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| Other Sizes |
| Targets |
The primary target of A-395 hydrochloride is the polycomb protein EED, an essential component of the Polycomb repressive complex 2 (PRC2). PRC2 is responsible for the methylation of histone H3K27, a mark associated with transcriptional repression. A-395 binds to EED in vitro with a Ki value of 0.4 nM, inhibiting the allosteric stimulation of PRC2 catalytic activity. It inhibits the PRC2 complex with an IC₅₀ value of 34 nM for methylation of H3K27.
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| ln Vitro |
The Polycomb Repressive Complex 2 (PRC2) includes the crucial member known as Embryonic Ectoderm Development (EED) protein. A-395 inhibits EED's ability to bind H3K27me3. In the H3K27me3 binding pocket of EED, A-395 binds, inhibiting the allosteric stimulation of PRC2 catalytic activity. With an IC50 of 7 nM, A-395 competes with EED for the binding of the H3K27me3 peptide. A-395 selectively and efficiently lowers the H3K27 methyl mark in cells to influence PRC2 activity, but not A-395N, its near chemical counterpart. H3K27me2 and H3K27me3 are both inhibited by A-395 treatment, with IC50 values of 390 nM and 90 nM, respectively. Moreover, human tumor cell lines susceptible to SAM-competitive EZH2 inhibitors were unable to proliferate after receiving A-395 therapy [1].
In vitro, A-395 hydrochloride demonstrates potent and selective inhibition of the PRC2 complex. It binds to EED with a Ki of 0.4 nM and inhibits the methylation of H3K27 with an IC₅₀ of 34 nM. The compound shows >100-fold selectivity over other histone methyltransferases and non-epigenetic targets. In cellular assays, such as in the RD rhabdoid tumor cell line, A-395 inhibits the PRC2 complex with an IC₅₀ of 90 nM, inhibiting the formation of H3K27me3. |
| ln Vivo |
The Polycomb Repressive Complex 2 (PRC2) includes the crucial member known as Embryonic Ectoderm Development (EED) protein. A-395 inhibits EED's ability to bind H3K27me3. In the H3K27me3 binding pocket of EED, A-395 binds, inhibiting the allosteric stimulation of PRC2 catalytic activity. With an IC50 of 7 nM, A-395 competes with EED for the binding of the H3K27me3 peptide. A-395 selectively and efficiently lowers the H3K27 methyl mark in cells to influence PRC2 activity, but not A-395N, its near chemical counterpart. H3K27me2 and H3K27me3 are both inhibited by A-395 treatment, with IC50 values of 390 nM and 90 nM, respectively. Moreover, human tumor cell lines susceptible to SAM-competitive EZH2 inhibitors were unable to proliferate after receiving A-395 therapy [1].
In vivo, A-395 hydrochloride has been used as a chemical probe to study the role of PRC2 in various biological processes. Its potent and selective inhibition of EED makes it a valuable tool for investigating the function of PRC2 in transcriptional repression and its involvement in diseases such as cancer. The compound's in vivo efficacy and pharmacokinetic properties have been characterized in preclinical studies. |
| Enzyme Assay |
In vitro binding studies for A-395 hydrochloride measure its affinity for EED. Techniques such as surface plasmon resonance (SPR) or fluorescence polarization are used to determine the dissociation constant (Kd) or inhibition constant (Ki). The compound's ability to inhibit the methylation of H3K27 by PRC2 is assessed using enzymatic assays with recombinant PRC2 complex and histone substrates. These assays confirm its mechanism as a potent and selective EED inhibitor.
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| Cell Assay |
In vitro cell-based assays for A-395 hydrochloride evaluate its effects on PRC2 activity in cells. Cells are treated with the compound, and the level of H3K27me3 is measured by Western blot or immunofluorescence. The compound's effect on cell proliferation and gene expression can also be assessed. In RD rhabdoid tumor cells, A-395 inhibits the formation of H3K27me3 with an IC₅₀ of 90 nM.
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| Animal Protocol |
In vivo animal studies for A-395 hydrochloride are conducted to evaluate its pharmacological effects. The compound is typically administered via intraperitoneal or oral routes. Pharmacodynamic studies assess the inhibition of H3K27me3 in target tissues. Efficacy studies may be performed in disease models where PRC2 is implicated, such as cancer. Pharmacokinetic studies determine the compound's half-life and bioavailability.
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| ADME/Pharmacokinetics |
The pharmacokinetic properties of A-395 hydrochloride have been characterized in preclinical studies. The compound is soluble in water at 10 mg/mL and should be stored at 2-8°C in a desiccated environment. Its molecular weight is 486.65 (free base basis). For research use, it is supplied as a white to light brown powder. Further details on its half-life, Cmax, and bioavailability are available from the primary research literature.
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| Toxicity/Toxicokinetics |
The toxicity profile of A-395 hydrochloride has been evaluated in preclinical studies. It is classified as a dangerous substance with hazard statements H301 (toxic if swallowed), H315 (causes skin irritation), and H319 (causes serious eye irritation). The compound is for research use only and is not intended for human consumption. Standard safety precautions for handling hazardous chemicals should be followed.
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| References | |
| Additional Infomation |
Additional information: A-395 hydrochloride is a potent and selective chemical probe for EED, developed through a partnership with the Structural Genomics Consortium (SGC). It is an epigenetic research tool. The compound is available as a high-purity (≥98%) product. A-395N is the negative control for the active enantiomer. This product is for research use only and is not approved for clinical or therapeutic applications.
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| Molecular Formula |
C26H38CL3FN4O2S
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|---|---|
| Molecular Weight |
596.02792596817
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| Exact Mass |
486.246
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| CAS # |
2089148-72-9
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| Related CAS # |
2138326-06-2 (R-isomer);2089148-72-9;2089148-71-8;2138319-85-2 (S-isomer)
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| PubChem CID |
123132213
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| Appearance |
White to yellow solid powder
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| LogP |
3.1
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
34
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| Complexity |
789
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| Defined Atom Stereocenter Count |
2
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| SMILES |
Cl.Cl.Cl.S(C)(N1CCN(CC1)C1C=CC(=CC=1)[C@H]1CN(C2C3C(=CC=CC=3CC2)F)C[C@@H]1N(C)C)(=O)=O
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| InChi Key |
REVJNSVNICWODC-KIDMSAQOSA-N
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| InChi Code |
InChI=1S/C26H35FN4O2S/c1-28(2)25-18-30(24-12-9-20-5-4-6-23(27)26(20)24)17-22(25)19-7-10-21(11-8-19)29-13-15-31(16-14-29)34(3,32)33/h4-8,10-11,22,24-25H,9,12-18H2,1-3H3/t22-,24?,25+/m1/s1
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| Chemical Name |
(3R,4S)-1-(7-fluoro-2,3-dihydro-1H-inden-1-yl)-N,N-dimethyl-4-(4-(4-(methylsulfonyl)piperazin-1-yl)phenyl)pyrrolidin-3-amine trihydrochloride
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| Synonyms |
A395 A 395A-395 hydrochlorideA-395 A-395 HCl
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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 |
| 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 (~205.49 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 0.83 mg/mL (1.71 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 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 0.83 mg/mL (1.71 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 8.3 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: ≥ 0.83 mg/mL (1.71 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 | 1.6778 mL | 8.3888 mL | 16.7777 mL | |
| 5 mM | 0.3356 mL | 1.6778 mL | 3.3555 mL | |
| 10 mM | 0.1678 mL | 0.8389 mL | 1.6778 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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