Discovery of Covalent Inhibitors Targeting the Transcriptional Enhanced Associate Domain Central Pocket

Article abstract of DOI:10.1021/acs.jmedchem.0c01275

Transcriptional enhanced associate domain (TEAD) transcription factors together with coactivators and corepressors modulate the expression of genes that regulate fundamental processes, such as organogenesis and cell growth, and elevated TEAD activity is associated with tumorigenesis. Hence, novel modulators of TEAD and methods for their identification are in high demand. We describe the development of a new "thiol conjugation assay"for identification of novel small molecules that bind to the TEAD central pocket. The assay monitors prevention of covalent binding of a fluorescence turn-on probe to a cysteine in the central pocket by small molecules. Screening of a collection of compounds revealed kojic acid analogues as TEAD inhibitors, which covalently target the cysteine in the central pocket, block the interaction with coactivator yes-associated protein with nanomolar apparent IC50 values, and reduce TEAD target gene expression. This methodology promises to enable new medicinal chemistry programs aimed at the modulation of TEAD activity.

Full text of DOI:10.1021/acs.jmedchem.0c01275

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Article
Discovery of Covalent Inhibitors Targeting the Transcriptional
Enhanced Associate Domain (TEAD) Central Pocket
Hacer Karatas, Mohammad Akbarzadeh, Hélène Adihou, Gernot Hahne, Ajaybabu V. Pobbati, Elizabeth
Yihui Ng, Stéphanie M. Guéret, Sonja Sievers, Axel Pahl, Malte Metz, Sarah Zinken, Lara Dötsch, Christine
Nowak, Sasikala Thavam, Alexandra Friese, Congbao Kang, Wanjin Hong, and Herbert Waldmann
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 10 Sep 2020
Downloaded from pubs.acs.org on September 10, 2020
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Discovery of Covalent Inhibitors Targeting the Transcriptional Enhanced
Associate Domain (TEAD) Central Pocket
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Hacer Karatas, Mohammad Akbarzadeh, Hélène Adihou, Gernot Hahne, Ajaybabu V. Pobbati,
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Elizabeth Yihui Ng, Stéphanie M. Guéret, Sonja Sievers, Axel Pahl, Malte Metz, Sarah
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Zinken, Lara Dötsch, Christine Nowak, Sasikala Thavam, Alexandra Friese, CongBao Kang,
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Wanjin Hong, Herbert Waldmann*
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Department of Chemical Biology, Max Planck Institute of Molecular Physiology,
Otto-Hahn Straße 11, 44227 Dortmund, Germany.
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AstraZeneca-Max Planck Institute Satellite Unit, Department of Chemical Biology, Otto-
Hahn-Strasse 11, 44227 Dortmund, Germany.
Medicinal Chemistry, Research and Early Development Cardiovascular, Renal and
Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, SE-431 83, Sweden.
Institute of Molecular and Cell Biology, A*STAR, 61 Biopolis Drive, 138673 Singapore,
Singapore.
Experimental Drug Development Centre (EDDC), Agency for Science, Technology and
Research (A*STAR), 10 Biopolis Road, Chromos, #05-01, Singapore 138670
Faculty of Chemistry and Chemical Biology, Technical University Dortmund, Otto-Hahn-
Straße 6, 44227, Dortmund, Germany.
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ABSTRACT: Transcriptional enhanced associate domain (TEAD) transcription factors together
with coactivators and corepressors modulate the expression of genes that regulate fundamental
processes, as organogenesis and cell growth, and elevated TEAD activity is associated with
tumorigenesis. Hence novel modulators of TEAD and methods for their identification are in high
demand. We describe the development of a new “thiol conjugation assay” for identification of
novel small molecules that bind to the TEAD central pocket. The assay monitors prevention of
covalent binding of a fluorescence turn-on probe to a cysteine in the central pocket by small
molecules. Screening of a collection of compounds revealed kojic acid analogs as TEAD
inhibitors, which covalently targets the cysteine in the central pocket, blocks the interaction with
coactivator Yes-Associated Protein (YAP) with nanomolar apparent IC50 values and reduces
TEAD target gene expression. This methodology promises to enable new medicinal chemistry
programs aimed at the modulation of TEAD activity.
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Introduction
Transcriptional enhanced associate domain (TEAD) transcription factors play significant roles
during development, tissue regeneration, cell growth and migration, and in human disease
progression.1-4 Elevated TEAD expression has been observed in solid tumors such as prostate,5
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lung, colorectal, gastric, and breast cancers . A number of TEAD target genes including cell
surface receptor tyrosine kinase Axl , connective tissue growth factor CTGF11,12, apoptosis
inhibitor survivin13 and tumor marker mesothelin14 are also frequently associated with
tumorigenesis. Therefore, TEAD transcription factors are potential therapeutic targets in cancer
therapy.
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There are four TEAD homologs (TEAD14) in mammalian cells that are expressed in a tissue
and development stage specific manner. TEAD1 is involved in cardiogenesis, while TEAD2 is
critical for neural development and TEAD4 is necessary for embryo implantation.15-18 Thus,
pharmacological modulation of TEAD activity may enable novel medicinal chemistry approaches
both in oncology and regenerative medicine.19,20 Accordingly, the development of small molecule
and peptidic modulators of TEAD function has been approached.1,20-23
TEAD homologs 14 share two highly conserved N- and C-terminal domains. The N-terminal
DNA binding domain forms a homeodomain and the C-terminal transactivation domain adopts a
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β-sandwich capped with a helix-turn-helix motif (Figure 1A, left panel, cyan). The transactivation
domain maintains the interaction with transcriptional co-modulators, i.e. coactivators YAP (Figure
1
A, left panel, pale yellow), transcriptional coactivator with PDZ-binding motif (TAZ), Vestigial-
like (Vgll) family proteins, as well as corepressor Vgll4 (Figure S1).24,25
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Cys367
Central pocket
HO
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B
Cl
H
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CF3
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CF3
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CF3
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(Flufenamic acid)
ITC_K : 73 µM
2 (Niflumic acid)
ITC_K : 28 µM
3 (TED-347)
d
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H
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S
H
N
H
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Cl
4 (K-975)
5 (DC-TEADin02)
6 (MGH-CP1)
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OH HN
N
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N
N
N
O
N
O
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S
O
H
N
N
H
F
HN
7
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F
Cl
Figure 1. hTEAD4 transactivation domain. (A) Structure of TEAD transactivation domain (cyan)
and the acylated cysteine residue in the central pocket (PDB ID: 5OAQ). Cofactor YAP (PDB ID:
3
KYS) is shown (pale yellow). Right panel zoomed into the acylated cysteine residue (Cys367).
(
B) Small molecules that bind the TEAD central pocket.
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TEADs contain a large hydrophobic central pocket within the globular β-sandwich to which
the cofactors bind. The pocket embodies a conserved cysteine residue that can be acylated with
palmitic or myristic acid (Figure 1A).26-28 Recent studies suggest that TEAD acylation is
dynamically regulated by auto-palmitoylation and depalmitoylases26,29, pointing to a possibility of
targeting the TEAD central pocket via the non-acylated form (Figure S2).
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Indeed, the non-acylated hydrophobic central pocket can be targeted by small molecules that
bind the lipidation site (Figure 1B). Nonsteroidal anti-inflammatory drugs flufenamic acid (1, Kd=
7
3 µM) and niflumic acid (2, K = 28 µM) bind the TEAD central pocket and inhibit expression of
d
3
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TEAD-YAP target genes and growth of cancer cell lines. Furthermore, a chloromethylketone
analog of flufenamic acid (3, TED-347) was developed as a covalent TEAD inhibitor that can
31
allosterically inhibit the TEAD-YAP interaction (K = 10.4 µM). Another covalent TEAD
i
inhibitor (4, K-975) showed efficacy in human malignant pleural mesothelioma xenograft
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2
models. More recently, a vinylsulfonamide derivative (5 , DC-TEADin02) was developed via
structure based virtual screening and was shown to covalently bind to TEAD in cells transfected
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3
with Flag-TEAD4. Triazole 6 efficiently inhibits TEAD palmitoylation in intestinal epithelium
3
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in vivo and compound 7 was identified via screening a DNA-Encoded Library with indole-
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focused Ugi-peptidomimetics. Furthermore, the hydroxyquinoline analog 8 was reported to bind
the central pocket, however, it activates TEAD function.36 A recently identified TEAD
palmitoylation inhibitor (9) mimics the palmitate binding mode, activates TEAD to act as a
transcriptional repressor independent from YAP and inhibits tumor growth in a xenograft model.37
Overall, the number and diversity of small molecules that target the TEAD central pocket has
been growing, and any methodology that will aid the development of novel TEAD modulators will
be invaluable.
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We describe the development of a new methodology for the identification of novel small
molecule inhibitors binding to the TEAD central pocket. By means of a newly developed “thiol
conjugation assay” we identified kojic acid derivatives that covalently target the palmitoylatable
cysteine in the TEAD central pocket and inhibit the TEAD-YAP interaction with nanomolar
apparent IC values. By means of a representative analog, we have shown the TEAD engagement
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in cell lysates and the inhibition of the expression of TEAD target gene ANKRD1. The novel
methodology in general promises to enable new medicinal chemistry programs aimed at the
discovery of covalent or non-covalent TEAD inhibitor classes.
RESULTS AND DISCUSSION
Development of a “Thiol Conjugation Assay”
In order to develop a robust, homogeneous and rapid binding assay for the TEAD central
pocket, we utilized the thiol reactive pro-fluorescent probe, N-(4-(7-diethylamino-4-
methylcoumarin-3-yl) phenyl) maleimide (CPM) that has been used for site specific protein
labeling, thermal stability analysis of membrane proteins and assaying N-acyltransferase activity.
3
8-42
The fluorescence in CPM is quenched due to the maleimide substitution on the phenyl group
that modulates the resonance between the coumarin carbonyl and 7-amino groups. However, upon
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1
reaction with a thiol, CPM fluorescence increases strongly (Figure 2A). We hypothesized that
reaction of a free cysteine residue in the TEAD central pocket would yield a fluorescence signal,
and that small molecules binding to the TEAD central pocket with appreciable potency would
prevent covalent labeling of the cysteine by CPM.
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A
B
8
102
N
O
O
HS
N
O
O
6
102
O
O
N
N
410
2
hTEAD4 (non-acylated) + CPM
CPM
S
O
O
102
2
CPM
CPM-Thiol Conjugate
0
Ex: 380 nm
Em: 470 nm
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CPM: (7-Diethylamino-3-(4'-Maleimidylphenyl)-4-Methylcoumarin)
Minutes
O
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OH
NH
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OH
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Flufenamic acid
Niflumic acid
Palmitic acid
N
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2
1
0 (IC50 = 3.2 µM)
11 (IC50 = 8.7 µM)
12 (IC50 = 43.1 µM)
O
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OH
OH
OH
-20
HO
HO
HO
-2
-1
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N
O
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O
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S
[
Compound] (Log M)
HN
OH
O
O
N
N
1
3 (IC50 > 100 µM)
14 (IC50 > 100 µM)
15 (IC50 > 100 µM)
Figure 2: Targeting the central pocket with small molecule inhibitors. (A) Thiol conjugation assay
principle. The free cysteine residue in the TEAD central pocket reacts with CPM yielding a
fluorescence signal. (B) Kinetic measurement of hTEAD4 (non-acylated) treatment with CPM
(
purple square) yields a fluorescence signal, while CPM alone (black square) has only a minimal
signal. (C) Competitive inhibition of hTEAD4 (non-acylated)-CPM with known non-covalent
central pocket binders. (D) Selected screening hits (1012) and inactive analogs (1315) with
similar structures.
For assay development, hTEAD4 was delipidated with hydroxylamine to cleave the covalent
thioester bond between the fatty acid and the cysteine (Cys367 for hTEAD4, Figure 1A right
panel). Delipidation was monitored and confirmed with whole-protein electron spray ionization
(
ESI)-mass spectrometry (Figure S3). Through expression, a mixture of the three recombinant28
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non-acylated hTEAD4 (MW: 26,495) and hTEAD4 covalently bound to either myristic acid (C14-
fatty acid, MW:26,705) or palmitic acid (C16-fatty acid, MW:26,733) (Figure S3A) was converted
to a homogeneous non-acylated hTEAD4 protein (MW: 26,495) (Figure S3B).
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Addition of the non-acylated hTEAD4 to 1 equivalent CPM yielded a significant increase in
the fluorescence signal, which reached a plateau in 30-40 min (Figure 2B). Analysis of the reaction
mixture with ESI-mass spectrometry indicated that two cysteine residues might have been labeled
with CPM (Figure S4A). Subsequent tandem mass spectrometry (MS-MS) analysis showed that
Cys367 and Cys330, both of which are located in the hTEAD4 central pocket, were covalently
modified with CPM (Figures S4B and S5). These findings suggested that monitoring of CPM
binding to the TEAD central pocket and its prevention by small molecules could be used to identify
novel TEAD central pocket targeting inhibitors.
For validation, we investigated inhibition of CPM binding and fluorescence decrease by the
known TEAD central pocket binders, flufenamic acid (1), niflumic acid (2), palmitic acid and the
covalent inhibitor 3 (TED-347). As shown in Figure 2C for non-covalent binders and in Figure S6
for TED-347, these compounds inhibited the signal increase in a dose dependent manner. To our
best knowledge, this is the first example that CPM is used for a binding assay. Since a cysteine
thiol is necessary in the reaction with CPM, we termed this novel assay as “Thiol Conjugation
Assay”.
Discovery of Small Molecule Inhibitors of the TEAD Central Pocket
Investigation of a collection of 14,000 compounds using the newly established thiol
conjugation assay identified 329 compounds, which showed > 50% inhibition at a concentration
of 12.5 µM (2.3 % primary hit rate). Kojic acid analogs 1012 (Figure 2D) were among the most
potent hits. Analogues 1315 lacking aryl substituents on the methyl amine, however, were
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inactive (Figure 2D) such that for initial structure-activity relationship (SAR) investigations a
1
3
phenyl group at both R and R positions was retained (Scheme 1). Compounds 1944 were
4
3
synthesized using a one pot Betti reaction starting from kojic acid (16), amines 17 and aldehydes
8 (Scheme 1). In addition, the 2-hydroxyl group in compounds 19 and 10 was methylated to yield
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compounds 45 and 46, respectively.
Scheme 1. Synthesis of kojic acid analogs.
O
O
O
OH
H
a
R3
OH
+
N
+
R3
R1
R2
OHC
HO
HO
O
N
R1
R2
16
17
18
10, 19-44
O
O
R4
b
10, 19
HO
R3
N
R1
R2
45-46
Reagents and conditions: (a) EtOH, rt, 2-20 days. (b) MeI, K CO , DMF, 24h, rt.
2
3
Direct binding of compounds 1946 to the TEAD central pocket was monitored by means of
a fluorescence polarization (FP)-based competitive binding assay employing a FITC-labelled
palmitate tracer (Scheme S1), which bound with K = 28 nM to non-acylated hTEAD4 (Figure
d
S7A). The FP assay was validated employing the previously reported competitive inhibitor
niflumic acid (2) as a positive control (Figure S7B).
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
IC values obtained from the thiol conjugation assay and the FP-based competitive binding
5
0
assay are shown in Table 1. Not surprisingly, due to the different assay principles (i.e. competition
with the covalent cysteine binder CPM, versus competition with non-covalent binder FITC-
palmitate) and protein concentrations, the IC values obtained from the FP and thiol conjugation
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
assays differ for all kojic acid analogues, consistently 5-10 fold.
1
2
Initial SAR investigations focused on modifications of amine substituents R and R (Table
1
). Replacement of the phenyl group in 19 by an isopropyl group (20) led to a 7-fold increase in
IC values for both assays. Although, a benzyl group (21) was slightly (2-fold) less favorable than
5
0
the phenyl group (19), the introduction of the constrained bis-benzyl moiety in 22 lowered the
1
affinity nearly 5-fold. A fluorine group at the meta-position of R (23) did not influence TEAD
binding, and introduction of an electron deficient 2-pyridine ring (24) gave a similar IC value as
5
0
for a phenyl group (19). Substitution of the 2-pyridine ring (25) with a methyl group on the 5-
position also did not alter the binding. However, the introduction of 2-pyrimidine- (26), 2-thiazole-
(
27) and 2-benzimidazole (28) groups resulted in more than 20-fold increase in the IC values.
50
1
Equipment of the phenyl ring at R with an electron withdrawing 4-carboxamide (29) did not
influence the affinity.
3
Replacement of the benzylic phenyl group at the R position with an aliphatic cyclohexyl-
(
30) or isopropyl group (31) or with a hydrogen (32) dramatically (100-fold) reduced TEAD
binding in the thiol conjugation assay showing the importance of an aromatic residue at the R3
position. Introduction of an electron withdrawing carboxamide substituent at the para-position
(
33) slightly (2-fold) improved binding. Hydrophobic substituents at the para-position (3438)
did not change binding significantly, and similarly introduction of a fluorine group (39) at the
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ortho-position was tolerated. Replacement of the phenyl ring with an electron deficient 2-pyridine
group (40), however, lowered the inhibitory activities by 2-3-fold in both assays.
1
3
Additional analogs with 2-pyridine residues at R and R (4144), in particular the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
combination of two 2-pyridines at both R and R positions (43) lowered the affinity significantly.
4
The importance of a 2-OH group at the R position was proven by investigation of compounds
45 and 46, i.e. the 2-OMe analogs of compounds 19 and 10, respectively. Both compounds were
inactive in the thiol conjugation assay and had > 200-fold lower affinity in the FP assay, showing
the critical influence of the 2-OH group for TEAD binding (Figure 3A-3C).
We additionally confirmed the TEAD binding of selected analogs (19, 27, 42, 45) using
differential scanning fluorimetry (Figure 3D). Compound 19 lowered the melting point (Tm) of
1
3
non-acylated hTEAD4 by 5 °C, and its 2-pyrido (R ), 4-bromo (R ) analog (42) induced a smaller
Tm shift (-2 °C) aligning with the binding experiments (Table 1). Consistent with the competitive
binding experiments, compounds 27 and 45 did not induce any change in the Tm of non-acylated
hTEAD4.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. SAR studies of the kojic acid analogs.
O
O
R4
N
HO
R3
R2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R1
Apparent IC (µM)
50
Compound
ID
R1
R2
R3
R4
Thiol
Fluorescence
Conjugation
Polarization
-H
19
-OH
-OH
2.3 ± 0.3
0.2 ± 0.04
2
0
-H
-H
16.8 ± 0.9
1.5 ± 0.2
NH
21
-OH
-OH
4.0 ± 0.6
0.4 ± 0.03
2
2
13.2 ± 2.7
0.9 ± 0.3
F
2
2
2
2
2
3
4
5
6
7
-H
-H
-H
-H
-H
-OH
-OH
-OH
-OH
-OH
1.5 ± 0.4
3.1 ± 0.6
2.4 ± 0.3
> 100
0.2 ± 0.05
0.3 ± 0.03
0.3 ± 0.08
13.4 ± 3.3
5.6 ± 0.9
N
N
N
N
S
53.4 ± 11.5
N
N
28
29
-H
-OH
> 100
6.4 ± 0.9
NH
O
-H
-H
-OH
-OH
1.7 ± 0.2
> 100
0.3 ± 0.09
22.6 ± 2.8
NH2
3
0
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3
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1
2
-H
-H
-OH
> 100
> 100
38.1 ± 8.4
H
-OH
36.4 ± 11.9
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NH2
33
-H
-OH
1.5 ± 0.2
0.3 ± 0.1
3
3
3
4
5
6
-H
-H
-H
-OH
1.3 ± 0.3
1.9 ± 0.2
1.2 ± 0.4
0.2 ± 0.03
0.3 ± 0.01
0.2 ± 0.06
F
-OH
Br
-OH
3
3
7
8
-H
-H
-H
-H
-OH
-OH
1.0 ± 0.3
1.1 ± 0.3
0.7 ± 0.1
18.9 ± 6.2
0.2 ± 0.04
0.2 ± 0.05
0.1 ± 0.03
0.5 ± 0.1
39
-OH
F
4
0
-OH
N
41
O
-H
-OH
9.2 ± 2.1
1.1 ± 0.2
N
NH2
Br
4
2
-H
-H
-OH
3.7 ± 0.1
3.2 ± 0.6
0.4 ± 0.06
0.3 ± 0.03
N
N
10
-OH
F
4
4
4
3
4
5
-H
-H
-H
-OH
> 100
> 100
> 100
16.0 ± 2.6
14.7 ± 3.9
41.6 ± 7.3
N
N
N
-OH
N
-OCH3
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
6
-H
-OCH3
> 100
49.9 ± 4.8
5.9 ± 1.1
N
F
HO
O
H
N
CF3
2
41.9 ± 10.5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
O
Cl
H
3
N
CF3
0.8 ± 0.4
14.1 ± 4.0
(
TED-347)
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A
B
C
O
O
R
O
1
00
50
0
TC
FP
100
50
0
1
9
19
45
HO
45
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R
NH
19 -H
4
5
-CH3
-50
-50
-3
-2
-1
0
1
2
-3
-2
-1
0
1
2
[
Compound] (Log M)
[Compound] (Log M)
D
1
00
O
O
1
9
(Tm: 36C)
OH
NH
Br
OH
0
42
(Tm: 39 C)
(Tm: 41C)
(Tm: 41C)
HO
HO
O
O
2
7
S
NH
4
5
-
100
200
N
N
DMSO (Tm: 41C)
42
27
-
0
20
40
60
80
100
Temperature
Figure 3: Target engagement studies with compound 19 and its inactive analog 45 in cell-free
systems. (A) Structures of the compounds 19 and 45. Representative inhibition curves in the (B)
thiol conjugation assay, and (C) FP-based competitive binding assay. (D) Differential scanning
fluorimetry measurements of hTEAD4 (non-acylated) with selected derivatives.
Covalent Binding to the TEAD Central Pocket
In light of the observation that a non-modified, acidic 2-OH group in kojic acid (pKa = 7.9) 44
is required for activity, we reasoned that the compounds of type 19 might undergo a retro-Mannich
reaction to yield a Michael acceptor, which then could covalently react with Cys367 in non-
acylated hTEAD4 (Figure 4A). In agreement with this hypothesis, methylation of the 2-OH group
(
45, 46) led to compounds inactive in the thiol conjugation assay (Figure 3B, Table 1). Indeed,
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
treatment of non-acylated hTEAD4 with 8-equivalents of compound 19 or 45, and subsequent
analysis of the mixture with whole protein ESI-MS spectrometry revealed that- consistent with our
hypothesis- modification of the protein (+230 Da) was observed upon incubation with 19 (Figure
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
A-4B), while modification of TEAD was not observed upon co-incubation with 45 (Figure 4C).
The hypothesis that compound 19 specifically targets Cys367 that is lipidated under
physiological conditions was further strengthened by the observation that incubation of 19 with
acylated (i.e. not treated with hydroxylamine) hTEAD4 did not result in formation of an acylated
hTEAD4-19 adduct (Figures 4D and S3A).
In addition, mutants of mouse TEAD4 (mTEAD4) in which the reactive cysteine was replaced
with alanine or serine and that should not react with compound 19, were investigated in the thiol
conjugation assay. As shown in Figure 4E, compound 19 cannot inhibit an increase in the
fluorescence signal when the Ser or Ala mutant of mTEAD4 is used. Collectively, these
observations show that kojic acid analog 19 covalently and specifically binds non-acylated
TEAD4.
To confirm the covalent reaction between compound 19 and non-acylated hTEAD4, thiol
conjugation and fluorescence polarization assays were used to determine the time-dependent
inhibition (TDI) of hTEAD4. Compound 19 showed a low-micromolar inhibition constant (K =
i
2.6 µM), which describes the potency of reversible binding between the compound and the protein
before the covalent bond formation (Figure S8A-S8B). Compound 19 inhibited non-acylated
hTEAD4 with kinact/K = 8.3 µM in the thiol conjugation assay and with k /K = 4.0 µM in the
i
inact
i
FP assay (Figures S8B and S8D).
Investigation of the specificity of compound 19 for binding to TEAD homologs by means of
the thiol conjugation assay showed that 19 inhibits hTEAD14 with similar low micromolar IC50
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values (2.6-6.9 µM, Figure S9) consistent with the high homology (72.0-87.9 %) of the
transactivation binding domains of TEAD14.
TEAD (non-acylated)
O
A
H
N
NH2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
O
O
O
OH
S
OH
NH
O
SH
HO
HO
HO
H+
N
H
O
19
+
230
B
hTEAD4 (non-acylated) + Compound 19
C
hTEAD4 (non-acylated) + Compound 45
+
230
hTEAD4 (non-acylated)
O
O
CH3
O
hTEAD4 (non-acylated)
HO
NH
45
D
hTEAD4 (acylated) + Compound 19
E
Protein
+ CPM
Protein + Compound 19 + CPM
600
400
200
0
+
+
Myristic acid (C14 fatty acid)
Palmitic acid (C16 fatty acid)
CPM
hTEAD4 (non-acylated)
4
4
)
4
a
4
d
D
D
e
D
)
D
r
e
l
a
A
A
lat
A
ted
A
E
A
0
6
E
E
E
S
T
T
y
T
T
0
h
c
h
l
m
6
3
m
3
a
y
c
s
-
s
y
n
a
y
o
(
C
C
n
(
Figure 4. Mechanism of action studies with kojic acid analogs. (A) Hypothesized mechanism of
action of compound 19. Whole protein ESI-MS analysis of hTEAD4 (non-acylated) treatment with
kojic acid analog (B) compound 19 and (C) compound 45. Compound 19 is a covalent binder for
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
nd
non-acylated hTEAD4 and the -OH group at the 2 position is required for the predicted reaction
with a thiol group. The -OMe analog (45) is not a covalent TEAD binder. (D) Whole protein ESI-
MS analysis of hTEAD4 (acylated) incubation with 19. No protein adduct with compound 19 was
observed when Cys367 in hTEAD4 is lipidated. (E) Comparison of thiol conjugation inhibition
with 10 µM compound 19 in the presence of hTEAD4 (non-acylated), hTEAD4 (acylated) or Cys
to Ala/Ser mutants of mTEAD4. When the cysteine residue is replaced with alanine or serine,
compound 19 cannot inhibit the thiol conjugation.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Binding Mode Analysis
1
15
To get insight into the binding mode and binding site of the new covalent inhibitors, H- N-
HSQC spectra of mTEAD4 in the absence and presence of compound 19 or 45 were recorded, and
4
5
signals were assigned based on the corresponding analysis of the transactivation domain of
mTEAD4. Compound 19, induced line broadening of several residues (Figure S10A), including
Phe386 (F386) and Phe408 (F408). These amino acids are in close proximity to the palmitoylation
site. Conversely, the negative control 45 did not induce a significant change compared to the
DMSO control (Figure S10B). These findings support the biochemical data suggesting that
compound 19 binds the central pocket in TEAD.
Covalent TEAD Binders Inhibit the TEAD-YAP Interaction
The chloromethyl ketone flufenamic acid analog (3, TED-347) that binds the TEAD central
pocket covalently, allosterically inhibits TEAD and YAP interaction and consequently YAP
3
1
mediated target gene expression. In order to determine whether the new inhibitor class can also
disrupt this protein-protein interaction, a fluorescence polarization assay that monitors the binding
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of fluorescently-labelled YAP peptide to non-acylated hTEAD4 was used. Pre-incubation with
compound 19 for 10 min potently inhibited the interaction of non-acylated hTEAD4 with human
YAP50-100 with an IC value of 70 nM. In contrast, and in agreement with the covalent inhibition
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mechanism proposed above, the OMe analog 45 had > 100-fold lower activity (IC = 9.5 µM)
5
0
(Figure 5A), and covalent inhibitor 19 did not inhibit the interaction of YAP with acylated
hTEAD4 and mTEAD4 with Cys360Ser or Cys360Ala mutations (Figure 5B-5D) (saturation
binding curves of FITC-YAP50-100 with the TEAD4 proteins are shown in Figure S11A-D).
3
1
Overall, these results are in accordance with previous findings for covalent allosteric inhibition
of TEAD and show that the covalent and potent TEAD central pocket binding small molecules
identified here can block the TEAD-YAP interaction efficiently.
A
B
hTEAD4 (non-acylated)-YAP interaction
hTEAD4 (acylated)-YAP interaction
100
100
5
0
50
0
0
-50
-50
-4
-3
-2
-1
0
1
2
-4
-3
-2
-1
0
1
2
[Compound] (Log M)
[Compound] (Log M)
C
D
mTEAD4 _Cys360Ser-YAP interaction
mTEAD4 _Cys360Ala-YAP interaction
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Figure 5. Inhibition of TEAD-YAP interaction as determined with an FP-based competitive
binding assay using FITC labeled hYAP50-100. Compound 19 (blue triangle) and compound 45
(black square) were tested. (A) Compound 19 can prevent YAP interaction with hTEAD4 (non-
acylated), but not with (B) hTEAD4 (acylated), mTEAD4 with (C) Cys360Ser or (D) Cys360Ala
mutation.
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TEAD Engagement in Cellular Environment
Target engagement in HCT116 cell lysates was proven by means of a Cellular Thermal Shift
Assay (CETSA). Treatment of the lysates with compound 19 for 10 mins increased the melting
temperature of hTEAD4 from 49.8 °C to 53.1 °C (Tm = 3.3 °C), providing evidence for the
interaction with hTEAD4 in a biologically relevant environment.
TEADs, together with the coactivators (e.g. YAP), regulate the expression of genes involved in
2
critical processes as cardiogenesis, cell growth and migration. Small molecules that bind the
TEAD central pocket have been shown to modulate the TEAD transcriptional activity, such as,
inhibition of the expression of well-established TEAD target genes (i.e. ANKRD1, CTGF and
CYR61).34,37 To evaluate the influence of kojic acid analogs on TEAD transcriptional activity,
HEK293 cells were incubated with compound 19, negative control 45 or TED-347. Treatment with
compound 19 for 6 h resulted in 90 % reduction of the mRNA level of the TEAD target gene
ANKRD1 compared to the DMSO control, while compound 45 had only a minimal effect at the
same concentration, further supporting the on target activity of the kojic acid analogs.
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A
B
36.9
40.1 44.1 47.9 58.0 51.0 54.4
61.3
64.5 67.0
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DMSO
hTEAD4 + 19,
Tm: 53.1
hTEAD4 + DMSO, Tm: 49.8
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ANKRD1
C
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Temperature [C]
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)
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µM
µM
M
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-
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E
T
Figure 6. TEAD inhibitory activity of 19 in cellular environment. (A, B) Target engagement in
HCT116 cell lysates. (A) Representative immunoblots of hTEAD4 treated with vehicle control
(
DMSO) or compound 19. (B) CETSA melting curves of hTEAD4 treated with vehicle control
(DMSO) or compound 19. (C) Inhibition of TEAD target gene ANKRD1 expression by compound
9 in comparison to the control compounds 45 and TED-347.
1
CONCLUSION
We have developed a new “thiol conjugation assay” that enables the systematic identification of
small molecule inhibitors of the TEAD central pocket. By means of this assay, kojic acid analogs
were identified as a new class of TEAD inhibitors. Our data show that these inhibitors covalently
bind to the Cys residue in the TEAD central pocket that is S-acylated under physiological
conditions. The representative kojic acid analog 19 inhibited the interaction of YAP with non-
acylated hTEAD4, but not with the corresponding acylated protein and mutants, in which the Cys
had been replaced by a serine or an alanine. Furthermore, CETSA in lysate provided evidence for
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compound 19 interacting with hTEAD4 in a biologically relevant medium. Consistent with the
binding experiments, kojic acid analog 19 inhibited TEAD target gene expression. Taken together
we have established a novel methodology that could be a guide for the development of novel
classes of TEAD inhibitors interacting with the protein covalently or non-covalently.
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EXPERIMENTAL SECTION
Plasmids. hTEAD4 plasmid was isolated from cDNA clone of hTEAD4 (Dharmacon, clone ID:
3
913870). Plasmid with mTEAD4 with Cys360Ala mutant was obtained through site-directed
mutagenesis using mTEAD4 wild-type template (Dharmacon, clone ID: 40142595). All plasmids
were verified by DNA sequencing.
Cloning, expression and purification of recombinant proteins. Sequences of the proteins used
in the study are shown in Table S1.
2
17-434
N-His hTEAD4: Plasmid of N-terminal His -tagged hTEAD4
was subcloned into pOPIN-
6
neo vector, which was used to transform E. coli BL21-CodonPlus (DE3)-RIPL (Agilent, 230280).
The transformed cells were grown in TB medium supplemented with 0.01 % alpha-lactose
monohydrate, 2 mM MgSO , 100 µg/ml ampicillin and 50 µg/ml chloramphenicol at 37 C for 4
4
h. Then the temperature was reduced to 25 C and the culture was incubated for a further 20-24 h.
The cells were harvested, and the pellets were taken in lysis buffer containing 50 mM Hepes, 300
mM NaCl, 20 mM imidazole, 1 mM TCEP, pH 8 and lysed by sonication followed by
ultracentrifugation. Then the cleared lysate was loaded on to the ÄktaXpress (GE Healthcare) for
affinity purification (His trap FF, GE Healthcare) which was followed by size exclusion
chromatography (SD75 26/60, GE Healthcare) using 20 mM HEPES, 100 mM NaCl, 1 mM TCEP,
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mM MgCl , 5 % glycerol, pH 8. The purified protein was concentrated using Amicon Ultra
2
Centrifugal Filters, 10K (Merck), aliquots were snap frozen and stored at -80 C.
N-His mTEAD4_Cys360Ser: A method provided earlier was used for the protein expression using
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45
1
mM IPTG. The harvested cell pellets were lysed, and the protein was purified as described
above for N-His hTEAD4.
2
09-427
N-His-MBP mTEAD4_Cys360Ala: cDNA of N-terminal His - and MBP-tagged mTEAD4
6
with Cys360Ala mutation was used to transform E. coli BL21-CodonPlus (DE3)-RIPL (Agilent,
30280). The protein was expressed, and the collected pellets were lysed as described for N-His
2
hTEAD4 above. The cleared lysate was loaded on to the ÄktaXpress (GE Healthcare) for affinity
purification (His trap FF, GE Healthcare) which was followed by size exclusion chromatography
(SD200 16/60, GE Healthcare) using 25 mM HEPES, 150 mM NaCl, 1 mM TCEP, pH 7.2. The
purified protein was concentrated using Amicon Ultra Centrifugal Filters, 50K (Merck), aliquots
were snap frozen and stored at -80 C. The storage buffer was exchanged with pH 8 Tris-HCl (20
mM), NaCl (100 mM) MgCl (2 mM) using Amicon Ultra Centrifugal Filters (10K) for the binding
2
studies.
2
09-426
N-His-GST hTEAD1: Plasmid of N-terminal His -tagged GST-hTEAD1
was subcloned into
pOPIN-neo vector, which was used to transform E. coli BL21-CodonPlus (DE3)-RIPL (Agilent,
30280). The transformed cells were grown in TB medium supplemented with 2 mM MgSO , 100
6
2
4
µg/ml ampicillin and 50 µg/ml chloramphenicol at 37 C until the optical density reached 0.6.
Then the temperature was reduced to 18 C and the culture was incubated for a further 16 h. The
cells were harvested, and the pellets were taken in lysis buffer containing 50 mM Hepes, 300 mM
NaCl, 20 mM imidazole, 1 mM TCEP, pH 8 and lysed by sonication followed by
ultracentrifugation. Then the cleared lysate was loaded on to the ÄktaXpress (GE Healthcare) for
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affinity purification (His trap FF, GE Healthcare) which was followed by size exclusion
chromatography (SD75 26/60, GE Healthcare) using 20 mM HEPES, 100 mM NaCl, 1 mM TCEP,
2 mM MgCl , 5 % glycerol, pH 8. The purified protein was concentrated using Amicon Ultra
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Centrifugal Filters, 10K (Merck), aliquots were snap frozen and stored at -80 C.
2
17-447
N-His hTEAD2: Plasmid of N-terminal His -tagged hTEAD4
was subcloned and the protein
6
was expressed as described for N-His hTEAD4 above.
216-434
N-His hTEAD3: N-terminal His -tagged hTEAD4
was expressed using the protocol
6
described for hTEAD4 earlier.36
Deacylation (depalmitoylation) of TEAD. To 500 µl 105 µM (2.8 mg/ml) recombinant hTEAD4
(
acylated) in an eppendorf tube, 30 µl of freshly prepared 105 mM hydroxyl amine solution was
added slowly and the mixture was gently shaken at room temperature for 3-4 h. The deacylation
process was monitored with whole protein ESI-Mass spectrometry. The protein was then purified
over two consecutive size exclusion columns (i) Amicon, 0.5 ml 10K (Merck) ii) Zebaspin, 7K
(
Thermo Fisher) using Tris (20 mM pH 8), NaCl (100 mM), MgCl (2 mM) as the final storage
2
buffer. 5 µL aliquots were snap frozen and stored at -80 °C.
4M hydroxylamine solution: 0.46 g (6.67 mmol) hydroxylamine HCl was dissolved in 0.53 ml of
H O in a glass vial. Then, 0.33 ml of diethylamine was added dropwise, followed by additional
2
0
.2 diethylamine and 0.63 ml H O addition. Freshly prepared 4M hydroxylamine solution was
2
further diluted to 105 mM with MilliQ water.
Binding assays. All the experiments were performed using black, low volume, round-bottom, non-
binding, 384-well plate plates (Corning 4514). The plates were imaged using Tecan Spark.
Spectramax Paradigm (Molecular Devices) was used for imaging the plates during low-throughput
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screening. 10 mM stock solutions of the compounds in DMSO were used. The K of the tracers
d
and the IC value of the inhibitors were calculated using GraphPad Prism 7 software.
5
0
Binding assays for TEAD central pocket:
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a) Thiol conjugation assay: To serially diluted solutions of the tested compounds in 5 µl assay
buffer (Hepes (25 mM), NaCl (150 mM) pH 6.5), freshly prepared 5 µl 0.45 µM protein solution was
added and incubated at room temperature for 10 mins followed by 5 µl of 0.45 µM CPM solution
addition, giving a 15 µl final assay volume, 150 nM protein and 150 nM CPM. The assay plate
was shaken at room temperature for 1h before measuring the fluorescence (Ex/Em: 380/470 nm).
Compound 19 was used as an internal standard to determine 100 % inhibition.
b) Thiol conjugation assay for low-through-put screening: A library of 14,000 compounds were
screened in 1536-well plates with using pH 6.5 MES buffer. To the pre-incubated solution of non-
acylated hTEAD4 (0.15 µM final concentration) and compound solution (12.5 µM final
concentration), CPM (0.15 µM final concentration) was added, and the assay plates were incubated
at room temperature for 50 mins before measuring the fluorescence (Ex/Em: 360/465 nm) using
Spectramax Paradigm (Molecular Devices). Z’ = 0.6-0.8 (10-plates).
c) Saturation binding experiment: To a serially diluted solution of non-acylated hTEAD4 in 12
µl assay buffer (Hepes (25 mM), NaCl (150 mM) pH 6.5), 8 µl of 25 nM of FITC-Palmitate solution
was added, giving 20 µl final assay volume with 10 nM tracer. The assay plate was incubated at
room temperature for 1h before measuring fluorescence polarization (Ex/Em: 485/535 nM).
d) FP-based competitive inhibition experiments: To serially diluted solution of the tested
compounds in 5 µl assay buffer (Hepes (25 mM), NaCl (150 mM) pH 6.5 buffer), freshly prepared 5
µl 0.15 µM protein solution was added and incubated at room temperature for 10 mins followed
by 5 µl of 30 nM FITC-Palmitate solution addition, giving 15 µl final assay volume, 50 nM protein
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and 10 nM tracer. The assay plate was shaken at room temperature for one hour before measuring
the fluorescence polarization (Ex/Em: 485/535 nm).
Binding assays for TEAD-YAP interaction: All the TEAD-YAP binding experiments for were
performed in PBS (pH 7.5).
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a) Saturation binding experiments: To a serially diluted solution of the protein in 12 µl PBS, 8
µl of FAM-YAP solution was added giving 20 µl final assay volume. The assay plate was
incubated at room temperature for 1h before measuring fluorescence polarization (Ex/Em: 485/530
nM). The final FAM concentrations are as following: hTEAD4 (non-acylated): 2 nM, hTEAD4
(acylated): 2 nM, mTEAD4_Cys360Ser: 10 nM, mTEAD4_Cys360Ala: 10 nM.
b) FP-based competitive inhibition experiments: To serially diluted solution of the tested
compounds in 5 µl PBS, freshly prepared 5 µl protein solution was added and incubated at room
temperature for 10 mins followed by 5 µl of FAM-YAP50-100 solution addition. The assay plate
was shaked at room temperature for one hour before measuring the fluorescence polarization
(Ex/Em: 485/535 nm). Final protein and FAM concentrations are as following (protein/FAM):
hTEAD4 (non-acylated): 5 nM/2 nM, hTEAD4 (acylated): 3 nM/15 nM, mTEAD4_Cys360Ser:
1
0 nM/50 nM, mTEAD4_Cys360Ala: 10 nM/50 nM.
Inhibition efficiency experiments: Time-dependent inhibition of hTEAD4 (non-acylated) by
compound 19 was monitored in FP (central pocket binding) and thiol conjugation assays at 5-7
different concentration (0.15-40 µM) following 1, 2, 4, and 24 h incubation at 4°C (mean ± SD; n
= 2). By fitting the percent of inhibition versus preincubation time at different concertation, kobs
was calculated. Next, kobs at was plotted against concentration and was further fitted to a non-linear
regression model to determine the kinetic parameters of covalent inhibition of hTEAD4, where
-1
plateau is showing kinact (min ) and t is K (µM).
1/2
i
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Differential Scanning Fluorometry (DSF). Non-acylated hTEAD4 (20 μM) was incubated in the
absence or presence of inhibitors (80 μM) at 25°C for 10 min. Then, SYPRO orange (Sigma
Aldrich) at a final concentration of 5x was added and the mixture was heated at a ramp rate of
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°C/min in a qPCR machine (Applied Biosystems). Derivative of the SYPRO orange fluorescent
intensity is plotted as a function of temperature.
Whole protein ESI-mass spectrometry. The protein solution (1 µl 5 µM) was injected to UPLC-
MS (ThermoScientific_UltiMate 3000 LC coupled to a Velos Pro MS) through a desalting column
(MassPrepTM Online desalting, 2.1×10 mm). For reactions, 1 µM of the protein was incubated at
room temperature for one hour with 8 equiv. of the compound in Hepes (25 mM), NaCl (150 mM)
pH 6.5 buffer. Then, the mixture was centrifuged before injecting 5 µl. The deconvolution of m/z
values was performed using Promass Xcalibur software.
Tandem mass spectrometry experiments
Non-acylated hTEAD4-CPM reaction: To 5 µl assay buffer (Hepes (25 mM), NaCl (150 mM)
pH 6.5), 5 µl 0.6 µM non-acylated hTEAD4 and 5 µl 0.6 µM CPM solution was added and
incubated at room temperature for 1h in a 384-well plate (Corning 4514). The reaction mixtures
were collected from 16-replicates and transferred to a non-binding eppendorf tube followed by co-
incubating with 10 µl 60 µM DTT for 30 mins to quench the unreacted CPM. The protein mixture
was then purified through Amicon (10K) to exchange the buffer with 50 mM Tris-HCl (pH 7.5).
In solution digestion and purification: To 50 µl of the protein sample in 50 mM Tris-HCl (pH
7.5), 250 µl denaturizing/reducing buffer (10 M urea, 1.2 mM TCEP, 50 mM Tris, pH 7.5) was
added and shaken at room temperature (350 rpm, 30 mins). Next, 33.3 µl 50 mM alkylation
solution (iodoacetamide prepared in denaturizing/reducing buffer) was added and shaken (350
rpm) at room temperature for 30 mins. Subsequently, 23.5 µl of 1 ng/µl solution of LysC (Wako,
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25-05061) was added at 37 C, and shaken (350 rpm) for 1h, followed by 7.8 µl of 6 ng/ µl
Trypsin (Roche, 03708969001) addition. The mixture was shaked (350 rpm) at 37 C overnight).
The digested samples were stage tip purified using C18 (octadecyl) tips (3M High Performance
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Extraction Discs, Empore) using a protocol described earlier. Bovine serum albumin (BSA) (50
µl, 0.5 µM prepared in 50 mM Tris-HCl, pH 7.5) was used in parallel as a control for the digestion-
purification experiments and treated as described above. The peptide solution was injected to
nanoLC-MS (ThermoScientific_UltiMate 3000 nanoLC coupled to a QExactive Plus or QExactive
HF MS) through a nano column (ThermoScientific PEPMAP C18 2UM 75UMX500MM).
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mTEAD4 (non-acylated) HSCQ Experiments. The C/ N-labeled mTEAD4 was prepared
45
following a protocol established earlier, and the protein was deacylated as described above for
1
15
hTEAD4. H- N-HSQC spectra in the presence of DMSO or the test compound (0.5 mM) were
collected at 37 °C on a Bruker 600 MHz magnet equipped with a cryo-probe in 20 mM HEPES,
pH 7.3 buffer containing 150 mM NaCl and 1 mM DTT. The spectra were processed and overlaid.
Cellular Thermal Shift Assay (CETSA) Experiments
Lysate preparation: HCT116 cells were grown in DMEM (Sigma Aldrich, #P04-03550)
containing 10% FBS, 1 x NEAA (Sigma Aldrich, # P08-32800) and 1 mM sodium pyruvate (Sigma
Aldrich, #S8636) until they reached 90% confluency. Cells were detached, transferred to two
separate tubes and washed three times with PBS. Then 0.6 mL PBS containing 0.04% NP-40 were
added to each tube and cells were lysed by means of freeze and thaw. Lysates were centrifuged
(Beckman Optima MAX-TL) at 100,000 g, 4°C for 25 min and protein concentration was
determined using the DC™ Protein Assay Kit I (BioRad, #5000111). 1.5 mL of aliquots containing
1 mg/mL protein were stored at -80°C until further usage.
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Page 29 of 61
Journal of Medicinal Chemistry
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CETSA: 50 µM of compound 19 (2.75 µL 10 mM stock) or DMSO (2.75 µL) were added to 550
µL of HCT116 lysates (1 mg/mL), samples were mixed and incubated for 10 min at room
temperature. Treated and non-treated lysates were divided into ten aliquots, each 50 µL in PCR
tubes. The aliquots were individually heated at different temperatures (Eppendorf Mastercycler ep
Gradient S). After the heat treatment the cell lysates were directly, completely transferred to
polycarbonate tubes and centrifuged (Beckman Optima MAX-TL) at 100,000 g, 4°C for 25 min.
To 16 µL of each supernatant was added 4 µL of 5x loading buffer and incubated for 5 min at
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5°C, before samples were analyzed by Immunoblotting. Proteins were separated by SDS-PAGE,
and transferred to PVDF membrane using wet transfer. The membranes were blocked with
Odyssey Blocking Buffer (TBS; Li-Cor) for 1 h and incubated with the primary antibody (Mouse
Anti-TEAD4 antibody corresponding to amino acids 151-261 of Human TEAD4 - ChIP Grade
(ab58310)) in Odyssey blocking buffer containing 0.2% Tween-20 at 4°C overnight. After
washing with TBS-T (TBS containing 0.1 % Tween-20) the membrane was incubated with the
secondary antibody coupled to IRDye® 800CW (goat anti-mouse IgG, Li-COR) for 1 h, in
Odyssey blocking buffer containing 0.2 % Tween-20 and 0.1 % SDS at room temperature.
Membranes were washed with TBS-T, then TBS before images were taken (Bio-Rad ChemiDoc™
MP Imaging System).
Analysis: Images were analyzed with ImageJ (FUJI). Normalization and all calculations were done
with GraphPad Prism 7 software. (Curves: IC variable slope  turning points correspond to
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melting temperature in obtained curves).
RNA purification and RT-qPCR. HEK293 cells were treated with DMSO or the indicated
compound for the indicated duration. RNA was extracted using the RNeasy Plus Mini Kit
(QIAGEN, Hilden, Germany) following the manufacturer’s procedure. DNA was removed on
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