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| Targets |
MYF-03-69 targets all four members of the TEAD transcription factor family, with IC₅₀ values of 385 nM for TEAD1, 143 nM for TEAD2, 558 nM for TEAD3, and 173 nM for TEAD4. Additionally, MYF-03-69 inhibits TEAD transcriptional activity with an IC₅₀ of 56 nM. The compound irreversibly modifies TEAD proteins by covalently binding to the conserved cysteine in the TEAD palmitate-binding pocket, thereby disrupting YAP-TEAD interaction.
TEAD family proteins (TEAD1, TEAD3, TEAD4). The compound acts as a pan-TEAD inhibitor by covalently binding to a conserved cysteine residue (Cys359 in TEAD1) within the central lipid-binding pocket of the TEAD protein, which is critical for its folding, stability, and interaction with co-activators YAP/TAZ. This covalent binding irreversibly disrupts the TEAD-YAP/TAZ complex and downstream gene expression. |
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| ln Vitro |
MYF-03-69 selectively inhibits the proliferation of Hippo signaling-defective mesothelioma cells (e.g., NCI-H226, MSTO-211H) while showing little cytotoxicity to normal mesothelial cells or Hippo signaling-intact cancer cells. At concentrations of 0.1-10 μM for 48 hours, MYF-03-69 induces G1 phase cell cycle arrest in NCI-H226 and MSTO-211H cells. At 2 μM, MYF-03-69 inhibits TEAD protein palmitoylation, downregulates YAP-TEAD target genes (such as CTGF, CYR61, and ANKRD1), and upregulates the pro-apoptotic gene BMF. Incubation of recombinant TEAD2 YBD protein with a 10-fold molar excess of MYF-03-69 at room temperature for 1 hour achieves 100% labeling efficiency. PRISM screening across 903 cancer cell lines reveals a high correlation between TEAD-YAP dependency and sensitivity to MYF-03-69.
In biochemical assays, MYF-03-176 inhibited TEAD transcriptional activity with an IC50 of 11 nM in a reporter assay. It directly inhibited TEAD1, TEAD3, and TEAD4 with IC50 values of 47, 32, and 71 nM, respectively. In Hippo signaling-deficient NCI-H226 malignant pleural mesothelioma cells, the compound treatment at 20-500 nM for 24h downregulated YAP target genes (CTGF, CYR61, ANKRD1) and inhibited cell growth with an IC50 of 9 nM. |
| ln Vivo |
Further optimization of MYF-03-69 led to MYF-03-176, an orally bioavailable compound that demonstrates strong antitumor efficacy in a malignant pleural mesothelioma mouse xenograft model via oral administration. In cell viability screens, MYF-03-69 shows selective antiproliferative effects against Hippo signaling-defective mesothelioma cells (such as NCI-H226 and MSTO-211H), with less effect on Hippo signaling-intact mesothelioma cells (NCI-H2452) and non-cancerous mesothelial cells (MeT-5A).
In a human mesothelioma NCI-H226 cell-derived xenograft (CDX) mouse model, MYF-03-176 administered orally at doses of 30-75 mg/kg twice daily for 28 consecutive days exhibited potent antitumor activity, significantly reducing tumor volume. In the SW1753 xenograft model, combination oral treatment with 100 mg/kg twice daily plus AMG510 (a KRAS inhibitor) effectively reduced tumor volume without affecting body weight. |
| Enzyme Assay |
TEAD Palmitoylation Inhibition Assay: 1 μM TEADs-YBD recombinant protein is incubated with indicated concentrations of MYF-03-69 at 37°C for 2 hours, followed by the addition of palmitoyl alkyne-coenzyme A in a total volume of 50 μL. After a 30-minute reaction, 5 μL of 10% SDS and 5 μL of click reaction reagents are added to initiate the click reaction for 1 hour. After adding 4× loading buffer, samples are subjected to Western blot analysis using IRDye 800CW Streptavidin for biotin detection and anti-His-tag antibody.
Mass Spectrometry Labeling Assay: Recombinant TEAD2 YBD protein is incubated with a 10-fold molar excess of MYF-03-69 at room temperature for 1 hour, followed by mass spectrometry analysis to detect labeling efficiency. Results show 100% labeling after 1 hour incubation.
Co-crystallization Assay: 300 μM TEAD1 pre-incubated with 600 μM MYF-03-69 for 1 hour is dispensed in crystallization buffer (3 M (NH₄)₂SO₄ and 0.1 M Tris pH 9.0) and incubated against 25 μL of crystallization buffer in a 384-well hanging-drop vapor diffusion microtiter plate at 20°C for 3 days to obtain crystals.
Molecular Docking: Docking of MYF-03-69 is performed with the covalent docking protocol from Schrödinger suite software (release 2019-02) with default parameters in the TEAD2 structure (PDB code:).
Cellular Thermal Shift Assay (CETSA) : CETSA is used to detect MYF-03-69 binding to TEAD proteins in cells.
A standard biochemical assay for TEAD inhibitors is a fluorescence polarization (FP) binding assay using a fluorescently labeled palmitate probe (e.g., BODIPY-C16) and recombinant TEAD protein. The test compound is serially diluted and incubated with the TEAD protein and probe. The degree of polarization is measured to calculate the binding affinity and confirm that the inhibitor displaces the probe from the palmitoylation pocket. |
| Cell Assay |
Cell Proliferation Inhibition Assay: NCI-H226, MSTO-211H, NCI-H2452 mesothelioma cells, and MeT-5A mesothelial cells are seeded in culture plates and treated with 0-10 μM MYF-03-69 for 5 days. Cell viability is assessed by CellTiter-Glo or MTT assays to calculate GR₅₀ values.
Cell Cycle Analysis: NCI-H226 and MSTO-211H cells are treated with 0.1-10 μM MYF-03-69 for 48 hours, and cell cycle analysis is performed by flow cytometry to detect G1 phase cell cycle arrest.
Western Blot Analysis: Cells are treated with 2 μM MYF-03-69 for indicated times and lysed. Protein expression levels of TEAD target genes (CTGF, CYR61, ANKRD1) and the pro-apoptotic gene BMF are detected by Western blot.
TEAD Transcriptional Activity Reporter Assay: A stable cell line is established using a TEAD luciferase reporter lentivirus to monitor TEAD transcriptional activity. Cells are treated with MYF-03-69, and TEAD transcriptional inhibition is assessed by luciferase activity detection (IC₅₀ = 56 nM).
PRISM High-Throughput Screening: PRISM screening is performed on 903 barcoded cancer cell lines. Log2(AUC) values are calculated to assess sensitivity to MYF-03-69, and correlation analysis is conducted with CRISPR knockout scores from the DepMap database.
To assess cellular activity, a luciferase reporter gene assay is commonly used. Cells (e.g., NCI-H226) are transfected with a plasmid containing a TEAD-responsive element (e.g., from the CTGF promoter) driving firefly luciferase expression. After transfection, cells are treated with varying concentrations of MYF-03-176 for 24-48 hours. TEAD transcriptional activity is quantified by measuring luminescence and normalized to a constitutive Renilla luciferase control. |
| Animal Protocol |
MPM Xenograft Mouse Model: A CDX mouse model is established using NCI-H226 human malignant pleural mesothelioma cells. The optimized MYF-03-69 analog MYF-03-176 is administered orally to evaluate antitumor efficacy.
The in vivo efficacy of MYF-03-176 is typically tested in a xenograft model. Nude mice are subcutaneously implanted with tumor cells (e.g., NCI-H226 or SW1753) and allowed to reach a certain size. Mice are then randomized into groups and treated with vehicle control or the test compound by oral gavage once or twice daily for several weeks. Tumor volumes and body weights are measured regularly to assess antitumor efficacy and tolerability. |
| ADME/Pharmacokinetics |
MYF-03-69 has a molecular weight of 443.42 and the molecular formula C₂₂H₂₀F₃N₅O₂. It is soluble in DMSO at 80 mg/mL (180.42 mM). The optimized analog MYF-03-176 has good oral bioavailability.
MYF-03-176 is an orally active compound with good bioavailability. In the described animal studies, the compound was administered at various doses (30-100 mg/kg) via oral gavage, indicating suitability for oral administration. Its metabolism and excretion properties have not been detailed in the literature reviewed, but as a covalent inhibitor, it is designed to have a long-lasting pharmacodynamic effect due to irreversible target binding. The HCl salt form enhances its solubility and stability. |
| Toxicity/Toxicokinetics |
Limited toxicity data is available, but in the animal studies described, treatment with MYF-03-176 was well-tolerated. In the SW1753 xenograft model, the combination treatment with MYF-03-176 did not affect the body weight of the nude mice, suggesting no overt acute toxicity at the therapeutic doses. Long-term and off-target toxicity profiles have not been reported, as the compound is primarily a research tool. Extended toxicology studies would be required for clinical development.
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| References |
[1]. Covalent Disruptor of YAP-TEAD Association Suppresses Defective Hippo Signaling. BioRxiv. doi: https://doi.org/10.1101/2022.05.10.491316. Now published in eLife doi: 10.7554/eLife.78810
[2]. Structure-Based Design of Y-Shaped Covalent TEAD Inhibitors. J Med Chem. 2023 Apr 13;66(7):4617-4632. [3]. YAP/TAZ mediates resistance to KRAS inhibitors through inhibiting proapoptosis and activating the SLC7A5/mTOR axis. JCI Insight. 2024 Dec 20;9(24):e178535. |
| Additional Infomation |
MYF-03-176 is a research tool compound derived from a medicinal chemistry campaign to develop selective, cysteine-covalent TEAD inhibitors. It was identified through structure-based design and is also known as compound 22. The compound is a potent inhibitor that significantly suppresses TEAD-regulated gene expression and proliferation of TEAD-dependent cancer cell lines, including those derived from mesothelioma and liposarcoma. It is not yet approved for clinical use and is solely intended for preclinical research.
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| Molecular Formula |
C19H19F4N4O2CL
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|---|---|
| Molecular Weight |
446.83
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| CAS # |
2857937-59-6
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| Appearance |
White to off-white solid powder
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| Synonyms |
MYF-03-176 HCl; MYF03-176 HCl
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: 100 mg/mL (243.68 mM)
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| Solubility (In Vivo) |
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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2380 mL | 11.1899 mL | 22.3799 mL | |
| 5 mM | 0.4476 mL | 2.2380 mL | 4.4760 mL | |
| 10 mM | 0.2238 mL | 1.1190 mL | 2.2380 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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