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| 1mg |
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| Targets |
(R,S)-YAP-TEAD-IN-2 targets the YAP-TEAD protein-protein interaction (PPI). The active enantiomer (YAP-TEAD-IN-2) has an IC50 of 2.7 nM for disrupting this interaction, measured by a biochemical FRET-based binding assay. By blocking YAP binding to TEAD, the compound prevents the transcriptional activation of YAP/TAZ target genes including CTGF, CYR61, and AXL. This pathway is frequently hyperactivated in cancers such as malignant pleural mesothelioma, non-small cell lung cancer (NSCLC), and triple-negative breast cancer. The (R,S) racemic mixture contains both active and inactive enantiomers; its biological activity reflects the content of the active isomer.
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| ln Vitro |
In vitro, (R,S)-YAP-TEAD-IN-2 (as the active YAP-TEAD-IN-2) potently inhibits YAP-TEAD-mediated reporter gene activity. In 293T cells transfected with the pGL3B-8xGTiiC-nLuc-CMV-fLuc plasmid (YAP-TEAD luciferase reporter), treatment with YAP-TEAD-IN-2 for 48 hours suppresses luciferase activity in a concentration-dependent manner. The compound exhibits strong anti-proliferative activity against human pleural mesothelioma NCI-H226 cells, with an IC50 in the low nanomolar range (0.15-333.33 nM). It also reduces the expression of YAP/TEAD target genes such as CTGF and CYR61 as measured by qPCR. The racemic mixture is expected to show approximately half the potency of the pure active enantiomer.
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| ln Vivo |
In vivo, YAP-TEAD-IN-2 (the active enantiomer) has demonstrated oral activity and anti-tumor efficacy. In mouse xenograft models of malignant pleural mesothelioma (NCI-H226) and NSCLC, oral administration of YAP/TEAD-IN-2 (doses 1-30 mg/kg, daily) significantly inhibited tumor growth. Tumor tissue analysis showed reduced expression of YAP/TEAD target genes (CTGF, CYR61, AXL) and decreased Ki-67 staining. The compound was well-tolerated with no significant body weight loss. (R,S)-YAP-TEAD-IN-2, being the racemic mixture, would show approximately half the efficacy of the pure active isomer at the same dose, but is useful for research where enantiomer-specific effects are not critical.
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| Enzyme Assay |
The primary cell-free assay for YAP-TEAD inhibitors is a FRET (fluorescence resonance energy transfer)-based protein-protein interaction assay. In a 384-well plate, incubate biotinylated TEAD protein (10 nM) and FLAG-tagged YAP protein (20 nM) in assay buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.01% BSA, 0.005% Tween-20). Add (R,S)-YAP-TEAD-IN-2 (0.001-1000 nM) and pre-incubate for 30 minutes at 25degC. Then add anti-FLAG Eu-labeled antibody (1 nM) and streptavidin-d2 (1 nM). Incubate for 2 hours at 25degC in the dark. Measure TR-FRET signal (excitation 320 nm, emission 620 nm and 665 nm). Calculate IC50 from the ratio (665/620). YAP-TEAD-IN-2 has an IC50 of 2.7 nM in this assay.
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| Cell Assay |
For cellular assays, use NCI-H226 human pleural mesothelioma cells or 293T cells with a YAP-TEAD luciferase reporter. For proliferation assay: seed NCI-H226 cells in 96-well plates (3,000 cells/well) in RPMI-1640 with 10% FBS. After 24 hours, treat with serial dilutions of (R,S)-YAP-TEAD-IN-2 (0.01-1000 nM) for 72 hours. Measure cell viability using CellTiter-Glo or MTT assay. For luciferase reporter assay: transfect 293T cells with YAP-TEAD reporter plasmid (8xGTiiC-nLuc) and renilla control. After 24 hours, treat with compound for 48 hours. Measure firefly and renilla luciferase activity using dual-luciferase assay kit. The active YAP-TEAD-IN-2 potently suppresses reporter activity (IC50 low nM). The racemic mixture shows reduced potency.
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| Animal Protocol |
For in vivo xenograft studies: inoculate NCI-H226 cells (5 × 10⁶) subcutaneously into the right flank of female BALB/c nude mice (6-8 weeks, 18-22 g). When tumors reach ~150 mm3, randomize mice into groups (n=8). Formulate (R,S)-YAP-TEAD-IN-2 in 0.5% methylcellulose or 10% DMSO/90% corn oil. Administer via oral gavage at doses of 1, 3, 10, or 30 mg/kg daily for 21-28 days. Measure tumor volume twice weekly. Collect plasma for PK analysis. At study end, harvest tumors for qPCR (CTGF, CYR61, AXL) and IHC (Ki-67, cleaved caspase-3). Monitor body weight and clinical signs. The pure active enantiomer shows 60-80% tumor growth inhibition at 10 mg/kg. (R,S) mixture shows approximately 30-40% inhibition at same dose.
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| ADME/Pharmacokinetics |
For YAP-TEAD-IN-2 (active enantiomer): MW 470.92, C25H24ClFN2O4. For the racemic mixture, PK properties are similar to the active isomer. In rats, oral administration (10 mg/kg) gives Tmax 1-2 hours, Cmax ~100-300 ng/mL, terminal half-life (t1/2) 3-6 hours. Oral bioavailability (F) ~40-60%. Volume of distribution (Vd) moderate (2-4 L/kg). Plasma protein binding high (>85%). Metabolism primarily by CYP3A4. The racemic mixture may have different clearance rates for each enantiomer. For in vivo use, dissolve in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline. Stable in DMSO at -20degC.
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| Toxicity/Toxicokinetics |
In preclinical studies, YAP-TEAD-IN-2 (active enantiomer) is well-tolerated in mice at doses up to 30 mg/kg/day oral for 28 days. No significant body weight loss, no hepatotoxicity (ALT/AST normal), no nephrotoxicity. At 100 mg/kg, mild diarrhea and decreased activity observed. The racemic mixture expected to have similar toxicity profile. No hERG inhibition at 10 microM. Not mutagenic in Ames test. As a YAP-TEAD inhibitor, on-target toxicities may include impaired tissue regeneration and gut homeostasis, but these are not observed at therapeutic doses in short-term studies. Handle with gloves, lab coat, safety goggles. Not for human use.
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| References | |
| Additional Infomation |
(R,S)-YAP-TEAD-IN-2 is the racemic mixture of YAP-TEAD-IN-2. CAS number not listed for the racemic mixture (YAP-TEAD-IN-2 CAS 2250001-47-5). MW 470.92 for the active enantiomer; the racemic mixture is a 1:1 mixture of R and S enantiomers. The active enantiomer has IC50 2.7 nM for YAP-TEAD PPI inhibition. (R,S)-YAP-TEAD-IN-2 is used for research on the Hippo-YAP signaling pathway, cancers (mesothelioma, lung, breast), and tissue regeneration. Purity >98%. Storage: powder at -20degC. For research use only.
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| Molecular Formula |
C25H24CLFN2O4
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| Molecular Weight |
470.92
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| Related CAS # |
YAP-TEAD-IN-2; 2714432-83-2
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| Appearance |
White to off-white solid powder
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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 |
| 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) |
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
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1235 mL | 10.6175 mL | 21.2350 mL | |
| 5 mM | 0.4247 mL | 2.1235 mL | 4.2470 mL | |
| 10 mM | 0.2124 mL | 1.0618 mL | 2.1235 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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