Discovery of a cryptic site at the interface 2 of TEAD – Towards a new family of YAP/TAZ-TEAD inhibitors

Article abstract of DOI:10.1016/j.ejmech.2021.113835

The Hippo pathway is involved in organ size control and tissue homeostasis by regulating cell growth, proliferation and apoptosis. It controls the phosphorylation of the transcription co-activator YAP (Yes associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif) in order to control their nuclear import and their interaction with TEAD (Transcriptional Enhanced Associated Domain). YAP, TAZ and TEADs are dysregulated in several cancers making YAP/TAZ-TEAD interaction a new emerging anti-cancer target. We report the synthesis of a set of trisubstituted pyrazoles which bind to hTEAD2 at the interface 2 revealing for the first time a cryptic pocket created by the movement of the phenol ring of Y382. Compound 6 disrupts YAP/TAZ-TEAD interaction in HEK293T cells and inhibits TEAD target genes and cell proliferation in MDA-MB-231 cells. Compound 6 is therefore the first inhibitor of YAP/TAZ-TEAD targeting interface 2. This molecule could serve with other pan-TEAD inhibitors such as interface 3 ligands, for the delineation of the relative importance of VGLL vs YAP/TAZ in a given cellular model.

Full text of DOI:10.1016/j.ejmech.2021.113835

European Journal of Medicinal Chemistry 226 (2021) 113835
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of a cryptic site at the interface 2 of TEAD e Towards a new
family of YAP/TAZ-TEAD inhibitors
Manon Sturbaut a, Fabrice Bailly a, Mathilde Coevoet a, Pasquale Sileo a,
Martine Pugniere b, Maxime Liberelle a, Romain Magnez c, Xavier Thuru c,
a
a
d
d
ꢀ ꢀ
Marie-Christine Chartier-Harlin , Patricia Melnyk , Muriel Gelin , Frederic Allemand ,
,
, e, *
Jean-François Guichou d **, Philippe Cotelle a
a Univ Lille, INSERM, CHU Lille, UMR-S 1172, Lille Neuroscience and Cognition Research Center, F-59000, Lille, France
Institut de Recherche en Cancerologie de Montpellier (IRCM), INSERM U1194 e ICM, UM 208 rue des Apothicaires, F-34298, Montpellier Cedex 5, France
b
ꢀ
c Univ. Lille, CNRS, Inserm, CHU Lille, IRCL, UMR9020 e UMR1277 - Canther e Cancer Heterogeneity, Plasticity and Resistance to Therapies, Platform of
Integrative Chemical Biology, F-59000, Lille, France
d Centre de Biologie Structurale (CBS), CNRS, INSERM, Univ Montpellier, Montpellier, France
e ENSCL-Centrale Lille, CS 90108, F-59652, Villeneuve d’Ascq Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2021
Received in revised form
31 August 2021
Accepted 3 September 2021
Available online 6 September 2021
The Hippo pathway is involved in organ size control and tissue homeostasis by regulating cell growth,
proliferation and apoptosis. It controls the phosphorylation of the transcription co-activator YAP (Yes
associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif) in order to control their
nuclear import and their interaction with TEAD (Transcriptional Enhanced Associated Domain). YAP, TAZ
and TEADs are dysregulated in several cancers making YAP/TAZ-TEAD interaction a new emerging anti-
cancer target. We report the synthesis of a set of trisubstituted pyrazoles which bind to hTEAD2 at the
interface 2 revealing for the first time a cryptic pocket created by the movement of the phenol ring of
Y382. Compound 6 disrupts YAP/TAZ-TEAD interaction in HEK293T cells and inhibits TEAD target genes
and cell proliferation in MDA-MB-231 cells. Compound 6 is therefore the first inhibitor of YAP/TAZ-TEAD
targeting interface 2. This molecule could serve with other pan-TEAD inhibitors such as interface 3 li-
gands, for the delineation of the relative importance of VGLL vs YAP/TAZ in a given cellular model.
© 2021 Elsevier Masson SAS. All rights reserved.
Keywords:
TEAD inhibition
TEAD cryptic binding pocket
Interface 2
Binding assays
Hippo pathway
1. Introduction
Enhanced Associated Domain) [1].
The phosphorylation of S127 of YAP (S89 in TAZ) promotes its
The Hippo pathway is involved in organ size control and tissue
homeostasis by regulating cell growth, proliferation and apoptosis.
It consists in a cascade of kinases including Mst1/2 (mammalian
ste20-like protein kinase), Sav1 (scaffold protein Salvador), Lats1/2
(large tumor suppressor kinase) and Mob proteins (mps one binder
kinase), which regulates the phosphorylation of the transcription
co-activator YAP (Yes associated protein) and TAZ (Transcriptional
coactivator with PDZ-binding motif) in order to control their nu-
clear import and their interaction with TEAD (Transcriptional
cytoplasmic retention by the protein 14-3-3 and the phosphoryla-
tion of S381 (S311 in TAZ) induces its degradation. Conversely,
unphosphorylated, YAP and TAZ enter into the nucleus, interact
with TEAD and drive the target gene expressions in charge of cell
proliferation and apoptosis avoidance such as CTGF (connective
tissue growth factor), Cyr61 (cysteine-rich angiogenic protein),
Survivin (also known as Birc-5) and AXL.
In the nucleus, YAP and TAZ compete with other natural TEAD
ligands, namely VGLL (Transcriptional cofactor Vestigial like pro-
tein family) which are nuclear regulators of the transcriptional
activity of TEADs [2]. More recently FAM181A and FAM181B, two
new TEAD interactors have been identified [3]. These proteins are
expressed most prominently in neural tissues, where Fam181A is
exclusively expressed during embryonic development [4].
* Corresponding author. Univ Lille, INSERM, CHU Lille, UMR-S 1172, Lille Neuro-
science and Cognition Research Center, F-59000, Lille, France.
** Corresponding author.
E-mail addresses: guichou@cbs.cnrs.fr (J.-F. Guichou), philippe.cotelle@univ-
lille.fr (P. Cotelle).
Several stimuli such as mechanical force, cell adhesion, serum
https://doi.org/10.1016/j.ejmech.2021.113835
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

M. Sturbaut, F. Bailly, M. Coevoet et al.
European Journal of Medicinal Chemistry 226 (2021) 113835
starvation, or energy stress promote activation of kinases and
regulate YAP/TAZ localization. In contrast, osmotic stress, high cell
density and cell detachment induce cytoplasmic translocation of
TEAD [5]. A dysregulation of this equilibrium brings to abnormal
and excessive proliferation leading to cancer where YAP/TAZ and
TEAD are overexpressed [6]. Thus, inhibition of YAP/TAZ-TEAD
complexes is a pertinent strategy for cancer therapy [7].
YAP/TAZ and TEADs form a complex through the interaction of
the N-terminal domain of YAP (and TAZ) (TEAD Binding Domain:
TBD) and the C-terminal domain of TEAD (YAP/TAZ Binding
Domain: Y/TBD). In their pioneer work, Li et al. [8] (Fig. 1) defined
the three interfaces of contact between TEAD1209-426 and YAP50-171
and their respective importance in the binding as follows: interface
3 (in red on Fig. 1A) > interface 2 (in green) > interface 1 (in blue).
However, the minimal fragment of YAP or TAZ which gives a
nanomolar range binding constant corresponds to interfaces 2 and
3 [9], while, Kd's of mVGLL127-56 are in the same nanomolar range
than YAP and TAZ although slightly superior while VGLL do not
interact with TEAD at the interface 3 [10]. Protein fragments only
discovery of the presence of palmitate in the internal pocket of
TEAD, Pobbati et al. [21] identified niflumic, bromoflufenamic and
flufenamic acids (Fig. 2), as TEAD ligands of the central pocket.
While niflumic acid has a KD of 28
mM for TEAD4 (measured by
isothermal calorimetry), it only presents cellular effect at 150
mM.
Bum-Erdene et al. [22] reported a small molecule inhibiting YAP-
TEAD complex transcriptional activity through the presumed for-
mation of a covalent bond with the cysteine residue in the central
pocket. Using fluorescence polarization experiments, the authors
were able to measure the inhibitory activity of their compounds for
the YAP-TEAD4 interaction. TED-347 (Fig. 2) possessed an EC50 of
5.9 mM and a similar IC50 in a HEK293 cell-based assay. Not sur-
prisingly, TEAD-347 was found to be toxic in EGFR-mutant NSCLC
cell lines and replaced by another covalent TEAD ligand which
bears an acrylamide moiety [23]. This new compound bearing a
vinylamide moiety (MYC-01-037 (Fig. 2)), is supposed to covalently
bind to C359 (TEAD1) in the palmitate pocket as another vinyl-
sulfamide (DCTEAD-02 [24]). and K-379 [25], Fig. 2). But the first
ligand of the palmitate pocket reported so far is MGH-CP1 (Fig. 2)
[26,27] which was very recently followed by two new compounds
(Compounds 1 and 2, Fig. 2) developed by Genentech and Roche
Pharma [28].
composed of the
Whereas the YAP
U
-loop present only micromolar affinities [3].
U
-loop is considered to be the “hot spot” of the
YAP-TEAD interaction, it was shown that the folding of the YAP a-
helix is firstly formed (interface 2) before interface 3 formation [11].
Interfaces 2 and 3 correspond to the external predicted druggable
sites in green and red (Fig. 1B) [12]. In 2016, was reported for the
first time, that Y/TBD of TEAD2 and TEAD3 are palmitoylated in an
internal hydrophobic pocket [13,14]. This palmitoylation is required
for the stability of TEAD, the interaction with YAP or TAZ and reg-
ulates the output of the Hippo pathway. This internal pocket clearly
appears as the third druggable site of TEAD (in purple, Fig. 1B).
Chemical control of the Hippo pathway can be divided into three
main strategies: (i) favor the phosphorylation [15] or the nuclear
import of YAP or TAZ, (ii) physically inhibit YAP/TAZ-TEAD inter-
action, (iii) interfer on the YAP/TAZ-TEAD transcriptional targets.
These different strategies have been reviewed in detail by
Pobbati and Hong [16].
During the preparation of this manuscript, inhibitors of hTEAD
autopalmitoylation patented by Vivace Therapeutics [29,30] have
been reported. Interestingly, VT103 (Fig. 2) is the first selective
inhibitor of hTEAD1 autopalmitoylation while VT107 (Fig. 2) is 50
fold less active than its enantiomer [31].
A fragment based-approach allowed the identification of one hit
(fragment 1, Fig. 2) which binds to YAP-binding interface 2 of TEAD
[32]. Its affinity for mTEAD4 is very low (300e1400 mM) and it has a
detectable cellular activity only at very high concentration (750
@ 33%).
mM,
In spite of a growing research activity as attested by the number
of recently published inhibitors, the design of TEAD ligands is still
in its infancy with micromolar activities and only one selective
TEAD1 ligand was reported to date [31].
Considering the second strategy, modified linear and cyclic YAP-
like peptides and a peptide composed of the VGLL4 interface 2
domain and the YAP interface 3 domain (super-TDU) were firstly
developed to compete with YAP for binding TEADs [17e19]. These
modified peptides present high affinity for TEAD. Only recently, the
first non-peptidic inhibitor, CPD3.1 (Fig. 2), targeting the interface 3
was published [20]. As a non-selective YAP-TEAD inhibitor, CPD3.1
has an IC50 ranging between 33 and 44 mM on the four members of
TEAD family as measured in HeLa cells transfected by a Gal4-luc
reporter together with the vectors for Gal4-TEADx. Prior to the
Here, we report the first TEAD inhibitors of the interface 2 which
allowed us to identify for the first time a cryptic site of TEAD C-
terminal domain.
We discovered a series of trisubstituted pyrazoles (Scheme 1)
which bind to hTEAD2 in an unapparent/unrevealed cryptic pocket
situated at the end of one of the alpha-helix of TEAD implicated in
the interface 2. The crystallographic structures of five complexes
between hTEAD2 and our ligands were resolved at high resolution.
Differential scanning fluorimetry (NanoDSF) experiments allowed
us to define a specific profile of edF/dT ¼ f(T) curves. Affinity
Fig. 1. (A): The 3D-structure model built by superimposition of hYAP250-171-hTEAD1209-426 complex (PDB code 3KYS) and hTEAD2217-447 (PDB code 5EMV) with interface 1 (blue), 2
(green) and 3 (red); (B): The three predicted druggable sites of TEAD (interface 2 (green), interface 3 (red) and internal pocket (purple).
2

M. Sturbaut, F. Bailly, M. Coevoet et al.
European Journal of Medicinal Chemistry 226 (2021) 113835
Fig. 2. A set of the most important TEAD ligands reported to date.
Scheme 1. Structures of previous hits leading to our new compounds.
3

M. Sturbaut, F. Bailly, M. Coevoet et al.
European Journal of Medicinal Chemistry 226 (2021) 113835
constants for hTEAD2 were measured in cell lysate by microscale
thermophoresis (MST). Some of our compounds proved to be effi-
cient in cellular assays (TEAD transcriptional activity in HEK-293T
cells and TEAD target gene expression in breast cancer cell lines).
readily available compound 1 (Scheme 2) with functionalized alkyl
chains which yielded 1,3,4-trisubstituted pyrazoles as the major
isomers.
Compound
1
was synthesized from ethyl 3-(3,4-
dichlorophenyl)-3-oxopropanoate and N,N-dimethylformamide
ꢁ
dimethylacetal according to Senica procedure [35]. After saponifi-
2. Results
cation, the resulting carboxylic acid reacted with benzylamine or
phenethylamine, in the presence of EDCI and HOBt to give the N-
benzyl and N-phenethyl-3-(3,4-dichlorophenyl)-1H-pyrazole-4-
carboxamide 2 and 3, respectively. Compounds 1e3 were then
2.1. Synthesis of a series of TEAD ligands
We previously screened a protein-protein interface inhibitors
enriched library (175000 chemical compounds) against the inter-
face 3, using the first X-ray structure of the hYAP-hTEAD1 complex
(PDB 3KYS) and identified a first hit 1 (Scheme 1) with inhibitory
properties in the micromolar range (IC50 ¼ 6.5
gene reporter assay [33]. This hit 1 was optimized into hit 2
(Scheme 1) which presents an IC50 of 1.7 M in the same luciferase
alkylated using N-(
4e12 were obtained after treatment with hydrazine hydrate.
Compounds 2 and 3 were also alkylated with ethyl -bromopro-
u-bromoalkyl)phthalamides and the amines
u
pionate or butanoate and the resulting esters were saponified to
give the acids 13e16.
mM) in a luciferase
m
assay [34]. To overcome solubility problems we use deconstruct
strategy and decided to keep the 3,4-dichlorophenyl ring and
suppress the isatin moiety and replace the central triazole ring by a
diazole ring to create a small library of 3-(3,4-dichlorophenyl)-1H-
pyrazole-4-carboxylates or carboxamides where N-1 was
substituted by alkylamine or alkylcarboxylic acid arms (Scheme 1).
Our chemical strategy was based on the substitution of the
2.2. In solution, our TEAD ligands bind to hTEAD2 at an external
interface
We firstly measured the thermal stability of hTEAD2217-447 by
NanoDSF in the presence or in the absence of our compounds.
NanoDSF is based on the intrinsic fluorescence of aromatic residues
of the protein and avoids the possible competition of a drug with
Scheme 2. i) a. DMF-DMA (1.1 eq.), toluene, reflux, 3 h; b. NH2-NH2.H2O (1 eq.), EtOH, 70 ꢀC, 5 h; ii) NaOH (10 eq.), EtOH, 78 ꢀC, 16 h; iii) EDCI (1.2 eq.), HOBt (1.2 eq.), benzylamine
or phenethylamine (1 eq.), DMF, rt, 16 h; iv)
u
-Bromoalkyl esters or N-(
u
-bromoalkyl)phthalimides (1 eq.), K2CO3 (2 eq.), anhydrous ACN, 82 ꢀC, 16 h; v) NH2-NH2.H2O (10 eq.), EtOH,
78 ꢀC, 3 h.
4

M. Sturbaut, F. Bailly, M. Coevoet et al.
European Journal of Medicinal Chemistry 226 (2021) 113835
the dye used in classical TSA (Thermal Shift Assay). For hTEAD2, we
observed two thermal transitions with two distinct melting tem-
peratures (Tm) (Fig. 3, red curve). According to Mesrouze et al. [36]
the first Tm value (44.6 0.5 ꢀC) was attributed to non-acylated
complexes (compounds 6, 7, 13, 14 and 15) were obtained with
resolutions ranging from 2.00 to 2.22 Å (Fig. 5A, Supplement
Tables 1 and 2 and Supplement Figures 4-8). The structures were
solved in space group C2 with two TEAD per asymmetric unit. In all
the internal pocket of the TEAD units, a myristate/palmitate
molecule is present either free, or bonded to the sulfur atom of
C380 (thioester) or to the nitrogen atom of K357 (amide) as pre-
viously reported [8,14,22]. hTEAD2 crystallized as a dimer in the
asymmetric unit cell, all the complexes were obtained with only
one compound because the second cryptic pocket is not accessible
due to the crystal packing. The five compounds fitted hTEAD2 in a
very similar manner at the larger end of the binding groove formed
hTEAD2 and the second Tm value (56.8
0.5 ꢀC) to acylated
hTEAD2. With a compound which binds to hTEAD2 in the palmitate
pocket, we expect to observe only one Tm. Conversely with a
compound which binds to hTEAD2 at an external interface, we
expect to observe two new shifted Tm values. In our hand, niflumic
acid (Fig. 3, cyan curve) gave an intermediate Tm value of 50.3 ꢀC.
Moreover, Tang et al. [31] found the same tendency with their re-
combinant TEAD proteins with all their TEAD ligands (which bind
to the internal pocket). In the presence of our compounds, the
thermal stability of the protein was changed and negative or pos-
itive thermal shifts were observed for each peak of the first derived
curve (-dF/dT ¼ f(T)). The profiles of the curves are characteristic of
those of TEAD-ligands which bind to TEAD on the external surface
of the protein.
The biological matrix is known to influence the affinity of a drug
for its target. For example, Wienken et al. [37] measured a 400-fold
reduction for the affinity of quercetin for its kinase PKA in human
serum vs in buffer. In order to reflect more accurately the affinity of
our drugs for hTEAD2 in complex medium, the interactions be-
tween our compounds and hTEAD2 were quantitatively measured
using microscale thermophoresis (MST) on GFP-labeled
hTEAD2217e447 in CHO-K1 cell lysate [35]. hYAP50e102, which is
the fragment which interacts with hTEAD in interfaces 2 and 3 was
used as a control for MST experiments.
by hTEAD2
interface 2 with YAP
a
3 and
a
4 helices (residues 381e405) involved in the
1 helix (residues 61e73) (Fig. 5A).
a
A cryptic pocket (Fig. 5C and D) is created where the 3,4-
dichlorophenyl moiety is perfectly inserted in. This new pocket is
due to the flipping of Y382 side chain. The ligand's position is at the
amide bond between L65 and D64 residues of YAP. The unsub-
stituted nitrogen of the pyrazole is engaged in a hydrogen bond
with the alcohol function of S349 of TEAD2 and therefore replaces
the hydrogen bond made by the carboxylate function of D64 of YAP
and the alcohol function of S349. The phenyl ring of compounds 7
and 13e15 does an angle of about 80ꢀ with the phenol ring of Y382
and the acidic function of compounds 13e15 is oriented towards
the terminal ammonium of K352 (Fig. 5B and E) but without
making any hydrogen bond with this residue. The bottom of the
cryptic pocket consists in the isobutyl group of L383 which is
specific of hTEAD2 (other TEADs have a methionine residue at this
position (M362 for TEAD1, M371 for TEAD3 and M370 for TEAD4).
Analysis of other crystal structures of hTEAD2 (5EMV for example)
and other hTEADs showed the cryptic pocket pre-exists and is
masked by Y382. The shape does not significantly differ amongst
the different TEADs.
The affinity of hYAP50e102 for GFP-labeled hTEAD2217e447 is
evaluated through the Kd value (96 nM) which is in accordance
with the literature [38]. We measured the affinity of the com-
pounds 6e7 and 13e15. 7 and 13 were found to produce residual
fluorescence and we were unable to properly measure their Kd
values. However, 14 and 15 presented a micromolar affinity with Kd
of 4.6 and 5.1
m
M respectively while 6 had a lower affinity than 14
2.3. Compound 6 inhibits TEAD-dependent target gene expression
We firstly measured the TEAD transcriptional activity in
or 15 for hTEAD2 (Kd ¼ 35
mM) (Fig. 4, and Supplement figures 1e3).
hTEAD2217-447 crystals were soaked with compounds 4e16. Five
Fig. 3. Representative thermograms obtained by NanoDSF for hTEAD2217-447 protein (5
m
M) in the absence (red) or in the presence of tested compounds 6 (orange), 7 (green), 14
(violet) and niflumic acid (light blue). The melting temperatures (Tm) were obtained by plotting the first derivative of the fluorescence emission (F) as a function of the temperature
(edF/dT). The curve minimum corresponds to Tm. Acidic ligands have a better affinity towards hTEAD2 than basic ligands.
5

M. Sturbaut, F. Bailly, M. Coevoet et al.
European Journal of Medicinal Chemistry 226 (2021) 113835
Fig. 4. Titration of eGFP-hTEAD2217e447 (30 nM) by compound 6 in CHO-K1 cell lysate; LED intensity: 100%; MST power: 40%. All the experiments have been made in triplicate on
three independent cell cultures (mean SD, n ¼ 3). Our TEAD ligands bind to TEAD at a cryptic pocket situated in the interface 2.
Fig. 5. (A) Superimposition of the crystal structure of hTEAD2217-447 in complex with compounds 6, 7, 13, 14 and 15 (PDB codes: 6S6J, 6S66, 6S64, 6S60 and 5S69, respectively) and of
hTEAD1210-426 in complex with hYAP50-100 (PDB code: 3KYS); (B) Zooming on the compound 14 environment; (C) and (D) The cryptic pocket: zooming on the pocket created (PDB
code: 6S60) (in tan) and superimposition of compound 14 on hTEAD2217-447 (PDB code: 5 EMV) (in blue, Y382 is in pink); (E) Principal interactions of compound 14 with hTEAD2.
6

M. Sturbaut, F. Bailly, M. Coevoet et al.
European Journal of Medicinal Chemistry 226 (2021) 113835
transfected HEK293T cells in the presence of our compounds (5e9
cells reached a plateau. Compound 6 decreased the cell prolifera-
and 11e16, 10
mM, 16 h) using a previously used TEAD reporter
tion by 40% at 5 mM and by 60% at 10 mM after 48 h (Fig. 8).
luciferase assay [33]. We used Dasatinib [39] at a concentration of
100 nM and MGH-CP1, a patented compound that was reported to
bind to TEAD in the palmitate pocket, at a concentration of 10
mM
3. Discussion and conclusion
[27], as references. We measured the -galactosidase activity in
b
order to normalize the luciferase activities and to qualitatively es-
timate the cytotoxicity of the tested compounds. In case of signif-
icant decrease of b-galactosidase signal after 24 h post transfection,
the luciferase activity result was not retained. The cell viability was
visually controlled after treatment. The reporter activities are given
on Fig. 6. Compounds 4 and 10 were found to be too unstable to be
We herein report the discovery of the first inhibitors of the YAP/
TAZ-TEAD interactions targeting the interface 2. The structures of
five complexes with a high resolution allowed us to discover and
characterize for the first time a cryptic site on the common surface
of the YAP/TAZ/VGLL-binding domain of TEAD [40,41]. The phenol
moiety of Y382, which, generally points towards the interface 2 (in
the direction of S349) groove in almost all previous crystal struc-
tures of TEAD2, moved away. Re-analysis of all the TEAD units
found in published crystallographic structures showed that, in two
monomers (A and C) of mTEAD4 of the crystal complex of TAZ-
mTEAD4 (PDB code: 5 GNO), this tyrosine residue (Y362 (Y382 in
hTEAD2)) pointed towards C360 making a hydrogen bond with the
thiol hydrogen atom when C360 is not covalently bonded to
palmitate and in the two other monomers (B and D) pointed to-
wards S329 (S349 in hTEAD2) (Fig. 9). It would be of interest in the
future to evaluate if Y382 plays a role in the palmitoylation of TEAD.
The deep pocket created is occupied by the 3,4-dichlorophenyl
ring of our compounds. The free nitrogen of the central pyrazole
of our drugs replaced the phenolic group of Y382 and created a
hydrogen bond with the hydroxyl group of S349. This residue is also
involved in a specific hydrogen bond with the imidazole ring of H44
of mVGLL1 [39] or with the carboxylate function of D64 of hYAP
[42]. We placed at position 4 an ethyl ester, a benzylamide or a
tested in cells. In our series, we found that all the amides with a
aminoalkyl chain (7-12) presented a dramatic decrease in
u
b
-
-
galactosidase activity which reflects an intrinsic toxicity and the
amides with a -carboxyalkyl chain (13e16) were almost inactive
u
in spite of a good affinity for 14 and 15 measured by MST. Finally,
aminoesters (5e6) were found to be the most active compounds of
the series. The affinity was measured on eGFP-TEAD2 in lysate
whereas the TEAD transcriptional activity was measure in cells, the
differences between 6 and 14e15 may be due to a lower nucleus
penetration for the acidic 14e15 than for the basic 6.
Dose-response curves for compound
6 gave an IC50 of
4.5 1.5 M (Supplement Figure 9). Here again, we found some
m
discrepancies between the affinity for eGPF-TEAD2 and the inhi-
bition of TEAD transcriptional activity which could be attributed to
differences between the method and the target. By MST we used
hTEAD2217-447 while HEK293T cells expressed TEAD2 at very low
levels [31]. We measured the effects of compound 6 (10 mM) and
Dasatinib (100 nM) on the RNA and expression of AXL, CTGF and
Cyr61 and protein expression of AXL, CTGF, Cyr61, and Survivin
(Birc-5) (Supplement Figure 10) in MDA-MB231 cells. Similar ef-
fects were also found in HeLa cell lines (Supplement Figure 11) but
not on SH-SY5Y cell lines which do not express YAP or TAZ (data not
shown). The results are reported on Fig. 7. In good correlation with
the reporter assay, compound 6 inhibited the expression of the four
target proteins. The same tendency was found with the RNA
expression of AXL, CTGF and Cyr61 after only 24 h of treatment.
Finally, we measured the effect of compound 6 on the prolifer-
ation of MDA-MB-231 cells at low confluence. Cells were plated at
low confluence and exposed to compound 6 at different concen-
trations. Images were made every 3 h until control DMSO-treated
phenethylamide group and at position 1 a u-aminoalkyl or u-car-
boxyalkyl chain. All these variations we introduced were found in
the compounds engaged in a complex with TEAD2 suggesting that
the main structural element of this new class of TEAD ligand is the
3-(3,4-dichlorophenyl)-1-substituted pyrazole-4-carboxylate or
carboxamide moiety. The
u-aminoalkyl or u-carboxyalkyl arm is
supposed to help N-2 to engage hydrogen bond with the hydroxyl
group of S349 through its electro-donating effect and head away
the interface 2. We previously demonstrated the importance of the
3,4-dichlorophenyl moiety [34] and we planned in the future to
delineate the relative importance of each chlorine atom of this
moiety.
Kaan et al. [32] have previously reported the discovery of a
fragment that targets the mTEAD4 interface 2 (pdb code: 5XJD).
Fig. 6. TEAD reporter luciferase activity observed in HEK293T cells treated with compounds 5e9 and 11e16 (10 mM), MGH-CP1, or dasatinib after 16 h post transfection. Data are
representative of at least three independent experiments in tri biological replicates; mean SD, n ¼ 3. p values were calculated using Kruskal-Wallis tests. ns ¼ not significant,
* ¼ p < 0.05, ** ¼ p < 0.01, *** ¼ p < 0.001.
7

M. Sturbaut, F. Bailly, M. Coevoet et al.
European Journal of Medicinal Chemistry 226 (2021) 113835
Fig. 7. Effects of Dasanitib and compound 6 on protein production of AXL, CTGF, Cyr61, and Survivin in MDA-MB-231 cells after 48 h exposure and RNA expression of AXL, CTGF and
Cyr61 in MDA-MB-231 cells after 24 h exposure. Data are representative of at least three independent experiments in tri biological replicates; mean SD, n ¼ 3. p values were
calculated using Kruskal-Wallis tests. ns ¼ not significant, * ¼ p < 0.05, ** ¼ p < 0.01, *** ¼ p < 0.001.
Fig. 8. Effect of compound 6 on the kinetic cell growth of MDA-MB-231 cells at low confluence (0.25 ꢁ 104 cells/well) (mean SD, n ¼ 3).
8

M. Sturbaut, F. Bailly, M. Coevoet et al.
European Journal of Medicinal Chemistry 226 (2021) 113835
compounds in the interface 2 but was found higher inhibitor of
VGLL4 than YAP and therefore activates YAP-TEAD interaction, in-
creases mRNA target genes levels in cardiomyocytes and acceler-
ates wound healing of RKO cells. Superimposition of the crystal
structures (6SBA and 6S60) showed our molecules only overlap at
the end of interface 2 with one end of one of the helix of the
eicosapeptide (Supplement figure 13).
4. Experimental section
Chemistry. General details. All reagents and solvents were
purchased from Aldrich-Chimie (Saint-Quentin-Fallavier, France) of
ACS reagent grade and were used as provided. All reagents and
solvents were purchased and used without further purification.
Reactions were monitored by TLC performed on Macherey Nagel
Alugram® Sil 60/UV254 sheets (thickness 0.2 mm). Some purifi-
cation of products was carried out by flash column chromatography
(FC) using Macherey Nagel silica gel (230e400 mesh). Melting
points were determined on a BÜCHI B-540 apparatus and are un-
corrected. NMR spectra were recorded on a Bruker Avance 300
spectrometer operating at 300 MHz (1H) or 75 MHz (13C). Chemical
shifts are in parts per million (ppm) and were referenced to the
residual proton peaks in deuterated solvents. Chemical shifts are
Fig. 9. Superimposition of the crystal structure of hTEAD2217-447 in complex with
compound 6 (PDB codes: 6S6J, in violet), of hTEAD2217-447 (PDB code: 5 EMV) (in
green) and of mTEAD4210-427 in complex with hTAZ24-57 (PDB code: 5GN0, unit D in
tan, unit C in blue).
This fragment symmetrically binds to two molecules of TEAD. It is
located closely to K389 at the third loop of
compounds bind to a pocket created by the flipping of Y382 (first
loop of 3 helix) (Supplement Figure 12). Computational ligand-
a3 helix while our
reported in
d units (ppm) and are assigned as singlets (s), doublets
(d), doublets of doublets (dd), triplets (t), quartets (q), quintets
(quin), sextuplets (sext), multiplets (m), and broad signals (br).
Mass spectra were recorded with an LCMS (Waters Alliance
Micromass ZQ 2000). LCMS analysis was performed using a Waters
a
mapping study allowed Kaan et al. to identify putative cryptic
binding sites in mTEAD4. However, it is difficult for us to know their
exact positions and if this study predicted our cryptic pocket.
As clearly shown before, expression of TEADs by Escherichia coli
afforded a mixture of acylated and non-acylated TEADs. A ligand
which binds to the palmitate pocket of TEAD gives rise to only one
thermal transition while a ligand which binds to the external sur-
face of TEAD affords two thermal transitions. NanoDSF allowed us
to observe two thermal transitions with distinct melting temper-
atures and confirm our compounds bind externally to TEAD YBD.
Affinity constants for e-GFP-TEAD2 in cell lysate were measured
for three of the five compounds which afforded crystal structures.
Measured affinity are in the micromolar range.
XBridge C18 column (5
mm particle size column, dimensions
50 mm ꢁ 4.6 mm). Reported m/z correspond to the most abundant
isotope (35Cl in the case of chlorine). A gradient starting from 98%
H2O/formate buffer 5 mM (pH 3.8) and reaching 100% CH3CN/
formate buffer 5 mM (pH 3.8) within 4 min at a flow rate of 2 mL/
min was used followed by a return to the starting conditions within
1 min. Purity of final tested compounds (5e9 and 11e16) was >95%
(except for 14, purity >94%) as determined by high pressure liquid
chromatography (HPLC) column: C18 Interchrom UPTISPHERE.
Analytical HPLC was performed on a Shimadzu LC-2010AHT system
equipped with a UV detector set at 254 nm and 215 nm. Com-
pounds were dissolved in 50 mL acetonitrile and 950 mL buffer B,
and injected into the system. The following eluent systems were
used: buffer A (H2O/TFA, 100:0.1) and buffer B (CH3CN/H2O/TFA,
80:20:0.1). HPLC retention times (HPLC tR) were obtained at a flow
rate of 0.2 mL/min for 35 min using the following conditions: a
gradient run from 100% of buffer A over 1 min, then to 100% of
buffer B over the next 30 min. Purity of final compounds was >95%
as determined by HPLC (see Supporting Information).
Compound 6 inhibits TEAD-dependent transcriptional activity
in HEK293T cells transfected with the reporter construct (8xGTIIC-
Luciferase) with an IC50 of 4.5
capability to induce a 50% inhibition of proliferation of MDA-MB-
231 cells at 5 M at the same level than in MDA-MB-231 cells.
mM which is comparable to its
m
Compound 6 effectively inhibits expression of proliferation, sur-
vival and anti-apoptotic YAP-TEAD target genes (Cyr61, CTGF, AXL
and Survivin) at protein and mRNA levels in the same cell line.
These molecules could serve with other pan-TEAD inhibitors
such as CPD3.1 [20] which is considered to target interface 3, for the
delineation of the relative importance of VGLL vs YAP/TAZ in a given
cellular model. VGLLs are known to be antagonists of YAP/TAZ-
TEAD complexes but may be tumor suppressor [43,44] or associ-
ated with a poor prognostic [45e48]. For example, VGLL4 is a tumor
suppressor in lung, gastric and colorectal cancers but high VGLL4
correlates with poor clinical outcomes in non-small-cell lung can-
cer [49]. Compound 6 represents a solid structural basis to be
optimized for the development of new therapies for the treatment
of cancer particularly where YAP or TAZ are overexpressed [6].
During the preparation of this article, was reported the design
and characterization of a stabilized protein tertiary structure that
acts as an inhibitor of the interaction between the transcription
factor TEAD and its co-repressor VGLL4 [50]. This eicosapeptide
linked to Tat sequence through a PEG2 linker presents a crosslink
between the acid function of a glutamic residue and the ammo-
nium function of a lysine residue. It binds to mTEAD4 as our
Ethyl 5-(3,4-dichlorophenyl)-1H-pyrazole-4-carboxylate (1).
A stirred solution of ethyl 3-(3,4-dichlorophenyl)-3-oxopropanoate
(2.00 g, 7.66 mmol) and N,N-dimethylformamide dimethyl acetal
(1.00 g, 8.43 mmol) was heated at 90 ꢀC for 3 h. After evaporation of
the excess of N,N-dimethylformamide dimethyl acetal under
reduced pressure, the crude enaminone was dissolved in EtOH
(14 mL) with hydrazine monohydrate (0.38 g, 7.66 mmol) and
heated at 70 ꢀC for 2 h. After concentration under vacuum, the
residue was taken up in EtOAc, washed with water. The organic
phase was dried over MgSO4 and concentrated under vacuum. The
residue was purified by FC (DCM/MeOH 98/2) to afford 1 (1.50 g,
69%) as a white solid (Mp ¼ 136e138 ꢀC). 1H NMR (300 MHz,
CDCl3):
d
1.32 (t, 3H, 3J ¼ 7.1 Hz), 4.30 (q, 2H, 3J ¼ 7.1 Hz), 7.51 (d, 1H,
3J ¼ 8.0 Hz); 7.63 (dd, 1H, 3J ¼ 8.4 Hz, 4J ¼ 2.0 Hz), 7.89 (d, 1H,
4J ¼ 2.0 Hz), 8.17 (s, 1H). 13C NMR (75 MHz, CDCl3):
d 14.2 (CH3), 60.6
(CH2), 111.9 (CIV), 128.5 (CH), 130.0 (CH), 131.0 (CH), 131.9 (CIV), 132.4
(CIV), 132.5 (CIV), 137.7 (CH), 148.3 (CIV), 168.9 (CO). LC-MS (ESI): m/z
Calculated: 284.01, Found: 285.10, [MþH]þ, 283.10, [M ꢂ H]-,
9

M. Sturbaut, F. Bailly, M. Coevoet et al.
European Journal of Medicinal Chemistry 226 (2021) 113835
tR ¼ 2.9 min.
organic layer was dried over MgSO4, concentrated under vacuum
and purified by FC (DCM/MeOH 9/1) to afford the desired alkyl
amine 4e12.
Ethyl 1-(2-aminoethyl)-3-(3,4-dichlorophenyl)-1H-pyrazole-
4-carboxylate (4). Following the general procedure, the compound
4 was isolated by FC (108 mg, 88%); 1H NMR (300 MHz, CDCl3):
N-Benzyl-5-(3,4-dichlorophenyl)-1H-pyrazole-4-
carboxamide (2). A mixture of ethyl 5-(3,4-dichlorophenyl)-1H-
pyrazole-4-carboxylate (2.30 g, 8.07 mmol) and NaOH (3.20 g,
80.70 mmol) in ethanol (90 mL) was stirred at reflux for 16 h. After
concentration under vacuum, the residue was taken up in water
and extracted with DCM. The aqueous layer was acidified with aq.
1.0 M HCl solution and extracted with EtOAc. The organic layer was
dried over MgSO4, concentrated under vacuum and used without
purification in the next step.
d
1.31 (t, 3H, 3J ¼ 7.1 Hz), 3.26 (m, 2H), 4.26 (m, 4H), 7.47 (d, 1H,
3J ¼ 8.4 Hz), 7.70 (dd, 1H, 3J ¼ 8.4 Hz, 4J ¼ 2.0 Hz), 7.95 (d, 1H,
4J ¼ 2.0 Hz), 8.07 (s,1H). 13C NMR (75 MHz, CDCl3):
d 14.3 (CH3), 41.6
(CH2), 55.5 (CH2), 60.4 (CH2),111.9 (CIV),128.6 (CH),129.8 (CH),131.1
(CH), 131.9 (CIV), 132.4 (CIV), 132.5 (CIV), 136.0 (CH), 150.9 (CIV), 162.8
(CO). LC-MS (ESI): m/z Calculated: 327.05, Found: 328.10, [MþH]þ,
tR ¼ 2.4 min.
5-(3,4-Dichlorophenyl)-1H-pyrazole-4-carboxylic acid (1.00 g,
3.89 mmol), EDCI (0.72 g, 4.67 mmol) and HOBt (0.63 g, 4.67 mmol)
were dissolved in DMF (100 mL). After cooling to 5 ꢀC, benzylamine
(1 eq., 0.42 g, 0.42 mL, 3.89 mmol) was added to the mixture and
the solution was stirred overnight at room temperature. After
concentration under vacuum, the residue was taken up in EtOAc
and washed with water. The organic layer was dried over MgSO4,
concentrated under vacuum and purified by FC with cyclohexane/
EtOAc (10:0 / 50:50, v/v) to afford 2 (0.77 g, 57%) as an orange
Ethyl 1-(2-aminopropyl)-3-(3,4-dichlorophenyl)-1H-pyrazole-
4-carboxylate (5). Following the general procedure, the compound
5 was isolated by FC (107 mg, 83%); 1H NMR (300 MHz, DMSO‑d6):
d
1.23 (t, 3H, 3J ¼ 7.1 Hz), 2.14 (quint, 2H, 3J ¼ 6.9 Hz), 2.77 (t, 2H,
3J ¼ 7.0 Hz), 4.19 (q, 3J ¼ 7.2 Hz, 2H), 4.32 (t, 2H, 3J ¼ 6.9 Hz), 7.68 (d,
1H, 3J ¼ 8.4 Hz), 7.76 (dd, 1H, 3J ¼ 8.4 Hz, 4J ¼ 1.8 Hz), 8.03 (d, 1H,
4J ¼ 1.8 Hz), 8.10 (brs, 3H) 8.52 (s, 1H). 13C NMR (75 MHz, DMSO‑d6):
powder (Mp ¼ 174e176 ꢀC). 1H NMR (300 MHz, DMSO‑d6):
d 4.40
(d, 2H, 3J ¼ 5.8 Hz), 7.30 (m, 5H), 7.61 (d,1H, 3J ¼ 8.4 Hz), 7.70 (s, 1H),
7.80 (dd,1H, 3J ¼ 8.4 Hz, 4J ¼ 1.8 Hz), 8.09 (d,1H, 4J ¼ 1.8 Hz), 8.29 (d,
1H, 3J ¼ 1.1 Hz), 8.65 (t, 1H, 3J ¼ 5.8 Hz). 13C NMR (75 MHz,
d 14.6 (CH3), 27.9 (CH2), 36.6 (CH2), 49.3 (CH2), 60.4 (CH2), 111.2
(CIV), 129.5 (CH), 130.6 (CH), 131.01 (CH), 131.04 (CIV), 131.4 (CIV),
133.3 (CIV), 137.4 (CH), 149.5 (CIV), 162.7 (CO). LC-MS (ESI): m/z
Calculated: 341.07, Found: 342.10, [MþH]þ, tR ¼ 2.5 min.
DMSO‑d6): d 42.7 (CH2), 115.3 (CIV), 127.2 (CH), 127.7 (2 CH), 128.7 (2
CH), 128.9 (CH), 130.4 (CH), 130.5 (CH), 130.9 (2 CIV), 132.0 (CH),
134.5 (CIV), 140.1 (CIV), 147.8 (CIV), 163.5 (CO). LC-MS (ESI): m/z
Calculated: 345.05, Found: 346.10, [MþH]þ, 344.20, [M ꢂ H]-,
tR ¼ 2.7 min.
Ethyl 1-(4-aminobutyl)-3-(3,4-dichlorophenyl)-1H-pyrazole-4-
carboxylate (6). Following the general procedure, the compound
6 was isolated by FC (196 mg, 73%); 1H NMR (300 MHz, DMSO‑d6):
d
1.23 (t, 3H, 3J ¼ 7.2 Hz),1.56 (quint, 2H, 3J ¼ 6.9 Hz),1.86 (quint, 2H,
5-(3,4-Dichlorophenyl)-N-phenethyl-1H-pyrazole-4-
3J ¼ 6.9 Hz), 2.79 (t, 2H, 3J ¼ 7.2 Hz), 4.20 (t, 3J ¼ 7.0 Hz, 2H), 4.22 (q,
carboxamide
(3).
5-(3,4-dichlorophenyl)-1H-pyrazole-4-
3J ¼ 7.2 Hz, 2H), 7.67 (d, 1H, 3J ¼ 8.4 Hz), 7.76 (dd, 1H, 3J ¼ 8.4 Hz,
carboxylic acid (1 eq., 1.30 g, 5.05 mmol), EDCI (1.2 eq., 0.94 g,
6.07 mmol) and HOBt (1.2 eq., 0.92 g, 6.07 mmol) were dissolved in
DMF (125 mL). After cooling to 5 ꢀC, phenethylamine (1 eq., 0.61 g,
0.64 mL, 5.05 mmol) was added and the mixture and was stirred
overnight. After concentration under vacuum, the residue was
taken up in EtOAc and washed with water. The organic layer was
dried over MgSO4, concentrated under vacuum and purified by FC
with cyclohexane/EtOAc (10:0 / 50:50, v/v) to afford 3 (1.54 g,
85%), as yellow solid (Mp ¼ 201e202 ꢀC). 1H NMR (300 MHz,
4J ¼ 1.8 Hz), 8.02 (brs, 3H), 8.03 (d, 1H, 4J ¼ 1.8 Hz), 8.49 (s, 1H). 13
C
NMR (75 MHz, DMSO‑d6):
d 14.6 (CH3), 24.5 (CH2), 26.8 (CH2), 38.6
(CH2), 51.6 (CH2), 60.3 (CH2), 111.1 (CIV), 129.5 (CH), 130.5 (CH), 131.0
(CH), 131.3 (CIV), 133.4 (CIV), 137.2 (CIV), 137.4 (CH), 119.3 (CIV), 162.8
(CO). LC-MS (ESI): m/z Calculated: 355.09, Found: 356.10, [MþH]þ,
tR ¼ 2.5 min.
1-(2-Aminoethyl)-N-benzyl-3-(3,4-dichlorophenyl)-1H-pyr-
azole-4-carboxamide (7). Following the general procedure, the
compound 7 was isolated by FC (70 mg, 27%); 1H NMR (300 MHz,
DMSO‑d6):
d
2.82 (t, 2H, 3J ¼ 7.4 Hz), 3.44 (t, 2H, 3J ¼ 7.4 Hz),
(CD3)2CO):
d
3.63 (t, 2H, 3J ¼ 6.1 Hz), 4.38 (t, 2H, 3J ¼ 6.1 Hz), 4.51 (d,
7.20e7.32 (m, 5H), 7.62 (d, 1H, 3J ¼ 8.4 Hz), 7.78 (d, 1H, 4J ¼ 1.2 Hz),
2H, 3J ¼ 6.0 Hz), 7.28 (m, 5H), 7.52 (d, 1H, 3J ¼ 8.4 Hz), 7.75 (brs, 1H),
8.10 (s, 1H) 8.20 (dd, 1H), 13.34 (brs, 1H). 13C NMR (75 MHz,
7.86 (dd, 1H, 3J ¼ 8.4 Hz, 4J ¼ 2.0 Hz), 8.17 (s, 1H), 8.18 (d, 1H,
DMSO‑d6):
d
36.05 (CH2), 41.2 (CH2), 116.0 (CIV), 126.9 (CH), 129.1
4J ¼ 2.0 Hz). 13C NMR (75 MHz, (CD3)2CO):
d 42.6 (CH2), 50.6 (CH2),
(CIV), 129.3 (CIV), 129.5 (CH), 130.8 (2 CH), 130.9 (CIV), 130.9 (2 CH),
130.9 (CIV), 132.2 (CH), 134.9 (CH), 140.3 (CH), 148.0 (CIV), 165.9
(CO); LC-MS (ESI): m/z Calculated: 359.09, Found: 360.16, [MþH]þ,
358.27, [M ꢂ H]þ, tR ¼ 2.8 min.
53.4 (CH2), 126.8 (CH), 127.5 (2 CH), 128.3 (2 CH), 128.5 (CH), 129.8
(CH), 130.4 (CH), 130.8 (CIV), 131.1 (CIV), 133.2 (CH), 133.3 (CIV), 134.2
(CIV), 139.7 (CIV), 147.6 (CIV), 162.8 (CO). LC-MS (ESI): m/z Calculated:
388.08, Found: 389.20, [MþH]þ, tR ¼ 2.5 min.
General procedure for the synthesis of ethyl 1-(
u
-amino-
1-(3-Aminopropyl)-N-benzyl-3-(3,4-dichlorophenyl)-1H-
pyrazole-4-carboxamide (8). Following the general procedure, the
compound 8 was isolated by FC (220 mg, 97%); 1H NMR (300 MHz,
alkyl)-3-(3,4-dichlorophenyl)-1H-pyrazole-4-carboxylates or 1-
-aminoalkyl)-N-benzyl-3-(3,4-dichlorophenyl)-1H-pyrazole-
(
u
4-carboxamides. A solution of N-benzyl-5-(3,4-dichlorophenyl)-
1H-pyrazole-4-carboxamide (1 eq., 0.30 g, 0.87 mmol), K2CO3 (2
eq., 0.24 g, 1.73 mmol) and the convenient N-(u-bromoalkyl)
CD2Cl2):
d
2.00 (m, 2H), 2.72 (t, 2H, 3J ¼ 6.6 Hz), 4.26 (t, 2H,
3J ¼ 6.9 Hz), 4.51 (d, 2H, 3J ¼ 5.8 Hz), 6.02 (t, 1H, 3J ¼ 5.8 Hz),
7.25e7.93 (m, 5H), 7.45 (d, 1H, 3J ¼ 8.3 Hz), 7.59 (dd, 1H, 3J ¼ 8.3 Hz,
4J ¼ 2.0 Hz), 7.89 (d, 1H, 4J ¼ 2.0 Hz), 7.90 (s, 1H). 13C NMR (75 MHz,
phthalimide (1 eq., 0.87 mmol) in anhydrous acetonitrile (15 mL)
and under a nitrogen atmosphere was stirred at reflux overnight
and concentrated under reduced pressure. The residue was taken
up in EtOAc and washed with water. The organic layer was dried
over MgSO4, concentrated under vacuum and purified by FC (DCM/
MeOH 98/2).
Hydrazine monohydrate (10 eq., 0.33 g, 0.32 mL, 6.71 mmol) and
N-benzyl or phenethyl-5-(3,4-dichlorophenyl)-1-[2-(1,3-dioxo-
2,3,3a,7a-tetrahydro-1H-isoindol-2-yl)ethyl]-1H-pyrazole-4-
carboxamide (1 eq., 0.67 mmol) were dissolved in EtOH (6 mL). The
mixture was stirred at reflux for 3 h then concentrated under
reduced pressure, taken up in EtOAc and washed with water. The
CD2Cl2): d 33.3 (CH2), 38.7 (CH2), 43.5 (CH2), 50.0 (CH2), 115.8 (CIV),
127.4 (CH), 127.6 (2 CH), 128.2 (CH), 128.6 (2 CH), 130.3 (CH), 130.5
(CH), 132.2 (CIV), 132.3 (CIV), 132.6 (CH), 133.0 (CIV), 138.4 (CIV), 147.4
(CIV), 162.7 (CO). LC-MS (ESI): m/z Calculated: 402.10, Found:
403.20, [MþH]þ, tR ¼ 2.5 min.
1-(4-Aminobutyl)-N-benzyl-3-(3,4-dichlorophenyl)-1H-pyr-
azole-4-carboxamide (9). Following the general procedure, the
compound 9 was isolated by FC (45 mg, 66%); 1H NMR (300 MHz,
(CD3)2CO):
d
1.63 (quint, 2H), 1.98 (m, 2H), 3.25 (t, 2H, 3J ¼ 6.7 Hz),
4.25 (t, 2H, 3J ¼ 7.0 Hz), 4.52 (d, 2H), 7.15e7.42 (m, 5H), 7.54 (d, 1H,
3J ¼ 8.4 Hz), 7.93 (dd, 1H, 3J ¼ 8.4 Hz, 4J ¼ 2.0 Hz), 8.23 (d, 2H), 8.25
10

M. Sturbaut, F. Bailly, M. Coevoet et al.
European Journal of Medicinal Chemistry 226 (2021) 113835
(s, 1H). 13C NMR (75 MHz, DMSO‑d6):
d
26.8 (CH2), 27.4 (CH2), 38.8
1-yl)propanoic acid (13). Following the general procedure, the
ester was obtained in a 52% yield and immediately converted into
its acid. Compound 13 was isolated (26 mg, 43%); 1H NMR
(CH2), 40.0 (CH2), 51.4 (CH2), 115.2 (CIV), 126.7 (CH), 127.3 (2 CH),
128.3 (CIV), 128.4 (2 CH), 128.5 (CIV), 129.9 (CH), 130.0 (CH), 130.5
(CH), 133.1 (CH), 133.7 (CIV), 139.6 (CIV), 147.0 (CIV), 162.7 (CO). LC-
MS (ESI): m/z Calculated: 416.12, Found: 417.30, [MþH]þ,
tR ¼ 2.5 min.
(300 MHz, DMSO‑d6):
d
2.85 (t, 2H, J ¼ 6.6 Hz), 4.37 (m, 4H), 7.28 (m,
5H), 7.61 (d, 1H, 3J ¼ 8.4 Hz), 7.76 (dd, 1H, 3J ¼ 8.4 Hz, 4J ¼ 2.0 Hz),
8.04 (d, 1H, 4J ¼ 2.0 Hz), 8.28 (s, 1H), 8.66 (t, 1H, 3J ¼ 5.7 Hz, NH),
1-(2-Aminoethyl)-3-(3,4-dichlorophenyl)-N-phenethyl-1H-
pyrazole-4-carboxamide (10). Following the general procedure,
the compound 10 was isolated by FC (9 mg, 22%); 1H NMR
12.41 (brs, 1H). 13C NMR (75 MHz, DMSO‑d6):
d 34.6 (CH2), 42.7
(CH2), 48.2 (CH2), 115.6 (CIV), 127.2 (CH), 127.7 (2 CH), 128.8 (2 CH),
128.9 (CH), 130.4 (CH), 130.5 (CH), 130.7 (CIV), 130.9 (CIV), 131.0 (CIV),
133.7 (CH), 134.1 (CIV), 147.6 (CIV), 160.0 (CO), 172.7 (CO); LC-MS
(ESI): m/z Calculated: 417.27, Found: 418.30, [MþH]þ, tR ¼ 2.5 min.
4-(4-(Benzylcarbamoyl)-3-(3,4-dichlorophenyl)-1H-pyrazol-
1-yl)butanoic acid (14). Following the general procedure, the ester
was obtained in a 23% yield and immediately converted into its
acid. Compound 14 was isolated (57 mg, 91%); 1H NMR (300 MHz,
(300 MHz, DMSO‑d6):
d
2.81 (t, 2H, 3J ¼ 6.9 Hz), 3.09 (t, 2H,
3J ¼ 6.1 Hz), 3.42 (t, 2H, 3J ¼ 6.5 Hz), 4.16 (t, 2H, 3J ¼ 6.1 Hz),
7.20e7.32 (m, 5H), 7.61 (d, 1H, 3J ¼ 8.4 Hz), 7.73 (dd, 1H, 3J ¼ 8.4 Hz,
4J ¼ 2.0 Hz), 8.08 (s, 1H), 8.16 (d, 1H, 4J ¼ 2.0 Hz), 8.23 (t, 1H). 13
C
NMR (75 MHz, DMSO‑d6):
d 35.6 (CH2), 40.7 (CH2), 41.7 (CH2),
54.9(CH2), 115.9 (CIV), 126.6 (CH), 127.7 (2 CH), 128.8 (2 CH), 129.1
(CH), 130.3 (CH), 130.5 (CH), 130.6 (CIV), 131.0 (CIV), 133.0 (CH), 134.1
(CIV), 139.9 (CIV), 147.4 (CIV), 163.1 (CO). LC-MS (ESI): m/z Calculated:
402.10, Found: 403.20, [MþH]þ, tR ¼ 2.6 min.
DMSO‑d6):
d
2.03 (m, 2H), 2.26 (t, 2H, 3J ¼ 7.1 Hz), 4.18 (t, 2H,
3J ¼ 6.9 Hz), 4.39 (d, 2H, 3J ¼ 5.9 Hz), 7.30 (m, 5H), 7.61 (d, 1H,
3J ¼ 8.3 Hz), 7.77 (dd, 1H, 3J ¼ 8.4 Hz, 4J ¼ 2.0 Hz), 8.06 (d, 1H,
4J ¼ 2.0 Hz), 8.27 (s, 1H), 8.66 (t, 1H, 3J ¼ 5.9 Hz, NH), 12.19 (brs, 1H,
1-(3-Aminopropyl)-3-(3,4-dichlorophenyl)-N-phenethyl-1H-
pyrazole-4-carboxamide (11). Following the general procedure,
the compound 11 was isolated by FC (240 mg, 63%); 1H NMR
OH). 13C NMR (75 MHz, DMSO‑d6):
d 25.5 (CH2), 30.9 (CH2), 42.7
(CH2), 51.4 (CH2), 115.7 (CIV) 127.2 (CH), 127.7 (2 CH), 128.7 (2 CH),
128.9 (CH),130.4 (CH),130.5 (CH),130.7 (CIV),130.9 (CIV),133.5 (CH),
134.1 (CIV), 140.0 (CIV), 147.5 (CIV), 163.1 (CO), 174.2 (CO); LC-MS
(ESI): m/z Calculated: 431.30, Found: 432.20, [MþH]þ, tR ¼ 2.8 min.
3-(3-(3,4-Dichlorophenyl)-4-(phenethylcarbamoyl)-1H-pyr-
azol-1-yl)propanoic acid (15). Following the general procedure,
the ester was obtained in a 52% yield and immediately converted
into its acid. Compound 15 was isolated (40 mg, 64%); 1H NMR
(300 MHz, DMSO‑d6):
d
1.90 (m, 2H); 2.81 (t, 2H, 3J ¼ 7.0 Hz), 3.19
(brs, 2H), 3.42 (m, 2H), 4.22 (t, 2H, 3J ¼ 7.0 Hz), 7.19e7.26 (m, 5H),
7.30 (d,1H, 3J ¼ 8.4 Hz), 7.72 (dd,1H, 3J ¼ 8.4 Hz, 4J ¼ 2.0 Hz); 8.05 (s,
1H), 8.17 (d, 1H, 4J ¼ 2.0 Hz), 8.25 (brs, 1H). 13C NMR (75 MHz,
DMSO‑d6):
d 26.5 (CH2), 35.6 (CH2), 40.6 (CH2), 47.4 (CH2), 49.9
(CH2), 115.9 (CIV), 126.6 (CH), 128.7 (2 CH), 128.8 (CH), 129.1 (2 CH),
130.2 (CH), 130.5 (CH), 130.6 (CIV), 131.0 (CIV), 133.2 (CH), 134.2 (CIV),
139.9 (CIV), 147.2 (CIV), 163.1 (CO); LC-MS (ESI): m/z Calculated:
416.12, Found: 417.10, [MþH]þ, tR ¼ 2.4 min.
(300 MHz, DMSO‑d6):
d
2.68 (t, 2H, 3J ¼ 6.9 Hz), 2.80 (t, 2H,
3J ¼ 7.0 Hz), 3.39 (q, 2H, 3J ¼ 6.9 Hz), 4.30 (t, 2H, 3J ¼ 6.7 Hz),
7.14e7.32 (m, 5H), 7.60 (d, 1H, 3J ¼ 8.4 Hz), 7.71 (dd, 1H, 3J ¼ 8.4 Hz,
4J ¼ 2.0 Hz), 8.02 (d, 1H, 3J ¼ 2.0 Hz), 8.16 (s, 1H), 8.21 (t, 1H,
1-(4-Aminobutyl)-3-(3,4-dichlorophenyl)-N-phenethyl-1H-
pyrazole-4-carboxamide (12). Following the general procedure,
the compound 12 was isolated by FC (13 mg, 72%); 1H NMR
3J ¼ 5.7 Hz, NH). 13C NMR (75 MHz, DMSO‑d6):
d 34.3 (CH2), 35.6
(300 MHz, DMSO‑d6):
d
1.56 (quint, 2H, 3J ¼ 7.0 Hz), 1.88 (m, 2H),
(CH2), 49.2 (CH2), 62.2 (CH2), 116.1 (CIV), 126.5 (CH), 128.0 (CH),
128.8 (2 CH), 129.1 (2 CH), 130.3 (CH), 130.5 (CH), 130.9 (CIV), 132.3
(CIV), 132.8 (CIV), 133.4 (CH), 139.9 (CIV), 146.0 (CIV), 163.0 (CO), 170.2
(CO); LC-MS (ESI): m/z Calculated: 431.30, Found: 432.20, [MþH]þ,
tR ¼ 2.8 min.
2.82 (m, 2H), 3.43 (m, 4H), 4.20 (t, 2H, 3J ¼ 7.0 Hz), 7.11e7.31 (m,
5H), 7.63 (d, 1H, 3J ¼ 8.5 Hz), 7.74 (dd, 1H, 3J ¼ 8.5 Hz, 4J ¼ 2.0 Hz),
7.92 (brs, 1H), 8.06 (d, 1H, 4J ¼ 2.0 Hz), 8.23 (s, 1H), 8.33 (t, 1H). 13
C
NMR (75 MHz, DMSO‑d6):
d 24.2(CH2), 26.5 (CH2), 35.2 (CH2), 40.6
(CH2), 42.4 (CH2), 51.1 (CH2), 115.6 (CIV), 126.1 (CH), 128.3 (2 CH),
128.6 (2 CH), 129.7 (CIV), 130.1 (CH), 130.3 (CH), 130.6 (CH), 130.65
(CIV), 132.9 (CH), 133.6 (CIV), 139.4 (CIV), 146.9 (CIV), 162.6 (CO). LC-
MS (ESI): m/z Calculated: 430.13, Found: 431.20, [MþH]þ,
tR ¼ 3.3 min.
4-(3-(3,4-Dichlorophenyl)-4-(phenethylcarbamoyl)-1H-pyr-
azol-1-yl)butanoic acid (16). Following the general procedure, the
ester was obtained in a 52% yield and immediately converted into
its acid. Compound 16 was isolated (36 mg, 56%); 1H NMR
(300 MHz, (CD3)2CO):
d
2.02 (m, 2H), 2.24 (t, 2H, 3J ¼ 6.8 Hz), 2.77 (t,
General procedure for the synthesis of [4-(benzylcarbamoyl)-3-
(3,4-dichlorophenyl)-1H-pyrazol-1-yl]alkanoic acids.
2H, 3J ¼ 7.1 Hz), 3.44 (m, 2H), 4.07 (t, 2H, 3J ¼ 7.0 Hz), 7.08 (brs, 1H),
7.12e7.30 (m, 5H), 7.39 (dd, 1H, 3J ¼ 8.2 Hz, 4J ¼ 2.0 Hz), 7,65 (d, 1H,
3J ¼ 8.2 Hz), 7.67 (d, 1H, 4J ¼ 2.0 Hz), 7.88 (s, 1H). 13C NMR (75 MHz,
Ethyl [4-(benzylcarbamoyl)-3-(3,4-dichlorophenyl)-1H-pyr-
azol-1-yl]alkanoates. A solution of N-benzyl or phenethyl-5-(3,4-
dichlorophenyl)-1H-pyrazole-4-carboxamide (1 eq., 0.30 g,
0.87 mmol), K2CO3 (2 eq., 0.24 g, 1.73 mmol) and the convenient
(CD3)2CO):
d 24.9 (CH2), 35.6 (CH2), 40.3 (CH2), 40.4 (CH2), 48.3
(CH2), 116.9 (CIV), 126.1 (CH), 128.3 (2 CH), 128.6 (2 CH), 130.2 (CIV),
130.3 (CH), 130.4 (CH), 131.7 (CIV), 132.4 (CH), 132.6 (CIV), 138.1 (CH),
139.6 (CIV), 141.0 (CIV), 161.8 (CO), 172.9 (CO). LC-MS (ESI): m/z
Calculated: 445.10, Found: 446.20, [MþH]þ, tR ¼ 2.7 min.
Protein expression and purification. The human TEAD2
sequence (residue 217 to 447) was expressed and purified accord-
ing to Ref. [34].
Crystallization and structure determination. Crystals of
hTEAD2217e447 were grown at 20 ꢀC using the hanging-drop vapor-
diffusion method with a reservoir solution containing 0.1 M HEPES
(pH 7.2) and 2.8 M sodium formate. The crystals were cryo-
protected with reservoir solution supplemented with 25% glyc-
erol and then flash-cooled in liquid nitrogen. X-ray diffraction data
were collected at the ALBA Synchrotron in Barcelona, Spain, on
beamline BL13-XALOC. Data were integrated and processed using
XDS [51]. The crystals belong to the space group C2 with two
monomers in the asymmetric unit. The structures were solved by
molecular replacement using PDB entry 5EMV as the search model.
ethyl
u-bromoalkylcarboxylate (1 eq., 0.87 mmol) in anhydrous
acetonitrile (15 mL) and under a nitrogen atmosphere was stirred at
reflux overnight and concentrated under reduced pressure. The
residue was taken up in EtOAc and washed with water. The organic
layer was dried over MgSO4, concentrated under vacuum and pu-
rified by FC (DCM/MeOH 98/2).
[4-(Benzylcarbamoyl)-3-(3,4-dichlorophenyl)-1H-pyrazol-1-
yl]alkanoic acids. A solution of ethyl [4-(benzylcarbamoyl)-3-(3,4-
dichlorophenyl)-1H-pyrazol-1-yl]alkanoate (1 eq., 0.15 mmol) and
NaOH (10 eq., 1.52 mmol) in EtOH (2 mL) was stirred at reflux for
16 h and concentrated under reduced pressure. The residue was
taken up in water and extracted with DCM. The aqueous layer was
acidified with aq. 1.0 M HCl solution and extracted with EtOAc. The
organic phase was dried over magnesium sulfate and concentrated
under vacuum to afford 13e16.
3-(4-(Benzylcarbamoyl)-3-(3,4-dichlorophenyl)-1H-pyrazol-
11

M. Sturbaut, F. Bailly, M. Coevoet et al.
European Journal of Medicinal Chemistry 226 (2021) 113835
Bound ligands were manually identified and fitted into FoeFc
electron density using Coot [52]. Files CIF format for ligand were
generated using Grade Server (http://grade.globalphasing.org/cgi-
bin/grade/server.cgi). The structures were refined by rounds of
rebuilding in Coot and refinement using Phenix [53]. Data collec-
tion and refinement statistics for crystal structures are presented in
Supplement Tables 1 and 2
NanoDSF Assay. NanoDSF assay were conducted according to
Ref. [34].
Microscale Thermophoresis. Microscale Thermophoresis ex-
periments were conducted according to Ref. [33].
PCR amplicons were run in 3% w/v agarose gel electrophoresis and
visualized by ethidium bromide staining. The primers for AXL were
designed by using Oligo 7 (version 7.60) and sequence specificity
checked using-BLAST software, the primers for CTGF were taken
from Nagaraja et al. [54] and Cyr61 from Chen et al. [55]. They were
manufactured by Eurogentec. Primer sequences are listed below.
The relative expression value of each target gene (AXL, CTGF and
DDCT
Cyr61) was utilizing the 2ꢂ
method using MAN2B1 for
normalization. All experiments were performed at least in
triplicates.
Primers
Forward
Reverse
AXL
CTGF
Cyr61
50-GGAGCCCAACAACTTCTGAGG-30
50-AATGCTGCGAGGAGTGGGT-30
50-GAGTGGGTCTGTGACGAGGAT-30
50-GGACTTTCTTCAGCCTGCGTG-30
50-GGCTCTAATCATAGTTGGGTCT-30
50-GGTTGTATAGGATGCGAGGCT-30
Cell Cultures. Cell cultures were made according to [33].
Luciferase Reporter Assay. Luciferase reporter assay was made
as described in Ref. [34].
Author contributions
M.S. took part in all the described experiments; F.B. supervised
the chemical synthesis; M.C. performed most of the biological as-
says; P. S. performed RT-qPCR measurements; M.P. performed
NanoDSF experiments; M.L., R.M. and X.T. helped at the expression
of GFP-TEAD2 in CHO cells; M.G. and F.A. produced and purified
hTEAD2217e447 protein and performed crystallization and structure
determination; J.-F.G. supervised all the X-ray crystallography,
NanoDSF studies; J.-F.G., P.M., and P.C. co-supervised all the project;
P.C. supervised writing the article.
4.1. Western blotting
The MDA-MB-231 cell line was cultivated in DMEM media
containing 0.2% heat-inactivated fetal bovine serum (FBS), L-
glutamine (2 mM), and penicillin/streptomycin (100 U mLꢂ1
/
0.1 mg mLꢂ1). Total extracts of cells were obtained with a RIPA
based buffer containing protease and phosphatase inhibitors
(Roche). Western blots were carried out using 20 mg of protein
lysates with the NuPage Electrophoresis and Iblot transfer systems
(Life Technologies). GADPH was used as loading control for total
extracts.
Notes
The authors declare no competing financial interest.
Accession numbers
4.2. Kinetic cell growth assay
The atomic coordinates and structure factors for hTEAD2-6,
hTEAD2-7, hTEAD2-13, hTEAD2-14, and hTEAD2-15 have been
deposited in the PDB with the codes PDB: 6S6J, 6S66, 6S64, 6S60
and 5S69, respectively. Authors will release the atomic coordinates
and experimental data upon article publication.
The effect of 6 on MDA-MB-231 cell growth was studied using a
kinetic cell growth assay. MDA-MB-231 cells were plated on 96-
well TPP plates in triplicate at low densities (2.5 ꢁ 103 cells/well)
in low serum conditions (0.2% SVF). 6 at different concentrations
was added 24 h after plating and cell number was monitored with
Incucyte Live-Cell imaging System and software (Essen In-
struments). Cell number was observed every 3 h for 72 h. The assay
was performed in independent triplicates.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
4.3. RNA extraction and quantitative RT-PCR
After the treatment of the MDA cells with DMSO (negative
control), Dasatinib (positive control) and compound 6 (tested
drug), total RNA was purified using NucleoSpin RNA followed by
NucleoSpin RNA Clean-up XS2 step (Macherey-Nagel). The integrity
of the extracted RNA was tested by using 1% w/v agarose gel elec-
trophoresis visualized by ethidium bromide staining. 250 ng RNA
was reverse-transcribed into cDNA using High-Capacity cDNA
Reverse Transcription Kit (Life Technologies) in accordance to the
manufacturer's instructions. Quantitative RT-PCR was executed
Acknowledgments
ꢂ
This work was financially supported by grants from le Ministere
de l’Education et de la Recherche and the University of Lille (M. S.
and P.S.) and fundings from le Canceropole Nord-Ouest (Mobility
Support Program for Young Researchers, M. S.). We express our
thanks to the NMR facility (Lille University), the Region Hauts-de-
ꢀ
^
ꢂ
France (France), the Ministere de la Jeunesse, de l’Education
Nationale et de la Recherche (MJENR) and the Fonds Europeens de
ꢀ
using PowerUP SYBR Green (Thermo Fisher Scientific) 1
mL of the
ꢀ
ꢀ
reverse-transcript was added to a 10
m
L PCR mixture for 40 cycles.
Developpement Regional (FEDER) and the Lille University Molec-
ular Interactions facility. This work was also supported by the
French Infrastructure for Integrated Structural Biology (FRISBI)
ANR-10-INSB-05-01. These experiments were performed at BL13-
Each cycle included 95 ꢀC for 15 s, 10 cycles of Touch Down PCR
from 70 ꢀC to 60 ꢀC for 15 s, and 72 ꢀC for 30 s, followed by 30 cycles
of 60 ꢀC for 15 s and 72 ꢀC for 30 s, to conclude with 5 min at 72 ꢀC.
12

M. Sturbaut, F. Bailly, M. Coevoet et al.
European Journal of Medicinal Chemistry 226 (2021) 113835
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SUPPLEMENTAL INFORMATION
Supplemental Information can be found with this article online
at.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113835.
ABBREVIATIONS
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CTGF
Cyr61
connective tissue growth factor
cysteine-rich angiogenic protein
DMF-DMA N,N-dimethylformamide dimethylacetal
nano-DSF differential scanning fluorimetry
EDCI
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride;
[17] Z. Zhang, Z. Lin, Z. Zhou, H.C. Shen, S.F. Yan, A.V. Mayweg, Z. Xu, N. Qin,
J.C. Wong, Z. Zhang, Y. Rong, D.C. Fry, T. Hu, Structure-based design and
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interaction, ACS Med. Chem. Lett. 5 (2014) 993e998, https://doi.org/10.1021/
ml500160m.
HOBt
Lats1/2
Mob
1-hydroxybenzotriazole
large tumor suppressor kinase
mps one binder kinase
MST
microscale thermophoresis
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W. Wang, X. Wang, T. Guo, P. Li, Y. Zhao, H. Ji, L. Zhang, Z. Zhou, A peptide
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Mst1/2
Nif Ac
Sav1
TAZ
TBD
TEAD
TSA
VGLL
YAP
Y/TBD
mammalian ste20-like protein kinase
niflumic acid
scaffold protein Salvador
transcriptional coactivator with PDZ-binding motif
TEAD binding domain
transcriptional enhanced associated domain
thermal shift assay
(Transcriptional cofactor Vestigial like protein family)
Yes associated protein
YAP/TAZ binding domain
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H.E. Shannon, B.J. Bailey, T.W. Corson, K.E. Pollok, C.D. Wells, S.O. Meroueh,
Small-molecule covalent modification of conserved cysteine leads to allosteric
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