Recent updates on Wnt signaling modulators: a
patent review (2014-2020)

Vishalgiri G. Goswami & Bhumika D. Patel
To cite this article: Vishalgiri G. Goswami & Bhumika D. Patel (2021): Recent updates on Wnt
signaling modulators: a patent review (2014-2020), Expert Opinion on Therapeutic Patents, DOI:
10.1080/13543776.2021.1940138
To link to this article: https://doi.org/10.1080/13543776.2021.1940138
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Publisher: Taylor & Francis & Informa UK Limited, trading as Taylor & Francis Group
Journal: Expert Opinion on Therapeutic Patents
DOI: 10.1080/13543776.2021.1940138
Recent updates on Wnt signaling modulators: a patent review (2014-2020)
Vishalgiri G. Goswami, Bhumika D. Patel
Department of Pharmaceutical Chemistry, Institute of Pharmacy, Nirma University, Ahmedabad,
Gujarat, India 382481
* Address for Correspondence:
Bhumika. D. Patel
Department of Pharmaceutical Chemistry,
Institute of Pharmacy, Nirma University,
Sarkhej-Gandhinagar Highway, Chharodi,
Ahmedabad – 382481
Phone No: +91 9662058019
[email protected]
[email protected]

Abstract
Introduction:
Wnt signaling is a signal transduction pathway that plays a vital role in embryonic development
and normal tissue preservation. Dysfunction of this pathway gives rise to many diseased
conditions like cancer, Alzheimer’s, metabolic and skeletal disorders, kidney and liver disease,
etc. Thus, targeting the Wnt pathway can be a potential approach to design and develop novel
therapeutic classes.
Areas covered:
Authors provided an overview of Wnt modulators from 2014 to 2020. Different heterocyclic
scaffolds and their pharmacology from a total of 104 PCT applications have been summarized.
Expert opinion:
The scientific community is working extensively to bring first in the class molecule to the market
which targets the Wnt pathway. Lorecivivint, Wnt inhibitor, for the treatment of knee
osteoarthritis and SM-04554, Wnt activator, for the treatment of androgenetic alopecia are
currently under Phase III. Other molecules like LGK-974, RXC-004, ETC-159, CGX-1321, PRI724, CWP-232291 and BC-2059 are also under different stages of clinical development for the
treatment of cancer. Antibody based Wnt modulator, OTSA101-DTPA-90Y is currently under
Phase I for the treatment of relapsed or refractory synovial sarcoma while OMP-18R5 is under
Phase I for metastatic breast cancer. Ongoing preclinical and clinical trials will define the role of
the Wnt pathway in different therapeutic areas and have opened new opportunities.
Keywords: β-Catenin, cancer, osteoarthritis, porcupine, Wnt modulators, Wnt signaling.

Article highlights:
• This review provides an overview and analysis of Wnt modulators patented as small
molecules and as antibodies from 2014 to 2020.
• The review also provides detailed insight about Wnt modulators which are currently
under clinical development.
• The role of the Wnt signaling pathway in different diseases is briefly summarized.
• A total of 104 PCT applications, published during 2014-2020 by 32 different
organizations, have been covered in detail with the emphasis on their heterocyclic
scaffold, SAR, and key biological activity.
• Though great progress has been made in the discovery of Wnt modulators, the field is
awaiting the ultimate success in the clinic. Ongoing and future clinical trial results will
help to understand the role of Wnt modulators in various human diseases.

1. Introduction
Since the discovery of Wnt genes in 1982[1], it has captivated the attention of scientists from
different backgrounds. The first gene int-1 was isolated from mouse mammary tumor integration
site-1 [2]. Int-1 is highly conserved in multiple species (similar to drosophila gene wingless) and
plays a crucial role in the development of wing, axis body formation, segmentation, and
evolution of flight movement [3, 4]. Hence, the name “Wnt” comes from the names wingless and
Int-1 [5, 6]. A total of 19 Wnt genes have been identified in humans [7].
Wnt is a lipid-modified glycoprotein with 350-400 amino acids in length [8]. It acts as a
secretary ligand and interacts with the Frizzled receptor (seven-trans membrane protein –
primary receptor) on the cell surface to activate the intracellular Wnt pathway [9]. Apart from
Wnt and Frizzled interaction, low-density lipoprotein-related-protein (LRP) acts as a co-receptor
and is required to mediate Wnt signaling [10, 11]. Once activated, the signal is transduced by
pathway activating protein, Disheveled (Dsh) [12]. The Dsh protein acts as a key switch for the
Wnt signaling pathway which further downstream into mainly two pathways A) canonical
pathway (β-catenin dependent) and B) Non-canonical pathway (Ca2+/PCP pathway) (Figure 1)
[11]. Overall, Wnt signaling is a signal transduction pathway that controls various biological cell
events like proliferation, cell fate determination, apoptosis, and cell migration [13, 14].
1.1 Canonical and Non-canonical Wnt pathway
1.1.1 Canonical/ β-catenin pathway: Inside the cell, β-catenin stability in the plasma is
dependent on a complex of different proteins, known as destruction complex, made up of
scaffolding protein, Axin [15]; tumor suppressor, adenomatous polyposis coli (APC) [16];
Glycogen synthase kinase 3 (GSK-3) and casein kinase 1 (CK1).
In the absence of the Wnt ligand, β-catenin binds to the destruction complex and gets
phosphorylated through CK1 and GSK-3. Subsequently, phosphorylated β-catenin is
ubiquitinated and degraded through the proteasome. (Figure 1)
While in the presence of Wnt secretary ligand which acts through the Frizzled receptor and its
co-receptor LRP; phosphorylation of β-catenin and its subsequent degradation is inhibited,
resulting in accumulation of β-catenin into the cytoplasm. Accumulated β-catenin then enters the
nucleus and binds to the T-cell factor/lymphoid enhancing factor (TCF/LEF) which leads to
transcription of Wnt target genes. This Wnt-inspired target gene plays a vital role in various
biological functions like microtubule formation, cell proliferation, and development.
Overall, the Canonical Wnt signaling pathway is well understood and widely explored for
targeting its different components for the treatment of diseases associated with the Wnt signaling
pathway.
1.1.2 Non-canonical pathway:
Apart from the canonical pathway, Wnt can also activate additional signaling pathways that are
independent of β-catenin. These are called non-canonical pathways which are further categorized
into two categories (a) Planar cell polarity (PCP) pathway and (b) Wnt/Ca2+ pathway.
1.1.2.1 PCP pathway:
The first step in PCP pathway activation is binding of Wnt to Frizzled receptor and co-receptors
like PTK-07 or ROR2 which is independent of LRP co-receptor (LRP co-receptor plays an active
role in the canonical pathway) [11, 17]. The other protein components of the pathway are Dsh,
Daam1, etc. Together this protein phosphorylates JNK (c-Jun NH2-terminal kinase, is a member
of the mitogen-activated protein kinases) which leads to cytoskeletal scaffolding and
arrangement of cells in the developmental process like embryonic heart induction, tissue
segregation, neuronal arrangement, etc. JNK also further activates protein transcription and
translation [18, 19].
1.1.2.2 Wnt/Ca2+ pathway
The main role of the Wnt/Ca2+ pathway is to regulate intracellular calcium levels from the
endoplasmic reticulum. Like other Wnt signaling pathways, the Ca2+ pathway requires the
binding of the Wnt ligand to the Frizzled receptor. The activated pathway further activates Dsh
and phospholipase C (PLC) and stimulates downstream effector, inositol 1,4,5-triphosphate (IP3),
and diacylglycerol (DAG) to bring out the intracellular release of calcium [20, 21]. The released
calcium further activates calmodulin-dependent kinase II (CAMKII) and protein kinase C (PKC).
Together they stimulate the nuclear factor of activated T-cells (NFAT). NFAT inspired genes
further play their role in cell fate determination [20, 22].
1.2 Role of the Wnt signaling pathway in various disorders [23]
1.2.1 Skeletal disease
Osteoarthritis (OA) is the most common chronic joint condition that resulted in cartilage loss and
structural changes including the formation of osteophytes and sclerosis. All these together
contribute to pain and loss of function. Wnt signaling pathway regulates osteoblasts and
chondrocytes differentiation and up regulation of Wnt pathway is observed in Osteoarthritis.
Many past studies have already proved the role of Wnt inhibitor in Osteoarthritis and
Osteoporosis. [24, 25].
1.2.2 Colorectal cancer (CRC)
Aberrant hyper activation of Wnt signaling is observed in CRC. APC is widely accepted as
tumor suppressor gene in CRC. Mutation or inactivation of this gene is a key early event in
colorectal tumorigenesis. APC truncation is a major driver of colorectal cancer. This indicates
the role of APC mediated canonical signaling pathway in colorectal cancer. APC mutation often
occurs in the mutation cluster region (MCR) which accounts for 10% of the entire coding region
in the APC gene. Consistent with this hypothesis, more than 80% of colorectal patients show
APC mutation [26, 27]. A study concluded that 28 out of 43 somatic mutations in colorectal
cancer cells occur in the MCR, which inhibit β-catenin ubiquitination, degradation, and
ultimately lead to unrestricted transcription of cell proliferation genes. β-catenin mutation also
plays a key role in colorectal cancer. Approximately 10% of CRC carry mutations in the GSK3β
phosphorylation site located in the N-terminus of β-catenin [28]. Alexander A. et. al described
that majority of β-catenin gene CTNNB1 mutation in CRC is homozygous and restricted to
mutation at codon 41 and 45 [29]. The same finding was also proved by Laura et.al where the
author suggested that CTNNB1 mutation is common in MSI-H (high level microsatellite
instability) colorectal carcinoma [30]. These types of specific mutations prove that the right level
of β-catenin stabilization is essential for carcinogenesis. This finding suggests an important role
of β-catenin in targeting colorectal cancer.
1.2.3 Breast and hepatocellular cancer
Around 45% of hepatocellular cancer cells overexpress LRP6 and found to have elevated βcatenin levels. Hyperactivity of Wnt signaling pathway is responsible for hepatocellular
carcinoma [31]. Similarly, up regulation of Wnt pathway due to alteration of many of its
components plays an essential role in breast cancer pathogenesis. Overexpression of LRP6 is
also observed in triple-negative breast cancers [32].
1.2.4 Alzheimer disease (AD)
The synaptic decline is a common observation in aging and Alzheimer’s disease. Wnt signaling
pathway plays its major role in the regulation of synaptic plasticity and in AD, decreased levels
of canonical Wnt signaling has been observed. Loss of cognition is also supported by the loss of
Wnt signaling. Enhancing Wnt signaling can boost synaptic function during aging and AD
patients. [33].
1.2.5 Metabolic diseases
It has been proved after many studies that each segment of the Wnt pathway is involved in
pancreatic cell proliferation, lipid metabolism, and insulin secretion [34]. Accumulation of
reactive oxygen species (ROS) leads to an increase in nuclear forkhead box O (FOXO – mammal
proteins that mediate the inhibitory action of insulin or insulin-like growth factor on functions
involved in cell metabolism, growth, differentiation, oxidative stress etc.) which leads to reduced
Wnt activity. [35]. Types of ROS, their cell type and tissue environment contribute to
maladaptive response which further leads to metabolic diseases and inflammatory signaling.
Metabolism of glucose also produce ROS via different metabolic pathways like sorbitol
metabolism, hexosamine metabolism, α-ketoaldehyde production and oxidative phosphorylation
[36]. ROS also contributes to regulation of vascular tone and inflammatory signaling in diabetes
mellitus and obesity. Modulation of Wnt signaling pathway governed by ROS production affects
overall activities like stem cell differentiation, angiogenesis, VEGF signaling, vascular cell
migration etc. [37]. Role of ROS in different signaling pathway and its effect on metabolic
disorder is area of research to understand it in more detail.
1.2.6 Cardiovascular disease
Both canonical and non-canonical Wnt signaling pathways contribute to the normal homeostatis
of cardiovascular system [38]. Mutation of the LRP6 gene plays a major role in early coronary
disease, hypertension, and hyperlipidemia. Also, an increased level of plasma DKK1 (Dickkopfrelated protein 1, An antagonist of the Wnt signaling pathway which acts by isolating LRP6 coreceptor so that it cannot act in activating the Wnt signaling pathway) is observed in patients
with coronary artery disease. [39, 40].
1.2.7 Liver disease
β-catenin plays important role in fibrotic human liver tissue. An increased level is also associated
with collagen production and proliferation. The depletion of β-catenin and other Wnt
components, (downregulation of Wnt signaling pathway) is responsible for delayed liver
regeneration following partial hepatectomy [41]. Mutation in regulatory genes of the Wnt
signaling pathway is characteristic of hepatobiliary tumors [42].
1.2.8 Kidney disease
Activation of the Wnt signaling pathway is required for tubular repair and regeneration after
acute kidney injury. Nevertheless, sustained activation (upregulation of the Wnt signaling
pathway) results in the advancement of acute kidney injury to chronic disease. Dysregulation of
Wnt signaling is observed in a wide variety of kidney disorders like fibrosis, cystic formation,
proteinuria [43, 44].
1.2.9 Lung disease
Developmental Wnt signaling pathway is essential pathway for lung development and altered
Wnt signaling activity contributed in pathogenesis of chronic lung disease and idiopathic
pulmonary fibrosis [45]. In lung inflammation, increased levels of matrix metalloproteinase
(MMPs) and inflammatory cytokines are common features. Activation of the canonical Wnt
signaling pathway leads to increased expression of various MMPs in mice which proved the role
of the Wnt signaling pathway in the regulation of lung inflammation [46].
1.2.10 Androgenetic alopecia
Therapy based on the Wnt signaling pathway also plays a crucial role in the field of
dermatology. Androgenetic alopecia, also known as male baldness, is due to hair follicular
miniaturization. Studies proved that inhibition of Wnt activity in the hair cycle is responsible for
alopecia. Activating the Wnt pathway showed a marked increase in hair growth [47]. SM-04554
is a topical scalp treatment Wnt activator that is currently under clinical evaluation of phase-III
for the treatment of androgenetic alopecia.
1.2.11 Tendinopathy
Out of total population of musculoskeletal disease in US, 30% of muscular tendinopathy is
related to either sports injury or injury resulted from daily tasks [48]. Modulation of Wnt
signaling pathway can affect tendon development as well as repair. This modality helps in
regeneration of the injured tendon. Novel molecule SM-04755, currently under phase I clinical
trial, inhibits intranuclear kinases and modulate Wnt activity for tenocyte differentiation [49].
Overall, it also reduces tendon destroying proteases and reduces inflammatory marker formation
[50].
1.3 Wnt modulators under clinical trials
Currently, a total of 13 molecules (10 small molecules and 3 antibodies) as Wnt modulators have
entered the clinical trial phase. Table 1 represents the overview of all the molecules under
clinical trials and Figure 2 summarized the Wnt protein components targeted by respective
inhibitor/activators. Out of 10 small molecules, Samumed LLC, USA is ahead with a total of 3
molecules. Lorecivivint (SM-04690) is a Wnt inhibitor into the phase-III clinical trial for knee
osteoarthritis (NCT04520607) [51, 52]. SM-04554, Wnt activator, is in its phase-III trial for
androgenetic alopecia (NCT03742518) [53]. SM-04755 acts as a Wnt inhibitor and under phaseI clinical trial for the treatment of tendinopathy [49]. LGK-974 developed by Novartis is under
Phase I for the treatment of Wnt-dependent malignancies[54, 55]. Array Biopharma is
developing the same molecule LGK-974 in combination with Cetuximab (anti-epidermal growth
factor receptor (EGFR) monoclonal antibody) for metastatic colorectal cancer [56]. RedX
Pharma is developing RXC-004 as a porcupine inhibitor for advanced malignancies which is
under phase-I clinical trial [57]. Another porcupine inhibitor ETC-159 developed by A*STAR
Research Entities for the treatment of advanced solid tumors is in its phase-1 clinical trial [55,
58]. CGX-1321 which acts as a Wnt inhibitor is under phase-I clinical trial, developed by
Curegenix Inc for advanced gastrointestinal tumors [59]. Prism pharma is also developing PRI724 as a Wnt inhibitor for advanced myeloid malignancies and is also in a phase-I clinical trial
[55, 60]. The same molecule is also under evaluation in combination with Gemcitabine
(pyrimidine nucleoside antimetabolite-cytotoxic agent) for metastatic pancreatic adenocarcinoma
[61]. Tegavivint (BC-2059) developed by Iterion pharmaceuticals is in its phase-I clinical trial
which acts as Wnt inhibitor for patients with unresectable desmoid tumor [62, 63]. CWP232291, developed by JW pharmaceuticals, is in its phase-I clinical trial and acts as Wnt
inhibitor through β-catenin for acute myeloid leukemia [64]. OTSA101-DTPA-90Y is an antiFrizzled Homolog 10 (FZD10) monoclonal antibody under phase I evaluation for relapsed or
refractory synovial sarcoma [65]. OMP-18R5 is also a monoclonal antibody against FZD
receptor under phase I evaluation for the treatment of solid tumors [66]. OMP-54F28 is FZD8
decoy receptor antibody under phase I evaluation for solid tumors [67].
2. Patented Wnt modulators (2014-2020)
2.1 Organization of the review
This patent review mainly focuses on small molecules as Wnt modulators that act via different
components/proteins of the Wnt signaling pathway and covers all applications from 2014 to the
present. Only published PCT applications have been considered in this review. During the last
five years, significant progress has been made in the area of Wnt modulators. We are describing
Wnt modulator patents from 25 major big pharma and academic institutes with coverage of
around ~104 published PCT on small molecule applications from 2014 to 2020. We also tried to
include ~12 patents on antibodies that act through Wnt modulation from 2014 to 2020.
Samumed explored different heterocyclic scaffolds based on indazole, 1H-pyrazolo[3,4-
b]pyridine, 1H-pyrazolo[3,4-c]pyridine, 1H-pyrazolo[4,3-b]pyridine where 5th position is
substituted with pyridine-3-yl. The organization tried these structural analogs for Wnt-related
disorders which act through modulation of one or more components of the Wnt signaling
pathway. Other derivatives containing 6-methylisoquinoline and 7-methylquinazoline type of
carboxamide scaffold have been explored as Wnt modulator for the treatment of diseases linked
to overexpression of DYRK1A.
During compiling this review, it was also observed that biaryl, bipyridine, and bicyclic
heterocyclic carboxamide scaffolds have been extensively explored by Bayer, ASTAR group,
Redx pharma, Huihan medical technology etc. Because multiple and diverse chemical scaffolds
have been explored and reported for Wnt modulation in various patents, it was found difficult to
categorize the patents based on a particular chemotype. Therefore, we have organized the key
compounds based on the maximum number of patents published by different organizations in the
area of Wnt modulation. The discussion begins with Samumed LLC with 37 patents and then
moves on to Bayer Pharma with 9 patents and likewise. At last, we tried to cover total 12 patents
published on antibody based Wnt modulators.
2.2 Key organizations targeting small molecule based Wnt signaling modulators
2.2.1 Samumed LLC, USA
Samumed LLC, a biopharmaceutical company based in San Diego, California, USA, is working
aggressively on the Wnt signaling pathway and had published more than 37 PCT in this area
since 2014. As a result, currently, three molecules that act through the Wnt signaling pathway are
under clinical development: SM-04690, SM-04554, and SM-04755. Table 2 represents an
overview of patents published by Samumed with the details like Patent number, the total number
of molecules covered in the patent, structure, and biological activity of the key compound, and
important comments.
First published PCT from Samumed WO2014110086 covers 3-(benzimidazol-2-yl)-indazole
derivatives as inhibitors of the Wnt signaling pathway for the treatment of Wnt-related disorders
[68]. The patent disclosed a variety of indazole compounds sufficient to inhibit the Wnt signaling
pathway which can correct genetic disorders due to mutation in Wnt signaling components. The
most promising molecule Lorecivivint, SM-04690 (Compound 6, Table 2) is currently under
Phase-III clinical trial for knee osteoarthritis. Wnt signaling pathway is a pivotal pathway in
Osteoarthritis (OA) which controls bone modulation, chondrocytes differentiation and protease
production. Increased Wnt activity in OA results into production of Osteoblast and protease
which cause thinning of the cartilage and ultimately results into OA. A novel Wnt signaling
pathway inhibitor SM-04690 inhibits Wnt pathway, prevents osteophytes formation and blocks
protease-mediated cartilage degradation by targeting cdc-like kinase 2 (CLK2) and DYRK1A.
As a therapeutic agent, it increases chondrocyte differentiation and function, slows down or
reverses the degenerative process of cartilage breakdown, causes generation of cartilage and
reduces the inflammation during the treatment of Osteoarthritis.
WO2014130869 disclosed gamma-diketone chemotype as a β-catenin activator of the Wnt
signaling pathway. Out of 767 derivatives based on 2,3-dihydrobenzo[b][1,4]dioxine ring
(compound 7, Table 2), the most promising Wnt activator, SM-04554, is currently under PhaseIII evaluation for Androgenetic alopecia [69]. Mammalian hair cycle regularly generates hair
follicles using stem cell-mediated process. The Hairless (Hr) gene is essential for hair follicle
generation. Gerard et al. proved in their study the exact role of Wnt activation and timing of hair
follicle regeneration [47]. Based on that mechanism, SM-04554 promotes follicular neogenesis
or follicle proliferation and hair growth through β-catenin mediated Wnt activation.
WO2015143380 disclosed 5-substituted indazole-3-carboxamides derivatives and explored for
the antiproliferative activity against human fibroblast LL29 cells. Patent focused on indazole
derivatives as Wnt modulators for treatment of disorders related to neurological conditions
linked to overexpression of DYRK1A. Various in vitro screening results of best compound 8 is
shown in Table 2 [70].
In 2016, WO2016040180 and WO2016040181 disclosed azaindazole derivatives where both
patents separately claims 1477 molecules and evaluated them for Wnt inhibitory activity [71,
72]. Most promising compound 9 (Table 2) showed antiproliferative activity against human
fibroblast LL29 cells with EC50 0.009nM. Indazole chemotype was subsequently explored
systematically by Samumed and as a result of that, a total of 25 PCT’s were published with an
exploration of different heteroaryl substitutions on Indazole moiety. Key compounds and
biological results are discussed in Table 2. (Compound 11-36) [73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98].
In continuous efforts from Samumed on exploring different chemotypes for Wnt modulation,
isoquinoline carboxamide was the second choice. The most promising compounds and biological
results are discussed in Table 2 (Compound 37-40) [99, 100, 101, 102].
A much larger patent WO2019089835 covered a total of 4658 molecules based on
diazanaphthalene-3-yl carboxamide scaffold as Wnt modulators for the treatment of neurological
conditions linked to overexpression of DYRK1A. (Table 2 Compound 41) [103].
WO2019241540 includes macrocyclic indazole heteroaryl derivatives as modulators of Wnt
signaling pathway. Total of 115 molecules have been covered of which promising compound
42, discussed in Table 2, decreased TGF-β1-induced fibrosis in primary human lung fibroblast
LL29 derived from idiopathic pulmonary fibrosis patients (EC50 = 0.019 µM) [104]. According
to the recently published PCT WO2020150545 in July 2020 from Samumed, Organization is
working now on pyrazole derivatives as Wnt modulators where modulation of overall Wnt
activity is linked to overexpression of DYRK1A. A total of 230 molecules were covered by the
patent, of which most promising Compound 43 (Table 2) reduced IL-6 production in human
peripheral blood mononuclear cells with EC50 1.024 μM in HTRF (Homogeneous TimeResolved Fluorescence) assays [105].
2.2.2 Bayer Pharma, Germany
In 2014, Bayer published a PCT WO2014147182 and disclosed the synthesis of substituted Nbiphenyl-3-acetylamino-benzamides derivatives and N-[3-(acetylamino)phenyl]-biphenylcarboxamides derivatives as novel β-catenin/Wnt inhibitors for the treatment of
hyperproliferative disorder [106]. The patent disclosed a total of 222 molecules with 127
exemplified molecules with detailed experimental procedures and 1
H-NMR and LC-MS data.
Wnt inhibitory activity was confirmed by observing inhibition on the constitutive active
colorectal cancer cells (CRC) in Super Top Flash (STF) assay (cells were generated by
transfection of the CRC cell line HCT116 with Super Top Flash vector). Selected compounds
were further assessed in STF assay for Wnt inhibitory activity using HEK293 cells. The patent
was about the exploration of the various aliphatic linker with an aryl amide scaffold. Wnt
inhibitory activity and IC50 value of key compound 44 are summarized in Figure 3.
Bayer published another PCT WO2014147021 with the same Markush structure and
exemplification of a total of 294 molecules, of which 177 molecules have been disclosed in
detail with synthetic experimental data [107]. In this PCT, various types of substitutions were
extensively explored like; fluorinated biaryl, phenyl pyridine, phenyl thiadiazole, etc. Wnt
inhibitory activity of all molecules was assessed using similar biological assays discussed above
for WO2014147182. Promising compound 45 (Figure 3) was a racemic mixture, which was
further isolated as a separate enantiomer using Chiral Prep HPLC. A significant difference
during in-vitro screening has been observed for both enantiomers. Compound 45R inhibited
Wnt signaling pathway in HCT116 cancer cells transfected with Super Top Flash and FOP
control vectors with IC50 0.014 µM and 20.2 µM respectively while Compound 45S showed
IC50 0.051 µM and 83.5 µM respectively. Thus, compound 45R is 3-4 fold more active than
compound 45S. Similarly, in wild-type Wnt signaling pathway in HEK293 cells transfected with
Super Top Flash and FOP control vectors, IC50 for compound 45R was 0.009 µM and 82 µM as
compared to 0.130 µM and 81 µM for compound 45S, which also reflected that compound 45R
was 14 fold more potent as compared to compound 45S. (Figure 3)
Inspired by promising results of compound 45, Bayer published another two PCT
WO2015140195 & WO2015140196 exactly after one year from the above-discussed patents
with the same Markush structure as inhibitors of the Wnt signaling pathway. WO2015140195
disclosed a total of 119 molecules based on different hetero aryl scaffolds like; pyridinepyrimidine, pyridine-thiadiazole, bipyridyl, pyrazine-thiazole, thiazole-thiadiazole, etc. [108].
While amide substitutions were mostly substituted methyl piperazine acetamide and morpholino
acetamide. It’s interesting to note that the most promising compound 46 (Figure 3), having only
a pyridine ring on the left-hand side of the ring structure compared to the simple aryl ring in
compound 45, showed improved biological activity; even though it was tested as a racemic
mixture. However, the isolation of individual enantiomers and their biological results were not
disclosed for compound 46 in the original patent document.
WO2015140196 disclosed synthesis and Wnt inhibitory activity for 63 novel compounds[109].
Patent mainly explored aryl-pyrazine, aryl-pyridazine & pyridyl-pyrazine moieties in its
pharmacophore. Molecules were evaluated for in-vitro inhibitory activity against constitutive
active colorectal cancer cell line HCT116, where promising compound 47 showed an IC50 value
of 0.013 µM. Compound 47 also showed Wnt inhibitory activity using mammalian cell line
HEK293 with IC50 >50 µM in a cellular reporter assay.
In 2016, Bayer published another three patents WO2016131794, WO2016131808, and
WO2016131810 as inhibitors of the Wnt signaling pathway [110, 111, 112]. WO2016131794
was about 3-Carbamoylphenyl-4-carboxamide and isophtalamide derivatives as Wnt inhibitors.
The patent disclosed a total of 63 exemplified compounds out of which the most promising
compound 48 (Figure 4) inhibited constitutive Wnt signaling pathway in mammalian cell line
HEK293 with IC50 of 0.0917 µM and human colon HCT116 cancer cells with IC50 of 28 µM in
cellular reporter gene assays. WO2016131808 disclosed 1,3,4-Thiadiazol-2-yl-benzamide
derivatives where a total of 34 derivatives were characterized, evaluated for Wnt inhibition.
Promising compound 49 (Figure 4) showed improved inhibitory activity compared to
compound 48. Compound 49 inhibited the Wnt signaling in mammalian cell line HEK293 and
human constitutive active colorectal cell line HCT116 with IC50 0.0415 µM and >50 µM
respectively. Another similar patent WO2016131810 disclosed 39 N-Phenyl-(morpholin-4-yl or
piperazinyl)acetamide derivatives as inhibitors of the Wnt signaling pathway. The most
promising compound 50 (Figure 4) showed 30-fold improved activity compared to compound
48 in the HEK293 TopFlash assay. However, it is interesting to note that compound 50 showed
a quite structural similarity with earlier explored promising molecules like compounds 44-46,
which reflects consistent and improved SAR results from long efforts puts in by Bayer pharma.
Recently a patent WO2019063704 published by Bayer disclosed 3-phenyl quinazoline-4(3H)-
one derivative as Wnt inhibitors [113]. Patent summarized that phenyl quinazoline-4(3H)-one
scaffold was earlier evaluated for obesity, diabetes, depression, and anxiety. However, it was
never explored earlier as a Wnt inhibitor. By bringing novelty into Phenyl quinazoline-4(3H)-
one core structure, a total of 149 molecules were exemplified of which promising compound 51
(Figure 4) showed IC50 6.1 nM (HEK293 TOP/FOP assay). A similar patent published on the
same date WO2019063708 was having the same Markush structure as that of WO2019063704
[114]. However, the 2nd position of quinazoline-4(3H)-one scaffold was extensively explored
using different secondary aliphatic cyclic amines. Out of 63 synthesized molecules, the most
promising compound 52 (Figure 4) showed IC50 48.3 nM (HEK293 TOP/FOP assay). Thus, no
significant improvement in terms of potency was observed as compared to compound 51. Both
Compounds 51 and 52 are quite similar structurally. Compound 52 is having a cyclopropyl
group in place of methyl on the amidic side chain and 6-oxa-3-azabicyclo[3.1.1]heptane ring in
place of morpholine ring.
2.2.3 Agency for Science, Technology, and Research (A*STAR), Singapore
ASTAR published a total of 6 patents from 2014 to 2020 on a different heterocyclic derivative as
Wnt modulators. WO2014175832 briefly summarized the role of the Wnt signaling pathway and
its association with various diseases like carcinoma, pulmonary fibrosis, diabetic retinopathy,
rheumatoid arthritis, etc. [115]. As a need of compound that modulate Wnt signaling pathway,
the agency reported a total of 139 heterocyclic carboxamide derivatives with a general structural
formula like Ar1-Ar2-X-C(R1 R2)-C(=O)-N(3)-Ar3-Ar4; [all Ar (1-4) were aryl or heteroaryl, R1-
R3 were hydrogen, alkyl or alkene group, X was ‘O’, ‘N’ or ‘CH2’]. In-vitro Wnt inhibitory
activity of all synthesized molecules was primarily assessed using luciferase assay (HEK293-
STF3A cell line was modified to express Wnt3A) followed by in-vivo screening using the mouse
mammary tumor virus (MMTV)-WNT1 animal model. Promising compounds 53 and 54
(Figure 5) showed IC50 < 0.1 µM in the in-vitro assay. Compound 53 reduced average tumor
volume from 950 mm3 to 380 mm3 and compound 54 reduced tumor volume from 950 mm3 to
300 mm3 after 21 days at 30mg/Kg oral dose.
A second application from ASTAR filed simultaneously, based on Purine-dione scaffold, was
WO2014189466 with a similar screening strategy [116]. The patent was mainly focused on
changing different hetero aryl substitutions while keeping the alkyl-substituted purine-dione
scaffold common. A total of 94 molecules were exemplified and screened. Representative
compound 55 (Figure 5) inhibited phosphorylated LRP6 (Ser1490) levels by 50-60% and 20%
at 2nM and 3.3nM, respectively in human pancreas HPAF-II cancer cells [LRP5 acts as a coreceptor with LRP6 and both are highly homologous single-pass transmembrane proteins of the
low-density lipoprotein receptor, upon Wnt binding, LRP is phosphorylated on multiple sites like
Thr1490, Ser1490 and Thr1493]. Compound 55 reduced average tumor volume from 1350 mm3
to <300 mm3 after 19 days with an oral dose of 30mg/kg using the (MMTV-WNT1 tumor animal
model. Compound 55 (also known as ETC 159) is currently under phase I evaluation for safety
and tolerability in advanced solid tumors [58].
ASTAR also developed a maleimide scaffold where the 3rd amidic position was extensively
explored using aryl and heteroaryl functionality in WO201594118 [117]. Out of 158 synthesized
molecules, promising compound 56 (Figure 5) suppressed palmitoylation of Wnt3A in human
lung HeLa cancer cells at 100 nM in Wnt3A palmitoylation assay. Details of the assay were well
covered by Yap et al [118]. Additionally, IC50 value of compound 56 in human pancreas
adenocarcinoma cells using soft agar assay is shown in Figure 5 (To evaluate cellular
transformation in-vitro, soft agar assay is widely used). Compound 56 was further evaluated invivo into MMTV-WNT1 animal tumor model at three different doses (1mg/kg, 3 mg/kg, and
10mg/kg) – where decreased tumor growth was observed in all treated mice at a dose of 3 mg/kg
and 10 mg/kg.
WO201594119 was subsequently published by ASTAR with dihydropyrazolo[1,5-a]pyrimidine
derivatives as Wnt modulators [119]. Compounds were evaluated with an extension of
methylene linker between two amide functional groups with a new scaffold as
dihydropyrazolo[1,5-a]pyrimidine. Out of 171 reported molecules, the most promising
compound 57 (Figure 5), displayed an IC50 value of less than 0.1 µM in HEK293-STF3A cells
expressing Wnt3A activity in luciferase assays. Compound 57 also suppressed the
palmitoylation of Wnt3A in human cervical HeLa cancer cells at 100 nM in combination with
alkyne palmitate. Compound 57 was also evaluated in vivo in MMTV-WNT1 mice tumor model
at three different doses – where decreased tumor growth was observed at an oral dose of 3mg/kg
and 10 mg/kg.
Lastly, in December 2015, ASTAR published another patent WO2015187094 with Phthalimide
derivatives as Wnt modulators [120]. Inspired by promising results from the maleimide scaffold
(WO201594118), Agency brings novelty through aromatization of maleimide moiety which led
to novel Phthalimide scaffold. Out of 129 synthesized derivatives compound 58 (Figure 5)
displayed an IC50 value of less than 0.1 µM in HEK293-STF3A cells expressing Wnt3A in
luciferase assays. Compound 58 also dose-dependently reduced tumor growth in a MMTVWNT1 mice tumor model at 1, 3, and 10 mg/kg.
In 2019, agency identified selective Wnt modulator by appending 1,3-dimethyl-3,4,5,7-
tetrahydro-1H-purine-2,6-dione core structure to amidic linker and published as patent
WO2019054941 [121]. The patent disclosed synthesis and extensive biological screening of only
one key compound 59 (Figure 5). Compound 59 displayed the IC50 value of 0.009 µM in
HEK293-STF3A cells expressing Wnt3A in luciferase assay with 42% bioavailability (%F).
Upon single dosing to male BALB/c mice, it showed the pharmacokinetic parameter values as
below: a) at 1 mg/kg i.v.: t1/2 = 4.24 h and AUC (infinite) = 1487.95 h·ng/mL; b) at 5 mg/kg p.o.:t1/2
= 3.26 h and AUC (infinite) = 3116.66 h·ng/mL
2.2.4 H. Lee Moffitt Cancer Center and Research Institute, Inc. USA
H. Lee Moffitt Cancer Center & Research Institute, established in 1981, is a nonprofit cancer
treatment and research center located in Tampa, Florida. A total of six patents were published
from 2014-2020 on the Wnt modulators. The focus for all patents was based on the conclusion
that the formation of β-catenin/B-cell lymphoma 9 (BCL9) complex in the cell nucleus is the
penultimate step of canonical Wnt signaling and so the aberrant formation of this protein-protein
complex is a major driving force for triple-negative breast cancer (TNBCs) tumorigenesis.
The first patent WO2019118961 was about finding a selective inhibitor for the β-catenin protein
interaction. Patent exemplified only five molecules from which promising compound 60
(Figure 6) suppressed wild-type β-catenin/BCL9 interactions (Ki = 8 µM) in Alpha Screen
assays [122].
A subsequent patent published in the next month, WO2019139961, disclosed only two
compounds that were modified by inserting amide functionality in place of the quinoline ring
while the tetrazole ring was appended with the replacement of 4-fluoro aryl moiety [123]. Key
compounds 61 and 62 (Figure 6) along with their biological screening results are summarized in
Figure 6.
In April 2020, H. Lee Moffitt's research center published another two PCT WO2020081917 and
WO2020081918 for evaluation as β-catenin/BCL9 interaction inhibitors. WO2020081917
exemplified a total of 32 derivatives with detailed synthetic experimental protocols [124]. In this
patent, the inventor tried to evaluate piperazine core with terminal cyclohexane ring and small
heteroaryl moiety appended to 3-fluoroaryl functionality. Most promising compound 63
(Figure 6) inhibited His6-tagged full-length β-catenin/biotinylated human BCL9 protein-protein
interaction with Ki = 12.34-25.37 µM in competitive Alpha Screen assays. In WO2020081918
patentee kept all structural aspects as like earlier published patents, except different aliphatic
amide functionality were appended and published a total of 16 molecules [125]. Compound 64
(Figure 6) inhibited wild-type full-length His6-tagged β-catenin and N-terminal biotinylated
human BCL9 protein interaction with Ki = 61-37 µM in competitive Alpha Screen assays.
Apart from the above-discussed inhibitors of β-catenin/BCL-9 interaction, H. Lee Moffitt's
research center also published another two different PCTs with different chemotypes and
approaches towards it. First, WO2019191410, where patentee had systematically identified
binding site followed by its bio isosteric replacement results into selective compound 68
(Figure 7) with Ki = 0.83 ± 0.15 µM as compared to its initial fragment compound 65 with Ki =
990 ± 36 µM [126]. In WO2019191410, the patentee disclosed the only overview of designing
strategies like binding site identification, followed by bio isosteric replacement with different
moiety to achieve better Ki value. One can refer to different publications of the same inventor
where they had used a similar designing approach [127, 128, 129]. Second, PCT
WO2020118179 was about to find peptidomimetic inhibitors of β-catenin/TCF protein-protein
interaction [130]. Out of 57 synthesized peptide molecules, compound 69 (Figure 7) inhibited
human β-catenin/TCF protein-protein interactions with Ki = 0.44 µM in fluorescence
polarization (FP) competitive inhibition assay. The detailed designing strategy of compound 73
was well covered by Z Wang et al[131].
2.2.5 Merck, USA and Cancer Research Technology Limited, United Kingdom.
Merck in collaboration with Cancer Research Limited published a total of 3 patents in the area of
Wnt modulation. All patents provided background information about the importance of the
canonical Wnt signaling pathway and its relevance to different types of cancers. Cancer Research
Institute had published independently its first patent on the Wnt signaling pathway in 2010
(WO2010041054), where pyridine and pyrimidine scaffolds were extensively explored,
however, the major limitation was about high human hepatic clearance.
To identify key Wnt modulators and to address earlier key limitations, the organization published
a patent WO2014063778 as 2-aminopyridine compounds [132]. Group had systematically
evaluated different spiral ring scaffolds appended to the 4th position of a pyridine ring structure.
To get a quick overview of SAR, key compounds 70-81 along with their IC50 (using LS174T-L5
reporter assay; where LS174T is human colon cancer cell line) values are summarized in Figure
8. Subsequently, the same group published a much large patent WO2014086453 with the
exemplification of a total of 300 molecules based on Aza heterobicyclic compounds. Key
compounds of interest, compounds 82-85 having IC50 below 0.05 µM are summarized in Figure
9. Compounds (82-85) inhibited the Wnt pathway in human colorectal adenocarcinoma HT-29
cells with IC50 < 0.05 µM in luciferase reporter gene assay [133].
In 2015, Merck published another patent WO2015144290 with an exploration of 100 Pyridyl
piperidine derivatives for the treatment of Wnt-dependent multiple disorders like cancer,
inflammatory disorders, and degenerative diseases [134]. Most promising compound 86 (Figure
9) inhibited Wnt signaling in HEK-293 cells with IC50 3.2 nM in luciferase reporter gene assays.
Compound 89 (Figure 9) showed the highest IC50 0.075 nM. However, compound 89 showed
high hepatic clearance.
2.2.6 Duke University, USA
Duke University is a private research university in Durham, North Carolina, founded in 1838. In
2016, University published a patent WO2016210247 on diseases associated with dysregulation
of the Wnt signaling pathway [135]. The patent disclosed a Total of 5 heterocyclic amide
molecules along with 3 commercially procured marker molecules. Molecules were further
evaluated for Wnt inhibitory activity using TopFlash reporter assay. Key compounds 91-93 and
biological screening results are summarized in Figure 10.
Another patent was published on the same date WO2016210289 as Wnt modulators[136]. A
total of 80 molecules were synthesized and evaluated for Wnt inhibitory activity. Key
compound 94 (Figure 10) showed an IC50 value of 0.23 µM in TopFlash reporter assay.
Different PK parameters of key compound 94 were further evaluated through in-vivo study in
mice using once-a-day oral dosing of 200mg/kg in corn oil-based formulation (Compound 94 is
lipophilic, ClogP 4-5 hence oil-based formulation was used). Cmax, AUC, and duration of action
of compound 94 were found to be significantly increased even at one-third dose as compared to
parent compound 91(Niclosamide) itself. (In-vivo PK results of Niclosamide were not disclosed
in WO2016210289 but similar PK results from Duke University were published by T.Osada et
al.[137]) Results are summarized in Figure 10.
Recently in 2020, University published WO2020028392 disclosing Niclosamide analogs and
their therapeutic use [138]. A total of 8 molecules were synthesized and evaluated. Standard
Niclosamide analogs were improvised biologically by synthesizing trifluoromethyl
benzimidazole analogs in-place of the 4-chlorophenol moiety. Comparative in-vitro screening
data of compound 99 with standard Niclosamide (compound 91) are summarized in Figure 11.
Promising compound 99 was further evaluated in-vivo in CRC tumor growth animal model.
NOD/SCID mice bearing CRC240PDX tumors were dosed orally daily for 11 days with
compound 99 at 1 mg/kg and Niclosamide (compound 95) at 72 mg/kg dose. Tumor size and
body weight were measured at day 0, 4, 8 and 11. Compound 99 demonstrated similar antitumor
activity as Niclosamide (Compound 91, dose 72mg/kg) but at a significantly lower dose.
2.2.7 Curegenix Ltd, USA
Curegenix has been always trying to discover and develop first-in-class innovative drugs with
operations in the San Francisco Bay Area and Guangzhou, China. The company is mainly
focusing on the discovery and development of novel drugs targeting the WNT signaling pathway
for cancer. Curegenix published two patents WO2014165232 and WO2014159733. The
WO2014165232 patent claims compounds as inhibitors of the Wnt signal transduction pathway
and used for the treatment of cancer while the WO2014159733 patent exclusively disclosed
compounds used to treat Fibrosis [139, 140]. Total 112 same compounds have been claimed by
the inventor for different therapeutic purposes. The structure of key compounds 100-102 and
biological results are summarized in Figure 12. Curegenix developed its first molecule CGX-
1321 from patent WO2014165233, which is currently under Phase-I clinical trial for advanced
gastrointestinal tumors [59].
2.2.8 New York University, USA
In 2016, Dasgupta et al. published a patent WO2016081451 and disclosed novel oxazole
derivatives as β-catenin modulators of the Wnt pathway which stabilized the pool of β-catenin.
In 2012, University published a patent US20128252823 where they disclosed substituted
mercaptomethyl oxazole compounds as β-catenin modulators. WO2016081451 patent claims a
total of 80 exemplified molecules, of which promising compounds 103-105 and their biological
activity are discussed in Figure 13. The same authors further explored their research on oxazole
derivatives and published a patent WO2017152032, where along with oxazole, thiazole
derivatives were also explored as β-catenin modulators. Out of a total of around 364 compounds,
key compounds 106 and 107 along with their biological results are discussed in Figure 13 [141,
142] where thiazole derivative compound 106 is the most active compound amongst all key
compounds from both patents.
2.2.9 The Board of Regents of the University of Texas system, USA
The University of Texas System (UT System) is an American government entity of the state
of Texas and ranks top 10 most innovative academic institutions in the world. In 2014,
University published a patent, WO2014186450 as Porcupine inhibitors to treat disorders like
cancer, degenerative disorder, type-II diabetes, and osteoporosis. Patent summarized earlier work
done by the inventors using high throughput screening (HTS) for the identification of the IWP
series of compounds. Lead molecule IWP 02 (Figure 14) was further iterated to build SAR.
Here, we summarized key compounds 108-114 in Figure 14 out of 117 exemplified compounds
along with their IC50 value measured using human kidney HEK293 cancer cells in Luciferase
reporter Super Top Flash assay. It showed that the biaryl or pyridyl-aryl ring system improved
activity. Key compound 114 blocks the phosphorylation of the cytoplasmic Wnt pathway at 2.5
µM concentration in human kidney HEK293 cancer cells. Compound 114 inhibited regeneration
of the tailfin of juvenile zebrafish at 5 µM which is a Wnt-dependent process. Compound 114
was further evaluated for its plasma stability in mouse, rat, and human plasma and found little
degradation in human plasma as compared to rat and mouse plasma. Team also published their
article where detailed SAR and biological results were discussed [143, 144].
To address the limited factor of IWP type compounds like pharmacokinetic properties and
potency, the inventor published another patent WO2018045182 on disubstituted and
trisubstituted triazole derivatives as Wnt inhibitors. Out of 36 exemplified molecules, herein we
summarized key molecules 115-119 with their biological results in Figure 14. Compound 115
(IWP-01) inhibited Wnt signaling in L-Wnt-STF cells (EC50 = 0.08 nM) in firefly luciferase
assays. It also suppressed the phosphorylation of both Dishevelled 2/3 (Dvl2/3) and low-density
lipoprotein receptor-related protein 6 (LPR6) in human cervical HeLa cancer cells determined by
Western blot analysis[145].
2.2.10 Redx Pharma Plc., United Kingdom
Redx Pharma is a Drug discovery company mainly focusing on areas of anti-cancer and fibrosis.
It aggressively works on the Wnt signaling pathway. As a result, the company was able to push
its first molecule RXC-004 in a clinical trial. (RXC-004, porcupine inhibitor for advanced
malignancies, is currently in its phase-I clinical trial [57]) Herein, we discussed two PCT
published by Redx Pharma WO2016055786 and WO2016055790 [146, 147]. WO2016055786
is about N-pyridinyl acetamide derivatives as porcupine inhibitors. The patent disclosed an
extension of work related to heterocyclic molecules earlier reported in WO2010101849,
US2014/0038922, and WO2012/003189. It claims a total of 111 novel molecules along with the
common synthetic procedure of key molecules. Best compound 120 (Figure 15) inhibited Wnt
signaling pathway in mice L cells in luciferase reporter gene assays with IC50 of 0.05 nM.
WO2016055790, published on the same date, disclosed N-pyridinyl acetamide derivatives as a
Porcupine inhibitor. However, this time inventor tried to append substituted imidazopyridine and
imidazopyrimidine rings in place of monosubstituted imidazole ring with the exemplification of
a total of 31 molecules. The most promising compound 121 (Figure 15) inhibited Wnt signaling
pathway in mice L cells in the luciferase reporter gene assay with 0.32 nM IC50.
2.2.11 Hangzhou Rex Pharmaceutical Co. Ltd, China.
In 2017, a patent published by Hangzhou Rex Pharma WO2017097215 was about the
development of urea derivatives as a Wnt inhibitor [148]. The inventor appended urea moiety
onto the compound 120 disclosed by RedX Pharma. Out of 29 reported molecules, the most
promising compound 122 (Figure 15) was further evaluated for human and mouse plasma
stability study and PK study in BABL/C mice. It was observed that compound 122 reduced
tumor growth in nude mice bearing human colon tumor CR3150 (TGI = 99.52%, T/C = 11.81%)
at 5 mg/kg p.o. dose b.i.d. for 28 days. Compound 122 displayed a half-life (t1/2) of 1.99 h and
oral bioavailability (F) of 93.8% at 10 mg/kg p.o. in BALB/c mice.
Another published patent on that same date WO2017097216 was about heterocyclic amides as
Wnt inhibitors [149]. Different five-membered heterocycles like pyrazole, thiazole, isoxazole,
1,3,4-thiadiazole were evaluated with a total synthesis of 56 molecules. Promising compound
123 (Figure 15) displayed a half-life time (t1/2) of 1.62 h and an oral bioavailability (F) of 50.6%
at 10 mg/kg p.o. dose in BALB/c mice.
2.2.12 Suzhou Research Park, China.
Zhang X et al. published two patents with heterocyclic amide derivatives as a Wnt inhibitor. In
WO2017062688 inventor disclosed a total of 202 molecules based on different heterocyclic
scaffolds like quinoline, 1,7-naphthyridine, 1,5-naphthyridine appended on pyridine and
pyrimidine scaffold (Figure 16) [150]. Most promising compound 129 inhibited Wnt signaling
pathway in HEK-293 cells transfected with TCF-luciferase reporter plasmid (IC50 = 0.04 nM) in
luciferase reporter gene assays. The inventor also published another patent WO2017167150
based on the same heterocyclic scaffold with 38 synthesized compounds [151]. Key compounds
132-134 (Figure 16) showed IC50 value of 0.04 to 0.06 nM in the luciferase reporter gene assay.
2.2.13 Miscellaneous
In this section, we have discussed patents from major Pharma companies/Universities that had
contributed in the area of small molecule Wnt modulators with a maximum of one patent.
2.2.13.1 Shanghai Institutes for Biological Sciences, and group, China.
Group published a patent WO2014169711, disclosing 15-Oxospiramilactone derivatives as
inhibitors of the Wnt signaling pathway mainly for the treatment of tumors. They stated that the
signal transduction pathway is vastly different between normal cells and tumor cells, which itself
allows the development of a selective Wnt modulator. The inventor claimed a total of 93
different heterocyclic compounds conjugated with 15-Oxospiramilactone moiety. Potent
compound 135 (Figure 17) inhibited Wnt signaling pathway in HEK-293 cells with an IC50
value of 7.83 µM in the reporter gene assay. In-vitro biological results on different cell lines
were summarized in Figure 17 [152].
2.2.13.2 National Cancer Center of Japan and Carna Biosciences, Inc, Japan.
Both groups together published a patent WO2015083833 on novel quinazoline derivatives as
Wnt inhibitors [153]. Most of the compounds from the patent are based on quinazoline
derivatives where the 7th position of quinazoline ring is substituted with cyclohexane-diol and 2nd
position with benzimidazole-6-amine. Out of 46 molecules, compound 136 (Figure 17)
inhibited Wnt/β-catenin signaling in transfected HEK293 cells (IC50 = 0.3 µM or less) in TCFluciferase reporter gene assay. Compound 136 also showed concentration-dependent inhibition
of AXIN2 and c-MYC gene expression in human colon HCT-116 cancer cells at 3 and 10 µM
concentration in gene expression assay. During in-vivo studies, it dose-dependently inhibited
tumor growth in nude mice bearing HCT-116 xenografts by 42 and 70% at 20 and 80 mg/kg,
p.o., b.i.d. dose, for 14 days [153].
2.2.13.3 Prism Bio Lab Co. Ltd, Japan, and Eisai R & D Management Co. Ltd, Japan.
Prism Bio Lab and Eisai combinely published a PCT WO2015098853 with a chemical moiety of
(6S,9aS)-N-Benzyl-6-[(4-hydroxyphenyl)methyl]-4,7-dioxo-8-(methyl)-2-(prop-2-en-1-yl)-
octahydro-1H-pyrazino[2,1-c][1,2,4]triazine-1-carboxamide and related compounds as Wnt
modulating agents for the treatment of cancer and fibrosis [154]. The patent claimed 29 multistep
synthetic schemes to synthesize molecules with detailed experimental and characterization data.
The most promising molecule compound 137 (Figure 17) inhibited Wnt signal activity in HEK293 cells with an IC50 value of 0.06 µM in a luciferase assay. It did not show any kind of visual
tumor in mice model of human leukemia K562 subcutaneous transplantation at 75 mg/kg p.o.
b.i.d. dose for 5 days in combination with Dasatinib (5 mg/kg p.o. q.d. dose x 5d).
2.2.13.4 Sichuan Haisco Pharmaceutical Co., Ltd, China.
WO2015135461 is based on substituted dihydro benzofuran-piperidine-ketone derivatives as
Wnt modulators capable of inhibiting tumor cell proliferation and tumor metastasis [155]. The
patent disclosed synthesis of only 7 molecules, of which most promising compound 138 (Figure
17) inhibited Wnt signaling pathway in HEK-293 cells transfected with Super Top Flash with an
IC50 value of 1.51 nM in luciferase reporter gene assays.
2.2.13.5 Kyowa Kirin Co., Ltd, Japan.
WO2015016195 disclosed fused heterocyclic compounds as Wnt signaling inhibitors for the
treatment of cancer, pulmonary fibrosis, fibromatosis, and osteoarthritis [156]. The patent
disclosed the synthesis and biological screening of 53 molecules. Key compound 139 (Figure
17) inhibited T-cell factor (TCF)-response luciferase reporter (91% at 1µM) as Wnt pathway
index, expressed in human colon DLD-1 cancer cells [156].
2.2.13.6 University of Maryland, USA.
University published a patent WO2017151786 disclosing compounds as Wnt inhibitors for the
treatment of Wnt-related disorders [157]. The patent disclosed a total of 149 molecules based on
aryl 1,2,3-triazole carboxamide moiety, where different heterocyclic rings were appended on
carboxamide functionality. Out of 149 exemplified molecules, key compounds (140-143) with
Wnt/β-catenin inhibitory activity using luciferase gene reporter assay are summarized in Figure
18.
2.2.13.7 Tottori University, Japan.
University published a patent WO2017047762 and reported suppression and regeneration
promoting effect of low molecular weight compounds on cancer and fibrosis as a novel treatment
for a malignant tumor or fibrosis [158]. The patent disclosed hexahydro-4H-pyrazino[1,2-
a]pyrimidine-4,7(6H)-dione derivatives and the key Compound 144 (Figure 18) displayed antiproliferative activity against human hepatic Huh-7 cancer cells (IC50 25.95 µM) in WST assay.
Compound 144 also inhibited the growth of the human oral squamous HSC-2 cell line (71.4% at
25 µM). Compound 144 suppressed tumor volume (mm3
in CD44-positive Huh-7 xenograft
mice at 50 mg/kg i.p. once a day dose for 18 days compared to 5-fluorouracil) Compound 144
reduced tumor volume up to 1.5 mm3
compared to 5-Fluorouracil 4.5 mm3
. (Tumor volume of
control/vehicle group prepared in DMSO is 13 mm3
. It also decreased fibrosis area in carbon
tetrachloride (CCl4)-induced fibrosis in C57BL/6 male mice model at 10.6 mg/kg i.p. 3x/1 wk x
4 wks dose.
2.2.13.8 Southern Research Institute, USA & the UAB Research Foundation, USA.
WO2017132511 disclosed benzimidazole derivatives as inhibitors of the Wnt signaling pathway
in cancer [159]. Out of 38 synthesized molecules, promising compounds 145 and 146 (Figure
18) inhibited Wnt signaling in HEK-293 cells expressing human LRP6 with IC50 value 0. 95 nM
and 0.93 nM in luciferase reporter gene assays.
2.2.13.9 University of California, Oakland.
WO2019152536 disclosed 120 compounds capable of modulating the Wnt/β-catenin
pathway[160]. Structurally, compounds are related to 3,5-di((E)-benzylidene)-piperidin-4-one
derivatives with various substitutions on the piperidine nitrogen atom. Most promising
compound 147 (Figure 19) significantly inhibited tumor growth in nude mice bearing human
colon SW-480 cancer xenografts (at 10 mg/kg i.p. 5d/week x 10 d) with no significant change in
body weight. (IC50 values on different cell-lines for Wnt inhibitory activity are summarized in
Figure 19)
2.2.13.10 Universite de Lausanne, Switzerland.
Pyrazole derivates were explored by Universite de Lausanne in WO2019166616 as Wnt
inhibitors for the treatment of cancer [161]. Promising compound 148 (Figure 19) inhibited the
Wnt pathway (IC50 = 11 µM) in TopFlash assay reporter assay. It inhibited DVL phosphorylation
and decreased total β-catenin levels in mouse L-fibroblasts and active β-catenin levels in human
triple-negative breast cancer HCC 1395 cells at 50 µM.
2.2.13.11 Huihan Medical Technologies Ltd. Co., Shandong, China.
Recently published patent in June 2020, WO2020125759 disclosed 5,6 bicyclic and 5,6-
heterocyclic bicyclic amino compounds as Wnt signaling inhibitors for diseases related to
dysfunction of Wnt signaling pathway[162]. Patent exemplified a total of 107 compounds and
evaluated for Wnt inhibitory activity. Key compounds 149-152 and their biological results are
summarized in Figure 19.
2.2.13.12 Kyoto Pharmaceutical University, Japan.
Recently published in June 2020, the WO2020130119 invention is related to a novel Wnt
signaling pathway inhibitor to be useful for the treatment of cancer, coronary artery disease,
acute coronary syndrome, and osteoarthritis[163]. Patent exemplified a total of 34 compounds
out of which compound 153, Figure 20 significantly inhibited Wnt/β-catenin-mediated
transcription in HEK293 cells (at 10 and 30 µM) in TopFlash luciferase reporter gene assays.
Compound suppressed the growth of various cancer cells in a concentration-dependent manner
(at 10-40 µM) in WST-8 assay. It induced apoptosis in human leukemic cell line MV-4-11 and
bone marrow KG-1a cancer cells at 9 and 14 µM concentration respectively using Western blot.
It effectively inhibited the proliferation of human breast MDA-MD-231 cancer stem cells
compared with MDA-MD-231 cancer cells (at 5-25 µM) in Cell Titer-Glo 3D cell viability
assay.
2.2.13.13 Nantbio Inc, USA
Nantbio has recently published a patent in April 2020 WO2020072540 and claimed amidic
heterocyclic 1,1-dioxidoisothiazolidin derivatives as a dual inhibitor of Wnt/β-catenin and sonic
hedgehog signal transduction pathway [164]. A common synthesis procedure for only two key
compounds was provided. Promising compound 154 (Figure 20) suppressed mWnt3a-mediated
phosphorylation of LRP6 and ERK in 293H cells (at 25 µM) in Western blot assay.
2.3 Key organizations targeting antibody based Wnt modulators
2.3.1 Surrozen INC, USA
Surrozen INC, a biopharmaceutical company based in South San Francisco, California, USA, is
working on selective activation of the Wnt pathway using targeted antibodies. In June 2019, two
patents were published by the organization; WO2019126398A1 and WO2019126401A1.
WO2019126398A1 disclosed Wnt pathway agonists for the treatment of Wnt-related diseases.
A selected product, R2M3-26 consisted of a human monoclonal IgG antibody (R2M3) targeting
the extracellular domain (ECD) of Fzd1, fused via 5 amino acid linker at the N-terminal light
chain to two antibody-derived binding fragments (Fabs) targeting LRP6. Administration of
R2M3-26 (i.p.) to ovariectomy-induced osteoporosis mice model, resulted in a rapid and
sustained increase in bone mineral density (BMD) and bone volume, as measured by dual-energy
x-ray absorptiometry (DEXA). Also, administration of R2M3-26 into C57BL6/J mice (i.p.10
mg/kg) resulted in a significant increase in the liver to body weight ratio, suggesting promotion
of liver regeneration. After PK/PD study, patent concluded that R2M3-26 showed high stability,
bioavailability, and therapeutic activity [165].
WO2019126401A1 is about anti-LRP5/6 antibodies useful for the treatment of Wnt-related
disorders. An exemplified product 18R5:009S was a bispecific Wnt surrogate construct,
comprising a single-chain variable fragment. 18R5:009S targets the Frizzled (Fzd) receptor,
fused via 6 amino acid linkers at the N-terminus to alanine amino acid residue. In an in vitro
assay, incubation of human melanoma A375 cells with 18R5:009S-E04 resulted in a dosedependent increase in activation of the Wnt signaling pathway[166].
WO2020167848A1 provides methods of treating ocular disorders with Wnt modulators. The
patent claims for a method of treating retinopathy using engineered Wnt signaling modulators. A
selected product 4SD1-03 is a bispecific Wnt surrogate construct comprising a human
monoclonal IgG antibody (R2M3) targeting the extracellular domain (ECD) of Fzd4, fused via 5
amino acid linker at the N-terminal light chain to two antibody-derived binding fragments
targeting LRP6. In an in vivo assay, administration of the 4SD1-03 (intravitreal) into oxygeninduced retinopathy Sprague-Dawley rats, resulted in significant inhibition of neovascular tuft
formation [167].
WO2020185960A1 is about the modulation of Wnt signaling for the treatment of gastrointestinal
disorders, specifically inflammatory bowel disease including Crohn’s disease (CD) and
ulcerative colitis (UC). In STF assay, antibody R2M3-26 effectively activated Wnt signaling in
human hepatoma (HUH7) cells. During in vivo screening in female mice model, twice weekly
administration of R2M3-26 improved body weight, repaired damaged colon epithelium and
decreased TNF-α. C14-mutRSPO2 is a Wnt surrogate construct, comprising a mutant human Rspondin 2 (mutRSPO2) and harboring F105R/F109A mutations at the furin-2 binding domain,
fused via linker at the C-terminus heavy chain of the monoclonal IgG antibody (C14). It targets
MUCIN 13 (MUC-13, high-molecular-weight transmembrane glycoprotein). Treatment with
C14-mutRSPO2 was able to maintain human small intestine organoid growth which confirmed
its intestine specific Wnt signaling enhancing activity [168].
2.3.2 Board of Regents, The University of Texas System, USA
Recently, Board published two patents on antibodies acting via the Wnt signaling pathway.
WO2020081579A1 disclosed monoclonal antibodies against human dickkopf3 (DKK3) and
claimed it to be potentially useful for the treatment of cancer. A promising product JM6-6-1 was
a neutralizing monoclonal antibody targeting human DKK3. In an in vitro assay, incubation of
JM6-6-1 with human pancreatic stellate cells (HPSC) resulted in the inhibition of growth (70 to
80 fold) and migration (5 to 11 fold) of these cells. In an in vivo assay, administration of JM6-6-
1 (i.p., 5 mg/kg) to pancreatic cancer mouse model resulted in significant inhibition of tumor
growth and improved the survival rate (43%) in the treated mice [169].
WO2020160532A1 disclosed monoclonal antibodies and antibody fragments for the treatment of
cancer that target dickkopf1 (DKK1) and human leukocyte antigens (HLA-A2) peptide complex.
In an in vitro assay, a promising candidate, A2-DKK1 bound to prostate cancer cell, as
determined by the live imaging system. In order to study the mechanism of A2-DKK1 in vivo,
immunodeficient mice were xenografted subcutaneously with U266 myeloma cells followed by
treatment with A2-DKK1 resulted in decreased tumor volume and increased survival rate [170].
2.3.3 Yale University, USA
WO2017074774A1 published by Yale University, USA is about humanized anti-DKK2
antibodies, claimed to be potentially useful for the treatment of multiple cancers. A selected
product, 5F8-HXT1-V2, is a humanized monoclonal IgG1 kappa antibody that demonstrated
very strong binding to human DKK2 in an in vitro ELISA assay. In an in vivo assay, the selected
humanized antibody significantly reduced tumor volume in C57/BL mice grafted with murine
colon adenocarcinoma (MC38) cells at a dose of 16 mg/kg for 15 days [171].
WO2018174984A1 patent claimed for methods of treating cancer by the administration of
antibodies that blocks the interaction between DKK2 and LRP5. An exemplified mouse
monoclonal antibody targeting human DKK-2, 5F8 was found to bind specifically to DKK2
antigen in a dose-dependent manner, as determined by in vitro ELISA. In another in vitro assay,
5F8 inhibited the binding of DKK2 to LRP5 expressing HEK293 cells. During in vivo screening,
treatment of 5F8 (i.p., 10 mg/kg, 1x/3 days) to C57BL mice grafted with MC38 cells (s.c.)
resulted in increased apoptosis and increased number of granzyme B-positive cells, as
determined by histological analysis. Furthermore, these mice also showed inhibited tumor
growth and prolonged survival rate [172].
2.3.4 Miscellaneous
In this section, we have discussed patents from major Pharma companies/Universities that had
contributed in the area of antibody based Wnt signaling modulation with a maximum of one
patent.
2.3.4.1 Institute for research in biomedicine, Barcelona, and other applicants
WO2017069628A1 disclosed bispecific antibodies targeting the proteins associated with the
Wnt signaling pathway, such as epidermal growth factor receptor (EGFR), human epidermal
growth factor receptor-3 (HER3), zinc and ring finger-3 (ZNRF3), and leucine-rich repeatcontaining G-protein coupled receptor (LGR) and claimed them potentially useful for the
treatment of cancer. A promising molecule, PB-10651 was a bispecific human monoclonal IgG1
antibody, targeting the ECD of human EGFR and LGR5. In an in vitro assay, PB-10651 (10
µg/ml) was observed to bind and block the signaling of EGFR in colon toroid cells, resulting in
tumor growth inhibition. PB-10651 was also observed to bind with LGR5-expressing CHO-K1
cells with an EC50 value of 156 ng/ml. In xenograft BALB/c nude mice model of colorectal
cancer (CR2519 and CR0193), treatment with PB-10651 (i.p., 0.5 mg/mouse) significantly
inhibited tumor growth. In a non-GLP repeated dose toxicity study, neither skin nor
gastrointestinal (GI) tract toxicity was observed after repeated administration of PB-10651 to
cynomolgus monkeys at an i.v. dose of 25 mg/kg. Also, there was no change observed in organ
weight and no adverse macroscopic or microscopic findings observed in the treated monkeys,
indicating the safety of the selected antibody [173].
2.3.4.2 Boehringer Ingelheim international GMBH, Germany
WO2017093478A1 claimed for polypeptides targeting LRP5/6 useful in the treatment of cancer.
The promising candidate, F-013500571 inhibited Wnt signaling by decreasing the over
expression of human Wnt1 and Wnt3a with an IC50 value of 0.05 nM as determined via Wnt1
and Wnt3a reporter assays. Intravenous dose-dependent administration of F-013500571 (2-10
mg/kg up to 21 days) to xenograft MMTV-Wnt1 transgenic mice resulted in tumor growth
inhibition up to 128% [174].
2.3.4.3 Antlera therapeutics INC, Canada
WO2020/250156A1 patent claimed for multivalent binding molecules comprising FZD2/7
binding domain attached to Fc, for activating Wnt signaling pathway. The promising antibody
FP+P-L61+3 effectively activated β-catenin signaling in HEK293 and RKO cells and interacted
with Fc receptors. In vivo administration of FP+P-L61+3 at 10 mg/kg/day dose, i.p., rescued
LGR5 expression in the crypt cells of mice, indicating that this antibody had enough
bioavailability and half-life for enabling β-catenin activation, leading to intestinal stem cell selfrenewal even in the absence of Wnt ligands [175].
2.3.4.4 The regents of the University of California, USA
University published a patent WO2020263862A1, claiming potential antibody useful for the
treatment of neurodegenerative disease including AD or Parkinson’s disease. An exemplified
product, Ex 5 was a monoclonal IgG antibody targeting the Wnt binding domain of human
tyrosine kinase (Ryk). During in vitro assay, anti-Ryk antibody pre-treated hippocampal neurons
(isolated from mice) when challenged with amyloid-β oligomer, resulted in a negligible
reduction of synapse number which indicated its beneficial effect in AD. In vivo, intracerebral
administration of the Ex 5 for 2 weeks, significantly rescued the number of synapses in a
transgenic AD mouse model. The results indicated that antibody-mediated Ryk-blockade
significantly inhibited amyloid-β oligomer-induced synapse loss [176].
3. Conclusion
A large number of patents illustrate continued and widespread interest in exploring Wnt
signaling pathway modulators for indications like a different type of cancer, osteoarthritis, skin
disease, metabolic disorder, etc. by different pharmaceutical industries. In the last six years, a
total of 92 patent applications on small molecules by twenty-five different organizations were
published. This wide number of patents cover different structural motifs as Wnt modulators with
distinct chemical classes like 1H-pyrazolo[3,4-b]pyridine, heterocyclic pyridine amidic type
compounds, purine 2,6-dione, spirocyclic indoline molecules, heterocyclic naphthyridine,
phthalazinone, and many more. As a result of extensive efforts by different research labs, a total
of nine molecules reached clinical trials out of which Samumed LLC is ahead with its molecules
Lorecivivint (SM-04690) and SM-04554. Antibodies targeting specific components of Wnt
signaling pathway are also a good choice of therapy. Total of 12 patents by seven different
organizations have been published recently. Similar to small molecules, antibodies also target
different therapeutic areas like gastrointestinal, ocular disorders, cancer and Alzheimer disease.
Though small molecules are ahead in clinical evaluation, few antibodies also reached to an early
phase of clinical trials like DKK1, by Leap therapeutics, USA for advanced solid tumors and
BHQ-880 by Novartis, Switzerland for multiple myeloma. Overall, Wnt signaling pathway is
becoming the most promising target for multiple organizations to actively pursue their research
and come up with a first in class Wnt modulators.
4. Expert opinion
The huge number of patents, published by various researchers around the globe, in the area of
Wnt modulation, supports its emerging role in various therapeutic indications. A total of ten
small molecules and three antibodies as Wnt modulators are under clinical development. SM04690 is the first molecule from Samumed LLC, USA of the Wnt inhibitor category at its highest
phase-III clinical trial for Osteoarthritis. The company is also developing two more molecules
SM-04554 and SM-04755 for androgenetic alopecia and tendinopathy respectively, for which
these molecules will turn out as a novel therapeutic approach. LGK-974 is working through
porcupine inhibition and is under Phase-I trial for malignancies. Redx Pharma is another
company working aggressively on Wnt signaling. Its molecule, Porcupine inhibitor RXC-004 is
under Phase-I for advanced malignancies. Curegenix is developing CGX-1321 as a Porcupine
inhibitor for GI tumors. Prism Pharma is developing PRI-724 as a Wnt signaling inhibitor, while
Iteration therapeutics is targeting the desmoid tumor with its molecule, Tegavivint (BC-2059), βcatenin inhibitor, which is under phase-I trial.
Small molecules targeting Wnt signaling components do not limit to any chemical class.
Chemical scaffolds explored by various organizations were mainly based on their previously
explored results and to make the molecule more potent like Bayer pharma, ASTAR group,
University of Texas, Redx Pharma for which common chemotype was biaryl amide-type
heteroaryl derivatives. Depending upon therapeutic class different chemotype has been observed
like molecules explored for Osteoarthritis (benzimidazole, indazole type bicyclic heteroaryl
derivatives) is different from molecules being explored for androgenetic alopecia (aryl 1,4-
diketone type moiety). Samumed also explored macrocyclic indazole chemotype of derivatives
for disease related to Wnt activation. While Merck widely explored 2-aminopyridine derivatives
with spirocyclic ring explored at 4th position of pyridine moiety for treatment of
hyperproliferative diseases like cancer, inflammation, and neurodegeneration. Scientists at H.
Lee. Moffitt institute focused to develop a novel type of selective inhibitors of β-catenin/T-cell
factor protein-protein interaction for the treatment of cancer using computation tools like binding
site identification, bio isosteric replacement, etc. Existing SAR analogy with different
chemotypes being explored for the particular therapeutic class will provide more opportunities
for selectively targeting Wnt signaling pathway and its components.
Another approach, apart from small-molecule Wnt inhibitor, is the direct delivery of molecules
into the bloodstream of patients like an antibody/gene therapy. OTSA101-DTPA-90Y is an antiFrizzled Homolog 10 (FZD10) monoclonal antibody under phase I evaluation for relapsed or
refractory synovial sarcoma [65]. OMP-18R5 is the antibody against FZD receptor and is
currently under phase I evaluation by Oncomed Pharmaceuticals in collaboration with Mereo
Biopharma for solid tumors [66]. OMP-54F28, developed by Oncomed Pharmaceuticals in
collaboration with Bayer, is the FZD8 decoy receptor antibody under phase I evaluation for solid
tumors [67]. While, antibody is the best option to target Wnt signaling pathway because of its
high specificity and reproducible results, it also comes with few limitations like high production
cost, considerable time and efforts for synthesis, therapeutic variability from rodents to higher
species etc.
Despite many patents on the Wnt pathway, investing in the research of the Wnt signaling
pathway is still considered a challenging journey. Wnt signaling is a complex pathway to target
because of the role of its different components like Wnt ligands, Frizzled receptors, and
additional Wnt binding proteins like CK1, GSK3, AXIN, APC, DVL, etc. and its interconnected
role in different pathophysiology. Thus, achieving selectivity in targeting the Wnt component is
a major challenge nowadays. Understanding of binding site of different Wnt components and its
dynamics is the key aspect to target this complex pathway. Targeting signal transduction
pathways can have harmful effects on embryonic pattern as well [177]. Thus, targeting a
pathway which is crucial for development of normal as well as cancer cell remains ‘Jekyll and
Hyde’ type of situation. Stabilizing efficacy of various Wnt modulators with on-target toxicity is
the biggest challenge. Studies suggest that systemic Wnt inhibition may cause defects in gut
homeostasis [177] and can also cause neurodegeneration [178] [179]. Due to the crucial role of
Wnt signaling in osteoblast and osteoclast differentiation, its inhibition can also negatively affect
bone homeostasis and may results into the bone loss [180, 181]. Due to all these challenges
related to interplay of Wnt signaling with other signaling cascades, even though the pathway has
been known for more than three decades, not a single small molecule has reached the market
which itself proved complexity in getting selectively. However, since the last decade, the role of
the Wnt signaling pathway has been emerged and extensively explored in the area of various
therapeutics like, Osteoarthritis, Androgenetic alopecia, cancer, etc. The role of different
components of the Wnt signaling pathway in different diseases will provide opportunities for
targeting the Wnt signaling pathway with better selectivity [182].
Acknowledgments
The authors are thankful to Nirma University, Ahmedabad, India for this work, which is a part of
the Doctor of Philosophy (PhD) research work of Vishalgiri Goswami, to be submitted to Nirma
University, Ahmedabad, India.
Funding
This paper was not funded.

Declaration of interests
The authors have no relevant affiliations or financial involvement with any organization or entity
with a financial interest in or financial conflict with the subject matter or materials discussed in
the manuscript. This includes employment, consultancies, honoraria, stock ownership or options,
expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures
Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

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Figures and Table Captions:
Table 1. Wnt modulators under clinical development
Table 2. A summary of PCT published by Samumed LLC on WNT signaling pathway.
Figure 1 Overview of the Wnt signaling pathway.
Figure 2 Overview of the Wnt signaling targets and respective inhibitors/activators
Figure 3 Representative molecules from Bayer Pharma, Germany
Figure 4 Representative molecules from Bayer Pharma, Germany
Figure 5 Representative molecules from A*STAR, Singapore
Figure 6 Representative molecules from H. Lee Moffitt Cancer Center and Research Institute,
Inc. USA
Figure 7 Representative molecules from H. Lee Moffitt Cancer Center and Research Institute,
Inc. USA
Figure 8 Representative molecules from Merck, United Kingdom
Figure 9 Representative molecules from Merck, United Kingdom
Figure 10 Representative molecules from Duke University, USA
Figure 11 Representative molecules from Duke University, USA
Figure 12 Representative molecules from Guangzhou Curegenix Ltd, China
Figure 13 Representative molecules from New York University, USA
Figure 14 Representative molecules from the board of regents of the University of Texas, USA
Figure 15 Representative molecules from Redx pharma, United kingdom, and Hangzhou Rex
pharma, China
Figure 16 Representative molecules from Suzhou, China
Figure 17 Representative molecules from Shanghai Institutes for Biological Sciences, China,
Carna bioscience, Japan, Prism bio lab, Japan, Sichuan Haisco Pharmaceutical Co., Ltd, China,
and Kyowa Ltd, Japan.
Figure 18 Representative molecules from the University of Maryland, USA and Tottori
University, Japan, and Southern Research Institute, USA
Figure 19 Representative molecules from the University of California, Oakland, University of
Lausanne, Switzerland, and Huihan medical technologies co. Ltd, China.
Figure 20 Representative molecules from the University of Kyoto pharma, Japan, and Nantbio
INC, USA.

List of abbreviations used:
Wnt : Wingless-related integration site
LRP : Low-density lipoprotein-related-protein
Dsh : Dishevelled
APC : Adenomatous polyposis coli
GSK3 : Glycogen synthase kinase 3
CK1 : Casein kinase 1
TCF : T-cell factor
LEF : Lymphoid enhancing factor
PCP : Planar cell polarity
PTK-07 : Protein kinase – 7
ROR2 : Tyrosine-protein kinase transmembrane receptor
JNK : C-Jun N-terminal kinase
PLC : Phospholipase C
IP3 : Inositol 1,4,5-triphosphate
DAG : Diacyl glycerol
CAMKII : Calmodulin-dependent kinase II
PKC : Protein kinase C
NFAT : Nuclear factor of activated T-cells
OA : Osteoarthritis
MCR : Mutation cluster region
LRP6 : Low-density lipoprotein receptor-related protein 6 precursor
FOXO : Forkhead box transcription factors
DKK1 : Dickkopf-related protein 1
MMPS : Matrix metalloproteinase
MMTV : Mouse mammary tumor virus
BCL9 : B-cell lymphoma 9
DYRK1A : Dual specificity tyrosine-phosphorylation-regulated kinase 1A
CTNNB1 : Catenin beta-1 gene
MSI : Microsatellite instability

Fig 1
Fig 2

Table-01: Wnt modulators under clinical development
Compo
und Structure Company Wnt
Target
Therape
utic
indicatio
n
Pha
se
Clinical
trial
identifier
Ref.
Lorecivi
vint
(SM04690)
Samumed
USA
Wnt
inhibito
r
through
CLK2
and
DYRK1
A
inhibiti
on
Knee
Osteoart
hritis
III NCT0452
0607 51,52
SM04554
Structure was
not disclosed
yet
Samumed
USA
Wnt/ βcatenin
activato
r
Androge
netic
Alopecia
III NCT0374
2518 53
SM04755
Structure was
not disclosed
yet
Samumed
USA
Wnt
signalin
g
pathwa
y
inhibito
r
Tendinop
athy I NCT0322
9291 49
LGK974
Novartis
Switzerlan
d
Porcupi
ne
inhibito
r
Cancer I NCT0135
1103
54,55
,56
RXC004
Structure was
not disclosed
yet
Redx
pharma
UK
Porcupi
ne
inhibito
r
Advance
d
Malignan
cies
I NCT0344
7470 57
ETC159
A*STAR
Singapore
Porcupi
ne
inhibito
r
Advance
d solid
tumors
I NCT0252
1844 55,58
CGX1321
Structure was
not disclosed
yet
Curegenix
Inc, USA
Porcupi
ne
inhibito
r
Advance
d GI
tumor
I NCT0267
5946 59
PRI-724
Prism
Pharma,
Japan
Wnt
signalin
g
pathwa
y
inhibito r
Advance
d solid
tumors
I NCT0130
2405
55,60
,61
Tegaviv
int
(BC2059)
Iterion
Therapeuti
cs, USA
β
-
catenin
inhibito r
Desmoid
tumor
I NCT0345
9469 62,63
CWP232291
Structure was
not disclosed
yet
JW
Pharmaceu
ticals
Wnt/
β
-
catenin
inhibito
r
Acute
myeloid
leukemia
I NCT0139
8462 64
OTSA1
01-
DTPA90Y
Monoclonal
antibody
Oncotherap
y science,
LLC
AntiFrizzled
Homolo
g 10
(FZD10
)
Monocl
onal
Antibod
y
Relapsed
or
refractor
y
synovial
sarcoma
I NCT0417
6016 65
OMP18R5
Monoclonal
antibody
Oncomed
pharmaceut
icals, Inc.
Monocl
onal
antibod
y
against
FZD
receptor
s
Solid
tumors I NCT0134
5201 66
OMP54F28
Monoclonal
antibody
Oncomed
pharmaceut
icals, Inc.
FZD8
decoy
receptor
Solid
tumors I NCT0160
8867 67

Table-02: Summary of PCT published by Samumed, USA on WNT signaling pathway
Structure of
key compound
/Patent number
Nos. of
molecul
es
covered
in the
Patent
Biological activity (nM)
Patent specific
biological assay
results/Comme
nts
Ref
.
Cell
TiterBlue cell
viability
assays in
human
colon
SW480
cancer
cells
(EC50 in
nM)a
Wnt
inhibitor
y
activity
in Sp5
luciferas
e
reporter
gene
assay
(EC50 in
nM)b
Reductio
n of
TGF-β1
induced
fibrosis
in
human
lung
fibroblas
ts LL29
(EC50 in
nM)c
283
Compou
nd was
not
evaluated
in this
assay
Compou
nd was
not
evaluated
in this
assay
Compoun
d was not
evaluated
in this
assay
• An
exemplified
compound 6
inhibited Wnt
signaling in
human colon
SW480
cancer cells
with an IC50
value of 2
nM.
68
767 1
Compou
nd was
not
evaluated
in this
assay
Compoun
d was not
evaluated
in this
assay
• An
exemplified
compound
enhanced
Wnt signaling
activity with
in Bright-Glo
luciferase
reporter gene
assays
69
1377
5
(Values
are in
IC50)
Compou
nd was
not
evaluated
in this
assay
51
(Values
are in
IC50)
• EC50 = 0.002
µM
(DYRK1A
activity in
human
neuroblastom
a SH-SY5Y
cells)
• It induced
chondrogenes
70
is (by 51%
relative to
TGFβ
3 as a
positive
control) in
primary
human
mesenchymal
cell at 300
nM
1477
4.5
(Values
are in
IC50
)
Compou
nd was
not
evaluated
in this
assay
0.009
• It induced
chondrogenes
is (by 100%
relative to
TGFβ
3) in
primary
human
mesenchymal
cell at 30 nM
71
1477
46
(Values
are in
IC50
)
Compou
nd was
not
evaluated
in this
assay
Compoun
d was not
evaluated
in this
assay
• It induced
chondrogenes
is (by 100%
relative to
TGFβ
3) in
primary
human
mesenchymal
cell at 30 nM
• It activated
trans
differentiation
of human
retinal
pigmented
epithelium
ARPE-19
cells into eye
neuronal cells
with an EC50
value of 0.005
µM in assays
measuring
PAX6
expression.
72
2290
15
(Values
are in
IC50)
Compou
nd was
not
evaluated
in this
assay
Compoun
d was not
evaluated
in this
assay
• It activated
trans
differentiation
of human
retinal
pigmented
epithelium
ARPE-19
cells into eye
neuronal cells
(EC50 = 0.002
µM) in assays
measuring
PAX6
expression.
73
N
NH
N
N
N HN
NH
O
F
12
WO2016040184
1477
59.7
(Values
are in
IC50)
Compou
nd was
not
evaluated
in this
assay
0.009
• Wnt
signaling
inhibitors
reported to
be useful for
the treatment
of cancer,
pulmonary
fibrosis,
idiopathic
pulmonary
fibrosis,
osteoporosis,
osteoarthritis
, bone or
cartilage
disorders.
74
2413
0.4
(Values
are in
IC50)
Compou
nd was
not
evaluated
in this
assay
0.009
• It induced
chondrogenes
is (by 100%
relative to
TGFβ
3) in
primary
human
mesenchymal
cell at 300
nM.
75
1404
71.8
(Values
are in
IC50)
Compou
nd was
not
evaluated
in this
assay
0.009
• Wnt
signaling
inhibitors
reported to
be useful for
the treatment
of cancer,
pulmonary
fibrosis,
idiopathic
pulmonary
fibrosis,
osteoporosis,
osteoarthritis
, bone or
cartilage
disorders.
76
1446
3.7
(Values
are in
IC50)
Compou
nd was
not
evaluated
in this
assay
0.009
• It induced
chondrogenes
is in primary
human
mesenchymal
cells at 300
nM relative
to positive
control TGF- β3.
77
1406
13
(Values
are in
IC50)
Compou
nd was
not
evaluated
in this
assay
0.009
• Wnt
signaling
inhibitors
reported to
be useful for
the treatment
of cancer,
idiopathic
pulmonary
fibrosis,
osteoporosis,
osteoarthritis
, bone or
cartilage
disorders and
degenerative
intervertebral
disc
disorders.
78
1151 322 1275 93
• Compound
reduced
LPS-induced
IL-6
production in
human acute
monocytic
leukemia
THP-1 cells
(EC50 > 10
µM) in
homogeneou
s timeresolved
fluorescence
(HTRF)
assays
79
1011 1363 530 67
• Compound
inhibited
LPS-induced
IL-6
production in
human acute
monocytic
leukemia
THP-1 cells
(EC50 = 0.1
µM) in HTRF
assays.
80
990 1290 937 141
• Compound
inhibited
LPS-induced
IL-6
production in
human acute
monocytic
leukemia
THP-1 cells
(EC50 = 2.468
µM) in HTRF
assays.
81
990 159 97 19
• Compound
showed and
EC50 greater
than 10 µM
in LPSinduced IL-6
production in
human acute
monocytic
leukemia
THP-1 cells
in HTRF
assays.
82
990 652 416 99
• Compound
inhibited
LPS-induced
IL-6
production in
human acute
monocytic
leukemia
THP-1 cells
(EC50 = 0.169
µM) in HTRF
assays.
83
990 1729 1850 9
• Compound
inhibited
LPS-induced
IL-6
production in
human acute
monocytic
leukemia
THP-1 cells
(EC50 = 0.425
µM) in HTRF
assays.
84
1037 1096 1232 21
• Compound
reduced LPSinduced IL-6
production in
acute
monocytic
leukemia
THP-1 cells
(EC50 = 0.732
85
µM) in HTRF
assays
990 261 56 76
• Compound
reduced LPSinduced IL-6
production in
human acute
monocytic
leukemia
THP-1 cells
(EC50 = 0.101
µM) in HTRF
assays.
86
1106 97 41 815
• Compound
reduced LPSinduced IL-6
production in
human acute
monocytic
leukemia
THP-1 cells
(EC50 = 0.125
µM) in HTRF
assays
87
1152 593 1978 80.8
• It reduced
LPS-induced
IL-6
production in
human acute
monocytic
leukemia
THP-1 cells
(EC50 =
1.8357 µM)
in HTRF
assays.
88
1037 290 239 38
• Compound
reduced LPSinduced IL-6
production in
human acute
monocytic
leukemia
THP-1 cells
(EC50 = 0.068
µM) in HTRF
89
assays.
N N
H
N
HN
N
N
N
H
O
28
WO2017023996
990 2427 1260 1557
• Compound
reduced LPSinduced IL-6
production in
acute
monocytic
leukemia
THP-1 cells
(EC50 = 0.063
µM) in HTRF
assays.
90
990 1269 590 33
• Compound
reduced LPSinduced IL-6
production in
acute
monocytic
leukemia
THP-1 cells
(EC50 = 0.103
µM) in HTRF
assays.
91
990 345 104.5 86
• Compound
inhibited
LPS-induced
IL-6
production in
human acute
monocytic
leukemia
THP-1 cells
(EC50 = 0.107
µM) in HTRF
assays
92
992 93 50 19
• It inhibited
LPS-induced
IL-6
production in
human acute
monocytic
leukemia
THP-1 cells
(EC50 = 0.034
µM) in HTRF
assays.
93
993 94 45 62
• Compound
suppressed
LPS-induced
IL-6
production in
human acute
monocytic
leukemia
THP-1 cells
(EC50 = 0.045
µM) in HTRF
assays.
94
1152 84 47 97
• Compound
reduced LPSinduced IL-6
production in
human acute
monocytic
leukemia
THP-1 cells
(EC50 = 0.250
µM) in HTRF
assays.
95
994 89 22 45
• Compound
reduced LPSinduced IL-6
production in
human acute
monocytic
leukemia
THP-1 cells
(EC50 = 0.083
µM) in HTRF
assays.
96
990 498 3346 199
• It reduced
LPS-induced
IL-6
production in
human acute
monocytic
leukemia
THP-1 cells
(EC50 = 2.36
µM) in HTRF
assays
97
1037 >10000 27 13
• Compound
reduced LPSinduced IL-6
production in
human acute
monocytic
leukemia
THP-1 cells
(EC50 = 0.044
µM) in HTRF
assays.
98
465 57.8 139 30
• Compound
inhibited
recombinant
DYRK1A
and GSK-3 βkinase
activity (EC50
= 0.0017 and
3.649 µM,
respectively)
in FRET
assays.
• It inhibited
LPS-induced
IL-6
production in
human
peripheral
blood
mononuclear
cells (EC50 =
0.293 µM) in
HTRF assays.
99
233 67 98 145
• Compound
inhibited
recombinant
DYRK1A
activity (EC50
= 0.002 µM)
with
selectivity
over
recombinant
GSK-3beta
(EC50 = 0.238
100
µM) in FRET
assays.
• It decreased
Tau
phosphorylati
on at Ser396
in human
neuroblastom
a SH-SY5Y
cells (EC50 =
0.259 µM) in
ELISA
assays.
• It also
inhibited
LPS-induced
IL-6
production in
human
peripheral
blood
mononuclear
cells (EC50 =
0.280 µM) in
HTRF assays.
70 14 35 94
• inhibited
recombinant
DYRK1A
activity (EC50
= 0.002 µM)
with
selectivity
over GSK-3β
(EC50 = 1.480
µM) in FRET
assays.
• It reduced
LPS-induced
IL-6
production in
human
peripheral
blood
mononuclear
cells (EC50 =
0.089 µM) in
101
HTRF assays
205 24 31 32
• It reduced
LPS-induced
IL-6
production in
human
peripheral
blood
mononuclear
cells (EC50 =
0.087 µM) in
HTRF assays
• Compound
inhibited
recombinant
DYRK1A
activity (EC50
= 0.002 µM)
with
selectivity
over
recombinant
GSK-3β
(EC50 = 4.803
µM) in FRET
assays.
102
4658 88 95 163
• It reduced
LPS-induced
IL-6
production in
human
peripheral
blood
mononuclear
cells (EC50
>10 µM) in
HTRF assays.
• Compound
inhibited
recombinant
DYRK1A
activity (EC50
= 0.001 µM)
with
selectivity
over GSK-3β
103
(EC50 = 0.995
µM) in FRET
assays.
115 42 49 19
• Inhibited
recombinant
DYRK1A
and GSK-3β
activity (EC50
= 0.003 and
0.001 µM,
respectively)
in FRET
assays
• It reduced
LPS-induced
IL-6
production in
human
peripheral
blood
mononuclear
cells (EC50 =
0.065 µM) in
HTRF assays.
104
230 25 339 18
• Inhibited
recombinant
DYRK1A
and GSK-3β
activity (EC50
= 0.042 and
0.005 µM,
respectively)
in FRET
assays
• It reduced
LPS-induced
IL-6
production in
human
peripheral
blood
mononuclear
cells (EC50 =
1.024 µM) in
HTRF assays.
105
a = Effect on cell viability (EC50 in nM) in CellTiter-Blue cell viability assays in human colon
SW480 cancer cells.
b = Wnt inhibitory activity (EC50 in nM) in Sp5 luciferase reporter gene assay.
c = Suppression measurement (EC50 in nM) of TGF-β1 induced fibrosis in human lung
fibroblasts LL29 derived from Idiopathic pulmonary fibrosis (IPF) patients. ETC-159