Targeting Transcriptional Enhanced Associate Domains (TEADs

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Targeting Transcriptional Enhanced Associate Domains (TEADs) Floriane Gibault, Manon Sturbaut, Fabrice Bailly, Patricia Melnyk, and Philippe Cotelle J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00879 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 18, 2017

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Journal of Medicinal Chemistry

TARGETING TRANSCRIPTIONAL ENHANCED ASSOCIATE DOMAINS (TEADs)

Floriane Gibault,§,† Manon Sturbaut,§,† Fabrice Bailly,† Patricia Melnyk† and Philippe Cotelle*†,‡ §

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

†

. Univ. Lille, Inserm, CHU Lille, UMR-S 1172 – JPArc - Centre de Recherche Jean-

Pierre Aubert Neurosciences et Cancer, F-59000, Lille, France ‡

. ENSCL, F-59000, Lille, France

ABSTRACT: ( 90%) and selective TEAD antibodies are therefore directed towards 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 ACS Paragon Plus Environment

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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, nineteen 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.

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 fourth has been deposited at the PDB but the authors are still working on. 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 TEAD-DBD 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 a DNA-ligand interaction assay, the studied DBD-TEAD was ACS Paragon Plus Environment

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found to bind to numerous muscle-CAT-like DNA sequences and the authors selected a 12-mer for further NMR study. Thanks to the analysis of the chemical shift perturbations of TEAD resonances in 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 ∆L1-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, ∆L1-TEAD-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 muscleCAT DNA element (13 base pairs) and the fourth structure corresponds to a hTEAD1(31104)-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 TEAD-DNA complexes (PDB code 5GZB32 and 5NNX33) (Figure 4) shows a clear conformational change of H3 for a better fit in the major groove. Independent biochemical characterization of the residues implicated in the TEADDNA interaction was 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 ACS Paragon Plus Environment

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

TEAD’s COACTIVATOR BINDING DOMAIN The YAP/TAZ/VgLL binding domain of TEAD (YBD) has been firstly 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 seleno-methionine 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 ACS Paragon Plus Environment


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(4785) 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 Nterminal 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 to side chains of R89 and S94 of YAP to strengthen the interactions by creating hydrogen bonds with D264 carboxylate and Y421 phenolate and E255 carboxylate of TEAD1. Mutations of YAP (M86A, R89A, L91A and F95A) as well as TEAD residues (Y421A or H) ACS Paragon Plus Environment

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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:

66

PQTV70P) where the alcoholic oxygen atom of

hydrogen bond with the hydrogen atom of the amide

385

68

T created a

N. 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 complex40 , 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 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 multi-angle light scattering. The comparison of mTEAD4(210-427)-mYAP(35-92) ACS Paragon Plus Environment


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 region for YAP and TAZ can be reduced to the αhelix:linker:Ω-loop 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 than 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 ACS Paragon Plus Environment

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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 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 coregulate 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-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 ACS Paragon Plus Environment


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. Since VgLL lack 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) than hYAP(61-99) or hTAZ(24-56).51 Comparison of human and mouse VgLL1 fragments affinity towards 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 has still to be investigated.

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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 stoechiometry not found with YAP, while it contributes to the YAP binding to TEAD.

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 on 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 two last 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 ACS Paragon Plus Environment


sensible to hydrolysis. In these two publications, 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 2mercaptoethanol, 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 were 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 Km of palmitoyl-CoA in TEAD2 autopalmitoylation was estimated to be around 0.8 µM55 and palmitoylation strongly stabilizes TEAD as attested by the ∆Tm observed between hydroxylamine-treated and untreated TEAD-YBD 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 palmitate-CoA 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. 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


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was the most preferred, followed by C14 and C13 and C18 which were incorporated in a much lesser and variable extent. Despite 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 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 non-acylated-TEAD, Myr-TEAD4 and Palm-TEAD4. As demonstrated by thermal shift assay (TSA) and by SPR, the acylation seems to be require to ensure fully active conformation but dispensable for the YAP/TAZ binding (measured Kd are in the nanomolar range and only third time lower with acyl-TEAD4 than with non-acyl-TEAD4).

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 ACS Paragon Plus Environment


to inhibit growth of human glioma in vitro without light activation.57 1 down-regulates YAP-TEAD classical downstream signaling 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).

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 a 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 ACS Paragon Plus Environment

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code 4RE1)(entry 5, Table 3). The C-terminal part of YAP was not resolved and the 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 Super-TDU composed of a large fragment comprising the Vg motif of 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(7499) (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 towards YAP(and TAZ)-driven human cancers. Super-TDU efficacy was finally 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 high-throughput screening strategy63-66 targeting interface 3 (in purple on figure 5B), Inventiva identified a family of small molecules and patented67 for the

treatment

of

malignant

mesothelioma.

Diversely

substituted

3-(2-

benzylidenehydrazinyl)benzo[d]isothiazole-1,1-dioxides, (11) (scheme 3) were studied for ACS Paragon Plus Environment


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 US 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 YAPTEAD target genes such as Axl and NF2, on HEK293 cells. Xu et al. screened at 5µM concentration a small molecule library of 50000 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 dose-dependently and efficiently the expression of CTGF and Cyr61 in several cancer cell lines (HeLa, JHH7, HuH7) and in a lesser extent in MCF10A cells. The co-crystal structure (entry 9, Table 3) ACS Paragon Plus Environment

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of 15 bound to TEAD2-YBD showed that this compound fits 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 Co-crystallization 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.

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 upregulate 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 ACS Paragon Plus Environment


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 which binds interface 2. The multimeric aspects of the interaction of TEAD with its coactivators must be however 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” where are implicated (YAP, TAZ or VgLL), 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 which targets 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, was reported a p38 MAPK-induced cytoplasmic translocation of TEAD under environmental stresses (osmotic stress, high cell density or cell detachment).73 p38 binds to the DBD of TEADs and the sequence alignment of TEAD with canonical p38 substrates suggests an ACS Paragon Plus Environment

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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.

AUTHOR INFORMATION

Corresponding author * Phone: 362 28 36 94; E-mail: [email protected]

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’ degrees in organic chemistry at the Rennes University. She has just obtained her Ph.D thesis 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 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 PhD in chemistry in 1992. He was appointed as assistant-professor at the University of Lille in 1993. He worked on peptide-intercalant hybrid, then on the synthesis of ovothiol-diselenides and finally worked on the synthesis of antiviral agents with Prof. Cotelle. Presently, his main topic of research is heterocyclic protein-protein inhibitors’ synthesis.

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 co-founded 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.


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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 moves on the area of protein-protein interactions and is interested by developing new drugs/chemical tools targeting the final effectors of the Hippo pathway.

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 n° 2016/09). We wish to thank Dr Nicolas Renault from the molecular modelling 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 reply to our numerous questions. We also wish to acknowledge the reviewers of the manuscript for their valuable suggestions, remarks and corrections always aimed at improving the readability of this review.

ABBREVIATIONS CBP

CREB-binding protein

CREB

C-AMP Response Element-binding protein

CTGF

Connective Tissue Growth Factor ACS Paragon Plus Environment


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 3-Hydroxy-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 Kinases PDB

Protein Data Bank

PDE

Phosphodiesterase

SCD1

Stearoyl-CoA-Desaturase 1

SIRT1

Sirtuin 1

SPR

Surface Plasmon Resonance


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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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(67) Barth, M.; Contal, S.; Montalbetti, C.; Spitzer, L. Preparation of new 4-(1,1-Dioxo1,2-benzothiazol-3-yl)hydrazono]methyl]-2-methoxyphenols

as

Inhibitors

of

the

YAP/TAZ-TEAD Interaction and their Use in the Treatment of Malignant Mesothelioma, PCT Int. Appl., WO 2017064277 A1 20170420, 2017. (68) Pobbati, A.V.; Han, X.; Hung, A.W.; Weiguang, S.; Huda, N.; Chen, G.Y.; Kang, C.; Chia, C.S.; Luo, X.; Hong, W.; Poulsen, A. Targeting the central pocket in human transcription factor TEAD as a potential cancer therapeutic strategy. Structure 2015, 23, 206-2086. (69) Xu, W. TEAD Transcription Factor Autopalmitoylation Inhibitors PCT Int. Appl., WO 2017053706 A1 20170330, 2017. (70) Kaan, H.Y.K.; Sim, A.Y.L.; Tan, S.K.J.; Verma, C.; Song, H. Targeting YAP/TAZTEAD protein-protein interactions using fragment-based and computational modeling approaches. PLoS One 2017, 12, e0178381. (71) Mao, Y.; Chen, X.; Xu, M.; Fujita, K.; Motoki, K.; Sasabe, T.; Homma, H.; Murata, M.; Tagawa, K.; Tamura, T.; Kaye, J.; Finkbeiner, S.; Blandino, G.; Sudol, M.; Okazawa, H.

Targeting

TEAD/YAP-transcription-dependent

necrosis,

TRIAD,

ameliorates

Huntington's disease pathology. Hum. Mol. Genet. 2016, 25, 4749-4770. (72) Kaneko, K.J.; De Pamphilis, M.L. TEAD4 establishes the energy homeostasis essential for blastocoel formation. Development 2013, 140, 3680-3690. (73) Lin, K.C.; Moroishi, T.; Meng, Z.; Jeong, H.S.; Plouffe, S.W.; Sekido, Y.; Han, J.; Park, H.W.; Guan, K.L. Regulation of Hippo pathway transcription factor TEAD by p38 MAPK-induced cytoplasmic translocation. Nat. Cell Biol. 2017, 19, 996-1002.



(74) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera-A visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605−1612.


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Figures, tables and schemes TEAD DNA-BINDING DOMAIN hTEAD1

28

DNDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVL97

hTEAD2

38

hTEAD3

28

hTEAD4

36

mTEAD4

29

GPDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVL107 DNDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVL97 DNDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRNELIARYIKLRTGKTRTRKQVSSHIQVL105 DNDAEGVWSPEIERSFQEALAIYPPCGRRKIILTEEGKMYGRNELIARHIKLRTGKTRTRKQVSSHIQVL98 Helix 1

Loop 1

hTEAD1

98

ARRKSRDFHSKLK----DQTAKDKALQHMAAMSSAQIVSA133

hTEAD2

108

ARRKSREIQSKLK----DQVSKDKAFQTMATMSSAQLISA143

hTEAD3

98

ARKKVREYQVGIKAMNLDQVSKDKALQSMASMSSAQIVSA137

hTEAD4

106

ARRKAREIQAKLK----DQAAKDKALQSMAAMSSAQIISA141

mTEAD4

99

ARRKAREIQAKLK----DQAAKNKALQSMAAMSSAQIVSA134

Helix 2

Loop 2

Helix 3

TEAD COACTIVATOR BINDING DOMAIN hTEAD1

206

WQGRSIGTTKLRLVEFSAFLEQQRDPDSYNKHLFVHIGHANHSYSDPLLESVDIRQIYDKFPEKKGGLKE275

hTEAD2

218

WQARGLGTARLQLVEFSAFVEPPDAVDSYQRHLFVHISQHCPSPGAPPLESVDVRQIYDKFPEKKGGLRE287

hTEAD3

215

WQDRTIASSRLRLLEYSAFMEVQRDPDTYSKHLFVHIGQTNPAFSDPPLEAVDVRQIYDKFPEKKGGLKE284

hTEAD4

214

WQGRSVASSKLWMLEFSAFLEQQQDPDTYNKHLFVHIGQSSPSYSDPYLEAVDIRQIYDKFPEKKGGLKD283

mTEAD4

207

WQGRSIASSKLWMLEFSAFLERQQDPDTYNKHLFVHISQSSPSYSDPYLETVDIRQIYDKFPEKKGGLKE278

hTEAD1

276

LFGKGPQNAFFLVKFWADLNCNIQ-DDAGA--------FYGVTSQYESSENMTVTCSTKVCSFGKQVVEK336

hTEAD2

288

LYDRGPPHAFFLVKFWADLNWGPSGEEAGAGGSISSGGFYGVSSQYESLEHMTLTCSSKVCSFGKQVVEK357

hTEAD3

285

LYEKGPPNAFFLVKFWADLNSTIQ-EGPGA--------FYGVSSQYSSADSMTISVSTKVCSFGKQVVEK345

hTEAD4

284

LFERGPSNAFFLVKFWADLNTNIE-DEGSS--------FYGVSSQYESPENMIITCSTKVCSFGKQVVEK344

mTEAD4

277

LFERGPSNAFFLVKFWADLNTNID-DEGSA--------FYGVSSQYESPENMIITCSTKVCSFGKQVVEK337

hTEAD1

337

VETEYARFENGRFVYRINRSPMCEYMINFIHKLKHLPEKYMMNSVLENFTILLVVTNRDTQETLLCMACV406

hTEAD2

358

VETERAQLEDGRFVYRLLRSPMCEYLVNFLHKLRQLPERYMMNSVLENFTILQVVTNRDTQELLLCTAYV427

hTEAD3

346

VETEYARLENGRFVYRIHRSPMCEYMINFIHKLKHLPEKYMMNSVLENFTILQVVTSRDSQETLLVIAFV415

hTEAD4

345

VETEYARYENGHYSYRIHRSPLCEYMINFIHKLKHLPEKYMMNSVLENFTILQVVTNRDTQETLLCIAYV414

mTEAD4

338

VETEYARYENGHYLYRIHRSPLCEYMINFIHKLKHLPEKYMMNSVLENFTILQVVTNRDTQETLLCIAYV408

hTEAD1

407

FEVSNSEHGAQHHIYRLVKD426

hTEAD2

428

FEVSTSERGAQHHIYRLVRD447

hTEAD3

416

FEVSTSEHGAQHHVYKLVKD435

hTEAD4

415

FEVSASEHGAQHHIYRLVKE434



409

mTEAD4

Page 40 of 50

FEVSASEHGAQHHIYRLVKE427

Table 1: Sequence alignments of DBD and YBD of crystallized TEADs. 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/.

COACTIVATOR TEAD-BINDING DOMAIN hYAP

50

AGHQIVHV-RG--DSETDLEALFNAVMNPKTANVPQTVPMRLRKLPDSFFKPPEPK102

mYAP

35

hTAZ

13

mTAZ

13

AGHQVVHV-RG—-DSETDLEALFNAVMNPKTANVPQTVPMRLRKLPDSFFKPPE85 PGQQVIHV-TQ--DLDTDLEALFNSVMNPK----PSS----WRKKILPESFFKE55 PGQQVIHV-TQ--DLDTDLEALFNSVMNPK----PSS----WRKKILPESFFKE55 25

mVgLL1 mVgLL4

203

hVgLL4

206

AGSVIFT-YFEG-DINSMVDEHFSRAL-RN51

DPVVEEHFRRSLGKNYKEPEPAPNSVSIT----G----S-VDDHFAKALGDTWLQIKAAKDSA256 DPVVEEHFRRSLGKNYKEPEPAPNSVSIT----G----S-VDDHFAKALGDTWLQIKAAKDGA259 Interface 0

Interface 1

Super-TDU

Interface 2

Interface 3

SVDDHFAKSLGDTWLQIGGSGNPKTANVPQTVPMRLRKLPDSFFKPPE

Table 2: Sequence alignments of TBD of TEADs coactivators. 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/.

Entry

Protein

PDB code

resolution

1

hTEAD1

2HZD

2

hTEAD1

4Z8E

2.09

3

hTEAD1

5NNX

3.29

4

hTEAD1

3KYS

5

hTEAD1

6

Other partners

method

position

Ref

NMR

28-104

[29]

X-ray

28-104

[30]

DNA (18-mer)

X-ray

31-104

[33]

2.80

hYAP1 (50-171)

X-ray

225-421

[38]

4RE1

2.20

YAP engineered disulfide

X-ray

209-421

[60]

hTEAD2

3L15

2.00

X-ray

217-447

[35]

7

hTEAD2

5DQ8

2.31

Flufenamic acid

X-ray

217-447

[68]

8

hTEAD2

5DQE

2.18

Bromofenamic acid

X-ray

217-447

[68]

9

hTEAD2

2.32

MGH-CP-1

X-ray

217-447

[69]

10

hTEAD2

5HGU

2.05

Palmitate

X-ray

217-447

[55]

11

hTEAD2

5EMV

2.00

Palmitate

X-ray

218-446

[54]

12

hTEAD3

5EMW

2.55

Palmitate

X-ray

219-435

[54]

13

hTEAD4

5GZB

2.70

Muscle CAT-DNA (13-mer)

X-ray

36-139

[32]

14

hTEAD4

5OAQ

1.95

hYAP(60-100)

X-ray

217-434

[40]


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and myristate 15

mTEAD4

3JUA

3.00

mYAP(47-85)

X-ray

210-427

[39]

16

mTEAD4

4EAZ

2.80

mVgLL1 (20-51)

X-ray

210-427

[45]

17

mTEAD4

4LN0

2.90

mVgLL4 (203-256)

X-ray

209-427

[50]

2,2’-oxybis(ethan-1-ol) 18

mTEAD4

5GN0

2.90

mTAZ (25-57)

X-ray

210-427

[42]

19

mTEAD4

5XJD

2.20

Fragment 16

X-ray

210-427

[70]

Table 3: Available structures of TEAD fragments resolved by NMR or X-ray diffraction

YAP Stoechiometries found in the 1:1 different

TAZ

VgLL1

VgLL4

1:1

2:2

1:1

2:1

crystallographic

structures (TEAD:PARTNER) Interface 0

absent

absent

absent

absent

present

Interface 1 (β -sheet)

present

absent

absent

present

present

Interface 2 (α-helix)

present

present

present

present

present

Linker

present

present

present

absent

absent

(folded)

(folded)

(elongated)

PXXΦP motif

present

absent

absent

absent

absent

Interface 3 (Ω loop)

present

present

present

absent

absent

Table 4: Stoechiometries and interfaces found in the different crystal structures of TEAD with YAP, TAZ and VgLL.



D

Q

W

Y

DNA-Binding Domain

TEAD1 TEAD2 TEAD3 TEAD4

30 40 30 38

WW

100

13

421 426 442 430 429 WW

204

230

WW

57

124

Activation Binding Domain

263 276

472

504

Activation Binding Domain

157

208

381 400

Vg

27

53

258 Vg

85 VgLL3

110

317

Vg

88 VgLL4

171

TEAD-BD

VgLL1 VgLL2

206 218 215 214

TEAD-BD

50 TAZ

YAP/TAZ/Vgll Binding Domain

120 130 124 128

YAP1-2γ

Page 42 of 50

112

326 Vg

206

Vg

249

290

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

Figure 2: Effect of the truncation of Loop1 on the interaction between the helixes of hTEAD1(28-104). A: lowest energy structure from 2HZD(left); B: One of the three molecules of ∆L1-hTEAD1(28-104) from 4Z8E. All structural figures were generated with Chimera 1.10.2 (UCSF).74


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Figure 3: A: Superimposition of hTEAD4(36-139)-DNA (13-mer)(PDB code 5GZB in grey) 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.

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

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.

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

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).


Page 44 of 50

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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).

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 Phenylalanine and 1 Tyrosine) and an aliphatic zone (1 Methionine, 1 Isoleucine, 4 Leucine, 4 Valine and 2 Alanine).



Figure 10: The α1 helix of YAP and the entry of the palmitate pocket. A: the 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).

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)


Page 46 of 50

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Scheme 1: TEAD ligands: its localization and its partners



Page 48 of 50

H3COOC Cl H3COOC

NH HN

O

Cl

N

HO O

N

N F

OH O HO

COOR

N

S

N

OH C19 5

COOR

Cerivastatin 4

Verteporfin 1 N N Cl

N

N

H N

N

N OH

H2N O

S

S

N H

O

O

N N

N

N

Pazopanib 3 Dasatinib 2 CF3

N

N N S

O

N

CF3

N N

N

S

OH XAV939 7

MF-438 6

O

N O HN

N NH

O O N

H N

N

I-BET151 8 Scheme 2: Hippo pathway inhibitors


Panobinostat 9

N H

OH

Page 49 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 3: Structures of some TEAD ligands.



Table of Contents Graphic


Page 50 of 50

Targeting Transcriptional Enhanced Associate Domains (TEADs

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