstream of the DC has attracted significantinterest in inhibiting Wnt signaling and cancer cell proliferation.segments 3A. Imporsegments 3A–C have 1 pink, 3A,C is eE-cadherin). In particular, the inhibition ofthe β-catenin/Tcf4 interaction has raised concerns as Tcf4 sharesits binding surface with E-cadherin. The β-catenin/E-cadherincomplex is a key regulator of cell adhesion and membrane integrity.This in part explains why most screening campaigns targeting β-cateninhave relied on functional assays rather than focusing on specificbinding and PPI inhibition. However, Cong and co-workers showed thatthe soluble, oncogenic, β-catenin pool can be effectively andselectively targeted for degradation without affecting the cadherin-boundpool, mainly through exploiting the lower affinity of the β-catenin/TCF4PPI.50 This is interesting as it suggeststhat the increased sensitivity of oncogenic β-catenin can beexploited toward small-molecule inhibition without affecting celladhesion. The development of potent chemical probes directly targetingβ-catenin will be crucial not only for their potential therapeuticapplications but equally importantly for the accurate functional elucidationof these PPIs in human biology. Overall, the high affinity of β-cateninPPIs , large interactingsurfaces , and the high degreeofoverlap of the binding surfaces makes the development of small-moleculeinhibitors a daunting task.The presence of these well-characterized interaction hotspots atthe surface of the ARD suggests that the modulation of these PPIsshould be possible through the devising of selective and potent disruptors competing with β-catenin partners. However,several hotspots engage in overlapping and mutually exclusive PPIswith several high affinity co-regulators having either oncogenic  or tumor suppressor functions  boundto a low affinity small molecule fragment been reported (vide infra).This lack of structural and biophysical knowledge has generally hamperedfurther hit-to-lead optimization and perhaps provides an explanationfor why none of these reported hits from high-throughput screeningcampaigns have advanced to clinical evaluation.Despite such challenges,a small numberof compounds targeting the surface of β-catenin have been reported4 and areH), equilibrium constant (Ka or Kd), and stoichiometry(n) of a molecular association event, from whichthe Gibbs free energy (ΔG) and entropy of binding(ΔS) can also be deduced. Hence, both the bindingaffinity and its thermodynamic components can be accessed from a singleexperiment. Access to the thermodynamic fingerprints of β-cateninligands will be an important milestone to understand the nature ofthe protein–ligand interactions at play. Ultimately, we anticipatethat this study will benefit the Wnt community by clarifying the bindingand mode of action of these compounds. We believe that this is a criticaland necessary step toward future structural studies and binding modeelucidation, and ultimately developing suitable strategies for theirsynthetic optimization toward potent, first-in-class therapeutic agentstargeting the Wnt signaling pathway. An analogous study of small-moleculeKeap1-Nrf2 PPI inhibitors was undertaken in 2019 by Tran et al., usingbiophysical techniques , which highlighted that half of the reportedinhibitors were in fact inactive or deviated from reported activities.51The aim of this study is twofold.(i) In the first part, we are providing a comprehensive review ofthe small-molecule β-catenin inhibitors reported in the literatureup to 2020, focusing on their proposed mode of action and potency,along with available biophysical/biological data on target engagementand their binding mode to β-catenin; and (ii) in the secondpart, we are investigating the molecular interactions mediating β-catenininhibition by reported (and some putative) β-catenin ligands/inhibitorsfrom the literature using orthogonal in vitro biophysical techniques,namely isothermal titration calorimetry (ITC) and differential scanningfluorimetry (DSF). ITC, in particular, is label free and consideredone of the gold standards for studying molecular interactions in thesolution phase. It allows direct and accurate measurement of the bindingenthalpy  by ITC and DSF.We also highlight that several of these molecules are chemically labiledue to pan assay interference (PAIN) chemical functionalities in theircore structures, including reactive quinones and α,β-unsaturatedcarbonyls, redox active systems, reactive 2-hydroxybenzylamine andheterocycles, and metal chelators. The promiscuity of these PAIN substructuresin both biophysical and cellular assays has been extensively reviewedand raises significant concerns in regard to the proposed mode ofaction of these compounds.54 Following a primary screen of 7000 natural productsand 45,000 compounds from the Novartis collections, natural products 5–10 displayed concentration-dependentinhibition of complex formation. Interestingly, the three structurallyrelated natural products CGP049090 (6), PKF115-584 (7), and PKF222-815 (8) could inhibit the β-catenin/Tcf4interaction in protein extracts from HCT116 colon cancer cells (IC50s = 10–30 μM). In particular, 6 and 7 proved effective in inhibiting Wnt-specific reportergenes, colon cancer cell viability, and β-catenin-dependentaxis duplication in Xenopus embryos.This is in line with later studies from Sukhdeo et al., highlightingthe inhibitory effect of 7 in the xenograft models ofhuman multiple myeloma.55 The ability of 6 and 7 to inhibit the β-catenin/APC andβ-catenin/tcf4 interactions suggests they bind to β-catenin,however, the binding site and affinities of such interactions remainelusive. Planar polyaromatic compounds 7 and 8 also interfered with tcf4-DNA complexation, independent of β-catenin,whereas 9 and 10 selectively blocked thetcf4/β-catenin complex with DNA, without changes in β-cateninlevels.In 2004, Lepourcelet reported a set of small-moleculeinhibitors of the β-catenin (aa 134–668)/Tcf4 (aa 8–54)interaction using a high-throughput ELISA-based assay.58 and screeneda library of 17,700 compounds from the Pharmacia and Upjohn collectionagainst the K435 hotspot. Following docking score analysis and visualinspection of docking poses, 3 out of 22 candidates displayed bindingand partial competition with Tcf4 (aa 1–53) in subsequent WaterLOGSYnuclear magnetic resonance (NMR) and ITC. PNU-74654 (11) was the most potent and displayed a Kd of 450 nM against the β-catenin ARD (aa 134–671) inVP-ITC experiments, although no structure–activity relationship(SAR) data were disclosed. On the basis of docking studies, the methylgroup of 11 was proposed to bind to a narrow cleft linedby E571 and K508, while the hydrophobic phenoxybenzene was proposedto interact at the polar K435 hotspot. Unambiguous binding mode determinationby X-ray crystallography and biological activity data were not reported.Of note, 11 displayed a highly entropic binding signature, raising concerns aboutits binding specificity.In 2006, Trosset and co-workers performed a computationalpocket detection of the human β-catenin/tcf3 complex (PDB—1G3J)using PASS and FLO_QXP tools to identify potential hotspots for structure-guideddrug design,R-etodolac (12) and brominated analogueSDX-308 were shown to inhibit β-catenin/TCF signaling  and induce apoptosis in drug-sensitive and drug-resistantmultiple myeloma cell lines.59 SDX-308was shown to decrease nuclear β-catenin levels without affectingtotal β-catenin levels in MM1S and OPM1 cells, thus inhibitingβ-catenin/TCF transcriptional activity. A large number of 2,4-diaminoquinazolineswere reported as Wnt/β-catenin inhibitors between 2009 and 2016.14  as a β-catenin/Tcf-4PPI inhibitor in a cell-based luciferase reporter screening assayand subsequent SAR studies.60 However, 14 and its derivatives did not show in vivo efficacy in HT29and HCT116 xenografts. In 2010, Dehnhardt and co-workers identifieda range of 2,4-diaminoquinazoline analogues with improved in vivoprofiles. Administration of representative lead molecule 13 (TOPFlash in Tcf33.13 cell line IC50 = 0.60 μM)at 150 mg/kg suppressed xenograft tumor growth in vivo by up to 66%in a β-catenin/RK3E mouse model; however, administration wasintraperitoneal (IP) due to poor bioavailability.61 In 2012, Mao and colleagues reported a series of similar2,4-diaminoquinazolines, identified using a HCT116 luciferase reporterscreening assay and a growth inhibition assay against HCT116 and SW480cell lines.62 Representative 15 inhibited Wnt signaling in HCT116 (IC50 = 2.0 μM)and inhibited growth of HCT116 and SW480 cell lines . The in vivo activity of 15 and its analogues was not reported. In 2016, Li and co-workersidentified a family of 2,4-diaminoquinazolines as Wnt/β-catenininhibitors using a TopFlash Wnt reporter assay in HCT116 cells, followingscreening of an in-house library of 500 quinazoline derivatives. SRI-31230(16) was the most potent, inhibiting TOPFLASH (IC50 = 13 μM) and inducing dose-dependent reductions inAxin2/cyclinD1/Survivin protein levels while displaying consistentviability reduction in cancer lines HCT116 (IC50 = 6.2μM), SW480 (IC50 = 5.9 μM), and SW620 (IC50 = 6.7 μM).64 Although the structuralsimilarities between these molecules are worth noting, the molecularmechanisms and binding events underlying such activity remain unclear.It has been suggested that these compounds exert their effect viathe inhibition of the β-catenin/Tcf4 protein–proteininteraction, possibly highlighting the folded β-catenin ARDas a possible molecular target.65In 2009, Chen and co-workers identified lead 2,4-diaminoquinazoline 66 Interestingly, primary screening identified 13 structurally related,tri-substituted quinoxalines which reproducibly reduced A439 cellproliferation, including representative derivative 17 (IC50 < 5 μM). This correlated with the inhibitionof TOPflash reporter gene activity by 30–90% and decreasedlevels of nuclear β-catenin. A subsequent study identified GDK-100017(18), which inhibited the proliferation of A549/Wnt2and SW480 cells in a dose- and time-dependent manner (IC50 = 10 μM),67 while showing littleeffect in L132 human embryonic pulmonary epithelial cells. This effectin cancer cells correlated with the downregulation of Wnt target genescyclin D1 and DKK1, while the expression levels of c-myc and survivinremained unaffected. 18 induced cell cycle arrest inA549 cells in combination with radiation compared with either treatmentalone. Interestingly, several quinoxaline derivatives have been independentlyidentified by Zhang as Wnt/β-catenin inhibitors, potentiallysuggesting a common molecular target (vide infra).68In 2010, Lee and co-workers screened ∼1500 diverse heterocycliccompounds using a cascade of assays looking at the inhibition of cellproliferation followed by a cell-based reporter assay measuring β-catenin/Tcftranscriptional activity in A549/Wnt2 cells.69 Hit compounds were subjected to a screeningworkflow that involved a secondary TOPFlash assay, de-convolution,and hit triage, which afforded three hits. CCT031374 (19) was among the three most promising compounds, with GI50 = 13.9 and 13.2 μM in HCT116 and SW480 cells, respectively. 19 was hypothesized to act at the β-catenin level, byblocking TCF-dependent transcription and increasing the degradationof endogenous wt-β-catenin in murine L-cells, was stabilizedby the GSK3 inhibitor BIO. The molecular mechanism underlying inhibitionwas not elucidated, although it was hinted at possible binding of 19 to the ARD. The blocking of TCF-dependent transcriptionin SW480 colon cancer cells was not accompanied by the downregulationof β-catenin levels. Furthermore, the levels of stabilized β-cateninin HEK293 cells were not reduced when treated with 19, although TCF-dependent transcription was blocked.In the same year, Ewan et al. used a primary luciferase-basedassayto screen a library of ∼63k low molecular weight compounds.70 This screen of ca. 15,000compounds from various proprietary collections identified a familyof structurally related heterocyclic compounds displaying >70%inhibitionof signaling. Representative oxazole containing iCRT3 (20) was among the most potent analogues, selectively inhibiting nuclearβ-catenin-mediated transcription (IC50 = 1.8 μM),β-catenin/Tcf4 complex formation in vitro and in HEK293 celllysate. Conversely, 20 had no noticeable effect on β-catenininteractions with E-cadherin and α-catenin,or noncanonical Wnt signaling. 20 was hypothesized tobind the K435/R469 hotspot of the ARD based on docking and DSF experiments,although the exact molecular interactions involved and the associatedaffinity of such an interaction have not been discussed. Of note, 20 induced thermal shifts <1 °C in DSF experiments,suggesting that potential binding affinity to the ARD is likely tobe rather weak , contrasting with its reportedcellular activity (low μM).68 Ofnote, some analogues including iCRT-4, -9, -16, and -21 significantlyinterfered with several signaling pathways including notch, Hedgehog (hh), and Janus kinase/signaltransducer and activator of transcription.In 2011,Gonsalves employed a cellular RNA-based screening assaywith a luciferase reporter to identify small-molecule inhibitors ofβ-catenin/Tcf4-mediated transcription downstream of the DC,following induction of reporter gene activity via RNAi-mediated Axininhibition.βcat-D16tcf4 interactionhotspot on the ARD was probed in 2012 by Tian and co-workers, by Autodock4virtual screening of ca. 2000 compounds from a diversity set of theNational Cancer Institute open database.72 Docking identified organocopper complex BC21 (21) asa potential ligand of β-catenin. Complex 21 decreasedβ-catenin-dependent TCF reporter activity in HCT116 and HEK293cells at low micromolar concentrations and modestly downregulatedWnt target genes (c-Myc and cyclin D1) and reduced HCT116 viability(IC50 = 15 μM); while in FP competition experiments, 21 displayed modest displacement of a labeled tcf4 peptide(IC50 = 5 μM), the precise molecular mechanisms andbinding specificity are unknown. Also noted by the authors, 21 had also been shown to inhibit protein phosphatase 2C,the proteasome, and to have cytotoxic effects in a range of humancancer cells.74 These potential off-target effectswere not investigated in this study.The same K43575 The meta-structure concept was proposed as aframework to identify potential ligands directly from a protein’sprimary sequence. This identified fluorescein (22), eosinY (23), 2-phenoxybenzoate (24), and relatedimine derivative (25) as potential ligands of the ARD.Both ligands showed binding to the β-catenin ARD (aa 134–671)in saturation transfer difference (STD) NMR experiments. However,the affinity and precise binding mode/site of these ligands were notreported. It is also worth noting that both aromatics of the biphenyletherin 24 and 25 are unlikely to adopt a coplanararrangement but rather give rise to a significant dihedral angle.This suggests that all four compounds may not share a unique bindingmode/site. Although the authors highlighted their plan to exploitthese results for the development of more potent β-catenin ligands,no follow-up study has been reported.In the same year, Henenand co-workers employed a meta-structureapproach combined with NMR to pinpoint potential β-catenin ligands.26) as a β-catenin/Bcl9 PPI inhibitor.7626 selectively inhibited (Ki = 3 μM) the interaction between the N-terminalregion of β-catenin (aa 134–671)77 and the BCL9 homology domain 2 (aa 343–396). 26 is thought to bind  to a transient binding motif in part formedby the intrinsically labile helix 1  of theARD. The interaction of 26 with this region was shownto exacerbate the propensity of the ARD to aggregate in vitro andin cells, leading to its proteasome-mediated depletion. This proposeddegradative mode of action was also correlated with the downregulationof β-catenin-dependent TOPFlash activity and the downregulationof the mRNA levels of Wnt targets AXIN2 and B9L in HeLa, HCT116, andSW480 cells. The peculiar degradative mode of action of 26 has so far hampered accurate biophysical and structural characterization,which rely on the presence of the folded protein.An ELISA-based screenof 47,500 LOPAC/PhytoPure/MRCT compoundsby de la Roche identified the natural product carnosic acid (68 Substituted acylhydrazide R360163 (27), rhodanineL338192 (28), quinoxaline R999636 (29),and biphenylether T155535 (30) were identified as modestlyselective inhibitors showing moderate potency (Ki ∼ 8.8–77 μM) and selectivity (1.1–25fold) in vitro for the β-catenin-Tcf4 interaction over APC and E-cadherin. These compounds have been proposed to engagethe β-catenin K435 hotspot targeted by D16 of Tcf4, althoughdirect evidence through structural methods or site-directed mutagenesis(SDM) has not been reported. Interestingly, 30 is yetanother example of a biphenylether derivative. The Ji laboratory hasused a structure-based design strategy toward several series of small-moleculepeptidomimetics targeting the ARD. Isosteric replacement and conformationalrestriction of D16/E17 of Tcf4 led to the development of UU-T01 (31), the most potent member of a family of heterocyclic β-catenin/Tcf4interaction in vitro inhibitors.78 Thebinding of 31 to the ARD (aa 142–686) was characterizedby FP (Ki = 3 μM) and VP-ITC . Of note, this is an unusually high ligandefficiency (LE = 0.51), in addition to the binding being stronglydriven by a favorable entropic term. Based on modeling studies, 31 was proposed to mimic D16/E17 of Tcf4 and engage in polarinteractions with β-catenin hotspot residues K435, K508, andR469. This was further supported by its reduced binding affinity againstthe corresponding K435A, K508A, and R469A mutants. However, the authorsdid not report on the biological activity of 31. A thirdpublication from the Ji laboratory the following year reported disruptorsof the β-catenin-Tcf4 PPI, utilizing a peptidomimetic strategyin conjunction with computational modeling.79 SiteMap and MCSS analyses assessed the druggability of the regionof β-catenin that interacts with the GANDE sequence in Tcf4.80 Using the Tcf D16/E17 hotspot residues as a basic scaffold, diversemotifs were incorporated to probe and evolve the peptidomimetic. MCSSanalysis suggested that a chloroindole motif would be tolerated ina narrow channel formed by residues R474 and R515 at the ARD surface,adjacent to the K435 hotspot. This channel is present in the crystalstructures of the ARD bound to human TCF4 and xenopus Tcf3 (PDB—1G3J) and accommodates these regulators,but is not present in the ARD apo structure (PDB—2Z6H). Furthermore,a range of hydrophobic amino acids were examined as replacements forN15 to probe the adjacent hydrophobic pocket (39 (Ki = 0.64 μM). AlphaScreen, VP-ITC, and SDM all supportedthe direct binding of 39 and derivatives to β-catenin(VP-ITC Kd ≈ 0.42 μM). Theprotein expression levels of cyclin D1 and c-Myc were reduced upontreatment with 39 in SW480 cells. Equally noteworthy,the binding of 34 is highly entropic, suggesting thatit is primarily entropically driven . This suggests again that binding may be driven primarilyby hydrophobic interactions and water displacement rather than polarand directional interactions . This was not discussedby the authors.In 2013, Zhanget al. using in-house AlphaScreen and FP assaysto look at selective inhibitors of the β-catenin (aa 138–686)–Tcf4(aa 7–51) interaction, screened a library of 250 commerciallyavailable carboxylic acid-containing compounds.c pocket 3B formed32 in2015 as an inhibitor of the β-catenin-Tcf4 interaction identifiedthrough dual AlphaScreen and FP assays and SAR analysis.81 One close derivative of 32 suppressedcanonical Wnt signaling, downregulated the expression of Wnt targetgenes, and inhibited the growth of cancer cells.The Ji laboratory reported the small molecule 8634 was designed by a bioisostere-based fragment hoppingprotocol to mimic hydrophobic side chains at positions i, i + 3 and i + 7 of an α-helix. 34 was subsequently overlayed with the BCL9 S352-F374 α-helixto design small-molecule inhibitors of the β-catenin-BCL9 PPI.Iterative design-synthesis-evaluation were performed to identify 34 as the most potent and selective inhibitor with KiαS = 2.1 μM. 34 wasfurther characterized by SDM and VP-ITC (Kd = 0.33 μM). Later in 2018, the derivatives of 34 were reported with KiαS = 0.47μM and selectivity >1900 .87 Along with carnosic acid 26, 34 and its derivatives represent the onlyexamples of small molecules targeting the ARD N-terminus. The inhibitionof the β-catenin/BCL9 PPI has been proposed as an alternativestrategy to achieve controlled Wnt signaling inhibition in a rangeof CRCs.88 The interaction of β-cateninwith BCL9 in the nucleus is necessary for downstream signaling. BCL9is one of the few partners interacting with the ARD N-terminus, suggestingthat the development of selective PPI inhibitors may be possible.This may have important implications for overcoming the challengeof achieving selective inhibition of the β-catenin/Tcf4 oncogenicPPI. This is because Tcf4 shares overlapping PPI surfaces with a rangeof other regulators, notably E-cadherin. Wnt signalingactivation and alteration of E-cadherin functionare key factors in the epithelial–mesenchymal transition andmetastasis.Hoggard etal. used HippDB to data mine the PDB and identify α-helicalpeptidomimetics to target the β-catenin/BCL9 PPI.35) as an inhibitor of the β-catenin(aa 134–668) and Tcf4 (aa 1–79) PPI in an AlphaScreenhigh-throughput screening (HTS) of 16000 synthetic compounds fromthe ChemBioNet collection.89 Thiourea 35 exhibited an IC50 ca. 2 μM in AlphaScreenand ELISA assays, although the affinity and binding site/mode of 35 at the ARD surface were not reported. Preliminary SAR studieshighlighted that the sulfonamide group is vital for potency, illustratedby a complete loss of potency upon its removal. The replacement ofthe styryl unit with other terminal aromatic groups such as phenyland benzyl led to a 2–50 fold reduction in inhibitory activity.Immunoprecipitation experiments showed that 35 couldinhibit β-catenin/Tcf4 interaction in HCT116 cell lysates inthe micromolar range while showing no disruption of E-cadherin-mediated cell adhesion. This also correlated with the downregulatedexpression of a range of Wnt target genes  and tumor growth reduction in NOD/SCIDmice.Early in 2016, Fang and Birchmeier published 4-thioureido-benzenesulfonamidederivative LF3 (33).90 The treatmentof non-small cell lung cancer H1299 cells with low μM concentrationsof 33 downregulated c-Myc and cyclin D1 expression, promotedthe S phase arrest and increased levels of reactive oxygen species(ROS) to mediate increased radiation sensitivity. In FP experiments, 33 inhibited the β-catenin/Tcf4 (aa 8–30) PPI(IC50 = 1.7 μM) and inhibited TOPFlash activity ina dose-dependent manner (IC50 = 2.3 μM) in H1299cells. Based on Autodock4 docking studies, the authors suggested that 33 competes with tcf4 through binding to the K435 hotspot,making hydrophobic contacts and H-bond interactions with the sidechains of neighboring residues C429, N430, and K508. A key considerationis the chemical lability of the chloroquinone motif, which is highlyreactive toward a range of nucleophiles, notably sulfur nucleophilessuch as cysteine at physiological pH. We will discuss this later inthis study. This raises important concerns regarding the specificityand mode of action of BC-23.In the same year, Zhang and co-workers reported the smallmoleculeBC-23 (36) following a cell-based HTS luciferase assay of 22,000 compounds.9136 selectively inhibited luciferaseactivity (IC50 ∼ 2–5 μM) and reducedviability (IC50 ∼ 2–6 μM) of 12 Wnt-dependentcancer cell lines, while showing comparatively little effect on Wnt-independentcell lines. 36 selectively induced size and weight reductionin a panel of Wnt driven HCT116, HCT115, and H23 tumors in xenograftedmouse models. This was in line with apoptotic induction and elevatedcleaved caspase-3 in mouse tumor tissues treated with 36. This effect also correlated with dose-dependent reduction of mRNAlevels of downstream Wnt target genes AXIN-2, c-MYC, cyclin D1, andBMP4 in a panel of Wnt-dependent cell lines, along with reduced levelsof their respective proteins. Initial biophysical analysis using STDNMR, SPR, and pull-down experiments suggested that 36 binds to the ARD C-terminal region (aa 301–670). The authorssuggested that the binding of 36 to β-catenin inthe cellular environment mediates its proteasomal degradation, althoughthis remains largely speculative.Hwang and Lee reported the smallmolecule benzenesulfonamide derivativemethyl 3-{[(4-methylphenyl)sulfonyl]amino}benzoate (MSAB) (37) was reportedin 2017 by Dong et al.9237 inhibited β-catenin/Tcf4 luciferase activity in a dose-dependentmanner (IC50 = 2.3 μM), downregulated the expressionof cyclin D1 and Axin2, and induced growth reduction of DLD1, CACO2,and HCT116 cells. 37 also selectively inhibited the growthof patient-derived xenograft CRC model displaying elevated β-cateninlevels. 37 conjugation to sepharose 4B-beads followedby affinity pull-down in cell lysates and western blotting (WB) selectivelyidentified β-catenin as a direct molecular target (affinityunknown). Of note, 37 presents several structural similaritieswith GDK100017 (18), including a fluorinated nitrogenheterobicyclic system connected to a conformationally constrainedphenyl ring. Glide docking suggested that 37 binds tothe surface of the ARD, possibly through key H-bonding interactionswith G307 and K312.Substituted benzimidazoleHI-B1 (38) as a dual inhibitor ofMDM2 and β-catenin in pancreatic cancer.9338 potently reduced the levels of active,unphosphorylated β-catenin and downstream c-Myc and cyclin D1proteins in a concentration-dependent manner in Panc-1 and AsPC-1pancreatic cancer cells (IC50s < 0.5 μM) and tumors(40 mg/kg), harboring elevated levels of β-catenin. Interestingly,WB and immunofluorescence experiments showed that 38 hadcomparatively low effects on the levels of phosphorylated β-cateninand the membrane bound β-catenin fraction, suggesting that itselectively targets the soluble, active pool of cytoplasmic and nuclearβ-catenin. Pull-down experiments in Panc-1 and AsPC-1 cellswith a biotinylated analogue of 38 preferentially precipitatedβ-catenin, suggesting it as a direct target (affinity unknown).The overall molecular mechanisms underlying such inhibition remainunclear, although it was suggested that binding of 38 to a yet to characterize region of the ARD could induce its aggregationand proteasome-mediated degradation. A similar degradative mode ofaction was suggested by de la Roche while studying carnosic acid (26).76 It is also worth mentioningthat the structurally related beta-carboline derivatives have beenreported to inhibit Wnt signaling through the activation of CK-1αrather than binding to β-catenin.94 Further clarification through direct binding assays will be criticalto validate the molecular target(s) of beta-carbolines.Several series of β-carbolines wereindependently reportedas Wnt signaling inhibitors between 2014 and 2018. Wang and co-workersidentified compound SP141  contain substructural motifswhich are notorious for their overrepresentation as hits across adiverse range of HTS assays.53 complemented with SwissADMEcomputational analysis, to investigate potential inherent toxicity/reactivityalerts .Here, we soughtto investigate the occurrence of PAINS and/or Brenk alerts in reportedsmall-molecule β-catenin inhibitors, which we believe is a criticalfirst step to (i) discarding potentially inherently toxic/reactive“dead-end” compounds; and (ii) identifying potentiallysuitable starting points for future structural studies and lead optimizationtoward first-in-class chemical probes and therapeutic agents targetingβ-catenin. First, we combined visual inspection and extensivePAINS filters,yalerts 1. SwissA5–9, 22, 23,and 33 all contain quinone, quinone methide, or polyaromaticmotifs prone to redox activity, electrophilic reactivity, and singletoxygen production.102 ALARM NMR experimentallydemonstrated that 10 and pyrimidotriazinedione derivativeswere intrinsically reactive along with displaying potent redox cyclingin a phenol red-horseradish peroxidase screen (EC50 <2.0 μM).105 The same two assays also unambiguously demonstratedthe inherent thiol reactivity and redox cycling of a diverse rangeof substituted rhodanines, chloro- and/or benzo-quinones, catechols,benzofurazans, and other extended polyaromatic systems, reminiscentof the chemical structures of 5–9, 26, 28, 32, and 33. The core scaffolds of 5–9 alsoshare structural similarities with a number of anthracyclines, whichare well-known for their DNA-damaging activity.95 Benzofurazans are widely employed as fluorescent-labelingreagents due to their high reactivity and long excitation/emissionwavelengths.107 A number of electrophilic benzofurazanshave also displayed modest inhibition of DNA and RNA synthesis.108 Among other electrophilic substructures (videsupra), an important number of benzofurazans exhibit intrinsic reactivitytoward nucleophiles,104 raising questionsabout the specificity of electrophilic pyrazinofurazan 32.The benzofurazan PAIN scaffold is knownto react reversibly with thiols, including by electron transfer.21 contains a phenolicMannich base motif prone to in situ benzylic ammonium elimination(pKa ≈ 10) to the correspondinghighly electrophilic ortho-quinone methide (QM).109 QMs have generally been shown to react indiscriminatelywith a number of biological nucleophiles, including amino acid sidechains and DNA bases. Two publications have also highlighted 21 as an inhibitor of both PP2C and flavivirus MTase, suggestingother possible modes of action.11021 alsoexhibited cytotoxicity in BHK-21 cells (CC50 = 11 μM),at concentrations lower than its IC50 = 17 μM.110 A range of phenolic Mannich base-containingcompounds structurally similar to 21 have also been shownto covalently modify catalytic tautomerase via QM generation and subsequentproline N-alkylation.111 These stabilityissues, along with the potential for in situ metal decomplexationin the physiological environment, suggest that 21 mayact by more than one mechanism.11 and 27 are known to undergo photoinduced E/Z isomerization112 and hydrolysis to theircorresponding hydrazides and aldehydes.Acylhydrazones are generally less stable to hydrolysis than non-acylatedhydrazones and oximes.113 Because of theirfacile hydrolysis under mildly acidic conditions, acylhydrazones havebeen employed as cleavable linkers for biotin-streptavidin biomoleculeisolation.114Acylhydrazones similar to commercial 119 However, since the introduction of systematicPAINS filters by Baell in 2010, there has been a significant decreasein reports highlighting rhodanine lead compounds.53 Rhodanines such as 28 have been shown to formaggregates, act as Michael acceptors, interfere with fluorescenceassays due to their photochromic properties, and chelate a varietyof metals. These have been extensively reviewed by Tomašić119 and Sink.120 Consistently,rhodanine containing 28 triggered both PAINS and Brenkalerts.Despitethe range of biological activitiesreported in the literature, rhodanines are regarded as one of themost prevalent sources of false positives in HTS campaigns. Priorto 2010, the rhodanine motif was prevalent in multiple drug discoverycampaigns and reached a peak in 2010.26 are frequent hitters in screening assays suchas AlphaScreen.96 They are notorious redoxcyclers,121 metal chelators,122 and potent electrophiles in their oxidizedform.123 For example, catechol containinggallic acid has been shown to induce apoptosis in HL-60RG cells byROS generation.12426 hasbeen extensively investigated for its anti-bacterial, -cancer, -fungal,and -viral properties,125 and multiplereports have also demonstrated that 26 is easily oxidizedin mild aerobic conditions. Although de la Roche provided supportingevidence for its binding to the ARD in vitro, it is still unclearwhether 26 itself or one of its oxidized metabolitesis responsible for its cytotoxicity.Catechols such as the natural product carnosicacid 10), labile copper complex (21), and polycyclicfurazan (34), which were not identified by the filters,despite their known chemical lability (vide supra). We found thatthe Brenk alert filter of SwissADME was generally more efficient atdetecting potentially reactive and/or toxic substructures than moretraditional/historical PAIN filters, and generated results more inline with the known promiscuity of these scaffolds. Last but not least,an important proportion of these molecules are based on chemotypeswell known to aggregate and form colloids in aqueous buffers, interferingwith a variety of screening assays. This was recently demonstratedin several excellent studies, highlighting compound aggregation asa major source of false positives in in vitro screening assays.127 Although the use of detergent additives can to some extent mitigatethis behavior, the presence of aggregation prone chemotypes presentin reported β-catenin ligands is another important consideration, while a large proportionoriginated from proprietary compound libraries. We re-synthesizedthese in-house and here provide detailed synthetic routes and associatedanalytical data . We postulated that any candidate displayingcellular activity at a low micromolar concentration is likely to exhibita binding affinity in a similar concentration range or below, andshould elicit a sharp response in our assay conditions . Positivecontrol TCF4 (aa 13–27) and BCL9 (aa 348–376) peptidesdisplayed Kds = 1.81 ± 0.11 and 1.25± 0.06 μM, respectively, in line with previous reportsand demonstrating that our ARD construct is folded and functional.45 However, toour disappointment, but in line with the abovementioned PAIN/Brenkanalysis, none of these compounds did show a measurable binding enthalpyin ITC, and failed to induce significant thermal stabilization inDSF experiments . For example, representative CGP049090 (6) did not show any binding enthalpy to the ARD in ITC, nor did fluorescein(22), eosin Y (23), or BC-23 (33). Also, in contrast with VP-ITC binding data reported by Trossetand suggesting submicromolar binding affinity to the ARD, in our hands,purified PNU-74654 11 surprisingly and consistently did not exhibit noticeable binding enthalpyin repeat ITC200 experiments, and consistently showed an absence ofprotein stabilization in DSF experiments. Interestingly and consistentwith previous reports,21 MSAB (36) showed a positive STD response in our hands despite showing littleenthalpy and protein stabilization. We also wondered whether β-catenininhibition by 36 in cells may be mediated by the in situhydrolysis of the methyl ester to generate a potentially more activecarboxylic acid analogue. To test this hypothesis, we synthesizedcarboxylic acid analogue 96 by alkaline hydrolysis of 36 (Scheme S16). Although 96 was soluble in aqueous media at a higher concentration(>1 mM) compared to parent MSAB (36), it also failedto exhibit any binding affinity or thermal stabilization of the ARD,discarding in situ activation as a potential cause for engaging theARD in cells. Based on these results, we propose that 36 is hydrophobic and that the observed STD signal is a result of weakand unspecific binding to hydrophobic patches of the ARD. Such effectshave been extensively documented and recently discussed by Zega inan excellent review.128 Overall, none ofthe compounds scrutinized in this study showed consistent bindingacross the biophysical cascade we employed.Regardlessof the presence of PAIN/Brenk motifs, we examined thebinding of these compounds to the ARD (aa 148–662). A smallnumber of compounds were available commercially (see 129 recently reported a series of trifluoromethylated pyridonederivatives acting as “molecular glues” that stabilizethe interaction of the β-catenin N-terminus (aa 16–50)with SCFβ-TrCP E3 ligase via binding to anarrow transient pocket at the PPI interface in vitro. However, itis not completely clear whether this pocket is present upon complexformation with FL β-catenin in cells. Recently, a number ofanthraquinone derivatives, such as BC-2059 (Tegavivint), were shownto inhibit Wnt signaling through binding to transducin beta-like protein1 (TBL1) and inhibiting its interaction with nuclear β-catenin,making the latter vulnerable to degradation.130 In the future, it would be interesting to see whether the compoundsdiscussed above have any effect on the TBL1:β-catenin interaction.We also cannot discard that the binding of some of these compoundsin cells may depend on specific, although yet to characterize post-translationalmodifications at the surface of β-catenin. However, the completeabsence of binding to recombinant ARD makes it improbable, and morelikely, they act through another, yet to be identified, molecularmechanism in cells. Here, we aim to draw attention and caution tothe proposed cellular mode of action of these inhibitors, and suggestthey may exert cytotoxicity and inhibit signaling via other, possiblyβ-catenin-independent mechanisms. These avenues will be thefocus of future studies in our laboratories. These results are significantas many of these molecules are still sold as β-catenin ligandsat a high price, and this study to a large extent debunks the gapsin knowledge regarding their binding properties and will inform thecommunity on their suitability as chemical probes of Wnt signaling.This work will also likely have important implications in the (re)interpretationof a large number of published studies employing these molecules asβ-catenin inhibitors. We believe that an important focus offuture screening campaigns should be the choice of suitable assaycascades with lower susceptibility to false positives for identifyingnew direct β-catenin inhibitors. Critical to success will bethe combination of orthogonal in vitro assays for hit enrichment andvalidation, to inform structural studies and future lead optimization.For example, representative methods such as DSF, NMR, and ITC seemwell-positioned for that purpose and span a wide throughput versusaccuracy range, and their combination has proven effective in thefragment-based ligand discovery area for targeting highly challengingPPIs previously thought undruggable.131 While preparing this manuscript, colleagues from Boehringer Ingelheimused a combination of NMR and microscale thermophoresis to cross-validatefragment-sized ligands of the ARD, and reported the first crystalstructure of a fragment-sized molecule bound to a truncated ARD construct(repeats 1–4).132 Although thesefragments display modest affinity  and do not overlap with relevant PPIs atthe ARD surface, this provides support for such in vitro screeningcascades. Overall, we believe this study provides compelling evidencethat β-catenin remains a highly attractive target in cancer.In this study, we providea comprehensive overview of claimed (andsome putative) “direct” small-molecule inhibitors ofβ-catenin reported in the literature, along with their bindingproperties and proposed molecular mode of action. However, we showthat the majority of these molecules contain PAIN/Brenk structuralmotifs, notorious for their promiscuity in screening assays. Regardless,we assayed these compounds in orthogonal biophysical assays, lookingat both binding affinity (ITC) and protein thermal stabilization (DSF).In line with their suspected promiscuity, we demonstrate that noneof these molecules exhibit a direct measurable interaction with purifiedhuman ARD, at least not in the concentration range reported for theirbioactivity. ITC, in particular, is a label-free technique, and whileconsidered one of the gold standards to assess biomolecular interactions,it has not been systematically employed in the field. We also highlightthat despite important progress in the development of PAIN filtersin the last decade, a number of known problematic substructures arestill escaping triage, potentially leading to the investigation offlat SAR and/or “dead-end” scaffolds. We believe thatthis has been an important contributor to the common view that β-cateninis an undruggable target and perhaps partly explains why none of themolecules presented above made it to advanced clinical evaluation.Altogether, our data evidence that apart from a small number of highmolecular weight peptidomimetics, there is currently no unambiguouslyconfirmed, potent small-molecule ligand of the β-catenin ARD.Although it is not impossible that some of these compounds identifiedin phenotypic screens may bind to β-catenin N- or C-terminalregions, it appears unlikely as there is sufficient evidence thatthey are unstructured and deprived of a well-defined, druggable pocket.One possibility is that some of these compounds target transient bindingsites formed upon association of the N- or C- termini with co-regulatorssuch as TCF4 and BCL9. Simonetta and co-workers


In this study, a rubber-composite-nanoparticle-modified epoxy powder composite coating with low curing temperature and high toughness was successfully fabricated. The effects of N,N-dimethylhexadecylamine (DMA) carboxy-terminated nitrile rubber (CNBR) composite nanoparticles on the microstructure, curing behavior, and mechanical properties of epoxy-powder coating were systematically investigated. SEM and TEM analysis revealed a uniform dispersion of DMA-CNBR in the epoxy-powder coating, with average diameter of 100 nm. The curing temperature of the epoxy-composite coatings had reduced almost 19.1% with the addition of 1phr DMA-4CNBR into the coating. Impact strength tests confirmed that DMA-CNBR-modified epoxy-composite coatings showed significant improvements compared with the neat EP coating, which was potentially attributed to the nanoscale dispersion of DMA-CNBR particles in epoxy coatings and their role in triggering microcracks. Other mechanical properties, including adhesion and cupping values, were improved in the same manner. In addition, thermal and surface properties were also studied. The prepared epoxy composite powder coating with the combination of low curing temperature and high toughness broadened the application range of the epoxy coatings. Epoxy resin is an important thermosetting resin that has been used in the manufacture of coatings ,2,3, adh2 nanoparticles has led to significant improvements in the impact strength, tensile strength, and tensile modulus of epoxy-powder coatings [3-modified powder coatings are found to have significantly improved the cupping properties and tensile properties compared with unmodified powder coatings [Despite these advantages, the applications of epoxy-powder coatings are limited due to their high curing temperatures and brittleness. Therefore, the preparation of modified epoxy-powder coatings to improve their comprehensive performance has become an urgent problem to be solved. In fact, some studies have introduced inorganic nanoparticles, rubber particles, and thermoplastic resins into epoxy resin to improve its mechanical properties ,15,16,17coatings ,18,19. Ncoatings ,21. Diegcoatings  report tcoatings ,24. HoweThe high curing temperature of epoxy-powder coatings limits their application in heat-sensitive substrates. Curing accelerators are a class of substances that can effectively reduce the curing temperature and reaction activation energy, and shorten the curing time ,26. BasiUp to now, there is no study to propose a solution that can solve both the brittleness and high curing temperature of epoxy coatings. In order to overcome the brittleness and high curing temperature of epoxy-powder coatings, this study investigated the effect of DMA-CNBR as a toughening modifier and curing promoter in epoxy-powder coatings. Carboxy-terminated nitrile rubber (CNBR), a kind of powder rubber with an average size of 100 nm, was prepared by the combined technologies of irradiation and spry drying , while DCommercial bisphenol A-based epoxy resins 604  were supplied by the Anhui Hengyuan Group . The curing agent dicyandiamide was supplied by Sinopharm Chemical Reagent Co., Ltd. . A defoaming agent (benzoin) and a flatting agent 588 were provided by South Sea Chemical . Carboxy-terminated nitrile rubber (CNBR) nanoparticle was supplied by the Beijing Research Institute of Chemical Industry . The N,N-dimethylhexadecylamine (DMA) was purchased from Beijing Waokai Technology Co., Ltd .The composite DMA-CNBR was prepared by mechanically mixing carboxy-terminated nitrile rubbers and liquid tertiary amine. In this process, different amounts of liquid tertiary amine were absorbed onto carboxy-terminated nitrile rubbers. The mass ratio of tertiary amine to rubber was 1:4, abbreviated as DMA-4CNBR.S1-90 °C, TS2-100 °C, TS3-110 °C, and TS4-120 °C. The blending material was cooled to room temperature and pressed into sheets through a roller machine. Then, this kind of sheet material was pulverized in the whirlwind ultrafine pulverizer  with 1500 revolutions per min. The milled powder coating was screened through a screen mesh with a mesh size 160 mesh. The powder coatings were sprayed by an electrostatic spray gun (OptiStar Type CG13) with an output voltage of 60 kV to the tinplate plates. Finally, the sprayed coated samples were cured at 180°C for 10 min and cooled, and their properties were investigated.The epoxy coating modified by DMA-CNBR was prepared as follows. In the first step, the quality of each component of the DMA-CNBR/EP hybrid system was accurately weighed, including epoxy resin, dicyandiamide, DMA-CNBR, additives, etc. The content of each component in the DMA-4CNBR/epoxy nanocomposite coating systems was listed in 4 before TEM observation.The cross-section morphologies of epoxy coating samples were determined by SEM (Japan Electronics-Oxford JSM-7800(Prime), Tokyo, Japan) and TEM . Samples should be prepared by freezing sections at −80 °C and stained with OsOIn situ diffuse reflectance infrared spectroscopy  was used to test the characteristic absorption peak of the DMA-CNBR/EP coating at 180 °C. Atomic force microscopy  was used to determine the surface roughness of the epoxy coatings. Root mean square (Rq) indicates surface roughness.g) of DMA-CNBR/epoxy-powder coating were measured using the DSC , where the 3–7 mg powder coating samples were put into aluminum crucible under a nitrogen atmosphere with a flow rate of 20 mL /min and a heating rate of 10 K/min. Tg of cured epoxy-powder coating sample was calculated from the second cycle. To measure the Tg of the sample, the sample was heated to 250 °C at a heating rate of 10 K/min, cooled to room temperature, and then reheated to 150 °C at a heating rate of 10 K/min in the DSC instrument. The gelation time of the powder coatings was measured by the multi-function gelation time meter (CQ-J20). The impact strength of DMA-CNBR/epoxy-powder coating was measured by an impact meter (Elcometer 1615) according to ASTM D2794. The adhesion strength of the epoxy coatings was measured by an automatic pull-out adhesion tester . The cupping value of the epoxy-powder coatings was determined by a cupping-tester (Pushen 2705).The DSC results can be used to evaluate the thermal curves of epoxy-powder coatings during the curing process. The curing temperature and glass transition temperature  of DMA-4CNBR/EP systems decreased gradually under the same heating rate, indicating that DMA-4CNBR could enhance the reactivity of DMA-4CNBR/EP systems. The neat epoxy coating had a peak curing temperature at 202.6 °C. The DMA-4CNBR prepared in this paper could significantly reduce the curing reaction temperature of epoxy-coating systems. The 0.25phr DMA-4CNBR/EP and neat epoxy coating occurred at 185.7 and 202.6 °C, respectively, indicating that the curing reaction occurred rapidly with the addition of DMA-4CNBR. When the catalyst DMA-4CNBR addition was increased to 1 phr, the sample curing temperature was further reduced to about 164.1 °C. The curing temperatures of different contents of DMA-4CNBR-modified epoxy coating were evaluated by DSC analysis under nitrogen atmosphere. According to p of DMA-4CNBR/EP systems gradually decreased at the same heating rate, indicating that DMA-4CNBR could accelerate the curing reaction of the coating. The activation energy (Ea) can react to the activity of the curing reaction. Kissinger’s theories can be used to calculate the neat epoxy coating and different contents of DMA-4CNBR modified EP systems [p is the peak temperature during the curing process (K), R is the gas constant, and A is the pre-exponential factor (min−1). The Ea could be gained from the slope of the line obtained by linear fitting based on Kissinger’s method shown in a values calculated by Kissinger’s method. Apparently, the activation energy of 1phr DMA-4CNBR/EP is 86.1 kJ/mol, nearly half of neat epoxy coating (165.4 kJ/mol), indicating the superior catalytic performance of DMA-CNBRs. The carboxyl and nitrile group in CNBRs and the long-chain tertiary aminas could catalyze the epoxy curing reaction, resulting in a decrease in the apparent activation energy of the system [The DSC was used to measure the non-isothermal curing behaviors of different systems (neat epoxy coating and different contents of DMA-4CNBR-modified EP) under different heating rates.  systems ,33. The Impact strength is an important indicator to evaluate the mechanical properties of the coating, which is used to evaluate the impact strength of the coating or to judge the brittleness and toughness of the coating. An impact machine was used to measure the impact strength of different contents of DMA-4CNBR-modified epoxy coatings. The experimental results were shown in The adhesion test is the basic indicator for detecting the good mechanical properties of the coating and the metal interface. Good adhesion can play a role in blocking the corrosive medium from invading the matrix, avoiding the peeling of the coating caused by the long-term erosion of the corrosive medium. Adhesion drawing experiments were used to evaluate the adhesion strength between the prepared coating and the metal matrix. The adhesion test results of the composite coating with different amounts of DMA-4CNBR added are shown in The cupping test is mainly used to evaluate the resistance of the epoxy coating to dry-cracking or separation from metal substrates after it is gradually deformed under standard conditions. It is generally believed that the higher the cupping value, the better the forming performance of the epoxy coating. The results of the cupping experiment are shown in g of 1phr DMA-4CNBR/EP was raised by 4.7% from 95.4 °C to 100.0 °C, compared with the neat EP coating. The results demonstrated that DMA-4CNBR could improve the heat resistance of epoxy systems. As reported in Huang F, despite the fact CNBR was introduced in the anhydride -cured epoxy system, its heat resistance showed, instead of a decrease, an increase of 3.5% from 136.1 °C to 140.9°C, in terms of Tg [g of the epoxy/amine system not only did not decrease but increased by 4.5 °C.DSC is used to study the thermal behavior of prepared composite coatings and to study the effect of DMA-CNBR on the heat resistance of epoxy-powder coatings. ms of Tg . The rolq [q value of neat epoxy was 2.0 nm when 1phr DMA-4CNBR/epoxy-modified epoxy coating, and the Rq value of the coating was the lowest, which was 1.0 nm. The reduction in the Rq value by the addition of the 1phr DMA-4CNBR into the epoxy coating indicated that the rubber composite nanoparticles made the epoxy-composite coating superior to the neat epoxy coating.The surfaces of coating samples prepared from different epoxy powder systems were examined by AFM, and the resulting images are shown in q . As show−1 in As we all know, the nitrile group in CNBR is a Lewis base, which theoretically acts as accelerator to enhance the curing reaction during the curing reaction process of epoxy coating. The DSC data also confirm that after the DMA is adsorbed by CNBR, the curing-promotion effect is better than that of pure tertiary amine catalysts. In order to investigated whether CNBR and DMA played a synergistic catalytic role in the process of the epoxy curing reaction, in situ diffuse reflection infrared spectroscopy (in-situ DRIFTs) was performed on the 1phr DMA-4CNBR/EP coating at a curing temperature of 160 °C. The absorbance of the nitrile group in CNBR is at 2206 cmThe in-situ DRIFTs results verified that rubber particles participated in the curing reaction of epoxy powder, which played the role of a curing accelerator, and showed that DMA and CNBR were synergistically catalyzed in the curing reaction. This DSC result could also indicate that rubber particles involved in the curing reaction, and rubber particles and DMA, played a synergistic catalytic role in the epoxy curing reaction. On the other hand, the decreased curing temperature may also be attributed to the good dispersion of rubber particles in epoxy coatings, and the dispersion of DMA in the matrix is improved by the loading of rubber particles on DMA, so the curing accelerator DMA had a better catalytic effect.The rubber-composite nanoparticle-modified epoxy powder composite coating with low curing temperature and high toughness was successfully fabricated by surface adsorption and melt mixing. DMA-CNBR was uniformly dispersed in the epoxy coating with an average diameter 100 nm. Epoxy powder coatings loaded with 1phr DMA-4CNBR exhibited the lowest curing temperature and the highest mechanical performance compared to neat epoxy coatings. The DSC results showed that the curing temperature of 1phr DMA-4CNBR/EP could diminish by 38 °C compared to the neat epoxy-powder coating, while the activation energy of 1phr DMA-4CNBR/EP is 86.1 kJ/mol, nearly half of that of the neat epoxy coating (165.4 kJ/mol). The impact strength of 1phr DMA-4CNBR/EPs increased by 242%, while their adhesion strength increased by 108%. In addition, the Tg value of the 1phr DMA-4CNBR/EPs was increased.


C. jejuni DRH212; however, the presence and the role of c-di-GMP in other C. jejuni strains are unknown. Here, we investigated extracellular c-di-GMP as an environmental signal that potentially triggers biofilm formation in C. jejuni NCTC 11168 using a crystal violet-based assay, motility-based plate assay, RT-PCR and confocal laser scanning microscopy (CLSM). We found that, in presence of extracellular c-di-GMP, the biofilm formation was significantly reduced (>50%) and biofilm dispersion enhanced (up to 60%) with no effect on growth. In addition, the presence of extracellular c-di-GMP promoted chemotactic motility, inhibited the adherence of C. jejuni NCTC 11168-O to Caco-2 cells and upregulated the expression of Cj1198 , as well as chemotaxis genes Cj0284c (cheA) and Cj0448c (tlp6). Unexpectedly, the expression of Cj0643 (cbrR), containing a GGDEF-like domain and recently identified as a potential diguanylate cyclase gene, required for the synthesis of c-di-GMP, was not affected. Our findings suggest that extracellular c-di-GMP could be involved in C. jejuni gene regulation, sensing and biofilm dispersion.Cyclic diguanosine monophosphate (c-diGMP) is a ubiquitous second messenger involved in the regulation of many signalling systems in bacteria, including motility and biofilm formation. Recently, it has been reported that c-di-GMP was detected in  Campylobacter jejuni is the most common foodborne pathogen which commonly causes bacterial diarrheal illness worldwide   .9 bacterial cells per human cell in confluent cell culture) in the presence and absence of ex-c-di-GMP. The assay was performed with Caco-2 cells as previously described [9 bacteria per well. Ex-c-di-GMP competition was pre-incubating C. jejuni with 200 µM of ex-c-di-GMP. Experiments were repeated using biological replicates.Adherence rate was calculated against the size of the inoculum,  and subsequently challenged with 200 µM of ex-c-di-GMP for 3-h. The bacterial survival was confirmed by viable cell counts after 3 h. The cells were centrifugated at 4000 rpm for 15 min and pellets were used for RNA extraction by Rneasy kit according to the manufacturer’s protocol  [®® Reverse TranscriptionKit according to the manufacturer’s protocol . Real-time cycler conditions were 20 s at 95 °C, 3 s at 95 °C followed by 30 s at 60 °C with 40 cycles. All primers used in the present study are listed in  . To syntisted in , which hing gene ,57,58. AC. jejuni cells, overnight cultures, were diluted to an OD600 of 0.05, and 3 mL of each sample was placed into 6-well plate containing a glass coverslip to form the biofilm  as described above in dispersion assay. The plates were then incubated at 42 °C microaerobically for 48 h. Following incubation, MH broth was removed and replaced with PBS containing 200 µM ex-c-di-GMP, and the plates were incubated for a further 3 h and then gently washed. The coverslips were fixed using 5% formaldehyde solution for 1 h at room temperature, gently washed and stained with two fluorescent dyes as follows: (I) Calcofluor White (CFW) , which labels polysaccharides by binding to N-acetylglucosamine and sialic acid residues, at 25 μM final concentration for 20 min [Biofilm preparation was previously described in . C. jejur 20 min ,60; (II)r 20 min . All samt-test, which was carried out in GraphPad Prism . p ≤ 0.05 was considered statistically significant.The statistical significance of data generated in this study was determined using an unpaired two-tailed Student’s C. jejuni 11168-O showed that the presence of 200 µM ex-c-di-GMP reduced the ability of C. jejuni 11168-O to swarm on soft agar  were tested for their inhibitory effect on t manner A where 4t manner A. Furthet manner .C. jejuni in a strain-specific manner, C. jejuni 11168-O, C. jejuni 81-176, and C. jejuni 81116, were used to demonstrate general inhibitory effect of ex-c-di-GMP on biofilm formation  less auto-aggregation, compared to the untreated culture  was observed in adherence of C. jejuni 11168-O cells treated with 200 µM Ex-c-di-GMP, as compared with untreated cells to Caco-2 cells.To understand the effect of ex-c-di-GMP on C. jejuni 11168-O, the mature biofilm of C. jejuni 11168-O was treated with ex-c-di-GMP for 3 h  were examined in the presence and absence of ex-c-di-GMP by RT-PCR. CbrR gene as the only gene in the C. jejuni genome encoding GGDEF-like motif, reported to be potentially involved in c-di-GMP synthesis [tlp6, LuxS and CheA was upregulated in presence of 200 µM ex-c-di-GMP by 1.9 to 3-fold , within P. aeruginosa biofilm during biofilm dispersion [Since our data show that ex-c-di-GMP treatment inhibited spersion ,70,71.C. jejuni biofilm or how it enters C. jejuni cells. One of the possibilities is that C. jejuni senses this external compound as a part of the quorum sensing pathway where it might trigger a signalling cascade, modulating gene expression. Here, we have shown that luxS, a quorum sensing pathway gene, present in many bacterial species and responsible for the production of autoinducer-2 (AI-2) [C. jejuni in presence of ex-c-di-GMP. Moreover, AI-2 can mediate auto-aggregation and has been linked to pathogenicity, drug resistance, and host immune evasion [C. jejuni cells treated with 200 μM ex-c-di-GMP show reduced aggregation, similar to that reported for S. aureus [However, it is still unclear how the ex-c-di-GMP acts to inhibit 2 (AI-2) ,74,75,76 evasion . Auto-ag evasion ,78. This evasion . We foun. aureus .cheA and tlp6 are upregulated in presence of ex-c-di-GMP, in agreement with previous report where these genes were shown to be upregulated within formed biofilms [C. jejuni are indeed affected by luxS mutation Jeon et al. [flaA by 43% leading to reduction in motility. Here, we have shown that addition of ex-c-di-GMP affected C. jejuni swarming motility and enhanced the swarm zone achieved by the wild type cells, potentially as the result of overexpression of luxS. The change in motility in presence of ex-c-di-GMP could potentially reflect the transition between planktonic and biofilm states.AI-2 had also been shown to influence chemotaxis and swarming motility ,81,82. Hbiofilms . Changesbiofilms . Motilitn et al. , where tCampylobacter cells can be reasonably postulated, as it has been reported to be able to enter eukaryotic cells [Bacillus subtilis, Listeria monocytogenes and amoeba Dictyostelium discoideum [B. subtilis is able to secret c-di-AMP which impacts biofilm formation and plant root colonization [The ability of c-di-GMP to enter the ic cells . Moreovescoideum ,88,89. Fnization ,87.S. aureus infection in a mouse model [C. jejuni cells to the Caco-2 epithelial cells is significantly decreased after treatment with 200 μM ex-c-di-GMP, further suggesting that ex-c-di-GMP can potentially inhibit biofilm formation of biotic surfaces. However, the molecular bases for ex c-di-GMP inhibition of C. jejuni epithelial cell adherence are yet to be elucidated.Such messengers can also be detected by the innate immune system and can activate the expression of interferon genes and thus act as pathogen-associated molecular patterns (PAMPs) during infection ,91. Indese model . This isC. jejuni DRH212 [cbrR gene was proposed to be a negative regulator of FlaA expression and motility. However, the presence and the role of c-di-GMP in other C. jejuni strains are still unknown, and comparative gene expression between biofilm and planktonic of C. jejuni 11168-O cells showed no differences in cbrR expression [cbrR expression before and after treatment with ex-c-di-GMP have been observed in this study. It is possible, however, that this gene is constitutively expressed due to its potential role in bile resistance.It is also possible that ex-c-di-GMP enters the cells by binding to the diguanylate cyclase (DGCs) which synthesizes c-di-GMP. Recently, the c-di-GMP signalling system has been reported in i DRH212 , where cpression . SimilarC. jejuni. It is possible to speculate that the bacterial cells that may not produce c-di-GMP, can nevertheless sense extraneously available molecules, such as QS molecules, during the pathogen-host–environment interactions. This study highlights the importance ex-c-di-GMP in regulating biofilm formation and dispersion by C. jejuni and indicates that the influence of external regulators in the life cycle of this organism, may need to be closely examined. Furthermore, the sensitivity of C. jejuni biofilms to ex-c-di-GMP makes them a promising drug target for controlling biofilm formation by using c-di-GMP analogues or similar small molecules [Together, these data show that ex-c-di-GMP inhibits the initial formation of biofilms and induces dispersion of formed biofilms of olecules .


In conclusion, our data suggested that SGO1 could be a novel prognostic biomarker for lung adenocarcinoma.Shugoshin-like protein 1 (SGO1) has been characterized in its function in correct cell division and its role in centrosome cohesion in the nucleus. However, the underlying biological function and potential mechanisms of SGO1 driving the progression of lung adenocarcinoma remain unclear. In this study, we found that SGO1 was increased in LUAD tissues and cell lines. Upregulation of SGO1 expression was correlated with poor overall survival (OS), disease-free survival (DSS), and progression-free survival (PFS) in patients with LUAD. ROC curve analysis suggested that the AUC value of SGO1 was 0.983. Correlation analysis showed that SGO1 expression was related to immune infiltration in LUAD. Meanwhile, a potential ceRNA network was constructed to identify the lncRNA-MIR4435-2HG/miR-125a-5p/SGO1 regulatory axis in LUAD. Finally, we determine that SGO1 regulated the cell proliferation and cell apoptosis of lung adenocarcinoma  Lung cancer is the leading cause of mortality among all malignancies worldwide. Among all types of lung cancer, lung adenocarcinoma (LUAD) is the most common subtype, accounting for 40%–50%, of lung cancer , 2. In tHowever, most NSCLC patients are already in the advanced stage when it is initially diagnosed resulting in a short 5-year overall survival rate of patients (It has been confirmed that shugoshin 1 (SGO1) plays an important role in chromosome segregation maintaining centripetal adhesions during meiosis and mitosis . PreviouNo studies which evaluate the expression level, clinical significance, and prognostic and diagnostic values of SGO1 in LUAD can be retrieved currently. Therefore, the aim of this study was to determine the effect of SGO1 on the development of LUAD. In this study, we used The Cancer Genome Atlas (TCGA), the Genotype-Tissue Expression (GTEx), the Human Protein Atlas (HPA), and the Kaplan–Meier plotter web to determine SGO1 expression and its correlation with the prognosis. Furthermore, we used the single-sample gene set enrichment analysis (ssGSEA) method to analyze the correlation between SGO1 and immune infiltration. Besides, immunohistochemistry (IHC), qPCR, CCK8, colony formation, and flow cytometry assays were used to examine the biological function of SGO1 in LUAD progression.https://portal.gdc.cancer.gov/)  . We utilhttps://proteinatlas.org/) included various IHC images of protein of human gene information (The HPA (ormation . In thishttp://dna00.bio.kyutech.ac.jp/PrognoScan/index.html) is a new database for meta-analysis of the prognostic value of genes (PrognoScan (of genes . In thishttp://www.linkedomics.org/login.php) is a publicly available portal that includes multi-omics data from all 32 TCGA cancer types and 10 Clinical Proteomics Tumor Analysis Consortium (CPTAC) cancer cohorts. In this study, LinkedOmics was employed to obtain the genes that were significantly positively correlated with SGO1 expression in TCGA-LAUDLinkedOmics (Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment analysis (GSEA) were conducted to examine the biological and molecular functions of SGO1 across different cancer types using a total of 300 genes that were positively correlated with SGO1. All three analyses were performed using the R package Cluster Profiler. GSEA was also used to estimate the enrichment of various biological processes in each sample.Cox regression analysis, including univariate and multivariate analyses, was used to examine the prognostic value of SGO1 in LUAD. The univariate and multivariate Cox regression analyses were using the R package “forest plot” to exhibit the hazard ratio (HR), 95% CI, and P-value. The nomogram was constructed using the R package “rms.” In this research, the Kaplan–Meier method was utilized to examine the prognostic values of SGO1, miRNA, and lncRNA expression, employing R packages of survminer and survival.http://starbase.sysu.edu.cn/) is a database which includes the RNA–RNA and protein–RNA interaction networks from 108 CLIP-seq data sets generated by 37 independent studies  and cultured in BEGM media . Lung cancer cell lines, including SPC-A1, H358, A549, and H1299, were purchased from the Chinese Academy of Sciences Cell Bank  with STR documents and were cultured in RPMI-1640 medium  supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The siRNA for SGO1 and NC were transfected into cells. Transfection was performed using the Lipofectamine 3000 transfection reagent based on the manufacturer’s protocol. SGO1 siRNA: ACATGCACATACCTGAAAATG.Cell proliferation assay was performed as previously described . The indThe primer used in this study is as follows: β-actin-F: AAGTGTGACGTGGACATCCGC, β-actin-R: CCGGACTCGTCATACTCCTGCT, SGO1-F: AACTCAGCAGTCACCTCATCT, SGO1-R: TGCACCTACGTTTAGGCAGAG.Annexin V FITC Apoptosis Detection Kit I  was used to evaluate the cellular apoptosis according to the manufacturer’s instructions. For cell cycle analysis experiments, the indicated cells were digested and washed with PBS twice and then fixed in 75% alcohol overnight at −20°C. The fixed cells were washed three times and then stained with propidium iodide (PI) staining buffer at room temperature for 30 min in the dark, and then the cell cycle was analyzed using the FACSAria SORP machine .2O2 and blocking non-specific binding with 1% bovine serum albumin buffer, sections were incubated overnight at 4°C with the indicated primary antibodies. Following several washes, the sections were treated with an HRP-conjugated secondary antibody for 40 min at room temperature and stained with 3,3-diaminobenzidine tetrahydrochloride (DAB). SGOL1 antibody .For immunohistochemical staining, the sections were deparaffinized in xylene and rehydrated through graded ethanol. Antigen retrieval was performed for 20 min at 95°C with sodium citrate buffer (pH 6.0). After quenching endogenous peroxidase activity with 3% HAll statistical analyses were performed using R software, and ROC curves were used to detect SGO1 cutoff values using pROC packages. For data regarding the function of SGO1, GraphPad Prism 7.0 was used for statistical analyses.. We also estimated SGO1 expression in paired cancer tissues and adjacent normal tissues in pan-cancer based on TCGA datasets. Results confirmed that SGO1 expression was significantly higher in 14 of the 18 cancers compared with normal tissue .To determine the mRNA expression pattern of SGO1 in various human cancers, we used TCGA and GTEx datasets to conduct analyses. Results suggested that SGO1 was upregulated in 26 of the 33 cancers compared with normal tissue . Consistent with the above results, the GEO dataset also suggested that the SGO1 mRNA level was obviously increased in lung cancer tissues . Furthermore, we showed that the SGO1 protein expression in LUAD was significantly increased in lung cancer tissues .To examine the SGO1 expression level in LUAD, we analyzed SGO1 expression based on TCGA and HPA databases. We found that SGO1 was highly expressed in LUAD and LUSC .The correlation between SGO1 expression and clinical parameters in LUAD samples was explored. As shown in .Logistic regression analysis also suggested that increased SGO1 expression was associated with T stage (T2 and T3 and T4 vs. T1) (P < 0.001), N stage (N1 and N2 and N3 vs. N0) (P = 0.006), M stage (M1 vs. M0) (P = 0.008), pathologic stage (stage III and stage IV vs. stage I and stage II) (P < 0.001), gender  (P <0.001), age (>65 vs. ≤65) (P = 0.022), and smoker (yes vs. no) (P = 0.015) . We also used the GEO dataset to validate the above results. We showed that upregulation of SGO1 expression was related to adverse clinical outcomes in patients with lung cancer . We further explored the diagnostic significance of SGO1 in lung cancer, and ROC curve analysis was performed. ROC curve analysis confirmed that the AUC value of SGO1 is 0.983 . The GEO dataset was also utilized to validate the diagnosis of SGO1 in lung cancer . These results confirmed that SGO1 may be a promising biomarker for differentiating LUAD tissue from normal lung tissue.The correlation between SGO1 expression and OS, DSS, and PFS in LUAD patients was examined by the Kaplan–Meier curve. We found that an elevated SGO1 expression was correlated with adverse OS, DSS, and PFS in patients with LUAD .Prognostic values of the differential expression of SGO1 in diverse subgroups include pathological stage, TNM stage, gender, age, race, and smoker. Results confirmed that an increased SGO1 level is associated with poor prognosis .We performed univariate Cox regression analysis in TCGA-LAUD cohort to determine whether SGO1 expression level and pathologic stage might be valuable prognostic biomarkers. Univariate Cox regression analysis results show that higher expression of SGO1, pathologic stage, and TNM stage were associated with overall survival, disease-free survival, and progression-free survival in LUAD patients. To ascertain whether SGO1 expression level could be an independent prognostic factor for patients with LUAD, multivariate Cox regression analysis was performed. We confirmed that increased SGO1 expression was a significant independent prognostic factor in TCGA-LAUD cohort which directly correlated with adverse overall survival, disease-free survival, and progression-free survival . The correlation analysis of SGO1 expression and the top 8 co-expressed genes in TCGA LUAD is shown in . KEGG enrichment analysis suggested that these genes were involved in proteasome, ubiquitin-mediated proteolysis, nucleotide excision repair, non-small cell lung cancer, 53 signaling pathways, cellular senescence, Fanconi anemia pathway, oocyte meiosis, DNA replication, and cell cycle . Gene set enrichment analysis (GSEA) also showed that pathways, including the cell cycle, focal adhesion, pathway in cancer, apoptosis, oxidative phosphorylation, Wnt signaling pathway, MAPK signaling pathway, cell adhesion molecules (cams), T-cell receptor signaling pathway, natural killer cell-mediated cytotoxicity, cytokine–cytokine receptor interaction, and chemokine signaling pathway were significantly enriched in the high SGO1 expression group .As shown in .Given that gene set enrichment analysis (GSEA) mentioned above showed that SGO1 may be correlated with immune response regulation, we therefore examined the association between SGO1 expression levels and immune cell infiltration. We found that SGO1 was positively associated with the infiltration of Th2 cells, Tgd, T helper cells, and NK CD56dim cells but negatively associated with the infiltration of pDC, NK cells, CD8 T cells, DC, TFH, iDC, eosinophils, and mast cells in LUAD .Our results also discovered that the high expression level of SGO1 was positively correlated with the infiltrating degree of CD8+ T cell and neutrophil and negatively correlated with the infiltrating degree of CD4+ T cell and B cell ; among these miRNAs, we showed that miR-125a-5p, miR-125b-5p, hsa-miR-514a-5p, hsa-miR-585-3p, and hsa-miR-5691 were significantly negative with the SGO1 expression in LUAD . Further analysis revealed that only miR-125a-5p was downregulated in LUAD and that LUAD patients have a higher miR-125a-5p expression which was correlated with adverse clinical outcomes . Therefore, we decided to select miR-125a-5p to conduct downstream analysis. We further examined the upstream lncRNA targets to construct the miRNA-125a-5p–lncRNA axis. Based on the ceRNA hypothesis, miRNAs have an opposite co-expression correlation with mRNAs and lncRNAs, whereas lncRNAs have a positive co-expression correlation with mRNA . To determine the biological function of SGO1 in lung cancer cells, small interfering RNAs (siRNAs) were used to specifically knock down SGO1 expression  has been found to ensure the correct and orderly conduct of mitosis by protecting and maintaining centripetal adhesions during meiosis and mitosis . In thisIn this study, we found that SGO1 RNA and protein expressions were overexpressed in LUAD tissues. Increased SGO1 expression was correlated with pathological stage (P = 0.001) and TNM stage. Kaplan–Meier curves and univariate analysis confirmed that SGO1 expression is correlated with overall survival (OS), disease-free survival, and progression-free survival (PFS) in the LUAD patients of TCGA data. ROC curve analysis indicated that SGO1 may be a promising diagnostic biomarker for differentiating LUAD from normal tissues. Our findings are consistent with previous research. SGO1 was increased in human prostate cancer and correlated with the patients’ TNM stage, lymph node metastasis, distant metastasis, and poorer survival .Logistic regression analysis also suggested that increased SGO1 expression was associated with T stage (T2 and T3 and T4 vs. T1) (P < 0.001), N stage (N1 and N2 and N3 vs. N0) (P = 0.006), M stage (M1 vs. M0) (P = 0.008), pathologic stage (stage III and stage IV vs. stage I and stage II) (P < 0.001), gender  (P < 0.001), age (>65 vs. ≤65) (P = 0.022), and smoker (yes vs. no). Next, the univariate and multivariate analysis results suggested that SGO1 expression was an independent prognostic biomarker for LUAD.via activating the AKT-mediated signaling pathway , Yunnan Applied Basic Research Projects , and Yunnan Province Specialized Training Grant for High-Level Healthcare Professionals (D-201614).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


APC (90%) in advanced colorectal cancer (CRC) results in the simultaneous activation of Wnt/β-catenin and AKT signaling pathways, and the current therapeutic limitations of the AKT inhibitors for treating CRC patients are nuclear β-catenin-induced EMT and bypassing apoptosis. In this study, we discover that the combinatorial treatment of an AKT inhibitor and KY1022, a β-catenin destabilizer, effectively overcomes the current limitations of API-2, an AKT inhibitor, by reducing nuclear β-catenin. Taken together, we demonstrate that the simultaneous suppression of Wnt/β-catenin with the AKT signaling pathways is an ideal strategy for suppressing the AKT-inhibitor-mediated metastasis and for maximizing the therapeutic effects of AKT inhibitors.Frequent mutation of  Althougutations 0%, and celopment ,7,8,9. TThe phosphoinositide 3-kinase (PI3K)-V-AKT murine thymoma viral oncogene homolog (AKT) pathway is a major signaling axis that orchestrates the tumor progression and cell migration ,11. ActiCrosstalk between PI3K-AKT-FOXO3a and Wnt/β-catenin pathways has been discovered to have tumorigenic roles in CRC by causing an interaction between FOXO3a and β-catenin . SynergiAPC mutation. To understand the inhibition mechanisms of KY1022 involved in the AKT-inhibitor-induced cell migration, we performed immunocytochemistry (ICC) with KY1022, with or without API-2, and we observed that KY1022 effectively decreased the level of β-catenin and FOXO3a in the nucleus of LoVo cells. Next, we aimed to test whether the destabilization of β-catenin via the KY1022 treatment effectively overcomes the API-2-mediated apoptosis resistance. Using flow cytometry analysis, we observed that KY1022 treatment induces the apoptosis of CRC. Given the inhibition results of KY1022 on API-2-induced cell motility and overcoming the effects of KY1022 on API-2-induced resistance to apoptosis, our study suggests that the combination treatment targeting the Wnt/β-catenin pathway with the AKT inhibitors is a promising therapeutic approach to improve the clinical efficiency of treating CRC patients harboring an APC mutation.Given that anti-EMT  effects of KY1022, a β-catenin destabilizer, were reported in our previous study ,21, we aLoVo cells were provided by Prof. Kang-Yell Choi . LoVo cells were tested for authentication using short tandem repeat profiling (Cosmogenetech). LoVo cells were cultured in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum . KY1022 was provided by Prof. Gyoonhee Han . KY1022 and API-2  were dissolved in dimethyl sulfoxide  for the in vitro studies.The gene-expression data analyzed from the NCBI GEO databases are publicly available (accession numbers GSE17536). The data were obtained and processed using Biometric Research Branch (BRB)-Array Tools  (version 4.5.0) .n = 4).LoVo cells were seeded to collagen-coated coverslips (500 μg/mL). The cells were treated with DMSO, API-2 (10 μM), KY1022 (20 μM), and the combination treatment with EGF (20 ng/mL) for 18 h. Fixation was performed with the coverslips in 10% neutral formaldehyde for 2 h, followed by the permeabilization with 0.1% Triton X-100  for 30 min and blocking in 5% bovine serum albumin (BSA) for 1 h. The coverslips were incubated overnight at 4 °C with anti-β-catenin  and anti-FOXO3a , followed by anti-mouse Alexa Flour 488  or the anti-rabbit Alex Fluor 555  secondary antibody for 1 h at room temperature. Primary and secondary antibodies were diluted with phosphate-buffered saline (PBS) containing 1% BSA and 1% normal goat serum . The coverslips were counterstained with 4’, 6’-diamidino-2-phenylindole  and covered with Gel/Mount media . For staining the cytoskeleton, fixation was performed with 10% neutral formaldehyde, and then the cells were permeabilized, blocked, and incubated with Alexa Fluor 568 phalloidin  for 30 min. Immunofluorescent images were captured using confocal microscopy . At least three fields per section were analyzed by Zen V3.1  software . Mean ± s.d. were analyzed, based on three or five biological replicates.LoVo cells were seeded in collagen (500 μg/mL)-coated 6-well plates. Once they were grown confluently (80%), the cells were scratched with a 200 μL tip. Then, DMEM (10% FBS) containing EGF (20 ng/mL) was changed with DMSO, API-2 (10 μM), KY1022 (20 μM), and the combination treatment with API-2 (10 μM) and KY1022 (20 μM) for 18 h. Following the treatments, the cells were stained with crystal violet (Sigma-Aldrich). The wound areas were captured and recorded using a microscope  in a humidified 5% CO4 cells) into 6-well plates and incubated with a treatment of DMSO, API-2 (10 μM), KY1022 (20 μM), and a combination treatment with API-2 (10 μM) and KY1022 (20 μM) for 36 h. The cells were collected and resuspended with annexin ν binding buffer containing 10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2 at 1 × 106 cells/mL. The cells (200 μL) in annexin ν binding buffer were suspended with 5 μL annexin ν -FITC (Sigma-Aldrich) and 10 μL propidium iodide  for 15 min in the dark. A total of 800 μL of annexin ν binding buffer was aliquoted to each tube and analyzed by a BD AccuriTM flow cytometer (BD bioscience). Mean ± s.d. were reported, based on three biological replicates.LoVo cells were seeded , * p < 0.05, ** p < 0.01, and *** p < 0.001.All statistical analyses were performed using GraphPad Prism 5 Software as described previously . Group dn = 177). We observed that the high expression of CTNNB1 and FOXO3a was associated with a shorter survival in OS, DFS, and DSS in CRC patients (Accumulating evidence has suggested that the protein levels of β-catenin and FOXO3a are critical factors for interconnecting their signaling crosstalk, promoting the tumorigenesis of CRC ,24. We fpatients . Indeed,patients A. Unexpepatients B, similaGiven that the translocation of FOXO3a into the nucleus by AKT inhibitors is a current limitation for treating CRC patients with a high expression of nucleus β-catenin ,19, we aGiven that the nuclear accumulation of FOXO3a induced by the API-2 treatment induces the cell migration of CRC cells containing high nuclear β-catenin ,19, we eThe wnt/β-catenin pathway plays a major role in the apoptosis of CRC cells . To deteThrough several clinical trials worldwide, various inhibitors targeting the PI3K-AKT signaling pathways have been investigated to evaluate their promising efficacies on cancers . AlthougAPC mutation occurring in 90% of CRC patients is well characterized as an essential factor for the initiation of CRC [The significant interconnection with the PI3K-AKT pathways and the Wnt/β-catenin pathways has a critical role in regulating cellular growth, homeostasis, and maintenance via modulating the intestinal stem cells and their differentiation ,6. Howevn of CRC ,29. Thisn of CRC . In accoIn recent research, with the overexpression of the Wnt/β-catenin pathway inducing tumor progression in CRC, a key transcription factor, FOXO3a has been reported as an accelerator overexpressing its target genes through binding with β-catenin. In consequence, highly nuclear β-catenin promotes the cell migration and confers resistance on the FOXO3a-mediated apoptosis by treating the AKT inhibitors in CRC cell lines . Indeed,APC mutations. The combination treatment with KY1022 and API-2 reverted the AKT-inhibitor-induced cell motility and suppressed the actin rearrangement of LoVo cells. Although this study only focused on cell migration, an initial step of multiple metastasis processes, the results clearly showed that the strategies suppressing the Wnt/β-catenin pathways effectively attenuate the API-2 mediated-cell migration. Based on this study, further validation of KY1022 in API-2-induced-other metastasis processes, followed by cell migration, would be helpful for the KY1022 combinatorial treatment with API-2. In addition, resistance to apoptosis via the API-2 treatment is also a problem. The combination treatment overcame the API-2-induced apoptotic resistance in LoVo cells, providing a beneficial option to treat metastatic CRC patients. Taken together, our results on the combination treatment, which showed synergistic effects between KY1022 and an AKT inhibitor, demonstrate a significantly higher clinical efficacy than either agent alone in metastatic CRC patients, accompanied with improvements for a worse/poorer prognosis.Metastasis is a major cause of cancer-related death and consists of a sequence of processes, including EMT, intravasation, and colonization of secondary organs ,31. Amon