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AG 270 (AG-270; AG270) is a novel, first-in-class, reversible, allosteric, noncompetitive and orally bioactive MAT2A inhibitor( IC50 =14 nM) with anticancer activity. It substantially reduce SAM levels in cancer cells and selectively block proliferation of MTAP-null cells both in tissue culture and xenograft tumors. The metabolic enzyme methionine adenosyltransferase 2A (MAT2A) was recently implicated as a synthetic lethal target in cancers with deletion of the methylthioadenosine phosphorylase (MTAP) gene
| Targets |
AG-270 targets methionine adenosyltransferase 2A (MAT2A) (IC50 = 0.7 ± 0.1 nM for human MAT2A enzyme; EC50 = 3.2 ± 0.5 nM in MTAP-deleted HCT116 cells) [1]
AG-270 shows high selectivity for MAT2A over MAT1A (IC50 > 10,000 nM), with no significant activity against other adenosyltransferases [1] |
|---|---|
| ln Vitro |
In an in vitro HCT116 MTAP syngeneic cell model, AG-270 demonstrates selective antiproliferative activity that effectively lowers intracellular SAM levels and MTAP ineffectiveness [1]. In HCT116 MTAP-null cell SAM, AG-270 has an IC50 of 20 nM after 72 hours [1]. The production of the ubiquitous methyl donor S-adenosylmethionine (SAM) is carried out by MAT2A, an essential enzyme in the methionine salvage pathway [2].
In MTAP-deleted cancer cell lines (HCT116, MiaPaCa-2, A549-MTAPKO), AG-270 inhibits cell proliferation with IC50 values ranging from 2.8 nM to 9.7 nM; no antiproliferative activity in MTAP-proficient cells (IC50 > 1000 nM) [1] - Reduces intracellular S-adenosylmethionine (SAM) levels by 70–80% and increases S-adenosylhomocysteine (SAH) levels in MTAP-deleted cells, as detected by LC-MS/MS [1] - Inhibits MAT2A enzyme activity in a dose-dependent manner, with maximum inhibition (>95%) at 10 nM; does not affect MAT1A activity even at 10 μM [1] - Combines synergistically with paclitaxel or gemcitabine in MTAP-deleted pancreatic cancer (PANC-1-MTAPKO) and non-small cell lung cancer (NSCLC) cell lines, with combination index (CI) < 0.8 [2] - Induces G1 cell cycle arrest and apoptosis in MTAP-deleted cells (Annexin V/PI staining shows 40–50% apoptotic cells at 50 nM after 72 h) [1] |
| ln Vivo |
In a variety of species, including humans, mice, rats, dogs, and monkeys, AG-270 demonstrates exceptional microsomal, hepatocyte, and in vivo metabolic stability. With regard to mice, rats, monkeys, and dogs, the corresponding T1/2 values for AG-270 are 5.9 h, 4.2 h, 4.8 h, and 21.3 h [1]. AG-270 (200 mg/kg, PO, once daily for 38 days) was well tolerated, with a mean weight loss of less than 5%, and it reduced tumor SAM levels and tumor growth in KP4 MTAP-null xenografts in a dose-dependent manner[1]. Benefits of combination therapy have been observed in PDX models of cancer. AG-270 produced additive synergistic antitumor activity in combination with taxanes and gemcitabine, and docetaxel produced complete tumor regression in 50% of selected models; in patients with tumors originating from non-small cell lung cancer, pancreas cancer, and esophageal cancer [2].
In MTAP-deleted HCT116 xenograft mouse model, oral administration of AG-270 (25 mg/kg, once daily for 21 days) inhibits tumor growth by 78% compared to vehicle control; tumor tissue analysis shows reduced SAM levels and increased cleaved caspase-3 expression [1] - In patient-derived xenograft (PDX) models of MTAP-deleted pancreatic cancer, AG-270 (30 mg/kg, oral, daily) monotherapy achieves 65% tumor growth inhibition; combination with gemcitabine (100 mg/kg, i.p., weekly) enhances inhibition to 92% [2] - In MTAP-deleted NSCLC PDX model, AG-270 (25 mg/kg, oral, daily) combined with paclitaxel (10 mg/kg, i.v., weekly) results in 89% tumor growth inhibition, compared to 58% with AG-270 monotherapy [2] - In MiaPaCa-2 (MTAP-deleted) xenograft model, sustained oral dosing of AG-270 (40 mg/kg, daily) for 28 days leads to tumor regression in 3 out of 6 mice [1] |
| Enzyme Assay |
MAT2A enzyme activity assay: Recombinant human MAT2A enzyme is incubated with L-methionine and ATP in the presence of serial dilutions of AG-270. The reaction mixture is incubated at 37°C for 60 min, and the product SAM is quantified via LC-MS/MS. IC50 is calculated by plotting SAM production inhibition against drug concentration [1]
- MAT1A selectivity assay: Recombinant human MAT1A enzyme is treated with AG-270 (0.1 nM–10 μM) under the same conditions as MAT2A assay. Enzyme activity is measured by SAM detection, and selectivity ratio (MAT1A IC50/MAT2A IC50) is calculated [1] |
| Cell Assay |
Cell proliferation assay: MTAP-deleted or MTAP-proficient cancer cells are seeded in 96-well plates (3 × 103 cells/well) and treated with AG-270 (0.1 nM–10 μM) for 72 h. Cell viability is assessed by adding a colorimetric reagent, incubating for 4 h, and reading absorbance at 570 nm. IC50 values are derived from dose-response curves [1]
- Intracellular metabolite analysis: MTAP-deleted cells are treated with AG-270 (10–100 nM) for 24 h, then lysed with cold methanol. Lysates are centrifuged, and supernatants are analyzed by LC-MS/MS to quantify SAM and SAH levels [1] - Apoptosis and cell cycle assay: HCT116 cells are treated with AG-270 (50 nM) for 72 h, harvested, and stained with Annexin V-FITC/PI (apoptosis) or propidium iodide (cell cycle). Samples are analyzed by flow cytometry to determine apoptotic cell percentage and cell cycle distribution [1] - Combination proliferation assay: MTAP-deleted pancreatic cancer cells are treated with AG-270 (0.5–10 nM) in combination with gemcitabine (1–20 nM) or paclitaxel (0.1–2 nM) for 72 h. Cell viability is measured, and combination index is calculated using the Chou-Talalay method [2] |
| Animal Protocol |
Animal/Disease Models: Pancreatic KP4 MTAP-null xenograft mouse model [1].
Doses: 10-200 mg/kg. Route of Administration: po (po (oral gavage)) one time/day for 38 days. Experimental Results: Resulting in a dose-dependent reduction in tumor SAM levels and tumor growth in KP4 MTAP-null xenografts (TGI = 36% (10 mg/kg), 48% (30 mg/kg), 66% (100 mg/kg) ), 67% (200 mg/kg). Xenograft tumor model: Female nude mice (6–8 weeks old) are subcutaneously injected with 5 × 106 MTAP-deleted HCT116 or MiaPaCa-2 cells. When tumors reach 100–150 mm3, mice are randomized into vehicle and treatment groups (n = 7 per group). AG-270 is formulated as an oral suspension in 0.5% hydroxypropyl methylcellulose/0.1% Tween 80 and administered at 25–40 mg/kg once daily for 21–28 days. Tumor volume is measured every 3 days [1] - PDX model (monotherapy): Patient-derived MTAP-deleted pancreatic cancer tissue is implanted subcutaneously into nude mice. Once tumors reach 150–200 mm3, mice receive AG-270 (30 mg/kg, oral, daily) for 28 days. Tumor weight and volume are recorded, and tumor tissue is collected for metabolite analysis [2] - PDX combination model: MTAP-deleted NSCLC or pancreatic cancer PDX mice are treated with AG-270 (25 mg/kg, oral, daily) plus paclitaxel (10 mg/kg, intravenous, weekly) or gemcitabine (100 mg/kg, intraperitoneal, weekly) for 4 weeks. Vehicle control groups receive either single-agent treatment or combination vehicle. Tumor growth inhibition is calculated relative to control [2] |
| ADME/Pharmacokinetics |
In mice, the bioavailability of oral AG-270 (25 mg/kg) was 78 ± 6%, reaching Cmax = 2.8 ± 0.4 μM 1.5 hours after administration [1] - The plasma half-life (t1/2) was 4.2 ± 0.7 hours (mice), 6.8 ± 1.1 hours (rats) and 9.5 ± 1.3 hours (dogs); the AUC0–24h in mice was 12.6 ± 1.8 μM·h [1] - Tissue distribution analysis in mice showed high accumulation of the drug in the liver (tissue/plasma ratio = 5.3 ± 0.8), moderate accumulation in the kidney (3.1 ± 0.5) and tumor (2.7 ± 0.4), and low accumulation in brain tissue (0.2 ± 0.1) [1] - Human liver microsomal metabolism studies showed that AG-270 It is mainly metabolized by CYP3A4 and CYP2C9, and has no significant inhibitory effect on major CYP450 isoenzymes [1]
- Approximately 15% of the total drug clearance is excreted by the kidneys of rats [1] |
| Toxicity/Toxicokinetics |
In a 28-day repeated-dose toxicity study in rats (oral doses of 10, 30, and 100 mg/kg/day), AG-270 did not cause significant weight loss or death; mild increases in ALT and AST (≤1.5 times the upper limit of normal) were observed at a dose of 100 mg/kg [1]. In dogs (28-day study, doses of 5, 15, and 50 mg/kg/day), no adverse effects on hematological parameters, renal function, or histopathology were observed at doses up to 50 mg/kg/day [1]. The plasma protein binding rates of AG-270, as determined by balanced dialysis, were 91 ± 2% in human plasma, 89 ± 3% in rat plasma, and 90 ± 2% in canine plasma [1]. In dogs, no significant prolongation of the QT interval was observed at doses up to 50 mg/kg/day [1].
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| References | |
| Additional Infomation |
AG-270, an oral small molecule methionine adenosine transferase IIα (MAT2A) inhibitor, has potential antitumor activity. AG-270 inhibits the activity of MAT2A after administration. MAT2A is a metabolic enzyme responsible for the production of S-adenosine-L-methionine (SAM). SAM is the main donor of methyl groups in cellular transmethylation reactions that regulate gene expression, cell growth, and differentiation. MAT2A activity is particularly important in cancer cells deficient in methylthioadenosine phosphorylase (MTAP). MTAP is a key enzyme in the methionine rescue pathway and is lost in some human cancers. Inhibition of MAT2A may suppress the growth of tumor cells in MTAP-deficient cancers that heavily depend on SAM synthesis. AG-270 is a first-in-class oral MAT2A inhibitor designed to treat MTAP homozygous deficient tumors in which SAM biosynthesis depends on MAT2A[1].
- Its mechanism of action involves the depletion of SAM, a key cofactor in DNA and histone methylation, leading to transcriptional dysregulation and cell death in MTAP-deficient tumor cells [1]. - AG-270 has shown clinical potential for treating MTAP-deficient solid tumors (pancreatic cancer, non-small cell lung cancer, colorectal cancer) as a monotherapy or in combination with taxanes or gemcitabine [2]. - In preclinical studies, AG-270 exhibited extremely low off-target activity, which gives it a favorable toxicity profile [1]. |
| Molecular Formula |
C30H31N5O2
|
|---|---|
| Molecular Weight |
493.5994
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| Exact Mass |
489.216
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| CAS # |
2201056-66-6
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| Related CAS # |
2201056-66-6 (free);2761546-65-8 (sodium);
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| PubChem CID |
134307820
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| Appearance |
White to off-white solid powder
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| LogP |
5.6
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| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
6
|
| Rotatable Bond Count |
6
|
| Heavy Atom Count |
37
|
| Complexity |
997
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
O=C1C(C2C([H])=C([H])C(=C([H])C=2[H])OC([H])([H])[H])=C(N([H])C2=C([H])C([H])=C([H])C([H])=N2)N([H])C2([H])C([H])(C3=C([H])C([H])([H])C([H])([H])C([H])([H])C3([H])[H])C([H])(C3C([H])=C([H])C([H])=C([H])C=3[H])N([H])N21
|
| InChi Key |
LSOYYWKBUKXUHQ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C30H27N5O2/c1-37-23-17-15-21(16-18-23)26-28(32-24-14-8-9-19-31-24)33-29-25(20-10-4-2-5-11-20)27(34-35(29)30(26)36)22-12-6-3-7-13-22/h3,6-10,12-19,34H,2,4-5,11H2,1H3,(H,31,32)
|
| Chemical Name |
3-(cyclohexen-1-yl)-6-(4-methoxyphenyl)-2-phenyl-5-(pyridin-2-ylamino)-1H-pyrazolo[1,5-a]pyrimidin-7-one
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| Synonyms |
AG-270AG 270AG270
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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 : ~4 mg/mL (~8.17 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 4.75 mg/mL (9.70 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 47.5 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: ≥ 4.75 mg/mL (9.70 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 47.5 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 | 2.0259 mL | 10.1297 mL | 20.2593 mL | |
| 5 mM | 0.4052 mL | 2.0259 mL | 4.0519 mL | |
| 10 mM | 0.2026 mL | 1.0130 mL | 2.0259 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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