Getting a Grip on the Undrugged: Targeting β-Catenin with Fragment-Based Methods

Article abstract of DOI:10.1002/cmdc.202000839

Aberrant WNT pathway activation, leading to nuclear accumulation of β-catenin, is a key oncogenic driver event. Mutations in the tumor suppressor gene APC lead to impaired proteasomal degradation of β-catenin and subsequent nuclear translocation. Restoring cellular degradation of β-catenin represents a potential therapeutic strategy. Here, we report the fragment-based discovery of a small molecule binder to β-catenin, including the structural elucidation of the binding mode by X-ray crystallography. The difficulty in drugging β-catenin was confirmed as the primary screening campaigns identified only few and very weak hits. Iterative virtual and NMR screening techniques were required to discover a compound with sufficient potency to be able to obtain an X-ray co-crystal structure. The binding site is located between armadillo repeats two and three, adjacent to the BCL9 and TCF4 binding sites. Genetic studies show that it is unlikely to be useful for the development of protein–protein interaction inhibitors but structural information and established assays provide a solid basis for a prospective optimization towards β-catenin proteolysis targeting chimeras (PROTACs) as alternative modality.

Full text of DOI:10.1002/cmdc.202000839

Accepted Article
Title: Getting a Grip on the Undrugged: Targeting β-Catenin with
Fragment-Based Methods
Authors: Dirk Kessler, Moriz Mayer, Stephan Zahn, Markus Zeeb,
Simon Wöhrle, Andreas Bergner, Jens Bruchhaus, Tuncay
Ciftci, Georg Dahmann, Maike Dettling, Sandra Döbel, Julian
E. Fuchs, Leonhard Geist, Wolfgang Hela, Christiane Kofink,
Roland Kousek, Franziska Moser, Teresa Puchner, Klaus
Rumpel, Maximilian Scharnweber, Patrick Werni, Bernhard
Wolkerstorfer, Dennis Breitsprecher, Philipp Baaske, Mark
Pearson, Darryl B. McConnell, and Jark Böttcher
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000839
Link to VoR: https://doi.org/10.1002/cmdc.202000839
01/2020

10.1002/cmdc.202000839
ChemMedChem
COMMUNICATION
Getting a Grip on the Undrugged: Targeting β-Catenin with
Fragment-Based Methods
Dirk Kessler[a] #, Moriz Mayer[a] #, Stephan Zahn[a] #, Markus Zeeb[b], Simon Wöhrle[a], Andreas
Bergner[a], Jens Bruchhaus[a], Tuncay Ciftci[b] , Georg Dahmann[b] , Maike Dettling[a] , Sandra Döbel[a] ,
Julian E. Fuchs[a], Leonhard Geist[a], Wolfgang Hela[a], Christiane Kofink[a], Roland Kousek[a], Franziska
Moser[b], Teresa Puchner[a], Klaus Rumpel[a], Maximilian Scharnweber[a], Patrick Werni[a], Bernhard
Wolkerstorfer[a], Dennis Breitsprecher[c],[d], Philipp Baaske[c], Mark Pearson[a], Darryl B. McConnell[a],
Jark Böttcher*[a]
[a]
[b]
[c]
[d]
#
Boehringer Ingelheim RCV GmbH & Co KG, Dr.-Boehringer-Gasse 5-11, 1121 Vienna, Austria
Boehringer Ingelheim Pharma GmbH & Co KG, Birkendorfer Straße 65, 88397 Biberach, Germany
NanoTemper Technologies GmbH, Floessergasse 4, 81369 Muenchen, Germany
present address: Leica Microsystems AG, Max Schmidheiny-Strasse 201, 9435 Heerbrugg, Schweiz
equally contributing
E-mail: jark.boettcher@boehringer-ingelheim.com
Supporting information for this article is given via a link at the end of the document.
Abstract: Aberrant WNT pathway activation, leading to nuclear
accumulation of β-catenin, is a key oncogenic driver event. Mutations
in the tumor suppressor gene APC lead to impaired proteasomal
degradation of β-catenin and subsequent nuclear translocation.
Restoring cellular degradation of β-catenin represents a potential
therapeutic strategy. Here, we report the fragment-based discovery of
Genetic inhibition of β-catenin impairs growth of WNT pathway driven
in vitro and in vivo tumor models[9-12], highlighting pharmacological
inhibition of β-catenin as a promising opportunity for targeted therapy
of WNT pathway driven cancers. However, despite decades of efforts
attempting to modulate the function of β-catenin, direct targeting of
the protein has remained an unreachable goal for conventional small
molecule discovery. Phenotypic screening approaches lead to the
identification of a number of indirect modulators of β-catenin[13]. The
most progressed small molecules bind to the N-terminal domain of the
coactivator, CBP, thereby antagonizing its interaction with β-
catenin[14]. One of these, PRI-724, has reached phase II of clinical
a
small molecule binder to β-catenin, including the structural
elucidation of the binding mode by x-ray crystallography. The difficulty
in drugging β-catenin was confirmed as the primary screening
campaigns identified only few and very weak hits. Iterative virtual and
NMR screening techniques were required to discover a compound
with sufficient potency to be able to obtain an x-ray co-crystal structure. development, outside of the cancer setting, for Hepatitis B and C
The binding site is located between armadillo repeats two and three,
adjacent to the BCL9 and TCF4 binding sites. Genetic studies show
that it is unlikely to be useful for the development of protein-protein-
interaction inhibitors but structural information and established assays
provide a solid basis for a prospective optimization towards β-catenin
proteolysis targeting chimeras (PROTACs) as alternative modality.
derived liver cirrhosis[15-16]
. In addition to classical screening
approaches, virtually designed peptide derived macrocycles were
reported, inhibiting the interaction of the N-terminal region of TCF and
β-Catenin, resulting in downregulation of target genes[17]. A validated,
chemistry-derived, binder to β-catenin, ideally with molecular
interactions elucidated by x-ray crystallography would offer options to
target β-catenin directly. Until recently, the only molecules fulfilling
these criteria for β-catenin were hydrocarbon-stapled peptides, based
on its natural binding partner Axin, bringing the burden of high
molecular weight[18-19]. Encouraged by the report of a short β-catenin
protein variant (β-catenin141-305) comprising only the first four Armadillo
repeats (Fig. 1a) and amenable for protein NMR applications[20], we
set out to apply fragment-based screening methods to the identify
novel small molecule binding sites on β-catenin.
Introduction: The WNT signal transduction cascade is a key
regulatory pathway during embryonic development and adult tissue
homeostasis. In the canonical WNT pathway, the armadillo repeat
protein β-catenin serves as the central signaling molecule, linking
WNT ligand mediated pathway activation to target gene induction in
the nucleus[1]. In the absence of WNT ligands, signal transduction is
tightly controlled by rapid proteasomal turnover of β-catenin, mediated
by a destruction complex consisting of the scaffolding proteins APC
and Axin and two serine-threonine kinases (CK1alpha and
GSK3alpha/beta)[2-3]. In cancer, aberrant WNT pathway activation is
frequently observed. In particular, loss-of-function mutations in the
tumor suppressor gene APC are present in approximately 80% of
colorectal cancers, resulting in impaired recruitment of β-catenin to
the destruction complex and WNT ligand-independent stabilization of
β-catenin[1, 4]. Upon nuclear translocation, β-catenin interacts with
transcription factors of the TCF/LEF family to induce WNT target gene
expression via recruitment of transcriptional co-activators, such as
Results: We applied two parallel fragment-based screening
approaches using recombinant β-catenin141-305 and a proprietary
fragment library composed of 1899 compounds, respectively. In one
cascade, we used saturation transfer difference nuclear magnetic
resonance (STD-NMR) spectroscopy and consecutive confirmation in
two-dimensional 1H/15N-transverse relaxation optimized spectroscopy
(TROSY) NMR experiments. Initially, the fragments were screened in
mixtures of four components per well with STD NMR, and 145 hits
were identified (Supplementary Fig. 1). From the primary hits 34
were confirmed in 2D 15N TROSY NMR experiments, where only
minor chemical shift perturbations (CSPs) could be detected. To
evaluate potential binding to the BCL9 site we mapped the epitope of
CBP and BCL9[5-8]
.
1
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the BCL9347-392 peptide by transferring the assignment from de la
Roche et al.[20] (Supplementary Fig. 2). However, as shown for
compound 1 (Fig. 1b, Supplementary Fig. 3) the observed CSPs are
non-overlapping to the BCL9 site.
In order to progress hit finding in the absence of structural information,
an automated workflow for ligand-based virtual screening was
employed. All identified hits were used as query structures in multiple
parallel similarity searches against the Boehringer Ingelheim (BI)
corporate compound collection, utilizing a panel of different similarity
search methods and metrics succeeded by data fusion[22], this serves
as BI’s “SAR by catalog” framework[23]. In total, 200 virtual screening
hits were selected and analyzed in 15NTROSY NMR detected titration
experiments. The racemic mixture of compound 3 showed the
strongest CSPs and was titrated to yield an average Kd of 1100 µM,
which had to be extrapolated because the solubility of the compound
was limited at 1000 µM (Table 1, Supplementary Fig. 5). Compound
3 was therefore used as novel query structure in a follow-up search
using the same approach as described above. The racemic mixture
of compound 4 and 5 was identified, maintaining the CSP pattern. For
further analysis the enantiomers where separated and subjected to
15N NMR detected titrations, yielding the eutomer compound 4
(NMR; Kdꢀ=2260 +/-1260ꢀµM; Table 1, Supplementary Fig. 6) and
the distomer compound 5 (NMR; Kdꢀ> 10mM, Table 1) and their
solubility was determined to be > 2000 µM, allowing for a higher
titration.
Figure 1. (a) Representation of the protein constructs used in this study based
on the crystal structure of the β-catenin-BCL9-Tcf4 complex (pdb ID: 2GL7).
Proteins are shown as cartoon (BCL9 in violet, TCF4 in marine, and β-catenin
Table 1. Molecular structure, solubility and affinity data for
compounds 1-7.
Compound
Solub.
pH 7.5
[µM]
HSQC
SPR Kd
[µM] [b]
(MST*) Kd
[µM][a]
1
2
3
4
5
6
7
>500
>10.000
n.d.
n.d.
n.d.
in wheat). The first four Armadillo repeats (AR) comprised in β-catenin141-305 are
labeled respectively. (b) Sections of superimposed 2D 15N TROSY spectra of
β-catenin141-305 in the absence (black) or presence of 500 µM compound 1 (red)
with cross peaks exhibiting observable minor chemical shift perturbation (full
spectrum shown in Supplementary Fig 2). Assignments were transferred from
de la Roche et al.[18] . (c) MST screening data of 50 nM fluorescently labeled β-
catenin141-305 in the presence of 1% DMSO as negative control (black) or 500
µM compound 2 (red). Time points of fluorescence readings for Fnorm
calculation are highlighted in blue and orange, respectively. (d) MST Kd
determination of compound 2; 50 nM labeled β-catenin141-305 with increasing
concentrations of 2.
>500
820+/-320*
1100+/-255
1000
>2000
>2000
>2000
>2000
2260+/-1260 >10.000
As a second fragment finding approach, we applied Microscale
Thermophoresis (MST). Initially, the screening assay was validated
by a titration with recombinant BCL9347-392 revealing a Kd of 360 nM
(Supplementary Fig. 4) which is in good agreement with the
previously determined binding affinity using ITC (Kd = 540 nM)[21]. The
outcome of the MST detected FBS was comparable to the STD-NMR
screen, yielding 134 primary hits. Compound 2 was identified as a
binder to β-catenin141-305 with a significant binding signal of Fnom of
7.5‰ compared to standard deviation with DMSO of 1.8‰ (Fig. 1c)
Subsequent titration experiments confirmed 16 hits as dose
dependent binders with affinities in the high three digit micromolar to
millimolar range, as shown for compound 2 (MST; Kdꢀ= 820+/-320 µM,
Table 1, Fig. 1d). Despite extensive co-crystallization experiments
with hits from both screening cascades, no co-crystal structure with β-
catenin141-305 could be obtained.
>10.000
915+/-303
>10.000
>10.000
1390+/-46
>10.000
[a] MST and NMR Kd values originate from single titrations, mean Kd´s
obtained from curves of selected cross peaks ± standard deviations.
[b]SPR Kd values reported as mean of 3 independent experiments ±
standard deviations.
2
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Given the lack of detailed structural information at this stage of the
project, conventionally provided by x-ray crystallography, we
performed ligand-based NMR techniques – GEM (group epitope
mapping) by STD[24] and WaterLOGSY-titration experiments[25-27] – to
evaluate the binding mode of compound 4. STD and WaterLOGSY
experiments provide information about protein-buriedness and
space. Hence a small series of phenyl derivatives were synthesized.
The racemic mixture of compound 6 and 7 showed an improved CSP
pattern and was subsequently subjected to chiral separation. The
consecutive NMR Kd titration revealed that eutomer 6 exhibits
improved affinity (NMR; Kdꢀ= 915+/- 303ꢀµM, Table 1, Fig. 2c and 2d)
whereas distomer 7 did not show any CSPs at a concentration of 500
µM (Supplementary Fig. 8).
solvent-exposure of ligand protons in the bound state. Figure
2
shows a comparison between STD GEMs (Fig. 2a), extracted from
STD build-up curves, and LOGSY-factors (Fig. 2b, Supplementary
Fig. 7) extracted from titration experiments for compound 4.
Compound 6 finally enabled the co-crystal structure determination in
complex with β-catenin141-305 at a resolution of 1.98 Å (Fig. 3a,
Supplementary Fig. 9). The ligand was found to bind to a solvent-
exposed pocket in a loop region between Armadillo repeats 2
(residues 205-210) and 3 (residues 243-251) (Fig. 3b)[28]
.
Figure 3. (a) Binding mode of compound 6 observed in the crystal structure in
complex with β-catenin141-305. Compound 6 is shown as stick model, color coded
by atom type with carbon shown in green. The refined 2Foꢀ–ꢀFc electron density
shown in blue is contoured at 0.8ꢀσ (b) Superposition of compound 6 from the
crystal structure (protein not shown) with the crystal structure of the β-catenin-
BCL9-Tcf4 complex (pdb ID: 2GL7) (C RMSDꢀ=ꢀ0.46 Å). Proteins are shown
as cartoon (BCL9 in violet, TCF4 in marine, and β-catenin in wheat), compound
6 is shown as spheres, color coded by atom type with carbon shown in green.
The first four Armadillo repeats (AR) comprised in β-catenin141-305 are labeled
respectively.
Figure 2. (a) STD-GEMs and (b) LOGSY-factors for compound
Superposition of two-dimensional 15N NMR spectra of 80ꢀµM uniformly 15
labeled β-catenin141-305 in absence (red) or presence (blue) of 500ꢀµM compound
6. (d) Titration curves of selected cross peaks yielding resulting in an averaged
Kd of 915+/- 303 µM (x-axis: (µM), y-axis δav(1H,15N) (ppm)).
4 (c)
N
The ligand’s distinct three-dimensional shape complements the
pocket while orienting the N-methyl lactam functionality towards the
receptor. Thereby, the carbonyl moiety of compound 6 forms a
hydrogen bond to the hydroxyl group of Ser246. Additionally, the
fused aryl ring forms CH-π interactions with the backbone CH of
Ser246 while orienting an ortho aryl CH towards the sulfur lone pairs
of Met243. The meta-cyano-phenyl substituent orients orthogonal to
the ligand’s bicyclic core, binding to a hydrophobic area involving
Val208 and Val248. The cyano substituent is solvent-exposed,
potentially acidifying the para CH via electron withdrawal and thus
strengthening its interactions with the backbone carbonyl of Asn206.
The larger β-catenin135-663 construct was used, as STD- and
WaterLOGSY-experiments are more efficient with larger proteins and
the observed ligand signals are not affected by underlying protein
signals, usually observed with small protein constructs. There is a
good consensus between the two methods, regarding the proton that
is most deeply buried and has the closest contact to the protein, as
indicated by the highest percentages. Both the STD GEMs and the
LOGSY-factors indicated that the phenyl ring is only partly buried and
there might be an opportunity to grow into unoccupied binding site
3
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COMMUNICATION
Based on the strong validation of the eutomer(6)-distomer(7) pair, we
attempted SPR analysis, which could serve as a high-throughput
assay for consecutive optimization cycles. The assay was validated
using BCL9347-392 as a positive control (SPR; Kd = 225 nM +/- 3.5,
Supplementary Fig. 10) In comparison, the Kd for eutomer 6 is weak
(SPR: 6; Kd = 1390 +/-46 µM) and data could only be acquired up to
valuable tools as they focused the area of chemical space explored
during the analoging process. Compound 6 binds to β-catenin with an
affinity of 915 µM and the co-crystal structure provides a path for
further optimization. The novel, unexplored binding site was
characterized with respect to potential interference with binding of the
transcription factors TCF4 and BCL9, using co-immunoprecipitation
studies. The experiments suggest that the identified binding site is
non-functional, and thereby is unlikely to be applicable for the
development of protein-protein-interaction inhibitors of TCF4 for
example which binds tightly to β-catenin (~20 nM)[30]. As oncogenic
mutations stabilize β-catenin and thereby lead to elevated protein
levels, restoring cellular degradation is a potential therapeutic strategy.
As oncogenic mutations stabilize β-catenin and thereby lead to
elevated protein levels, restoring cellular degradation is a potential
therapeutic strategy. This concept lead to the discovery of molecular
glue-like small molecules enhancing the interaction of mutant β-
catenin and the natural E3 ligase β-TrCP in engineered mutant β-
catenin cells [31]. Proteolysis targeting chimeras (PROTACs) are a
promising strategy to degrade and target β-catenin. In-vivo proof of
concept for this approach was recently achieved using stapled peptide
based PROTACs recruiting the E3 ligase VHL[32]. The discovered
eutomer-distomer pair provides a high level of confidence that can
support ligand optimization and consecutive enablement of β-catenin
targeting PROTACs.
a
concentration of 1 mM and the fit curve is extrapolated
(Supplementary Fig. 11a). The sensorgrams show contribution from
non-specific binding and an overstoichiometric binding response is
observed. Interestingly, distomer 7 shows a comparable non-specific
binding effect in the absence of any saturable behavior (SPR: 7; Kd >
10 mM, Supplementary Fig. 11b).
The overlay of the co-crystal structure of compound 6 with the crystal
structure of a β-catenin-BCL9-Tcf4 complex[21] (pdb ID: 2GL7)
suggests that binding of compound 6 does not interfere with binding
of the selected transcription factors TCF4 and BCL9 (Fig. 3b). To
assess the potential functional relevance of the compound binding site,
TOPFlash reporter gene assays were performed (Supplementary
Fig. 12a). The TOPFlash reporter plasmid comprises a TCF-driven
firefly luciferase, allowing for luminescence based readout of β-
catenin/TCF transcriptional activity.
Transfection of β-catenin
(control) or β-catenin variants harboring substitutions in the
compound binding site (A211F, S246V, A211F_S256V) resulted in
comparable protein expression levels in HEK293 cells
(Supplementary Fig. 12a). In the reporter gene assay, β-catenin
variants showed similar transcriptional activity, in comparison to the
β-catenin control construct (Supplementary Fig. 12a). In particular,
comparable reporter gene activation to the β-catenin control was
observed for the A211F_S256V double mutant variant. In β-catenin
co-immunoprecipitation studies with TCF4 and BCL9, we further
tested whether the compound binding site is relevant with respect to
PPI interactions with co-activators. To this end, β-catenin (control) or
compound binding site variants (A211E, A211F_S246V,
A211E_S246E, A211I_S246F) were co-expressed in HEK293 and
co-immunoprecipitation of TCF4 and BCL9 was monitored upon pull-
down of β-catenin using immunoblotting (Supplementary Fig. 12b).
In comparison to β-catenin (control), all variants showed similar co-
immunoprecipitation of TCF4 and BCL9, indicating that interactions of
β-catenin with TCF4 or BLC9 were not affected by introduction of
amino acid substitutions in the compound binding site. In line with
previous structural and functional studies[28], the results of the reporter
gene and co-immunoprecipitation studies imply that addressing this
binding site might not affect β-catenin function.
Data availability: The authors declare that the data supporting the
findings of this study are available within the publication and its
supplementary information. The coordinates of the crystal structure
of β-catenin141-305 in complex with compound 6 have been deposited
in the RCSB Protein Data Bank (PDB; http://www.rcsb.org) with the
accession number 7AFW.
Competing interests: D. K., M. M., S. Z., M. Z., S. W., A. B., J. Br.,
T. C., G. D. , M. D., S. D., J. E. F., L. G., W. H., C. K., R. K., F. M., T.
P., K. R., M. S., P. W., B. W., M. P., D. B. M., J. Bö.. are employees
of Boehringer Ingelheim.
Keywords: β-catenin • Fragment-based Screening • Microscale
Thermophoresis • WaterLOGSY • PROTAC
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10.1002/cmdc.202000839
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We report the fragment-based discovery of a small molecule binder to the key cancer target β-catenin. Iterative virtual screening and
ligand-based NMR techniques enabled hit optimization in absence of structural information. The co-crystal structure of a derivative in
complex with a short -catenin variant revealed a novel small molecule binding site between armadillo repeats two and three.
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