Discovery and biological evaluation of vinylsulfonamide derivatives as highly potent, covalent TEAD autopalmitoylation inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.111767

Transcriptional enhancer associated domain family members (TEADs) are the most important downstream effectors that play the pivotal role in the development, regeneration and tissue homeostasis. Recent biochemical studies have demonstrated that TEADs could undergo autopalmitoylation that is indispensable for its function making the lipid-binding pocket an attractive target for chemical intervention. Herein, through structure-based virtual screen and rational medicinal chemistry optimization, we identified DC-TEADin02 as the most potent, selective, covalent TEAD autopalmitoylation inhibitor with the IC₅₀ value of 197 ± 19 nM while it showed minimal effect on TEAD-YAP interaction. Further biochemical counter-screens demonstrate the specific thiol reactivity and selectivity of DC-TEADin02 over the kinase family, lipid-binding proteins and epigenetic targets. Notably, DC-TEADin02 inhibited TEADs transcription activity leading to downregulation of YAP-related downstream gene expression. Taken together, our findings proved the validity of modulating transcriptional output in the Hippo signaling pathway through irreversible chemical interventions of TEADs autopalmitoylation activity, which may serve as a qualified chemical tool for TEADs palmitoylation-related studies in the future.

Full text of DOI:10.1016/j.ejmech.2019.111767

Journal Pre-proof
Discovery and biological evaluation of vinylsulfonamide derivatives as highly potent,
covalent TEAD autopalmitoylation inhibitors
Wenchao Lu, Jun Wang, Yong Li, Hongru Tao, Huan Xiong, Fulin Lian, Jing Gao,
Hongna Ma, Tian Lu, Dan Zhang, Xiaoqing Ye, Hong Ding, Liyan Yue, Yuanyuan
Zhang, Huanyu Tang, Naixia Zhang, Yaxi Yang, Hualiang Jiang, Kaixian Chen, Bing
Zhou, Cheng Luo
PII:
S0223-5234(19)30919-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.111767
EJMECH 111767
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 August 2019
Revised Date: 24 September 2019
Accepted Date: 6 October 2019
Please cite this article as: W. Lu, J. Wang, Y. Li, H. Tao, H. Xiong, F. Lian, J. Gao, H. Ma, T. Lu, D.
Zhang, X. Ye, H. Ding, L. Yue, Y. Zhang, H. Tang, N. Zhang, Y. Yang, H. Jiang, K. Chen, B. Zhou,
C. Luo, Discovery and biological evaluation of vinylsulfonamide derivatives as highly potent, covalent
TEAD autopalmitoylation inhibitors, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.111767.
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Discovery and Biological Evaluation of Vinylsulfonamide Derivatives as
Highly Potent, Covalent TEAD Autopalmitoylation Inhibitors
a,b,l,#
a,d,#
b,c,#
a,e,#
c
f
Wenchao Lu
, Jun Wang , Yong Li , Hongru Tao , Huan Xiong , Fulin Lian ,
g
a,h
a,d
a,i
a,j
a
Jing Gao , Hongna Ma , Tian Lu , Dan Zhang , Xiaoqing Ye , Hong Ding , Liyan
a
a
b,c
f
b,c
Yue , Yuanyuan Zhang , Huanyu Tang , Naixia Zhang , Yaxi Yang , Hualiang
a,b
a,b,k
b,c,*
a,b,k,*
Jiang , Kaixian Chen , Bing Zhou , Cheng Luo
a
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
b
University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China
c
Department of Medicinal Chemistry, State Key Laboratory of Drug Research, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203,
China
d
Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University
of Chinese Medicine, 138 Xianlin Road, Nanjing 210023, China
e
Department of Chemistry, College of Sciences, Shanghai University, 99 Shangda Road,
Shanghai 200444, China
f
Department of Analytical Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
g
Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai
2
01203, China
h
Department of Pharmacy, Guiyang University of Traditional Chinese Medicine, South Dong
Qing Road, Guizhou 550025, China
i
j
Key Laboratory of Guizhou for Fermentation Engineering and Biomedicine, School of
Pharmaceutical Sciences, Guizhou University, Guizhou 550025, China
College of Life Sciences, Zhejiang Sci-Tech University, 928 No.2 Street, Hangzhou 310018,
China

k
Open Studio for Druggability Research of Marine Natural Products, Pilot National Laboratory
for Marine Science and Technology (Qingdao), 1 Wenhai Road, Aoshanwei, Jimo, Qingdao
2
66237, China
l Now in the Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02215,
#
USA These authors contributed equally.
Highlights:
1
2
3
4
5
. DC-TEADin02 is the most potent TEAD autopalmitoylation inhibitor ever reported.
. DC-TEADin02 inhibits TEADs transcription activity and downstream gene expression.
. DC-TEADin02 shows minimal reactivity with biologically relevant nucleophiles.
. DC-TEADin02 is selective over other lipid-binding proteins
. The study demonstrates how virtual screen cooperating with rational optimization.

ABSTRACT:
Transcriptional enhancer associated domain family members (TEADs) are the most
important downstream effectors that play the pivotal role in the development, regeneration and
tissue homeostasis. Recent biochemical studies have demonstrated that TEADs could undergo
autopalmitoylation that is indispensable for its function making the lipid-binding pocket an
attractive target for chemical intervention. Herein, through structure-based virtual screen and
rational medicinal chemistry optimization, we identified DC-TEADin02 as the most potent,
selective, covalent TEAD autopalmitoylation inhibitor with the IC value of 197±19 nM while it
5
0
showed minimal effect on TEAD-YAP interaction. Further biochemical counter-screens
demonstrate the specific thiol reactivity and selectivity of DC-TEADin02 over the kinase family,
lipid-binding proteins and epigenetic targets. Notably, DC-TEADin02 inhibited TEADs
transcription activity leading to downregulation of YAP-related downstream gene expression.
Taken together, our findings proved the validity of modulating transcriptional output in the Hippo
signaling pathway through irreversible chemical interventions of TEADs autopalmitoylation
activity, which may serve as a qualified chemical tool for TEADs palmitoylation-related studies in
the future.
KEYWORDS:
Hippo pathway; TEAD transcription factor; Covalent inhibitor; Palmitoylation inhibitor.

1
. Introduction
The Hippo signaling pathway is an evolutionarily conserved signaling network that plays
essential role in cell proliferation, organ size control, stem cell self-renewal and homeostasis
maintenance[1, 2]. Transcriptional enhancer associated domain family members (TEADs) are
transcriptional factor families that act as downstream effectors in the regulation of transcriptional
output of the Hippo signaling pathway[3]. The transcriptional activity of TEADs could be
dynamically regulated by well-characterized transcriptional coactivators YAP/TAZ and other
indispensable Hippo-independent coactivators like the vestigial-like (VGLL) protein family and
the p160 coactivator family in the context of Hippo signaling[4, 5].
In recent years, a plethora of evidence suggests TEADs could undergo S-palmitoylation
under physiological conditions, which is required for its stability and function[6]. In 2016, PuiYee
Chan and co-workers demonstrated that palmitoylation-deficient TEAD mutants showed impaired
YAP-induced transcriptional activities with minimal effect on TEADs localization[7]. Although
the study showed far-reaching importance of TEAD palmitoylation and the lipid-interaction
proteome was generally considered with broader ligandability compared to the whole fraction of
proteome, which showed great opportunities for pharmacological intervention, little progress has
been achieved in the development of small molecule inhibitors[8]. Tremendous efforts have been
devoted to the drug discovery of TEAD-YAP inhibitors[9-15]. However, only a handful of TEAD
autopalmitoylation inhibitors have been reported. The current reported palmitoylation inhibitors
including 2-BP, cerulenin lack specificity and potency. In 2015, Pobbati et al. discovered
flufenamic acid, a FDA-approved non-steroidal anti-inflammatory drug, as the moderate TEAD
autopalmitoylation inhibitor with the K value of 73 µM, which occupied two distinct sites of
D

TEAD-YBD with minimal effect on TEAD-YAP interaction[16]. Further chemistry modifications
led to the identification of the allosteric inhibitor TED-347 based on the scaffold of flufenamic
acid, which could inhibit TEAD-YAP interaction and TEAD autopalmitoylation activity
simultaneously[13]. Thus, it still holds enormous potential to develop more potent, selective
TEAD autopalmitoylation inhibitors with novel chemotype and well-established mechanisms of
action for the illumination of biological processes and future therapeutic innovation[17].
Herein, starting with the non-covalent scaffold identified using computational approaches, we
described the discovery, optimization and biological characterization of DC-TEADin02 as the
most potent, selective TEAD autopalmitoylation inhibitor targeting the conserved cysteine within
the palmitate binding pocket while it showed minimal effect on TEAD-YAP interaction. Further
biochemical selectivity profiling demonstrated its specific thiol reactivity and selectivity over
epigenetic targets, kinases family and lipid-binding proteins. In cells, DC-TEADin02 inhibited
TEADs transcription activity leading to downregulation of YAP-related downstream gene
expression, making it a very useful chemical probe for TEAD palmitoylation-related studies in the
future.
2
. Results and discussion
Structure-based virtual screening. Exploitation and characterization of potential
drug-actionable pocket is the cornerstone in drug discovery studies[18]. Thus, SiteMap panel
integrated in Schrödinger program suite was used to identify the potential drug-binding pocket of
TEAD4. The results revealed that the hydrophobic central cavity was more druggable than
TEAD-YAP interface with the Dscore value of 1.364, which showed reasonable size and enclosure
for small-molecule binding (Table S1). Since there is no high-throughput assay available for the

identification of palmitoylation inhibitors, molecular docking-based virtual screen of in-house
diversity-oriented compound collections containing approximately 22,000 compounds was
conducted using Standard Precision mode in Glide panel (Figure 1A). The top-ranked 1,000 hits
were picked out for further cluster analysis and visual inspection. Considering the diversity of hit
scaffolds, 50 candidates were eventually chosen for further bio-orthogonal reaction-based
validation.
Figure 1. The discovery of DC-TEADin01 as the moderate TEAD4 autopalmitoylation inhibitor.
(A) The flowchart of the structure-based virtual screening campaign. (B-C) DC-TEADin01 and

flufenamic acid dose dependently inhibited TEAD4 autopalmitoylation activity. The quantification
results of band density were analyzed in ImageJ (NIH).
Hit Validation. Activity-based protein profiling (ABPP) has been widely applied to
characterize palmitoylated proteins by providing quantitative readouts in native biological
systems[19-22]. Thus, we sought to use Cu-catalyzed azide-alkyne cycloaddition (CuAAC)
reactions to evaluate the inhibitory effects of 50 candidate compounds against TEAD
autopalmitoylation activity via the ‘clickable’ palmitoyl alkyne-coenzyme A handle and biotin
azide. Among them, DC-TEADin01 showed moderate inhibitory activity with the IC value of 32
5
0
µM, which was more potent than the previously reported TEAD autopalmitoylation inhibitor
flufenamic acid (Figure 1B-C).
Upon the preliminary evaluation using enzymatic assays, the direct binding was further
investigated via a series of additional biochemical and biophysical assays. As evidenced in Figure
2
A, DC-TEADin01 favored protein folding and induced a positive T shift for over 4℃ in a dose
m
dependent manner, demonstrating that the observed inhibitory activity against TEAD4 was due to
entropically driven binding instead of substantial destabilization of protein structure.[23] Then,
ligand-based NMR spectroscopy was applied to detect the binding[24]. In the
Carr-Purcell-Meiboom-Gill (CPMG) experiments, the addition of TEAD4-YBD protein led to a
significant decrease of DC-TEADin01 NMR signal (Figure 2B). Moreover, enhanced resonance of
the ligand signal was also observed in the STD spectrum (Figure 2C). We also performed
competition experiments with palmitoyl CoA, a known binder in the pocket, to exclude the
possibility of non-specific binding. As illustrated in Figure 2D, palmitoyl CoA partially displaced
DC-TEADin01 validating its specific interaction. Surface plasmon resonance (SPR) experiments

also indicated that DC-TEADin01 bound to TEAD4-YBD with a K of 41 µM whereas flufenamic
D
acid bound to TEAD4-YBD with a K of 84 µM under our assay conditions (Figure 2E, Figure
D
S1). Additionally, the molecular docking studies demonstrated that DC-TEADin01 inserted into
the deep substrate tunnel of TEAD4-YBD, making extensive hydrophobic contact with the
surrounding residues including F393, F415, V389, L377, F373 and M366 (Figure 2F). Taken
together, we identified DC-TEADin01 as the bona fide hit, which could be efficiently optimized
into more potent ligands[25].
Figure 2. Biochemical and biophysical methods demonstrated the direct binding of
DC-TEADin01. (A) The protein thermal shift assays. Synthetic YAP peptide was used as the
positive control with the ratio of 1:5. (B-C) CPMG and STD NMR spectrum assays. (D)
Competitive NMR binding assay using 1 µM palmitoyl CoA. (E) SPR assays. (F) The predicted
binding mode of DC-TEADin01 with TEAD4-YBD in molecular docking studies.
Design and SAR Analysis of Covalent Inhibitors. The development of irreversible
covalent inhibitors has generated considerable interest and has been encouraged by the clinical

success of BTK inhibitor Ibrutinib and EGFR inhibitor Afatinib for the treatment of chronic
lymphocytic leukemia and non-small-cell lung cancer[26]. To further develop more potent small
molecular TEAD inhibitors, we try to rationally design the covalent inhibitor dependent on the
nucleophilic cysteine adjacent to the pocket, which is 5.4 Å away from the amine group of
DC-TEADin01 (Figure 2F).
Then a series of compounds featuring a vinylsulfonamide moiety were synthesized and
evaluated using in vitro palmitoylation assays (Table 1). Notably, the addition of the covalent
moiety yields DC-TEADin03 with significant improvement on its inhibitory activity. The
replacement of the oxygen atom with carbon atom in the linker region (DC-TEADin10) was still
tolerable, while variation of the length of the linker (DC-TEADin06) led to a moderate decrease
on its activity. Considering the hydrophobicity of the pocket, the phenyl group was replaced with
more hydrophobic naphthalene moiety to provide DC-TEADin02, which showed significant
inhibitory activity at 800 nM and 200 nM. Further introduction of the water-soluble substituents
led to the loss of activity (DC-TEADin07-DC-TEADin12). Compared with DC-TEADin02,
DC-TEADin04 and DC-TEADin13 showed the minimal effect on TEAD4 palmitoylation. Thus,
the more property-like inactive analog DC-TEADin13 were used as a negative control in the
following studies.
Table 1. The inhibitory activities of vinylsulfonamide derivatives.
ID
Structure
Inh@800nM
Inh@200nM

DC-TEADin02
DC-TEADin03
91.6%±5.2%
94.8%±2.9%
72.5%±7.3%
62.2%±6.3%
DC-TEADin04
DC-TEADin05
DC-TEADin06
25.2%±6.5%
1.1%±2.5%
52.5%±6.2%
-4.1%±1.2%
8.4%±3.3%
21.0%±2.8%
DC-TEADin07
DC-TEADin08
10.1%±3.6%
35.0%±3.7%
0.3%±4.8%
15.6%±5.2%
DC-TEADin09
DC-TEADin10
DC-TEADin11
25.4%±7.0%
92.5%±5.6%
92.1%±3.8%
5.4%±1.4%
59.2%±6.2%
44.3%±4.7%

DC-TEADin12
DC-TEADin13
82.5%±5.2%
6.5% ±1.8%
18.9%±1.2%
1.2% ±2.1%
Binding mode analysis. To further delineate the binding mode of DC-TEADin02 and
elucidate the mode-of-action of the series of compounds, covalent docking was performed using
Covalent Docking panel integrated in Maestro 9.2. The results show that DC-TEADin02 adopts
the similar conformation with endogenous myristic acid (Figure 3A). Additionally, the sulphamide
group adjacent to the opening of the pocket forms the hydrogen bonds with K344 and C367,
which shares the similar binding mode to the carboxyl group of flufenamic acid with TEAD2[16]
(
Figure 3B). The naphthalene moiety inserts into the deep substrate tunnel, making extensive
hydrophobic interactions with adjacent residues in the β-sandwich motif including F229, M370,
I374, F393, I395, F415 (Figure 3C-D). Consistent with the lack of inhibitory activity, the phenyl
and piperidine moiety of DC-TEADin04 and DC-TEADin13 would be expected to clash sterically
with F229, which renders it improbable for potent binding (Figure 3E). Taken together, the

docking results rationalized the structure-activity relationship and established a solid binding
mode for further structural decoration (Figure 3F).
Figure 3. In silico docking of DC-TEADin02 and its derivatives into the palmitoylation site of
TEAD4. (A-B) The alignment of DC-TEADin02 (green) with myristic acid (yellow) (PDB code:
5
OAQ)[27] and flufenamic acid (yellow) (PDB code: 5DQ8)[16]. (C) The detailed interactions
between DC-TEADin02 and TEAD4. (D) Schematic representation of interactions between
+
DC-TEADin02 and TEAD4 using LigPlot [28].The hydrophobic interactions and hydrogen bonds
were depicted in red arcs and green dotted lines, respectively. (E) The superposition of
DC-TEADin02 (white) with the negative compound DC-TEADin13 (green). (F) Local
surface view of the binding pocket.
Characterization
of
DC-TEADin02
as
Potent,
Covalent
TEAD4
autopalmitoylation inhibitor. For in vitro palmitoylation assay, DC-TEADin02 inhibited
TEAD autopalmitoylation activity with the IC50 value of 197±19 nM whereas DC-TEADin13
showed minimal effect at 50 µM (Figure 4A-B). In order to demonstrate that DC-TEADin02
formed the covalent linkage to TEAD4-YBD, mass spectrometry was conducted to analyze the
protein mass change upon compound binding. A higher mass peak was detected with the
difference in mass by +323 Da, which was in accordance with the molecular weight of
DC-TEADin02 via a direct Michael addition (Figure 4C). Additionally, the HPLC-MS/MS
spectrum of a peptide from DC-TEADin02 treated sample [IHRSPLC(C19H17NO2S) EYMINFIHK]
demonstrated the specific modification on Cys367 of TEAD (Figure S2). Notably, the interactions
of DC-TEADin02 with wild-type TEAD4-YBD resulted in a significant thermal transition with

the △T value of ~7 ℃ at the ratio of 1:1 while the reference compound TED-347 showed
m
substantial destabilization of protein structure indicating different mechanism of action (Figure 4D,
Figure S3). To further clarify the specific modification of DC-TEADin02 on Cys367, all cysteines
of TEAD4-YBD were individually mutated to alanine for thermal shift evaluation. Compared with
other TEAD4 mutants, only TEAD4-YBD C367A mutant significantly abolished the compound
effect on protein thermal stability (Figure 4E, Figure S4). We also explored the compound effect
on TEAD-YAP interaction in the fluorescence polarization (FP) assay using fluorescein
isothiocyanate (FITC)-labeled YAP peptide61-100. The results indicated that DC-TEADin02 showed
minimal effect on TEAD-YAP interaction making it a very qualified chemical tool for TEAD
palmitoylation-related studies, which is in consistent with the mechanism of action of previously
reported TEAD autopalmitoylation chemical probe flufenamic acid (Figure S5).
Considering palmitoyl CoA used in CuAAC-based click chemistry methods is the
biologically active thiol, we then tested DC-TEADin02 for its ability to form covalent adducts
with CoA, which may also inhibit TEAD4 autopalmitoylation. As evidenced in Figure 4F, after 8
h incubation, no covalent adducts have been detected by UPLC-MS. Similarly, DC-TEADin02 is
unreactive with GSH, which excludes its non-proteinaceous thiol reactivity (Figure 4G).
Additionally, the inhibitory activity of DC-TEADin02 was unaffected in the presence of excess
bovine serum albumin (BSA), which can mitigate thiol reactivity (Figure 4G). Collectively, these
results demonstrated the specific thiol reactivity of DC-TEADin02.
It has been well characterized in the chemoproteomics studies that kinases harboring many
targetable, active site-proximal cysteines account for the off-target effect of covalent inhibitors
owing to its high nucleophilicity[29, 30]. Thus, we conducted kinome profiling to evaluate

compound selectivity. The results showed that it did not inhibit the activity of kinases at 10 µM
Figure 4H, Table S2). Additionally, it was selective over epigenetic targets in the biochemical
(
selectivity profiles (Table S3). Considering the fact that the non-covalent scaffold of
DC-TEADin02 may bind to other lipid binding proteins, we tested the compound effect on the
FABP3-5 using thermal shift assays. The results showed that DC-TEADin02 showed minimal
effect on FABP3-5 demonstrating the specific interaction with TEAD lipid-binding pocket (Figure
S6).

Figure 4. The biophysical and biochemical characterization of DC-TEADin02. (A) The chemical
s structures of DC-TEADin02 and its property-like inactive analog DC-TEADin13. (B) The
inhibitory activity of DC-TEADin02 and DC-TEADin13 through CuAAC-based click chemistry.
The band intensity was quantified in ImageJ (NIH). The experiments were performed in triplicate
and the data was shown as mean ± SD. (C) Mass spectrometry demonstrated the covalent
modification of DC-TEADin02 with TEAD4-YBD protein. (D-E) DC-TEADin02 significantly
improved the thermostability of TEAD and its mutant protein except C367A mutant. Synthetic
YAP peptide was used as the positive control with the ratio of 1:5. (F) DC-TEADin02 was
incubated up to 8ꢀh with 5-fold excess GSH/CoA for additional adduct analyses in UPLC-MS. (G)
DC-TEADin02 inhibited TEAD autopalmitoylation in the presence of excess BSA. (H) Kinase
profiling with the compound concentration of 10 µM. Each experiment was performed with one
replicate to ensure data quality.
Cellular Activity of DC-TEADin02. In order to further evaluate the inhibitory effect of
compounds in cells, we performed TEADs specific luciferase reporter assays with DC-TEADin02
and the negative compound DC-TEADin13. As expected, the treatment of cells transfected with
the TEAD luciferase reporter with DC-TEADin02 led to the significant reduction in TEAD
reporter activity compared with DC-TEADin13. Additionally, the treatment of DC-TEADin02
showed similar levels versus control cells in TEADs-independent reporter systems demonstrating
the specific regulation on Hippo pathway (Figure 5A). We also evaluated TEAD downstream gene
expression patterns in colorectal cancer cells HCT 116, which has been evidenced with relative
high expression of TEAD4 in the web-accessible GENT database (Figure S5)[31, 32]. Consistent
with the inhibition of TEAD transcriptional activity, the treatment of DC-TEADin02 suppressed

downstream gene CTGF and CYR61 expression at a dose-dependent manner while it showed
minimal effect on TEAD-YAP interaction, which was in accordance with the findings in vitro
(
Figure 5B-C). In order to further demonstrate the direct interaction between DC-TEADin02 and
TEAD4 and evaluate the cellular uptake of the compound, we performed the cellular thermal shift
assay (CETSA) in intact HCT 116 cell. The results showed that the detected soluble fraction of
TEAD4 was increased upon the compound treatment, demonstrating its cellular uptake and direct
binding on TEAD4 (Figure 5D).

Figure 5. DC-TEADin02 inhibited the TEAD transcriptional activity downregulating the
downstream gene expression. (A) The effect of DC-TEADin02 and DC-TEADin13 on
TEAD-dependent/independent reporter system. (B) Quantitative RT-PCR analysis demonstrated
the downregulation of TEAD-specific genes upon compound treatment. (C) The pull-down assay
to detect TEAD-YAP interaction. (D) HCT 116 cells were treated with 50 µM DC-TEADin02 for
4
h in CETSA experiments to demonstrate the direct binding of DC-TEADin01 with TEAD4 in
intact cells. The results shown are mean ± SD of three replicates (*P<0.05, **P < 0.01, ***P <
.001, ****P<0.0001, N.S. P>0.05). (E) The modification of TEAD4 upon 50 µM DC-TEADin02
0
treatment for 4 hr was detected by MS experiments.
Moreover, HEK293T cells transfected with Flag-TEAD4 was treated with 50 µM
DC-TEADin02 for 4 hr. Then Flag-TEAD4 protein was extracted by affinity purification with
anti-flag resin from the cell lysate and submitted for MS analysis. As expected, the direct
modification on Cys367 of Flag-TEAD4 was detected demonstrating the on-target effect in cells
(
Figure 5E).
3
. Conclusion
In eukaryotic organisms, fatty acylation is increasingly recognized as one of the most
well-characterized post-translational modifications that could dynamically and spatiotemporally
regulate protein stability, localization, trafficking and protein-membrane interactions[33]. Global
studies demonstrate that in mammalian cells proteins could be modified by at least six types of
lipids [34]. Among them, S-palmitoylation is the uniquely reversible fatty acylation that
orchestrates diverse biological processes[35].

Emerging evidence demonstrates that pharmacologically antagonize protein palmitoylation
may have great potential for the treatment of a variety of malignant diseases[36]. In 2013, Petrova
et al. developed Hhat inhibitors based on high-throughput screen, which could specifically block
Shh palmitoylation overcoming drug resistance[37]. In 2018, Hansen and Haag et al.
demonstrated that covalent inhibition of STING palmitoylation could significantly inhibit STING
signaling cascade and inflammatory cytokine production, which shed light on the treatment of
STING-dependent autoinflammatory disease[38-40]. Taken together, these evidences suggest
targeting protein palmitoylation could be a valid option for future therapeutic development.
In mammalian cells, a variety of key signaling proteins could be lapidated including RAS,
Hedgehog, Shh, Wnt etc[36]. And numerous palmitoyl-proteomic studies have provided many
useful resources for related studies in this area[41]. In 2016, Chan and co-workers demonstrated
that TEADs transcription family could also undergo autopalmitoylation through a reversible
thioester bond, which is sensible to hydrolysis under physiological conditions. The enzymatic
S-palmitoylation is indispensable for YAP-induced TEADs transcriptional activities, making it an
attractive target for chemical intervention. Although recent years have witnessed massive gain in
our understanding of its biochemical function, the development of TEAD autopalmitoylation
inhibitors lags behind and is still at its infancy[42].
Herein, based on integrated structure-based virtual screen and CuAAC-based click chemistry
biorthogonal evaluation, we identified DC-TEADin01 as the moderate TEAD4 autopalmitoylation
inhibitor. A series of biochemical and biophysical methods were adopted to validate the reliability
of its scaffold. Further rational chemical optimization led to the identification of DC-TEADin02 as

the most potent, selective, cell active covalent TEAD autopalmitoylation inhibitor bearing a
previously unexplored chemotype with the IC50 value of 197±19 nM with minimal effect on
TEAD-YAP interaction making it a useful chemical probe for TEAD palmitoylation-related
studies. DC-TEADin02 did not interact with GSH and CoA and its inhibitory activity was not
comprised in the presence of excessive BSA with specific thiol activity. Additionally, it showed
great selectivity against kinase family harboring function cysteines, lipid binding proteins and
epigenetic families demonstrating the specificity of both covalent and non-covalent motifs. In
cells, it could covalently bind to the Cys367 of TEAD4 protein, leading to the inhibition of
TEADs transcriptional activity and downstream gene expression. As the starting point,
DC-TEADin02 may serve as a qualified lead compound to develop more potent, subtype-
selective irreversible TEAD autopalmitoylation inhibitors, which shed light on future therapeutic
innovations.
These results provide an efficient paradigm of how virtual screening cooperating with rational
optimization could efficiently promote the drug discovery progress. The similar paradigm could be
easily and broadly extended to other undruggable targets besides transcription factors. Our results
demonstrate the feasibility and the value of using small molecule inhibitors to selectively target
protein lipidation in cell signaling, which represent a major advance on the road for functional
study in the context of Hippo signaling.
4
. Material and methods
.1 Chemistry
Unless otherwise stated, all materials and anhydrous solvents were purchased from
commercial suppliers and used without further purification. All products were confirmed by NMR
4

spectra and high-resolution mass spectra. NMR spectra were obtained on a Bruker Avance 400
MHz or Varian Inova 400 MHz spectrometer using tetramethylsilane as an internal reference.
Coupling constants (J values) were reported in Hertz (Hz) and proton coupling patterns were
described as broad (b), singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m).
High-resolution mass spectra were conducted on a triple TOF 5600+ MS/MS system (AB Sciex,
Concord, Ontario, Canada) in the negative or positive ESI mode. Reactions were monitored by
thin-layer chromatography or UPLC-MS (Waters Acquity UPLC H-class with Waters SQ Detector
2
) analysis. Column chromatography was performed on silica gel (200-300 mesh). All tested
compounds were purified to ≥ 95% purity as determined by ultra performance liquid
chromatography.
4
.1.1 Synthesis of 4-(p-tolyloxy)aniline (DC-TEADin01)
Scheme1. Synthesis scheme of DC-TEADin01
Reagents and conditions: 1a) K CO , DMSO, 90 ℃ , 8 h. 1b) H , Pd/C, MeOH, 6 h.
2
3
2
General synthesis procedure 1a：p-Cresol (200 mg, 1.85 mmol), K CO (767 mg, 5.55 mmol)
2
3
and 4-Fluoronitrobenzene (261 mg, 1.85 mmol) were added into DMSO (15 mL). The mixture
was stirred at 90 ℃ for 8 h. The reaction mixture was diluted and extracted with EtOAc. The
organic layer was washed with water for 5 times then evaporated. The residue was
chromatographed
(silicagel,
Petroleum
ether/EtOAc=100:1)
to
give

1
1
-methyl-4-(4-nitrophenoxy)benzene (410 mg, 97%) as a white solid, H NMR (400 MHz,
Chloroform-d) δ 8.24-8.17 (m, 2H), 7.28-7.22 (m, 2H), 7.04-6.97 (m, 4H), 2.41 (s, 3H).
General synthesis procedure 1b：1-methyl-4-(4-nitrophenoxy)benzene (DC-TEADin01-1, 100
mg, 0.44 mmol), Pd/C (10 mg) were added in methanol with several drops of HCl and stirred at
room temperature under H for 6 h. The reaction mixture was filtered using Celite and evaporated.
2
The residue was chromatographed (silicagel, Petroleum ether/EtOAc=20:1) to give
1
4
-(p-tolyloxy)aniline (DC-TEADin01, 71 mg, 81%) as a white solid. H NMR (400 MHz,
Chloroform-d) δ 7.09 (d, J = 8.2 Hz, 2H), 6.89-6.81 (m, 4H), 6.70- 6.64 (m, 2H), 3.56 (s,2H), 2.31
+
(
s, 3H). HRMS (ESI): m/z [M + H] calculated for C H NO – 200.107, found 200.1074.
1
3
14
4
.1.2 Synthesis of N-(4-(naphthalen-2-ylmethyl)phenyl)ethenesulfonamide (DC-TEADin02)
Br
NO2
2a
1b
NO2
2b
NH2
O
O
OH
B
S
N
H
OH
DC-TEADin02-1
DC-TEADin02-2
Scheme 2.
DC-TEADin02
Scheme2. Synthesis scheme of DC-TEADin02
Reagents and conditions: 2a) 1,1-Bis(diphenylphosphino)-ferro-cene]dichloropalladium (II) ,
DCE:2M aqueous Na CO (v/v) =3:1, 100 ℃ , 2 h. 1b) H , Pd/C, MeOH, 6 h. 2b) Pyridine,
2
3
2
Anhydrous Dichloromethane, 2-Chloroethanesulfonyl chloride, 0 ℃ , 2 h
General synthesis procedure 2a ： 4-Nitrobenzyl bromide (500 mg, 2.43 mmol),
2
1
-Naphthaleneboronic
acid
(500
mg,
2.91
mmol),
,1-Bis(diphenylphosphino)-ferro-cene]dichloropalladium (II) (0.05 mmol, 34 mg) were dissolved
in degassed 1,2-Dichloroethane (15 mL) and 2 M sodium carbonate solution (5 mL). The mixture
was stirred at 100 ℃ under argon for 2 h. The reaction mixture was diluted with DCM and filtered

using Celite, then saturated aqueous NH Cl was added to the filtrate and extracted with EtOAc.
4
The combined organic layers were dried over Na SO and evaporated. The residue was
2
4
chromatographed (silicagel, Petroleum ether/EtOAc=200:1) to give 2-(4-nitrobenzyl)naphthalene
1
(
DC-TEADin02-1，550 mg, 90%) as a white solid. H NMR (400 MHz, Chloroform-d) δ
8
.18-8.13 (m, 2H), 7.86-7.75 (m, 3H), 7.64 (s, 1H), 7.52-7.43 (m, 2H), 7.41-7.35 (m, 2H), 7.27
dd, J = 8.4, 1.7 Hz, 1H), 4.24 (s, 2H).
General synthesis procedure 1b：Starting with 2-(4-nitrobenzyl)naphthalene (DC-TEADin02-1),
(
4
-(naphthalen-2-ylmethyl)aniline (DC-TEADin02-2) was obtained as a white solid by using
1
general synthesis procedure 1b (yield 90%). H NMR (400 MHz, Methanol-d ) δ 7.85-7.77 (m,
4
3
H), 7.70 (s, 1H), 7.50-7.42 (m, 4H), 7.37-7.31 (m, 3H), 4.22 (s, 2H). Hydrogens of NH were not
2
found.
General synthesis procedure 2b：4-(naphthalen-2-ylmethyl)aniline (DC-TEADin02-2, 80 mg,
0
2
.34 mmol) was stirred with pyridine (0.5 mL) in DCM (10 mL) at 0 ℃ , then
-Chloroethanesulfonyl chloride (56 mg, 0.343 mmol) which was diluted with DCM (2 mL) was
slowly added into the mixture. The reaction mixture was stirred for 2 h then evaporated. The
residue was diluted and extracted with EtOAc then washed with water for 3 times. The organic
layers were dried over Na SO and evaporated. The residue was chromatographed (silicagel,
2
4
Petroleum ether/EtOAc=5:1) to give N-(4-(naphthalen-2-ylmethyl)phenyl)ethenesulfonamide
1
(
DC-TEADin02) (93 mg, 85%) as a white solid. H NMR (400 MHz, Chloroform-d) δ 7.78 (m,
3
H), 7.61 (s, 1H), 7.51-7.39 (m, 2H), 7.28 (d, J = 8.4 Hz, 1H), 7.18 (d, J = 8.3 Hz, 2H), 7.10 (d, J
8.4 Hz, 2H), 6.63 (s, 1H), 6.54 (dd, J = 16.5, 9.9 Hz, 1H), 6.26 (d, J = 16.5 Hz, 1H), 5.94 (d, J =
=

+
9
.9 Hz, 1H), 4.10 (s, 2H). HRMS (ESI): m/z [M + H] calculated for C H NO S 324.1053, found
19 18 2
3
24.1062.
4
.1.3 Synthesis of 4-phenoxyphenyl ethenesulfonate (DC-TEADin03)
Scheme 3. Synthesis scheme of the DC-TEADin03
Reagents and conditions: 3a) 4-phenoxyphenol, TEA, Anhydrous Dichloromethane,
-Chloroethanesulfonyl chloride, 0 ℃, 2 h.
2
General synthesis procedure 3a：4-phenoxyphenol (186 mg, 1 mmol) was stirred with TEA (278
µL, 2 mmol) in Anhydrous DCM (10 mL) at 0 ℃, then 2-Chloroethanesulfonyl chloride (105 µL,
1
mmol) which was diluted with DCM (2 mL) was slowly added into the mixture. The reaction
mixture was stirred for 2h then evaporated. The residue was diluted and extracted with EtOAc
then washed with water for 3 times. The organic layers were dried over Na SO and evaporated.
2
4
The residue was chromatographed (silicagel, Petroleum ether/EtOAc=3:1) to give
1
4
-phenoxyphenyl ethenesulfonate (DC-TEADin03) (193 mg, 70%) as a colorless oil. H NMR
(
400 MHz, Chloroform-d) δ 7.36 (t, J = 7.9 Hz, 2H), 7.22-7.10 (m, 3H), 7.05-6.95 (m, 4H), 6.68
dd, J = 16.6, 10.0 Hz, 1H), 6.39 (d, J = 16.7 Hz, 1H), 6.18 (d, J = 10.0 Hz, 1H).
(
4
.1.4 Synthesis of N-(2-phenoxyphenyl)ethenesulfonamide (DC-TEADin04)
Scheme 4. Synthesis scheme of the DC-TEADin04

General
synthesis
procedure
3a
：
Starting
with
2-phenoxyaniline,
N-(2-phenoxyphenyl)ethenesulfonamide (DC-TEADin04) was obtained as a colorless solid by
1
using general synthesis procedure 3a (yield 76%). H NMR (400 MHz, Chloroform-d) δ 7.59 (dd,
J = 7.6, 1.9 Hz, 1H), 7.36 (t, J = 7.9 Hz, 2H), 7.16 (t, J = 7.4 Hz, 1H), 7.08 (pd, J = 7.5, 1.7 Hz,
2
H), 6.98 (d, J = 8.2 Hz, 2H), 6.88 (s, 1H), 6.86 (dd, J = 7.7, 1.7 Hz, 1H), 6.51 (dd, J = 16.5, 9.9
+
Hz, 1H), 6.28 (d, J = 16.5 Hz, 1H), 5.93 (d, J = 9.9 Hz, 1H). HRMS (ESI): m/z [M + H]
+
calculated for C H NO S−324.1053, found 324.1062. HRMS (ESI): m/z [M + H] calculated for
1
9
18
2
C H NO S 276.0689, found 276.0692.
1
4
14
3
4
.1.5 Synthesis of N-(naphthalen-2-yl)ethenesulfonamide (DC-TEADin05)
Scheme 5. Synthesis scheme of the DC-TEADin05
General
synthesis
procedure
3a
：
Starting
with
naphthalen-2-amine,
N-(naphthalen-2-yl)ethenesulfonamide (DC-TEADin05) was obtained as a white solid by using
1
general synthesis procedure 3a (yield 80%). H NMR (400 MHz, Chloroform-d) δ 7.79 (t, J = 9.1
Hz, 3H), 7.65 (d, J = 1.9 Hz, 1H), 7.52-7.41 (m, 2H), 7.31 (dd, J = 8.7, 2.2 Hz, 1H), 6.99 (s, 1H),
6
.61 (dd, J = 16.5, 9.9 Hz, 1H), 6.32 (d, J = 16.5 Hz, 1H), 5.95 (d, J = 9.9 Hz, 1H). HRMS (ESI):
+
m/z [M + H] calculated for C H NO S 234.0583, found 234.0584.
1
2
12
2
4
.1.6 Synthesis of N-(4-(benzyloxy)phenyl)ethenesulfonamide (DC-TEADin06)

Scheme 6. Synthesis scheme of the DC-TEADin06
synthesis procedure 3a Starting with 4-(benzyloxy)aniline,
General
：
N-(4-(benzyloxy)phenyl)ethenesulfonamide (DC-TEADin06) was obtained as a white solid by
1
using general synthesis procedure 3a (yield 75%). H NMR (400 MHz, Chloroform-d) δ 7.37 (dq,
J = 21.1, 7.3, 6.5 Hz, 5H), 7.14 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.9 Hz, 2H), 6.54 (dd, J = 16.5,
9
.9 Hz, 1H), 6.40 (s, 1H), 6.20 (d, J = 16.6 Hz, 1H), 5.92 (d, J = 9.9 Hz, 1H), 5.04 (s, 2H). HRMS
+
(
ESI): m/z [M + H] calculated for C H NO S 290.0845, found 290.0854.
1
5
16
3
4
.1.7 Synthesis of N-(1,2,3,4-tetrahydroisoquinolin-6-yl)-4-(vinylsulfonamido)benzamide
(DC-TEADin07)
Scheme7. Synthesis scheme of the DC-TEADin07
Reagents and conditions: 7a) EDCI, 4-DMAP, DCM, overnight. 1b) H , Pd/C, MeOH, 6 h. 2b)
2
Pyridine, Anhydrous Dichloromethane, 2-Chloroethanesulfonyl chloride, 0 ℃, 2 h. 7b) HCl (4 M
solution in 1,4-Dioxane), 2 h.
General
-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (248 mg, 1 mmol), 4-nitrobenzoic acid (167
mg, 1 mmol), EDCI (212 mg, 1.1 mmol), and catalytic quantity of 4-DAMP are added into DCM
synthesis
procedure
7a
，
1b
and
2b
：
Tert-butyl
6

(
10 mL) and the mixture were stirred for 8 h. The reaction mixture was diluted with DCM and
filtered using Celite, then evaporated. Starting with the residue which was not purified, Tert-butyl
-(4-(vinylsulfonamido)benzamido)-3,4-dihydroisoquinoline-2(1H)-carboxylate
6
(
(
(
DC-TEADin07-1) was obtained as a white solid by using general synthesis procedure 1b and 2b
1
yield 45%). H NMR (400 MHz, Methanol-d ) δ 7.87 (d, J = 8.9 Hz, 2H), 7.55-7.41 (m, 2H), 7.28
4
d, J = 8.9 Hz, 2H), 7.11 (d, J = 8.5 Hz, 1H), 6.71 (dd, J = 16.5, 10.0 Hz, 1H), 6.24 (d, J = 16.5 Hz,
1
H), 6.01 (d, J = 9.9 Hz, 1H), 5.48 (s, 2H), 4.53 (s, 2H), 3.63 (t, J = 5.9 Hz, 2H), 2.83 (t, J = 5.9
Hz, 2H), 1.49 (s, 9H).
General synthesis
-(4-(vinylsulfonamido)benzamido)-3,4-dihydroisoquinoline-2(1H)-carboxylate
DC-TEADin07-1, 30 mg, 0.07 mmol) was added into a flask, then HCl (4 M solution in
,4-Dioxane, 3 mL) was added. The mixture was stirred for 2 h. Then the reaction mixture was
procedure
7b
：
Tert-butyl
6
(
1
evaporated
to
get
N-(1,2,3,4-tetrahydroisoquinolin-6-yl)-4-(vinylsulfonamido)benzamide
1
(
DC-TEADin07) as a HCl salt. H NMR (400 MHz, DMSO-d ) δ 10.50 (s, 1H), 10.23 (s, 1H),
6
9
.57 (s, 2H), 7.93 (d, J = 8.5 Hz, 2H), 7.68 (s, 1H), 7.61 (d, J = 8.6 Hz, 1H), 7.26 (d, J = 8.5 Hz,
H), 7.17 (d, J = 8.5 Hz, 1H), 6.85 (dd, J = 16.4, 9.9 Hz, 1H), 6.21 (d, J = 16.4 Hz, 1H), 6.10 (d, J
2
=
9.9 Hz, 1H), 4.19 (s, 2H), 3.34 (s, 2H), 3.00 (t, J = 5.9 Hz, 2H). (as a HCl salt). HRMS (ESI):
+
m/z [M + H] calculated for C H N O S 358.122, found 358.1211.
1
8
20
3
3
4
.1.8 Synthesis of N-(4-(vinylsulfonamido)phenyl)-2-naphthamide (DC-TEADin08)

Scheme 8. Synthesis scheme of the DC-TEADin08
General synthesis procedure 7a：Starting with 2-naphthoic acid (172 mg, 1 mmol) and (208 mg,
1
mmol), tert-butyl (4-(2-naphthamido)phenyl)carbamate (DC-TEADin08-1) was obtained as a
1
white solid by using general synthesis procedure 7a (yield 85%). H NMR (400 MHz, DMSO-d )
6
δ 10.31 (s, 1H), 9.30 (s, 1H), 8.55 (s, 1H), 8.10 – 7.94 (m, 4H), 7.73 – 7.55 (m, 4H), 7.43 (d, J =
8
.6 Hz, 2H), 1.47 (s, 9H).
General synthesis
4-(2-naphthamido)phenyl)carbamate
procedure
7b
and
2b
：
Starting
with
tert-butyl
(
(DC-TEADin08-1),
N-(4-(vinylsulfonamido)phenyl)-2-naphthamide (DC-TEADin08) was obtained as a white solid
1
by using general synthesis procedure 7b and 2b (yield 70%). H NMR (400 MHz, DMSO-d ) δ
6
9
7
1
.88 (s, 1H), 8.55 (s, 1H), 8.09-7.96 (m, 4H), 7.74 (dd, J = 9.0, 2.5 Hz, 2H), 7.66-7.58 (m, 2H),
.15 (d, J = 8.9 Hz, 2H), 6.76 (dd, J = 16.4, 9.9 Hz, 1H), 6.07 (d, J = 16.4 Hz, 1H), 6.01 (d, J =
+
0.0 Hz, 1H). One hydrogen of NH was not found. HRMS (ESI): m/z [M + H] calculated for
C H N O S 353.0954, found 353.0962.
1
9
17
2
3
4
.1.9
Synthesis
of
N-(2-benzoyl-1,2,3,4-tetrahydroisoquinolin-6-yl)ethenesulfonamide
(DC-TEADin09)

Scheme 9. Synthesis scheme of the DC-TEADin09
Reagents and conditions: 2b) Pyridine, Anhydrous Dichloromethane, 2-Chloroethanesulfonyl
chloride, 0 ℃ , 2 h. 7b) HCl (4M solution in 1,4-Dioxane), 2h. 3a) Benzoyl chloride, TEA,
Anhydrous Dichloromethane, 0 ℃ , 2 h.
General
synthesis
-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate,
-(vinylsulfonamido)-3,4-dihydroisoquinoline-2(1H)-carboxylate
procedure
2b
：
Starting
with
tert-butyl
tert-butyl
6
6
(DC-TEADin09-1)
was
1
obtained as a white solid by using general synthesis procedure 2b (yield 70%). H NMR (400
MHz, Chloroform-d) δ 7.01 (dd, J = 24.2, 9.7 Hz, 3H), 6.70 (s, 1H), 6.55 (dd, J = 16.5, 9.9 Hz,
1
H), 6.27 (d, J = 16.5 Hz, 1H), 5.95 (d, J = 9.9 Hz, 1H), 4.52 (s, 2H), 3.61 (t, J = 5.2 Hz, 2H), 2.79
t, J = 5.7 Hz, 2H), 1.48 (s, 9H).
General synthesis procedure
-(vinylsulfonamido)-3,4-dihydroisoquinoline-2(1H)-carboxylate
N-(2-benzoyl-1,2,3,4-tetrahydroisoquinolin-6-yl)ethenesulfonamide
obtained as a white solid by using general synthesis procedure 7b and 3a (yield 60%). H NMR
400 MHz, DMSO-d ) δ 9.91 (s, 1H), 7.47-7.38 (m, 5H), 7.23-6.86 (m, 3H), 6.74 (dd, J = 16.4,
(
7b
and
3a
：
Starting
with
(DC-TEADin09-1),
(DC-TEADin09) was
tert-butyl
6
1
(
6
1
0.0 Hz, 1H), 6.08 (d, J = 16.4 Hz, 1H), 6.00 (d, J = 9.9 Hz, 1H), 4.58 (d, J = 82.8 Hz, 2H), 3.65

+
(
d, J = 124.2 Hz, 2H), 2.78 (s, 2H). HRMS (ESI): m/z [M + H] calculated for C H N O S
18 19 2 3
3
43.1111, found 343.1118.
4
.1.10 Synthesis of N-(4-benzylphenyl)ethenesulfonamide (DC-TEADin10)
Scheme 10. Synthesis scheme of the DC-TEADin10
General
synthesis
procedure
3a
：
Starting
with
4-benzylaniline,
N-(4-benzylphenyl)ethenesulfonamide (DC-TEADin10) was obtained as a pale yellow solid by
1
using general synthesis procedure 3a (yield 70%). H NMR (400 MHz, Chloroform-d) δ 7.34-7.09
(m, 9H), 6.57 (dd, J = 16.5, 9.9 Hz, 1H), 6.46 (s, 1H), 6.29 (d, J = 16.5 Hz, 1H), 5.97 (d, J = 9.9
+
Hz, 1H), 3.97 (s, 2H). HRMS (ESI): m/z [M + H] calculated for C H NO S 274.0896, found
1
5
16
2
2
74.0899.
4
.1.11 Synthesis of N-(3-hydroxy-4-(naphthalen-2-ylmethyl)phenyl)ethenesulfonamide
(DC-TEADin11)