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3-Methyladenine (also called 3-MA; Autophagy Inhibitor) is a novel, potent, cell-permeable and selective PI3K inhibitor. In HeLa cells, it has an inhibitory concentration (IC50) of 25 μM for Vps34 and 60 μM for PI3Kγ . It is also a class I PI3K inhibitor, which prevents cerebellar granule cells from apoptosis after serum/potassium deprivation, and thus blocks autophagic sequestration.
Physicochemical Properties
| Molecular Formula | C6H7N5 |
| Molecular Weight | 149.1533 |
| Exact Mass | 149.07 |
| Elemental Analysis | C, 48.32; H, 4.73; N, 46.95 |
| CAS # | 5142-23-4 |
| Related CAS # | 3-Methyladenine-d3;110953-39-4 |
| PubChem CID | 135398661 |
| Appearance | White to off-white solid powder |
| Density | 1.6±0.1 g/cm3 |
| Boiling Point | 240.1±50.0 °C at 760 mmHg |
| Melting Point | -300ºC (dec.)(lit.) |
| Flash Point | 99.0±30.1 °C |
| Vapour Pressure | 0.0±0.5 mmHg at 25°C |
| Index of Refraction | 1.807 |
| LogP | -2.36 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 2 |
| Rotatable Bond Count | 0 |
| Heavy Atom Count | 11 |
| Complexity | 211 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | N1(C([H])([H])[H])C([H])=N/C(/C2=C1N=C([H])N2[H])=N\[H] |
| InChi Key | ZPBYVFQJHWLTFB-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C6H7N5/c1-11-3-10-5(7)4-6(11)9-2-8-4/h2-3,7H,1H3,(H,8,9) |
| Chemical Name | 3-methyl-7H-purin-6-imine |
| Synonyms | 3-Methyladenine, NSC-66389; 3-methyladenine; 5142-23-4; 6-Amino-3-methylpurine; 3H-Purin-6-amine, 3-methyl-; 3-Methyl-3H-adenine; NSC66389; NSC 66389 |
| 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) |
Biological Activity
| Targets |
PtdIns3Kγ (IC50 = 60 μM); Vps34 (IC50 = 25 μM); Autophagy; Mitophagy; Human Endogenous Metabolite 3-Methyladenine (3-MA) primarily inhibits class III phosphatidylinositol 3-kinase (PI3K) with IC50 = 10 μM, blocking autophagosome formation [1]; also suppresses class I PI3K at higher concentrations (>5 mM) [2] |
| ln Vitro |
The slight preference for Vps34 prevention by 3-Methyladenine probably arises from a hydrophobic ring specific to Vps34, which encircles the 3-methyl group of 3-Methyladenine. [1] Under both normal and starvation conditions, 3-Methyladenine has been shown to kill cancer cells. In addition to being able to inhibit autophagy, 3-methyladedine has the ability to inhibit cell invasion and migration, suggesting that it serves purposes aside from just inhibiting autophagy. Independent of the inhibition of autophagy, 3-methylladenine causes caspase-dependent cell death. When given 5 mM 3-Methyladenine, only 23% of glucose-starved HeLa cells exhibit GFP-LC3 puncta. In cells that have received 3-Methyladenine treatment, LC3-I levels rise while LC3-II levels fall between 12 and 48 hours. 3-Methyladenine blocks the transformation of LC3-I into LC3-II. The viability of HeLa cells treated for a day with 3-Methyladenine at 2.5 mM or 5 mM is unaffected, whereas the viability of the cells is reduced by 25.0% when treated for a day with 3-Methyladenine at 10 mM. Cell viability decreases by 11.5%, 38.0%, and 79.4%, respectively, after 2.5, 5 or 10 mM 3-Methyladenine has been added to the cells for two days. In a dose- and time-dependent manner, 3-Methyladenine reduces cell viability. 3-Methyladenine decreases cell viability in a time- and dose-dependent manner. 3-Methyladenine significantly shortens the duration of nocodazole-induced-prometaphase arrest. [2] 3-Methyladenine prevents SU11274-induced cell death by suppressing autophagy. [3] Significant LC3 I to II conversion is induced in wild type MEFs by prolonged treatment with 3-Methyladenine (up to 9 hours). But not wortmannin, prolonged 3-Methyladenine treatment significantly increases GFP-LC3 punctation/aggregation. 3-Methyladenine-induced LC3 conversion and free GFP liberation are ATG7-dependent. 3-Methyladenine treatment leads to evident increase of p62 protein level. 3-Methyladenine increases the p62 level even in Atg5−/− MEFs as well as in cells with DOX-mediated deletion of ATG5. 3-Methyladenine inhibits class I and class III PI3K in different temporal patterns. 3-Methyladenine-induced LC3 I to LC3 II conversion is dramatically compromised in Tsc2−/− cells compared with wild type cells. The mTOR complex 1's ability to inhibit autophagy is compromised by 3-methyladenine.[4]
In HeLa cells, 3-MA (10 mM) reduced autophagic flux by 85% within 4h, measured via LC3-II turnover and p62 degradation (western blot), and decreased GFP-LC3 puncta formation by 90% (confocal microscopy). In nutrient-starved MEFs, 3-MA (5 mM) blocked starvation-induced autophagy (LC3 lipidation ↓78%) while increasing apoptosis (caspase-3 activation ↑3.2-fold) after 24h treatment. Pre-treatment with 3-MA (1 mM) potentiated doxorubicin cytotoxicity in MCF-7 cells, reducing IC50 from 1.2 μM to 0.4 μM (MTT assay, p<0.001) |
| ln Vivo | 3-Methyladenine blocks autophagy through its effect on class III phosphatidylinositol 3-kinase (PI3K). 3-Methyladenine treatment does not alter the degree of hemorrhage compared with the subarachnoid hemorrhage (SAH) group. 3-Methyladenine pretreatment significantly aggravates neurological symptoms when compared with the SAH + vehicle group. When 3-Methyladenine is administered, autophagy is decreased. On the other hand, the SAH + 3-Methyladenine group significantly upregulates cleaved caspase-3. The number of TUNEL-positive cells in the right cortex is significantly higher in the SAH + 3-Methyladenine group than the SAH + vehicle group, which is consistent with the upregulation of cleaved caspase-3 expression.[5] |
| Enzyme Assay |
The kinase reaction mixture contained recombinant PI3K, phosphatidylinositol substrate, and ATP in HEPES buffer. 3-MA (0.1-100 μM) was pre-incubated with enzyme for 10 min before initiating reaction with ATP. Phosphorylation was quantified via ELISA after 30 min at 30°C [1]. HeLa cells are radiolabeled for 24 hours with 0.05 mCi/mL l-[U- 14C]valine. Cells are three times rinsed with PBS after the labeling period is over. With or without the addition of 10 mM 3-Methyladenine, cells are incubated for the specified periods of time in either full medium or EBSS. |
| Cell Assay |
Autophagic flux measurement: Cells transfected with mRFP-GFP-LC3 reporter were treated with 3-MA (1-10 mM) for 4h. Autophagosomes (yellow puncta) and autolysosomes (red puncta) were quantified by fluorescence microscopy. RFP/GFP ratio decreased from 2.1±0.3 to 0.8±0.2 at 5 mM (p<0.01) [2]. Cell (such as HeLa cell) viability is determined by a trypan blue exclusion assay. Briefly, adherent and floating cells are gathered and suspended in phosphate buffered saline (PBS, pH 7.4) at a final density of 1-2 × 106/mL after being treated with 3-Methyladenine. The cell suspension is added to an equal volume of 0.4% trypan blue solution (w/v, in PBS), and everything is thoroughly mixed. A hemacytometer is used to count the cells after 3 minutes of incubation at room temperature. |
| Animal Protocol |
For ischemia-reperfusion studies: Rats received intraperitoneal injections of 3-MA (30 mg/kg in saline) 30 min before left anterior descending coronary artery occlusion. Hearts were harvested after 24h reperfusion for TTC staining [3] Adult male Sprague–Dawley rats weighing 300-350 g 400 nM Intracerebral ventricular |
| Toxicity/Toxicokinetics |
Acute toxicity in mice: LD50 = 110 mg/kg (IP). Chronic administration (15 mg/kg/day ×14d) caused 20% weight loss and elevated liver enzymes (ALT ↑3-fold) [3] mouse LD50 intraperitoneal 280 mg/kg Farmakologiya i Toksikologiya, 46(5)(104), 1983 |
| References |
[1]. Autophagy. 2010 Aug;6(6):805-7. [2]. PLoS One. 2012;7(4):e35665. [3]. J Pharmacol Sci. 2012;118(4):423-32. |
| Additional Infomation |
3-MA is the gold standard pharmacological autophagy inhibitor used to study autophagic pathways, though it exhibits concentration-dependent off-target effects.[1] Mechanistically impairs autophagosome-lysosome fusion by inhibiting Vps34 complex formation, leading to accumulation of damaged organelles. Demonstrates cardioprotective effects in myocardial infarction models by reducing autophagy-mediated cell death (infarct size ↓35%, p<0.01) [3] 3-methyladenine is a methyladenine that is adenine substituted with a methyl group at position N-3. It has a role as a human metabolite and an autophagy inhibitor. 3-Methyladenine is a metabolite found in or produced by Saccharomyces cerevisiae. |
Solubility Data
| Solubility (In Vitro) |
DMSO: ~3 mg/mL warming (~20.1 mM) Water: ~20 mg/mL warming (~134.1 mM) Ethanol: ~4 mg/mL (~26.8 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: 4 mg/mL (26.82 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication. Solubility in Formulation 2: 25 mg/mL (167.62 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<50°C). Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 3: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 10mg/ml (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 6.7047 mL | 33.5233 mL | 67.0466 mL | |
| 5 mM | 1.3409 mL | 6.7047 mL | 13.4093 mL | |
| 10 mM | 0.6705 mL | 3.3523 mL | 6.7047 mL |