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| Targets |
Cardiomyocyte proliferation promoting agent-1 (GA-002) targets the protein kinases LATS1 (large tumor suppressor kinase 1) and LATS2 (large tumor suppressor kinase 2). LATS1 and LATS2 are core components of the Hippo signaling pathway, which regulates organ size, tissue homeostasis, and cell proliferation. In the Hippo pathway, activated MST1/2 phosphorylate and activate LATS1/2, which in turn phosphorylate and inhibit the transcriptional coactivators YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif). Phosphorylated YAP/TAZ are retained in the cytoplasm and degraded. GA-002 is a potent inhibitor of LATS1/2 kinases (IC50 values of 3.93 nM and 3.87 nM, respectively), preventing YAP/TAZ phosphorylation and promoting their nuclear translocation. In the nucleus, YAP/TAZ bind to TEAD transcription factors to activate the expression of genes involved in cell proliferation, including cyclins and survival factors. The compound promotes cardiomyocyte proliferation, which is normally suppressed postnatally. It operates within the Hippo-YAP signaling pathway. [21L7-L10][8L4-L7]
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| ln Vitro |
In vitro, Cardiomyocyte proliferation promoting agent-1 (GA-002) promotes the proliferation of primary neonatal rat ventricular cardiomyocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). A typical protocol: isolate NRVMs from 1-3 day old rat pups, culture in DMEM with 10% FBS. Treat with GA-002 at 0.1, 0.5, 1, 5, 10 microM (DMSO ≤0.1%) for 24-72 h. Measure proliferation by EdU incorporation (10 microM EdU added for the last 4 h of culture, then fixed and stained) and by Ki67 and phospho-histone H3 (pH3) immunofluorescence staining. GA-002 increases the percentage of EdU+, Ki67+, and pH3+ cardiomyocytes compared to vehicle control. The compound also induces YAP nuclear translocation (immunofluorescence, YAP localization) and reduces YAP phosphorylation at Ser127 (Western blot). It inhibits LATS1/2 kinase activity in cell lysates, as measured by LATS1-mediated phosphorylation of YAP (Western blot). No significant cytotoxicity is observed up to 10 microM in cardiomyocytes. Vehicle control (0.1% DMSO). Positive control: XMU-MP-1 (a MST1/2 inhibitor, 1-10 microM) or YAP activator. [21L7-L10]
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| ln Vivo |
In vivo activity data are not publicly detailed for Cardiomyocyte proliferation promoting agent-1. Based on its mechanism of promoting cardiomyocyte proliferation via LATS1/2 inhibition and YAP activation, the compound is expected to have efficacy in mouse models of myocardial infarction (MI). A typical study would involve inducing MI by permanent ligation of the left anterior descending (LAD) coronary artery in adult C57BL/6 mice. GA-002 (formulated in 10% DMSO, 40% PEG300, 5% Tween 80, 45% saline) is administered intraperitoneally (IP) or orally at doses of 5-30 mg/kg daily starting immediately after MI for 7-28 days. Endpoints: echocardiography (ejection fraction, fractional shortening), histology (infarct size, fibrosis by Masson's trichrome, cardiomyocyte proliferation by Ki67/EdU/pH3), and survival. Not performed in available sources. For research use only. [21L7-L10]
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| Enzyme Assay |
For non-cellular LATS1/2 kinase inhibition assay, purify recombinant active human LATS1 or LATS2 protein (full-length or kinase domain, commercially available). Prepare 20 microL reaction mixture containing 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT, 0.01% Triton X-100, 10 microM ATP, and LATS1/2 (5-10 ng). Add fluorescent YAP peptide substrate (e.g., FITC-labeled peptide containing LATS phosphorylation site, 1-5 microM) and GA-002 (0.001-1000 nM, 3-fold serial dilutions, DMSO ≤1%). Incubate at 30degC for 30-60 min. Stop reaction with EDTA. Detect phosphorylated substrate by capillary electrophoresis or by homogeneous time-resolved fluorescence (HTRF) using anti-phospho-YAP (Ser127) antibody. Calculate IC50 from dose-response curve using four-parameter logistic regression. Positive control: LATS1/2 inhibitor (e.g., XMU-MP-1, IC50 ~10-100 nM for MST1/2, not directly for LATS). Negative control: DMSO only. All conditions in duplicate or triplicate. [21L7-L10]
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| Cell Assay |
For in vitro cell assays, isolate primary neonatal rat ventricular cardiomyocytes (NRVMs) from 1-3 day old Sprague-Dawley pups using enzymatic digestion (trypsin/collagenase). Pre-plate cells for 2 h to remove fibroblasts, then seed on laminin-coated plates: 1×10⁵ cells/well in 24-well plates for immunostaining, or 2×10⁶ cells/well in 6-well plates for Western blot. Culture in DMEM with 10% FBS, 10% horse serum, 1% P/S, 1% L-glutamine. After 48 h, switch to serum-free medium containing GA-002 at 0.1, 0.5, 1, 5, 10 microM (0.1% DMSO). For EdU incorporation, add 10 microM EdU to culture for the last 4 h of treatment. Fix in 4% paraformaldehyde, permeabilize with 0.5% Triton X-100, and stain with EdU Click-It kit (Alexa Fluor 488 or 647). For immunofluorescence, stain with primary antibodies: anti-Ki67 (1:200), anti-phospho-histone H3 (pH3, Ser10, 1:400), anti-YAP (1:200), anti-cTNT (cardiac troponin T, 1:500) as cardiomyocyte marker. Secondary antibodies: Alexa Fluor 488/594-conjugated. Counterstain nuclei with DAPI. Image with fluorescence microscopy. For YAP localization, quantify nuclear vs. cytoplasmic YAP intensity ratio. For Western blot, treat cells for 24 h, lyse in RIPA buffer, blot for phospho-YAP (Ser127, 1:1000), total YAP (1:1000), LATS1 (1:1000), and beta-actin (1:5000). Vehicle control (0.1% DMSO). All experiments in triplicate. [21L7-L10]
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| Animal Protocol |
No in vivo animal protocols are publicly available for Cardiomyocyte proliferation promoting agent-1. A typical study for myocardial infarction would use male C57BL/6J mice (8-10 weeks, 20-25 g, n=10-15/group). Perform left anterior descending (LAD) coronary artery ligation under isoflurane anesthesia to induce MI (permanent occlusion). Immediately after LAD ligation, randomly assign mice to treatment groups: vehicle control, GA-002 10 mg/kg, 20 mg/kg, 30 mg/kg, and positive control (e.g., YAP activator or XMU-MP-1 10 mg/kg). Formulate GA-002 in 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline to a concentration of 1-3 mg/mL. Administer intraperitoneally (IP) once daily for 14 days. Perform echocardiography (VEVO 2100) at days 7 and 14 post-MI to measure ejection fraction (EF%), fractional shortening (FS%), and left ventricular end-diastolic dimension (LVEDD). At endpoint (day 14), euthanize, perfuse with PBS, and harvest hearts. Process for histology: paraffin sections stained with Masson's trichrome for fibrosis; fluorescent sections for EdU (injected 2 h prior to sacrifice), Ki67, and pH3 to assess cardiomyocyte proliferation. For vehicle control, use same volume of formulation without GA-002. Statistical analysis by two-way ANOVA and t-test. Not performed in available sources. For research use only. [21L7-L10]
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| ADME/Pharmacokinetics |
Specific pharmacokinetic data for GA-002 are not publicly available. The compound has a molecular weight of 341.40 and molecular formula C19H23N3O3. Based on its structure, it is likely to have moderate to good oral bioavailability (F% 30-70%) in rodents. After oral or intraperitoneal administration, Tmax is expected at 0.5-2 h. Terminal half-life (t½) is estimated to be 2-6 h, supporting once-daily dosing. Volume of distribution (Vd) likely >1 L/kg. Clearance (CL) is likely hepatic via CYP450 metabolism. For storage, powder at -20degC for up to 3 years; in DMSO at -80degC for 6 months. Solubility: DMSO (10 mM). In vivo formulation: 10% DMSO, 40% PEG300, 5% Tween 80, 45% saline. For research use only. [21L10-L13]
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| Toxicity/Toxicokinetics |
No formal toxicity data are available for Cardiomyocyte proliferation promoting agent-1. As a LATS1/2 inhibitor and YAP activator, potential on-target adverse effects include uncontrolled cell proliferation in non-cardiac tissues, particularly in organs where YAP activation is oncogenic (e.g., liver, lung, skin). Chronic activation of YAP has been associated with tumorigenesis in animal models. However, short-term administration for cardiac regeneration may have a favorable safety profile. In cell viability assays, no significant cytotoxicity is observed in cardiomyocytes at concentrations up to 10 microM. Standard laboratory safety precautions: avoid inhalation, ingestion, skin/eye contact; use PPE (gloves, lab coat, safety goggles); work in a chemical fume hood. For research use only-not for human use. Dispose of waste according to local hazardous waste regulations. [21L10-L13]
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| References | |
| Additional Infomation |
CAS: 2351906-71-1. Molecular formula: C19H23N3O3, molecular weight: 341.40. Also known as (E/Z)-GA-002. GA-002 (single E isomer) IC50 values: LATS1: 3.93 nM, LATS2: 3.87 nM. Appearance: white to off-white solid powder. Purity: 99.87% by HPLC. Storage: powder at -20degC for 3 years; 4degC for 2 years; in DMSO at -80degC for 6 months. Solubility: DMSO. Shipping: ambient temperature. Research areas: heart disease, myocardial regeneration, Hippo-YAP signaling. Target: LATS1, LATS2. Not for human use. For research only. [21L10-L13][8L4-L7]
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| Molecular Formula |
C19H23N3O3
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| Molecular Weight |
341.40
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| Exact Mass |
341.174
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| CAS # |
2351906-71-1
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| PubChem CID |
146736742
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
4
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
25
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| Complexity |
463
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC/C(=N/NC(=O)C(C)NC1=CC=CC=C1)/C2=C(C(=C(C=C2)O)C)O
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| InChi Key |
RJHQUBJDRHLKEX-PGMHBOJBSA-N
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| InChi Code |
InChI=1S/C19H23N3O3/c1-4-16(15-10-11-17(23)12(2)18(15)24)21-22-19(25)13(3)20-14-8-6-5-7-9-14/h5-11,13,20,23-24H,4H2,1-3H3,(H,22,25)/b21-16-
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| Chemical Name |
2-anilino-N-[(Z)-1-(2,4-dihydroxy-3-methylphenyl)propylideneamino]propanamide
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| Synonyms |
(E/Z)-GA-002
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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, avoid exposure to moisture. |
| 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 | 2.9291 mL | 14.6456 mL | 29.2912 mL | |
| 5 mM | 0.5858 mL | 2.9291 mL | 5.8582 mL | |
| 10 mM | 0.2929 mL | 1.4646 mL | 2.9291 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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