Direct and selective pharmacological disruption of the YAP-TEAD interface by IAG933 inhibits Hippo-dependent and RAS-MAPK-altered cancers.

The YAP-TEAD protein-protein interaction mediates YAP oncogenic functions downstream of the Hippo pathway. To date, available YAP-TEAD pharmacologic agents bind into the lipid pocket of TEAD, targeting the interaction indirectly via allosteric changes. However, the consequences of a direct pharmacological disruption of the interface between YAP and TEADs remain largely unexplored. Here, we present IAG933 and its analogs as potent first-in-class and selective disruptors of the YAP-TEAD protein-protein interaction with suitable properties to enter clinical trials. Pharmacologic abrogation of the interaction with all four TEAD paralogs resulted in YAP eviction from chromatin and reduced Hippo-mediated transcription and induction of cell death. In vivo, deep tumor regression was observed in Hippo-driven mesothelioma xenografts at tolerated doses in animal models as well as in Hippo-altered cancer models outside mesothelioma. Importantly this also extended to larger tumor indications, such as lung, pancreatic and colorectal cancer, in combination with RTK, KRAS-mutant selective and MAPK inhibitors, leading to more efficacious and durable responses. Clinical evaluation of IAG933 is underway.

mRNA Display Identifies Potent, Paralog-Selective Peptidic Ligands for ARID1B.

The ARID1A and ARID1B subunits are mutually exclusive components of the BAF variant of SWI/SNF chromatin remodeling complexes. Loss of function mutations in ARID1A are frequently observed in various cancers, resulting in a dependency on the paralog ARID1B for cancer cell proliferation. However, ARID1B has never been targeted directly, and the high degree of sequence similarity to ARID1A poses a challenge for the development of selective binders. In this study, we used mRNA display to identify peptidic ligands that bind with nanomolar affinities to ARID1B and showed high selectivity over ARID1A. Using orthogonal biochemical, biophysical, and chemical biology tools, we demonstrate that the peptides engage two different binding pockets, one of which directly involves an ARID1B-exclusive cysteine that could allow covalent targeting by small molecules. Our findings impart the first evidence of the ligandability of ARID1B, provide valuable tools for drug discovery, and suggest opportunities for the development of selective molecules to exploit the synthetic lethal relationship between ARID1A and ARID1B in cancer.

Optimization of a Class of Dihydrobenzofurane Analogs toward Orally Efficacious YAP-TEAD Protein-Protein Interaction Inhibitors.

The inhibition of the YAP-TEAD protein-protein interaction constitutes a promising therapeutic approach for the treatment of cancers linked to the dysregulation of the Hippo signaling pathway. The identification of a class of small molecules which potently inhibit the YAP-TEAD interaction by binding tightly to the Ω-loop pocket of TEAD has previously been communicated. This report details the further multi-parameter optimization of this class of compounds resulting in advanced analogs combining nanomolar cellular potency with a balanced ADME and off-target profile, and efficacy of these compounds in tumor bearing mice is demonstrated for the first time.

Design and Biochemical Characterization of Peptidic Inhibitors of the Myb/p300 Interaction.

The Myb transcription factor is involved in the proliferation of hematopoietic cells, and deregulation of its expression can lead to cancers such as leukemia. Myb interacts with various proteins, including the histone acetyltransferases p300 and CBP. Myb binds to a small domain of p300, the KIX domain (p300KIX), and inhibiting this interaction is a potential new drug discovery strategy in oncology. The available structures show that Myb binds to a very shallow pocket of the KIX domain, indicating that it might be challenging to identify inhibitors of this interaction. Here, we report the design of Myb-derived peptides which interact with p300KIX. We show that by mutating only two Myb residues that bind in or near a hotspot at the surface of p300KIX, it is possible to obtain single-digit nanomolar peptidic inhibitors of the Myb/p300KIX interaction that bind 400-fold tighter to p300KIX than wildtype Myb. These findings suggest that it might also be possible to design potent low molecular-weight compounds to disrupt the Myb/p300KIX interaction.

Biochemical and Structural Characterization of a Peptidic Inhibitor of the YAP:TEAD Interaction That Binds to the α-Helix Pocket on TEAD.

The TEAD transcription factors are the most distal elements of the Hippo pathway, and their transcriptional activity is regulated by several proteins, including YAP. In some cancers, the Hippo pathway is deregulated and inhibitors of the YAP:TEAD interaction are foreseen as new anticancer drugs. The binding of YAP to TEAD is driven by the interaction of an α-helix and an Ω-loop present in its TEAD-binding domain with two distinct pockets at the TEAD surface. Using the mRNA-based display technique to screen a library of in vitro-translated cyclic peptides, we identified a peptide that binds with a nanomolar affinity to TEAD. The X-ray structure of this peptide in complex with TEAD reveals that it interacts with the α-helix pocket. Under our experimental conditions, this peptide can form a ternary complex with TEAD and YAP. Furthermore, combining it with a peptide binding to the Ω-loop pocket gives an additive inhibitory effect on the YAP:TEAD interaction. Overall, our results show that it is possible to identify nanomolar inhibitors of the YAP:TEAD interaction that bind to the α-helix pocket, suggesting that developing such compounds might be a strategy to treat cancers where the Hippo pathway is deregulated.

N-terminal ß-strand in YAP is critical for stronger binding to scalloped relative to TEAD transcription factor.

The yes-associated protein (YAP) regulates the transcriptional activity of the TEAD transcription factors that are key in the control of organ morphogenesis. YAP interacts with TEAD via three secondary structure elements: a ß-strand, an α-helix, and an Ω-loop. Earlier results have shown that the ß-strand has only a marginal contribution in the YAP:TEAD interaction, but we show here that it significantly enhances the affinity of YAP for the Drosophila homolog of TEAD, scalloped (Sd). Nuclear magnetic resonance shows that the ß-strand adopts a more rigid conformation once bound to Sd; pre-steady state kinetic measurements show that the YAP:Sd complex is more stable. Although the crystal structures of the YAP:TEAD and YAP:Sd complexes reveal no differences at the binding interface that could explain these results. Molecular Dynamics simulations are in line with our experimental findings regarding ß-strand stability and overall binding affinity of YAP to TEAD and Sd. In particular, RMSF, correlated motion and MMGBSA analyses suggest that ß-sheet fluctuations play a relevant role in YAP53-57 ß-strand dissociation from TEAD4 and contribute to the lower affinity of YAP for TEAD4. Identifying a clear mechanism leading to the difference in YAP's ß-strand stability proved to be challenging, pointing to the potential relevance of multiple modest structural changes or fluctuations for regulation of binding affinity.

The First Class of Small Molecules Potently Disrupting the YAP-TEAD Interaction by Direct Competition.

Inhibition of the YAP-TEAD protein-protein interaction is an attractive therapeutic concept under intense investigation with the objective to treat cancers associated with a dysregulation of the Hippo pathway. However, owing to the very extended surface of interaction of the two proteins, the identification of small drug-like molecules able to efficiently prevent YAP from binding to TEAD by direct competition has been elusive so far. We disclose here the discovery of the first class of small molecules potently inhibiting the YAP-TEAD interaction by binding at one of the main interaction sites of YAP at the surface of TEAD. These inhibitors, providing a path forward to pharmacological intervention in the Hippo pathway, evolved from a weakly active virtual screening hit advanced to high potency by structure-based design.

Long-range structural preformation in yes-associated protein precedes encounter complex formation with TEAD.

Yes-associated protein (YAP) is a partly intrinsically disordered protein (IDP) that plays a major role as the downstream element of the Hippo pathway. Although the structures of the complex between TEA domain transcription factors (TEADs) and the TEAD-binding domain of YAP are already well characterized, its apo state and the binding mechanism with TEADs are still not clearly defined. Here we characterize via a combination of different NMR approaches with site-directed mutagenesis and affinity measurements the intrinsically disordered solution state of apo YAP. Our results provide evidence that the apo state of YAP adopts several compact conformations that may facilitate the formation of the YAP:TEAD complex. The interplay between local secondary structure element preformation and long-range co-stabilization of these structured elements precedes the encounter complex formation with TEAD and we, therefore, propose that TEAD binding proceeds largely via conformational selection of the preformed compact substates displaying at least nanosecond lifetimes.

The role of lysine palmitoylation/myristoylation in the function of the TEAD transcription factors.

The TEAD transcription factors are the most downstream elements of the Hippo pathway. Their transcriptional activity is modulated by different regulator proteins and by the palmitoylation/myristoylation of a specific cysteine residue. In this report, we show that a conserved lysine present in these transcription factors can also be acylated, probably following the intramolecular transfer of the acyl moiety from the cysteine. Using Scalloped (Sd), the Drosophila homolog of human TEAD, as a model, we designed a mutant protein (Glu352GlnSd) that is predominantly acylated on the lysine (Lys350Sd). This protein binds in vitro to the three Sd regulators-Yki, Vg and Tgi-with a similar affinity as the wild type Sd, but it has a significantly higher thermal stability than Sd acylated on the cysteine. This mutant was also introduced in the endogenous locus of the sd gene in Drosophila using CRISPR/Cas9. Homozygous mutants reach adulthood, do not present obvious morphological defects and the mutant protein has both the same level of expression and localization as wild type Sd. This reveals that this mutant protein is both functional and able to control cell growth in a similar fashion as wild type Sd. Therefore, enhancing the lysine acylation of Sd has no detrimental effect on the Hippo pathway. However, we did observe a slight but significant increase of wing size in flies homozygous for the mutant protein suggesting that a higher acylation of the lysine affects the activity of the Hippo pathway. Altogether, our findings indicate that TEAD/Sd can be acylated either on a cysteine or on a lysine, and suggest that these two different forms may have similar properties in cells.

Biochemical properties of VGLL4 from Homo sapiens and Tgi from Drosophila melanogaster and possible biological implications.

The TEAD (Sd in drosophila) transcription factors are essential for the Hippo pathway. Human VGLL4 and drosophila Tgi bind to TEAD/Sd via two distinct binding sites. These two regions are separated by few amino acids in VGLL4 but they are very distant from each other in Tgi. This difference prompted us to study whether it influences the interaction with TEAD4/Sd. We show that the full-length VGLL4/Tgi proteins behave as intrinsically disordered proteins. They have a similar affinity for TEAD4/Sd revealing that the length of the region between the two binding sites has little effect on the interaction. One of their two binding sites (high-affinity site) binds to TEAD4/Sd 100 times more tightly than to the other site, and size exclusion chromatography experiments reveal that VGLL4/Tgi only form trimeric complexes with TEAD4/Sd at high protein concentrations. In solution, therefore, VGLL4/Tgi may predominantly interact with TEAD4/Sd via their high-affinity site to create dimeric complexes. In contrast, when TEAD4/Sd molecules are immobilized on sensor chips used in Surface Plasmon Resonance experiments, one VGLL4/Tgi molecule can bind simultaneously with an enhanced affinity to two immobilized molecules. This effect, due to a local increase in protein concentration triggered by the proximity of the immobilized TEAD4/Sd molecules, suggests that in vivo VGLL4/Tgi could bind with an enhanced affinity to two nearby TEAD/Sd molecules bound to DNA. The presence of two binding sites in VGLL4/Tgi might only be required for the function of these proteins when they interact with TEAD/Sd bound to DNA.

Study of the TEAD-binding domain of the YAP protein from animal species.

The Hippo signaling pathway, which plays a central role in the control of organ size in animals, is well conserved in metazoans. The most downstream elements of this pathway are the TEAD transcription factors that are regulated by their association with the transcriptional coactivator YAP. Therefore, the creation of the binding interface that ensures the formation of the YAP:TEAD complex is a critical molecular recognition event essential for the development/survival of many living organisms. In this report, using the available structural information on the YAP:TEAD complex, we study the TEAD-binding domain of YAP from different animal species. This analysis of more than 400 amino acid sequences reveals that the residues from YAP involved in the formation of the two main contact regions with TEAD are very well conserved. Therefore, the binding interface between YAP and TEAD, as found in humans, probably appeared at an early evolutionary stage in metazoans. We find that, in contrast to most other animal species, several Actinopterygii species possess YAP variants with a different TEAD-binding domain. However, these variants bind to TEAD with a similar affinity. Our studies show that the protein identified as a YAP homolog in Caenorhabditis elegans does not contain the TEAD-binding domain found in YAP of other metazoans. Finally, we do not identify in non-metazoan species, amino acid sequences containing both a TEAD-binding domain, as in metazoan YAP, and WW domain(s).

A new perspective on the interaction between the Vg/VGLL1-3 proteins and the TEAD transcription factors.

The most downstream elements of the Hippo pathway, the TEAD transcription factors, are regulated by several cofactors, such as Vg/VGLL1-3. Earlier findings on human VGLL1 and here on human VGLL3 show that these proteins interact with TEAD via a conserved amino acid motif called the TONDU domain. Surprisingly, our studies reveal that the TEAD-binding domain of Drosophila Vg and of human VGLL2 is more complex and contains an additional structural element, an Ω-loop, that contributes to TEAD binding. To explain this unexpected structural difference between proteins from the same family, we propose that, after the genome-wide duplications at the origin of vertebrates, the Ω-loop present in an ancestral VGLL gene has been lost in some VGLL variants. These findings illustrate how structural and functional constraints can guide the evolution of transcriptional cofactors to preserve their ability to compete with other cofactors for binding to transcription factors.

An Early Association between the α-Helix of the TEAD Binding Domain of YAP and TEAD Drives the Formation of the YAP:TEAD Complex.

The Hippo pathway is an evolutionarily conserved signaling pathway that is involved in the control of organ size and development. The TEAD transcription factors are the most downstream elements of the Hippo pathway, and their transcriptional activity is regulated via the interaction with different co-regulators such as YAP. The structure of the YAP:TEAD complex shows that YAP binds to TEAD via two distinct secondary structure elements, an α-helix and an Ω-loop, and site-directed mutagenesis experiments revealed that the Ω-loop is the "hot spot" of this interaction. While much is known about how YAP and TEAD interact with each other, little is known about the mechanism leading to the formation of a complex between these two proteins. Here we combine site-directed mutagenesis with pre-steady-state kinetic measurements to show that the association between these proteins follows an apparent one-step binding mechanism. Furthermore, linear free energy relationships and a Φ analysis suggest that binding-induced folding of the YAP α-helix to TEAD occurs independently of and before formation of the Ω-loop interface. Thus, the binding-induced folding of YAP appears not to conform to the concomitant formation of tertiary structure (nucleation-condensation) usually observed for coupled binding and folding reactions. Our findings demonstrate how a mechanism reminiscent of the classical framework (diffusion-collision) mechanism of protein folding may operate in disorder-to-order transitions involving intrinsically disordered proteins.

Identification of FAM181A and FAM181B as new interactors with the TEAD transcription factors.

The Hippo pathway is a key signaling pathway in the control of organ size and development. The most distal elements of this pathway, the TEAD transcription factors, are regulated by several proteins, such as YAP (Yes-associated protein), TAZ (transcriptional co-activator with PDZ-binding motif) and VGLL1-4 (Vestigial-like members 1-4). In this article, combining structural data and motif searches in protein databases, we identify two new TEAD interactors: FAM181A and FAM181B. Our structural data show that they bind to TEAD via an Ω-loop as YAP/TAZ do, but only FAM181B possesses the LxxLF motif (x any amino acid) found in YAP/TAZ. The affinity of different FAM181A/B fragments for TEAD is in the low micromolar range and full-length FAM181A/B proteins interact with TEAD in cells. These findings, together with a recent report showing that FAM181A/B proteins have a role in nervous system development, suggest a potential new involvement of the TEAD transcription factors in the development of this tissue.

Discovery of Potent and Selective Antibody-Drug Conjugates with Eg5 Inhibitors through Linker and Payload Optimization.

Targeted antimitotic agents are a promising class of anticancer therapies. Herein, we describe the development of a potent and selective antimitotic Eg5 inhibitor based antibody-drug conjugate (ADC). Preliminary studies were performed using proprietary Eg5 inhibitors which were conjugated onto a HER2-targeting antibody using maleimido caproyl valine-citrulline para-amino benzocarbamate, or MC-VC-PABC cleavable linker. However, the resulting ADCs lacked antigen-specificity in vivo, probably from premature release of the payload. Second-generation ADCs were then developed, using noncleavable linkers, and the resulting conjugates (ADC-4 and ADC-10) led to in vivo efficacy in an HER-2 expressing (SK-OV-3ip) mouse xenograft model while ADC-11 led to in vivo efficacy in an anti-c-KIT (NCI-H526) mouse xenograft model in a target-dependent manner.

Structure-based design of potent linear peptide inhibitors of the YAP-TEAD protein-protein interaction derived from the YAP omega-loop sequence.

The YAP-TEAD protein-protein interaction is a potential therapeutic target to treat cancers in which the Hippo signaling pathway is deregulated. However, the extremely large surface of interaction between the two proteins presents a formidable challenge for a small molecule interaction disrupter approach. We have accomplished progress towards showing the feasibility of this approach by the identification of a 15-mer peptide able to potently (nanomolar range) disrupt the YAP-TEAD interaction by targeting only one of the two important sites of interaction. This peptide, incorporating non-natural amino acids selected by structure-based design, is derived from the Ω-loop sequence 85-99 of YAP.

Structural States of Hdm2 and HdmX: X-ray Elucidation of Adaptations and Binding Interactions for Different Chemical Compound Classes.

Hdm2 (human MDM2, human double minute 2 homologue) counteracts p53 function by direct binding to p53 and by ubiquitin-dependent p53 protein degradation. Activation of p53 by inhibitors of the p53-Hdm2 interaction is being pursued as a therapeutic strategy in p53 wild-type cancers. In addition, HdmX (human MDMX, human MDM4) was also identified as an important therapeutic target to efficiently reactivate p53, and it is likely that dual inhibition of Hdm2 and HdmX is beneficial. Herein we report four new X-ray structures for Hdm2 and five new X-ray structures for HdmX complexes, involving different classes of synthetic compounds (including the worldwide highest resolutions for Hdm2 and HdmX, at 1.13 and 1.20 Å, respectively). We also reveal the key additive 18-crown-ether, which we discovered to enable HdmX crystallization and show its stabilization of various Lys residues. In addition, we report the previously unpublished details of X-ray structure determinations for eight further Hdm2 complexes, including the clinical trial compounds NVP-CGM097 and NVP-HDM201. An analysis of all compound binding modes reveals new and deepened insight into the possible adaptations and structural states of Hdm2 (e.g., flip of F55, flip of Y67, reorientation of H96) and HdmX (e.g., flip of H55, dimer induction), enabling key binding interactions for different compound classes. To facilitate comparisons, we used the same numbering for Hdm2 (as in Q00987) and HdmX (as in O15151, but minus 1). Taken together, these structural insights should prove useful for the design and optimization of further selective and/or dual Hdm2/HdmX inhibitors.

Molecular and structural characterization of a TEAD mutation at the origin of Sveinsson's chorioretinal atrophy.

Four TEAD transcription factors (TEAD1-4) mediate the signalling output of the Hippo pathway that controls organ size in humans. TEAD transcriptional activity is regulated via interactions with the YAP, TAZ and VGLL proteins. A mutation in the TEAD1 gene, Tyr421His, has been identified in patients suffering from Sveinsson's chorioretinal atrophy (SCA), an autosomal dominant eye disease. This mutation prevents the YAP/TAZ:TEAD1 interaction. In this study, we measure the affinity of YAP, TAZ and VGLL1 for the four human TEADs and find that they have a similar affinity for all TEADs. We quantitate the effect of the mutation found in SCA patients and show that it destabilizes the YAP/TAZ:TEAD interaction by about 3 kcal·mol-1 . We determine the structure of YAP in complex with this mutant form of TEAD and show that the histidine residue adopts different conformations at the binding interface. The presence of this flexible residue induces the destabilization of several H-bonds and the loss of van der Waals contacts, which explains why the Tyr421HisTEAD1 mutation has such a large destabilizing effect on the formation of the YAP:TEAD complex. DATABASE: The crystallographic data have been deposited at the RSCB Protein Data Bank (PDB, www.pdb.org) with the access codes: 6HIL (wtYAP :Tyr421HisTEAD1 ), 6HIK (wtYAP :Tyr429HisTEAD4 ).

Discovery and Structural Characterization of ATP-Site Ligands for the Wild-Type and V617F Mutant JAK2 Pseudokinase Domain.

The oncogenic V617F mutation lies in the pseudokinase domain of JAK2, marking it as a potential target for development of compounds that might inhibit the pathogenic activity of the mutant protein. We used differential scanning fluorimetry to identify compounds that bind the JAK2 pseudokinase domain. Crystal structures of five candidate compounds with the wild-type domain reveal their modes of binding. Exploration of analogs of screening hit BI-D1870 led to the identification of compound 2, a 123 nM ligand for the pseudokinase domain. Interestingly, crystal structures of the V617F domain in complex with two unrelated compounds reveal a conformation that is characteristic of the wild-type domain, rather than that previously observed for the V617F mutant. These structures suggest that certain ATP-site ligands can modulate the V617F allosteric site, thereby providing a mechanistic rationale for targeting the pseudokinase domain and a structural foundation for development of more potent and pseudokinase-selective compounds.

Adaptation of the bound intrinsically disordered protein YAP to mutations at the YAP:TEAD interface.

Many interactions between proteins are mediated by intrinsically disordered regions (IDRs). Intrinsically disordered proteins (IDPs) do not adopt a stable three-dimensional structure in their unbound form, but they become more structured upon binding to their partners. In this communication, we study how a bound IDR adapts to mutations, preventing the formation of hydrogen bonds at the binding interface that needs a precise positioning of the interacting residues to be formed. We use as a model the YAP:TEAD interface, where one YAP (IDP) and two TEAD residues form hydrogen bonds via their side chain. Our study shows that the conformational flexibility of bound YAP and the reorganization of water molecules at the interface help to reduce the energetic constraints created by the loss of H-bonds at the interface. The residual flexibility/dynamic of bound IDRs and water might, therefore, be a key for the adaptation of IDPs to different interface landscapes and to mutations occurring at binding interfaces.