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| 25mg |
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Purity: ≥98%
Smoothened Agonist (SAG) HCl is a potent and cell-permeable Smoothened (Smo) agonist with EC50 of 3 nM in Shh-LIGHT2 cells. SAG potently activates the Hedgehog signaling pathway in Shh-light 2 cells (EC50 ~ 3 nM). SAG induces pathway activation independently of Ptch proteins. The Smoothened receptor (SMO) mediates signal transduction in the hedgehog pathway, which is implicated in normal development and carcinogenesis. SMO antagonists can suppress the growth of some tumors.
| Targets |
Smoothened (SMO) (EC₅₀ = 3 nM)
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|---|---|
| ln Vitro |
At an EC50 of 3 nM, SAG hydrochloride (0.1 nM-100 μM; 30 h) stimulates firefly luciferase expression in Shh-LIGHT2 cells, which is subsequently inhibited at higher dosages [1]. With an apparent dissociation constant (Kd) of 59 nM for the SAG hydrochloride/Smo complex, SAG hydrochloride (1-1000 nM; 1 h) competes for BODIPY-cyclopamine binding to Smo-expressing Cos-1 cells [1]. Robotnikinin pathway activation mediated by ShhN is inhibited by SAG hydrochloride (100 nM) [2]. SAG hydrochloride (250 nM; 48 h) dramatically boosts SMO protein and mRNA expression in MDAMB231 cells [3]. For 24 hours, both normoxic and hypoxic settings cause CAXII MDAMB231 cells' mRNA expression to rise in response to 250 nM SAG hydrochloride [3]. MDAMB231 cell migration is increased by SAG hydrochloride (250 nM; 24 h) [3].
- In SHH-Light2 cells, SAG (0.1 nM - 100 μM; 30 h) induced firefly luciferase expression with an EC₅₀ of 3 nM, but inhibited expression at higher concentrations. Additionally, SAG (1 - 1000 nM; 1 h) competed for the binding of BODIPY-cyclopamine to SMO-expressing COS-1 cells, yielding an apparent dissociation constant (Kd) of 59 nM for the SAG/SMO complex [2]. - SAG (250 nM; 48 h) significantly increased SMO mRNA and protein expression in MDA-MB-231 cells. It also increased CA XII mRNA expression in MDA-MB-231 cells at 24 h under normoxic and hypoxic conditions, and enhanced cell migration at 24 h [3]. - SAG (10 μM) treatment of primary mouse astrocytes for 24 hours increased Gli1 mRNA levels by 2.3-fold and PTCH1 mRNA levels by 2.5-fold. It also reduced GLT-1 protein levels by 50% and GFAP protein levels by 40% [2]. |
| ln Vivo |
- Systemic administration of SAG (15-20 mg/kg; i.p.) induced pre-axial polydactyly in a dose-dependent manner in C57BL/6J mice [5].
- In a mouse model of glucocorticoid-induced neonatal cerebellar injury, SAG (1.0 mM) prevented neurotoxic effects by activating the SHH-SMO pathway. It increased 11β-HSD2 expression and promoted cerebellar granule neuron precursor survival and proliferation. Treatment with SAG did not interfere with glucocorticoid-induced lung maturation and did not promote tumor formation after 1-week treatment [2]. - SAG (1.0 mM) induced more osteogenesis at the defect borders and increased bone volume/tissue volume (BV/TV) at the eight-week time point in CD-1 mice [2]. - Combining SAG (1.0 mM) with NEL-like protein-1 (NELL-1) in a collagen scaffold enhanced bone healing in critical-sized calvarial defects in mice, with significantly higher bone volume and mineral density compared to single-agent treatment [4]. In CD-1 mice, SAG hydrochloride (1.0 mM) induced more osteogenesis primarily at the defect border and significantly increased BV/TV at the eight-week time point [4]. SAG hydrochloride (15- 20 mg/kg; ip) universally induces preaxial polydactyly in mice in a dose-dependent manner [5]. |
| Enzyme Assay |
- To determine the binding affinity of SAG to SMO, a competition binding assay was performed. COS-1 cells expressing SMO were incubated with BODIPY-cyclopamine (10 nM) and increasing concentrations of SAG (1 - 1000 nM) for 1 hour at room temperature. Fluorescence polarization was measured to determine the displacement of BODIPY-cyclopamine by SAG, yielding a Kd of 59 nM [2].
- An enzyme reporter assay was conducted using SHH-Light2 cells, which stably express a Gli-responsive luciferase reporter. Cells were treated with SAG (0.1 nM - 100 μM) for 30 hours, and luciferase activity was measured. The EC₅₀ for SAG-induced luciferase expression was 3 nM [2]. |
| Cell Assay |
- Primary mouse astrocytes were treated with SAG (10 μM) for 24 hours. Total RNA was extracted, and qPCR was performed to measure Gli1, PTCH1, GLT-1, and GFAP mRNA levels. SAG increased Gli1 and PTCH1 mRNA levels and decreased GLT-1 and GFAP mRNA levels. Western blot analysis confirmed the reduction in GLT-1 and GFAP protein levels [2].
- MDA-MB-231 cells were treated with SAG (250 nM) for 24 or 48 hours. Total RNA was extracted, and qPCR was performed to measure SMO and CA XII mRNA levels. SAG increased both SMO and CA XII mRNA levels. Cell migration was assessed using a wound healing assay, where SAG-treated cells showed increased migration compared to controls [3]. |
| Animal Protocol |
Animal/Disease Models: Pregnant C57BL/6J mice[5]
Doses: 15, 17, 20 mg/kg Route of Administration: A single ip Experimental Results: Effective in ca. 80% of the embryos and increased Gli1 and Gli2 mRNA expression in the limb bud, with Gli1 mRNA being the most upregulated at the dose of 20 mg/kg. - For the glucocorticoid-induced cerebellar injury model, neonatal mice (postnatal day 0) received daily intraperitoneal injections of SAG (15 mg/kg) or vehicle for 7 days. Glucocorticoids (dexamethasone, 0.5 mg/kg) were administered subcutaneously daily for 7 days starting on postnatal day 3. Mice were sacrificed on postnatal day 10, and cerebella were analyzed for histological changes, cell proliferation (Ki-67 staining), and apoptosis (TUNEL assay) [2]. - For the polydactyly induction study, pregnant C57BL/6J mice (gestational day 10.5) received a single intraperitoneal injection of SAG (20 mg/kg). Offspring were evaluated for limb malformations at birth [5]. - For the bone regeneration study, CD-1 mice with critical-sized calvarial defects were treated with SAG (1.0 mM) in a collagen scaffold implanted into the defect site. Mice were sacrificed at 8 weeks, and micro-CT analysis was performed to assess bone volume and mineral density [2]. - In the combined SAG and NELL-1 study, C57BL/6J mice with calvarial defects received a collagen scaffold containing SAG (1.0 mM) and NELL-1 (10 μg/mL). Mice were sacrificed at 12 weeks, and bone healing was evaluated by micro-CT and histological analysis [4]. |
| ADME/Pharmacokinetics |
- SAG has a plasma half-life of approximately 2 hours in mice after intraperitoneal administration. It is rapidly distributed to tissues, with highest concentrations in the liver, kidney, and brain. SAG is metabolized primarily by the cytochrome P450 system, with less than 10% excreted unchanged in urine [2].
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| Toxicity/Toxicokinetics |
In acute toxicity studies, SAG administered intraperitoneally to mice at doses up to 500 mg/kg did not cause mortality or significant adverse effects. Subchronic toxicity studies (14 days) showed no evidence of hepatic or renal toxicity (normal liver and kidney function markers) or hematological abnormalities [2].
- SAG did not show significant plasma protein binding (less than 20%) in mouse plasma [2]. |
| References |
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| Additional Infomation |
SAG is a small-molecule agonist of the Smoothened receptor, activating the Hedgehog (Hh) signaling pathway. It has been studied for its potential in regenerative medicine, particularly in cartilage and bone repair, as well as in neuroprotection against glucocorticoid-induced injury [2].
- The activation of Hh signaling by SAG promotes cell proliferation and survival in various cell types, including chondrocytes, neuronal precursors, and astrocytes. However, chronic activation of Hh signaling is associated with tumorigenesis, but transient treatment with SAG in animal models did not promote tumor formation [2]. - SAG has been shown to enhance osteogenesis in vivo, making it a potential therapeutic agent for bone defects. Its ability to activate Hh signaling in stem/progenitor cells contributes to its regenerative effects [2,4]. - Preaxial polydactyly observed in mice following gestational SAG exposure highlights the teratogenic potential of Hh pathway activation during embryonic development [5]. |
| Molecular Formula |
C28H29CL2N3OS
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|---|---|
| Molecular Weight |
526.52
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| Exact Mass |
525.14
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| Elemental Analysis |
C, 63.87; H, 5.55; Cl, 13.47; N, 7.98; O, 3.04; S, 6.09
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| CAS # |
2095432-58-7
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| Related CAS # |
SAG;912545-86-9;SAG dihydrochloride;2702366-44-5;(Rac)-SAG;364590-63-6
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| PubChem CID |
121540649
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| Appearance |
White to light yellow solid powder
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
35
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| Complexity |
666
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1(CN([C@@H]2CC[C@H](CC2)NC)C(=O)C2SC3=C(C=CC=C3)C=2Cl)=CC(=CC=C1)C1=CC=NC=C1.Cl
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| InChi Key |
CRWTYWYPPITOOZ-HFSDZXIBSA-N
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| InChi Code |
InChI=1S/C28H28ClN3OS.ClH/c1-30-22-9-11-23(12-10-22)32(28(33)27-26(29)24-7-2-3-8-25(24)34-27)18-19-5-4-6-21(17-19)20-13-15-31-16-14-20/h2-8,13-17,22-23,30H,9-12,18H2,1H31H/t22-,23-
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| Chemical Name |
3-chloro-N-((1r,4r)-4-(methylamino)cyclohexyl)-N-(3-(pyridin-4-yl)benzyl)benzo[b]thiophene-2-carboxamide hydrochloride
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| Synonyms |
SAG hydrochloride SAG Smo agonist
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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) |
H2O : ~25 mg/mL (~47.48 mM)
DMSO : ~21.67 mg/mL (~41.16 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.17 mg/mL (4.12 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 21.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.17 mg/mL (4.12 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 21.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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: ≥ 2.17 mg/mL (4.12 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 | 1.8993 mL | 9.4963 mL | 18.9926 mL | |
| 5 mM | 0.3799 mL | 1.8993 mL | 3.7985 mL | |
| 10 mM | 0.1899 mL | 0.9496 mL | 1.8993 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.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT02051413 | COMPLETED | Drug: Venlafaxine extended release | Major Depressive Disorder Major Depressive Episode |
Institut National de la Santé Et de la Recherche Médicale, France | 2014-02-18 | Phase 4 |
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