Small-Molecule Covalent Modification of Conserved Cysteine Leads to Allosteric Inhibition of the TEAD?Yap Protein-Protein Interaction

Article

Small-Molecule Covalent Modiﬁcation of Conserved

Cysteine Leads to Allosteric Inhibition of the

TEAD,Yap Protein-Protein Interaction

Graphical Abstract

Authors

Khuchtumur Bum-Erdene,

Donghui Zhou,

Giovanni Gonzalez-Gutierrez, ...,

Karen E. Pollok, Clark D. Wells,

Samy O. Meroueh

Correspondence

smeroueh@iu.edu

In Brief

A small molecule that forms a covalent

bond with a conserved cysteine within the

palmitate binding pocket of TEADs was

found to inhibit the TEAD,Yap protein-

protein interaction through allostery. The

compound inhibited TEAD transcriptional

activity in mammalian cells and blocked

patient-derived glioblastoma cell

viability.

Highlights

d

Small molecules form a covalent bond with palmitate

cysteine

Covalent engagement of cysteine inhibited TEAD4,Yap1

protein-protein interaction

Inhibition of TEAD4,Yap1 in mammalian cells blocked TEAD

transcriptional activity

Small-molecule inhibition of TEAD4,Yap1 inhibited

glioblastoma cell viability

Bum-Erdene et al., 2019, Cell Chemical Biology 26, 1–12

March 21, 2019 ª 2018 Elsevier Ltd.

https://doi.org/10.1016/j.chembiol.2018.11.010

Please cite this article in press as: Bum-Erdene et al., Small-Molecule Covalent Modiﬁcation of Conserved Cysteine Leads to Allosteric Inhibition of the

TEAD,Yap Protein-Protein Interaction, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.11.010

Cell Chemical Biology

Article

Small-Molecule Covalent Modiﬁcation of Conserved

Cysteine Leads to Allosteric Inhibition

of the TEAD,Yap Protein-Protein Interaction

Khuchtumur Bum-Erdene,¹Donghui Zhou,¹Giovanni Gonzalez-Gutierrez,²Mona K. Ghozayel,¹Yubing Si,¹David Xu,³

Harlan E. Shannon,⁴Barbara J. Bailey,⁴Timothy W. Corson,^1,5Karen E. Pollok,⁴Clark D. Wells,¹and Samy O. Meroueh^1,6,

¹Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis,

IN 46202, USA

*

²Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA

³Department of BioHealth Informatics, Indiana University School of Informatics and Computing, Indianapolis, IN 46202, USA

⁴Department of Pediatrics, Herman B. Wells Center for Pediatric Research, Indiana University Simon Cancer Center, Indiana University

School of Medicine, Indianapolis, IN 46202, USA

⁵Eugene and Marilyn Glick Eye Institute, Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, IN 46202, USA

⁶Lead Contact

*Correspondence: smeroueh@iu.edu

https://doi.org/10.1016/j.chembiol.2018.11.010

SUMMARY

rounding extracellular matrix are factors that promote Yap/TAZ

(Aragona et al., 2013) to enter the nucleus where they co-activate

The Hippo pathway coordinates extracellular signals TEA domain (TEAD) transcription factors (Hong and Guan, 2012;

Mauviel et al., 2012; Pobbati and Hong, 2013; Yu and Guan,

2013). In mammals, there exist four highly conserved TEAD

onto the control of tissue homeostasis and organ

size. Hippo signaling primarily regulates the ability

of Yap1 to bind and co-activate TEA domain (TEAD)

transcription factors. Yap1 tightly binds to TEAD4

via a large ﬂat interface, making the development of

small-molecule orthosteric inhibitors highly chal-

lenging. Here, we report small-molecule TEAD,Yap

inhibitors that rapidly and selectively form a covalent

(also known as TEF) transcription factors, namely TEAD1–4 (El-

dridge et al., 1997). The average sequence identity among

TEADs is 73%, which is considered high (Noland et al., 2016).

TEADs possess similar domains: an N-terminal DNA binding

TEA/ATTS domain (Anbanandam et al., 2006) and a C-terminal

immunoglobulin-like b-sandwich fold (Pobbati et al., 2012).

TEADs alone cannot initiate gene expression; they rely on co-ac-

tivators such as Yap and its paralog TAZ, as well as vestigial-like

proteins (VGLL), and the p160 family of nuclear receptor co-ac-

bond with a conserved cysteine located within the

unique deep hydrophobic palmitate-binding pocket

of TEADs. Inhibition of TEAD4 binding to Yap1 by tivators (Hong and Guan, 2012; Pobbati and Hong, 2013). Activa-

tion of TEADs initiates expression of CCN matricellular growth

factors, such as connective tissue growth factor (CTGF),

these compounds was irreversible and occurred on

a longer time scale. In mammalian cells, the com-

pounds formed a covalent complex with TEAD4,

inhibited its binding to Yap1, blocked its transcrip-

tional activity, and suppressed expression of

connective tissue growth factor. The compounds in-

hibited cell viability of patient-derived glioblastoma

spheroids, making them suitable as chemical probes

Cyr61, epidermal growth factor receptor ligand amphiregulin,

and Axl receptor tyrosine kinase (Fridell et al., 1996; Mauviel

et al., 2012; Piccolo et al., 2013; Rosell et al., 2013; Yu et al.,

2012). Expression of these growth factors leads to cell growth,

apoptotic avoidance, and stem cell self-renewal (Hong and

Guan, 2012; Piccolo et al., 2013; Yu and Guan, 2013).

Despite substantial evidence that Yap1 promotes tumor

to explore Hippo signaling in cancer.

progression and metastasis through its TEAD-interaction

domain (Liu-Chittenden et al., 2012; Zhao et al., 2008), no small

molecules have been identiﬁed that directly disrupt this interac-

tion. The approved drug verteporﬁn, which was identiﬁed by

high-throughput screening to impair Hippo signaling (Liu-Chitten-

INTRODUCTION

The Hippo signaling pathway controls tissue homeostasis and den et al., 2012), has not been demonstrated to physically asso-

organ size (Wu et al., 2003). Hippo is triggered by an NF2/ ciate with either TEAD or Yap. Instead, recent work indicates

Merlin-Kibra-Expanded tumor suppressor complex that acti- that verteporﬁn works through other mechanisms, including the

vates the Ste20-like kinase, Hippo (Mst1/2 in mammals), which inhibition of p62 (Donohue et al., 2014; Donohue et al., 2013; Do-

then phosphorylates and activates the large tumor suppressor nohu e e t al., 2011). Cyclic Yap-like peptides(Zho u e t a l., 2015) and

kinases (Lats1/2). Lats1/2 kinases, in turn, phosphorylate the recombinant proteins such as VGLL have been used to disrupt the

transcriptional co-activators Yap and TAZ to promote their cyto- interaction of TEAD with Yap1 in vitro. VGLL, which directly com-

plasmic retention and degradation (Yu and Guan, 2013). Lack of petes with Yap1 for a common binding site on TEAD, was also

cell crowding coupled with mechanical loading such as stretch- found to negatively regulate TEAD/Yap activity and to suppress

ing, location at edges of an epithelial sheet, or stiffness of the sur- lung and gastric tumor activity (Zhang et al., 2014). Along these

Cell Chemical Biology 26, 1–12, March 21, 2019 ª 2018 Elsevier Ltd.

1

Please cite this article in press as: Bum-Erdene et al., Small-Molecule Covalent Modiﬁcation of Conserved Cysteine Leads to Allosteric Inhibition of the

TEAD,Yap Protein-Protein Interaction, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.11.010

lines, a peptide that mimics the TDU domain of VGLL4 was found (FDA)-approved drug ﬂufenamic acid 1 (TED-346) was previously

to suppress tumor growth in gastric cancer cells both in vitro and found to bind weakly to two sites on TEAD2, but did not inhibit

in vivo (Jiao et al., 2014). Small interfering RNA-lipid nanoparticles TEAD binding to Yap (Pobbati et al., 2015). One of the binding

that target Yap1 have also been shown to decrease liver tumor sites is located within the deep hydrophobic palmitate-binding

proliferation (Fitamant et al., 2015). While upstream regulators of pocket of the transcription factor and the other at the protein-pro-

Hippo signaling such as G-protein-coupled receptors and kinases tein interaction interface. The binding mode of 1 (TED-346) in the

are amenable to small-molecule inhibition (Fan et al., 2013; Reddy deep pocket of TEAD2 shows that the compound’s carboxylic

and Irvine, 2013; Yu et al., 2012), their prominence in other acid moiety is located near the thiol of a conserved cysteine res-

signaling pathways will likely result in off-target effects.

idue (Cys-380) that is the acylation site of a palmitoyl group (Chan

The three-dimensional structure of the TEAD,Yap complex et al., 2016). We hypothesized that modiﬁcation of the carboxylic

(Chen et al., 2010; Li et al., 2010) reveals that disruption of the acid to an electrophile may lead to covalent bond formation and

protein-protein interaction is expected to be difﬁcult. The modulation of the TEAD4,Yap1 protein-protein interaction.

interaction interface between TEAD,Yap is unusually large, Considering that the palmitate pocket is located outside the

2

˚

exceeding 1,000 A (Chen et al., 2010). It is devoid of a well- TEAD4,Yap1 protein-protein interaction interface, it was not

deﬁned druggable binding pocket. The large interface and lack obvious whether adduct formation would stabilize or inhibit the

of binding site likely explains the failure to develop agents that TEAD4,Yap1 protein-protein interaction. To explore the effect

competitively inhibit the TEAD,Yap interaction. Alternatively, a of adduct formation on the TEAD4,Yap1 interaction, we designed

deep hydrophobic palmitate binding pocket within all TEAD a derivative of compound 1, namely 2 (TED-347), which pos-

members has been shown to be important for their stability but sesses a chloromethyl ketone moiety that can form a covalent

not their biological activity (Chan et al., 2016; Noland et al., bond with Cys-367. We applied microsecond explicit-solvent mo-

2016). All TEAD paralogs are palmitoylated at a conserved lecular dynamics simulations to determine whether covalent bond

cysteine located within this pocket (Noland et al., 2016). Consid- formation at the cysteine residue affects TEAD4,Yap1 protein-

ering that the palmitate pocket is located away from the protein interaction. We carried out three separate simulations:

TEAD,Yap interface, it has been suggested that palmitoylation TEAD4,Yap1, [TEAD4,2],Yap1 non-covalent complex (Fig-

allosterically alters TEAD to stabilize an interaction with Yap ure 1C), and [TEAD4-2],Yap1 covalent complex (Figure 1D).

(Chan et al., 2016; Noland et al., 2016). Thus, targeting the palmi- Each simulation consisted of 50 separate 50-ns trajectories re-

tate binding pocket may be an effective strategy for modulating sulting in 2.5 ms (50 3 50 ns) of explicit-solvent molecular dy-

the interaction of TEAD with Yap.

namics simulations per complex. Structures sampled from these

Here we report the discovery of small molecules that bind to the simulations were collected to determine the free energy of binding

TEAD4 palmitate pocket, form a covalent bond with a conserved of TEAD4 to Yap1 using the widely used MM-GBSA free energy

cysteine, and disrupt the TEAD4,Yap1 protein-protein interac- calculation method (Figure 1E). We found that non-covalent bind-

tion. Starting with the structure of ﬂufenamic acid bound to ing of 2 (TED-347) to TEAD4 exhibited little change to the

TEAD2 (Pobbati et al., 2015), we designed a small molecule TEAD4,Yap1 binding afﬁnity (DDG_MMGBSA= 0.5 0.1 kcal/mol).

that can form a covalent bond with a conserved cysteine within However, covalent bond formation of 2 (TED-347) to TEAD4 led

the palmitate-binding pocket. Extensive explicit-solvent molecu- to substantially greater loss of TEAD4 afﬁnity to Yap1 by nearly

lar dynamics simulations revealed that covalent bond formation 10.9 0.1 kcal/mol (Figure 1F). The 20-fold reduction in the bind-

of this compound reduced the TEAD4,Yap1 binding afﬁnity. ing afﬁnity suggests that adduct formation at Cys-367 leads to

Synthesis of this compound and several derivatives followed allosteric inhibition of the TEAD4,Yap1 protein-protein interac-

by biochemical studies that characterized binding afﬁnity and tion. These results were conﬁrmed by repeating the calculations

inhibition kinetics conﬁrmed the computational results. The with compound 5 (TED-551). Non-covalent binding of the com-

structure of the covalent complex between TEAD and com- pound led to little change for the afﬁnity of the TEAD4,Yap1

pounds was revealed by X-ray crystallography. Compounds complex (DDG_MMGBSA= ꢀ0.5 0.1 kcal/mol), while covalent

were explored for their effect on TEAD4 protein-protein interac- bond formation led to substantial reduction in the MM-GBSA

tions and transcriptional activity in HEK-293 mammalian cells, binding afﬁnity to 5.3 0.1 kcal/mol (Figure 1F). These results

as well as in glioblastoma (GBM) cancer cell lines.

suggest that mere binding to the pocket is not sufﬁcient to disrupt

the protein-protein interaction, whereas covalent bond formation

with the cysteine residue may lead to inhibition of the interaction.

RESULTS

Molecular Dynamics Simulations Reveal that Covalent

Bond Formation at Allosteric Pocket Cysteine Reduces

TEAD4,Yap1 Afﬁnity

Compound 2 and Derivatives Form a Covalent Bond at an

Allosteric Site Cysteine and Inhibit TEAD4 Binding

to Yap1

The TEAD three-dimensional structure contains a 12-strand Chloromethyl ketone 2 (TED-347) (Figure 2) was prepared to

b-sandwich fold, ﬂanked by four short a helices (Figure 1A). The determine whether it formed a covalent complex with TEAD4.

N-terminal region of Yap1 (residues 61–100) forms an a helix (res- To explore direct binding of the compounds to TEAD4, we

idues 61–73), which binds between TEAD a3 and a4 helices, and developed a ﬂuorescence polarization (FP) assay that used a ﬂu-

an U loop (residues 85–99), which binds near TEAD a1 and b12 orescently labeled Yap1-derived peptide FAM-YAP_60–99(FAM-

(Figure 1A). Crystal structures of TEADs reveal the presence of DSETDLEALFNAVMNPKTANVPQTVPMCLRKLPASFCKPP).

a deep hydrophobic pocket that is occupied by palmitate (Fig- FAM-YAP_60–99includes the entire Yap1$TEAD4 binding inter-

ure 1B) (Pobbati et al., 2015). The Food and Drug Administration face (Figure 3A). The labeled peptide binds to TEAD4 with a K_D

2

Cell Chemical Biology 26, 1–12, March 21, 2019

Please cite this article in press as: Bum-Erdene et al., Small-Molecule Covalent Modiﬁcation of Conserved Cysteine Leads to Allosteric Inhibition of the

TEAD,Yap Protein-Protein Interaction, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.11.010

Figure 1. Three-Dimensional Structures and Free Energy Calculations

(A) Stereo view of the X-ray structure of the TEAD4,Yap1 complex (PDB: 3JUA). TEAD4 and Yap1 are shown in gray and cyan ribbon representation, respectively.

(B) Structure of the TEAD4,Yap1 complex depicting the deep hydrophobic pocket of TEAD4. The pocket is occupied by palmitate, which is shown as capped

sticks and color coded by atom type (yellow and red correspond to carbon and oxygen atoms, respectively). The pocket is shown in solvent-accessible surface

area and color coded by hydrophobicity (brown is hydrophobic). PLM corresponds to palmitate.

(C) Non-covalent complex of 2 (TED-347) bound to TEAD4. Compound 2 (TED-347) and surrounding amino acids are shown as capped sticks and color coded by

atom type (for 1 [TED-347], yellow, blue, red, cyan, and green correspond to carbon, nitrogen, oxygen, ﬂuorine, and chlorine respectively. Carbon atoms are

shown in green and sulfur atoms in gold for TEAD4).

(D) Covalent complex of 2 (TED-347) and TEAD4. Compound 2 (TED-347) and surrounding residues are shown as capped sticks and color coded by atom type

(for 1 [TED-347], yellow, blue, red, cyan, and green correspond to carbon, nitrogen, oxygen, ﬂuorine, and chlorine, respectively. Carbon atoms are shown in green

and sulfur atoms in gold for TEAD4).

(E) MM-GBSA free energy of binding of Yap1 to TEAD4 bound to compounds 2 (TED-347) or 5 (TED-551). MM-GBSA free energies and their components

correspond to the difference between TEAD4,Yap1 with TEAD4 in complex with a compound and TEAD4,Yap1 with TEAD4 in the apo state. All energies are

reported in kcal/mol; mean SE, n = 30,000 snapshots.

(F) The change in free energy for covalent and non-covalent complexes between TEAD4 and 1 (TED-347); mean SE, n = 30,000 snapshots. p values were

calculated using two-tailed t tests, ***p < 0.0005.

of 78.2 9.9 nM. Compound 1 (TED-346, ﬂufenamic acid) was valent bond, since the chlorine atom leaving group is replaced by

tested and we found that the drug did not inhibit the a methyl group (Figure 3B). A time-dependent study was per-

TEAD4,Yap1 interaction, consistent with previous studies (Fig- formed at 0.5, 6, 24, and 48 hr for 2 (TED-347) (Figure 3C),

ure 3B). The effects of other compounds on the TEAD4,Yap1 whereby 2 (TED-347) reached maximum inhibition of 80% at

interaction were tested using our FP assay. Following 24 hr incu- 48 hr. Based on the time- and concentration-dependent inhibi-

bation of TEAD4 with 2 (TED-347) at 4^ꢁC, the compound in- tion study of TEAD4 with 2 (TED-347) (Figure 3D), the rates of

hibited the TEAD4,Yap1 protein-protein interaction by 53% inactivation were calculated for the compounds and several

with an apparent EC₅₀of 5.9 0.4 mM (Figure 3B). Compound derivatives (Table S1). The maximum rate of inactivation of 2

3 (TED-550) did not inhibit, as the compound cannot form a co- (TED-347) was calculated to be 0.038 0.003 hr^ꢀ1(Figure 3D),

Cell Chemical Biology 26, 1–12, March 21, 2019

3

Please cite this article in press as: Bum-Erdene et al., Small-Molecule Covalent Modiﬁcation of Conserved Cysteine Leads to Allosteric Inhibition of the

TEAD,Yap Protein-Protein Interaction, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.11.010

Figure 2. Structures of Compounds

347), as well as peak broadening,

compared with the TEAD4 incubated with

DMSO, both of which suggest conforma-

tional change of the protein. Furthermore,

it is highly unlikely that Cys-367 oxidation

is responsible for the lack of 100% inhibi-

tion at longer times, since we have shown

that covalent bond formation is rapid and

complete within less than an hour. Also,

whole-protein mass spectrometry carried

out at 24 hr does not reveal the presence

of the oxidized species.

To further establish that 2 (TED-347)

speciﬁcally forms a bond with Cys-367

within the central pocket of TEAD4, we

tested its interaction with

a TEAD4

Cys367Ser mutant. Adduct formation

by 2 (TED-347) to the mutant TEAD4

Cys367Ser was analyzed by mass spec-

trometry. After 24 hr, 2 (TED-347) did not

form an adduct with the mutant protein

(Figure 4A). TEAD4 Cys367Ser mutant

showed no change in afﬁnity for FAM-

YAP_60–99peptide, with a K_Dof 49.1

corresponding to a t₁^N₌₂of 18.2 hr. To determine whether 2 (TED- 3.0 nM (Figure 4B). Subsequently, the compounds were tested

347) is a reversible or irreversible inhibitor, we incubated TEAD4 for inhibition of the peptide binding to TEAD4 Cys367Ser mutant.

with 50 mM compound for 24 hr at 4^ꢁC and then dialyzed it We found that 2 (TED-347) did not inhibit the mutant TEAD4

against buffer for 24 hr at 4^ꢁC, prior to interaction with the ﬂuo- Cys367Ser protein binding to the peptide, suggesting that cova-

rescently labeled Yap1 peptide (Figure 3E). Compound 2 (TED- lent bond formation by 2 (TED-347) is essential for its ability to

347) inhibited the TEAD4,Yap1 interaction even after dialysis, inhibit the protein-protein interaction (Figure 4C).

indicating that the compound is an irreversible inhibitor.

We explored whether mere covalent bond formation with

The formation of a covalent bond between compounds and conserved Cys-367 was sufﬁcient to inhibit the TEAD4,Yap1

TEAD4 was conﬁrmed by whole-protein electrospray ionization protein-protein interaction. ESI mass spectrometry was used

(ESI) mass spectrometry studies. Following incubation of to detect formation of adducts by iodoacetamide. In 30 min,

TEAD4 at 10 mM with 200 mM of 2 (TED-347) for 24 hr at 4^ꢁC, a we saw a concentration-dependent adduct formation up to

peak at 26,229 was observed, corresponding to the TEAD4-2 200 mM, whereby the protein was modiﬁed by a single adduct

(TED-347) adduct, while the peak at 25,952 corresponding to (Figure S1A). After 6 hr, the protein was modiﬁed by a single

TEAD4 disappeared (Figure 3F). As expected, compound 3 adduct at all concentrations of iodoacetamide (Figure S1B).

(TED-550) only showed a peak at 25,952, indicating no adduct We did not see the presence of a second reaction site until

formation. The covalent bond formation by 2 (TED-347) was rela- 24 hr at the highest tested concentration of 200 mM (Figure S1C).

tively fast (Figure 3G), reaching nearly 100% adduct formation To determine whether Cys-367 is the target of the single adduct,

after 30 min of incubation with TEAD4. Because the rate of inhi- we reacted TEAD4 Cys367Ser mutant with varying concentra-

bition developed over a longer time scale (Figure 3C), 2 (TED- tions of iodoacetamide for 24 hr. After 24 hr there was no

347) is proposed to induce a slow conformational change in modiﬁcation of the protein except for a small adduct that was de-

TEAD4 that inhibits its interaction with Yap1. To rule out the pos- tected only at 200 mM iodoacetamide concentration, which is

sibility that 2 (TED-347) induces slow aggregation of TEAD4, and consistent with the wild-type TEAD4 (Figure S1D). Although

not the proposed slow conformational change, we incubated iodoacetamide is able to react with TEAD4 Cys-367, it was

GST-TEAD4 with DMSO or 2 (TED-347) for 24 hr at 4^ꢁC, followed unable to inhibit the activity of the protein in the FP assay (Fig-

by injection into a size-exclusion chromatography (SEC) column ure S1E). Thus, merely reacting with the cysteine does not guar-

(Figure 3H). There is no signiﬁcant aggregation of GST-TEAD4 antee inhibition of activity.

after 24 hr of incubation, with or without 2 (TED-347). There

To further conﬁrm that 2 (TED-347) inhibited the interaction be-

was a slight increase in dimer formation for the TEAD4 sample tween TEAD4 and Yap1 peptide observed in our FP assay, we

incubated with 2 (TED-347) compared with the sample incu- applied biolayer interferometry (BLI) that used full-length Yap1

bated with DMSO. In addition, we noticed a slight shift in the protein. The binding afﬁnity between glutathione S-transferase

retention time of the TEAD4 sample incubated with 2 (TED- (GST)-tagged Yap1 and the TEAD4 protein was found to be

4

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Please cite this article in press as: Bum-Erdene et al., Small-Molecule Covalent Modiﬁcation of Conserved Cysteine Leads to Allosteric Inhibition of the

TEAD,Yap Protein-Protein Interaction, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.11.010

Figure 3. Compounds Inhibit TEAD4,Yap1 Protein-Protein Interaction

(A) Increasing concentration of TEAD4 incubated with 16 nM FAM-labeled Yap (FAM-Yap_60–99) peptide followed by measurements of changes in ﬂuorescence

polarization (FP) (mean SD, n = 3). Inset: an illustration of the FP assay, where the bound FAM-Yap_60–99is displaced from TEAD4 due to the interaction by an

inhibitor (TED), resulting in loss of polarization.

(B) TEAD4 was incubated with increasing concentration of compounds for 24 hr at 4^ꢁC followed by addition of FAM-Yap_60–99to measure changes in FP

(mean SD, n = 3).

(C) Time-dependent inhibition of TEAD4 by 2 (TED-347) was assessed by FP using FAM-Yap_60–99at 10 different concentrations (0.1–100 mM) following 0.5, 6, 24,

and 48 hr of incubation at 4^ꢁC (mean SD, n = 3).

(D) Time-dependent inhibition of TEAD4 by 2 (TED-347) was assessed by FP at 10 concentrations ranging from 0.1 to 100 mM following incubation at 0.5, 6, 24,

and 48 hr at 4^ꢁC (mean SD, n = 3). The observed rate of inactivation (k_obs) was calculated at each compound concentration using percent inhibition data at each

time point. The rate constant is plotted against the concentration of compound.

(E) TEAD4 was incubated with 50 mM 2 (TED-347) for 24 hr at 4^ꢁC, then dialyzed against PBS for 24 hr at 4^ꢁC, prior to addition of FAM-Yap_60–99for FP

measurements (mean SD, n = 3).

(F) TEAD4 (10 mM) was incubated with 200 mM compounds for 24 hr at 4^ꢁC, then analyzed by ESI mass spectrometry.

(G) TEAD4 (10 mM) was incubated with 2, 10, and 50 mM 2 (TED-347) for 0.5, 6, and 25 hr at 4^ꢁC and analyzed by ESI mass spectrometry. Percent ratio of the

adduct over total protein signal, quantiﬁed from the relative ion count, is plotted versus time.

(H) TEAD4 was incubated with DMSO or 2 (TED-347) followed by injection into a SEC column. No signiﬁcant aggregation was observed.

116.5 5.9 nM (Figure 4D), which was comparable with that of ceptor (uPAR) and its ligand urokinase (uPA) and (2) the a-sub-

the FAM-YAP_60–99peptide. As with the peptide FP assay, unit of the voltage-gated calcium channel Cav2.2 with its

TEAD4 was incubated with 2 (TED-347) for 24 or 48 hr at 4^ꢁC b-subunit Cavb₃. We have previously developed FP assays for

prior to studying its interaction with GST-Yap1 using BLI. As these interactions as we have reported for uPAR (Mani et al.,

with the FP assay, we observed dose- and time-dependent inhi- 2013) and Cavb3 (Chen et al., 2018). Compounds 2 (TED-347)

bition of TEAD4 binding to full-length Yap1 (Figure 4E).

and 3 (TED-550) showed no inhibition of uPAR$uPA or Cav2.2

Since the palmitate binding pocket and the Cys-367 residue is a$b protein-protein interactions (Figures 4H and 4I). Both pro-

conserved in all four human TEAD proteins, we tested whether 2 teins have cysteine residues capable of forming covalent bonds.

(TED-347) would be active in TEAD2. His-tagged TEAD2 protein These results further conﬁrm the selectivity of 2 (TED-347).

was tested in the FP binding assay, where it showed an apparent

K_Dof 27.6 1.7 nM (Figure 4F). Compounds 1–3 were incubated Structure of 2 in Complex with TEAD2

with TEAD2 for 24 hr at 4^ꢁC prior to the addition of the Yap1 pep- A TEAD2-2 complex was formed by soaking TEAD2 crystals with

˚

tide, and 2 (TED-347) was shown to inhibit TEAD2, while 1 (TED- 2 (TED-347). The crystal diffracted to 2.43-A resolution, and the

346) and 3 (TED-550) were inactive as expected (Figure 4G). We structure was solved in space group C2 with two TEAD2 per

expect that 2 (TED-347) will likely inhibit protein-protein interac- asymmetric unit (Table S2). The overall structure of TEAD2 in

tions of TEAD1 and TEAD3 with Yap1, considering their close complex with 2 (TED-347) is the same as previously published

˚

structural similarity to TEAD4. structures, with a Ca root-mean-square deviation of 0.59 A

The selectivity of the compounds was explored with two unre- compared with a previously published structure (PDB: 5DQ8).

lated protein-protein interactions between (1) the urokinase re- The density of 2 (TED-347) within the central binding pocket is

Cell Chemical Biology 26, 1–12, March 21, 2019

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Please cite this article in press as: Bum-Erdene et al., Small-Molecule Covalent Modiﬁcation of Conserved Cysteine Leads to Allosteric Inhibition of the

TEAD,Yap Protein-Protein Interaction, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.11.010

Figure 4. Compound 2 Forms an Adduct at Cys-367 on TEAD4

(A) TEAD4 Cys367Ser mutant (10 mM) was incubated with 200 mM compound for 24 hr at 4^ꢁC and then analyzed by ESI mass spectrometry.

(B) Increasing concentration of TEAD4 Cys367Ser mutant was mixed with 16 nM FAM-Yap_60–99peptide, and FP due to binding was measured (mean SD, n = 3).

(C) TEAD4 Cys367Ser mutant was incubated with increasing concentration of compounds for 24 hr at 4^ꢁC followed by addition of FAM-Yap_60–99for FP mea-

surements (mean SD, n = 3).

(D) Biotin-labeled GST-Yap1 was captured onto streptavidin-conjugated biolayer interferometry sensors, which were dipped into varying concentrations of

TEAD4. The binding of TEAD4 to the captured Yap was measured by biolayer interferometry (mean SD, n = 3).

(E) Biotin-labeled GST-Yap1 was captured onto streptavidin-conjugated biolayer interferometry sensors, which were dipped into solutions containing 100 nM

TEAD4, pre-incubated with varying concentrations of 2 (TED-347) for 24 or 48 hr at 4^ꢁC (mean SD, n = 3).

(F) Increasing concentration of HIS-TEAD2 was mixed with 16 nM FAM-Yap_60–99peptide, and FP due to binding was measured (mean SD, n = 3).

(G) HIS-TEAD2 was incubated with increasing concentration of compounds for 24 hr at 4^ꢁC followed by addition of FAM-Yap_60–99for FP measurements

(mean SD, n = 3).

(H) Urokinase receptor (uPAR) was incubated with varying concentrations of compounds for 24 hr at 4^ꢁC followed by addition of a urokinase-derived ﬂuorescently

labeled peptide AE147 for FP measurements, according to our previously established protocol (Mani et al., 2013) (mean SD, n = 3).

(I) The b-3 subunit of the voltage-gated calcium channel Cav2.2 was incubated with varying concentrations of compounds for 24 hr at 4^ꢁC followed by addition of

an a-subunit peptide that was ﬂuorescently labeled for FP measurement, according to our previously established protocol (Chen et al., 2018) (mean SD, n = 3).

weak (Figure 5A), possibly indicating less than 100% occupancy. a 3-step soaking experiment, whereby the crystal was ﬁrst

To conﬁrm that the observed density is 2 (TED-347) and not soaked in buffer containing DTT for 2 hr to remove the fatty

palmitate, we soaked out the fatty acid by incubating the crystal acid, then exchanged into buffer without DTT for 2 hr, and ﬁnally

in a buffer containing dithiothreitol (DTT) for 2 hr. There is no den- incubated with 2 (TED-347) for 3–4 hr. The crystal quality suf-

sity within the central pocket after this treatment. We performed fered after the treatment, but an unambiguous positive density

6

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TEAD,Yap Protein-Protein Interaction, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.11.010

Figure 5. Crystal Structure of TEAD2 in Com-

plex with 2

(A) Stereo image of 2 (TED-347) covalently bound to

Cys-380 in the central binding pocket of TEAD2.

The 2jFoj ꢀ jFcja_calcmap around 2 (TED-347) is

illustrated in black mesh. Compound 2 (TED-347)

and residues near the reaction site of 2 (TED-347)

are shown in sticks (green, carbon; red, oxygen;

blue, nitrogen; gold, sulfur) with accompanying

labels.

(B) Two-dimensional ligand interaction map of

covalently-bound 2 (TED-347) in the central pocket

of TEAD2.

the reactivity of the compound, as an

electron-withdrawing nitrogen atom was

added to the aromatic ring bearing the

chloromethyl ketone group. Compound 6

(TED-589) was designed to improve the af-

ﬁnity and selectivity of 2 (TED-347) against

TEAD4 by exploiting the nearby pocket.

Compounds 7 (TED-587) and 8 (TED-588)

were designed to improve the afﬁnity of

the compound based on docking studies.

After 24 hr of incubation with TEAD4 at

4^ꢁC, 4 (TED-548), 5 (TED-551), and 6

(TED-589) showed a maximum inhibition

of 31%, 81%, and 51% respectively, while

7 (TED-587) and 8 (TED-588) displayed

less than 20% inhibition (Figure S2A). Yet

the EC₅₀of compound 4 (TED-548) was

substantially lower (nearly an order of

magnitude) than its parent 2 (TED-347),

as well as the other derivatives. It is worth

noting that 5 (TED-551) also inhibited the

TEAD4 Cys367Ser mutant, in contrast to

4 (TED-548) and 6 (TED-589) (Figure S2B).

Compound 6 (TED-589) had improved

EC₅₀of 2.3 0.8 mM, while still being selec-

tive toward Cys-367. Whole-protein mass

spectrometry analysis of TEAD4 with the

was observed in the pocket covalently attached to Cys-380. compounds showed that 4 (TED-548), 6 (TED-589), 7 (TED-

Compared with the structure of TEAD2 in complex with 1 587), and 8 (TED-588) formed single adducts, consuming all of

(PDB: 5DQ8), the ﬁrst benzene ring of 2 (TED-347) is rotated the protein, while 5 (TED-551) formed more than one covalent

away from the direction of Val-347 by about 90^ꢁto allow the co- complex (Figure S2C). Furthermore, only 5 (TED-551) formed

valent bond to form. The second ring and the triﬂuoromethyl an adduct with the TEAD4 Cys367Ser mutant as evidenced by

group is shifted further into the hydrophobic pocket (Figures a minor peak corresponding to a mass of 26,217 (Figure S2D).

5A and 5B). However, due to our multi-step soaking experiment The lack of TEAD4 inhibition by 7 (TED-587) and 8 (TED-588) (Fig-

and the resulting positive density, we can visibly see that 2 (TED- ure S2A), while still forming 100% adduct with TEAD4 (Fig-

347) forms a covalent bond with TEAD2 at our proposed site of ure S2C), again demonstrates that mere binding and reaction

Cys-380.

to Cys-367 on TEAD4 is not sufﬁcient for inhibition of TEAD4 ac-

tivity, as demonstrated with iodoacetamide (Figure S1). The ﬁve

derivatives were also tested for inhibition of TEAD2 binding to

Yap1. Compound 5 (TED-551) showed similar inhibition of

Synthesis and Biochemical Studies of Compound 2

Derivatives

Five derivatives of 2 (TED-347) were synthesized (Figure 2). Com- TEAD2 binding to Yap1, while 4 (TED-548) and 6 (TED-589)

pound 4 (TED-548) was designed to more closely mimic palmi- were much weaker inhibitors of TEAD2 compared with TEAD4

tate, with an elongated polyethylene glycol-like moiety, which (Figure S2E).

we hypothesized could improve the binding afﬁnity of the

The three active derivatives showed concentration- and time-

compound. Compound 5 (TED-551) was designed to improve dependent inhibition of TEAD4 (Figures S2F–H). The rate of

Cell Chemical Biology 26, 1–12, March 21, 2019

7

Please cite this article in press as: Bum-Erdene et al., Small-Molecule Covalent Modiﬁcation of Conserved Cysteine Leads to Allosteric Inhibition of the

TEAD,Yap Protein-Protein Interaction, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.11.010

Figure 6. Compound 2 Inhibits TEAD Transcriptional Activity and Protein-Protein Interactions in Cell Culture

(A) The activity of the TEAD4 luciferase reporter was measured in HEK-293 cells treated with either vehicle or compound 2 (TED-347). CNYT corresponds to no

transfection; mean SD, n = 3 biological replicates.

(B) Co-immunoprecipitation of FLAG-tagged Yap1 and myc-tagged TEAD4 from lysates of HEK-293 cells treated with vehicle, 2 (TED-347), or a peptide (FAM-

Yap_60–99) containing the residues in Yap1 that directly bind to TEAD4.

(C) Average normalized values relative to lane A from three biologic replicates; mean SD, n = 3 biological replicates.

(D) Lysates from HEK-293 cells treated with 2 (TED-347) or 5 (TED-551) were treated with 9 (TED-549) followed-up by pull-down and detection of TEAD4. Proteins

in complexes that were retained by streptavidin pull-down were detected by immunoblot analysis using the indicated antibody.

(E) Average normalized values relative to lane A from three biological replicates; mean SD, n = 3 biological replicates.

(F) qRT-PCR analysis of CTGF levels following treatment of HEK-293 cells with compounds for 48 hr; mean SD, n = 3 biological replicates. p values were

calculated using two-tailed t tests.

*p < 0.05, **p < 0.005, ***p < 0.0005.

inactivation of 4 (TED-548) is lower than its parent at 0.010

signiﬁcant loss of co-immunoprecipitation of Myc-tagged

0.001 hr^ꢀ1, which resulted in a t₁^N₌₂of 67.3 hr (Figure S2I). The TEAD4 with FLAG-tagged Yap1 (Figures 6A–6C). To establish

rate of inactivation of 5 (TED-551) is slightly faster than its parent, that 2 (TED-347) forms a covalent bond with TEAD4 in cells, we

with a k_inactof 0.049 0.003 hr^ꢀ1corresponding to a half-life of synthesized a biotin-conjugated variant, termed 9 (TED-549).

14.3 hr (Figure S2J). The rate of inactivation of 6 (TED-589), Following addition of 9 (TED-549) to cell lysates, TEAD4 was spe-

k_inact= 0.034 0.003 hr^ꢀ1(t₁^N₌₂= 20 hr) (Figure S2K), was similar ciﬁcally detected by immunoblot analysis in a streptavidin pull-

to that of the parent 2 (TED-347).

down, consistent with 2 (TED-347) directly engaging TEAD4 in

cells in a covalent complex (Figures 6D, 6E, and S3B). The reduc-

tion in TEAD4 in 9 (TED-549) containing samples that were also

treated with higher concentrations of 2 (TED-347) or 5 (TED-

Small Molecules Inhibit TEAD Transcriptional Activity

and Protein Interactions in Cells

The effect of 2 (TED-347) on the intracellular transcriptional activ- 551) indicates that these compounds compete with 9 (TED-

ity of TEAD4 was compared with its effects on the interaction of 549) for binding to TEAD4 (Figures 6D and 6E, lanes 3 and 4).

TEAD4 with Yap (Figure 6). Treatment of cells transfected with To monitor endogenous TEAD activity, we measured the levels

a TEAD reporter over 48 hr with 2 (TED-347) at 5 mM resulted in of CTGF transcript (a well-established target of TEAD) by

over 70% reduction in reporter activity, whereas cells treated qRT-PCR from control cells and cells incubated with 2 (TED-

with 10 mM of 2 showed a complete loss of reporter activity. 347) or 5 (TED-551). Cells incubated with compounds 2 (TED-

Less activity is observed at 24 hr, suggesting time-dependent ac- 347) and 5 (TED-551) showed a signiﬁcant reduction in CTGF

tivity in cells (Figure S3A). To further establish the selectivity of the transcript levels versus control cells (Figure 6F). Cells incubated

small molecule, we repeated the TEAD4 transcriptional activity with compound 3 (TED-550), which lacks the reactive moiety

luciferase reporter assays using transfected Cys367Ser mutant. necessary to form an adduct with TEAD4, showed similar levels

We found that treatment of HEK-293 cells with 2 (TED-347) did of CTGF transcript versus control cells.

not result in the inhibition of TEAD4 transcriptional activity as

was observed for wild-type TEAD4 (Figure S3B). Consistent Compounds Inhibit GBM Cancer Cell Viability

with these effects being a result of disruption of the TEAD4,Yap1 Hippo signaling promotes tumor growth and invasion in a range

interaction, cells incubated with 5 mM of 2 (TED-347) showed a of cancers including GBM (Artinian et al., 2015; Orr et al., 2011;

8

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TEAD,Yap Protein-Protein Interaction, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.11.010

Figure 7. Compound

2 Inhibits Patient-

Derived GBM43 Glioblastoma Growth in 3D

Spheroids

(A) Spheroids of patient-derived GBM43 glioblas-

toma cell lines were grown and treated with 1 (TED-

346), 2 (TED-347), and 5 (TED-551); mean SD,

n = 3 biological replicates.

(B) The activity of the TEAD4 luciferase reporter was

measured in GBM43 cells treated with either vehicle

or compound 2 (TED-347). CNYT corresponds to no

transfection; mean SD, n = 3 biological replicates.

(C) qRT-PCR analysis of CTGF levels following

treatment of GBM43 cells with compounds; mean

SD, n = 3 biological replicates.

(D) Spheroids of patient-derived GBM43 glioblas-

toma cell lines were grown and treated with temo-

zolomide; mean SD, n = 3 biological replicates.

p values were calculated using two-tailed t tests.

*p < 0.05, **p < 0.005, ***p < 0.0005.

namic acid (Pobbati et al., 2015) shows

that the FDA-approved drug binds to two

sites: (1) the palmitate pocket and (2) the

TEAD,Yap interface. Flufenamic acid

Shah et al., 2014). We investigated the effects of 1 (TED-346), 2 binds weakly to TEAD2 and does not inhibit its interactions, as

(TED-347) and 5 (TED-551) on GBM cell viability using patient- reported earlier and conﬁrmed in this work.

derived GBM43 cells that were grown as three-dimensional

The three-dimensional structures of TEADs reveal a

spheroids (Figure 7A). Both 2 (TED-347) and 5 (TED-551) in- conserved cysteine (Cys-367) deep within the palmitate binding

hibited GBM43 cancer cell viability. At 10 mM, which is the pocket of TEAD4. Cys-367 spontaneously forms an adduct to

concentration used to demonstrate inhibition of TEAD4 activity palmitoyl-coenzyme A (Chan et al., 2016; Noland et al., 2016), re-

in cells, the compounds inhibit GBM43 cell viability by 30%. sulting in stability of the TEAD,Yap complex (Mesrouze et al.,

At this concentration, the compound did not show any effect 2017). Since palmitate does not come in contact with Yap, the

on cell viability of non-transformed normal astrocytes (Fig- process is believed to occur through an allosteric mechanism.

ure S4). Compound 1 (TED-346), which does not inhibit This prompted us to postulate that a small molecule that forms

TEAD4,Yap1, did not affect GBM43 cancer cell growth (Fig- a covalent bond with Cys-367 may modulate the TEAD pro-

ure 7A). Compound 2 (TED-347) also inhibited TEAD4 transcrip- tein-protein interaction with Yap. To test this, we modiﬁed ﬂufe-

tional activity in GBM43 cells (Figure 7B) in a concentration- namic acid by introducing a reactive chloromethyl ketone moiety

dependent manner. Similarly, as shown in Figure 7C, both 2 and used microsecond explicit-solvent molecular dynamics sim-

(TED-347) and 5 (TED-551) suppressed CTGF transcript levels, ulations followed by free energy calculations to investigate the

while 3 (TED-550) had no effect versus cells treated with vehicle. effect of adduct formation on the protein-protein complex. These

The potency of compounds 2 (TED-347) and 5 (TED-551) were substantial calculations revealed that non-covalent binding of

compared with temozolomide, which is the standard of care compounds led to negligible change in the MM-GBSA free en-

for patients with glioblastoma. Temozolomide inhibited GBM43 ergy of the TEAD4,Yap1 complex, whereas covalent bond for-

spheroid growth with a substantially higher EC₅₀of 244

24 mM (Figure 7D).

mation with Cys-367 led to substantial weakening of the interac-

tion. To test this hypothesis we prepared 2 (TED-347), which was

found to inhibit the protein-protein interaction in a time-depen-

dent manner. Biochemical studies conﬁrmed that 2 (TED-347)

formed a covalent bond with Cys-367 with a K_i= 10.3

DISCUSSION

The intense interest in Hippo signaling has highlighted the need 2.6 mM and an inactivation rate constant k_inact= 0.038

for small-molecule probes to explore the pathway in normal and 0.003 hr^ꢀ1, which corresponds to a half-life t₁^N₌₂of 18 hr. The

pathological processes. However, the development of orthos- compound is selective for TEADs as shown by its ability to inhibit

teric small-molecule inhibitors of the TEAD,Yap interaction has TEAD2 with the same efﬁcacy, while not being able to inhibit un-

not been successful, likely due to this interaction occurring related protein-protein interactions, uPAR$uPA and Cav2.2 a$b.

2

over a large and featureless binding interface (1,300 A ) with a Synthesis of several derivatives afforded a structure-activity

˚

K_Din the nanomolar range. While the drug verteporﬁn was study and the discovery of three compounds that exhibited:

initially proposed to inhibit the TEAD,Yap interaction, it has not higher afﬁnity but poorer rate of inactivation (compound 4), bet-

been shown to directly bind either TEAD or Yap. Its effects ter inhibition rate and similar afﬁnity (compound 5), and higher

have subsequently been attributed to interactions with other afﬁnity while maintaining a similar rate of inactivation (com-

unknown proteins. A crystal structure of TEAD2 bound to ﬂufe- pound 6). In each case, covalent modiﬁcation of Cys-367 led

Cell Chemical Biology 26, 1–12, March 21, 2019

9

Please cite this article in press as: Bum-Erdene et al., Small-Molecule Covalent Modiﬁcation of Conserved Cysteine Leads to Allosteric Inhibition of the

TEAD,Yap Protein-Protein Interaction, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.11.010

to inhibition of the TEAD4,Yap1 protein-protein interaction. with a conserved cysteine within the palmitate binding

Whole-protein mass spectrometry revealed that adduct forma- pocket of TEADs. This compound led to allosteric inhibition

tion of TEAD4 with 2 (TED-347) was complete within a few of the TEAD,Yap interaction. Considering the profound

minutes, in contrast to the hours required to inhibit the interest in Hippo, these compounds will serve as tools

TEAD4,Yap1 protein-protein interaction. The covalent bond for- to explore the role of Hippo in normal and pathological

mation is therefore not the event that leads to inhibition of the processes.

protein-protein interaction. This is further conﬁrmed by studies

with iodoacetamide as well as compounds 7 and 8, all of which

readily form adducts to TEAD4 but do not inhibit its interaction

with Yap1. Covalent bond formation likely leads to local confor-

STAR+METHODS

Detailed methods are provided in the online version of this paper

mational changes, followed by large-scale conformational and

and include the following:

dynamical changes that favor TEAD4 conformational states

d KEY RESOURCES TABLE

not suitable for complex formation with Yap1. Comparison of

the three-dimensional structure of the non-covalent and

covalent complex of 2 (TED-347) with TEAD4 shows that the

benzene ring bearing the reactive warhead adopts a different

conformation in the covalent complex. This structure may shift

the conformation of TEAD4 that leads to inhibition of the

TEAD4,Yap1 complex.

d CONTACT FOR REAGENT AND RESOURCE SHARING

d EXPERIMENTAL MODEL AND SUBJECT DETAILS

d METHOD DETAILS

B Luciferase Reporter Assay

B Covalent Pull Down of TEAD4

B Co-immunoprecipitation

B RNA Extraction and Real-Time PCR

B Sphere-Forming Assay

Compound 2 (TED-347) was then shown to functionally disrupt

the TEAD,Yap1 interaction in cells and to reduce the viability of

patient-derived glioblastoma cell lines. Previous studies have

shown that Hippo plays a major role in promoting GBM growth

and invasion (Artinian et al., 2015; Orr et al., 2011; Shah et al.,

2014). HEK-293 and low-passage patient-derived GBM43 cells

treated with 2 (TED-347) were found to have reduced TEAD4

transcriptional activity and to lack protein-protein complexes be-

tween TEAD4 and Yap1. Pull-down studies conﬁrmed that 2

(TED-347) binds and forms covalent bonds with TEAD4 in these

cells. Studies showing GBM43 cells treated with 2 (TED-347) and

5 (TED-551) had signiﬁcantly reduced rates of proliferation, sug-

gesting that our method to allosterically target the TEAD4,Yap1

interaction is a promising paradigm for the development of ther-

apeutics to treat GBM tumor growth in vivo. In fact, these com-

pounds may also be effective against other tumors, as studies

have shown several components of the Hippo pathway to act

as oncogenes in pancreatic ductal adenocarcinoma and breast

cancer.

B Protein Expression and Puriﬁcation

B Size-Exclusion Chromatography

B Fluorescence Polarization

B Crystallization of TEAD2 and Structure Reﬁnement

B Protein Mass Spectrometry

B Biolayer Interferometry

B In Silico Protein Preparation

B Covalent Docking

B Molecular Dynamics Simulations

B Free Energy Calculations

B Synthesis

d QUANTIFICATION AND STATISTICAL ANALYSIS

d DATA AND SOFTWARE AVAILABILITY

d ADDITIONAL RESOURCES

SUPPLEMENTAL INFORMATION

Supplemental Information includes four ﬁgures and two tables and can be

found with this article online at https://doi.org/10.1016/j.chembiol.2018.11.010.

In sum, the development of 2 (TED-347) and derivatives is a

signiﬁcant breakthrough as there are no existing inhibitors of

the TEAD4,Yap1 interaction to enable exploration of Hippo in

cell culture and in vivo. Furthermore, these compounds suggest

that pockets outside tight and challenging protein-protein inter-

action interfaces with nucleophilic residues may be suitable for

the development of allosteric small-molecule inhibitors.

ACKNOWLEDGMENTS

The research was supported by the American Cancer Society Research

Scholar grant RSG-12-092-01-CDD (S.O.M.), by an Indiana Drug Discovery

Alliance grant (S.O.M.), and by a Vera Bradley Foundation grant (K.B.-E.).

Computer time on the Big Red II, Karst, and Carbonate supercomputers at In-

diana University is supported in part by Lilly Endowment, through its support

for the Indiana University Pervasive Technology Institute, and in part by the In-

diana METACyt Initiative. We thank Dr. Jed Fisher for reading the manuscript

and for helpful discussions.

SIGNIFICANCE

The Hippo pathway controls tissue homeostasis and organ

size. Hippo signaling leads to phosphorylation of the tran-

scriptional co-activator Yap, which is sequestered in the

cytoplasm and degraded. In cancer, Yap phosphorylation

is inhibited, resulting in its entry into the nucleus and binding

to TEAD transcription factors. TEAD activation leads to the

expression of a range of proteins that results in cell growth,

apoptotic avoidance, and stem cell self-renewal. Here, we

report small-molecule inhibitors of the TEAD,Yap protein-

protein interaction following an innovative strategy that con-

sisted of developing a compound that forms a covalent bond

AUTHOR CONTRIBUTIONS

K.B.-E., D.Z., G.G.-G., M.K.G., Y.S., D.X., H.E.S., B.J.B., and S.O.M. per-

formed experiments. K.B.-E. and S.O.M. designed experiments and analyzed

data. K.E.P. and C.D.W. contributed reagents. K.B.-E., C.D.W., T.W.C., and

S.O.M. wrote the paper.

DECLARATION OF INTERESTS

The authors declare no competing interests.

10 Cell Chemical Biology 26, 1–12, March 21, 2019

Please cite this article in press as: Bum-Erdene et al., Small-Molecule Covalent Modiﬁcation of Conserved Cysteine Leads to Allosteric Inhibition of the

TEAD,Yap Protein-Protein Interaction, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.11.010

Received: April 29, 2018

function to estimate the binding afﬁnity of ligands in receptor complexes.

Revised: August 27, 2018

Accepted: November 15, 2018

Published: December 20, 2018

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12 Cell Chemical Biology 26, 1–12, March 21, 2019

Please cite this article in press as: Bum-Erdene et al., Small-Molecule Covalent Modiﬁcation of Conserved Cysteine Leads to Allosteric Inhibition of the

TEAD,Yap Protein-Protein Interaction, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.11.010

STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies

Monoclonal Anti-c-Myc antibody produced in mouse

Bacterial and Virus Strains

E. coli BL-21 (DE3)

Fisher

Cat# MA1980; RRID: AB_558470

Cat# C2527H

New England Biolabs

Chemicals, Peptides, and Recombinant Proteins

HRV-3C protease

ThermoFisher

BioVision

Cat# 88946

Cat# 7925

N/A

Thrombin Sepharose Beads

FAM-Yap(60-99) peptide: FAM- DSETDLEALFNAVM

NPKTANVPQTVPMCLRKLPASFCKPP

Synthesized by American

Peptide

Recombinant Protein: GST-TEAD4 (aa 217-434; ref#

NP_003204.3)

This study

N/A

Recombinant Protein: GST-TEAD4 C367S (217-434)

Recombinant Protein: GST-YAP1 (ref# NP_001123617.1)

This study

N/A

Recombinant Protein: HIS-TEAD2 (aa 217-447; ref#

NP_003589.1)

Fetal Bovine Serum- Premium

Atlanta Biologicals

Gibco

Cat# S11150

Cat# 11965-092

Cat# 11320-033

Cat# 12587-010

Cat# AF-100-15

Cat# AF-100-18B

Cat# DAL1100

N/A

DMEM (Dulbecco’s Modiﬁed Eagle Medium), high glucose

DMEM/F12 1:1

Gibco

B-27^TMSupplement (50X), minus vitamin A

Animal-Free Recombinant Human EGF

Animal-Free Recombinant Human FGF-basic

alamarBlue^TMCell Viability Reagent

DNA ﬁngerprint analysis

Gibco

Peprotech

Invitrogen

IDEXX BioResearch

Critical Commercial Assays

Dual-Glo Luciferase Assay System

GenJet Plus DNA In Vitro Tranfection Reagent

Deposited Data

Promega

SignaGen

Cat# E2920

Cat# SL100499

Crystal structure of human TEAD2-Yap binding domain

This paper

PDB: 6E5G

PDB: 5EMV

PDB: 3JUA

PDB: 5HGU

PDB: 5DQ8

covalently bound to 2

Crystal structure of the palmitoylated human TEAD2

transcription factor

Structural basis of YAP recognition by TEAD4 in the

Hippo pathway

Crystal structure of human transcription factor TEAD2

in complex with palmitate

Crystal structure of human transcription factor TEAD2

in complex with ﬂufenamic acid

Experimental Models: Cell Lines

HEK-293

ATCC

Cat# CRL-1573; sex of cell line

is not available.

GBM43

Mayo Clinic

Gift from Dr. Jann Sarkaria; cells

were obtained from male patient.

Oligonucleotides

CTGF Forward Primer-5’TTGGCCCAGACCCAACTA3⁰

CTGF Reverse Primer- 5’GCAGGAGGCGTTGTCATT3’

ß-actin Forward Primer-5’TTGGCAATGAGCGGTTCC3’

ß-actin Reverse Primer-5’GTTGAAGGTAGTTTCGTGGATG3’

This study

N/A

(Continued on next page)

Cell Chemical Biology 26, 1–12.e1–e13, March 21, 2019 e1

Please cite this article in press as: Bum-Erdene et al., Small-Molecule Covalent Modiﬁcation of Conserved Cysteine Leads to Allosteric Inhibition of the

TEAD,Yap Protein-Protein Interaction, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.11.010

Continued

REAGENT or RESOURCE

Recombinant DNA

SOURCE

IDENTIFIER

Plasmid: pGEX-6P-1 TEAD4 (217-434)

Plasmid: pGEX-6P-1 TEAD4 (217-434) C367S

Plasmid: pGEX-6P-1 Yap1

Plasmid: pET-28a TEAD2 (217-447)

Software and Algorithms

This study

N/A

XDS

Kabsch (2010)

http://xds.mpimf-heidelberg.mpg.de;

RRID: SCR_015652

PHENIX

PHASER

Coot

Adams et al. (2010)

McCoy et al. (2007)

Emsley et al. (2010)

http://www.phenix-online.org;

RRID: SCR_014224

http://www.phaser.cimr.cam.ac.uk;

RRID: SCR_014219

http://www2.mrc-lmb.cam.ac.uk/Personal/

pemsley/coot/; RRID: SCR_014222

¨

PyMOL by Schrodinger

PyMOL v1.8

https://pymol.org; RRID: SCR_000305

¨

Schrodinger

Small-Molecule Drug Discovery Suite v2017-4

https://www.schrodinger.com/suites/small-

molecule-drug-discovery-suite;

RRID: SCR_014879

Amber14 (Serial/GPU)

The Amber Project

http://ambermd.org/AmberMD.php;

RRID: SCR_014230

AmberTools16 (MPI/CPU)

http://ambermd.org/AmberTools.php;

RRID: SCR_014230

Gaussian 09

Gaussian, Inc

http://gaussian.com/; RRID: SCR_014897

SigmaPlot 13.0

Systat Software, Inc

https://systatsoftware.com/products/sigmaplot/;

RRID: SCR_003210

Other

GSTrap FF column

GE Life Science

PerkinElmer

Agilent

Cat# 17513101

Cat# 17525501

Cat# 28989336

Cat# 2102

HisTrap FF column

HiLoad 26/600 Superdex 200 pg SEC column

Envision Multilabel Plate Reader

Agilent 6520 Accurate Mass Q-TOF

Agilent 1200 LC-MS

Cat# 6520

Agilent

Cat# 1200

OctetRed 384

ForteBio

Cat# RED384

Cat# 3474

96-well Clear Flat Bottom Ultra-Low Attachment Microplate

Corning

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulﬁlled by the Lead Contact,

Samy O. Meroueh (smeroueh@iu.edu).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

HEK-293 and GBM43 cells were cultured in DMEM medium with glutamine (Cellgro, Manassas, VA) supplemented with 10% FBS

and 1% penicillin/streptomycin in 5% CO₂at 37^ꢁC.

E. coli BL-21 (DE3) strain was purchased from New England Biolabs (Ipswich, MA) and cultured and grown in Luria Broth media

at 37^ꢁC.

METHOD DETAILS

Luciferase Reporter Assay

HEK-293 or GBM43 cells were plated at 2.4310⁴cells/well in a 96-well microplate and were transfected after 24 hr with the a pGL3.1

reporter containing the CTGF promoter and a plasmid encoding TK-Renilla luciferase in combination with control vectors or vectors

that express Yap1 and TEAD4. After 48 hr, cells were treated with 0.5, 1.0, 5.0 or 10 mM of 2 (TED-347) for another 48 hr. Luciferase

e2 Cell Chemical Biology 26, 1–12.e1–e13, March 21, 2019

Please cite this article in press as: Bum-Erdene et al., Small-Molecule Covalent Modiﬁcation of Conserved Cysteine Leads to Allosteric Inhibition of the

TEAD,Yap Protein-Protein Interaction, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.11.010

activity was measured according to the Dual-Glo luciferase assay (Promega) instructions using a Biotek Synergy Neo2 plate reader.

Relative luciferase activity represents the ratio of ﬁreﬂy/renilla luminescence values.

Covalent Pull Down of TEAD4

HEK-293 cells transfected with the myc-TEAD4 construct were grown for 48 hr and then treated with DMSO or with 25 mM of 2 (TED-

347) for an additional 48 hr. Cells were then harvested in lysis buffer (50 mM Tris-HCl, pH 7.3, 150 mM NaCl, 0.5 mM EDTA, 1% Triton

X-100, PhosSTOP phosphatase inhibitor cocktail, and EDTA-free protease inhibitors cocktail). Cell lysates containing 2 mg of protein

were incubated with the indicated compounds or DMSO for 24 hr. Extracts were then incubated with Dynabeads M-280 Streptavidin

(Sigma-Aldrich) for 2 hr at 4^ꢁC. The Dynabeads were then washed and bound proteins were denatured and eluted according to the

manufacturer’s instructions. Relative levels of myc-TEAD4 from each complex were measured by immunoblot analysis with the

anti-c-Myc antibody (1:5,000, Sigma-Aldrich).

Co-immunoprecipitation

HEK293 cells transfected with Flag-YAP1 alone or in combination with myc-TEAD4 were incubated with DMSO or the indicated

amount of compounds for 48 hr. Cells were harvested in lysis buffer (50 mM Tris-HCl, pH 7.3, 150 mM NaCl, 0.5 mM EDTA, 1% Triton

X-100, PhosSTOP phosphatase inhibitor cocktail, and complete EDTA-free protease inhibitors cocktail). Extracts were immunopre-

cipitated with magnetic beads coupled to the M2 (anti-Flag) antibody (Sigma-Aldrich) for 4 hr at 4^ꢁC. The Dynabeads were then

washed and bound proteins were denatured and eluted according to the manufacturer’s instructions. Relative levels of myc-

TEAD4 from each complex was then measured by immunoblot analysis with the anti-c-Myc antibody (1:5,000, Sigma-Aldrich).

RNA Extraction and Real-Time PCR

HEK293 cells co-transfected the Flag-YAP and myc-TEAD4 constructs were incubated with DMSO or the indicated amount of com-

pounds for 48 hr. Total RNA was puriﬁed using the RNeasy plus mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s

instructions. Complementary DNA was synthesized from 500 ng total RNA with Oligo-dT primers and the Multi-Scribe reverse tran-

scriptase (Fisher, Waltham, MA) according to the manufacturer’s instructions.

Real-time PCR reactions utilized 100 ng cDNA, 200 nM gene speciﬁc primers and the Sensifast No-ROX mix (Bioline, Taunton, MA)

in a total volume of 20 mL. All measurements were carried out in triplicate using an Eppendorf Mastercyclerꢀ RealPlex2. The se-

quences of primers for CTGF were forward, 5⁰- TTGGCCCAGACCCAACTA-3; and reverse, 5⁰- GCAGGAGGCGTTGTCATT-3’. The

primer sequences for b-actin were forward, 5⁰-TTGGCAATGAGCGGTTCC-3; and reverse, 5⁰- GTTGAAGGTAGTTTCGTGGATG-3⁰.

Sphere-Forming Assay

The GBM43 xenograft tissue was a kind gift from Dr. Jann Sarkaria (Mayo Clinic, Rochester, MN), and tumors were expanded by passage

in the ﬂankofNOD/SCIDg^nullmice. TogenerateGBM43 celllines, tumorswere harvested, disaggregated, and maintainedin2.5%FBSfor

14 days on Matrigel-coated plates (BD Biosciences) to remove murine ﬁbroblasts. In-vitro GBM43 cell lines were propagated in DMEM

with 10% FBS for no more than 7 passages. Cell line identity was conﬁrmed by DNA ﬁngerprint analysis (IDEXX BioResearch) for species

and baseline short-tandem repeat analysis testing. GBM43 spheroids were generated by plating early-passage cells at 2.5 x 10⁴cells per

well in 96-well ultralow attachment plates (Corning Inc.) in DMEM/F12 (1:1; GIBCO) supplemented with 2% B27 supplement (GIBCO),

20 ng/mL epidermal growth factor (EGF), and 20 ng/mL ﬁbroblast growth factor (FGF) (Peprotech) for 2 days. The spheroids were then

treated with compounds 1 (TED-346), 2 (TED-347) and 5 (TED-551) and growth analyzed by Alamar blue staining.

Protein Expression and Puriﬁcation

TEAD4 (217-434), TEAD4 (217-434) Cys367Ser mutant, and Yap1 (Full-length) were expressed as GST-fusion proteins in BL-21 (DE3)

strain of E. coli from the pGEX-6P-1 vector. Transformed bacteria were grown in LB at 37^ꢁC until they reached an OD₆₀₀of 0.6 – 0.8.

Isopropyl-b-D-galactoside (IPTG) was added to a ﬁnal concentration of 0.5 mM and cells were then incubated at 16^ꢁC for 16 hr. Cell

pellets were re-suspended in a buffer containing 200 mM NaCl, 20 mM Tris, 2 mM dithiothreitol (DTT), pH 8.0, and lysed by passage

through a microﬂuidizer. Cell debris was removed by centrifugation at 35,000 x g for 1 hr. Clariﬁed lysates were loaded onto a pre-

equilibrated 5 mL GSTrap HP column at 1 ml/min. The column was washed with 5 column volumes of buffer and the protein was

eluted with 10 mM reduced glutathione in the same buffer. The protein was further puriﬁed on a HiLoad 26/600 Superdex 200 pg

SEC column (GE, Boston, MA) with 100 mM NaCl, 20 mM Tris, 2 mM DTT, pH 8.0 as buffer. The GST-tag was cleaved from proteins

by incubation with the HRV-3C protease (ThermoFisher, Waltham, MA) at 100:1 w/w ratio while dialyzing against PBS with 5 mM

b-mercaptoethanol for 48 hr at 4^ꢁC. The cleavage solution was passed through a GSTrap HP column to remove the cleaved GST

and the HRV-3C protease. Cleavage was veriﬁed by SDS-PAGE and mass spectrometry.

TEAD2 (217-447) was expressed as N-terminal HIS-fusion protein in BL-21 (DE3) strain of E. coli from the pET-28a vector. Trans-

formed bacteria were grown in Terriﬁc Broth at 37^ꢁC until they reached an OD₆₀₀of 0.6 – 0.8. IPTG was added to a ﬁnal concentration

of 0.5 mM and cells were then incubated at 16^ꢁC for 16 hr. Cell pellets were re-suspended in a buffer containing 500 mM NaCl, 50 mM

HEPES, 8 mM b-mercaptoethanol, pH 7.5 and lysed by multiple passages through a microﬂuidizer. Cell debris was removed by

centrifugation at 35,000 x g for 1 hr. Clariﬁed lysates were loaded onto a pre-equilibrated 5 mL HisTrap FF column at 1 mL/min.

The column was washed with 100 mL of buffer containing 300 mM NaCl, 25 mM HEPES, 1 mM TCEP, 5 % v/v glycerol, 30 mM imid-

azole, pH 7.5 prior to elution with the same buffer containing 300 mM imidazole. The protein was further puriﬁed on a HiLoad 26/600

Cell Chemical Biology 26, 1–12.e1–e13, March 21, 2019 e3

Please cite this article in press as: Bum-Erdene et al., Small-Molecule Covalent Modiﬁcation of Conserved Cysteine Leads to Allosteric Inhibition of the

TEAD,Yap Protein-Protein Interaction, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.11.010

Superdex 200 pg SEC column (GE, Boston, MA) with 150 mM NaCl, 25 mM HEPES, 1 mM TCEP, pH 7.5 as buffer. For crystallization

trials, the elute from the HisTrapFF afﬁnity chromatography was dialyzed against 150 mM NaCl, 50 mM Tris pH 8.0 for 2 hr, then

cleaved with 1:100 w/w thrombin at 4^ꢁC overnight. The cleaved protein was dialyzed against 300 mM NaCl, 25 mM HEPES,

1 mM TCEP, 5 % v/v glycerol, 10 mM imidazole, pH 7.5. The cleaved HIS-tag was removed by passing through the HisTrap FF col-

umn. TEAD2 without the HIS-tag was further puriﬁed on SEC, as above.

Size-Exclusion Chromatography

2 ml of 6.3 mM GST-TEAD4 in PBS was incubated with 100 mM 2 (TED-347) in 2 % v/v DMSO or DMSO without compound for 24 hr at

4^ꢁC. After the incubation, the samples were injected into a HiLoad 26/600 Superdex 200 pg SEC column, pre-equilibrated with PBS.

The elution proﬁle of the column was analyzed for protein aggregation.

Fluorescence Polarization

GST-TEAD4, GST-TEAD4 Cys367Ser mutant or HIS-TEAD2 interaction with Yap1 was investigated using a ﬂuorescently-labeled

peptide (FAM-Yap_60-99), consisting of FAM-labeled TEAD-binding peptide fragment of Yap1 (FAM-DSETDLEALFNAVMNPK

TANVPQTVPMCLRKLPASFCKPP), which has a disulﬁde bridge (American Peptide, Sunnyvale, CA). Addition of FAM-Yap_60-99to

the TEAD was followed by measurement of changes in polarization. 40 mL of 125 nM GST-TEAD4 WT or GST-TEAD4 Cys367Ser

in assay buffer (PBS with 0.01 % v/v Triton-X100) or 40 mL of 64 nM HIS-TEAD2 was added to a 384-well black polystyrene plate

(Cat. No. 262260; Nunc, Roskilde, Denmark) and incubated with 5 mL of 2 – 2000 mM serially diluted compounds in assay buffer sup-

plemented with 20 % v/v DMSO for 24 hr at 4^ꢁC. Finally, 5 mL of 160 nM FAM-Yap_60-99peptide was added, the plate centrifuged, and

the polarization was measured on an Envision Multilabel Plate Reader (PerkinElmer, Waltham, MA) using a ﬁlter set with excitation

and emission wavelengths of 485 and 535 nm, respectively. Percent inhibition was calculated as relative to a minimum inhibition con-

trol, which is without compound, and a maximum inhibition control, which is without a TEAD.

For the determination of the inhibition efﬁciency k_inact/K_I, the protein – compound incubation time was varied between 0.5 – 48 hr,

prior to the addition of the FAM-Yap_60-99peptide and ﬂuorescence polarization measurements. The progressive decrease in TEAD

activities were plotted against time for all 10 concentrations (0.2 – 100 mM) of the compounds and the observed rate of inhibition (k_obs)

was calculated by ﬁtting a simple exponential function. The observed rate of inhibition was then plotted against the concentration of

k_inact½Inhibitorꢂ

the compound and a polynomial function k_obs

=

was ﬁtted to determine the k_inactand K_Ivalues.

K_I+ ½Inhibitorꢂ

Crystallization of TEAD2 and Structure Reﬁnement

Puriﬁed TEAD2 was concentrated to ꢃ12 mg/mL and crystallized at 20^ꢁC using the hanging-drop vapor-diffusion method with a

reservoir solution containing 0.1 M HEPES (pH 7.2 – 7.4) and 2.4 - 2.8 M sodium formate. The crystals were soaked in reservoir so-

lution supplemented with 3 - 5 mM of 2 (TED-347) and 25 % v/v glycerol for 3 hr and were subsequently ﬂash-cooled in liquid nitrogen.

To rule out the possibility that the observed density of 2 (TED-347) was not the endogenous S-palmitoylation from protein expression

(Noland et al., 2016), some crystals were soaked in a cryo-protectant solution supplemented with 2 mM DTT for 2 hr to soak out the

fatty acid. The crystal structure of these crystals was solved and no extra electron-density was observed. Another batch of crystals

were soaked in three steps: (1) in a cryo-protectant solution supplemented with 2 mM DTT for 2 hr, (2) in a cryo-protectant solution

(wash) for 2 hr, and (3) in a cryo-protectant solution supplemented with 3-5 mM of 2 (TED-347) for 3 hr.

Data was collected at beamline 4.2.2 at the Advanced Light Source (ALS, Berkeley, CA, USA) and processed with XDS. All crystals

contained two molecules per asymmetric unit and the symmetry corresponded to space group C2. Molecular Replacement was used

to obtain the initial phases using Phaser and the crystal structure of TEAD2 transcriptional activation domain (PDB: 5EMV) as the

search model. Initial model building was carried out using Autobuild in PHENIX. The ﬁnal model (R_free0.268, with good geometry

and no Ramachandran outliers) was obtained by iterative cycles of manual building in Coot and reﬁnements with PHENIX-reﬁne.

Protein Mass Spectrometry

Compounds at 200 mM concentrations (unless otherwise speciﬁed) were incubated with 10 mM TEAD4 WT or TEAD4 Cys367Ser

mutant in 20 mM NH₄OAc for 24 hr (unless otherwise speciﬁed) at 4^ꢁC. The samples were centrifuged at 20,000 x g for 20 min to

remove precipitants prior to being injected into an empty column on an Agilent 1200 liquid chromatography system (Agilent, Santa

Clara, CA), using 80 % Buffer A (H₂O, 5 mM NH₄OAc) and 20 % Buffer B (ACN, 5 mM NH₄OAc), and the masses were detected on an

Agilent 6520 Accurate Mass Q-TOF.

Biolayer Interferometry

Biolayer Interferometry was measured on OctetRed 384 (ForteBio, Menlo Park, CA) using PBS with 0.025% v/v Tween-20 at 30^ꢁC

with constant shaking at 1000 rpm. Streptavidin-conjugated sensors (ForteBio, Menlo Park, CA) were loaded with 30 mg/ml biotin-

labeled GST-Yap or biocytin and were introduced to 1-1000 nM TEAD4. The sensors were regenerated with 5 mM HCl solution after

each interaction. For compound inhibition study, 100 nM TEAD4 was pre-incubated with 0.1 – 100 mM 2 (TED-347) in 2 % v/v DMSO

for 24 hr at 4^ꢁC prior to interaction with captured GST-Yap.

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In Silico Protein Preparation

The crystal structures of TEAD4$YAP (PDB: 3JUA), TEAD2$PLM (PDB: 5HGU, palmitic acid), and TEAD2$FLF (PDB: 5DQ8, ﬂufe-

¨

namic acid) were retrieved and prepared using the Protein Preparation Wizard in the Schrodinger software package (Schrodinger

LLC, New York, NY, 2017)(Greenwood et al., 2010; Sastry et al., 2013). Bond orders were assigned and hydrogen atoms were added.

Missing side chains and loops were introduced using the Prime module (Jacobson et al., 2004). The resulting protein and compound

structures were protonated at pH 7.0 using PROPKA (Olsson et al., 2011) and Epik (Shelley et al., 2007), respectively. The structure of

2 (TED-347) was generated by replacing the carboxylic acid on FLF with chloromethyl ketone. Subsequently, the binding modes of

¨

PLM and 2 (TED-347) to TEAD4 were obtained using the align function in PyMOL (Schrodinger, 2015).

Covalent Docking

The covalent structure of TEAD4$2 was generated using CovDock (Toledo Warshaviak et al., 2014). The chloromethyl ketone group

˚

of 2 was deﬁned as the reaction group for a nucleophilic substitution reaction with the TEAD4 Cys-367. Residues within 3.0 A of 2

were reﬁned during covalent docking. The covalent bond parameters from the OPLS force ﬁeld (Banks et al., 2005) were extracted.

Molecular Dynamics Simulations

The structures of TEAD4,Yap1, TEAD4,Yap1$PLM, non-covalent [TEAD4$2]$Yap1, and covalent [TEAD4-2]$Yap1 were used to run

molecular dynamics simulations using the AMBER14 software package (Case et al., 2014). The restrained electrostatic potential

(RESP) atomic charges (Bayly et al., 2013) of PLM and 2 in the covalent and non-covalent complexes were calculated at the HF/

6-31G* level (McWeeny and Diercksen, 1968; Petersson et al., 1988; Pople and Nesbet, 1954) using the Gaussian 09 package (Frisch

et al., 2009). In the covalent [TEAD4-2]$Yap1 complex, 2, Leu-366, Cys-367, and Glu-368 were extracted for RESP charge ﬁtting. The

atom charges of Cys-367 were replaced by RESP charges and the optimized parameters of bond length, bond angle, and dihedral

angle between Cys-367 and 2 were used to build new frcmod parameters. The a-carbon atom of 2 and sulfur atom of Cys-367 were

bonded using tleap program.

˚

Complexes were immersed in a box of TIP3P water molecules (Jorgensen et al., 1983). No atom on the complex was within 14 A of

any side of the box. The solvated box was further neutralized with Na⁺or Cl^-counterions using the tleap program. Simulations were

carried out using the GPU accelerated version of the pmemd program with ff14SB (Maier et al., 2015) and gaff force ﬁelds (Wang

et al., 2004) in periodic boundary conditions. All bonds involving hydrogen atoms were constrained by using the SHAKE algorithm

(Ryckaert et al., 1977), and a 2 femtoseconds (fs) time step was used in the simulation. The particle mesh Ewald (PME) method (Dar-

den et al., 1993) was used to treat long-range electrostatics. Simulations were run at 298 K under 1 atm in NPT ensemble employing

Langevin thermostat and Berendsen barostat. Water molecules were ﬁrst energy-minimized and equilibrated by running a short

simulation with the complex ﬁxed using Cartesian restraints. A series of energy minimizations were subsequently applied in which

-2

˚

the Cartesian restraints were gradually relaxed from 500 kcal∙A to 0 kcal∙A , and the system was subsequently gradually heated

to 298 K with a 48 ps molecular dynamics run. For each complex, we generated 50 independent simulations (replicates) that are each

50 ns in length. The initial velocity of each replicate was randomly assigned. In total, 2.5 ms of simulation was run for each complex.

Free Energy Calculations

In each of the 50 trajectories (50 ns in length), the ﬁrst 2 ns were discarded for equilibration. Snapshots were saved every 1 ps, yielding

48000 structures per trajectory. 30000 snapshots were selected at regular intervals for free energy calculations using the cpptraj pro-

gram (Roe and Cheatham, 2013). The Molecular Mechanics-Generalized Born Surface Area (MM-GBSA)(Still et al., 1990) method

was used to calculate the free energy using the MMPBSA.py script (Miller et al., 2012). The calculation using the GB method was

performed with sander and Onufriev’s GB model (Feig et al., 2004; Onufriev et al., 2004). Solvent-accessible surface area (SASA)

calculations were switched to the icosahedron (ICOSA) method, where surface areas are computed by recursively approximating

a sphere around an atom, starting from an icosahedron. Salt concentration was set to 0.1 M. The entropy was estimated by normal

mode calculations (Brooks and Karplus, 1983) with the mmpbsa_py_nabnmode module by selecting 150 of the 30000 snapshots

used in the free energy calculations at regular intervals. The maximum number of cycles of minimization was set to 10000. The

convergence criterion for the energy gradient to stop minimization was 0.5. In total, 30000 frames were used for each MM-GBSA

calculations while 150 frames were used for each normal mode analysis. All other parameters were left at default values.

The MM-GBSA binding free energy is expressed as:

DG_MM-GBSA= DE_GBTOTꢀ TDS_NMODE

where DE_GBTOTis the combined internal and solvation energies, T is the temperature (298.15 K). DS_NMODEis the entropy estimated by

normal mode calculations. The total enthalpy from the generalized Born model, DE_GBTOT, is the sum of 4 components:

DE_GBTOT= DE_VDW+ DE_ELE+ DE_GB+ DE_SURF

where DE_VDWand DE_ELEare the van der Waals and electrostatic energies, respectively, and DE_GBand DE_SURFare the polar and non-

polar desolvation energies, respectively. All binding energies are determined by:

DE = E^COM- E^REC- E^LIG

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where E^COM, E^RECand E^LIGare total energies corresponding to the complex, receptor, and ligand, respectively. The relative differ-

ence in free energy is determined by:

DDG = DG_COM- DG_APO

where DG_COMand DG_APOare the covalent or non-covalent complex and the unbound native apo complex, respectively.

Synthesis

All chemicals were purchased from either Aldrich or Acros and used as received. Column chromatography was carried out with silica

gel (25-63 m). Mass spectra were measured on an Agilent 6520 Accurate Mass Q-TOF instrument. ¹H NMR spectra were recorded in

CDCl₃or Methanol-d4 on a Bruker 500 MHz spectrometer. Chemical shifts are reported using residual CHCl₃or MeOH as internal

references. All compounds that were evaluated in biological assays had >95% purity by HPLC.

2-((3-(triﬂuoromethyl)phenyl)amino)benzoic acid (1, TED-346)

Purchased from a commercial source.

¹H NMR (400 MHz, CDCl₃). d 9.42 (s, 1H), 8.09-8.07 (dd, J=8.0Hz, J=1.2Hz, 1H), 7.51 (s, 1H), 7.49-7.40 (m, 3H), 7.36-7.34 (m, 1H),

7.27-7.25 (m, 2H), 6.87-6.83 (t, J=8.0Hz, 1H).

Synthesis of 2-chloro-1-(2-((3-(triﬂuoromethyl)phenyl)amino)phenyl) ethanone (2, TED-347)

A solution of 2-((3-(triﬂuoromethyl)phenyl)amino)benzoic acid (1, 200 mg, 0.71 mmol) in SOCl₂(6 mL) was reﬂuxed for 1 hr. The

mixture was evaporated to dryness. The residue was dissolved in MeCN (10 mL) and cooled to 0^ꢁC, followed by the addition of

TMSCHN₂(1.07 mmol, 0.5 mL). It was stirred for 1 hr, and conc. HCl (0.5 mL) was added to the mixture at 0^ꢁC. The mixture was stirred

for 0.5 hr. The mixture was quenched with NaHCO₃(aq) and diluted with water. The mixture was extracted with EA (15 mL x 2). The

combined organic layers were dried with Na₂SO₄, ﬁltered and concentrated. The residue was puriﬁed by Prep-TLC (PE / EA = 5 / 1) to

give 2-chloro-1-(2-((3-(triﬂuoromethyl)phenyl)amino)phenyl)ethanone (6.4 mg, 2.8%); ¹H NMR (400 MHz, CDCl₃): d 10.43 (s, 1H),

7.79-7.64 (dd, J=4.0Hz, J=0.8Hz, 1H), 7.51-7.47 (m, 1H), 7.45-7.41 (m, 1H), 7.38-7.36 (m, 3H), 7.29-7.26 (m, 1H), 6.85-6.82

(m, 1H), 4.75 (s, 1H); ¹³C NMR (400 MHz, CDCl₃) d 193.41, 147.80, 140.67, 135.66, 132.16, 131.86, 130.04, 125.86, 125.20,

+

122.49, 120.71, 120.67, 120.63, 119.33, 119.30, 119.26, 117.84, 116.87, 114.65, 46.53. LRMS calculated for C₁₅H₁₂ClF₃NO

[M+H]⁺, 314.05 found 314.0.

Synthesis of 1-(2-((3-(triﬂuoromethyl)phenyl)amino)phenyl)propan-1-one (3, TED-550)

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To a solution of N-methoxy-N-methyl-2-((3-(triﬂuoromethyl)phenyl)amino)benzamide (100 mg, 0.31 mmol) in THF (5 mL) was

added 1 M EtMgBr in THF (1.8 mL) at -78 ^oC under N₂. The resulting mixture was warmed to rt and stirred for 5 hr. The reaction mixture

was quenched with sat. NH₄Cl (20 mL), and extracted with EA (5 mL x 3). The organic phase was washed with brine, dried

over Na₂SO₄, and ﬁltered. The ﬁltrate was concentrated. The residue was puriﬁed by prep-TLC (PE/EA=20/1) to give 1-(2-((3-(triﬂuor-

omethyl)phenyl)amino)phenyl)propan-1-one (15 mg, 16.5%); ¹H NMR (400MHz, CDCl₃): d 10.68 (s, 1H), 7.89 (d, J=8.0Hz, 1H), 7.51

(s, 1H), 7.30-7.46 (m, 5H), 6.83 (t, J=7.2Hz, 1H), 3.07 (q, 2H), 1.24 (t, J=7.2Hz, 3H); ¹³C NMR (400 MHz, CDCl₃) d 204.20, 146.57,

141.44, 134.38, 131.59, 129.87, 125.03, 119.78, 119.72, 118.52, 118.49, 117.79, 114.56, 32.70, 8.69. LRMS calculated for

C₁₆H₁₅F₃NO⁺[M+H]⁺, 293.3 found 294.1.

Synthesis Scheme of 4 (TED-548)

Synthesis of 1-(2-((3-(2-methoxyethoxy)phenyl)amino)phenyl)ethanone (TED-548-2). A mixture of 3-(2-methoxyethoxy)aniline

(500 mg, 2.99 mmol), 1-(2-bromophenyl)ethanone (625 mg, 3.14 mmol), Pd₂(dba)₃(275 mg, 0.30 mmol) , xphos (286 mg,

0.60 mmol), and Cs₂CO₃(1.47 g, 4.50 mmol) in dioxane (10 mL) was heated to 90^ꢁC under N₂, and stirred for 2 hr. The reaction mixture

was cooled to room temperature, and ﬁltered over Celite. The ﬁltrate was concentrated. The residue was dissolved in EA (40 mL),

washed with brine, and dried over Na₂SO₄. The solution was ﬁltered, and the ﬁltrate was concentrated. The residue was puriﬁed

by column chromatography (PE-PE/EA=5/1) to give 1-(2-((3-(2-methoxyethoxy)phenyl)amino)phenyl)ethanone (710 mg, 83.2%);

¹H NMR (400MHz, CDCl₃): d 10.51 (s, 1H), 7.81 (d, J=8.0Hz, 1H), 7.30-7.31 (m, 2H), 7.23 (t, J=8.0Hz, 1H), 6.84-6.85 (m, 2H), 6.68-

6.76 (m, 2H), 4.11 (t, J=4.8Hz, 2H), 3.75 (t, J=4.8Hz, 2H), 3.45 (s, 3H), 2.64 (s, 3H). LRMS calculated for C₁₇H₂₀NO₃[M+H]⁺,

+

286.3 found 286.2.

Synthesis of 2-chloro-1-(2-((3-(2-methoxyethoxy)phenyl)amino)phenyl) ethanone (4, TED-548). To a mixture of 1-(2-((3-(2-methox-

yethoxy)phenyl)amino)phenyl)ethanone (50 mg, 0.18 mmol) in DCM (3 mL) were added DIEA (67 mg, 0.52 mmol) and TMSOTf (58 mg,

0.26 mmol) at 0^ꢁC under N_2.The resulting mixture was warmed to room temperature and stirred for 5 hr. NCS (24 mg, 0.18 mmol) was

added and the mixture was stirred for another 2 hr. It was quenched with water (10 mL), and the mixture was extracted with DCM

(5mL x 3). The organic phase was washed with brine, dried over Na₂SO₄, and ﬁltered. The ﬁltrate was concentrated. The residue

was puriﬁed by prep. TLC (PE/EA=5/1) to give 2-chloro-1-(2-((3-(2-methoxyethoxy)phenyl)amino)phenyl)ethanone (5.1 mg, 8.9%);

¹H NMR (400MHz, CDCl₃): d 10.35 (s, 1H), 7.77 (d, J=8.0Hz, 1H), 7.22-7.39 (m, 4H), 7.07 (d, J=2.8Hz, 1H), 6.84 (t, J=7.2Hz, 1H),

6.63-6.65 (m, 1H), 4.76 (s, 2H), 4.08 (t, J=4.8Hz, 2H) , 3.74 (t, J=4.8Hz, 2H) , 3.45 (s, 3H); ¹³C NMR (400 MHz, CDCl₃): d 193.13,

157.99, 147.15, 137.90, 135.36, 131.25, 130.56, 117.91, 117.49, 115.51, 110.85, 109.43, 70.95, 67.75, 59.25, 46.55. LRMS calcu-

lated for C₁₇H₁₉ClNO₃⁺[M+H]⁺, 320.8 found 320.0.

Synthesis Scheme of 5 (TED-551)

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Synthesis of 1-(3-((3-(triﬂuoromethyl)phenyl)amino)pyridin-2-yl)ethanone (TED-551-1). A mixture of 1-(3-bromopyridin-2-yl)etha-

none (200 mg, 1.00 mmol), 3-(triﬂuoromethyl)aniline (161 mg, 1.00 mmol), Pd₂(dba)₃(92 mg, 0.10 mmol), xphos (95 mg,

0.20 mmol), and Cs₂CO₃(489 mg, 1.50 mmol) in dioxane (10 mL) was heated to 90^ꢁC under N₂. The mixture was stirred for 2 hr.

The reaction mixture was cooled to room temperature, and ﬁltered through Celite. The ﬁltrate was concentrated. The residue was

dissolved in EA (40 mL), and the mixture was washed with brine, dried over Na₂SO₄, and ﬁltered. The ﬁltrate was concentrated.

The residue was puriﬁed by column chromatography (PE-PE/EA=20/1) to give 1-(3-((3-(triﬂuoromethyl)phenyl)amino)pyridin-2-yl)

ethanone (160 mg, 57.1%); ¹H NMR (400MHz, CDCl₃): d 10.43 (s, 1H), 8.13 (d, J=4.0Hz, 1H), 7.59 (d, J=8.0Hz, 1H), 7.47-7.51

(m, 2H), 7.39 (t, J=6.4Hz, 2H), 7.26-7.30 (m, 1H), 2.79 (s, 3H). LRMS calculated for C₁₄H₁₂F₃N₂O⁺[M+H]⁺, 281.3 found 281.1.

Synthesis of 2-chloro-1-(3-((3-(triﬂuoromethyl)phenyl)amino)pyridin-2-yl)ethanone (5, TED-551). The procedure was the same as 4

(TED-548) to give 5 (TED-551).

5 (TED-551). ¹H NMR (400MHz, CDCl₃): d 10.21 (s, 1H), 8.11-8.10 (dd, J=4.0Hz, J=1.2Hz, 1H), 7.61-7.58 (m, 1H), 7.53-7.49 (m, 2H),

7.44-7.39 (m, 2H), 7.34-7.31 (m, 1H), 5.20 (s, 2H). ¹³C NMR (400 MHz, CDCl₃): d 195.58, 143.74, 139.61, 139.07, 133.56, 132.42,

132.10, 130.32, 129.07, 126.07, 125.05, 122.34, 121.64, 121.47, 121.39, 119.51, 119.47, 48.03. LRMS calculated for

C₁₄H₁₁ClF₃N₂O⁺[M+H]⁺, 315.04 found 315.0.

Synthesis Scheme of 6 (TED-589)

Synthesis of methyl 4-methoxy-2-((3-(triﬂuoromethyl)phenyl)amino)benzoate (TED-589-1). A mixture of methyl 2-amino-4-methox-

ybenzoate (1.0 g, 5.5 mmol), 1-iodo-3-(triﬂuoromethyl)benzene (1.8 g, 6.6 mmol), Pd₂(dba)₃(504 mg, 0.55 mmol) , xphos (286 mg,

0.60 mmol), and Cs₂CO₃(3.6 g, 11.0 mmol) in dioxane (40 mL) was stirred at 90^ꢁC under N₂overnight. The reaction mixture was

cooled to room temperature, and ﬁltered over Celite. The ﬁltrate was concentrated. The residue was dissolved in ethyl acetate

(40 mL), washed with brine, and dried over Na₂SO₄. The solution was ﬁltered, and the ﬁltrate was concentrated. The residue was

puriﬁed by column chromatography (PE-PE/EA=5/1) to give methyl 4-methoxy-2-((3-(triﬂuoromethyl)phenyl)amino)benzoate

(1.12 g, 62.7%); ¹H NMR (400MHz, CDCl₃): d 9.75 (s, 1H), 7.95-7.92 (d, J=8.8 Hz, 1H), 7.52 (s, 1H), 7.44-7.43 (m, 2H), 7.32-7.30

(d, J=6.8 Hz, 1H), 6.73 (d, J=6.4 Hz, 1H), 6.39-6.36 (dd, J=9.2 Hz, J=2.4 Hz, 1H), 3.88 (s, 3H), 3.76 (s, 3H). LRMS calculated for

C₁₇H₂₀NO₃⁺[M+H]⁺, 326.1 found 326.1.

Synthesis of 4-methoxy-2-((3-(triﬂuoromethyl)phenyl)amino)benzoic acid (TED-589-2). To a mixture of methyl 4-methoxy-2-((3-(tri-

ﬂuoromethyl)phenyl)amino)benzoate (TED-589-1, 1.12 g, 3.38 mmol) in a mixture of dioxane/water (10 mL/10 mL) was added

LiOH.H₂O(1.43 g, 33.8 mmol) at rt and the mixture was stirred for 2 hr. The mixture was acidiﬁed to pH 6 with 1 M HCl, and the organic

phase was extracted with ethyl acetate (10 mL x 2). The organic phase was washed with brine, dried over Na₂SO₄, and ﬁltered. The

ﬁltrate was concentrated. The residue was puriﬁed by column chromatography (DCM/MeOH=10/1) to give 4-methoxy-2-((3-(triﬂuor-

omethyl)phenyl)amino)benzoic acid (1.07 g, 99% yield); LRMS calculated for C₁₇H₂₀NO₃⁺[M+H]⁺, 312.1 found 312.1.

Synthesis of N,4-dimethoxy-N-methyl-2-((3-(triﬂuoromethyl)phenyl)amino)benzamide (TED-589-3). To a solution of 4-methoxy-2-

((3-(triﬂuoromethyl)phenyl)amino)benzoic acid (TED-589-2, 1.07 g, 3.46 mmol), N,O-dimethylhydroxylamine hydrochloride

(503 mg, 5.19 mmol) and HATU (1.97 g, 5.19 mmol) in DMF (20 mL) was added N-ethyl-N-isopropylpropan-2-amine (880 mg,

7.0 mmol). The mixture was stirred at room temperature for 2 hr. After completion of the reaction, ethyl acetate was added to the

mixture. Then the mixture was washed with water (100 mL x 2) and brine (100 mL). The organic phase was dried over Na₂SO₄, ﬁltered

and concentrated. The residue was puriﬁed by column chromatography (PE/EA= 3:1) to give N,4-dimethoxy-N-methyl-2-((3-(triﬂuor-

omethyl)phenyl)amino)benzamide as a yellow oil (1.12 g, 91.3 % yield); ¹H NMR (400MHz, CDCl₃): d 8.44 (s, 1H), 7.54-7.52

(d, J=8.8 Hz, 1H), 7.41-7.40 (d, J=9.2 Hz,1H), 7.38-7.36 (d, J=8.0 Hz,1H), 7.29-7.26 (m, 1H), 7.20-7.18 (d, J=7.6 Hz,1H), 6.88

(d, J=2.8 Hz, 1H), 6.46-6.43 (dd, J=11.2 Hz, J=2.4 Hz, 1H), 3.77 (s, 3H), 3.60 (s, 3H), 3.36 (s, 3H). LRMS calculated for

C₁₇H₂₀NO₃⁺[M+H]⁺, 355.1 found 355.1.

Synthesis of 1-(4-methoxy-2-((3-(triﬂuoromethyl)phenyl)amino)phenyl)ethanone (TED-589-4). To a solution of N,4-dimethoxy-N-

methyl-2-((3-(triﬂuoromethyl)phenyl)amino)benzamide (TED-589-3, 400 mg, 1.13 mmol) in dry THF (20 mL) under N₂was added

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methylmagnesium bromide (10ml 1.0 M in THF, 10.0 mmol) at 0^ꢁC. The mixture was stirred at 0^ꢁC for 0.5 hr and then at room tem-

perature for 2 hr. The reaction mixture was quenched by the addition of saturated aqueous NH₄Cl. The organic phase was ex-

tracted with ethyl acetate. The combined ethyl acetate layers were washed with brine, dried over anhydrous sodium sulfate,

and concentrated. The residue was puriﬁed by column chromatography (PE/EA= 6:1) to give the desired product as a yellow

oil (310 mg, 88.5% yield); ¹H NMR (400 MHz, CDCl₃): d 10.90 (s, 1H), 7.80-7.77 (d, J=8.8 Hz, 1H), 7.55 (s, 1H), 7.46-7.34

(m, 1H), 7.35-7.33 (d, J=6.8 Hz, 1H), 6.70 (d, J=2.4 Hz, 1H), 6.38-6.35 (dd, J=8.8 Hz, J=2.4 Hz, 1H), 3.76 (s, 3H), 2.59 (s, 3H).

LRMS calculated for C₁₆H₁₃F₃NO⁺[M+H]⁺, 310.1 found 310.1.

Synthesis of 2-chloro-1-(4-methoxy-2-((3-(triﬂuoromethyl)phenyl)amino)phenyl)ethanone (6, TED-589). To a mixture of 1-(4-me-

thoxy-2-((3-(triﬂuoromethyl)phenyl)amino)phenyl)ethanone (TED-589-4, 310 mg, 1.0 mmol) in dichoromethane (DCM) (10 mL)

were added DIEA (256 mg, 2.0 mmol) and TMSOTf (266 mg, 1.2 mmol) at 0^ꢁC under N_2.The resulting mixture was warmed to

room temperature and stirred for 2 hr. NCS (24 mg, 0.18 mmol) was added and the mixture was stirred for another 2 hr. It was

quenched with water (10 mL), and the mixture was extracted with DCM (5mL x 3). The organic phase was washed with brine, dried

over Na₂SO₄, and ﬁltered. The ﬁltrate was concentrated. The residue was puriﬁed by prep. TLC (PE/EA=5/1) to give the crude product

(207 mg). The compound was further puriﬁed by reverse HPLC Gilson to afford the desired product as a yellow solid (78 mg, 22.6%

yield); ¹H NMR (400MHz, CDCl₃): d 10.72 (s, 1H), 7.73-7.70 (d, J=9.2 Hz, 1H),7.56 (s, 1H) 7.50-7.38 (m, 3H), 6.69 (s, 1H), 6.40-6.38

(d, J=8.8 Hz, 1H), 4.67 (s, 2H), 3.77 (s, 3H); ¹³C NMR (400MHz, CDCl₃): d 191.67, 165.44, 150.50, 140.54, 133.67, 132.44, 132.12,

131.79, 130.11, 126.22, 125.19, 122.49, 120.81, 120.78, 119.66, 119.62, 119.59, 110.91, 105.99, 97.20, 55.37, 46.22. LRMS calcu-

lated for C₁₇H₁₉ClNO₃⁺[M+H]⁺, 344.1 found 344.1.

Synthesis Scheme of 7 (TED-587)

Synthesis of 2-(4-nitrophenyl)thiophene (TED-587-1). A mixture of 1-bromo-4-nitrobenzene (1.0 g, 5.0 mmol), thiophen-2-ylboronic

acid (0.64 g, 5.0 mmol), Pd(PPh3)₄(580 mg, 0.5 mmol), and Na₂CO₃(1.1 g, 10.0 mmol) in dioxane (40 mL) and water (5 mL) was stirred

at 90^ꢁC under N₂overnight. The reaction mixture was cooled to room temperature, and ﬁltered over Celite. The ﬁltrate was concen-

trated. The residue was dissolved in ethyl acetate (40 mL), and the solution was washed with brine, and dried over Na₂SO₄. The

solution was ﬁltered, and the ﬁltrate was concentrated. The residue was puriﬁed by column chromatography (PE-PE/EA=10/1)

to give 2-(4-nitrophenyl)thiophene (0.54 g, 52.6% yield). LRMS calculated for C₁₀H₈NO₂S⁺[M+H]⁺, 206.0 found 206.0.

Synthesis of 4-(thiophen-2-yl)aniline (TED-587-2). To a mixture of 2-(4-nitrophenyl)thiophene (540 mg, 2.63 mmol) in EtOH (30 mL)

was added sat. NH₄Cl (5 mL), followed by iron powder (740 mg, 13.15 mmol). The resultant mixture was heated to reﬂux and stirred

for 30 min, and then cooled to rt. The mixture was ﬁltered over celite and the ﬁltrate was concentrated. The residue was dissolved in

EA. The solution was washed with brine, dried over Na₂SO₄, and ﬁltered. The ﬁltrate was concentrated. The residue was puriﬁed by

column chromatography (PE/EA=4:1) to afford 4-(thiophen-2-yl)aniline (360 mg, 78.3% yield). ¹H NMR (400 MHz, CDCl₃): d 7.42-7.40

(d, J=8.4 Hz, 2H), 7.16-7.15 (m, 2H), 7.03-7.01 (m, 1H), 6.69-6.67 (d, J=8.4 Hz, 2H), 3.72 (br, 2H). LRMS calculated for C₁₀H₁₀NS⁺

[M+H]⁺, 176.1 found 176.1.

Synthesis of 1-(2-((4-(thiophen-2-yl)phenyl)amino)phenyl)ethanone (TED-587-3). A mixture of 4-(thiophen-2-yl)aniline (360 mg,

2.06 mmol), 1-(2-bromophenyl)ethanone (405 mg, 2.06 mmol), Pd₂(dba)₃(190 mg, 0.21 mmol) , xphos (150 mg, 0.32 mmol),

and Cs₂CO₃(1.30 g, 4.0 mmol) in dioxane (30 mL) was stirred at 90^ꢁC under N₂overnight. The reaction mixture was cooled to

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room temperature, and ﬁltered over Celite. The ﬁltrate was concentrated. The residue was dissolved in ethyl acetate (40 mL), and the

solution was washed with brine, and dried over Na₂SO₄. The solution was ﬁltered, and the ﬁltrate was concentrated. The residue was

puriﬁed by column chromatography (PE-PE/EA=8/1) to give1-(2-((4-(thiophen-2-yl)phenyl)amino)phenyl)ethanone (130 mg, 21.5%

yield). LRMS calculated for C₁₈H₁₆NOS⁺[M+H]⁺, 294.1 found 294.1.

Synthesis of 2-chloro-1-(2-((4-(thiophen-2-yl)phenyl)amino)phenyl)ethanone (7, TED-587). To a mixture of 1-(2-((4-(thiophen-2-yl)

phenyl)amino)phenyl)ethanone (130 mg, 0.44 mmol) in DCM (10 mL) were added DIEA (120 mg, 0.9 mmol) and TMSOTf (150 mg,

0.66 mmol) at 0^ꢁC under N_2.The resulting mixture was warmed to rt and stirred for 2 hr. NCS (70 mg, 0.53 mmol) was added and

the mixture was stirred for another 2 hr. It was quenched with water (10 mL), and the mixture was extracted with DCM (5mL x 3).

The organic phase was washed with brine, dried over Na₂SO₄, and ﬁltered. The ﬁltrate was concentrated. The residue was puriﬁed

by prep. TLC (PE/EA=5/1) to give the crude product (207 mg). The compound was further puriﬁed by reverse phase HPLC Gilson to

afford the desired product as a yellow solid (54 mg, 35.2% yield); ¹H NMR (400 MHz, CDCl₃): d 10.42 (s, 1H), 7.76-7.74 (d, J = 8.0 Hz,

1H), 7.62-7.60 (d, J = 8.4 Hz, 2H), 7.39-7.30 (m, 2H), 7.28-7.25 (m, 4H), 7.09-7.07 (dd, J = 4.8 Hz, J = 3.6 Hz, 1H), 6.79-6.75

(t, J = 7.2 Hz, 1H), 4.76 (s, 2H); ¹³C NMR (400 MHz, CDCl₃): d 193.05, 148.54, 143.93, 139.12, 135.52, 131.30, 130.60, 128.07,

126.97, 124.50, 123.41, 122.72, 117.00, 116.26, 114.86, 46.62. LRMS calculated for C₁₈H₁₅ClNOS⁺[M+H]⁺, 328.1 found 328.1.

Synthesis Scheme of 8 (TED-588)

Synthesis of 2-(3-nitrophenyl)thiophene (TED-588-1). The method was the same as TED-587-1 to give 2-(3-nitrophenyl)thiophene

(650 mg, 63.1% yield); ¹H NMR (400 MHz, CDCl₃): d 8.45 (s, 1H), 8.13-8.11 (d, J=8.4 Hz, 1H), 7.92-7.90 (d, J=7.6 Hz, 1H), 7.57-7.53

(t, J=8.0 Hz, 1H), 7.44 (d, J=3.6 Hz, 1H), 7.39 (d, J=4.8 Hz, 1H), 7.15-7.13 (m, 1H). LRMS calculated for C₁₀H₈NO₂S⁺[M+H]⁺, 206.0

found 206.0.

Synthesis of 3-(thiophen-2-yl)aniline (TED-588-2). The method was same as TED-587-2 to give 3-(thiophen-2-yl)aniline (440 mg,

78.1% yield). LRMS calculated for C₁₀H₁₀NS⁺[M+H]⁺: 176.1, found 176.1.

Synthesis of 1-(2-((3-(thiophen-2-yl)phenyl)amino)phenyl)ethanone (TED-588-3). The method was same as TED-587-3 to give 1-(2-

((3-(thiophen-2-yl)phenyl)amino)phenyl)ethanone (1.4 g, 78.1% yield). LRMS calculated for C₁₈H₁₆NOS⁺[M+H]⁺: 294.1, found 294.1.

Synthesis of 2-chloro-1-(2-((3-(thiophen-2-yl)phenyl)amino)phenyl)ethanone (8, TED-588). The method was same as 7 (TED-587) to

give 2-chloro-1-(2-((3-(thiophen-2-yl)phenyl)amino)phenyl)ethanone (27.4 mg, 61.6% yield); ¹H NMR (400 MHz, CDCl₃): d 10.42

(s, 1H), 7.76-7.74 (d, J=8.0 Hz, 1H), 7.49 (s, 1H), 7.40-7.34 (m, 3H), 7.31-7.25 (m, 3H), 7.18-7.17 (d, J=7.2 Hz, 1H), 7.09-7.07 (m,

1H), 6.78-6.75 (m, 1H), 4.76 (s, 2H); ¹³C NMR (400 MHz, CDCl₃): d 193.09, 148.77, 143.75, 140.36, 135.82, 135.56,

131.30,129.97, 128.07, 125.51, 123.44, 122.31, 122.09, 116.95, 116.95, 116.20, 114.76, 46.62. LRMS calculated for C₁₈H₁₅ClNOS⁺

[M+H]⁺: 328.1, found 328.1.

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Synthesis Scheme of 9 (TED-549)

Synthesis of 5-(benzyloxy)pentan-1-ol (TED-549-1). To a solution of pentane-1,5-diol (5.0 g, 48.01 mmol) in DMF (50 mL) was added

60% of NaH (1.3 g, 33.61 mmol) at 0^ꢁC under N₂. The resulting mixture was warmed to room temperature and stirred for 30 min. BnBr

(5.7g, 33.61 mmol) was added at 0^ꢁC. The mixture was heated to 50^ꢁC under N₂, and stirred for 30 min. The reaction mixture was

cooled to room temperature, and it was quenched with water (100 mL). The mixture was extracted with EA (40 mL x 3). The organic

phase was washed with brine, dried over Na₂SO₄, and ﬁltered. The ﬁltrate was concentrated. The residue was puriﬁed by column

1

chromatography (PE-PE/EA=2/1) to give 5-(benzyloxy)pentan-1-ol (2.2 g, 33.7%); H NMR (400MHz, CDCl₃): d 7.26-7.34 (m, 5H),

4.50 (s, 2H), 3.64 (t, J=6.4Hz, 2H), 3.48 (t, J=6.4Hz, 2H), 1.56-1.67 (m, 4H), 1.41-1.49 (m, 2H), 1.32 (br, 1H).

Synthesis of 5-(benzyloxy)pentyl methanesulfonate (TED-549-2). To a mixture of 5-(benzyloxy)pentan-1-ol (TED-549-1, 2.2 g,

11.32 mmol) in DCM (30 mL) were added MsCl (1.4 g, 12.45 mmol) and TEA (2.3 g, 22.64 mmol) at 0^ꢁC under N_2.The resulting mixture

was warmed to room temperature and stirred for 1 hr. The reaction mixture was quenched with water (50 mL), and the mixture was

extracted with DCM (30mL x 3). The organic phase was washed with brine, dried over Na₂SO₄, and ﬁltered. The ﬁltrate was concen-

trated to give 5-(benzyloxy)pentyl methanesulfonate (2.5 g, 81.3%); ¹H NMR (400MHz, CDCl₃): d 7.26-7.35 (m, 5H), 4.50 (s, 2H), 4.23

(t, J=6.4Hz, 2H), 3.48 (t, J=6.4Hz, 2H), 2.99 (s, 3H), 1.76-1.80 (m, 2H), 1.63-1.71 (m, 2H), 1.48-1.54 (m, 2H).

Synthesis of 2-(2-((5-(benzyloxy)pentyl)oxy)ethoxy)ethanol (TED-549-3). To a mixture of 2,2’-oxydiethanol (2.9 g, 27.53 mmol) in THF

(50 mL) was added 60% of NaH (550 mg, 13.77 mmol) at 0^ꢁC under N₂. The resulting mixture was warmed to room temperature and

stirred for 30 min. A solution of 5-(benzyloxy)pentyl methanesulfonate (TED-549-2, 2.5 g, 9.18 mmol) in THF (10 mL) was added and

the mixture was reﬂuxed for 3 hr under N₂, and then cooled to room temperature. The reaction mixture was quenched with water

(150 mL), and the mixture was extracted with EA (40mL x 3). The organic phase was washed with brine, dried over Na₂SO₄, and

ﬁltered. The ﬁltrate was concentrated. The residue was puriﬁed by column chromatography (PE/EA=10/1 to 1/1) to give 2-(2-((5-(ben-

zyloxy)pentyl)oxy)ethoxy)ethanol (1.9 g, 73.4%); ¹H NMR (400MHz, CDCl₃): d 7.26-7.34 (m, 5H), 4.50 (s, 2H), 3.71-3.74 (m, 2H),

3.66-3.69 (m, 2H), 3.57-3.63 (m, 4H) , 3.47 (t, J=6.4Hz, 4H), 2.47 (br, 1H), 1.59-1.68 (m, 4H), 1.40-1.47 (m, 2H).

Synthesis of 2-(2-((5-(benzyloxy)pentyl)oxy)ethoxy)ethyl methanesulfonate (TED-549-4). To a mixture of 2-(2-((5-(benzyloxy)pentyl)

oxy)ethoxy)ethanol (TED-549-3, 1.9 g, 6.74 mmol) in DCM (50 mL) were added MsCl (0.92 g, 8.09 mmol) and TEA (1.4 g, 13.48 mmol)

at 0^ꢁC under N_2.The resulting mixture was warmed to room temperature and stirred for 1 hr. The reaction mixture was quenched with

water (100 mL), and the mixture was extracted with DCM (30 mL x 3). The organic phase was washed with brine, dried over Na₂SO₄,

1

and ﬁltered. The ﬁltrate was concentrated to give 2-(2-((5-(benzyloxy)pentyl)oxy)ethoxy)ethyl methanesulfonate (1.8 g, 74.1%); H

NMR (400MHz, CDCl₃): d 7.26-7.37 (m, 5H), 4.50 (s, 2H), 4.38 (t, J=4.4Hz, 2H), 3.76 (t, J=4.4Hz, 2H), 3.64-3.66 (m, 2H), 3.56-3.58

(m, 2H), 3.43-3.49 (m, 4H), 3.06 (s, 3H), 1.57-1.65 (m, 4H) , 1.38-1.46 (m, 2H).

Synthesis of N,N-diBoc-2-(2-((5-(benzyloxy)pentyl)oxy)ethoxy)ethan-1-amine (TED-549-5). A mixture of 2-(2-((5-(benzyloxy)pentyl)

oxy)ethoxy)ethyl methanesulfonate (TED-549-4, 1.8 g, 5.00 mmol), HNBoc₂(1.2 g, 5.5 mmol) and K₂CO₃(1.4 g, 10.00 mmol) in

DMF (30 mL) was heated to 100 ^oC under N₂and the mixture was stirred for 3 hr. It was then cooled to room temperature. The reaction

mixture was quenched with water (100 mL), and extracted with EA (40mL x 3). The organic phase was washed with brine, dried

over Na₂SO₄, and ﬁltered. The ﬁltrate was concentrated. The residue was puriﬁed by column chromatography (PE/EA=10/1 to

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3/1) to give N,N-diBoc-2-(2-((5-(benzyloxy)pentyl)oxy)ethoxy)ethan-1-amine (2.0 g, 83.1%); ¹H NMR (400MHz, CDCl₃): d 7.26-7.34

(m, 5H), 4.50 (s, 2H), 3.74-3.80 (m, 2H), 3.53-3.63(m, 6H), 3.42-3.48 (m, 4H), 1.58-1.65 (m, 4H), 1.49 (s, 18H), 1.39-1.45 (m, 2H).

Synthesis of N-(2-(2-((5-hydroxypentyl)oxy)ethoxy)ethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (TED-

549-6). A mixture of ditert-butyl (2-(2-((5-(benzyloxy)pentyl)oxy)ethoxy)ethyl)carbamate (TED-549-5, 2.0 g, 4.15 mmol) and 10% of

Pd/C (300 mg) in MeOH (100 mL) was stirred for 18 hr under H₂. The reaction mixture was ﬁltered through Celite and the ﬁltrate was

concentrated. The residue was dissolved in DCM (10 mL) and 6 M HCl in dioxane (5 mL) was added. The resulting mixture was stirred

at room temperature for 2 hr and then the solvent was removed. The residue was dissolved in DMF (15 mL), and TEA (1.03 g,

10.02 mmol) was added, followed by HATU (1.16 g, 3.06 mmol). The resulting mixture was stirred at room temperature for 2 hr,

and concentrated. The crude product was puriﬁed by prep-HPLC to give N-(2-(2-((5-hydroxypentyl)oxy)ethoxy)ethyl)-5-(2-oxohex-

ahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (120 mg, 6.9%); ¹H NMR (400MHz, CD₃OD): d 4.47-4.50 (m, 1H), 4.29-4.32

(m, 1H), 3.48-3.59 (m, 10H), 3.36 (t, J=5.6Hz, 2H), 3.18-3.23 (m, 1H), 2.90-2.95 (m, 1H), 2.70 (t, J=12.8Hz, 1H), 2.21 (t, J=7.2Hz,

2H), 1.54-1.75 (m, 8H), 1.40-1.48 (m, 4H). LRMS calculated for C₁₉H₃₆N₃O₅S⁺[M+H]⁺, 418.6, found 418.2.

Synthesis of tert-butyldimethyl(3-nitrophenoxy)silane (TED-549-7). To a solution of 3-nitrophenol (1.0 g, 7.20 mmol) in DCM (20 mL)

were added TBSCl (1.2 g, 7.92 mmol) and imidazole (979 mg, 14.40 mmol) at room temperature. The resulting mixture was stirred for

2 hr and quenched with water (40 mL). It was extracted with DCM (30 mL x 3). The organic phase was washed with brine, dried

over Na₂SO₄, and ﬁltered. The ﬁltrate was concentrated. The residue was puriﬁed by column chromatography (PE-PE/EA=2/1) to

give tert-butyldimethyl(3-nitrophenoxy)silane (1.3 g, 71.4%); ¹H NMR (400MHz, CDCl₃): d 7.83 (d, J=8.0Hz, 1H), 7.66 (t, J=2.4Hz,

1H), 7.38 (t, J=8.0Hz, 1H), 7.16 (d, J=8.0Hz, 1H), 1.00 (s, 9H), 0.24 (s, 6H), 1.41-1.49 (m, 2H), 1.32 (br, 1H).

Synthesis of 3-((tert-butyldimethylsilyl)oxy)aniline (TED-549-8). To a mixture of tert-butyldimethyl(3-nitrophenoxy)silane (TED-

549-7, 1.3 g, 5.14 mmol) in MeOH (20 mL) was added 10% of Pd/C (200 mg). The resulting mixture was stirred at room temperature

overnight under H₂(1 atm). The reaction mixture was ﬁltered through Celite and the ﬁltrate was concentrated to give 3-((tert-butyldi-

methylsilyl)oxy)aniline (1.1 g, 95.7%); LRMS calculated for C₁₂H₂₂NOSi⁺[M+H]⁺, 224.4, found 224.3.

Synthesis of 1-(2-((3-((tert-butyldimethylsilyl)oxy)phenyl)amino)phenyl)ethan-1-one (TED-549-9). The mixture of 3-((tert-butyldime-

thylsilyl)oxy)aniline (TED-549-8, 600 mg, 2.69 mmol), 1-(2-bromophenyl)ethanone (562 mg, 2.83 mmol), Pd₂(dba)₃(246 mg,

o

0.27 mmol) , xphos (257 mg, 0.54 mmol), and Cs₂CO₃(1.30 g, 4.04 mmol) in dioxane (15 mL) was heated to 90 C under N₂, and

it was stirred for 2 hr. The reaction mixture was cooled to room temperature, ﬁltered through Celite and the ﬁltrate was concentrated.

The residue was dissolved in EA (40 mL), washed with brine, and dried over Na₂SO₄. It was ﬁltered, and the ﬁltrate was concentrated.

The residue was puriﬁed by column chromatography (PE-PE/EA=20/1) to give 1-(2-((3-((tert-butyldimethylsilyl)oxy)phenyl)amino)

phenyl)ethan-1-one (410 mg, 44.6%); ¹H NMR (400MHz, CDCl₃): d 10.47 (s, 1H), 7.81 (d, J=8.4Hz, 1H), 7.28-7.33 (m, 2H), 7.18

(t, J=8.0Hz, 1H), 6.85 (d, J=7.6Hz, 1H), 6.71-6.74 (m, 2H), 6.61 (d, J=8.4Hz, 1H), 2.64 (s, 3H), 0.98 (s, 9H), 0.21 (s, 6H).

Synthesis of 1-(2-((3-hydroxyphenyl)amino)phenyl)ethan-1-one (TED-549-10). To a solution of 1-(2-((3-((tert-butyldimethylsilyl)oxy)

phenyl)amino)phenyl)ethan-1-one (TED-549-9, 100 mg, 0.29 mmol) in THF (2 mL) was added 1M TBAF in THF (0.35 mL, 0.35 mmol)

dropwise at room temperature. The resulting mixture was stirred for 1 hr and quenched with water (20 mL). The mixture was extracted

with EA (10 mL x 3), and the organic phase was washed with brine, and dried over Na₂SO₄. It was ﬁltered, and the ﬁltrate was concen-

trated. The residue was puriﬁed by prep-TLC (DCM/MeOH=10/1) to give 1-(2-((3-hydroxyphenyl)amino)phenyl)ethan-1-one (52 mg,

70.8%); ¹H NMR (400MHz, CDCl₃): d 10.49 (s, 1H), 7.82 (d, J=8.0Hz, 1H), 7.32 (d, J=7.6Hz, 2H), 7.20 (t, J = 8.0 Hz, 1H), 6.83

(d, J=8.0Hz, 1H), 6.73-6.77 (m, 2H), 6.58 (d, J=8.0Hz, 1H), 2.65 (s, 3H). LRMS calculated for C₁₄H₁₄NO₂⁺[M+H]⁺, 228.3 found 228.1.

Synthesis of N-(2-(2-((5-(3-((2-acetylphenyl)amino)phenoxy)pentyl)oxy)ethoxy)ethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-

yl)pentanamide (TED-549-11). To a mixture of N-(2-(2-((5-hydroxypentyl)oxy)ethoxy)ethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]

imidazol-4-yl)pentanamide (TED-549-6, 50 mg, 0.12 mmol), 1-(2-((3-hydroxyphenyl)amino)phenyl)ethan-1-one (TED-549-10,

35 mg, 0.16 mmol), and PPh₃(63 mg, 0.24 mmol) in dioxane (3 mL) was added DIAD (53 mg, 0.26 mmol) at 0 ^oC under N₂, The re-

sulting mixture was warmed to room temperature and stirred overnight. The reaction mixture was quenched with water (5 mL), and

extracted with EA (10 mL x 2). The organic phase was washed with brine, dried over Na₂SO₄, and ﬁltered. The ﬁltrate was concen-

trated. The residue was puriﬁed by prep-TLC (DCM/MeOH=5/1) to give N-(2-(2-((5-(3-((2-acetylphenyl)amino)phenoxy)pentyl)oxy)

ethoxy)ethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (42 mg, 46% yield); ¹H NMR (400MHz, CDCl₃):

d 10.50 (s, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.26-7.34 (m, 2H), 7.12-7.24 (m, 1H), 6.72-6.85 (m, 2H), 6.63-6.66 (m, 1H), 6.48-6.58 (m,

1H), 6.12 (s, 1H), 5.22 (s, 1H), 4.86-4.97 (m, 1H), 4.46-4.49 (m, 1H), 4.28-4.31 (m, 1H), 3.88-3.96 (m, 2H), 3.44-3.64 (m, 10H),

3.10-3.14 (m, 1H), 2.87-2.91 (m, 1H), 2.70-2.74 (m, 1H), 2.64 (s, 2H), 2.15-2.26 (m, 2H), 1.56-1.84 (m, 12H). LRMS calculated for

C₃₃H₄₇N₄O₆S⁺[M+H]⁺, 627.3 found 627.4.

Synthesis of N-(2-(2-((5-(3-((2-(2-chloroacetyl)phenyl)amino)phenoxy)pentyl)oxy)ethoxy)ethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]

imidazol-4-yl)pentanamide (9, TED-549). The method was the same as 4 (TED-548) to give N-(2-(2-((5-(3-((2-(2-chloroacetyl)

phenyl)amino)phenoxy)pentyl)oxy)ethoxy)ethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (11 mg); ¹H NMR

(400MHz, CDCl₃): d 10.33 (s, 1H), 7.71-7.80 (m, 2H), 6.72-6.85 (m, 4H), 6.60-6.62 (m, 3H), 6.30-6.39 (m, 2H), 5.44 (s, 2H), 4.88-

5.09 (m, 3H), 4.76 (s, 2H), 4.70 (s, 1H), 4.47 (s, 2H), 4.29 (m, 2H), 3.92-4.00 (m, 5H), 3.43-3.60 (m, 25H), 3.05-3.15 (m, 2H), 2.86-

2.91 (m, 2H), 2.65-2.74 (m, 3H), 2.21 (br, 6H). LRMS calculated for C₃₃H₄₆ClN₄O₆S⁺[M+H]⁺, 661.3, found 661.2.

e12 Cell Chemical Biology 26, 1–12.e1–e13, March 21, 2019

Please cite this article in press as: Bum-Erdene et al., Small-Molecule Covalent Modiﬁcation of Conserved Cysteine Leads to Allosteric Inhibition of the

TEAD,Yap Protein-Protein Interaction, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.11.010

QUANTIFICATION AND STATISTICAL ANALYSIS

Fluorescence polarization, biolayer interferometry, and cell biological studies results are representatives of at least three independent

studies, performed in duplicates.

Crystal structure statistics are available in Table S2.

DATA AND SOFTWARE AVAILABILITY

The accession number for the atomic coordinates of human TEAD2 Yap-binding domain covalently bound to compound 2, reported

in this paper, is PDB: 6E5G.

ADDITIONAL RESOURCES

N/A.

Cell Chemical Biology 26, 1–12.e1–e13, March 21, 2019 e13

Article Doi

Small-Molecule Covalent Modification of Conserved Cysteine Leads to Allosteric Inhibition of the TEAD?Yap Protein-Protein Interaction

DOI: 10.1016/j.chembiol.2018.11.010

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Article abstract of DOI:10.1016/j.chembiol.2018.11.010

Full text of DOI:10.1016/j.chembiol.2018.11.010

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