Discovery of a new class of reversible TEA domain transcription factor inhibitors with a novel binding mode
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Evaluation Summary:
Hu and colleagues describe the discovery and characterization of a new class of reversible palmitoylation (PLM) binding site TEAD inhibitors. X-ray co-crystallographic analysis reveals that the ligand class, identified from a screen of 30,000 small molecules, binds to a new site within the auto-PLM site. The TM2 lead compound inhibits the growth of NF2-deficient cell lines. The discovery has the potential to significantly impact the design and development of new effective TEAD inhibitors. Some clarification or additional data are required to support and justify some of the authors' claims regarding the molecular significance of this new class of inhibitors.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
The TEA domain (TEAD) transcription factor forms a transcription co-activation complex with the key downstream effector of the Hippo pathway, YAP/TAZ. TEAD-YAP controls the expression of Hippo-responsive genes involved in cell proliferation, development, and tumorigenesis. Hyperactivation of TEAD-YAP activities is observed in many human cancers and is associated with cancer cell proliferation, survival, and immune evasion. Therefore, targeting the TEAD-YAP complex has emerged as an attractive therapeutic approach. We previously reported that the mammalian TEAD transcription factors (TEAD1–4) possess auto-palmitoylation activities and contain an evolutionarily conserved palmitate-binding pocket (PBP), which allows small-molecule modulation. Since then, several reversible and irreversible inhibitors have been reported by binding to PBP. Here, we report a new class of TEAD inhibitors with a novel binding mode. Representative analog TM2 shows potent inhibition of TEAD auto-palmitoylation both in vitro and in cells. Surprisingly, the co-crystal structure of the human TEAD2 YAP-binding domain (YBD) in complex with TM2 reveals that TM2 adopts an unexpected binding mode by occupying not only the hydrophobic PBP, but also a new side binding pocket formed by hydrophilic residues. RNA-seq analysis shows that TM2 potently and specifically suppresses TEAD-YAP transcriptional activities. Consistently, TM2 exhibits strong antiproliferation effects as a single agent or in combination with a MEK inhibitor in YAP-dependent cancer cells. These findings establish TM2 as a promising small-molecule inhibitor against TEAD-YAP activities and provide new insights for designing novel TEAD inhibitors with enhanced selectivity and potency.
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Evaluation Summary:
Hu and colleagues describe the discovery and characterization of a new class of reversible palmitoylation (PLM) binding site TEAD inhibitors. X-ray co-crystallographic analysis reveals that the ligand class, identified from a screen of 30,000 small molecules, binds to a new site within the auto-PLM site. The TM2 lead compound inhibits the growth of NF2-deficient cell lines. The discovery has the potential to significantly impact the design and development of new effective TEAD inhibitors. Some clarification or additional data are required to support and justify some of the authors' claims regarding the molecular significance of this new class of inhibitors.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes …
Evaluation Summary:
Hu and colleagues describe the discovery and characterization of a new class of reversible palmitoylation (PLM) binding site TEAD inhibitors. X-ray co-crystallographic analysis reveals that the ligand class, identified from a screen of 30,000 small molecules, binds to a new site within the auto-PLM site. The TM2 lead compound inhibits the growth of NF2-deficient cell lines. The discovery has the potential to significantly impact the design and development of new effective TEAD inhibitors. Some clarification or additional data are required to support and justify some of the authors' claims regarding the molecular significance of this new class of inhibitors.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Activation of TEAD-dependent transcription by YAP/TAZ has been implicated in the development and progression of a significant number of malignancies. For example, loss of function mutations in NF2 or LATS1/2 (known upstream regulators that promote YAP phosphorylation and its retention and degradation in the cytoplasm) promote YAP nuclear entry and association with TEAD to drive oncogenic gene transcription and occurs in a significant majority of mesothelioma patients. High levels of nuclear YAP have also been reported for a number of other cancer cell types. As such, the YAP-TEAD complex represents a promising target for drug discovery and therapeutic intervention. Based on the essential functional role for TEAD palmitoylation at a conserved cysteine site, discovered by the authors of this manuscript, …
Reviewer #1 (Public Review):
Activation of TEAD-dependent transcription by YAP/TAZ has been implicated in the development and progression of a significant number of malignancies. For example, loss of function mutations in NF2 or LATS1/2 (known upstream regulators that promote YAP phosphorylation and its retention and degradation in the cytoplasm) promote YAP nuclear entry and association with TEAD to drive oncogenic gene transcription and occurs in a significant majority of mesothelioma patients. High levels of nuclear YAP have also been reported for a number of other cancer cell types. As such, the YAP-TEAD complex represents a promising target for drug discovery and therapeutic intervention. Based on the essential functional role for TEAD palmitoylation at a conserved cysteine site, discovered by the authors of this manuscript, several groups have successfully targeted this site using both reversible binding non-covalent TEAD inhibitors (i.e., flufenamic acid (FA), MGH-CP1, compound 2 and VT101~107), as well as covalent TEAD inhibitors (i.e., TED-347, DC-TEADin02, and K-975), which have been demonstrated to inhibit YAP-TEAD function and display anti-tumor activity in cells and in vivo.
Here, Hu et al. disclose the discovery of a new class of reversible TEAD inhibitors that putatively binds across a new site within the auto-PLM pocket across the TEAD family of proteins. To date, reported inhibitors that target this site (flufenamic acid (FA), TED-347, MYF-01-037, K975, VT103, MGH-CP1) function by binding to the same hydrophobic palmitate binding pocket (PBP) which is highly conserved. The novel TEAD inhibitor is identified from a screen of 30,000 compounds using recombinant TEAD2 and TEAD4 proteins and TEAD2 and TEAD4 click-ELISA assays. A molecule termed TM2 was found to show the strongest inhibition on TEAD auto-palmitoylation (IC50= 156nM for TEAD2 and 38nM for TEAD4) and is chosen for further characterization. A co-crystal structure of TEAD2 YAP binding domain in complex with TM2 was solved and revealed that TM2 not only occupied the familiar hydrophobic PLM site but also binds within a new pocket. Notably, the charge properties of this site might be compatible with the development of more drug-like analogs with enhanced hydrophilic properties. TM2 is profiled against several malignant pleural mesothelioma (MPM) cell lines (IC50 = 26-157nM) and activity correlates well with in vitro TEAD palmitoylation inhibition. Notably, consistent with results for related inhibitor classes, TM2 is not active as a monotherapy in cells beyond NF2 deficient cell lines, although cell type selective cytotoxicity is clearly shown.
Strengths:
This newly reported chemical series is highly tractable and could provide an excellent starting point for the development of effective TEAD inhibitors. The reported ability of TM2 to bind to a new site within the conserved TEAD auto-PBP site could enable this area of drug discovery.
Weaknesses:
The authors claim that TM2 is "more specific" than existing inhibitors, with respect to its ability to regulate YAP/TAZ-TEAD target gene expression. However, this statement is based on a comparison of unrelatable RNA-Seq datasets. TM2-regulated expression is evaluated in the context of cell (mono)culture following a continuous duration of target engagement. Comparison is made to the covalent inhibitor K975 based on a published dataset involving analysis of a drug-treated NCI-H226 tumor xenograft (i.e, in vivo drug treatment). Heterogeneity in cells, time point of analysis and duration and target engagement preclude the possibility of any reasonable comparison of these datasets. The ability of TM2 to bind within a new site is a significant finding. The comparison of TM2 binding mode to other inhibitors is based on structures of non-TEAD2 co-crystal structures. It will help significantly to clarify the differences in the side chains within this site across TEAD family members (i.e., TEAD1-4). The robustness of the proposed synergistic activity of TM2 in non-responsive cell lines is unclear.
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Reviewer #2 (Public Review):
Hu, Sun, Liu et al., identify and describe new small molecule inhibitors of the TEAD family of transcription factors, which direct the transcriptional activity of the Hippo pathway effectors YAP and TAZ. The study primarily focuses on characterizing the molecule, TM2, which shows strong inhibition of TEAD auto-palmitoylation in vitro and in cells, doing so by occupying the central hydrophobic pocket that other TEAD small molecule inhibitors are known to bind along with an unexplored adjacent hydrophilic region. TM2 was shown to suppress YAP-TEAD binding and gene expression and inhibit the proliferation of YAP-dependent cells in vitro, an effect that was exacerbated in combination with MEK inhibition.
Major Strengths:
The identification that new residues within the TEAD palmitate-binding pocket that may …Reviewer #2 (Public Review):
Hu, Sun, Liu et al., identify and describe new small molecule inhibitors of the TEAD family of transcription factors, which direct the transcriptional activity of the Hippo pathway effectors YAP and TAZ. The study primarily focuses on characterizing the molecule, TM2, which shows strong inhibition of TEAD auto-palmitoylation in vitro and in cells, doing so by occupying the central hydrophobic pocket that other TEAD small molecule inhibitors are known to bind along with an unexplored adjacent hydrophilic region. TM2 was shown to suppress YAP-TEAD binding and gene expression and inhibit the proliferation of YAP-dependent cells in vitro, an effect that was exacerbated in combination with MEK inhibition.
Major Strengths:
The identification that new residues within the TEAD palmitate-binding pocket that may contribute to YAP-TEAD function is very interesting, and while the study does not delve into the importance of these residues, the observations motivate future testing and modification of this class of molecules for potentially improved translation.Major Weaknesses:
The biological experiments testing the newly identified compounds are somewhat limited and statistical analysis is lacking for much of the data. -
