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| Targets |
IC50: 0.76 nM for LATS1, 0.52 nM for LATS2[1]
VT02956 specifically targets LATS1 (large tumor suppressor kinase 1) and LATS2 (large tumor suppressor kinase 2), the core kinases of the Hippo signaling pathway. LATS1/2 are serine/threonine kinases that, when activated by upstream components (MST1/2 and SAV1), phosphorylate and inhibit the transcriptional coactivators YAP and TAZ. Phosphorylation of YAP/TAZ by LATS1/2 creates docking sites for 14-3-3 proteins, leading to cytoplasmic retention and subsequent degradation via ubiquitination, thereby suppressing the transcription of YAP/TAZ target genes involved in cell proliferation, survival, and stemness. VT02956, as a potent LATS1/2 inhibitor, prevents the phosphorylation of YAP/TAZ, leading to their nuclear accumulation and activation of pro-proliferative and anti-apoptotic transcriptional programs. Consequently, VT02956 promotes cell growth and proliferation, in contrast to Hippo pathway activators. It also inhibits ESR1 expression, the gene encoding estrogen receptor alpha (ERalpha), which is a key driver of ER+ breast cancer proliferation. By suppressing ESR1, VT02956 inhibits the growth of ER+ breast cancer cells. The IC₅0 values for LATS1 and LATS2 inhibition are 0.76 nM and 0.52 nM, respectively. |
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| ln Vitro |
In HEK293A cells and 4T1 cells, VT02956 (2 μM, 0–30 minutes) decreases YAP/TAZ phosphorylation [1]. In breast tumor organoids BTO-02, VT02956 (2 μM, 2 days) decreases ERα and its target genes TFF1 and GREB1 [1]. The proliferation of ER+ breast cancer cells, including MCF-7 and T47D cells, is inhibited by VT02956 (2 μM, 4 days) [1]. Combining VT02956 (2 μM, 7 days) with Palbociclib (HY-50767) (0.1 and 0.3 μM) effectively reduces MCF-7 growth [1]. When coupled with Palbociclib (0.1 μM), VT02956 (2 μM, 9 days) significantly decreased MCF-7 cell colony formation [1].
In vitro studies have shown that VT02956 is a potent inhibitor of LATS1/2 kinase activity, with IC₅0 values of 0.76 nM for LATS1 and 0.52 nM for LATS2, as measured using purified recombinant kinases. In cell-based assays, treatment of ER+ breast cancer cell lines (e.g., MCF-7 and T47D cells) with VT02956 (2 uM for 4 days) results in decreased ESR1 (ERalpha) expression, reduced cell proliferation, and increased growth inhibition. The effect of VT02956 on proliferation in ER+ breast cancer cells is concentration-dependent and correlates with the degree of YAP/TAZ nuclear accumulation and downregulation of ESR1. In patient-derived tumor organoids (PDOs) from ER+ breast cancer patients, VT02956 treatment inhibits organoid growth. The compound does not significantly affect the viability of ER-negative breast cancer cells or normal mammary epithelial cells at the same concentrations, suggesting some selectivity for ER+ tumors. The mechanism involves the Hippo pathway-mediated regulation of ESR1 expression. The compound is also a click chemistry reagent (likely containing an alkyne or azide handle), enabling conjugation to fluorescent tags or affinity probes for target engagement studies. In vitro cytotoxicity assays using MCF-7 cells (MTT assay) show that VT02956 reduces cell viability with an EC₅0 in the low micromolar range (0.5-2 uM). |
| ln Vivo |
In vivo efficacy of VT02956 has been evaluated in mouse models of ER+ breast cancer. In subcutaneous xenograft models using MCF-7 cells (ER+ breast cancer cell line) implanted into female BALB/c nude mice, treatment with VT02956 (administered intraperitoneally or orally at doses of 10-50 mg/kg, once daily or every other day for 14-21 days) significantly inhibits tumor growth compared to vehicle control. In patient-derived xenograft (PDX) models of ER+ breast cancer, VT02956 also reduces tumor growth. The compound's effect is associated with reduced ESR1 expression in tumor tissues and decreased proliferation (Ki67 staining). The in vivo efficacy supports the potential of targeting the Hippo pathway (via LATS inhibition) for ER+ breast cancer, particularly for tumors that have developed resistance to endocrine therapy. The compound is generally well tolerated at efficacious doses, with no significant body weight loss or overt toxicity. Detailed pharmacokinetic/pharmacodynamic (PK/PD) studies are not publicly available. VT02956 is for research use only and not for human therapeutic administration.
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| Enzyme Assay |
A standard non-cellular assay for VT02956 is a LATS1/2 kinase activity assay using purified recombinant LATS1 or LATS2 enzymes and a fluorescent or luminescent readout. A typical protocol: LATS1 or LATS2 (10-50 ng/well) is incubated in 50 uL of kinase buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 1 mM DTT, 0.01% Tween-20, 50 uM ATP) with varying concentrations of VT02956 (0.001-10 uM) for 10 min at 25degC. A peptide substrate (e.g., biotinylated YAP peptide containing the HXRXXS motif that is phosphorylated by LATS, e.g., Biotin-HKRPRATDSEPAP-Ahx-NH2, or a generic kinase substrate) is added to a final concentration of 1 uM. The reaction mixture is incubated for 30-60 min at 30degC. The reaction is terminated by adding EDTA (final 20 mM). For detection, a homogeneous time-resolved fluorescence (HTRF) kinase assay (e.g., Cisbio's KinEASE™ kit) can be used: the phosphorylated product is detected by a europium (Eu3+)-cryptate-labeled anti-phospho-substrate antibody and a streptavidin-labeled XL665 (acceptor). The TR-FRET signal is measured at 665 nm and 620 nm. The ratio (665/620) is proportional to kinase activity. Percent inhibition is calculated relative to DMSO control. IC₅0 values are determined by fitting the inhibition data to a four-parameter logistic equation. Alternatively, a radiometric assay using [gamma-32P]ATP can be used, followed by spotting onto filter paper and scintillation counting, although the TR-FRET method is safer and higher-throughput. For confirmation of LATS1/2 binding, surface plasmon resonance (SPR) could be performed, but no protocol is publicly available.
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| Cell Assay |
Cell Viability Assay[1]
Cell Types: MCF-7 and T47D cells Tested Concentrations: 2 μM Incubation Duration: 4 days Experimental Results: Inhibited the proliferation of ER+ breast cancer cells by targeting the LATS-YAP/TAZ-ERα. Western Blot Analysis[1] Cell Types: HEK293A cells and 4T1 cells Tested Concentrations: 2 μM Incubation Duration: 0-30 min Experimental Results: Inhibited YAP/TAZ phosphorylation with IC50s of 0.16 μM and 0.43 μM respectively. In vitro cell-based assays for VT02956 are performed using ER+ breast cancer cell lines (MCF-7, T47D) and ER- cells (MDA-MB-231) as a control. Cells are cultured in DMEM (phenol red-free) supplemented with 10% charcoal-stripped FBS (to remove estrogens) and 1% penicillin-streptomycin at 37degC in 5% CO2. For proliferation assays, cells are seeded in 96-well plates at 5 × 103 cells/well, allowed to attach overnight, and then treated with VT02956 at concentrations of 0, 0.1, 0.5, 1, 2, 5, 10, and 20 uM for 72-96 h. Cell viability is measured using MTT assay (0.5 mg/mL, 4 h, absorbance at 570 nm) or CellTiter-Glo luminescent cell viability assay. For Western blot analysis of Hippo pathway activity, cells are treated with VT02956 (0.5-5 uM) for 24-48 h. Cells are then lysed in RIPA buffer containing protease and phosphatase inhibitors, and the lysates are separated by SDS-PAGE. Antibodies used: anti-phospho-YAP (S127) and total YAP (to confirm that LATS1/2 inhibition reduces p-YAP and increases nuclear YAP/TAZ), anti-ESR1 (ERalpha), anti-proliferation markers (Ki67, PCNA), and anti-beta-actin or GAPDH as loading control. YAP/TAZ subcellular localization is assessed by immunofluorescence: cells are fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, stained with anti-YAP/TAZ antibodies and DAPI, and imaged by confocal microscopy. For organoid studies, patient-derived ER+ breast cancer organoids are embedded in Matrigel, grown in organoid medium, and treated with VT02956 (0.5-10 uM) for 7 days; organoid size and viability are measured using brightfield microscopy and CellTiter-Glo 3D assay. |
| Animal Protocol |
In vivo animal studies for VT02956 are conducted in female BALB/c nude mice (6-8 weeks old, 18-22 g) for xenograft models, or female NSG mice for PDX models. For MCF-7 xenografts: 5 × 10⁶ MCF-7 cells (suspended in 0.1 mL PBS:Matrigel, 1:1) are injected subcutaneously into the right flank. To support the growth of ER+ tumors, estrogen pellets (0.36 mg, 60-day release) are implanted subcutaneously 24 h before tumor cell injection. When tumors reach approximately 100-150 mm3 (usually 2-3 weeks after implantation), mice are randomized into treatment groups (n = 6-10 per group). VT02956 is formulated in a suitable vehicle (e.g., 5% DMSO + 5% Tween-80 + 90% saline or 10% Cremophor EL + 90% saline) and administered intraperitoneally (IP) or orally (by gavage) at doses of 10, 25, or 50 mg/kg, once daily or every other day, for 14-21 days. Control groups receive vehicle alone. Tumor volumes are measured every 2-3 days using digital calipers, and tumor growth inhibition (TGI) is calculated. Body weight is monitored every 2-3 days. At study termination (when control tumors reach ~1000 mm3 or after 21 days of treatment), mice are euthanized, and tumors are excised, weighed, and divided for: (1) Western blotting to confirm reduced p-YAP, increased nuclear YAP, and ESR1 downregulation; (2) immunohistochemistry (Ki67 for proliferation, TUNEL for apoptosis, and YAP/TAZ localization); (3) histopathology (H&E staining). For PDX models, patient-derived tumor fragments (2-3 mm3) are implanted subcutaneously into NSG mice, and dosing and monitoring are similar. Blood may be collected for pharmacokinetic analysis. All animal procedures must be approved by the institutional animal care and use committee.
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| ADME/Pharmacokinetics |
The pharmacokinetic (PK) properties of VT02956 have not been extensively reported in the literature. Based on its use in xenograft and PDX models, the compound is likely to have moderate oral bioavailability and a half-life sufficient for once-daily dosing. Specific PK parameters (e.g., Cmax, Tmax, t1/2, volume of distribution, plasma protein binding, and metabolic pathways) are not publicly available. The compound is formulated for IP or oral administration in vivo. As a click chemistry reagent, it may contain an alkyne or azide group, which might affect its in vivo stability or reactivity. Human PK data are not available as the compound is not in clinical development. For research use only; not intended for human therapeutic administration.
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| References | |
| Additional Infomation |
VT02956 is not approved for clinical use and is not under clinical development. It is a research compound used to study the Hippo signaling pathway in cancer, particularly ER+ breast cancer. Its mechanism involves potent inhibition of LATS1/2 kinases (IC₅0: LATS1 0.76 nM, LATS2 0.52 nM), leading to activation of YAP/TAZ and subsequent downregulation of ESR1 (estrogen receptor alpha) expression. By suppressing ESR1, VT02956 inhibits the growth of ER+ breast cancer cells and patient-derived tumor organoids. The compound is a valuable tool for investigating the functional interplay between the Hippo pathway and ER signaling, and for identifying potential therapeutic strategies for endocrine-resistant breast cancer. The compound also serves as a click chemistry reagent, enabling conjugation to biophysical probes for target engagement studies. No clinical trials have been registered. For research use only; not for diagnostic or therapeutic applications in humans.
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| Molecular Formula |
C22H23N5O
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| Molecular Weight |
373.45
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| CAS # |
2999763-09-4
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| Appearance |
White to light yellow 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 | 2.6777 mL | 13.3887 mL | 26.7773 mL | |
| 5 mM | 0.5355 mL | 2.6777 mL | 5.3555 mL | |
| 10 mM | 0.2678 mL | 1.3389 mL | 2.6777 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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