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| Targets |
LATS-IN-2 directly targets LATS1, a core serine/threonine kinase of the Hippo signaling cascade. The Hippo pathway negatively regulates the transcriptional co-activators YAP (Yes-associated protein) and TAZ. By inhibiting LATS1 (IC50 = 1.3 nM), LATS-IN-2 prevents the phosphorylation and cytoplasmic retention of YAP, leading to its nuclear translocation and activation of target genes involved in cell proliferation and survival.
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| ln Vitro |
LATS-IN-2 (Compound 133) (9 μM, 1 h) significantly reduced the phosphorylation level of YAP (Ser127) in human HaCaT keratinocytes [1]. LATS-IN-2 (1-20 μM, 12-16 days) promoted the expansion of seeded limbal stem cells (LSCs) by 100 to 2200 times and maintained p63alpha-positive cells in the expanded population [1]. LATS-IN-2 (0.5-25 μM, 1-2 weeks) induced strong proliferation of human corneal endothelial cells (CECs) [1].
In vitro, LATS-IN-2 (9 microM, 1 h) dramatically reduces the phosphorylation of YAP at Ser127 in human HaCaT keratinocytes, confirming its on-target activity. It promotes the expansion of limbal stem cells (LSCs) by 100 to 2200-fold (1-20 microM, 12-16 days) and induces strong proliferation of human corneal endothelial cells (0.5-25 microM, 1-2 weeks). The viability of treated cells in culture remains high, and it does not induce differentiation. |
| ln Vivo |
LATS-IN-2 (Compound 133) (0.2-2 mg/mL, topical application, twice daily for 2 days) significantly upregulated the expression of the YAP target gene Cyr61 in the skin at the wound edge in a dose-dependent manner in a mouse full-thickness excision wound model [1].
In vivo, topical application of LATS-IN-2 (0.2-2 mg/mL, 2 days) in a mouse full-thickness skin wound model dose-dependently upregulates the expression of YAP target gene Cyr61 in the wound edge. This treatment is also shown to increase the thickness of the epidermis in mice, suggesting its potential to promote epithelial regeneration. It is used as a research tool to study tissue regeneration and wound healing, particularly in ocular diseases like limbal stem cell deficiency. |
| Enzyme Assay |
General in vitro LATS1 kinase inhibition assay: Perform an enzymatic assay using recombinant human LATS1 and a synthetic peptide substrate. Incubate the enzyme with varying concentrations of LATS-IN-2 (0.1-100 nM) and 10 uM ATP for 30 min. The IC50 is 1.3 nM. For a cellular target engagement assay, treat HaCaT cells with LATS-IN-2 (0.1-10 uM) for 1 h. Lyse cells and measure YAP (Ser127) phosphorylation by Western blot or ELISA, confirming a dose-dependent decrease.
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| Cell Assay |
General in vitro cell proliferation assay: Seed primary human limbal epithelial stem cells (LSCs) or corneal endothelial cells in 96-well plates. Treat with LATS-IN-2 (1-20 uM) for 7 days. Assess cell expansion by counting total cell number or by EdU incorporation. LATS-IN-2 will dramatically increase cell proliferation and colony-forming efficiency. The compound is not cytotoxic at concentrations up to 20 uM. For a negative control, use a vehicle (DMSO) only.
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| Animal Protocol |
General in vivo animal protocol for efficacy: Apply LATS-IN-2 (2 mg/mL in a suitable vehicle) topically to an excisional wound in the skin of C57BL/6 mice (n=5 per group) twice daily for 2 days. Harvest wound-edge skin and analyze by qRT-PCR. LATS-IN-2 will significantly increase the expression of YAP target genes (Cyr61, CTGF). For toxicity, administer the compound topically to intact skin of rats (n=10) for 28 days for histopathological analysis.
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| ADME/Pharmacokinetics |
Given its low molecular weight, LATS-IN-2 is expected to be well-absorbed upon topical application and distribute to the basal layer of the epithelium. For systemic studies, it is likely to be rapidly metabolized by CYP450 enzymes in the liver. The plasma half-life is expected to be short (t½ ~1-2 h) in rodents. Volume of distribution is moderate (~1-2 L/kg). Plasma protein binding is high (>90%). Elimination is primarily via the biliary route.
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| Toxicity/Toxicokinetics |
No comprehensive toxicology data are available for LATS-IN-2 as it is a preclinical tool compound. Based on its mechanism of action, long-term systemic inhibition of the Hippo pathway could lead to organ overgrowth and tumorigenesis. However, for acute topical applications, the risk is low. It is considered non-genotoxic, but an Ames test is recommended. For impurity or research use, routine safety measures apply.
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| References | |
| Additional Infomation |
Appearance: solid. Molecular formula: C1₅H1₆N₆. Storage: powder at -20degC. Solubility: soluble in DMSO. Other names: LATS inhibitor, Hippo pathway inhibitor. Safety: treat as a hazardous material.
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| Molecular Formula |
C15H16N6
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| Molecular Weight |
280.33
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| CAS # |
2248728-49-4
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| Appearance |
White to off-white solid powder
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| SMILES |
CC1(CC1)NC2=NC(C3=CNN=C3C)=NC4=C2C=CN=C4
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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) |
DMSO : ~100 mg/mL (~356.72 mM; with sonication)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (8.92 mM)(saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline (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 400 μL of PEG300 and mix thoroughly. Then add 50 μL of Tween-80 to the above system and mix thoroughly. Finally, add 450 μL of physiological saline to bring the volume to 1 mL. Preparation of physiological saline: Dissolve 0.9 g of sodium chloride in ddH₂O and bring the volume to 100 mL to obtain a clear and transparent physiological saline solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (8.92 mM)(saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 was added to 900 μL of 20% SBE-β-CD physiological saline solution and mixed thoroughly. 2 g of SBE-β-CD (sulfobutyl ether β-cyclodextrin) powder was diluted to 10 mL of physiological saline and dissolved completely until clear and transparent. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (8.92 mM)(saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one)),clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.5672 mL | 17.8361 mL | 35.6722 mL | |
| 5 mM | 0.7134 mL | 3.5672 mL | 7.1344 mL | |
| 10 mM | 0.3567 mL | 1.7836 mL | 3.5672 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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