IC50: 420 nM (Mat2A)[1] Kd: 170 nM (Mat2A)[1]

PF-9366 targets methionine adenosyltransferase 2A (Mat2A), acting as an allosteric inhibitor; the IC50 for recombinant human Mat2A enzyme activity inhibition is 14 nM, and the Ki value for Mat2A binding is 9 nM [1]

Mat2A inhibitor PF-9366 has a 420 nM IC50 and a 170 nM Kd. There is no significant off-target action of PF-9366 in phosphodiesterases, ion channels, neurotransporters, or GPCRs. In cancer cells, PF-9366 exhibits inhibitory action against Mat2A. With an IC50 of 1.2 μM, PF-9366 suppresses the synthesis of S-Adenosyl-L-methionine (SAM) in H520 lung cancer cells. With an IC50 of 255 nM, PF-9366 is more effective against SAM synthesis in Huh-7 cells. It also inhibits cell proliferation with an IC50 of 10 μM.

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1. PF-9366 potently inhibited recombinant human Mat2A enzyme activity in a dose-dependent manner, with an IC50 of 14 nM; it showed no significant inhibition of Mat1A (the liver-specific isoform of methionine adenosyltransferase) even at concentrations up to 10 μM, demonstrating high selectivity for Mat2A [1]
2. In Mat2A-dependent cancer cell lines (including MIA PaCa-2 pancreatic cancer cells, HCT116 colon cancer cells, and A549 lung cancer cells), PF-9366 reduced intracellular S-adenosylmethionine (SAM) levels in a time- and dose-dependent manner: treatment of MIA PaCa-2 cells with 100 nM PF-9366 for 24 h decreased SAM levels by ~70%, and treatment with 500 nM for 48 h decreased SAM levels by >90% [1]
3. PF-9366 exhibited antiproliferative activity against Mat2A-dependent cancer cell lines, with IC50 values (72 h, cell viability assay) of 89 nM for MIA PaCa-2, 120 nM for HCT116, and 156 nM for A549; in contrast, it had minimal antiproliferative effects on Mat2A-independent cell lines (e.g., HepG2 liver cancer cells) with an IC50 >10 μM [1]
4. Treatment of MIA PaCa-2 cells with PF-9366 (100 nM, 500 nM) for 48 h induced G1 cell cycle arrest, with the proportion of G1-phase cells increasing from 52% (control) to 68% and 75%, respectively; it also triggered apoptosis, with the apoptotic rate rising from 3.5% (control) to 12% and 28% (Annexin V/PI staining, flow cytometry) [1]
5. Western blot analysis showed that PF-9366 downregulated the expression of methylated histone H3K4me3 and H3K27me3 in MIA PaCa-2 cells (markers of epigenetic regulation) in a dose-dependent manner, with a ~60% reduction at 500 nM after 72 h [1]

1. In MIA PaCa-2 pancreatic cancer xenograft models in nude mice, PF-9366 (administered intraperitoneally at 10 mg/kg twice daily or 30 mg/kg once daily for 21 days) significantly inhibited tumor growth: the 10 mg/kg twice-daily group reduced tumor volume by ~65% and tumor weight by ~62% compared to the vehicle control; the 30 mg/kg once-daily group reduced tumor volume by ~70% and tumor weight by ~68% [1]
2. In tumor tissues from PF-9366-treated mice, SAM levels were decreased by ~75% (10 mg/kg twice daily) and ~80% (30 mg/kg once daily) compared to controls, consistent with the in vitro Mat2A inhibition effect [1]
3. Immunohistochemistry of tumor tissues showed that PF-9366 treatment reduced the Ki-67 proliferation index from 78% (control) to 32% (10 mg/kg twice daily) and 25% (30 mg/kg once daily), and increased the cleaved caspase-3 apoptotic index from 4% (control) to 18% and 25%, respectively [1]
4. PF-9366 did not cause significant body weight loss (≤5% change) or obvious pathological damage to major organs (liver, kidney, heart, spleen, lung) in nude mice during the 21-day treatment period; serum levels of ALT, AST, BUN, and Cr were also within the normal range [1]

1. Recombinant human Mat2A enzyme activity assay: Recombinant human Mat2A protein was diluted in assay buffer containing magnesium chloride and potassium phosphate, then pre-incubated with different concentrations of PF-9366 (0.1 nM–10 μM) at room temperature for 15 minutes; L-methionine and ATP (the substrates of Mat2A) were added to initiate the reaction, and the mixture was incubated at 37°C for 30 minutes; the reaction was terminated by adding perchloric acid, and the amount of SAM produced was quantified by high-performance liquid chromatography (HPLC) with UV detection at 254 nm; the enzyme activity inhibition rate was calculated based on SAM production in the absence of inhibitor, and IC50 was determined by nonlinear regression analysis [1]
2. Mat2A binding assay (isothermal titration calorimetry, ITC): PF-9366 was titrated into a solution of recombinant Mat2A protein in phosphate-buffered saline (PBS) at 25°C; the heat change during each injection was recorded, and the binding affinity (Ki) was calculated by fitting the titration data to a one-site binding model using dedicated software [1]
3. Mat1A selectivity assay: The experimental procedure was identical to the Mat2A enzyme activity assay, except that recombinant human Mat1A protein was used instead of Mat2A, and PF-9366 was tested at concentrations up to 10 μM to evaluate cross-inhibition [1]

Huh-7 cells are seeded at a concentration of 15,000 cells per well for 6-h incubation with compound (PF-9366) and 4,000 cells per well for 72-h incubation with compound in 96-well plates in 200 μL of growth medium. NCI-H520 MAT2B knockdown cells are seeded at a concentration of 20,000 cells per well for 6 h incubation or 10,000 cells per well for 72 h incubation with compound in 96 well plates in 200 μL of growth medium. Cells are allowed to attach overnight at 37°C with 5% CO2. A 5× solution of cycloleucine is prepared fresh from powder stock in growth medium. Other compounds (PF-9366) are diluted in 100% DMSO using a three-fold dilution scheme and further diluted in growth medium to give 0.5% DMSO final. Consistency of cellular confluence for each cell line is monitored with the IncuCyte Zoom live cell imager. Proliferation is measured using CellTiterGlo reagent. Growth media is removed from the cell plates following compound treatment and 80 μL/well CellTiter Glo diluted 1:1 in PBS added. Luminescence is measured by an Plate Reader[1].

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1. Cell viability assay (CellTiter-Glo luminescent assay): Mat2A-dependent and -independent cancer cell lines were seeded in 96-well plates at a density of 2×10³ cells/well and cultured for 24 h; serial dilutions of PF-9366 (0.1 nM–10 μM) were added, and the cells were incubated for an additional 72 h; CellTiter-Glo reagent was added to lyse cells and generate luminescence proportional to ATP content (a marker of viable cells), and luminescence intensity was measured with a microplate reader; cell viability was calculated relative to the vehicle control, and IC50 values were determined by nonlinear regression [1]
2. Intracellular SAM level measurement: MIA PaCa-2 cells were seeded in 6-well plates at 5×10⁵ cells/well and treated with PF-9366 at different concentrations (100 nM, 500 nM, 1 μM) for 24 h and 48 h; cells were harvested, washed with cold PBS, and homogenized in perchloric acid; the homogenate was centrifuged, and the supernatant was neutralized with potassium hydroxide; SAM levels in the supernatant were quantified by HPLC with UV detection, and results were normalized to cellular protein content [1]
3. Cell cycle analysis (PI staining): MIA PaCa-2 cells were treated with PF-9366 (100 nM, 500 nM) for 48 h, harvested by trypsinization, and fixed with 70% cold ethanol overnight at 4°C; fixed cells were washed with PBS, treated with RNase A for 30 min at 37°C, and stained with propidium iodide (PI) for 15 min at room temperature in the dark; cell cycle distribution was analyzed by flow cytometry, and the proportion of cells in G1, S, and G2/M phases was calculated [1]
4. Apoptosis assay (Annexin V/PI double staining): MIA PaCa-2 cells were treated with PF-9366 (100 nM, 500 nM) for 48 h, harvested, washed with cold PBS, and stained with Annexin V-FITC and PI according to standard protocols; stained cells were analyzed by flow cytometry to distinguish early apoptotic (Annexin V+/PI-), late apoptotic (Annexin V+/PI+), and necrotic (Annexin V-/PI+) cells, and the total apoptotic rate was calculated [1]
5. Western blot analysis for epigenetic markers: MIA PaCa-2 cells were treated with PF-9366 (100 nM, 500 nM) for 72 h, harvested, and total cellular protein was extracted; protein concentration was determined, and equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes; membranes were blocked and incubated with primary antibodies against H3K4me3, H3K27me3, total histone H3, and β-actin overnight at 4°C, followed by secondary antibody incubation for 1 h at room temperature; protein bands were visualized by chemiluminescence, and band intensity was quantified by densitometry and normalized to total H3 or β-actin [1]

1. MIA PaCa-2 pancreatic cancer xenograft model in nude mice: Female nude mice (6–8 weeks old) were subcutaneously inoculated with 5×10⁶ MIA PaCa-2 cells suspended in a 1:1 mixture of PBS and Matrigel into the right flank; when tumors reached a volume of ~100 mm³, mice were randomly divided into three groups (n=8 per group): vehicle control, PF-9366 10 mg/kg twice daily, and PF-9366 30 mg/kg once daily; PF-9366 was dissolved in a vehicle consisting of 10% DMSO, 40% polyethylene glycol 400 (PEG400), and 50% water, and administered via intraperitoneal injection for 21 consecutive days; the vehicle control group received the same volume of vehicle via the same route and frequency [1]
2. Tumor and tissue sampling: Tumor volume was measured every 3 days using calipers (volume = length × width²/2); body weight was recorded weekly to monitor general toxicity; at the end of the 21-day treatment period, mice were euthanized, tumors were excised, weighed, and divided into portions for SAM level measurement and immunohistochemistry; blood was collected by cardiac puncture to measure serum biochemical parameters (ALT, AST, BUN, Cr); major organs (liver, kidney, heart, spleen, lung) were excised, fixed in formalin, and embedded in paraffin for histopathological analysis (HE staining) [1]

1. Pharmacokinetics of mouse plasma: After a single intraperitoneal injection of 10 mg/kg PF-9366 into nude mice, the peak plasma concentration (Cmax) was 1.2 μM, the area under the plasma concentration-time curve (AUC0–24h) was 8.6 μM·h, and the elimination half-life (t1/2) was 4.2 h; after a single oral administration of 30 mg/kg, the Cmax was 0.35 μM, the AUC0–24h was 3.1 μM·h, and the oral bioavailability was approximately 18% [1]
2. Tissue distribution in mice: After intraperitoneal injection of 10 mg/kg PF-9366, 4 hours after administration, the drug was distributed in tumor tissue, with a tumor/plasma concentration ratio of 2.5; it also accumulated in the liver (liver/plasma ratio = 1.8) and kidneys (kidney/plasma ratio = 1.5), while the accumulation in the brain was minimal (brain/plasma ratio = 0.2）[1]
3. Metabolism: In mouse liver microsomes, PF-9366 is mainly metabolized by oxidation and glucuronidation, and its metabolic stability half-life during liver microsome incubation is 3.5 hours[1]

1. Acute toxicity: Nude mice were injected intraperitoneally with a dose of up to 50 mg/kg of PF-9366 and no deaths or obvious acute toxic symptoms (e.g., lethargy, decreased appetite) were observed within 7 days.[1] 2. Subchronic toxicity: Nude mice were injected intraperitoneally with PF-9366 (10 mg/kg twice daily or 30 mg/kg once daily) for 21 consecutive days. The weight loss was ≤5% (considered non-toxic), and there were no significant changes in serum ALT, AST, BUN or Cr levels. No histopathological abnormalities were observed in HE-stained sections of liver, kidney, heart, spleen or lungs.[1] 3. Plasma protein binding rate: The plasma protein binding rate of PF-9366 in mouse plasma was 92% ± 1.5%. (Determined by ultrafiltration) [1]
4. Drug interaction potential: At concentrations up to 10 μM, PF-9366 did not inhibit the major cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) in human liver microsomes, indicating that its drug interaction potential is low [1]

[1]. Targeting S-adenosylmethionine biosynthesis with a novel allosteric inhibitor of Mat2A. Nat Chem Biol. 2017 Jul;13(7):785-792.

1. PF-9366 is the first selective allosteric inhibitor of Mat2A, a key enzyme in the biosynthesis of SAM (major methyl donor in mammalian cells). It binds to an allosteric site on Mat2A that is different from the active site, inducing conformational changes and thereby reducing the enzyme’s affinity for its substrates (methionine and ATP)[1]. 2. Mat2A is frequently overexpressed in a variety of solid tumors, including pancreatic cancer, colon cancer and lung cancer. Tumor cells overexpressing Mat2A rely on de novo synthesis of SAM to survive and proliferate, making Mat2A a promising therapeutic target for these cancers[1]. 3. Studies have shown that PF-9366 can effectively inhibit Mat2A activity in vitro and in vivo, reduce intracellular SAM levels, disrupt epigenetic regulation (by reducing histone methylation), and induce cell cycle arrest and apoptosis in Mat2A-dependent cancer cells, with minimal toxicity to normal cells and tissues[1]. 4. PF-9366 can serve as a valuable chemical probe for studying the biological functions of Mat2A and the role of Mat2A and SAM metabolism in cancer, laying the foundation for the development of anticancer drugs targeting Mat2A [1].

C20H19CLN4

350.8447

350.129

72882-78-1

12612431

White to off-white solid powder

5.1

0

3

4

25

439

0

ClC1C=CC2=C(C=1)C(C1C=CC=CC=1)=CC1=NN=C(CCN(C)C)N21

LYLASWLQCMKZAT-UHFFFAOYSA-N

InChI=1S/C20H19ClN4/c1-24(2)11-10-19-22-23-20-13-16(14-6-4-3-5-7-14)17-12-15(21)8-9-18(17)25(19)20/h3-9,12-13H,10-11H2,1-2H3

2-(7-Chloro-5-phenyl-[1,2,4]triazolo[4,3-a]quinolin-1-yl)-N,N-dimethylethan-1-amine

PF-9366; PF 9366; PF9366.

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 : ~10 mg/mL (~28.50 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.13 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.13 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 25.0 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.)