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Discovery of AG-270, a First-in-Class Oral MAT2A Inhibitor for Treatment of Tumors

Discovery of AG-270, a First-in-Class Oral MAT2A Inhibitor for the Treatment of Tumors with Homozygous MTAP Deletion


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, which is adjacent to the CDKN2A tumor suppressor and codeleted with CDKN2A in approximately 15% of all cancers. Previous attempts to target MAT2A with small-molecule inhibitors identified cellular adaptations that blunted their efficacy. Here, they report the discovery of highly potent, selective, orally bioavailable MAT2A inhibitors that overcome these challenges. Fragment screening followed by iterative structure-guided design enabled >10 000-fold improvement in potency of a family of allosteric MAT2A inhibitors that are substrate noncompetitive and inhibit release of the product, S-adenosyl methionine (SAM), from the enzyme’s active site. They demonstrate that potent MAT2A inhibitors substantially reduce SAM levels in cancer cells and selectively block proliferation of MTAP-null cells both in tissue culture and xenograft tumors.


Graphical Abstract

The methylthioadenosine phosphorylase (MTAP) gene is located adjacent to the CDKN2A tumor suppressor and is codeleted with CDKN2A in approximately 15% of all cancers, leading to aggressive tumors with poor prognoses for which no effective molecularly targeted therapies exist.The metabolic enzyme methionine adenosyltransferase 2A (MAT2A) has an important role in metabolism and epigenetics because it is the primary producer of the universal methyl donor S-adenosyl methionine (SAM). Recent work has demonstrated that depletion of MAT2A using RNA interference leads to a selective antiproliferative effect in cancers with deletion of MTAP. A simple explanation for this selective vulnerability has been described, in which the activity of the SAM-utilizing type II protein arginine N-methyltransferase 5 (PRMT5) is inhibited by the MTAP substrate, 5′-methylthioadenosine (MTA), which accumulates when MTAP is deleted. Within this tumor environment, the catalytic activity of the PRMT5 enzyme is reduced, and it becomes vulnerable to further inhibition by reduction of SAM levels, whereas its activity in normal tissues and MTAP-proficient tumors remains largely unaffected.

Hit identification. (A) MAT2A enzyme inhibition IC50 (μM). Values are the mean of three experiments. (B) Surface plasmon resonance sensorgrams (the result of only one experiment each shown). (C) Mechanism of action study demonstrates compound 2 is noncompetitive with regard to ATP and l-methionine substrates; test concentrations of 2 were 0.5, 1.5, and 4.5 μM.


Although these results suggest that targeting MAT2A may prove beneficial in MTAP-deleted cancers, past efforts to devise effective MAT2A inhibitors have been challenging. Methionine analogues such as cycloleucine as well as stilbene derivatives have been reported in the literature to be inhibitors of MAT2A; however, their weak biochemical potency (>10 μM) and very weak cellular activity did not enable their development into useful therapeutics. The recent discovery of a moderately potent allosteric MAT2A inhibitor, PF-9366, demonstrates the potential to drug MAT2A via an allosteric mechanism. Unfortunately, PF-9366 treatment in cells induced cellular adaptation, particularly upregulation of MAT2A itself, which blunted cellular potency and led to inadequate antiproliferative effects.

Herein, they describe the drug discovery efforts that led to the identification of AG-270, a first-in-class, orally bioavailable MAT2A inhibitor currently in clinical development (ClinicalTrials.gov NCT03435250). This class of MAT2A inhibitors is allosteric, substrate noncompetitive, and inhibits MAT2A activity by preventing product release. They also report findings from preclinical studies describing the in vitro and in vivo characterization of this class of inhibitors with a focus on their clinical molecule AG-270.


Cocrystal structure of compound 2 with MAT2A and reaction product SAM (PDB code 7KCE). (A) Homodimeric form of MAT2A with two protein chains shown in green and cyan, respectively. SAM molecules, in yellow, are in the active site and compound 2, in magenta, is in an allosteric site. (B) Allosteric binding site of MAT2A. Compound 2, in magenta, and water interacting with residue Gly215 in red sphere. (C) Structural class pyrazolo[1,5-a]pyrimidin-7(4H)-one and all the substituents to be explored in the SAR campaign.


Cocrystal structure of MAT2A·SAM·AGI-24512. (A) Crystal structure of MAT2A SAM free form. (B) Close-up of the allosteric binding site, highlighting AGI-24512 and its interactions with MAT2A protein and a key water molecule. (C) The current crystal structure obtained with AGI-24512 in the presence of SAM. (D) Close-up of the apo-MAT2A region indicating the “open” form of the α-helix gate that is unstructured in the absence of SAM. (E) Close-up of the active site gate loop (protein in green and yellow, SAM in atom color).


Pharmacologic targeting of MAT2A with in vivo tool molecule selectively blocks growth of MTAP-null tumors in vivo. Fifteen mice per arm (total 30 animals) were used in this study. (A) Key in vitro parameters of AGI-25696. (B) PK data from the study after 3 b.i.d. doses. (C) Mean tumor volume in mice inoculated with KP4 MTAP-null pancreatic cells treated daily with 300 mg/kg AGI-25696 (black line) or vehicle (red line). (D) Mean body weight of the mice. Error bars show standard error of the mean. HLM, human liver microsomes; MLM, mouse liver microsomes.



Are tautomers responsible for the physical chemistry properties? Hypothesis: three possible tautomers of the pyrrolopyrimidinone scaffold (A–C) share the acidic proton in different locations of the scaffold, which can potentially increase the ability of each compound to bind to plasma proteins.



Intramolecular hydrogen bonding improves maximum percentage inhibition. (A) Cocrystal structure of MAT2A·SAM·34 identifies intramolecular hydrogen bond and new interaction with the MAT2A protein. (B) Cocrystal structure of MAT2A·SAM·35 confirms both the intramolecular hydrogen bond.



Cocrystal structure of AG-270. The crystal structure of AG-270 (in yellow) confirms the binding mode, the intramolecular hydrogen bond, and all the key interactions with MAT2A.


Dose-dependence study in KP4 xenograft mouse model reveals that maximal efficacy is related to SAM reduction. Twelve mice were used per arm (60 total animals) in this study. AG-270 was given orally q.d. (on days 24–38) to mice inoculated subcutaneously with KP4 MTAP-null cells, and both compound and SAM levels were monitored. (A) Plasma pharmacokinetics of AG-270 were monitored up to 24 h post last 200 mg/kg dose (solid blue line) and displayed very good coverage; tumor SAM levels were also monitored in the same time period and showed stable, low concentrations (blue dotted line). (B) Tumor volume over a 38-day study. (C) Tumor growth inhibition (TGI) and percentage tumor SAM reduction at various doses indicate that SAM reduction between 60 and 80% leads to the same level of TGI at ∼66%. Reduction in SAM concentration was dose dependent from 10 to 200 mg/kg and in tumor volume from 10 to 100 mg/kg. (D) Mean change in mouse body weight. Error bars show standard error of the mean.


  1. Discovery of AG-270, a First-in-Class Oral MAT2A Inhibitor for the Treatment of Tumors with Homozygous MTAP Deletion Zenon Konteatis, Jeremy Travins, Stefan Gross, Katya Marjon, Amelia Barnett, Everton Mandley, Brandon Nicolay, Raj Nagaraja, Yue Chen, Yabo Sun, Zhixiao Liu, Jie Yu, Zhixiong Ye, Fan Jiang, Wentao Wei, Cheng Fang, Yi Gao, Peter Kalev, Marc L. Hyer, Byron DeLaBarre, Lei Jin, Anil K. Padyana, Lenny Dang, Joshua Murtie, Scott A. Biller, Zhihua Sui, and Kevin M. Marks Journal of Medicinal Chemistry 2021 64 (8), 4430-4449 DOI: 10.1021/acs.jmedchem.0c01895