- TEAD1, TEAD2, TEAD3, TEAD4 (pan-TEAD inhibitor)[2] - IC50 values in TR-FRET biochemical assays for TEAD1-4 are: 15 ± 5 nM, 17 ± 1 nM, 26 ± 12 nM, 21 ± 15 nM, respectively[2] - Cellular TEAD transcriptional activity (NCI-H226 reporter assay): EC50 of 17 ± 5 nM[2]
TEAD1, TEAD3, TEAD4. MYF-03-176 acts by covalently binding to the palmitate-binding pocket of TEAD proteins, thereby blocking the interaction between YAP and TEAD and inhibiting downstream Hippo signaling pathway-mediated gene expression.

- Anti-proliferative Activity: MYF-03-176 shows potent anti-proliferative effects in various TEAD-dependent cancer cell lines. The IC50 is 24 nM in NCI-H226 (NF2-deficient mesothelioma) cells after a 5-day proliferation assay[2]. In another study, the IC50 is 16 nM in NCI-H226 cells[1]. It also effectively inhibits the proliferation of liposarcoma cells (e.g., 94T778) and cholangiocarcinoma cells (e.g., HuCCT1)[1]. - Inhibition of TEAD Transcriptional Activity: In a TEAD luciferase reporter assay using NCI-H226 cells, MYF-03-176 inhibits YAP-TEAD transcriptional activity with an IC50 of 11 nM after 72 hours of treatment. This is 3-fold more potent than the parent compound MYF-03-69 (IC50=45 nM)[1]. - Regulation of Downstream Gene Expression: MYF-03-176 significantly downregulates the expression of YAP/TEAD target genes (e.g., CTGF, CYR61, ANKRD1) and upregulates the pro-apoptotic gene BMF[1]. - Overcoming KRAS Inhibitor Resistance: In multiple resistant KRAS G12C-mutant (e.g., H2030, SW1573, KYSE410) and KRAS G12D-mutant (e.g., HPAFII, PANC1, SW1990) cancer cells, combining MYF-03-176 with KRAS inhibitors (e.g., AMG510, MRTX1133) shows a synergistic effect, significantly enhancing the anti-proliferative activity of the KRAS inhibitors. Mechanistically, it reverses resistance by inhibiting the YAP/TAZ/TEAD axis, leading to upregulation of pro-apoptotic proteins (BCL2L11, PUMA) and downregulation of SLC7A5 expression[3].

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MYF-03-176 inhibits TEAD transcriptional activity in NCI-H226 cells with an IC50 of 11 nM, and inhibits cell growth with an IC50 of 9 nM over 5 days. It significantly downregulates YAP target genes including CTGF, CYR61, and ANKRD1, while upregulating BMF expression. In combination with KRAS G12C and G12D inhibitors, it exhibits synergistic effects across multiple cell lines.

- Anti-tumor Efficacy: In an NCI-H226 (human mesothelioma) cell line-derived xenograft (CDX) mouse model, MYF-03-176 demonstrates significant anti-tumor activity. Tumor-bearing mice were orally administered the compound twice daily for 28 days. At a dose of 30 mg/kg, an average tumor regression of 54% was observed; at 75 mg/kg, an average tumor regression of 68% was observed. The anti-tumor activity between the two doses was comparable[1]. - Combination Therapy: In SW1573 (KRAS G12C-mutant) and H358R (acquired resistant KRAS G12C-mutant) xenograft models, the combination of MYF-03-176 with AMG510 more effectively inhibits tumor growth compared to monotherapy groups[3].

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In a human mesothelioma NCI-H226 cell-derived xenograft (CDX) model, oral administration of MYF-03-176 at 30-75 mg/kg twice daily for 28 days exhibits potent antitumor activity. In an SW1753 xenograft BALB/c nude mouse model, oral administration at 100 mg/kg twice daily for 21 days in combination with AMG510 effectively reduces tumor volume without affecting body weight.

- TR-FRET Assay: A TR-FRET biochemical assay was established using a FAM-labeled tracer, WZJ-10 (based on the reversible TEAD inhibitor VT-107). 100 nM of His-tagged TEAD recombinant YBD protein was pre-incubated with serial dilutions of MYF-03-176 in assay buffer (50 mM HEPES pH 7.5, 200 mM NaCl, 0.1% Pluronic F-68) for 5 hours. Subsequently, 800 nM of WZJ-10 tracer and 50 ng/mL of MAb Anti-6HIS Tb cryptate Gold were added. The TR-FRET signal (490/520 nm) was measured using a PHERAstar FSX plate reader to determine IC50 values[2]. - Intact Mass Spectrometry: TEAD1 protein was incubated with a 10-fold molar excess of MYF-03-176 for 1 hour at room temperature. The reaction was then analyzed by LC-MS to confirm covalent adduct formation. The mass spectra showed a mass shift consistent with the covalent binding of MYF-03-176 to the TEAD1 protein[2].

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Not explicitly detailed in reference sources. A typical assay for TEAD inhibitors involves incubating the test compound with recombinant TEAD proteins in a buffer system, adding a fluorescent-labeled YAP-derived peptide, and measuring the binding interaction using fluorescence polarization or TR-FRET techniques after a fixed incubation period at room temperature.

- Cell Proliferation Assay: NCI-H226 cells were seeded in 384-well white plates at a density of 100 cells per well in a total volume of 50 µL. Cells were treated with MYF-03-176 using an HP D300 digital dispenser for 5 days. On day 5, 12.5 µL of CellTiter-Glo reagent was added per well and incubated for 20 minutes. Luminescence was read using a PHERAstar FSX plate reader, and data were normalized to DMSO-treated samples[2]. - TEAD Reporter Gene Assay: NCI-H226 cells were transduced with a TEAD luciferase reporter lentivirus and selected with puromycin. Selected cells were seeded in 384-well white plates at 1000 cells per well in 40 µL. Cells were treated with MYF-03-176 using an HP D300 digital dispenser for 3 days. Luciferase activity was measured using One-Glo luciferase reagent, and cell viability was measured using CellTiter-Glo reagent. TEAD transcriptional activity was calculated by normalizing the luciferase signal to cell viability[2]. - Immunoblotting: After treatment with MYF-03-176, cell lysates were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with specific primary antibodies (e.g., anti-BCL2L11, anti-PUMA, anti-SLC7A5) overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using a chemiluminescent substrate[3]. - RT-qPCR: Total RNA was extracted from cells treated with MYF-03-176 using an RNA extraction kit and reverse-transcribed into cDNA. RT-qPCR was performed using SYBR Green master mix and specific primers (e.g., for CTGF, CYR61, BMF, SLC7A5) on a real-time PCR system. ACTB was used as an internal control, and relative expression levels were calculated using the ΔΔCT method[3]. - Clonogenic Assay: Cells were seeded in 6-well plates and treated with MYF-03-176 and/or KRAS inhibitors (e.g., AMG510, MRTX1133). After treatment, colonies were fixed, stained, and counted[3].

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Not explicitly detailed in reference sources. A standard cell-based assay for TEAD transcriptional activity uses NCI-H226 cells transfected with a TEAD-responsive luciferase reporter construct. Cells are treated with varying concentrations of MYF-03-176 for 72 hours, and luciferase activity is measured to determine the IC50 for transcriptional inhibition.

- In Vivo Efficacy Study (CDX model): 5×10^6 NCI-H226 cells mixed with 50% Matrigel were implanted subcutaneously into the right flank of female NSG mice. When tumors reached an average size of ~141.3 mm³, mice were randomized into treatment groups (n=8-9 per group). MYF-03-176 was formulated as a suspension in 10% DMSO, 10% Tween 80 in water. The compound was administered orally twice daily for 28 days. Control mice received vehicle alone. Tumor volumes (calculated as length × width^2 / 2) and body weights were measured twice weekly[1]. - In Vivo Combination Study: For SW1573 and H358R xenograft models, when tumors reached approximately 100-150 mm³, mice were randomized into groups. AMG510 and MYF-03-176 were administered orally, alone or in combination. Tumor volumes and body weights were monitored to assess efficacy and tolerability[3].

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Female BALB/c nude mice bearing NCI-H226 subcutaneous xenografts are orally administered MYF-03-176 at doses of 30, 45, or 75 mg/kg twice daily for 28 consecutive days. Tumor volumes and body weights are measured twice weekly. For combination studies, mice are orally dosed with 100 mg/kg twice daily for 21 days.

- Oral Bioavailability and Clearance: Compared to the earlier compounds MYF-01-37 and MYF-03-69, MYF-03-176 exhibits decent pharmacokinetic properties, including low clearance and high oral bioavailability[1]. - Metabolic Stability: Liver microsome stability was evaluated[1].

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MYF-03-176 has a molecular weight of 410.37. It is soluble in DMSO (≥100 mg/mL). For oral administration in animal studies, MYF-03-176 is formulated in 10% DMSO + 90% corn oil at a concentration of ≥2.5 mg/mL. It is an orally bioavailable compound.

- Body Weight Effect: In the NCI-H226 CDX in vivo efficacy study, the 30 mg/kg BID oral dose of MYF-03-176 was well tolerated, with average body weight gain comparable to the vehicle control. At the 75 mg/kg BID dose, an average weight loss of 5% was observed, and 3 out of 8 animals showed 12-14% body weight loss. The weight loss recovered once drug administration was stopped[1].

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Toxicity data for MYF-03-176 are not explicitly detailed in reference sources. In the NCI-H226 xenograft mouse model, oral administration of MYF-03-176 (30-75 mg/kg, twice daily) is well tolerated without significant body weight loss, suggesting a favorable safety profile at these doses.

[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.

- Mechanism of Action: MYF-03-176 is a Y-shaped, cysteine-targeting covalent TEAD inhibitor. Its co-crystal structure (PDB ID: 7LI5) shows that the acrylamide warhead forms a covalent bond with Cys359 of TEAD1, and the molecule adopts a Y-shape, fitting into the palmitate-binding pocket and interacting with surrounding hydrophilic and hydrophobic residues. By covalently binding to the conserved palmitoylation site cysteine on TEAD, it blocks the YAP/TAZ-TEAD interaction, thereby inhibiting downstream transcriptional activity of the Hippo pathway[1][2]. - Indications and Resistance: MYF-03-176 holds therapeutic potential for various YAP/TEAD-dependent cancers, including mesothelioma, liposarcoma, liver cancer, and lung cancer. Importantly, studies have established that targeting the YAP/TAZ-TEAD axis is an effective strategy to overcome both acquired and intrinsic resistance to KRAS inhibitors (e.g., sotorasib, adagrasib, MRTX1133), and MYF-03-176 serves as a potent tool compound for this strategy[3].

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MYF-03-176 is a research compound not yet approved for clinical use. It is specifically developed for TEAD-dependent cancers, particularly malignant pleural mesothelioma (MPM) with defective Hippo signaling. The compound has demonstrated synergistic antitumor activity when combined with KRAS G12C/G12D inhibitors, supporting its potential in combination therapy strategies.

C19H18F4N4O2

410.365437984467

410.1366

C, 55.61; H, 4.42; F, 18.52; N, 13.65; O, 7.80

2857937-59-6

164517031

White to off-white solid powder

3.1

1

9

6

29

575

2

FC(=C)C(N1C[C@H]([C@@H](C1)NC1N=CC=CN=1)OCC1C=CC(C(F)(F)F)=CC=1)=O

KVAWNRUJSYWYJX-HZPDHXFCSA-N

InChI=1S/C19H18F4N4O2/c1-12(20)17(28)27-9-15(26-18-24-7-2-8-25-18)16(10-27)29-11-13-3-5-14(6-4-13)19(21,22)23/h2-8,15-16H,1,9-11H2,(H,24,25,26)/t15-,16-/m1/s1

2-fluoro-1-[(3R,4R)-3-(pyrimidin-2-ylamino)-4-[[4-(trifluoromethyl)phenyl]methoxy]pyrrolidin-1-yl]prop-2-en-1-one

MYF-03-176; MYF03-176; 2857937-59-6; MYF-03 176; CHEMBL5433922; 2-Fluoro-1-((3R,4R)-3-(pyrimidin-2-ylamino)-4-((4-(trifluoromethyl)benzyl)oxy)pyrrolidin-1-yl)prop-2-en-1-one; 2-Fluoro-1-[(3R,4R)-3-(pyrimidin-2-ylamino)-4-[[4-(trifluoromethyl)benzyl]oxy]pyrrolidin-1-yl]prop-2-en-1-one;

2934.99.9001

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.

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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