TARGETING TRANSCRIPTIONAL ENHANCED ... - ACS Publications

Perspective Cite This: J. Med. Chem. 2018, 61, 5057−5072

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Targeting Transcriptional Enhanced Associate Domains (TEADs) Miniperspective Floriane Gibault,†,§ Manon Sturbaut,†,§ Fabrice Bailly,† Patricia Melnyk,† and Philippe Cotelle*,†,‡ †

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JPArc, Centre de Recherche Jean-Pierre Aubert, Neurosciences et Cancer, UMR-S-1172, INSERM, CHU Lille, Université de Lille, F-59000 Lille, France ‡ ENSCL, F-59000 Lille, France

ABSTRACT: Transcriptional enhanced associate domain (TEAD) proteins are the downstream effectors of the Hippo signaling pathway that regulate cell proliferation and stem cell functions. TEADs are unable to activate transcription and require the help of coactivators such as YAP, TAZ, VgLL, and p160 proteins. The expression of TEAD family is up-regulated in many cancer types including gastric, colorectal, breast, and prostate cancers, which is correlated with poor survival in patients. Pharmacological modulators of TEADs could therefore find application in cancer treatment and regenerative medicine. In this review, we present the very recent available structures of TEADs with or without coactivators or inhibitors and discuss the potential therapeutic application of their ligands.

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INTRODUCTION TEAD (transcriptional enhancer factor TEF with TEA/ATTS domain) transcription factors are the final effectors of the Hippo signaling pathway. Their functions are mediated by their interactions with the nuclear coactivators which can be classified into three groups: (1) YAP (yes associated protein) and its paralog TAZ (transcriptional coactivator with PDZ-binding motif) (also called WWTR1),1 (2) VgLLs, and (3) p160s proteins. YAP and TAZ are also regulated by other important cellular pathways including Wnt/β-catenin pathway. Hippo signaling pathway is a conserved regulator of organ size that is composed of a core kinase cascade. Phosphorylation of the downstream effectors YAP or TAZ changes their subcellular localization from the nucleus (unphosphorylated) to the cytoplasm (phosphorylated) (Scheme 1). When phosphorylated, YAP and TAZ are sequestered by 14−3−3 protein or degraded via the ubiquitin/proteasomal pathway, whereas unphosphorylated YAP/TAZ translocates to the nucleus and activates several nuclear transcriptional factors including TEADs. The activation of TEADs induces the expression of several Hippo pathway target genes including CTGF, Cyr61, and Axl. Wnt signaling pathway plays a crucial role during embryonic development and homeostasis in adults. In the absence of Wnt signaling, the destruction complex targets β-catenin and © 2017 American Chemical Society

promotes its degradation. Conversely, the presence of a Wnt ligand induces inactivation of the destruction complex yielding the accumulation of β-catenin and the liberation of YAP/TAZ which are a component of the destruction complex. This means that YAP/TAZ compounds are sequestered in the cytoplasm in the destruction complex, and in the presence of Wnt ligand YAP/TAZ compounds are liberated and activate TEAD too.1 It has been recently demonstrated that SCD1 (stearoyl-CoAdesaturase 1, the enzyme responsible for the production of palmitoleoyl-CoA which is the substrate for the O-palmitoleoylation of Wnt) regulates lung cancer stemness via stabilization and nuclear localization of YAP/TAZ.2 In the nucleus, it has been shown that TEAD interactions with YAP or VgLL4 can be controlled by an acetylation/ deacetylation equilibrium. In 31 hepatocellular carcinoma (HCC) tumor tissues, the levels of sirtuin 1 (SIRT1) were found to be significantly higher than that of the adjacent nontumor tissues, while YAP expression was found to be similar in tumor and nontumor tissues.3 Examination of the mRNA levels of SIRT1 and CTGF indicated that SIRT1 may regulate YAP-TEAD target genes in HCC. Indeed, YAP is acetylated by Received: June 15, 2017 Published: December 18, 2017 5057

DOI: 10.1021/acs.jmedchem.7b00879 J. Med. Chem. 2018, 61, 5057−5072

Journal of Medicinal Chemistry

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Scheme 1. TEAD Ligands: Its Localization and Its Partners

p300/CBP and deacetylated by SIRT1 in vitro and in vivo. Lysine to arginine mutations have revealed that the four lysine residues located in the TEAD domain (K76, K90, K97, and K102) are important for the regulation of YAP mediated by SIRT1. Finally, it was demonstrated that SIRT1 promoted the nuclear accumulation of YAP in response to cisplatin treatment in HepG2 cells and inhibited the induced apoptosis. The same response to alkylating agents was demonstrated in HeLa cells upon methyl methanesulfonate (MMS) treatment.4 However, in this study, it was demonstrated that p300/CBP mediates K494 acetylation of hYAP upon MMS treatment and SIRT1 is responsible for the deacetylation. The fetal cardiomyocyte proliferation is stimulated by overexpressed YAP which binds to TEAD1 and therefore induces heart growth. VgLL4 (vestigial-like protein 4) was found to inhibit this cardiomyocyte proliferation by targeting TEAD1 and induced its degradation by cysteine peptidases.5

VgLL4 activity is regulated by its interaction with p300 which acetylated hVgLL4 mainly at K219. However, the authors did not mention which histone desacetylase (HDAC) is implicated in the deacetylation of Ac-VgLL4. p160 coactivators were found to activate TEAD-dependent transcription in only one report.6 As no structure on this TEAD/ p160 complex is available, this review will not focus further on it. Dysregulations of the Hippo pathway have been reported in a wide range of cancers including in colorectal cancer in which YAP, TAZ,7 and TEAD8 are overexpressed, and VgLL49 and the C-terminal splicing isoform of TEAD10 are down-regulated. Whereas the central role of YAP and TAZ in cancers has been largely reviewed11−13 over the past years including the possible therapeutic interventions,14,15 only few articles have been devoted to the roles of TEAD and its coactivators.16 Several reviews have been recently published on different targets provided by the Hippo pathway.17−19 In 2012, Liu5058



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Scheme 2. Hippo Pathway Inhibitors

Chittenden et al.20 identified verteporfin (1) (Scheme 2) as the first FDA-approved drug targeting the Hippo pathway. 1 was then reported to up-regulate 14−3−3σ (a protein responsible for the cytoplasmic retention of YAP).21 Dasatinib (2) and pazopanib (3) (Scheme 2), two multireceptor tyrosine kinase inhibitors, induce YAP and TAZ phosphorylation and promote proteasomal degradation of YAP and TAZ (in the case of 3) in several cancer cells overexpressing YAP and/or TAZ.22,23 3Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of the mevalonate cholesterol biosynthesis pathway reducing geranylgeranyl pyrophosphate (GGPP), is required for membrane localization and activation of RHO GTPases. The mevalonate−RHO axis thus promotes nuclear accumulation of YAP and TAZ. Cerivastatin (4) (Scheme 2) (an inhibitor of HMG-CoA reductase) is able to sequester YAP and TAZ in the cytoplasm.24 From a screening of a series of new compounds designed for their action on the Hippo signaling pathway, a small molecule named C19 (5) (Scheme 2) was identified as inhibitor of Hippo, TGF-β, and Wnt pathways. Mechanistically, 5 activates the core kinase MST1 and Lats1, inducing in several cell lines the degradation of TAZ.25 Conversely, selective and reversible small inhibitors of MST1/MST2 kinases were developed to potentiate tissue repair and regeneration.26 MF-438 (6) (Scheme 2), an inhibitor of SCD1 (stearoyl-CoA-desaturase 1), down-modulates YAP and TAZ in lung cancer,2 and XAV939 (7), a tankynase inhibitor, reduces the expression of YAP/TEAD gene targets in various human cancers.27

Combination of epigenetic regulators (I-BET151 (8), a bromodomain and extra-terminal (BET) protein inhibitor, and panobinostat (9), an HDAC inhibitor) synergistically induces down-regulation of the AKT and Hippo pathways in melanoma cell lines. The YAP down-regulation by this combination appears to be transcriptional and not due to binding by cytoplasmic proteins.28 An impressive number of important papers have been published in the past years dealing with the structural aspects of TEAD isoforms and their partners, giving rise to new and original molecular approaches to target these downstream effectors. In this review, we will underline the most recent papers and advise the readers to consult the references cited in these papers. We will focus this review on the structure of TEAD proteins and their molecular interactions with their different partners. The post-traductional modifications of YAP, TAZ, and VgLL as well as the recent discovery of an alternative splicing of TEAD allowing a fine-tuning of the transcriptional response will be discussed. Finally, we will conclude on the perspectives offered by the different druggable sites of TEAD for cancer treatment or regenerative medicine applications and comment on the first patents on this exciting topic.

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TEAD1−4: PRIMARY STRUCTURES AND CELLULAR ROLES Mammalian TEAD protein family contains four members named TEAD1−4 encoded by four different genes that are 5059



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Figure 1. Domain architecture of the four TEAD isoforms, the YAP1−2γ isoform, TAZ, and VgLL1−4.

fourth has been deposited at the PDB but the authors are still working on it. The two first structures are from the Veeraraghavan’s group and correspond to the NMR structure in solution of hTEAD1(28−104)29 and to the crystal structure of the same DBD missing the loop 1 (52−63).30 The studied entire TEADDBD is a folded globular protein made of three α-helices (H1, H2, and H3) connected by a long loop (L1) and a shorter one (L2). H1 and H2 form an angle of 36° and pack on either side of the beginning of H3 with interhelix angles of about 110° (Figure 2A). Using electrophoretic mobility shift assay, the authors measured a nanomolar affinity close to that previously published for full-length TEAD and demonstrated that this fragment alone confers to TEAD its DNA-binding activity.31 By designing of a DNA−ligand interaction assay, the studied DBD-TEAD was found to bind to numerous muscle-CAT-like DNA sequences, and the authors selected a 12-mer for further NMR study. Because of the analysis of the chemical shift perturbations of TEAD resonances in the presence of 12-mer, it was established that the beginning of H3 (R87 and K88 of TEAD1) and some residues in the L2 immediately preceding H3 bind to DNA. Ten years later was reported the crystallographic structure and activity of TEAD-DBD mutant containing a truncated L1 loop.30 Model building and refinement show three molecules of ΔL1-TEAD-DBD. Reducing the L1 loop induces major modifications on the folding between H1 and H2 (Figure 2B). However, in spite of these major structural modifications, ΔL1TEAD-DBD is sufficient for binding to an isolated M-CAT-like DNA element. The third published structure is a hTEAD4(36−139)-DBD32 in complex with a muscle-CAT DNA element (13 base pairs), and the fourth structure corresponds to a hTEAD1(31−104)DBD33 in complex with a DNA fragment of 18 base-pairs (entries 3 and 13, Table 3). The two structures superimpose well (Figure 3A) and clearly show the insertion of H3 in the major groove and L1 in the minor groove. Comparison between the apo state of hTEAD1 (PDB code 2HZD)29 and the TEADDNA complexes (PDB codes 5GZB32 and 5NNX33) (Figure 4) shows a clear conformational change of H3 for a better fit in the

expressed in almost every type of tissues. Their specific functions have been deduced from gene inactivation studies performed in mice and reviewed.16 TEAD1 facilitates the expression of cardiac specific genes and is considered important for the differentiation of cardiac muscle. The role of TEAD2 is not well-established, but it may be involved in the gene regulation of neural development. The prime function of TEAD4 is linked to embryo implantation. The specific function of TEAD3 has not been reported so far. Nevertheless, almost all tissues express at least one of the TEAD genes and some express all four of them. The four TEADs present an overall homology ranging from 61% to 73% and are divided into a DNA-binding domain (DBD) at the N-terminus (about 80−90 aa) and a C-terminal YAP/ TAZ/VgLL binding domain (YBD) (about 220 aa) (Figure 1). Both domains are linked by a sequence of about 90−100 amino acids which has a low homology across the four isoforms. Individually, DBD and YBD present within the TEAD family a high homology (>90%), and selective TEAD antibodies are therefore directed toward sequences found in the central linker. The recently identified splicing isoform of TEAD410 corresponds to a C-terminal fragment which only includes YBD and the central linker. While full TEADs are exclusively nuclear, this spliced isoform, which has been detected in all tissues, is located in both nucleus and cytoplasm acting as a cytoplasmic scavenger of YAP and TAZ. Full alignment of the four isoforms of human TEAD has been already reported.15 Here we only reproduce the sequence alignment of DBD and YBD of crystallized TEADs (Table 1). To date, 19 structures (Table 3) including TEAD fragments have been resolved by high field NMR (entry 1) and X-ray. Entry 9 has still no Protein Data Bank code at the time of the submission of this review.

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DNA BINDING DOMAIN Four structures of N-terminal fragments of hTEAD are available at the Protein Data Bank (PBD) (entries 1, 2, 3 and 13, Table 3) which correspond to the DNA binding domain (DBD) of TEAD. Three of them have already been published while the 5060



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Table 1. Sequence Alignments of DBD and YBD of Crystallized TEADsa

a

Residues corresponding to the palmitate pocket are highlighted in purple, and residues of the interfaces 0, 1, 2, and 3 are highlighted in green, yellow, blue, and red, respectively. Residues in light grey are implicated in the palmitate pocket (side chain) and interface 1 (peptide chain). Residues in dark purple are implicated in the palmitate pocket and interface 2. Numbering of the different proteins can be found in http://www. uniprot.org/. 5061



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Figure 2. Effect of the truncation of loop 1 on the interaction between the helixes of hTEAD1(28−104): (A) lowest energy structure from 2HZD (left); (B) one of the three molecules of ΔL1-hTEAD1(28−104) from 4Z8E. All structural figures were generated with Chimera 1.10.2 (UCSF).74

Figure 3. (A) Superimposition of hTEAD4(36−139)-DNA (13-mer) (PDB code 5GZB in gray) and hTEAD1(31−104)-DNA (18-mer) (PDB code 5NNX in pink) complexes; (B) zoom on the basic residues of TEAD pointing in the major groove.

determined by electrophoretic mobility shift assay and ITC (isothermal titration calorimetry).32 TEAD4 mutations R64A, K65A, I66A, L68A, M74A, R95A, K96A, Q103A, and R111A increased the KD of TEAD4-DNA complexation, and S100A mutation almost abolished the TEAD4-DNA interaction (Figure 3B shows the side chain of the cited residues pointing into the major or the minor groove of DNA). Transfected HEK293 cells with TEAD4 mutants effectively decreased the expression of CTGF or Cyr61 and overexpression of TEAD4 mutants inhibited the growth of human gastric cancer cells (HGC-27), confirming that the TEAD-DNA interaction is indispensable for the oncogenicity of TEAD. To date, no inhibitor targeting the DNA binding domain (DBD) of TEAD has been reported. DBD-TEAD presents three potentially druggable domain depicted in Figure 5A. According to our analysis, the most promising one should be the pocket defined by the three α-helices (in cyan) with the possibility for a ligand to prevent a maximum of interactions between TEAD and DNA major groove. Targeting transcription factor DNA binding sites as an approach for controlling gene expression has recently

Figure 4. Structural comparison of TEAD in apo state (PDB code 2HZD) in gold and in complex with DNA (PDB codes 5NNX in red and 5GZB in purple).

major groove. Independent biochemical characterization of the residues implicated in the TEAD-DNA interaction was 5062



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Figure 5. Druggable cavities of TEAD protein. (A) Druggable cavities of the DNA-binding domain of TEAD. The structures used for this analysis are 5GZB and 5NNX: in cyan, a cavity surrounded by the three α-helices of TEAD; in purple, a cavity defined by L1, H1, and H3. (B) Druggable cavities of the coactivator binding domain of TEAD. The structures used for this analysis are 3KYS and 5DQ8. The cavities corresponding to the interface 3, interface 2, and palmitate pocket are in purple, blue, and pink, respectively.

been reviewed34 and is considered a valuable new strategy in the fields that need new therapeutic interventions such as the field of cancer. Designing inhibitors of the DNA-TEAD interfaces should be one of the future strategies against the downstream transcriptional factor of the Hippo pathway.

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TEAD’S COACTIVATOR BINDING DOMAIN The YAP/TAZ/VgLL binding domain of TEAD (YBD) has been first described by Tian et al. in 2010 (entry 6, Table 3).35 The structure of hTEAD2(217−447)36 has been determined from crystals of the selenomethionine modified protein (PDB code 3L15). TEAD2-YBD structure presents a central core with two β-sheets packing each other to form a β-sandwich. This central core is structurally closely related to phosphodiesterase δ (PDEδ), and the two structures overlay well.35

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TEAD-YAP/TAZ/VgLL Two groups have simultaneously published the crystal structures of TEAD-YAP complex in 2010. The first one corresponds to a hTEAD1(209−426)-hYAP(50−171)37 complex (PDB code 3KYS) (entry 4, Table 3) 38 and the second to a mTEAD4(210−427)-mYAP(47−85) complex (PDB code 3JUA) (entry 14, Table 3).39 A third crystal structure has just been published and corresponds to a hTEAD4(217−434)hYAP(60−100) complex (entry 14, Table 3).40 The natively unfolded TEAD-binding domain of YAP37 wraps around the compacted TEAD structure creating three interfaces (Figure 6). The interface 1 (present on 3KYS but not on 3JUA) is mediated by seven intermolecular hydrogen bonds between the peptide backbones of YAP β1 and TEAD β7 forming an antiparallel β sheet. Deletion of β1 in YAP did not alter the YAP-TEAD interaction as measured in an in vitro GST pull down assay.38 The interface 2 is created by the YAP α1 helix which is close to a groove formed by TEAD α3 and α4. The binding is here mediated by hydrophobic interactions, but mutations of L68A and F69A involved in this interface 2 did not reduce YAP-TEAD interactions in the GST pull down assay.38 This interface was also found with a shorter fragment of YAP (mYAP(47−85)) where the α1 helix is partially truncated at the N-terminal end.39 In the interface 3, the Ω-loop of YAP interacts with a deep pocket formed by β4, β11, β12, α1, and α4 of TEAD. The structure of the Ω-loop of YAP is due to internal hydrophobic interactions between M86, R87, L91, F95, and F96 allowing side chains of R89 and S94 of YAP to strengthen the interactions by creating hydrogen bonds with D264 carboxylate and Y421

Figure 6. Superimposition of YAP-TEAD complexes (PDB code 3KYS, TEAD in purple and YAP in green; PDB code 3JUA, TEAD in pink and YAP in yellow).

phenolate and E255 carboxylate of TEAD1. Mutations of YAP (M86A, R89A, L91A, and F95A) as well as TEAD residues (Y421A or H) confirmed the importance of this interface. Moreover, the TEAD1 Y421H mutation has been described as a cause of Sveinsson’s chorioretinal atrophy.41 Chen et al.39 noted an essential role for the YAP PXXΦP motif (P is proline, X is an amino acid, and Φ is a hydrophobic residue: 66PQTV70P) where the alcoholic oxygen atom of 68T created a hydrogen bond with the hydrogen atom of the amide 385N. Mutations of the proline residues to alanine or deletion of this motif was found to disrupt YAP-TEAD binding.39 In the third structure of TEAD-YAP complex,40 hTEAD4 is S-myristoylated (this will be discussed below) and the YAP(60−100) interacts with TEAD very similarly to the previous ones.38,39 In an NMR study35 of a large N-terminal hYAP(2−268) fragment including WW domains, Tian et al. recorded the heteronuclear single quantum coherence (HSQC) spectra of hYAP(2−268) with or without TEAD2(217−447). Whereas the signals belonging to WW domains remained unaffected, about 30 signals of YAP were abolished upon TEAD binding. In order to identify the domain corresponding to these signals, the authors synthesized two shortened YAP fragments and showed that hYAP(61−100) represented the essential TEAD-binding 5063



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Figure 7. (A) Superimposition of YAP-TEAD (PDB code 3JUA, TEAD in purple and YAP in yellow) and the 1:1 TAZ-TEAD complexes (PDB code 5GN0, TEAD in pink and TAZ in green). (B) Superimposition of YAP-TEAD (PDB code 3JUA, TEAD is missing and YAP in orange red) and 2:2 TAZ-TEAD complex (PDB code 5GN0, TEAD in pink and purple and TAZ in green and yellow).

Table 2. Sequence Alignments of TBD of TEADs Coactivatorsa

a

Residues of the interfaces 0, 1, 2, and 3 are highlighted in green, yellow, blue, and red, respectively. The acetylated lysine residues are highlighted in dark blue. Numbering of the different proteins can be found in http://www.uniprot.org/.

region for YAP and TAZ can be reduced to the α-helix:linker:Ωloop motifs where the α-helix and the Ω-loops are very similar but the linker regions are different. The α1 helix dramatically enhances the affinity of fragments YAP(61−99) or TAZ(24− 56) (α1 helix-linker-Ω-loop) vs YAP(85−99) or TAZ(43−56) (Ω loop). The most important difference between the primary sequence of YAP(61−99) or TAZ(24−56) is the linker. TAZ has no PXXΦP motif but presents the same affinity for TEAD as YAP. A systematic analysis of the relative importance of the residues implicated in the interfaces 2 and 3 of YAP and TEAD has been undertaken using a combination of single site-direct mutagenesis and double mutant analyses44 which confirmed the central role of F69 of YAP as a “hot-spot” residue at the interface 2. The guanidinium group of R89 of YAP and the carboxylate group D272 of hTEAD4 (D264 for hTEAD1) establishing a strong ionic interaction were also evidenced as essential in this study. VgLL proteins are transcription coactivators named after the Drosophila transcription coactivator vestigial (Vg). The four mammalian VgLL proteins named VgLL1−4 do not contain any DNA-binding domain (DBD) and as YAP or TAZ exert their transcriptional regulatory functions through interacting with TEAD via their Vg motifs (also named Tondu or TDU domain). VgLL1−3 share one Vg domain (highlighted in blue) located in the N-terminal regions, while VgLL4 has two Vg domains (highlighted in blue and green) located in the C-terminal region (Table 2). The precise function of the interactions between TEADs and VgLLs is still poorly understood. TEAD/VgLL1 could promote anchorage-independent cell proliferation in

domain of YAP through the measurement of the dissociation constant by ITC. The structure of mTEAD4(210−427)-mTAZ(25−57) complex (PDB code 5GN0) (entry 17, Table 3) was recently resolved by Kaan et al.42 The authors found two different binding modes: a classical 1:1 complex very similar to the already published YAP-TEAD structure39 and a 2:2 complex where two molecules of TAZ bind to and bridge two molecules of TEAD (Figure 7). This latter binding mode was validated by cross-linking and multiangle light scattering. The comparison of mTEAD4(210−427)-mYAP(35−92) complex (PDB code 3JUA) and mTEAD4(210−427)-mTAZ(25−57) complex (PDB code 5GN0) (Figure 7A) shows virtually superimposed interface 2 and interface 3, and the main differences lie in the linker between α1 helix and Ω-loop. In the 2:2 TAZ-TEAD complex, each TAZ binds to one TEAD with its α1 helix while it binds to the other TEAD with its Ω-loop (Figure 7B). For each TEAD the interactions of the α1 helix of one TAZ (yellow) and the Ω-loop of the other TAZ (green) superimpose well with one YAP (orange red). Here again the main difference is due to the shorter linker of TAZ. The PXXΦP motif of YAP making important interactions in the maintenance of YAP-TEAD complex is replaced in TAZ by a set of interactions with TEAD due to S41, S42, W43, and K46. Comparable dissociation constants were found for similar YAP fragment (hYAP(61−99)) by SPR (surface plasmon resonance) in a large study devoted to establish similarities and differences between TEAD-YAP and TEAD-TAZ protein− protein interactions.43 It was shown that the TEAD-binding 5064



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Table 3. Available Structures of TEAD Fragments Resolved by NMR or X-ray Diffraction entry

protein

PDB code

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

hTEAD1 hTEAD1 hTEAD1 hTEAD1 hTEAD1 hTEAD2 hTEAD2 hTEAD2 hTEAD2 hTEAD2 hTEAD2 hTEAD3 hTEAD4 hTEAD4 mTEAD4 mTEAD4 mTEAD4

2HZD 4Z8E 5NNX 3KYS 4RE1 3L15 5DQ8 5DQE

resolution

5HGU 5EMV 5EMW 5GZB 5OAQ 3JUA 4EAZ 4LN0

2.09 3.29 2.80 2.20 2.00 2.31 2.18 2.32 2.05 2.00 2.55 2.70 1.95 3.00 2.80 2.90

18 19

mTEAD4 mTEAD4

5GN0 5XJD

2.90 2.20

other partners

DNA (18-mer) hYAP1 (50−171) YAP engineered disulfide flufenamic acid bromofenamic acid MGH−CP-1 palmitate palmitate palmitate muscle CAT-DNA (13-mer) hYAP(60−100) and myristate mYAP(47−85) mVgLL1(20−51) mVgLL4(203−256) 2,2′-oxybis(ethan-1-ol) mTAZ(25−57) fragment 16

prostate cancer lines, suggesting a role in cancer progression.45 VgLL2 is expressed predominately in skeleton muscle and plays a role in muscle differentiation.46,47 VgLL3 potentially plays a role in cancers and interacts in the progression of soft-tissue sarcoma and in the development of myxoinflammatory fibroblastic sarcoma.48,49 VgLL4 expression is found to be significantly down-regulated in clinical colorectal carcinoma and positively associated with patient survival rate.9 It targets a TCF4-TEAD4 complex and co-regulate Wnt and Hippo pathways. The first structure of a VGLL-TEAD complex was published in 2012.45 Superimposition of hTEAD1-hYAP1 (PDB code 3KYS) and mTEAD4(210−427)-mVGLL1(20−56) (PDB code 4EAZ) (entry 15, Table 3) complexes (Figure 8A) shows that interfaces 1 and 2 superimpose well and revealed the absence of Ω-loop for VgLL1. Attempts to crystallize VgLL1-

method

position

ref

NMR X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray

28−104 28−104 31−104 225−421 209−421 217−447 217−447 217−447 217−447 217−447 218−446 219−435 36−139 217−434 210−427 210−427 209−427

29 30 33 38 60 35 68 68 69 55 54 54 32 40 39 45 50

X-ray X-ray

210−427 210−427

42 70

TEAD complexes with elongated VgLL1 at the C-terminus failed. However, whereas mutations at the interface 2 did not reduce YAP-TEAD interaction,38 H44A and F45A double mutations severely compromised VgLL1-TEAD interactions. A second complex was then described by Jiao et al. in 2014.50 The authors used a long fragment of VgLL4 which included the two Vg motifs. mVgLL4(203−256) forms with mTEAD4(210− 427) a 1:2 complex (PDB code 4LN0) (entry 16, Table 3) where each Vg motif interacts with one TEAD (Figure 8B). The C-terminal Vg motif (233−244) binds to one TEAD molecule in the same interface 2 as YAP, while the β1 sheet of VgLL4 is antiparallel to the two β7 sheets of the two TEAD molecules. Finally, the N-terminal Vg motif (204−214) forms an interface 0 (orthogonal to the Vg motif implicated in the interface 2) with the other TEAD molecule.

Figure 8. (A) Superimposition of YAP-TEAD (PDB code 3KYS, TEAD in purple and YAP in green) and mVgLL1-TEAD complex (PDB code 4EAZ, TEAD in pink and VgLL1 in yellow). (B) Superimposition of YAP-TEAD (PDB code 3KYS, TEAD in purple and YAP in green) and mVgLL4-TEAD complex (PDB code 4LN0, TEAD in pink and VgLL4 in yellow). 5065



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Table 4. Stoichiometries and Interfaces Found in the Different Crystal Structures of TEAD with YAP, TAZ, and VgLL YAP stoichiometries found in the different crystallographic structures (TEAD:PARTNER) interface 0 interface 1 (β-sheet) interface 2 (α-helix) linker PXXΦP motif interface 3 (Ω loop)

VgLL1

VgLL4

1:1

1:1

2:2

1:1

2:1

absent present present present (folded) present present

absent absent present present (folded) absent present

absent absent present present (elongated) absent present

absent present present absent absent absent

present present present absent absent absent

Since VgLL lacks Ω-loop, the key element that confers to YAP and TAZ a high affinity to TEAD, it would be expected that VgLL will present a lower affinity for TEAD than YAP or TAZ. However, Mesrouze et al. showed by TR-FRET and SPR comparable affinity for mVgLL1(27−51) compared with hYAP(61−99) or hTAZ(24−56).51 Comparison of human and mouse VgLL1 fragments affinity toward hTEAD4 revealed the importance of the presence of M40 and H44 in mVgLL1 (which are not conserved in hYAP) but also the critical roles of F45, R47, and A48.52 It was also demonstrated that hVgLL4 is a potent inhibitor of YAP in mice,53 suggesting at that moment that VgLL and YAP (and TAZ) are in competition in the nucleus for access to TEAD transcription factors. p300 acetylated hVgLL4 mainly at K219 (which corresponds to K216 of mVgLL4) which is situated in an apparently specific VgLL4-TEAD interface (Figure 8B). The exact implication of this post-transcriptional modification on the VgLL4-TEAD binding still has to be investigated. mVgLL1, mVgLL4, mYAP, hYAP, and mTAZ present the particularity to bind to TEAD in different manners (see Table 4): the stoichiometries found in the studied crystals are 1:1, 2:2 or 2:1 (TEAD:ligand), and five interfaces can be defined. The only common element for all the ligands is the interface 2 which could be the seat of the competition between the different ligands of TEAD. The Ω-loop is specific to YAP and TAZ but does not confer to these coactivators higher affinity to TEAD than VgLL. The main difference between TAZ and YAP lies in the linker and the PXXΦP motif present in YAP but not in TAZ. Its absence allows TAZ to be able to bind to two TEAD molecules yielding a 2:2 stoichiometry not found with YAP, while it contributes to the YAP binding to TEAD.

TAZ

Figure 9. hTEAD2 (in blue) and palmitate (in purple) (PDB code 5HGU). The entry is controlled by C380 and K357, and the hydrophobic pocket presents an aromatic area (with 3 phenylalanines and 1 tyrosine) and an aliphatic zone (1 methionine, 1 isoleucine, 4 leucines, 4 valines, and 2 alanines).

previously reported structures of human or mouse TEAD in complex with YAP, VgLL, or small cyclic protein were revisited and the same electron density was found to be present and wrongly attributed to 2-mercaptoethanol, glycerol, or 2,2′oxybis(ethan-1-ol). The entry of this internal pocket delineated by C359 (TEAD1) is masked by the YAP β1 sheet (interface 1) (Figure 10) or the VgLL4 β1 sheet (not shown). FLAG-TEAD was metabolically labeled with ω-alkynyl palmitate in HEK293T cell. After a FLAG immunoprecipitation, the alkyne-labeled palmitoylated proteins were submitted to Huisgen condition with a biotinylated azide. TEAD and palmitoylation status were monitored using convenient antibodies and dyes, and the authors concluded that all four hTEADs were found to show S-palmitoylation in mammalian cells. The apparent K m of palmitoyl-CoA in TEAD2 autopalmitoylation was estimated to be around 0.8 μM,55 and palmitoylation strongly stabilizes TEAD as attested by the ΔTm observed between hydroxylamine-treated and untreated TEADYBD of 12.3 °C.54 On the basis of evolutionarily conserved residues, cysteines 53, 327, and 359 of TEAD1 were mutated and the mutants were submitted to autopalmitoylation in the presence of palmitateCoA confirming that C359S mutation is the most critical in TEAD1 palmitoylation. Furthermore, FRET-based binding assay between TEAD1 and YAP showed a weaker association of TEAD1 mutant (C359S) than wt TEAD1.

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THE INNER POCKET: A DRUGGABLE SITE? Recently, the structure of hTEAD3(219−435) (entry 12, Table 3) has been solved and, surprisingly, the authors found electron density resembling to an aliphatic chain in a central hydrophobic pocket that was directly connected to C368 (TEAD3) (highlighted in purple and underlined in Table 1). 54 hTEAD2(217−447) structure (PDB code 5EMV) (entry 11, Table 3) presented a similar electron density connected to C380 (TEAD2). Almost concomitantly, during a program dedicated to explore palmitoylacyltransferases and autopalmitoylated proteins, Chan et al.55 identified more than 300 probe-labeled proteins including TEAD1 and TEAD3. Expressed and purified native hTEAD2(217−447) from E. coli was solved (PDB code 5HGU) (entry 10, Table 3) and revealed the same electron density in the same pocket adjacent to C380 (TEAD2) (Figure 9). The difference between the last two structures is the presence of a thioester bond in structure 5EMV (and 5EMW) and a free palmitate in structure 5HGU suggesting that the thioester bond is relatively sensible to hydrolysis. In these two publications, 5066



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Figure 10. YAP and the entry of the palmitate pocket: (A) TEAD structure (purple surface; PDB code 5HGU); (B) superimposition of hYAP (yellow surface; PDB code 3KYS) and the TEAD structure (purple surface; PDB code 5HGU).

molecules and up-regulates p38 MAPK. Since 1 was also reported to up-regulate 14−3−3 protein,21 we can conclude that 1 targets YAP in the cytoplasm via several modes of action. Truncated versions of 1 were synthesized without significant improvment.58 However, a symmetric divinyldipyrrine was found to be as active as 1 in the cellular assay (HEK293 cells transfected with TEAD luciferase plasmid).

Click chemistry was used to probe the depth and the selectivity of this lipophilic pocket. Transfected HEK293T cells with TEAD containing C-terminal FLAG-tag were incubated with ω-alkynyl fatty acids of various length. The authors54 observed that C16 fatty acid was the most preferred, followed by C14 and C13 and C18 which were incorporated in a much lesser and variable extent. Although palmitate does not directly interact with the TEAD surface connected to YAP, it has been suggested that palmitate allosterically regulates YAP binding.55 The palmitate cavity is constituted by conserved lipophilic residues (highlighted in purple on Table 1), and the bottom is lined with phenylalanine residues F299 and F416 (TEAD3) closely connected with two residues of the interface 3 (the most critical pocket for YAP/ TAZ binding), K298 and E417 (of TEAD3). The same authors showed that TEAD1 mutant (C359S) has weaker association with YAP than wt TEAD1 but this mutation has no effect on the formation of TEAD1-VgLL4 while a revisit of the crystallographic structures of VgLL-TEAD complexes suggested the presence of palmitate. Following the discovery of the palmitate pocket, Mesrouze et al.40 has recently brought the proof that TEAD can incorporate other fatty acid, confirming the previous observation.54 The purification of wt hTEAD4(217−434) yielded the nonacylatedTEAD, Myr-TEAD4, and Palm-TEAD4. As demonstrated by thermal shift assay (TSA) and by SPR, the acylation seems to be required to ensure fully active conformation but dispensable for the YAP/TAZ binding (measured Kd values are in the nanomolar range and only third time lower with acyl-TEAD4 than with nonacyl-TEAD4).

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SMALL MOLECULES TARGETING YAP(TAZ)/TEAD Analysis of the available structures of the TEAD coactivator binding domain provides three different druggable pockets (Figure 5B). Taking advantage of the absence of overlapping between VgLL and YAP in the shallow pocket of TEAD corresponding to interface 3 (in purple on Figure 5B), cyclic peptides were first designed on the basis of YAP(81−100) fragment.59 Ala-scan and truncation identified YAP(84−100) as the shortest peptide which kept inhibitory activity and a beneficial D93A mutation. Further modifications (M86 to 3-chlorophenylalanine, R87 to homocysteine, L91 to norleucine and F96C) (Scheme 3) and disulfide bridge formation to rigidify the peptide yielded a cyclic peptide 10 with an improved binding affinity for TEAD1 (Kd = 15 nM) and a IC50 of 25 nM. In a second article,60 the same authors synthesized an optimized cyclic heptadecapeptide corresponding to YAP(84−100) with the following mutations R87C, D93A, and F96C and the replacement of the leucine 91 residue by (S)-2-aminoheptanoic acid with higher IC50 and Kd values than the ones previously reported.59 They also reported the crystallographic structure of hTEAD1(209−426) in complex with the quadruple mutant hYAP(50−171)(R87C, D93A, F96C, and E100R) (PDB code 4RE1) (entry 5, Table 3). The C-terminal part of YAP was not resolved, and Figure 11A only shows the structure of hYAP(53−99) bound to hTEAD1. Probably the disulfide bond (Figure 11B) induces constraint that can be reduced by adding one carbon (change of a cysteine by a homocysteine residue). These cyclic peptides61 and other chimeric peptides62 targeting the interaction between TEAD and YAP or TAZ have been patented. Starting from the crystallographic structure of mVgLL4-mTEAD4 complex, Jiao et al.50 designed a hybrid octatetracontapeptide named SuperTDU composed of a large fragment comprising the Vg motif of

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TARGETING YAP/TEAD WITH VERTEPORFIN ANALOGUES In their pioneer article, Liu-Chittenden et al.20 identified 1 and two other porphyrins as YAP/TEAD interaction inhibitor. As 1 is highly photosensitive, exposure to light had to be stringently controlled during cell treatment. Several studies reported its proteotoxicity, and its effective interaction with YAP was subjected to controversy.56 Finally, 1 was reported to inhibit growth of human glioma in vitro without light activation.57 1 down-regulates YAP-TEAD classical downstream signaling 5067



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tested on primary gastric tumor cells which were derived from two patients.50 Several screening campaigns have been described. Using a dual fragment-based drug discovery approach and highthroughput screening strategy63−66 targeting interface 3 (in purple on Figure 5B), Inventiva identified a family of small molecules and patented them67 for the treatment of malignant mesothelioma. Diversely substituted 3-(2-benzylidenehydrazinyl)benzo[d]isothiazole 1,1-dioxides (11) (Scheme 3) were studied for their ability to inhibit malignant mesothelioma cell growth without showing any effect in the SW620 cell line, which is a YAP-independent cell line.67 Some months before the discovery of the presence of palmitate within the central pocket of TEAD, Pobbati et al. identified flufenamic acid (12) from the screening of an in-house fragment library and of a library containing U.S. FDA approved drugs.68 The structure of the TEAD2-flufenamic acid complex (PDB code 5DQ8) (entry 7, Table 3) revealed that flufenamic acid occupied two distinct positions: one in the palmitate pocket and another one in the shallow pocket of the interface 3. Similar structure was obtained with bromofenamic acid (13) (PDB code 5DQE) (entry 8, Table 3). The affinity for TEAD4 of eight fenamic acids and related compounds was evaluated by ITC, and niflumic acid (14) was identified as the most active of the series with a Kd of 28 μM. However, flufenamic or niflumic acids (Scheme 3) had no effect on the YAP-TEAD interaction, suggesting that these compounds did not cause any conformational change at the YAP-binding surface of TEAD. At high concentration (150 μM), flufenamic and niflumic acids significantly reduced the TEAD reporter activity, the cell proliferation, and the expression of YAP-TEAD target genes such as Axl and NF2, on HEK293 cells. Xu et al. screened at 5 μM concentration a small molecule library of 50 000 compounds using a HEK293A stable cell line expressing TEAD-binding element luciferase reporter and YAP.69 Among the compounds presenting an inhibition higher than 50%, compound MGH-CP169 (15) (Scheme 3) was found to inhibit YAP-TEAD interaction with an IC50 of 83 nM in a Gal4-TEAD1 reporter assay. It was found to inhibit dosedependently and efficiently the expression of CTGF and Cyr61 in several cancer cell lines (HeLa, JHH7, HuH7) and to a lesser extent in MCF10A cells. The cocrystal structure (entry 9, Table 3) of 15 bound to TEAD2-YBD showed that this compound fits

Scheme 3. Structures of Some TEAD Ligands

VgLL4 involved in the interface 2 (236−252) with TEAD and a large portion including the Ω-loop of YAP involved in the interface 3 with TEAD(74−99) (Table 2 for the primary sequence of this peptide). This super-TDU inhibited cell viability and growth of gastric cancer cell lines and downregulated expression of CTGF, Cyr61, and CDX2. Several types of gastric cancer cell lines were injected to mice to form tumors. Once palpable tumors were detected, Super-TDU was injected. Size and weight of tumors as well as YAP target genes were markedly decreased for Super-TDU treated mice.50 Pharmacological evaluation showed that Super-TDU is relatively safe and specific and may have a broad antitumor effect toward YAP (and TAZ)-driven human cancers. Super-TDU efficacy was finally

Figure 11. Structure of hTEAD1 in complex with a quadruple YAP mutant (PDB code 4RE1): (A) TEAD is in purple, YAP mutant in cyan, and the disulfide bridge in yellow. (B) Zoom on the created disulfide bridge by F96C and R87C mutations (PDB code 4RE1 in cyan and PDB code 3KYS in green). 5068



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suggests an interaction with the D domain (97LARRK101 of TEAD1). The antibody used in this study recognizes all TEAD, and the authors did not see obvious TEAD mitochondrial localization. The possible cytoplasmic localization of TEAD (full length or splicing form) which could depend on culture conditions (environmental stresses), cell lines, and period of development had to be taken into account in the design and the potential application of new TEAD ligands.

in the hydrophobic pocket (in pink on Figure 5B) occupied by palmitate. Kaan et al. identified a hit fragment 16 by screening 1000 fragments from the Maybridge Ro3 fragment library in a thermal shift assay (Scheme 3).70 Cocrystallization of 16 with mTEAD4(210−427) (entry 19, Table 3) showed that this ligand binds to TEAD4 at the interface 2 (in blue on Figure 5B). 16 binds to TEAD with a low affinity (300 μM) as measured by ITC and reduces the TEAD reporter activity in HEK293 cells by 33% at 750 μM.

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AUTHOR INFORMATION

Corresponding Author

CONCLUSION To date, several FDA-approved drugs have been identified as inhibitors of the Hippo pathway by controlling the nuclear localization of YAP or TAZ. 1 was reported to up-regulate 14− 3−3σ. Dasatinib and pazopanib induce YAP and TAZ phosphorylation and are supposed to promote proteasomal degradation of YAP and TAZ in several cancer cells that overexpress YAP and/or TAZ. Statins are able to sequester YAP and TAZ in the cytoplasm. C19 activates the core kinase MST1 and Lats1 and inhibits Hippo, TGF-β, and Wnt pathways. Conversely, selective and reversible small inhibitors of MST1/ MST2 kinases were developed to potentiate tissue repair and regeneration. Combination of epigenetic regulators synergistically induces down-regulation of the AKT and Hippo pathways in melanoma cell lines. As YAP and TAZ are known to interact with several other cytoplasmic and nuclear proteins, targeting the YAP or TAZ/ TEAD interactions should give more selective drugs against cancers caused by a dysregulation the hippo pathway. In this review, we presented the different options arising from the numerous available crystallographic structures. Interface 3 and palmitate pocket of TEAD are indeed druggable and give rise to promising results, still to be improved however, while only one article reports a molecule that binds interface 2. The multimeric aspects of the interaction of TEAD with its coactivators however must be clarified by studying the interaction of the full-length proteins in solution in order to avoid misinterpretation due to truncated proteins. As suggested by Kaan et al. in their conclusion,42 the structure of the “ménage à trois” is implicated (YAP, TAZ, or VgLL), and TEAD and their DNA targets should provide important information for the discovery of more efficient (and maybe selective) new drugs. The exact role of the TEAD autopalmitoylation/myristoylation has to be investigated. It is demonstrated that TEAD bearing a mutation of the cysteine residue linked (or close to) the carboxylate function of palmitate has a weaker affinity for YAP than wt TEAD38 while flufenamic or niflumic acids have no effect on the YAP-TEAD interaction.68 Screening of natural and chemical modified fatty acids may give important information on the depth of this pocket and could reveal subtle differences between TEAD1, TEAD3, and TEAD2/4. Finally, the report of the splicing isoform of TEAD410 opens the opportunity to develop molecules that target the cytoplasmic YAP/TAZ-TEAD-S interactions for tissue regeneration and neurodegenerative disease treatment such as Huntington’s disease.71 Mitochondrial localization of TEAD4 has previously been reported during blastocoel formation to prevent oxidative stress.72 During the revision of this article, a p38 MAPK-induced cytoplasmic translocation of TEAD under environmental stresses (osmotic stress, high cell density, or cell detachment) was reported.73 p38 binds to the DBD of TEADs, and the sequence alignment of TEAD with canonical p38 substrates

*Phone: 362 28 36 94. E-mail: [email protected]. ORCID

Philippe Cotelle: 0000-0003-0924-0433 Author Contributions §

F.G. and M.S. contributed equally to this review.

Notes

The authors declare no competing financial interest. Biographies Floriane Gibault obtained her Engineer degree at the National Graduate School of Engineering Chemistry of Rennes and her Master’s degree in Organic Chemistry at Rennes University. She has obtained her Ph.D. in Medicinal Chemistry at the University of Lille in the laboratory of Prof. Patricia Melnyk in Oct 2017. Her main research interest concerns the design and synthesis of new small molecules to inhibit protein−protein interaction and their evaluation. Manon Sturbaut obtained her Master’s degree in Drug Design at the University of Lille. She is currently a Ph.D student in Medicinal Chemistry at the University of Lille in the laboratory of Prof. Patricia Melnyk. Her main research interest concerns the design and synthesis of new small molecules to inhibit protein−protein interaction and their evaluation. Fabrice Bailly studied chemistry at National Graduate School of Engineering Chemistry of Lille where he obtained his Engineer’s degree (1989). He received his Ph.D. in Chemistry in 1992. He was appointed Assistant Professor at the University of Lille in 1993. He worked on peptide-intercalant hybrids, then on the synthesis of ovothioldiselenides, and finally on the synthesis of antiviral agents with Prof. Cotelle. Presently, his main topic of research is synthesis of heterocyclic protein−protein inhibitors. Patricia Melnyk earned her degree in Chemical Engineering from Chimie ParisTech (1989) and a Ph.D. in Organic Chemistry from the Institut de Chimie des Substances Naturelles (Paris 6 University, 1993). She is Professor of Chemistry at the Faculty of Pharmacy of Lille University and the team leader of “Onco and NeuroChemistry” team in the Jean-Pierre Aubert Research Center (Inserm UMR-S1172). Its major research interests focus on medicinal and biological chemistry in the area of neurodegenerative diseases and cancer. She cofounded AlzProtect, a start-up dedicated to the clinical development of drug candidates for Alzheimer and neurodegenerative diseases. She is at the origin of the discovery of AZP2006, a drug candidate in clinical phase I. Philippe Cotelle obtained his Ph.D. in Spectrochemistry at the University of Lille in 1987. He is Professor of Organic Chemistry at the National Graduate School of Engineering Chemistry of Lille, and his expertise is in medicinal chemistry and translational research. He worked for several years in the field of antiviral agents (HIV-1 integrase, HIV-1 and HBV ribonuclease H) and has coordinated several national projects in viral diseases. He has moved into the area of protein−protein interactions and is interested in developing new drugs/chemical tools targeting the final effectors of the Hippo pathway. 5069



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T. Acetylation of VGLL4 regulates Hippo-YAP signaling and postnatal cardiac growth. Dev. Cell 2016, 39, 466−479. (6) Belandia, B.; Parker, M. G. Functional interaction between the p160 coactivator proteins and the transcriptional enhancer factor family of transcription factors. J. Biol. Chem. 2000, 275, 30801−30805. (7) Corvaisier, M.; Bauzone, M.; Corfiotti, F.; Renaud, F.; El Amrani, M.; Monté, D.; Truant, S.; Leteurtre, E.; Formstecher, P.; Van Seuningen, I.; Gespach, C.; Huet, G. Regulation of cellular quiescence by YAP/TAZ and Cyclin E1 in colon cancer cells: Implication in chemoresistance and cancer relapse. Oncotarget 2016, 7, 56699−56712. (8) Liu, Y.; Wang, G.; Yang, Y.; Mei, Z.; Liang, Z.; Cui, A.; Wu, T.; Liu, C. Y.; Cui, L. Increased TEAD4 expression and nuclear localization in colorectal cancer promote epithelial-mesenchymal transition and metastasis in a YAP-independent manner. Oncogene 2016, 35, 2789− 2800. (9) Jiao, S.; Li, C.; Hao, Q.; Miao, H.; Zhang, L.; Li, L.; Zhou, Z. VGLL4 targets a TCF4-TEAD4 complex to coregulate Wnt and Hippo signaling in colorectal cancer. Nat. Commun. 2017, 8, 14058. (10) Qi, Y.; Yu, J.; Han, W.; Fan, X.; Qian, H.; Wei, H.; Tsai, Y. H.; Zhao, J.; Zhang, W.; Liu, Q.; Meng, S.; Wang, Y.; Wang, Z. A splicing isoform of TEAD4 attenuates the Hippo-YAP signaling to inhibit tumour proliferation. Nat. Commun. 2016, 7, ncomms11840. (11) Harvey, K. F.; Zhang, X.; Thomas, D. M. The Hippo pathway and human cancer. Nat. Rev. Cancer 2013, 13, 246−257. (12) Moroishi, T.; Hansen, C. G.; Guan, K. L. The emerging roles of YAP and TAZ in cancer. Nat. Rev. Cancer 2015, 15, 73−79. (13) Zanconato, F.; Cordenonsi, M.; Piccolo, S. YAP/TAZ at the roots of cancer. Cancer Cell 2016, 29, 783−803. (14) Johnson, R.; Halder, G. The two faces of Hippo: targeting the Hippo pathway for regenerative medicine and cancer treatment. Nat. Rev. Drug Discovery 2014, 13, 63−79. (15) Santucci, M.; Vignudelli, T.; Ferrari, S.; Mor, M.; Scalvini, L.; Bolognesi, M. L.; Uliassi, E.; Costi, M. P. The Hippo pathway and YAP/ TAZ-TEAD protein-protein interaction as targets for regenerative medicine and cancer treatment. J. Med. Chem. 2015, 58, 4857−4873. (16) Pobbati, A. V.; Hong, W. Emerging roles of TEAD transcription factors and its coactivators in cancers. Cancer Biol. Ther. 2013, 14, 390− 398. (17) Nakatani, K.; Maehama, T.; Nishio, M.; Goto, H.; Kato, W.; Omori, H.; Miyachi, Y.; Togashi, H.; Shimono, Y.; Suzuki, A. Targeting the Hippo signalling pathway for cancer treatment. J. Biochem. 2017, 161, 237−244. (18) Bae, J. S.; Kim, S. M.; Lee, H. The Hippo signaling pathway provides novel anti-cancer drug targets. Oncotarget 2017, 8, 16084− 16098. (19) Gong, R.; Yu, F. X. Targeting the Hippo pathway for anti-cancer therapies. Curr. Med. Chem. 2015, 22, 4104−4117. (20) Liu-Chittenden, Y.; Huang, B.; Shim, J. S.; Chen, Q.; Lee, S. J.; Anders, R. A.; Liu, J. O.; Pan, D. Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP. Genes Dev. 2012, 26, 1300−1305. (21) Wang, C.; Zhu, X.; Feng, W.; Yu, Y.; Jeong, K.; Guo, W.; Lu, Y.; Mills, G. B. Verteporfin inhibits YAP function through up-regulating 14-3-3σ sequestering YAP in the cytoplasm. Am. J. Cancer Res. 2016, 6, 27−37. (22) Oku, Y.; Nishiya, N.; Shito, T.; Yamamoto, R.; Yamamoto, Y.; Oyama, C.; Uehara, Y. Small molecules inhibiting the nuclear localization of YAP/TAZ for chemotherapeutics and chemosensitizers against breast cancers. FEBS Open Bio 2015, 5, 542−549. (23) Taccioli, C.; Sorrentino, G.; Zannini, A.; Caroli, J.; Beneventano, D.; Anderlucci, L.; Lolli, M.; Bicciato, S.; Del Sal, G. MDP, a database linking drug response data to genomic information, identifies dasatinib and statins as a combinatorial strategy to inhibit YAP/TAZ in cancer cells. Oncotarget 2015, 6, 38854−38865. (24) Sorrentino, G.; Ruggeri, N.; Specchia, V.; Cordenonsi, M.; Mano, M.; Dupont, S.; Manfrin, A.; Ingallina, E.; Sommaggio, R.; Piazza, S.; Rosato, A.; Piccolo, S.; Del Sal, G. Metabolic control of YAP and TAZ by the mevalonate pathway. Nat. Cell Biol. 2014, 16, 357−366.

ACKNOWLEDGMENTS This work was financially supported by grants from le Ministère de l’Education et de la Recherche and the University of Lille (F.G. and M.S.) and fundings from SIRIC OncoLille and le Cancéropôle Nord-Ouest (Grant 2016/09). We thank Dr. Nicolas Renault from the molecular modeling platform of the University of Lille for technical assistance and helpful discussions. We are also indebted to Pr. Cunningham, Dr. Pobbati, Pr. Song, and Pr. Guan for kindly replying to our numerous questions. We also acknowledge the reviewers of the manuscript for their valuable suggestions, remarks, and corrections always aimed at improving the readability of this review.

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ABBREVIATIONS USED CBP, CREB-binding protein; CREB, C-AMP response elementbinding protein; CTGF, connective tissue growth factor; Cyr61, cysteine-rich angiogenic protein 61; DBD, DNA binding domain; FDA, Food and Drug Administration; FRET, fluorescence resonance energy transfer; GGPP, geranylgeranyl pyrophosphate; GST, glutathione S-transferase; HCC, hepatocellular carcinoma; HDAC, histone deacetylase; HEK, human embryonic kidney; HGC, human gastric cancer; HMG-CoA, 3hydroxy-3-methylglutaryl coenzyme A; HSQC, heteronuclear single quantum coherence; ITC, isothermal titration calorimetry; Lats1/2, large tumor suppressor 1/2; MSS, methyl methanesulfonate; Mst1/2, mammalian sterile 20-like 1/2; NF2, neurofibromatose type II; NMR, nuclear magnetic resonance; p38 MAPK, p38 mitogen-activated protein kinase; PDB, Protein Data Bank; PDE, phosphodiesterase; SCD1, stearoyl-CoA-desaturase 1; SIRT1, sirtuin 1; SPR, surface plasmon resonance; TAZ, transcriptional coactivator with PDZ binding motif; TCF, trancription factor; TDU, Tondu domain; TEA, transcriptional enhanced associate; TEAD, transcriptional enhanced associate domain; TEAD-S, splicing isoform of transcriptional enhanced associate domain; TGF-β, transforming growth factor β; TR-FRET, time-resolved fluorescence resonance energy transfer; TSA, thermal shift assay; VgLL, vestigial-like protein; Wnt, wingless-related integration site; wt, wild-type; YAP, yes associated protein; YBD, YAP binding domain

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REFERENCES

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