MAT2A (methionine adenosyltransferase 2A) – enzymatic IC50 = 8 nM (0.0089 μM), enzymatic maximal inhibition = 91% [2]

AGI-24512 (0-1 μM; 96 hours) suppresses the growth of HCT116 cancer cells with an IC50 of 100 nM by blocking MTAP (methylthioadenosine phosphorylase)[1]. AGI-24512 suppresses PRMT5-mediated SDMA labeling in MTAP-/- cells with an IC50 of 95 nM, hence dramatically increasing MTAP-/. In HCT116 MTAP cells, AGI-24512 induces SAM (SAM). With an IC50 of 100 nM, the levels of S-adenosylmethionine) drop in a dose-dependent way [2].

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AGI-24512 treatment in HCT116 MTAP-null cells led to a dose-dependent decrease in S-adenosyl methionine (SAM) levels with an IC50 of 100 nM (0.100 μM) after 4 or 72 hours (Table 3 and text). [2]
AGI-24512 demonstrated MTAP-null selective antiproliferative activity in an HCT116 engineered isogenic cell model (no specific IC50 value provided). [2]
Mode-of-action studies confirmed that AGI-24512, like compound 2, is non-competitive with respect to both ATP and L-methionine substrates (Supporting Information Figure S2). [2]
Upon treatment with AGI-24512, upregulation of MAT2A protein was observed, consistent with previous results with PF-9366. However, this feedback mechanism did not prevent the antiproliferative activity, presumably due to the improved potency of AGI-24512. [2]

AGI-24512 showed limited barrier absorption and inhibitory half-life in malignancies [2].

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MAT2A enzymatic inhibition was measured using a phosphate release assay. The MAT2A homodimer was diluted to 1.25 μg/mL in assay buffer (50 mM Tris pH 8.0, 50 mM KCl, 15 mM MgCl2, 0.3 mM EDTA, 0.005% BSA). Test compound was prepared in 100% DMSO at 50× the final concentration. 1 μL of compound dilution was added to 40 μL of enzyme dilution and equilibrated for 60 min at 25 °C. The reaction was initiated by adding 10 μL of substrate mix (500 μM ATP pH 7.0, 400 μM L-methionine in 1× assay buffer) and incubated for another 60 min at 25 °C. The reaction was stopped, and the phosphate released stoichiometrically with SAM production was measured using a PiColorLock Gold kit. Absolute product amounts were determined by comparison with a standard curve of potassium phosphate buffer pH 8.0. [2]
For mechanism-of-action studies regarding L-methionine, substrate concentrations were varied from 6.25 to 400 μM while ATP was fixed at 100 μM. For ATP mechanism, ATP was varied from 15 μM to 1 mM while L-methionine was fixed at 50 μM. [2]
Surface plasmon resonance (SPR) analysis: MAT2A protein was immobilized onto CMS sensor chips via covalently attached anti-His IgG. The chip surface was activated with EDC/NHS. Penta-anti-His IgG was diluted in sodium acetate pH 5.0 and injected. Active sites were blocked with 1 mM ethanolamine-HCl. MAT2A protein was captured on the prepared surface after dilution in running buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10 mM MgCl2, 0.005% P20, 0.5 mM TCEP). Binding analysis used 100 mM compound stock diluted in running buffer. [2]
Crystallization: MAT2A-SAM complex was generated by mixing 20 mg/mL MAT2A with 2 mM SAM and incubating on ice for 2 h. Crystals were obtained by hanging-drop vapor diffusion with 2 μL of MAT2A-SAM complex mixed with 1 μL of well buffer containing 0.2 M LiCl, 0.1 M Tris-HCl pH 7.8-8.4, 16-22% PEG6000 and 10% ethylene glycol at 18 °C. Inhibitor complexes were obtained by cocrystallization mixing 20 mg/mL MAT2A with 2-25 mM compound, then adding 2 mM SAM and incubating on ice for 2 h. Crystals were cryoprotected in mother liquor with 10% DMSO and flash frozen. The structure of MAT2A-SAM-AGI-24512 was determined at 1.10 Å resolution (PDB code 7KCF). [2]

A cell-based assay using HCT116 MTAP-null cells was developed to assess MAT2A inhibition by measuring the abundance of the MAT2A product SAM following treatment with increasing concentrations of compounds. Cells were treated with AGI-24512 for 4 h or 72 h (as indicated in Tables 1-3) and SAM levels were quantified to determine IC50 values. [2]
Antiproliferative activity was assessed in HCT116 MTAP-null and MTAP-WT isogenic cell models. AGI-24512 showed selective growth inhibition of MTAP-null cells (specific IC50 not provided). [2]

AGI-24512 exhibited poor oral absorption and a short half-life in rats. [2]
Human liver microsomal extraction ratio (ER) for AGI-24512 was 0.66 (Table 4). [2]
Human plasma protein binding (PPB) of AGI-24512 was 99.9% bound, with a free fraction of 0.09% (Table 5). [2]

[1]. MAT2A Inhibition Blocks the Growth of MTAP-Deleted Cancer Cells by Reducing PRMT5-Dependent mRNA Splicing and Inducing DNA Damage. Cancer Cell. 2021 Feb 8;39(2):209-224.e11.

[2]. Discovery of AG-270, a First-in-Class Oral MAT2A Inhibitor for the Treatment of Tumors with Homozygous MTAP Deletion. J Med Chem. 2021 Apr 22;64(8):4430-4449.

AGI-24512 is an allosteric MAT2A inhibitor that is non-competitive with respect to both ATP and L-methionine. Its binding traps the reaction product SAM in the active site by stabilizing the closed conformation of the α-helix gate. The cocrystal structure (PDB code 7KCF) confirmed that the phenol substituent at the C-6 position replaces a water molecule (water-436) that interacts with Gly215. The piperidine group at the C-3 position is encased in a hydrophobic environment created by residues Phe18, Phe20, Phe139, Ser331, Phe333, and Leu315. AGI-24512 was used as an in vitro tool to enable further cellular studies evaluating MAT2A biology. Its improved potency relative to prior MAT2A inhibitors (e.g., PF-9366) allowed it to maintain antiproliferative effects despite cellular adaptation (MAT2A upregulation). [2]

C24H24N4O2

400.472965240479

400.189

2201066-53-5

134307780

White to gray solid powder

4.1

2

5

3

30

779

0

O=C1C(C2C=CC(=CC=2)O)=C(C)N=C2C(=C(C3C=CC=CC=3)NN21)N1CCCCC1

JDHPOXNOROPDTE-UHFFFAOYSA-N

InChI=1S/C24H24N4O2/c1-16-20(17-10-12-19(29)13-11-17)24(30)28-23(25-16)22(27-14-6-3-7-15-27)21(26-28)18-8-4-2-5-9-18/h2,4-5,8-13,25,29H,3,6-7,14-15H2,1H3

6-(4-hydroxyphenyl)-5-methyl-2-phenyl-3-(piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7(4H)-one

AGI-24512 AGI 24512 AGI24512.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~312.13 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.19 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 20.8 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.

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Solubility in Formulation 2: 2.08 mg/mL (5.19 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.19 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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