Received: 7 June 2020 | Revised: 30 August 2020 | Accepted: 5 January 2021

DOI: 10.1002/med.21787

REVIEW ARTICLE

Direct targeting of β‐catenin in the Wnt signaling

pathway: Current progress and perspectives

Zhen Wang1 | Zilu Li2 | Haitao Ji1,2

1
Department of Drug Discovery, H. Lee
Moffitt Cancer Center and Research Abstract
Institute, Tampa, Florida, USA
Aberrant activation of the Wnt/β‐catenin signaling circuit
2
Department of Chemistry, University of
South Florida, Tampa, Florida, USA
is associated with cancer recurrence and relapse, cancer
invasion and metastasis, and cancer immune evasion. Di-
Correspondence
rect targeting of β‐catenin, the central hub in this signaling
Haitao Ji, Department of Drug Discovery,
H. Lee Moffitt Cancer Center and Research pathway, is a promising strategy to suppress the hyper-
Institute, 12902 Magnolia Dr, Tampa,
active β‐catenin signaling but has proven to be highly
FL 33612, USA.
Email: Haitao.Ji@moffitt.org challenging. Substantial efforts have been made to discover
compounds that bind with β‐catenin, block β‐catenin‐
Funding information
mediated protein–protein interactions, and suppress
Floridian Breast Cancer Foundation,
Grant/Award Number: Scientific Grant/ β‐catenin signaling. Herein, we characterize potential
19012901; U.S. Department of Defense, small‐molecule binding sites in β‐catenin, summarize
Grant/Award Number: CDMRP BCRP
breakthrough award/W81XWH‐14‐1‐0083; bioactive small molecules that directly target β‐catenin,
National Cancer Institute, and review structure‐based inhibitor optimization,
Grant/Award Number: Cancer Center
Support Grant/P30 CA076292; Susan G. structure–activity relationship, and biological activities of
Komen, Grant/Award Number: Career reported inhibitors. This knowledge will benefit future in-
Catalyst Research Grant/CCR16380693
hibitor development and β‐catenin‐related drug discovery.

KEYWORDS
BCL9, binding site analysis, protein–protein interaction,
small‐molecule inhibitor, TCF, Wnt/β‐catenin signaling, β‐catenin

1 | INTRODUCTION

The Wnt/β‐catenin signaling pathway plays important roles in regulating embryogenesis, stem cell renewal, and
tissue maintenance.1‐3 β‐Catenin is the central mediator of this pathway, and Wnt/β‐catenin signaling is balanced
through precise control of β‐catenin levels in the cytosol (Figure 1).4 Without a Wnt signal, cytosolic β‐catenin is
actively phosphorylated by a destruction complex comprising adenomatous polyposis coli (APC), Axin, glycogen
synthase kinase 3β (GSK3β), casein kinase 1α (CK1α), and protein phosphatase 2A.5 Within this destruction

Zhen Wang and Zilu Li contributed equally to this study.

Med Res Rev. 2021;1–21. wileyonlinelibrary.com/journal/med © 2021 Wiley Periodicals LLC | 1

2 | WANG ET AL.

F I G U R E 1 Overview of the Wnt/β‐catenin signaling pathway in its on and off state. APC, adenomatous
polyposis coli; CBP, CREB‐binding protein; DVL, dishevelled; Fzd, frizzled; GSK, glycogen synthase kinase; LRP 5/6,
lipoprotein receptor‐related proteins 5 and 6; TCF, T‐cell factor; TLE, transducin‐like enhancer of split; β‐cat,
β‐catenin

complex APC and Axin serve as the scaffold that facilitates phosphorylation of β‐catenin at residue S45 by CK1α
and phosphorylation at residues S33, S37, and T41 by GSK3β. These phosphorylation events drive β‐catenin to
undergo ubiquitination and proteasome degradation. As a result, only a minimal amount of β‐catenin are main-
tained in unstimulated cells.6 Upon binding of a Wnt ligand to a member of the frizzled (Fzd) family of G‐protein‐
coupled receptors and one of two low‐density lipoprotein receptor‐related proteins 5 and 6, Axin and the adaptor
protein dishevelled (Dvl) are recruited to this membrane‐anchored protein complex, resulting in disassembly of the
destruction complex. This allows β‐catenin to be stabilized into the dephosphorylated state, accumulated in the
cytoplasm and translocated into the cell nucleus, where the active unphosphorylated β‐catenin displaces co‐
repressor Groucho/transducin‐like enhancer of split from the T‐cell factor (TCF)/lymphoid enhancer‐binding factor
(LEF) family of transcriptional factors, and recruits co‐activators B‐cell lymphoma 9 (BCL9) or BCL9‐like (BCL9L),
Pygopus (Pygo 1 or Pygo 2), CREB‐binding protein (CBP)/p300, and among others to transcribe Wnt/β‐catenin
target genes (Figure 1).7‐13 In addition to its involvement in Wnt signaling, β‐catenin acts as a structural component
of adherens junctions, where it binds to the cytoplasmic domain of E‐cadherin to recruit and organize actin
filaments.14 This pool of β‐catenin in most cases is highly stable and not involved in the Wnt pathway‐related
machinery.15
Hyperactive Wnt/β‐catenin signaling is implicated in the initiation and progression of various types of cancer,
such as colorectal cancer (CRC), noncolorectal gastrointestinal cancer, lung cancer, prostate cancer, leukemia, and
breast cancer. In most cases, oncogenic activation of Wnt/β‐catenin signaling is triggered by inactivation mutations
of the components of the destruction complex (e.g., APC and Axin), or activation mutations at the N‐terminal
phosphorylation domain of the β‐catenin gene CTNNB1,16‐26 driving transcription of Wnt/β‐catenin target genes
that induce cancer cell epithelial‐to‐mesenchymal transition, activate self‐renewal of stem‐like cancer cells, pro-
mote tumor cell invasion and metastasis, confer treatment resistance, and foster tumor immune evasion. Auto-
crine/paracrine activation of the upstream effectors (Fzd, Dvl, and Wnt ligands) of the Wnt/β‐catenin pathway and
epigenetic silence of Wnt antagonist genes also causes hyperactivation of this signaling pathway and has been
recorded in many types of cancers.27‐34 Several proof‐of‐concept studies targeting this pathway for the potential
treatment of cancer have been conducted. Restoration of APC was shown to promote cellular differentiation and
reestablish crypt homeostasis in CRC.35 Enforced expression of Axin suppressed proliferation and self‐renewal
capacity of chronic myelogenous leukemia cells that bear activated β‐catenin.36 Dicer‐substrate small interfering
RNAs‐mediated β‐catenin knockdown reduces liver tumor burden in vivo.37 In addition, collective research data
WANG ET AL. | 3

have indicated that β‐catenin signaling mediates immune escape of cancer cells and resistance to checkpoint
blockade immunotherapies.38‐44 These studies have demonstrated that suppression of Wnt/β‐catenin signaling
offers a new approach toward anticancer therapies.
Substantial efforts have been made to target this pathway by developing therapeutic agents against the
upstream effectors. OMP‐18R5 (vantictumab),45 a monoclonal antibody against Fzd receptors, has completed
phase I clinical trials (Clinicaltrials.gov IDs: NCT01345201, NCT02005315, NCT01957007, and NCT01973309).
OMP‐54F28,46 a recombinant protein of the Fzd 8 cysteine‐rich domain fused to the fragment crystallizable part of
immunoglobulin, acts as a decoy receptor and binds with all Wnt ligands to suppress Wnt/β‐catenin signaling.
OMP‐54F28 is also ready for phase II clinical trials (Clinicaltrials.gov IDs: NCT02069145, NCT02092363,
NCT02050178, and NCT01608867). Porcupine is a membrane‐bound O‐acyltransferase,47 and the inhibition of
this enzyme prevents the production of bioactive Wnt ligands to suppress Wnt/β‐catenin signaling. LGK974 and
ETC‐1922159 are two representative porcupine inhibitors that are in phase I clinical trials (LGK974, Clinicaltrials.
gov ID: NCT01351103; and ETC‐1922159, Clinicaltrials.gov ID: NCT02521844).48,49 Other proteins, such as
tankyrases 1/2, CK1α, CK1ε, GSK3β, which play important roles in Wnt/β‐catenin signaling are also actively being
targeted, but neither the inhibitors of tankyrase, CK1ε, and GSK3β nor the activators of CK1α have entered into
clinical trials.50‐58 Despite the progress, targeting of the upstream effectors might have a limited application due to
the following concerns: (1) cancer cells with more downstream genetic or epigenetic mutations, such as loss‐of‐
function mutations of the destructive complex or β‐catenin activation mutations, and upregulation of downstream
effectors such as β‐catenin by crosstalk with the other signaling pathways are anticipated to be resistant to such
agents; and (2) the upstream targets such as tankyrase, GSK3β, and CK1α are involved in multiple cellular pro-
cesses. The intervention of these targets increases the risks of undesired off‐pathway toxicities.51,59 As a con-
sequence, the β‐catenin‐containing transcriptional complex emerges as the most promising target for the
development of inhibitors, because this nuclear transcriptional complex is formed at the very late stage and
determines the transcriptional activities of the Wnt/β‐catenin signaling cascade.60 To date, the only reported drug‐
like inhibitors targeting this transcriptional complex are ICG‐00161 and its second‐generation prodrug derivatives
PRI‐724,62 CWP232228,63,64 and CWP232291.65 This series of compounds bind with the general coactivator CBP
for the disruption of the CBP/β–catenin interaction. The mechanism, hence, may less likely be Wnt pathway‐
specific, because CBP has been reported to be involved in many transcriptional processes.66 On the other hand,
direct targeting of β‐catenin is expected to result in specific inhibition of the pathway, but has been proven
challenging. Over years, dozens of β‐catenin‐containing protein complex structures have been reported, and the
potential binding sites of β‐catenin for native protein binding partners and inhibitors have been analyzed by
different research groups using different structural and biochemical methods. Herein, for the first time, we provide
a review to characterize possible small‐molecule binding sites in β‐catenin, summarize the efforts on the devel-
opment of bioactive small molecules that directly bind with β‐catenin, and assess their biological characteristics.
Structure‐based inhibitor design and structure–activity relationship (SAR) of the reported inhibitors are discussed
in the context to reveal new directions for future inhibitor development and drug discovery (Box 1).

2 | P O T E N T I A L SM A LL ‐ M O L E C U L E B I N D I N G S I T E S I N β ‐ C AT E N I N

Human β‐catenin is comprised of 781 amino acids with a central structural core of 12 armadillo repeats (residues
138–664) and the intrinsically disordered N‐ and C‐terminal regions.67 Structural biology studies reveal that β‐
catenin utilizes its armadillo repeat domain to interact with most of its known ligand proteins. These structures
include β‐catenin in complex with Wnt signaling suppressors APC,68‐70 Axin,71 and ICAT,72,73 transcription
activators LEF1,74 TCF3,75 and TCF4,76‐78 transcription coactivator BCL9,78 and Wnt signaling‐relevant
proteins E‐cadherin14 and LRH1,79 among which LEF/TCF and BCL9 interact with β‐catenin in the Wnt
transcription complex. Disruption of either of these two types of protein–protein interactions (PPIs)
4 | WANG ET AL.

Box 1 How does β‐catenin regulate Wnt target gene expression?

Upon activation of the Wnt/β‐catenin signaling pathway, β‐catenin is translocated into the cell nucleus
and activates transcription of Wnt/β‐catenin target genes. Due to the lack of the DNA binding domains in
β‐catenin, the DNA‐binding partners are necessary to bring β‐catenin to the promoter of its specific DNA
sequences.67 The TCF/LEF family of transcription factors is the main nuclear partner that guide β‐catenin
to its specific DNA loci. The core interaction region for β‐catenin binding with TCF/LEF transcriptional
factors is armadillo repeats 3–10 of β‐catenin.75 Meanwhile, the proteins of the chromatin remodeling
complex including CBP, p300, Tip60, SWI/SNF, ISWI, and so forth, are recruited to the C‐terminal domain
of β‐catenin. These proteins occupy a similar binding site in β‐catenin. The C‐terminal domain of β‐catenin
serves as a platform for the recruitment and sequential exchange of these transcriptional
co‐activators.129 The armadillo repeat 1 of β‐catenin interacts with the homology domain 1 (HD1) of
β‐catenin co‐activator, BCL9 or BCL9 paralog, BCL9L. The HD2 of BCL9 or BCL9L binds with Pygo which
will localize chromatin‐binding proteins to the modified nucleosomes.130

(i.e., β‐catenin/TCF PPI and β‐catenin/BCL9 PPI) would disassemble the transcriptional complex, resulting in in-
activation of Wnt/β‐catenin signaling. These PPIs are well‐characterized, and the distinct binding sites on
β‐catenin can be extracted by analyses of the crystallographic data of protein complex structures. Specifically, the
β‐catenin/TCF PPI interface covers approximately 4800 Å2, and this PPI interface structure offers four key binding
areas.80‐84 The first key binding area is around residues N426, K435, R469, H470, and K508 of
β‐catenin where these residues form a concave pocket to interact with residues D16, E17, L18, and I19 of TCF4 or
the equivalent residues in TCF1, LEF1, and TCF3. Mutations of these β‐catenin residues to alanine strongly
reduced the binding affinity with LEF1 and TCF4 in co‐immunoprecipitation (co‐IP) experiments.80 Residue D16 of
TCF4 forms salt bridge interactions with residue K435 of β‐catenin and a hydrogen bond with β‐catenin residue
H470, as well as hydrogen bond networks with structural water molecules. This aspartate residue is the most
important protruding hot spot of the TCF/LEF family of transcriptional factors by site‐directed mutagenesis studies
(Box 2 shows the definition of protruding hot spot, hot spot pocket, and hot spot interactions of PPIs).81‐84 For
example, the D16A mutation of TCF4 led to a significant decrease in binding affinity with β‐catenin in enzyme‐
linked immunosorbent assays (ELISA) and induced a ΔΔG of +2.4 kcal/mol in isothermal titration calorimetry (ITC)
experiments. The double mutation (D16A and E17A) of TCF4 further greatly reduced the TCF4 binding affinity,
while the binding affinity of the single E17A mutation was weakened by fourfold when compared to that of wild‐
type TCF4.81 The second key binding area is around β‐catenin residues K312 and K345 where TCF4 residues E24
and E29 bind. The dissociation constant (KD) of the PPI between β‐catenin and TCF4 E24A/E29A double mutant
was sixfold higher than that of the β‐catenin/wild‐type TCF4 PPI in surface plasmon resonance (SPR) studies, and
the half maximal inhibitory concentration (IC50) value of the TCF4 E24A/E29A double mutant for disrupting β‐
catenin/TCF4 PPI was increased by 62‐fold compared with that of wild‐type TCF4 in fluorescence polarization (FP)
competitive inhibition assays.85 The co‐IP experiments further demonstrated that mutations of all four glutamates
in this region (E24A, E26A, E28A, and E29A) abolished the binding with β‐catenin.77 However, no obvious binding
pocket was observed in this region of β‐catenin. The third binding area is around residues H578 and R582 of β‐
catenin where D11 of TCF4 interacts. Mutagenesis and ITC studies showed an approximately 30‐fold increase in
KD between wild‐type TCF4 and β‐catenin mutation H578A or R582A. The D11A mutation had a large effect on
TCF4 binding than the deletion of the entire TCF4 N‐terminal 9 or 12 residues.83 The fourth key binding site is a
hydrophobic area around β‐catenin residues F253, F293, and I296, where TCF4 residues L41, V44, and L48
adopted an α‐helical structure to bind with these residues through hydrophobic interactions. Mutation of the
WANG ET AL. | 5

Box 2 Hot spot interactions between proteins

For the PPI a small subset of residues at the protein–protein interface contributes to the majority of the
binding free energy. These residues are called hot spots.131 A hot spot is defined as a residue which
substitution by an alanine leads to a significant decrease in the free energy of binding (ΔΔGbinding > 1.5
kcal/mol). These hot spots tend to cluster and contact with each other to form an area of conserved
interactions called hot regions.132 The protruding hot spot of one protein (called the ligand–protein)
packs against the concave hot region of the other protein (called the target protein). The protruding hot
spot is also called the projecting hot spots. The concave hot region is often called the hot spot pocket.133

hydrophobic residues, L41A, V44A, and L48A, of TCF4, significantly reduced their binding affinity with
β‐catenin.81‐84
The contacting surface area of β‐catenin/BCL9 PPI is much smaller (∼1450 Å2), where BCL9 adopts an α‐helix
to bind with β‐catenin. Crystallographic and biochemical experiments identified two key binding areas at this PPI
interface. One is a hydrophobic pocket formed by residues L156, L159, V167, A171, M174, and L178 of β‐catenin,
which interacts with BCL9 residues L366, I369, and L373. The β‐catenin L156A/L159A double mutant loses its
binding affinity with BCL9 in pulldown experiments.78 Other double mutants of β‐catenin including L156S/L159S
and L156S/L178S also block their binding with BCL9 in both fluorescence anisotropy binding experiments and
AlphaScreen competitive binding assays.86 The protein pulldown experiments demonstrate the binding of BCL9
with β‐catenin was abrogated by BCL9 mutations L366K, L373A, and L366A/I369A.78,87 BCL9 mutations L366A,
I369A, and L373A showed no inhibition of the β‐catenin/wild‐type BCL9 PPI in FP competitive inhibition assays.88
The second key binding area is an acidic knob formed by β‐catenin residues D162, E163, and D164, which form salt
bridge interactions with BCL9 residues H358 and R359. The β‐catenin D162A mutant decreased its binding affinity
with BCL989 and the β‐catenin D164A mutant abolished its interaction with BCL9 or BCL9L.90 BCL9 H358A and
R359A mutants significantly reduced their binding affinity withβ‐catenin.78 The binding of BCL9 with β‐catenin was
358
fully abrogated by the BCL9 HRE360/358AKQ360 mutant.87

3 | I N H I B I T O R S O F T H E β ‐CATENIN/TCF P PI

Most efforts targeting β‐catenin have been focused on discovering the agents that antagonize the β‐catenin/TCF
PPI, and to date, various inhibitors have been reported. Peptide‐based inhibitors including hydrocarbon‐stapled
peptides and peptoid‐peptide macrocycles have been reported.91,92 On the basis of the observation that the β‐
catenin binding domain of Axin adopts an α‐helical structure to bind with β‐catenin and the binding site of β‐
catenin for Axin is overlapped with that for the TCF4 α‐helical domain, hydrocarbon‐stapled peptides were de-
signed by modifying the Axin sequence with an i/i + 4 side‐chain staple and then optimizing the sequence using
phage display. These efforts led to two stapled peptides, StAx‐35 (KD = 13 nM) and StAx‐35R (KD = 53 nM)
(Figure 2A). The direct binding of StAx‐35 and StAx‐35R with β‐catenin was demonstrated by the protein pulldown
assays using the cell lysates. N‐terminally acetylated StAX‐35 (aStAx‐35) was successfully co‐crystalized with β‐
catenin (residues 134–665) (Protein Data Bank [PDB] ID, 4DJS), offering the first and the only co‐crystal structure
of β‐catenin with its inhibitor (Figure 2B).91 Cell‐based studies indicated these stapled peptides can penetrate the
cell membrane, selectively suppress TOPFlash luciferase reporter activity while sparing FOPFlash luciferase re-
porter activity, and downregulate Wnt target genes LEF1, LGR5, and Axin2. The proliferation of Wnt‐hyperactive
cancer cells was blocked at the micromolar levels after 5‐day incubation. Further structural optimization was
6 | WANG ET AL.

F I G U R E 2 (A) Structures and the binding affinity of StAx‐35 and StAx‐35R. (B) Crystal structure of β‐catenin in
complex with aStAx‐35 (PDB ID, 4DJS) [Color figure can be viewed at wileyonlinelibrary.com]

performed to increase the cellular uptake and cell‐based potency. The nuclear localization sequence (NLS) of the
SV40 large T‐antigen that was previously shown to increase both cellular uptake and nuclear localization was
introduced to the N‐terminal end of the stapled peptide StAx‐h, in which all R residues of StAx‐35R were replaced
by homoarginine, resulting in NLS‐StAx‐h. The fluorescently labeled version of NLS‐StAx‐h exhibits a sevenfold
improvement in cellular uptake at 5 mM and considerable cytosolic distribution with respect to the fluorescently
labeled StAx‐35R. The core sequence of NLS‐StAx‐h (StAx‐h) was shown to bind with β‐catenin in pulldown
experiments using the lysate of DLD‐1 cells. Further studies reveal that NLS‐StAx‐h suppresses Wnt target gene
expression and inhibits proliferation and migration of CRC cells.93 It should be noted that disruption of the β‐
catenin/Axin PPI can potentially activate Wnt/β‐catenin signaling. Indeed, two Axin‐mimicking stapled peptides,
SAHPA1 and SAHPA2, were reported.94 SAHPA1 disrupted the interaction between β‐catenin and Axin, and
activated rather than suppressed Wnt/β‐catenin signaling. Similarly, a small molecule SKL2001 that disrupts the β‐
catenin/Axin PPI was demonstrated to be an agonist of the Wnt/β‐catenin signaling pathway.95 Using the Rosetta
suite of protein design algorithms, a series of cyclized peptide‐peptoid macrocycles was designed to bind with β‐
catenin. The most active macrocycle that potently disrupts the β‐catenin/TCF PPI also markedly decreases the
growth of prostate cancer cells and inhibits Wnt signaling in the in vivo zebrafish model.92
Some natural products including PKF115‐584,96,97 GCP049090,96 Henryin,98 organocopper compound
BC21,99 inhibitors of catenin‐responsive transcription (iCRT) series (iCRT3, iCRT5, and iCRT14),100
ZINC02092166 and its chemically stable derivatives,101 and compound LF3102 were reported to disrupt the
β‐catenin/TCF PPI and showed inhibitory activities in cell‐based studies. Compounds LF3 and iCRT3 have been
extensively characterized. LF3 was identified from 16,000 compounds via compound screening using AlphaScreen
and ELISA assays. LF3 suppressed Wnt/β‐catenin signaling in cells with high Wnt activity, and reduced expression
WANG ET AL. | 7

of Wnt target genes. LF3 also showed potent inhibitory activities against cancer cells related to Wnt signaling in
vitro and in a mouse xenograft model. Co‐IP assays using HCT116 colon cancer cells indicated that LF3 dose‐
dependently disrupted the β‐catenin/TCF4 interaction but spared the β‐catenin/E‐cadherin interaction, indicating
that this compound does not disturb β‐catenin‐mediated cell adhesion. iCRT3 was identified from 14,997 com-
pounds through an Axin RNA interference‐based chemical genetic screen. iCRT3 suppressed not only β‐catenin but
also androgen receptor signaling pathways. Both pathways are hyperactive in prostate cancer. Correspondingly,
iCRT3 inhibited the growth of prostate cancer cells in vitro and in an in vivo xenograft model.103 However, the
other compounds (PKF115‐584, GCP049090, iCRT5, iCRT14, and ZINC02092166) contain pan‐assay interference
compounds (PAINS) substructures that can cause frequent hits in biochemical assays.104‐106 More importantly, all
of these compounds have not been reported to directly bind with β‐catenin by biochemical or biophysical ex-
periments. Therefore, we only review in detail the following small‐molecule inhibitors, which were reported to bind
directly with β‐catenin.

3.1 | Inhibitor PNU‐74654 (2006)

PNU‐74654 in Table 1 was initially discovered by in silico docking of 17,700 compounds to a well‐defined binding
pocket around β‐catenin residues K435 and R469, followed by evaluation of 22 compounds with the best docking
scores using WaterLOGSY nuclear magnetic resonance (NMR) and competitive ITC studies.107 The direct binding of
PNU‐74654 with β‐catenin was assessed by the ITC experiment (KD = 450 nM). The binding mode between PNU‐
74654 and β‐catenin (around hot spot residues K435/R469) was investigated by extensive docking studies (Figure 5).
The results predicted that the methyl group on the furan ring and the phenyl moiety bound with two small pockets on
both sides of the hot spot pair, respectively. The contribution of these two groups to the binding affinity of PNU‐
74654 was supported by the experimental data of its two analogs, in which the methyl group on the furan ring and
the distal phenyl moiety were replaced by a proton and a piperidine ring, respectively, and both show an apparent
decrease in binding affinity. Neither selectivity nor cellular activity data were reported for PNU‐74654 although this
compound was claimed to specifically inhibit TCF4 transactivation in the luciferase reporter experiment. It is worth
noting that PNU‐74654 has a reactive functional group, acyl hydrozone, which has been recognized as a PAINS
moiety.104‐106 This PAINS substructure could be problematic when further optimizing this compound to improve
potency, selectivity, and physicochemical properties. One example for the modification of this linker group was the
optimization of ZINC02092166,101 in which the acyl hydrozone group of ZINC02092166 was substituted by a new
substructure, resulting in a series of chemically stable derivatives of ZINC02092166 as the inhibitors of the β‐
catenin/TCF PPI. The tetraheterocyclic ring of ZINC02092166 in Figure 3 contains 17 electrons and is not aromatic.
The substitution of the imine carbon atom with a nitrogen atom resulted in a new tetraheterocyclic ring with 18
electrons, which constitutes a stable large aromatic system. These new derivatives not only show selectivity for β‐
catenin/TCF PPI over β‐catenin/cadherin and β‐catenin/APC PPIs, but also suppress transactivation of Wnt/β‐catenin
signaling, downregulate Wnt target genes Axin2, cyclin D1, and c‐myc, and inhibit the growth of cancer cells. More
importantly, their cell‐based inhibitory activities are in agreement with that from the biochemical assays. On the
contrary, ZINC02092166 displayed higher inhibitory activities in cellular experiments than in biochemical studies.

3.2 | Inhibitor UU‐T01 (2013)

Compound UU‐T01 in Table 1 was designed by a hot spot‐based bioisostere replacement strategy.85 Specifically,
different bioisosteres were used to mimic the carboxylic acid groups of TCF4 hot spots D16 and E17, and the
selected fragments were merged with the assistance of a linker library. UU‐T01 was obtained by combining the
optimal fragments and linkers. The direct binding between β‐catenin and UU‐T01 was evaluated in the ITC
 8

TABLE 1 Small‐molecule inhibitors that were reported to directly bind with β‐catenin
|

TOP‐Flash,
Compound M. W. Discovery method Direct binding KD, µM Binding sites IC50, μM In vivo efficacy

PNU‐74654 320 Virtual screening NMR, ITC 0.450 K435, R469 N. D. N. D.

UU‐T01 230 Hot spot‐based design ITC, mutagenesis 0.531 K435, K469 N. D. N. D.

K508

UU‐T02 665 Peptidomimetic design ITC, mutagenesis 0.418 K435, R469 SW480: 232 N. D.

R474, K508

R515

MSAB 305 Luciferase‐reporter screening SPR, NMR N. D. residues 301‐670 HCT116: 0.58 20 mg/kg p. o., q. 2d.

HI‐B1 255 Rational design based on a hit Protein pull down N. D. K312 DLD1: 13 50 mg/kg i. p., q. d.

CACO2: 13

carnosic acid 332 Screening by in vitro ELISA NMR 5–20 ARD N‐terminus SW480: ∼25 Diet containing 0.1% or 1% carnosate

PNPB‐22 646 Hot spot‐based design ITC, mutagenesis 0.330 L156, L159, V167, A171, M174, L178 SW480: 13 N. D.
WANG

Abbreviations: ARD, armadillo repeat domain; ELISA, enzyme‐linked immunosorbent assay; ITC, isothermal titration calorimetry; M. W., molecular weight; N.D., not determined; NMR,
ET AL.

nuclear magnetic resonance.

WANG ET AL. | 9

F I G U R E 3 Optimization of ZINC02092166 as inhibitors of β‐catenin/T‐cell factor (TCF) protein–protein

interaction (PPI)

experiment (KD = 531 nM). The inhibition of β‐catenin/TCF4 PPI was demonstrated by FP (Ki = 3.1 μM) and
AlphaScreen (Ki = 7.6 μM) competitive inhibition assays. The binding mode was assessed using Autodock docking
(Figure 5). The indazole‐1‐ol group mimics the carboxylate moiety of TCF4 E17, makes salt bridge interactions with
residue K508 of β‐catenin, and forms cation−π interactions with the positively charged guanidino group of
β‐catenin R469. The tetrazole ring mimics the carboxylate moiety of TCF4 D16 and forms salt bridge and hydrogen
bond interactions with β‐catenin residues K435 and N430, respectively. The binding mode was supported by site‐
directed mutagenesis and SAR studies. For example, UU‐T01 shows a threefold to 7.6‐fold loss in activity against
β‐catenin K435A, R469A, and K508A mutations in ITC studies. Moreover, compounds containing the indazole‐1‐ol
moiety are more potent than those with the benzotriazole‐1‐ol ring in FP competitive inhibition assays. This
observation is consistent with the hypothesis that the cation−π interaction plays an important role in this series of
compounds to bind with β‐catenin residue R469. The indazole‐1‐ol ring has a higher π‐electron density than the
benzotriazole‐1‐ol ring due to the different electronegativities between the nitrogen and carbon atoms, and is
more favorable for forming the cation−π interaction. The other carboxyl acid bioisosteres, 5‐oxo‐1, 2, 4‐oxadiazole,
and 5‐thioxo‐1, 2, 4‐oxadiazole, are inferior to tetrazole for binding. The length of the linker between indazole‐1‐ol
and tetrazole is also critical for inhibitor binding affinity, and the CH2CH2 linker is superior to the CH2 and
CH2OCH2 linkers. This study not only presents a new hot spot‐based bioisosteric replacement approach to de-
signing small‐molecule PPI modulators, but also provides evidence demonstrating the ligandability of this binding
area (around residues K435 and K508), which is in consistent with the results of binding site analyses. It should be
noted that the selectivity and cell‐based activity data were not collected for UU‐T01.

3.3 | Inhibitor UU‐T02 (2014)

UU‐T02 in Table 1 was also designed to target the potential binding pockets surrounding β‐catenin residues K435
and R469.108 As described above, crystallographic studies indicated that β‐catenin utilized its armadillo repeat
domain to bind with a diverse group of protein partners such as TCF/LEF, BCL9, E‐cadherin, APC, Axin, and ICAT.
Available crystal structures14,69,70,74‐78 and biochemical studies82,109‐111 have indicated that the same surface area
of β‐catenin is used to bind with TCF, APC, and regions III and IV of E‐cadherin. Both in vitro and in vivo studies
indicate the binding mode of β‐catenin with TCF, APC, and E‐cadherin is mutually exclusive. Therefore, inhibitor
selectivity turns out to be the main concern when designing compounds to disrupt the β‐catenin/TCF PPI. UU‐T02
was designed to approach this problem (Figure 4). Specifically, although the most important protruding hot spot
D16 of TCF4 is also conserved in E‐cadherin (D830) and APC (D1486 of the APC‐R3 repeat), the binding features
adjacent to this critical residue are different between TCF, E‐cadherin, and APC when binding with
β‐catenin.75,80,84 For example, as described above, TCF4 E17 is relatively important for TCF4 binding with
10 | WANG ET AL.

F I G U R E 4 Discovery and optimization of UU‐T02. APC, adenomatous polyposis coli; LEF, lymphoid enhancer‐
binding factor; MCSS, multiple‐copy simultaneous search; PPI, protein–protein interaction; SAR, structure–activity
relationship; TCF, T‐cell factor

β‐catenin while the residues at the same position in E‐cadherin and APC‐R3 (S831 and T1487, respectively) do not
make direct interactions with β‐catenin. In addition, deletion experiments showed that the TCF4 G8‐A14 sequence
adjacent to hot spot D16 is important for TCF4 binding with β‐catenin.77,83,84 The E‐cadherin and APC‐R3 residues
at the same position of TCF4 G13AN15 do not bind with β‐catenin directly. The D16A/E17A TCF4 peptide cannot
disrupt the wild‐type β‐catenin/wild‐type TCF4 PPI in FP competitive inhibition assays, while the alanine mutations
of E‐cadherin P826YDS829 and APC‐R3 D1484ADT1487 sequences only leads to 10‐ and 30‐fold increase of IC50
values for disrupting the wild‐type β‐catenin/wild‐type E‐cadherin PPI and the wild‐type β‐catenin/wild‐type
APC‐R3 PPI, respectively.108 On the basis of these analyses, it was hypothesized that occupying the TCF4
G13ANDE17 binding area by capturing the binding features of G13ANDE17 would produce selective inhibitors for
the β‐catenin/TCF PPI. Careful analyses of the co‐crystal structures of β‐catenin with TCF4 reveal that this TCF4
G13ANDE17 binding area can be divided into four pockets: pocket A is formed by residues C429, N430, K435,
H470, S473, and R474 of β‐catenin; pocket B is lined with β‐catenin residues I507, K508, V511, L539, I569, and
C573, and this pocket is relatively deep; pockets C and D are shallow surface pockets with pocket C surrounded by
residues L519 and I579, and pocket D defined by residues E462, C466, L506, K508 (the side chain carbon atoms),
and A509. Pockets A and B are connected to pocket C through an arginine channel formed by residues R474 and
R515. Pockets B and D are connected through residues R469 and K508. With the assistance of multiple‐copy
simultaneous search (MCSS) and SiteMap analyses, new β‐catenin/TCF inhibitors were designed by the peptido-
mimetic approach based on TCF4 G13ANDE17 sequence. The critical residues D16 and E17 were kept in new
inhibitors to bind with pocket A and K508, while the 4‐OMe benzyl and indole moieties were initially selected to
occupy pocket B and the arginine channel toward pocket C, respectively. The first designed compound disrupts the
β‐catenin/TCF4 PPI with a Ki of 5.7 μM in FP competitive inhibition assays. SAR studies on this scaffold reveal that
the 5‐Cl indole moiety is the optimal substituent for the arginine channel and the larger hydrophobic group is
preferred for pocket B. The representative compound, UU‐T02, inhibits the β‐catenin/TCF4 interaction with a Ki of
WANG ET AL. | 11

F I G U R E 5 Predicted binding sites of the reported small‐molecule inhibitors with β‐catenin [Color figure can be
viewed at wileyonlinelibrary.com]

1.32 μM in FP assays and is 175‐ and 64‐fold selective against β‐catenin/E‐cadherin and β‐catenin/APC interac-
tions, respectively. The direct binding between UU‐T02 and β‐catenin was examined by ITC experiments (KD =
418 nM). The binding mode was assessed by Autodock and Glide docking (Figure 5). Two carboxylate groups of
UU‐T02 were predicted to form salt bridge interactions with β‐catenin residues K435 and K508. The naphthyl
group binds deeply with the hydrophobic pocket B, and the indole N‐H could form a hydrogen bond with C = O
group of N516 side chain, and the indole ring is designed to form cation–π interactions with R474 and R515. This
predicted binding model was supported by SAR and site‐directed mutagenesis studies. UU‐T02 exhibits low cell‐
based activity probably due to its poor cell membrane permeability which is caused by its two carboxylate groups.
Further optimization of UU‐T02 was performed to increase its activities in both biochemical and cell‐based
studies.112 Extensive SAR studies not only demonstrate that the naphthyl group, two carboxylate groups, and the
5‐Cl indole moiety are critical for maintaining the inhibitory activity, but also reveal that the methyl ester of
UU‐T02 can be replaced by the larger hydrophobic groups, and the carboxylate groups can be substituted by its
bioisosteres to improve activity. These efforts have led to new inhibitors with improved activity (Ki = 0.44 μM).112
Cell‐based studies showed that the last inhibitor in Figure 4 selectively disrupted β‐catenin/TCF over β‐catenin/
E‐cadherin and β‐catenin/APC interactions, dose‐dependently suppressed transactivation of Wnt/β‐catenin sig-
naling, inhibited growth of Wnt/β‐catenin signaling‐hyperactive cancer cells, and blocked migration and inva-
siveness of Wnt/β‐catenin‐dependent cancer cells.

3.4 | Inhibitor MSAB (2016)

Compound MSAB in Table 1 was identified from a library of 22,000 compounds using a TCF‐dependent luciferase
reporter system as the pilot assay.113 This compound selectively suppresses the transactivation of Wnt/β‐catenin
12 | WANG ET AL.

signaling and inhibits the growth of cancer cells with hyperactive Wnt/β‐catenin signaling. It also displays anti-
tumor effects in Wnt‐dependent xenograft tumor models in vivo. Mechanistic analyses indicated this compound
bound with β‐catenin, promoted its degradation, and selectively downregulated Wnt target genes expression. The
binding of MSAB with β‐catenin was first demonstrated by biotin‐based affinity purification combined with tandem
mass spectrometry showing that β‐catenin was the leading candidate among MSAB‐binding Wnt effectors proteins
and further confirmed by ligand‐observed NMR and SPR studies. Pull‐down and NMR experiments suggested that
MSAB likely bound to the second armadillo repeat of β‐catenin (residues K301‐Y670). The SAR analysis reveals
that the para substitution of the phenyl ring and the ester group of phenylsulfonamidobenzoates are important for
maintaining the inhibitory activity, which is in consistent with the predicted binding mode in Figure 5. Specifically,
the methyl and methoxy groups are favored for the para substitution of the phenyl ring while the fluoro or chloro
substituent at this position results in the complete loss of activity. The replacement of the ester group of phe-
nylsulfonamidobenzoates with amides also abolishes the inhibitory activity.

3.5 | Inhibitor HI‐B1 (2017)

HI‐B1 in Table 1 was designed by cyclization of resveratrol, a previously discovered Wnt inhibitor.114 This com-
pound inhibited the β‐catenin/TCF4 luciferase reporter activity, downregulated transcription and expression Wnt
target genes cyclin D1 and Axin2, and selectively suppressed the growth of Wnt‐dependent cancer cells in vitro and
in vivo. The direct binding of HI‐B1 with β‐catenin was demonstrated by incubation of HI‐B1‐sepharose 4B beads
with DLD cell lysates and then western blotting of the proteins that were pulled down. The binding of HI‐B1 with
β‐catenin led to the disruption of the β‐catenin/TCF4 PPI, which was confirmed by co‐IP experiments at both
biochemical and cellular levels. The binding mode was assessed based on the results of computer modeling
(Figure 5) and preliminary SAR studies, which suggested that the nitrogen atom in the imidazole ring of HI‐B1 may
form a hydrogen bond with β‐catenin K312. The importance of this nitrogen atom in the imidazole ring of HI‐B1 for
binding with β‐catenin was further evaluated by SAR studies.

3.6 | Inhibitors of β‐catenin/BCL9 PPI

Compared with β‐catenin/TCF PPI, β‐catenin/BCL9 PPI has a smaller contacting area (1450 Å2) and a moderate
binding affinity (KD = 470 nM), representing a promising alternative binding site for inhibitor design to suppress
Wnt/β‐catenin signaling. The roles of the β‐catenin‐BCL9‐Pygo axis in Wnt target gene transcription is described in
Box 1. Peptides‐based inhibitors including triazole‐ and hydrocarbon‐stapled peptides and sulfono‐γ‐AApeptide
have been designed to disrupt this PPI interface.115‐118 By mimicking the binding features of the α‐helical HD2
domain of BCL9, a stabilized α‐helix of BCL9 (SAH‐BCL9B) with the i, i + 4 side‐chain stapling was designed and
synthesized. SAH‐BCL9B can dissociate the β‐catenin/BCL9 complex in cells, selectively suppress Wnt transcrip-
tional activity, and inhibit proliferation, angiogenesis, and migration of β‐catenin‐hyperactive cancer cells. More-
over, in INA‐6 multiple myeloma and Colo320 colorectal carcinoma mouse models, SAH‐BCL9B was found to
suppress tumor growth, angiogenesis, invasion, and metastasis. The helical foldamer scaffold based on the un-
natural sulfono‐γ‐AApeptide scaffold was used to design and synthesize derivatives to disrupt β‐catenin/BCL9 PPI.
Although sulfono‐γ‐AApeptides do not faithfully mimic α‐helix, these sulfono‐γ‐AApeptide derivatives can capture
key structural features of BCL9 for binding with β‐catenin, disrupt β‐catenin/BCL9 PPI, and exhibit cell‐based
activity. The merit of using these sulfono‐γ‐AApeptides is that they are absolutely resistant against proteases and
proteinases when compared to peptide‐based inhibitors. A small‐molecule natural product, carnosic acid in Table 1,
was reported as an inhibitor of the β‐catenin/BCL9 PPI after screening two libraries containing 1250‐compounds
by in vitro ELISA assay to monitor the interaction between His6‐tagged BCL9 HD2 and the glutathione
WANG ET AL. | 13

S‐transferase tagged β‐catenin armadillo repeat domain that was immobilized on glutathione‐coated micro-
plates.119 NMR saturation transfer difference studies and heteronuclear single‐quantum correlation experiments
between 1H and 15N demonstrated that carnosic acid bound with β‐catenin at the first four armadillo repeats. NMR
and analytical ultracentrifugation analyses in combination with crystallographic analysis reveal an intrinsically
labile α‐helix adjacent to the BCL9‐binding site in β‐catenin. This labile α‐helix is responsive to the addition of
carnosic acid. The binding of carnosic acid promoted degradation of active β‐catenin, suppressed
Wnt/β‐catenin signaling transactivation, and regulated expression of Wnt target genes. It is noted that the catechol
moiety of carnosic acid is liable for oxidization and reactions with protein nucleophiles, and has been identified as a
PAINS moiety.104,105 This compound is also associated with many biological activities. The inhibitor optimization
based on carnosic acid is yet to be reported.
On the basis of the observation that the HD2 domain (residues S352‐F374) of BCL9 adopts an α‐helical
structure to bind with β‐catenin first armadillo repeat, and BCL9 residues L366 (i), I369 (i + 3), and L373 (i + 7)
serve as the protruding hot spots, a fragment‐size scaffold 3‐(4‐fluorophenyl)‐N‐phenylbenzamide (PNPB) was
designed as a generalizable scaffold to mimic the binding features of these three protruding α‐helical hot
spots at positions i, i + 3, and i + 7 and was used as the starting point to design inhibitors for disruption of the
β‐catenin/BCL9 PPI (Figure 6).86 The potency of the inhibitor was improved by introducing substituents to
this scaffold. Specifically, two positively charged pyrrolidino groups were introduced to make salt bridge
interactions with β‐catenin D145 and E155, respectively. One F atom was added to the 4‐fluorophenyl moiety
to enhance hydrophobic interactions in the pocket lined with β‐catenin residues L159, L160, V167, and A171.
An additional 4‐phenyl ring was introduced to occupy the pocket lined with residues Q177, L178, and K181 of
β‐catenin. This design strategy was assessed by SAR and site‐directed mutagenesis studies. The direct binding
of the representative compound (PNPB‐22, Ki = 2.1 μM, Table 1) with β‐catenin was demonstrated by ITC
experiments (Kd = 330 nM). PNPB‐22 selectively disrupted the β‐catenin/BCL9 interaction over the β‐catenin/
E‐cadherin interaction in biochemical and cell‐based experiments, inhibited Wnt/β‐catenin signaling

F I G U R E 6 Discovery and optimization of the PNPB series of small‐molecule β‐catenin/B‐cell lymphoma 9

(BCL9) inhibitors. PNPB, 3‐(4‐fluorophenyl)‐N‐phenylbenzamide
14 | WANG ET AL.

transactivation, regulated transcription and expression of Wnt target genes, and suppressed growth of
Wnt‐dependent cancer cells. A further modification was carried out to improve the activity and drug‐like
properties of this series of inhibitors. By the analysis of the predicted binding mode between PNPB‐22 and
β‐catenin (Figures 5 and 6), the tetrazole group was introduced to the PNPB scaffold to capture the additional
interaction with β‐catenin K181, resulting in a compound, PNPB‐29, with improved activity (Ki = 0.47 μM).120
The success of this strategy was again demonstrated by SAR and mutagenesis analyses. To improve drug‐like
properties of the PNPB series, a separate effort was made by introducing the piperazine linker to replace one
phenyl ring in PNPB‐22, followed by SAR studies.121

4 | CONCLUDING REMARKS A ND FUTURE PERS PECTIVES

Aberrant activation of β‐catenin signaling has strongly been implicated in initiation and progression of many types
of cancer. Collective evidence has suggested suppression of this signaling pathway circuit would offer a novel
strategy for the treatment of metastatic cancers and addressing cancer dormancy. Rather than regulating up-
stream effectors, direct targeting of oncogenic β‐catenin, a key signal hub of this pathway, is a biologically com-
pelling approach to suppress hyperactive β‐catenin signaling, but has proven to be a formidable challenge. Peptide‐
based inhibitors have been designed to bind with β‐catenin, but they commonly suffer from low protease and
proteinase stability and poor cell permeability. Small molecules are also reported to bind with β‐catenin and
suppress Wnt/β‐catenin signaling. The major lessons that we have learned from these discovery programs are
listed below.
First, although these small‐molecule inhibitors have been demonstrated to directly bind with β‐catenin using
various biophysical and/or biochemical experiments, none of them have been moved to the latter stages of
inhibitor development. One question to be addressed is how to optimize the compounds to obtain more potent
β‐catenin inhibitors. The co‐crystal structure of small molecules with its target protein often plays a crucial role in
inhibitor optimization, especially for challenging therapeutic targets such as PPIs.122 To date, only the co‐crystal
structure of N‐terminally acetylated 17‐mer stapled peptide aStAx‐35 in complex with β‐catenin (residues
134–665) was resolved (PDB ID, 4DJS, resolution = 3.03 Å, Rfree = 0.291, Figure 2B), highlighting the challenge in
obtaining the co‐crystal structure of small molecules with β‐catenin. One of the reasons behind this might be
suboptimal crystallization conditions. The second question to be addressed is the inferior physiochemical prop-
erties of some reported β‐catenin inhibitors. For example, some reported inhibitors contain PAINS substructures,
which were not identified in the early hit triage and characterization processes and have caused problems for
optimizing these compounds in the β‐catenin inhibitor campaigns. These undesired structures could cause severe
off‐target effects that contribute to the exaggerated pharmacological readouts, further complicating the inhibitor
optimization efforts. On one hand, medicinal chemistry efforts should continue to improve the potency and
physiochemical properties of the inhibitors. On the other hand, new technologies such as cryo‐electron microscopy
(cryo‐EM) might be a powerful additional tool in determining β‐catenin‐small molecule complex structures.123,124
Cryo‐EM was reported to be advantageous for determining the structures of ‘intractable’ targets for which X‐ray
and NMR do not work.
Second, in silico docking, NMR, and mutagenesis studies revealed that most of these inhibitors bind to a region
around hot spot residues K435, N426, H470, R469, and K508 of β‐catenin where residues D16, E17, L18, and I19
of TCF4 interact, implying this hot‐spot region might be a ligandable site. Future screening and structure‐based
design efforts could be focused on this site. Alternatively, the hot‐spot regions identified from the BCL9 binding
site in β‐catenin emerge as new potential sites for small‐molecule binding, given that the contacting area between
β‐catenin and BCL9 is much smaller (1450 Å2 for β‐catenin/BCL9 vs. 4800 Å2 for β‐catenin/TCF) and this PPI
displays moderate binding affinity (KD = 470 nM for β‐catenin/BCL9 PPI vs. 7−10 nM for β‐catenin/TCF PPI).
Encouragingly, carnosic acid and PNPB series were reported to occupy the BCL9 binding site of β‐catenin, and the
WANG ET AL. | 15

residues of β‐catenin for binding with these two series of compounds were determined by NMR and mutagenesis
studies, respectively. These results provide preliminary evidence supporting that the BCL9‐binding site in β‐catenin
can be ligandable. More efforts need to be made to diversify the chemical spaces of the inhibitors, identify more
potent inhibitors, and determine the druggability of this binding site.
Third, β‐catenin not only uses the same surface area for binding with TCF, E‐cadherin (regions III and IV), and
APC, but also use the same area for binding with BCL9 and region V of E‐cadherin. Therefore, selectivity is a
major concern when designing small molecules to disrupt β‐catenin/TCF or β‐catenin/BCL9 interaction. The
selectivity data have been obtained for some of the inhibitors discussed above. UU‐T02 and PNPB series
represent two examples of designing selective inhibitors for β‐catenin/TCF and β‐catenin/BCL9 interactions,
respectively. By analyzing the binding mode of the sequences flanking the hot‐spot residue D16 of TCF, a binding
area at the β‐catenin/TCF interface that is selective for β‐catenin/TCF over β‐catenin/E‐cadherin and β‐catenin/
APC PPIs was revealed by site‐directed mutagenesis studies. To occupy this selective binding site, small‐
molecule inhibitors were designed by a peptidomimetic strategy that includes SiteMap and MCSS technologies.
As a result, the representative compound, UU‐T02, displayed good selectivity for β‐catenin/TCF4 over β‐catenin/
E‐cadherin and β‐catenin/APC interactions. A generalizable PNPB scaffold was designed to mimic the hydro-
phobic side chains of α‐helical hot spots at positions i, i + 3, and i + 7. This scaffold was used to mimic the binding
mode of BCL9 hot spots L366, I369, and L373. Optimization of this scaffold was conducted by introducing two
positively charged pyrrolidino groups to interact with β‐catenin residues E155 and D145. These initial efforts led
to a collection of selective inhibitors of β‐catenin/BCL9 PPI with compound PNPB‐22 (the selectivity for
β‐catenin/BCL9 over β‐catenin/E‐cadherin PPIs = 125‐fold) being the representative. Further optimization was
conducted by introducing the tetrazole group to interact with another adjacent K181 of β‐catenin, resulting in a
compound with improved activity and selectivity. In addition to the selectivities between β‐catenin binding
partners, the specificities of β‐catenin inhibitors over off‐targets should also be assessed by designing appro-
priate rescuse experiments and using appropriate control compounds.125 The off‐target effects from
non‐β‐catenin targets could cause various adverse effects and hamper further development of these inhibitors as
cancer therapeutics.
Last but not least, human β‐catenin has 781 amino acids while only residues 140–664 (central armadillo
domain) have been targeted. The N‐terminal and C‐terminal domains are essential for β‐catenin phosphorylation,
stabilization, and target gene transcription. The binding of small molecules with these two intrinsically disordered
domains is expected to regulate Wnt/β‐catenin signaling, especially given that the areas in the central armadillo
domain occupied by TCF/LEF are very large.126 The challenge is how to make these two intrinsically disordered
regions form binding pockets for small‐molecule binding. In addition, proteolysis targeting chimeras (PROTAC)
technology can particularly be useful for targeting the abnormally accumulated β‐catenin in cancer cells.127 This
approach may be more interesting than the disruption of the challenging PPIs. A PROTAC peptide (xStAx‐VHLL)
has been reported by connecting stapled peptide StAx‐35R with the Von Hippel–Lindau (VHL) ligand.128 xStAx‐
VHLL achieved durable β‐catenin degradation and impaired Wnt/β‐catenin signaling in cancer cells. Moreover,
xStAx‐VHLL decreased CRC cell proliferation and tumor formation in nude mice and reduced the existing tumors
in the mouse models with APC gene mutation. xStAx‐VHLL also inhibited the survival of organoids derived from
patients with CRC. These results indicate the PROTAC might have the potential for developing therapeutic agents
to treat β‐catenin‐related cancer. On the other hand, it is worth noting that small molecules like MSAB and
carnosic acid promote the proteasomal degradation of β‐catenin.
Taken together, β‐catenin is a highly promising target for cancer drug discovery because it is a downstream
effector of the Wnt/β‐catenin signaling pathway and all upstream Wnt signaling pathways are convergent onto this
transcription coactivator. However, targeting this protein has proven challenging. A number of small‐molecule
inhibitors have been reported to bind with β‐catenin, disrupt β‐catenin‐mediated PPIs, and suppress Wnt/β‐catenin
signaling. Although none of these inhibitors go beyond preclinical studies, they serve as good starting‐points for
further inhibitor discovery.
16 | WANG ET AL.

A C K N O W L E D GM E N T S
This study was supported by the Department of Defense CDMRP BCRP breakthrough award W81XWH‐14‐1‐
0083, the Susan G. Komen Career Catalyst Research Grant CCR16380693, Floridian Breast Cancer Foundation
Scientific Grant (19012901), and the 2020 Moffitt Team Science Award. The H. Lee Moffitt Cancer Center &
Research Institute is an NCI‐designated Comprehensive Cancer Center, supported by the Cancer Center Support
Grant P30 CA076292.

C O NF L IC T O F IN T E R ES T S
The authors declare that there are no conflict of interests.

DATA AV AILA BILITY STATEMENT

Data sharing not applicable as no datasets were generated or analyzed during the current study.

ORCID
Haitao Ji http://orcid.org/0000-0001-5526-4503

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WANG ET AL. | 21

A U T H O R B IO G R A P H I E S

Zhen Wang received a Ph.D. degree in medicinal chemistry from the Guangzhou Institute of Biomedicine and
Health, University of Chinese Academy of Sciences. His Ph.D. project was the design and synthesis of small‐
molecule inhibitors targeting receptor kinase discoidin domain receptors. Currently, he is a postdoctoral fellow
at H. Lee Moffitt Cancer Center and Research Institute. He is working on design and synthesis of small‐
molecule inhibitors for β‐catenin/TCF and β‐catenin/BCL9 protein–protein interactions.

Zilu Li received a B.S. degree in pharmaceutical science from Shandong University. He is currently a Ph.D.
student at the University of South Florida and a research associate at H. Lee Moffitt Cancer Center and
Research Institute. He is working on design and synthesis of small‐molecule inhibitors to disrupt the β‐catenin/
BCL9 protein‐protein interaction.

Haitao Ji is an Associate Professor and Associate Member at H. Lee Moffitt Cancer Center and Research
Institute. He received his B.S. degree in pharmacy and Ph.D. degree in medicinal chemistry from the Second
Military Medical University. He received postdoctoral training with Prof. Richard B. Silverman at Northwestern
University. His research has been focused on developing new technologies to design and characterize
inhibitors for protein–protein interactions (PPIs), applying newly developed tools/techniques to design and
synthesize potent and selective inhibitors of important PPI targets, and developing new PPI inhibitors into
novel targeted therapies.

How to cite this article: Wang Z, Li Z, Ji H. Direct targeting of β‐catenin in the Wnt signaling pathway:
Current progress and perspectives. Med Res Rev. 2021;1–21. https://doi.org/10.1002/med.21787