PF-9366 (PF9366) is a novel, potent and allosteric inhibitor of human methionine adenosyltransferase 2A (Mat2A) with anticancer activity. It inhibits Mat2A with an IC50 of 420 nM and a Kd of 170 nM. Human methionine adenosyltransferase 2A (Mat2A), the extrahepatic isoform, is often deregulated in cancer. We identified a Mat2A inhibitor, PF-9366, binds an allosteric site on Mat2A that overlaps with the binding site for the Mat2A regulator, Mat2B. Studies exploiting PF-9366 suggested a general mode of Mat2A allosteric regulation. Allosteric binding of PF-9366 or Mat2B altered the Mat2A active site, resulting in increased substrate affinity and decreased enzyme turnover. These data support a model whereby Mat2B functions as an inhibitor of Mat2A activity when methionine or SAM levels are high, yet functions as an activator of Mat2A when methionine or SAM levels are low.

Physicochemical Properties

Molecular Formula	C20H19CLN4
Molecular Weight	350.8447
Exact Mass	350.129
CAS #	72882-78-1
PubChem CID	12612431
Appearance	White to off-white solid powder
LogP	5.1
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	4
Heavy Atom Count	25
Complexity	439
Defined Atom Stereocenter Count	0
SMILES	ClC1C=CC2=C(C=1)C(C1C=CC=CC=1)=CC1=NN=C(CCN(C)C)N21
InChi Key	LYLASWLQCMKZAT-UHFFFAOYSA-N
InChi Code	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
Chemical Name	2-(7-Chloro-5-phenyl-[1,2,4]triazolo[4,3-a]quinolin-1-yl)-N,N-dimethylethan-1-amine
Synonyms	PF-9366; PF 9366; PF9366.
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	IC50: 420 nM (Mat2A)[1] Kd: 170 nM (Mat2A)[1]
ln Vitro	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.
Cell Assay	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].
References	[1]. Targeting S-adenosylmethionine biosynthesis with a novel allosteric inhibitor of Mat2A. Nat Chem Biol. 2017 Jul;13(7):785-792.

Solubility Data

Solubility (In Vitro)

DMSO : ~10 mg/mL (~28.50 mM)

Solubility (In Vivo)

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

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.8503 mL	14.2515 mL	28.5030 mL
	5 mM	0.5701 mL	2.8503 mL	5.7006 mL
	10 mM	0.2850 mL	1.4252 mL	2.8503 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.