| Size | Price | Stock | Qty |
|---|---|---|---|
| 10mg |
|
||
| 25mg |
|
||
| 50mg |
|
||
| 100mg |
|
||
| 250mg |
|
||
| Other Sizes |
Purity: ≥98%
A-366 (A366) is a novel, potent and peptide-competitive inhibitor of the histone methyltransferase G9a with anticancer activity. It inhibits G9a with an IC50 3 nM and > 100-fold selectivity over other methyltransferases and other non-epigenetic targets. A-366 has been shown to inhibit H3K9 methylation in cells with an IC50 of 100 nM and exhibits minimal cellular toxicity compared with previous quinazoline-based probes. A-366 selectively inhibits G9a and the closely related GLP (EHMT1), but not other histone methyltransferases. A-366 has significantly less cytotoxic effects on the growth of tumor cell lines compared to other known G9a/GLP small molecule inhibitors despite equivalent cellular activity on methylation of H3K9me2.
| Targets |
G9a (histone H3 lysine-9 methyltransferase) – potently diminishes H3K9 dimethylation
Histamine H3 receptor (H3R) – functional antagonist: KB = 15 nM [2-150 nM] at human H3R (hH3R) determined from Schild analysis; also active at rat H3R (rH3R) with EC50 = 2.3 nM [0.4-14.9 nM] for shifting histamine activation in CRE-Luc assay Spindlin1 (epigenetic reader protein) – inhibitory activity[1] |
|---|---|
| ln Vitro |
A-366 (0.01-10 μM; 14 days) impacts the viability of MV4;11 cells and causes differentiation [4]. In PC-3 prostate cancer cells, A-366 (0.3-3 μM; 72 hours) decreases overall H3K9me2 levels in a concentration- and time-dependent manner, with a cellular EC50 of roughly 300 nM. A-366 (0.01-10 μM; 4 days; HL-60 cells) decreased differentiation and concomitant proliferation in a dose-dependent manner. A-366-treated HL-60 cells' DNA content analysis revealed an increase of G1 phase cells, which is consistent with cell arrest [4].
A-366 at 1 μM potently displaced [3H]N-methylhistamine from human H3R with inhibition comparable to pitolisant (approx. 90% inhibition based on Fig. 1, exact percentage not specified). [1] In a CRE-Luc reporter gene assay using HEK-293T cells stably expressing rat H3R, A-366 concentration-dependently shifted histamine-induced cAMP reduction, showing antagonist properties. The affinity (KB) at human H3R was calculated as 15 nM [2-150 nM] from Schild regression (slope 0.79±0.45, not significantly different from unity). [1] A-366 inhibited G9a enzymatic activity, as evidenced by reduced H3K9 dimethylation in an AlphaLISA-based assay (exact IC50 not provided). [1] A-366 also showed inhibitory activity against Spindlin1 (exact IC50 not provided). [1] Selectivity screening at 1 μM revealed that A-366 had significantly lower inhibition of radioligand binding to dopamine D1, D2, D3, D5 receptors and histamine H4 receptor compared to its inhibition at H3R (P<0.02), indicating subtype selectivity. Differences between A-366 and UNC-0642 at H3R, H4R, D1R, and D2R were not significant (P>0.22), but UNC-0642 showed higher displacement of [3H]spiperone from D2R and D3R than A-366 (P<0.01). [1] |
| ln Vivo |
Growth suppression occurs when MV4;11 xenografts are treated with A-366 (30 mg/kg; osmotic minipump; once daily for 14 days) [4].
In an MV4;11 AML flank xenograft model, administration of A-366 via osmotic mini-pump at 30 mg/kg/day for 14 days resulted in modest tumor growth inhibition (45% TGI) [4]. |
| Enzyme Assay |
G9a inhibition was examined using an AlphaLISA-based format. Compounds were incubated for 30 minutes on white 384-well microplates with 5 mM G9a enzyme, 100 mM histone H3 (1-21) fragment, and 15 μM S-adenosylmethionine (SAM) in assay buffer (50 mM Tris-HCl pH 9.0, 50 mM NaCl, 1 mM dithiothreitol, 0.01% Tween-20). The reaction was terminated by adding anti-H3K9me2 acceptor beads in detection buffer. After 60 minutes of incubation, streptavidin-coated donor beads were added for an additional 30 minutes. Luminescence was measured using an AlphaLISA luminescence filter with 1000 ms integration time. [1]
Spindlin1 inhibition was determined using a fluorescence polarization displacement assay. Twelve concentrations were measured in triplicates to obtain IC50 values (exact values for A-366 not provided). [1] Radioligand displacement assays for GPCRs: Membrane preparations of transfected HEK-293T cells expressing human H3R were incubated with A-366 (titration 0.003-1000 nM, duplicates) and 2 nM [3H]N-methylhistamine for 90 minutes. Non-specific binding was determined with 10 μM pitolisant. For off-target selectivity screening, 1 μM A-366 was incubated with respective receptors under conditions described in Table 2 (using [3H]SCH23390 for D1/D5, [3H]spiperone for D2/D3, [3H]histamine for H4R). Incubation was terminated by filtration onto GF/B filters pre-soaked with 0.3% polyethyleneimine, washed three times with 4°C water, dried, and counted by scintillation. [1] |
| Cell Assay |
Cell Viability Assay [4]
Cell Types: MV4; 11 cells Tested Concentrations: 0.01-10 μM Incubation Duration: 14 days Experimental Results: Resulted in inhibition of proliferation and diminished viability, corresponding to the dose response observed by CD11b staining. CRE-Luc reporter gene assays were performed in HEK-293T cells stably transfected with rat H3R cDNA and a luciferase vector containing a cAMP-response element. Cells were seeded into polyethyleneimine-coated 96-well plates at 2×10⁵ cells/200 μL per well in assay medium (DMEM without phenol red, 1% FBS) and allowed to attach for 24-48 hours. Then, forskolin (final concentration 3 μM) and serially diluted Nα-methylhistamine (10,000 to 0.01 mM) were added in the absence or presence of A-366 (10 to 100,000 nM) using a liquid handling robot. The mixture was incubated for 5 hours under culture conditions (37.0°C, 5.0% CO₂, 95% humidity). After removal of medium, cells were lysed with 80 μL lysis buffer (25 μM tricine, 10% glycerol, 2 μM egtaic acid, 1% Triton X-100, 5 μM MgSO₄·7H₂O, 1 μM dithiothreitol) for 30 minutes with shaking at 300 rpm. Lysate was transferred to white microplates, and luminescence was recorded immediately after injection of 40 μL assay buffer (25 mM glycylglycine, 15 mM MgSO₄·7H₂O, 15 mM KH₂PO₄, 4 mM egtaic acid, 2 mM dithiothreitol, 1 mM ATP, 50 μM coenzyme A, 0.02 mg/mL D-luciferin potassium salt) using an injector module, with 3000 ms integration time. Data were normalized to forskolin-only control (100%) and forskolin plus 10 μM Nα-methylhistamine (0%). [1] |
| Animal Protocol |
Animal/Disease Models: 6-8 weeks old SCID beige female mice (MV4; 11 xenografts) [4]
Doses: 30 mg/kg Route of Administration: via mini-osmotic pump; one time/day for 14 days Experimental Results: In In the model, A-366 treatment produced a modest 45% tumor growth inhibition. MV4;11 Flank Xenograft Efficacy Study: MV4;11 cells (5×10^6) mixed with Matrigel were injected subcutaneously into the flank of female SCID-beige mice. When tumors reached ~200 mm³, mice were allocated into groups (n=10/group). A-366 was formulated in 98% PEG-400 and 2% Tween-80 and delivered via osmotic mini-pump at 30 mg/kg/day for 14 days. Tumor volumes were measured twice per week with a digital caliper. Tumor growth inhibition (TGI) was calculated [4]. |
| ADME/Pharmacokinetics |
In tumor-bearing mice, A-366 showed significant accumulation in tumor tissue and other tissues after administration via osmotic mini-pump [4].
|
| Toxicity/Toxicokinetics |
Initial maximum tolerated dose (MTD) studies with intraperitoneal (IP) dosing of A-366 showed toxicity in animals at 10 mg/kg [4].
- In human PBMCs (non-transformed cells), A-366 had no impact on viability up to 10 μM [4]. |
| References |
|
| Additional Infomation |
A-366 is a spirocyclic 2-amino-3H-indole-based G9a inhibitor that is likely protonated at physiological pH due to an inherent amidine or aromatic guanidine functionality. It contains a 3-pyrrolidinopropoxy moiety, which mimics the lysine residue of histone H3K9 and blocks the lysine binding tunnel in the G9a active site. A-366 was identified as a potent H3R antagonist with high affinity (KB = 15 nM at human H3R) and selectivity over dopamine D1, D2, D3, D5 receptors and histamine H4 receptor. The compound shows dual inhibition of G9a and Spindlin1, making it a multi-target ligand. The study proposes that A-366 could potentially provide both symptomatic relief (via H3R antagonism) and causal intervention (via G9a-mediated restoration of imprinted genes) for Prader-Willi syndrome (PWS). The authors note that A-366 has advanced preclinical development status and is tolerated in mouse or rat models, suggesting its suitability for future in vivo investigations in PWS. [1]
|
| Molecular Formula |
C19H27N3O2
|
|---|---|
| Molecular Weight |
329.4366
|
| Exact Mass |
329.21
|
| CAS # |
1527503-11-2
|
| PubChem CID |
76285486
|
| Appearance |
Light yellow to yellow solid powder
|
| LogP |
3.057
|
| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
4
|
| Rotatable Bond Count |
6
|
| Heavy Atom Count |
24
|
| Complexity |
471
|
| Defined Atom Stereocenter Count |
0
|
| InChi Key |
BKCDJTRMYWSXMC-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C19H27N3O2/c1-23-16-12-14-15(21-18(20)19(14)6-4-7-19)13-17(16)24-11-5-10-22-8-2-3-9-22/h12-13H,2-11H2,1H3,(H2,20,21)
|
| Chemical Name |
5'-methoxy-6'-(3-(pyrrolidin-1-yl)propoxy)spiro[cyclobutane-1,3'-indol]-2'-amine
|
| Synonyms |
A 366A-366 A366
|
| 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 |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
DMSO : ~50 mg/mL (~151.77 mM)
|
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.59 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 25.0 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: ≥ 2.5 mg/mL (7.59 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 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.0355 mL | 15.1773 mL | 30.3545 mL | |
| 5 mM | 0.6071 mL | 3.0355 mL | 6.0709 mL | |
| 10 mM | 0.3035 mL | 1.5177 mL | 3.0355 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.
|
|
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
COA
