SAG hydrochloride

Alias: SAG hydrochloride SAG Smo agonist

Cat No.:V7879 Purity: ≥98%

COA Product Data Instructions for use SDS

Smoothened Agonist (SAG) HCl is a potent and cell-permeable Smoothened (Smo) agonist with EC50 of 3 nM in Shh-LIGHT2 cells.

SAG hydrochloride Chemical Structure CAS No.: 2095432-58-7
Product category: Smo

This product is for research use only, not for human use. We do not sell to patients.

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Cell 2020,183:1202-1218.e25.

Science Adv 2022:8,eabm9427.

Nature 2021,597(7874):119-125.

Cell 2020,183:1202-1218.e25.

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Purity & Quality Control Documentation

Current batch：

Purity: ≥98%

COA

MSDS

Instructions

Product Description
Bioactivity & Protocols
Physicochemical Properties
Solubility Data
Calculators
Clinical Trial Information
Biological Data

Product Description

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.

Biological Activity I Assay Protocols (From Reference)

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].
ln Vivo	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].
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.
References	[1]. Small molecule modulation of Smoothened activity. Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14071-6. [2]. A small molecule that binds Hedgehog and blocks its signaling in human cells. Nat Chem Biol. 2009 Mar;5(3):154-6. [3]. Inhibition of smoothened in breast cancer cells reduces CAXII expression and cell migration. J Cell Physiol. 2018 Dec; 233(12): 9799-9811. [4]. Combining Smoothened Agonist (SAG) and NEL-like protein-1 (NELL-1) Enhances Bone Healing. Plast Reconstr Surg. 2017 Feb 13. [5]. Preaxial polydactyly following early gestational exposure to the smoothened agonist, SAG, in C57BL/6J mice. Birth Defects Res A Clin Mol Teratol. 2016 Nov 1.

These protocols are for reference only. InvivoChem does not independently validate these methods.

Physicochemical Properties

Molecular Formula	C28H29CL2N3OS
Molecular Weight	526.52
Exact Mass	525.14
Elemental Analysis	C, 63.87; H, 5.55; Cl, 13.47; N, 7.98; O, 3.04; S, 6.09
CAS #	2095432-58-7
Related CAS #	SAG;912545-86-9;SAG dihydrochloride;2702366-44-5;(Rac)-SAG;364590-63-6
PubChem CID	121540649
Appearance	White to light yellow solid powder
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	6
Heavy Atom Count	35
Complexity	666
Defined Atom Stereocenter Count	0
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
InChi Key	CRWTYWYPPITOOZ-HFSDZXIBSA-N
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-
Chemical Name	3-chloro-N-((1r,4r)-4-(methylamino)cyclohexyl)-N-(3-(pyridin-4-yl)benzyl)benzo[b]thiophene-2-carboxamide hydrochloride
Synonyms	SAG hydrochloride SAG Smo agonist
HS Tariff Code	2934.99.9001
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)

Solubility Data

Solubility (In Vitro)	H2O : ~25 mg/mL (~47.48 mM) DMSO : ~21.67 mg/mL (~41.16 mM)
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. 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 corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.)

Solubility (In Vitro)

H2O : ~25 mg/mL (~47.48 mM)
DMSO : ~21.67 mg/mL (~41.16 mM)

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.
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 corn oil and mix evenly.

(Please use freshly prepared in vivo formulations for optimal results.)

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.

Calculator

Mass

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Molarity Calculator allows you to calculate the mass, volume, and/or concentration required for a solution, as detailed below:

Calculate the Mass of a compound required to prepare a solution of known volume and concentration
Calculate the Volume of solution required to dissolve a compound of known mass to a desired concentration
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See Example

An example of molarity calculation using the molarity calculator is shown below:
What is the mass of compound required to make a 10 mM stock solution in 5 ml of DMSO given that the molecular weight of the compound is 350.26 g/mol?

Enter 350.26 in the Molecular Weight (MW) box
Enter 10 in the Concentration box and choose the correct unit (mM)
Enter 5 in the Volume box and choose the correct unit (mL)
Click the “Calculate” button
The answer of 17.513 mg appears in the Mass box. In a similar way, you may calculate the volume and concentration.

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Dilution Calculator allows you to calculate how to dilute a stock solution of known concentrations. For example, you may Enter C1, C2 & V2 to calculate V1, as detailed below:

What volume of a given 10 mM stock solution is required to make 25 ml of a 25 μM solution?
Using the equation C1V1 = C2V2, where C1=10 mM, C2=25 μM, V2=25 ml and V1 is the unknown:

Enter 10 into the Concentration (Start) box and choose the correct unit (mM)
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Enter 25 into the Volume (End) box and choose the correct unit (mL)
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The answer of 62.5 μL (0.1 ml) appears in the Volume (Start) box

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Molecular Weight Calculator allows you to calculate the molar mass and elemental composition of a compound, as detailed below:

Note: Chemical formula is case sensitive: C12H18N3O4 c12h18n3o4
Instructions to calculate molar mass (molecular weight) of a chemical compound:

To calculate molar mass of a chemical compound, please enter the chemical/molecular formula and click the “Calculate’ button.

Definitions of molecular mass, molecular weight, molar mass and molar weight:

Molecular mass (or molecular weight) is the mass of one molecule of a substance and is expressed in the unified atomic mass units (u). (1 u is equal to 1/12 the mass of one atom of carbon-12)
Molar mass (molar weight) is the mass of one mole of a substance and is expressed in g/mol.

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Reconstitution Calculator allows you to calculate the volume of solvent required to reconstitute your vial.

Enter the mass of the reagent and the desired reconstitution concentration as well as the correct units
Click the “Calculate” button
The answer appears in the Volume (to add to vial) box

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal to make allowance for loss during the experiment)

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Step 2: Enter in vivo formulation (This is only a calculator, not the exact formulation for a specific product. Please contact us first if there is no in vivo formulation in the solubility section.)

% DMSO

% Tween 80

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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.

Note： (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.

Clinical Trial Information

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

Biological Data

SAG acts downstream of Ptch1 in the Hh pathway and counteracts cyclopamine inhibition of Smo. (A) Chemical structure of SAG and its activity in Shh-LIGHT2 cells. (B) SAG induces firefly luciferase expression in Shh-LIGHT2 cells with an EC50 of 3 nM and then inhibits expression at higher concentrations. For comparison, the luciferase activity induced by 2 nM ShhNp is indicated by the green line. (C) SAG induces β-galactosidase expression in P2Ptch1−/− cells treated with 100 nM KAAD-cyclopamine. Hh pathway activation in these cells is indicated by β-galactosidase activity, because expression of this reporter enzyme is under the control of the Ptch1 promoter, and Ptch1 itself is a transcriptional target of Hh signaling. Observed β-galactosidase activities in the absence of pharmacological modulation and with 100 nM KAAD-cyclopamine alone are indicated by the green and red lines, respectively. (D) SAG induces firefly luciferase expression in SmoA1-LIGHT2 cells treated with 1.5 μM KAAD-cyclopamine. Luciferase activities in the absence of small molecules and in the presence of 1.5 μM KAAD-cyclopamine alone are depicted by the green and red lines, respectively. [1].Chen JK, et al. Small molecule modulation of Smoothened activity. Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14071-6.

SAG binds directly to Smo heptahelical bundle. (A) Chemical structure of the photoaffinity reagent PA-SAG and its activity in Shh-LIGHT2 cells. (B) 125I-labeled PA-SAG cross-links the post-ER form of Smo-Myc3 (black arrowhead) expressed in Cos-1 cells upon photoactivation, and this reaction is inhibited by 150 nM SAG (Left). The ER-localized form of Smo-Myc3 (white arrowhead) is not detectably cross-linked, and cells expressing GFP as a control or SmoA1-Myc3 do not yield specifically cross-linked products. An endogenous Cos-1 protein that is nonspecifically labeled by PA-SAG is denoted by the asterisk. Expression levels of Smo-Myc3 and SmoA1-Myc3 as determined by Western analysis are shown for comparison (Right). (C) SAG competes for PA-SAG cross-linking of post-ER Smo-Myc3 (Left) in a manner similar to its ability to inhibit PA-cyclopamine cross-linking of Smo-Myc3 (see Fig. Fig.11G). Cellular levels of post-ER Smo-Myc3 are not affected by SAG (Right). [1].Chen JK, et al. Small molecule modulation of Smoothened activity. Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14071-6.

A bivalent model of SAG action. Hh pathway stimulation or inhibition by SAG at low or high concentrations, respectively, can be accounted for by bivalent binding of SAG to Smo and to a downstream effector. In this model, Hh pathway activation would normally involve the recruitment of a downstream effector (green) by a subpopulation of Smo molecules (blue). At subsaturating concentrations, SAG (red) can bind both Smo and the effector, thereby promoting Smo/effector association and increasing pathway activity levels. Higher concentrations of SAG, however, can inhibit the formation of this ternary complex by independently binding both proteins.[1].Chen JK, et al. Small molecule modulation of Smoothened activity. Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14071-6.