TEAD1 Palmitoylation

Endogenous TEAD1 and TEAD3 phospholipids are inhibited by VT107 (3 μmol/L; 20 hours; HEK293T cells) and endogenous TEAD4 phospholipids are efficiently inhibited as well [1]. On TEAD2 and TEAD4, VT107 is somewhat more potent than VT104. The removal of thermoplastic TEAD1 was produced by VT107, but it did not increase thermoplastic TEAD1. The thermoplastics TEAD3 and TEAD4 are decreased in VT107 without being increased in VT107. Additionally, VT107 prevents YAP and TAZ from reacting with TEAD1 and TEAD4. VT107 efficiently prevents NF2 mutant or defective cell lines from proliferating [1].

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Upon co-incubation with VT103, TEAD1 showed the highest increase in melting temperature—a shift of 8.3°C—compared with other members of the TEAD family. The thermal denaturation curves clearly showed two separate peaks for TEAD1 alone (red curve) and TEAD1+VT103 (blue curve), while the peaks of the ±VT103 curves remained largely overlapping for the other TEAD proteins (Fig. 4A, top). This is consistent with the finding from the functional palmitoylation assays that VT103 is a TEAD1-selective inhibitor. On the other hand, VT107, which was determined to be a pan-TEAD inhibitor by TEAD palmitoylation assays, significantly shifted the melting temperatures of all four TEAD family members (Fig. 4A). VT104 shifted the melting temperatures of all four TEAD family members, but higher shifts were observed for TEAD1 and TEAD3. VT106 only weakly shifted the melting temperatures of all four TEADs. [1]

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To determine whether our optimized compounds prevent YAP/TAZ–TEAD protein–protein interaction in the cell in TEAD-selective manner, we treated the NF2-mutant NCI-H2373 cells with VT103 or VT107 for 4 or 24 hours, immunoprecipitated the endogenous TEAD1 and TEAD4 protein using TEAD1-specific antibody and TEAD4-specific antibody (Fig. 5A and B), respectively, and probed the immunocomplexes with anti-YAP and anti-TAZ antibodies. Consistent with its selective binding to TEAD1 protein and selective inhibition of TEAD1 palmitoylation, VT103 reduced YAP interaction with TEAD1 but not TEAD4 after 4- and 24-hour treatment. The TAZ–TEAD1 interaction was disrupted with 4-hour treatment of VT103. In contrast, VT107 blocked YAP and TAZ interaction with both TEAD1 and TEAD4, with stronger effect at 24 than 4 hours (Fig. 5A and B). The less active enantiomer, VT106, failed to block the YAP/TAZ–TEAD4 interactions (Fig. 5B) and only weakly disrupted YAP/TAZ–TEAD1 interactions after 24-hour treatment (Fig. 5A). VT103 also selectively disrupted YAP–TEAD1 interaction in the NF2-deficient NCI-H226 cells after 4- and 24-hour treatment (Fig. 5C and D) [1].

VT107 (10 mg/kg; po) is an enantiomeric analog of VT104 [1].

Cell-free TEAD palmitoylation assay [1]
Purified recombinant TEAD1–YBD was first incubated with compounds and then with 2 μmol/L alkyne-palmitoyl-CoA. The reaction was quenched with 1% SDS followed by click chemistry reaction with biotin-azide as described previously. In some experiments, APCoA was added at different concentrations and in different sequence. Palmitoylated TEAD and total TEAD proteins were detected by streptavidin HRP and anti-TEAD1 antibody (Abcam) immunoblotting, respectively.

Western Blot Analysis[1]
Cell Types: HEK293T Cell
Tested Concentrations: 3 μmol/L
Incubation Duration: 20 hrs (hours)
Experimental Results: Inhibited palmitoylation of endogenous TEAD1 and TEAD3 proteins, and most effectively blocked palmitoylation of endogenous TEAD4 protein .

Animal/Disease Models: Mouse[1]
Doses: 10 mg/kg (pharmacokinetic/PK/PK analysis)
Route of Administration: Po
Experimental Results: Similar enantiomer to VT104.

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Mouse pharmacokinetics [1]
VT103, VT104, and VT107, formulated in 5% DMSO + 10% Solutol + 85% D5W, were dosed intravenously or orally at 7 or 10 mg/kg. Blood was drawn from the saphenous vein at indicated timepoints. Compounds were quantified by LC/MS-MS using a QTRAP 6500. Data were analyzed using Phoenix WinNonlin 6.3, and intravenously noncompartmental model 201, and orally noncompartmental model 200. The calculation method was linear/log trapezoidal.

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In vivo pharmacodynamic and efficacy studies [1]
All the procedures related to animal handling, care, and the treatment were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec or Crown Bioscience, Inc., following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The testing article formulated in dosing solution (5% DMSO + 10% solutol + 85% D5W; D5W = 5% glucose) was orally administrated daily at the indicated doses. Tumor volume and animal weights were monitored twice weekly.

VT103 is an analog of VT101, which has improved potency and good oral pharmacokinetics in mice (Fig. 2; Supplementary Table S1). VT104 is an analog of VT102, which has improved potency and good oral pharmacokinetics in mice (Fig. 2; Supplementary Table S1). VT105 is a more soluble analog of VT104 (Fig. 2), which was useful in TEAD X-ray crystallography experiments. VT106 and VT107 are enantiomers analogous to VT104; they have quite different potencies, making them useful mutual controls in biochemical and cellular experiments (Fig. 2).[1]

[1]. Small Molecule Inhibitors of TEAD Auto-palmitoylation Selectively Inhibit Proliferation and Tumor Growth of NF2-deficient Mesothelioma. Mol Cancer Ther. 2021; 20(6):986-998.

Mutations in the neurofibromatosis type 2 (NF2) gene that limit or abrogate expression of functional Merlin are common in malignant mesothelioma. Merlin activates the Hippo pathway to suppress nuclear translocation of YAP and TAZ, the major effectors of the pathway that associate with the TEAD transcription factors in the nucleus and promote expression of genes involved in cell proliferation and survival. In this article, we describe the discovery of compounds that selectively inhibit YAP/TAZ-TEAD promoted gene transcription, block TEAD auto-palmitoylation, and disrupt interaction between YAP/TAZ and TEAD. Optimization led to potent analogs with excellent oral bioavailability and pharmacokinetics that selectively inhibit NF2-deficient mesothelioma cell proliferation in vitro and growth of subcutaneous tumor xenografts in vivo These highly potent and selective TEAD inhibitors provide a way to target the Hippo-YAP pathway, which thus far has been undruggable and is dysregulated frequently in malignant mesothelioma and in other YAP-driven cancers and diseases. [1]

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Other than establishing the tolerability of our compounds in mice, the research described herein does not address any toxicity of the compounds, which could be related or unrelated to inhibition of TEAD palmitoylation. Formal toxicologic evaluation in multiple animal species will be required to characterize the safety of the small molecule compounds. If favorable, clinical evaluation of a TEAD palmitoylation inhibitor is warranted in NF2 mutant mesothelioma and cancers with activated YAP/TAZ-TEAD transcriptional activity as monotherapy or in combination with other targeted cancer therapies. [1]

C25H20F3N3O

435.44

435.155

C, 68.96; H, 4.63; F, 13.09; N, 9.65; O, 3.67

2417718-63-7

2417718-63-7 (S-isomer);2417718-64-8 (R-isomer);2417720-81-9 (Racemate);

153583246

White to off-white solid powder

5.5

2

6

4

32

634

1

C1(C(=O)N[C@@H](C)C2N=C(C=CC=2)N)=CC=C2C(C3C=CC(C(F)(F)F)=CC=3)=CC=CC2=C1

OBEXKSHQMHIUFP-HNNXBMFYSA-N

InChI=1S/C25H20F3N3O/c1-15(22-6-3-7-23(29)31-22)30-24(32)18-10-13-21-17(14-18)4-2-5-20(21)16-8-11-19(12-9-16)25(26,27)28/h2-15H,1H3,(H2,29,31)(H,30,32)/t15-/m0/s1

N-[(1S)-1-(6-aminopyridin-2-yl)ethyl]-5-[4-(trifluoromethyl)phenyl]naphthalene-2-carboxamide

VT107; 2417718-63-7; N-[(1S)-1-(6-amino-2-pyridinyl)ethyl]-5-[4-(trifluoromethyl)phenyl]-2-Naphthalenecarboxamide; VT-107; N-[(1S)-1-(6-aminopyridin-2-yl)ethyl]-5-[4-(trifluoromethyl)phenyl]naphthalene-2-carboxamide; CHEMBL5173358; OBEXKSHQMHIUFP-HNNXBMFYSA-N;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~229.65 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.74 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.74 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.74 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)