TEAD1 Palmitoylation

Since VT103 (HEK293T cells; 3 μM) does not prevent TEAD2, TEAD3, or TEAD4 from being palmitoylated, it seems to be TEAD1-selective. The YAP-TEAD1 connection is specifically disrupted by VT103 (NF2-deficient NCI-H226 cells; 3 mmol/L; 4 or 24 hours) [1]. Non-palmitoylated TEAD1 increases concurrently with the decrease of palmitoylated TEAD1 due to VT103 [1]. With an IC50 of 1.02 nM in the YAP reporter gene experiment, VT103 was demonstrated [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].

VT103 (0.3~10 mg/kg; administered orally once day) can decrease tumor growth even at a dose of 0.3 mg/kg [1]. Pharmacokinetics of VT103 in mice [1] Dose IV PO 7 mg/kg T1/2 (hour) Vdss (L/kg) CI (mL/min/kg) AUC 0-24 hours (μg*h/ mL) AUC 0- 24 hours (μg*h/mL) Oral availability (%) Cmax (ng/mL) C24 hours (ng/mL) 13.2 4.5 4.7 20.0 14.9 75 896 (1 hour) 340.

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VT103 and VT104 block growth of NF2-deficient mesothelioma xenografts [1]
With excellent oral bioavailability (≥75%) and long half-life (>12 hours) in mice (Fig. 2), VT103 and VT104 allowed us to evaluate target engagement and antitumor efficacy of TEAD auto-palmitoylation inhibition in human mesothelioma xenograft models in vivo. As shown in Fig. 6B, 4 hours after the third daily dose, VT103 significantly downregulated the expression of the Hippo pathway target genes, CTGF and CYR61, in the NF2-deficient NCI-H226 tumors in mice in a dose-dependent manner. Within the same NCI-H226-tumor bearing animals, VT103 also downregulated target gene expression in kidneys and livers in dose-dependent manner (Supplementary Fig. S6A). However, there did not seem to be any pathologic effect at the same time point as H&E images of kidneys and livers showed no difference between vehicle and drug-treated groups (Supplementary Fig. S6B). Bioanalysis showed that there was dose-dependent exposure of VT103 in circulation as well as in tumor tissues (Supplementary Fig. S6C). In addition, within the same animal, there appeared to be more compound accumulation in tumor than in circulation (Supplementary Fig. S6C). Examination of YAP and TEAD1 proteins in the VT103-treated NCI-H226 tumor tissues by IHC indicated no change in the cellular localization or levels of these proteins (Supplementary Fig. S6D).

VT103 significantly blocked NCI-H226 tumor growth in mice in dose-dependent manner (Fig. 6C). NCI-H226 tumor-bearing mice were randomized and daily oral administration of VT103 was initiated when tumors reached approximately 110 mm3. The last dose was administered on day 45. Plasma samples were collected on day 46. Bioanalysis showed the expected drug concentrations in circulation in dose-dependent manner (Supplementary Fig. S6E). VT103 exhibited strong antitumor efficacy, leading to tumor regression, when orally administered at 3 mg/kg once daily (TGI = 106.14%, P < 0.001, by day 46) in the NCI-H226 CDX model. Given at 1 and 0.3 mg/kg once daily, VT103 also significantly inhibited tumor growth (TGI = 83.79%, P < 0.001, and 47.95%, P = 0.001, respectively). No adverse effect on body weights were observed during such a long-term treatment with daily dosing of VT103 (Fig. 6C).

NCI-H2373 is a human mesothelioma cell line that harbors homozygous deletion of the NF2 gene. Although this cell line does not grow tumors well in mice (Supplementary Fig. S7 and described in detail below), we were able to perform a short-term drug treatment to assess VT103 target engagement and effect on pathway target gene expression in a second human mesothelioma model using this line. The parental NCI-H2373 cells were injected subcutaneously in mice, and when tumors reached approximately 300 mm3 in size, the mice were treated by oral administration of VT103 at 10 mg/kg once daily for 3 days. APEGS assay analysis of tumors harvested 4 hours after the third dose from these VT103-treated NCI-H2373 tumor bearing mice indicated that VT103 reduced the level of lipid-modified form of TEAD1 relative to total TEAD1 by 40.0±0.06% (Fig. 6D; Supplementary Fig. S8). In these NCI-H2373 tumors, 5-fold downregulation of the CTGF gene expression was observed (Fig. 6E; we did not check CYR61 expression in this model study).

Although measurable tumors were observed by day 7 after subcutaneous injection of NCI-H2373 cells in mice, these tumors grew very slowly for the following 4 weeks, with some regressing during this period (Supplementary Fig. S7). Thus, even though sufficient for short-term pharmacodynamic study, this was not a desirable model for drug efficacy study. To obtain a second working in vivo model for evaluating antitumor efficacy in long-term treatment, we serially passaged the NCI-H2373 tumors in mice and derived the NCI-H2373-Tu-P2 line, which was confirmed to be genetically related to NCI-H2373 by STR and sensitive to our TEAD inhibitors by in vitro cell proliferation assay. In this second human mesothelioma CDX model, NCI-H2373-Tu-P2, we also observed significant tumor regression when VT103 was orally administered once daily at 10, 3, and 1 mg/kg (TGI = 126.70%, 118.32%, 110.51%; P = 0.018, 0.022, and 0.030; Fig. 6F). Even at 0.3 mg/kg, VT103 blocked tumor growth in vivo, albeit not statistically significant (TGI value = 76.43%, P = 0.124). Animals in all four dose groups of VT103 had no body weight loss (Fig. 6F). Given the difficulty of growing NF2 mutant mesothelioma tumors in vivo, we were able to repeat the efficacy study twice before the model was eventually lost. The results indicated that VT103 is highly active in vivo at tolerated doses, and worthy of further evaluation.

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.

Cell-based TEAD palmitoylation assays [1]
Myc-TEAD expression plasmid transfected HEK293T cells were treated with DMSO or 100 μmol/L alkyne palmitate + DMSO/compound for 20 hours. Myc-TEAD protein was immunoprecipitated with anti-Myc antibody and subjected to click chemistry. Palmitoylated TEAD was detected by streptavidin immunoblotting. The Acyl-PEGyl Exchange Gel-Shift Assay was performed as described previously.

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Cell proliferation assay [1]
Cells treated for various time periods with compounds in dose titration starting from 3 μmol/L were assayed by CellTiter-Glo Luminescent Cell Viability Assay Kit according to the manufacturers' protocol. The IC50 and maximum inhibition % were calculated using dose response curves.

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Immunofluorescence [1]
After fixation with 4% paraformaldehyde for 10 to 15 minutes and permeabilization with 0.1% Triton X-100 in PBS, cells were blocked in 3% BSA in PBS for 1 to 2 hours at room temperature, stained with primary antibodies overnight at 4°C, and then with Alexa fluor-conjugated secondary antibodies for 2 to 3 hours at RT. Slides were mounted with prolong gold antifade reagent with DAPI. Images were captured with a Nikon Eclipse Ti confocal microscope.

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Immunoprecipitation [1]
Cells were washed with PBS and lysed [50 mmol/L Tris pH 7.5, 150 mmol/L NaCl, 1% Triton-X100, 50 mmol/L NaF, 1 mmol/L PMSF, protease inhibitor cocktail, phosphatase inhibitor]. After sonication and centrifugation, supernatant was collected and incubated with anti-TEAD, anti-YAP, or control antibodies, precipitated by Protein A/G beads, and analyzed by immunoblotting (see antibody information in Supplementary Table S3) using standard protocols.

Animal/Disease Models: NCI- H226 tumor-bearing mice [1]
Doses: 0.3~10 mg/kg
Route of Administration: Po one time/day
Experimental Results: Tumor growth can be prevented even at a dose of 0.3 mg/kg.

Mouse pharmacokinetics
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.

In vivo pharmacodynamic and efficacy studies
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]

C18H17F3N4O2S

410.413392782211

410.102

C, 52.68; H, 4.18; F, 13.89; N, 13.65; O, 7.80; S, 7.81

2290608-13-6

137534047

Off-white to gray solid powder

3.1

2

8

5

28

617

0

S(C1C=CC(=C(C2=CN(C)C=N2)C=1)NC1C=CC(C(F)(F)F)=CC=1)(NC)(=O)=O

LCVDQHLYHBAHR-UHFFFAOYSA-N

InChI=1S/C18H17F3N4O2S/c1-22-28(26,27)14-7-8-16(15(9-14)17-10-25(2)11-23-17)24-13-5-3-12(4-6-13)18(19,20)21/h3-11,22,24H,1-2H3

N-methyl-3-(1-methyl-1H-imidazol-4-yl)-4-((4-(trifluoromethyl)phenyl)amino)benzenesulfonamide

VT-103; VT 103; 2290608-13-6; N-Methyl-3-(1-methyl-1H-imidazol-4-yl)-4-((4-(trifluoromethyl)phenyl)amino)benzenesulfonamide; CHEMBL5198469; SCHEMBL20779435; LLCVDQHLYHBAHR-UHFFFAOYSA-N; QRD60813; VT103

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 : ~50 mg/mL (~121.83 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.09 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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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.1 mM) in 10% DMSO + 90% Corn oil, clear solution
For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 25 mg/mL of DMSO stock solution and add to 900 μL of corn oil, mix well (clear solution).

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