METTL3 (IC50 = 16.9 nM)

Compound 72, STM2457, has an IC50 of 8.699 μM, which inhibits MOLM13 cell proliferation[1].
we present the identification and characterisation of a highly potent and selective first-in-class catalytic inhibitor of METTL3 (STM2457) and its co-crystal structure bound to METTL3/METTL14. Treatment with STM2457 leads to reduced AML growth, and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect[2].

Following the positive evidence for strong pharmacological inhibition of METTL3 in vitro, we performed in vivo studies using clinically relevant AML models. Initially, we utilised 3 human AML patient derived xenografts (PDX) of different genotypes. Daily treatment with STM2457 led to impairment of engraftment and AML expansion in vivo and significantly prolonged the mouse lifespan (Fig. 4a-d and Extended Data Fig. 6a-e) with no overt toxicity or effect on mouse body weight (Extended Data Fig. 6f). The anti-leukaemic effect was also confirmed by the reduction of human CD45+ cells in bone marrow and spleen following treatment (Fig. 4e). Effective METTL3 target inhibition by STM2457 in vivo was demonstrated by the selective reduction of key METTL3 m6A substrates at the protein level while METTL3 levels remained unchanged (Extended Data Fig. 6g). Additionally, total m6A levels on poly-A+-enriched RNA were significantly reduced following treatment with STM2457 (Extended Data Fig. 6h). In parallel with the PDX models, we used a primary murine MLL-AF9/Flt3Itd/+ in vivo model with similar anti-leukaemic observations regarding reduction in the engrafted AML cells, reduction in spleen size, selective reduction of METTL3 biomarkers as well as reduction of m6A on poly-A+-enriched RNA (Extended Data Fig. 7a-d)[2].

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Finally, we evaluated the potential toxicity of the established anti-leukaemic dose of STM2457 in vivo. No significant changes were observed in the numbers of bone marrow-derived hematopoietic stem cells (HSCs) and early progenitors (Lin−, Sca1+, Kit+), peripheral blood counts or mouse body weight (Extended Data Fig. 8a-e). We also confirmed effective catalytic inhibition of METTL3 as the m6A levels on poly-A+-enriched RNA were significantly reduced after treatment with STM2457 (Extended Data Fig. 8f). These data suggest that small molecule inhibition of METTL3 is detrimental for the maintenance of AML, but has no significant or lasting impact on normal haematopoiesis[2].

METTL3/14 RF/MS methyltransferase assay[2]
The enzymatic assay was established to determine IC50 values for the inhibition of RNA methyltransferase activity. The enzyme used was full-length his-tagged METTL3 co-expressed with full length FLAG-tagged METTL14 in a baculovirus expression system. The enzyme complex was purified using standard affinity chromatography. Enzymatic reactions were performed at room temperature in 384-well plates using a final reaction volume of 20 μL containing 20 mM TrisCl pH 7.6, 1 mM DTT, 0.01% Tween-20. 5 nM final concentration of METTL3/14 was pre-incubated with various compound concentrations for 10 minutes, followed by addition of 0.2 μM final concentration synthetic RNA substrate (5’P-uacacucgaucuggacuaaagcugcuc-3’) and 0.5 μM final concentration S-adenosyl methionine (SAM). The reaction was incubated for further 60 minutes at room temperature, and then quenched by the addition of 40 μL 7.5% TCA with internal standard. After termination, plates were sealed, centrifuged and stored at 4°C until analysis.
METTL3 activity was assessed using the RapidFire™ mass spectrometry (RF/MS) platform to measure the S-adenosyl homocysteine (SAH) product. Stopped and stable assay plates were analyzed on the Agilent RF300 integrated autosampler/solid-phase extraction (SPE) system coupled to an ABSciex 4000 mass spectrometer for the quantification of the SAH and normalized to the ratio of signal of two internal standards. The mass transition for the product (SAH) was 384.9/135.9 Da. Transitions of the internal standard were used for normalization of matrix effects.[2]

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STM2457 selectivity profiling[2]
The selectivity profile of STM2457 was assessed by testing the level of inhibition in a panel of methyltransferases and kinases. Inhibition of 4 RNA methyltransferases was tested at Evotec AG, Hamburg using RFMS assays equivalent to the METTL3 assay described above. The IC50 was determined from a dilution series of STM2457 with a top concentration of 120 μM and the degree of inhibition at 10μM compound was inferred from this. Additionally, the level of inhibition of a panel of 41 DNA and protein methyltransferases was assessed in a radiometric assay measuring substrate methylation using tritiated SAM by Reaction Biology at 10 μM STM2457 in duplicate.

METTL3 cellular target engagement[2]
Cellular target engagement of STM2457 was measured by thermal shift using the InCell Pulse Assay (DiscoverX). HeLa cells were transfected with pICP-hMETTL3-eLP (human METTL3 Met1-Leu580) or pICP-mMETTL3-eLP (mouse METTL3 Met1-Leu580), using Fugene HD following the supplier’s protocol. 24 hours post transfection, cells were frozen and stored in liquid nitrogen until the day of the assay. On the day of the assay 100 nL compound dilutions (11 point 3-fold dilutions, top concentration in assay: 25 μM) were spotted into the assay plate (384-well, PP, transparent). The transfected cells were thawed in a water bath at 37°C and cryoprotectant was exchanged with Opti-MEM lacking phenol red. 20 μL of the cell suspension was added to each well of the assay plate to give a final cell number of 1360 cells/well. Plates were sealed with aluminium foil and incubated for 1 hour at 37°C. Afterwards, plates were incubated for 15 minutes upside down in a water bath at 45°C, further incubated for 10 minutes at room temperature, and finally centrifuged briefly to gather the liquid in the bottom of each well. Subsequently, 25 μL of detection solution (working solution: 16.7 v/v EA reagent, 16.7 v/v lysis buffer and 66.7% substrate reagent) was added to each well and the solution was transferred to the measurement plate (384-well, PS, black, Flat Bottom). The plate was sealed with aluminium foil and incubated at room temperature and slow shaking for 30 minutes. Finally, the assay plate was centrifuged for 1 minute at 100 x g and room temperature and luminescence was measured using an EnVision multimode plate reader. Dose response curves were obtained from three biological replicates.

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Flow cytometry analyses of AML cells[2]
Cells were treated with vehicle (DMSO) or STM2457 and stained at the indicated timepoints with anti-mouse CD11b PE/Cy5 and anti-human CD11b PE. Data were analyzed using LSRFortessa and FlowJo.
Apoptosis levels were measured in human and/or mouse AML cells treated with vehicle (DMSO) or STM2457 at indicated timepoints, using Annexin V. Data were analyzed by using LSRFortessa instruments.
Cell cycle levels were measured in human and/or mouse AML cells treated with vehicle (DMSO) or STM2457 at indicated timepoints, by using bromodeoxyuridine (BrdU) using the FITC BrdU Flow Kit or the APC BrdU Flow Kit. Data were analyzed by using LSRFortessa instruments.

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Western blot analysis[2]
Cells were treated with Vehicle (DMSO) or the indicated concentrations of STM2457 and after 72 hours cell pellets resuspended in whole cell lysis buffer (50 mM Tris-HCl pH=8, 450 mM NaCl, 0.1% NP-40, 1mM EDTA), supplemented with 1 mM DTT, protease inhibitors, and phosphatase inhibitors. Protein concentrations were assessed by Bradford assay and an equal amount of protein was loaded per track. Prior to loading, the samples were supplemented with SDS-PAGE sample buffer and DTT was added to each sample. 10-40 μg of protein was separated on SDS-PAGE gels, and blotted onto polyvinylidene difluoride membranes.

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Drug and Proliferation Assays[2]
All suspension cells were plated in 96-well plates in triplicate at 5,000–10,000 cells per well and treated for 72 hours with vehicle or the indicated concentrations of STM2457 and STM2120 (0.04-50 μM). On day 4, an equal volume for all wells was split using fresh media and compound, such that the resulting cell density in each well matched the initial seeding density. Plates were measured on day 6 using CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay in order to calculate the relative cell proliferation. All the compounds were dissolved in DMSO.

For PDX experiments related to Figure 4 and Extended Data 6, 6- to 10-week-old NSG female mice were injected with 106 patient-derived AML cells by intravenous injection. For primary transplants, indicated doses of STM2457 or vehicle were delivered to the mice via intraperitoneal injection (IP) on day 5 (PDX-2) or day 10 post-transplant (PDX1,3), once daily for total 12 or 14 days (12-14 treatments). Then bone marrow and spleen cells from these mice were freshly dissected (as mentioned above) and flow-cytometry analysis was performed after staining with various antibodies.[2]

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For primary and secondary transplantation experiments using primary murine MLL-AF9/Flt3ITD/+, 6- to 10-week-old NSG female mice were injected with 106 AML cells by intravenous injection. For primary transplants, indicated doses of STM2457 or vehicle were delivered to the mice via intraperitoneal injection (IP) on day 7 post-transplant, once daily for total 10 days. Then bone marrow cells from these mice was freshly dissected (as mentioned above) and blocked with anti-mouse CD16/32 (BD Pharmigen, cat. no. 101323) and 10% mouse serum. For the identification of L-GMP and CD93 populations, staining was performed using various antibodies.[2]

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Generation and bioluminescent imaging of primary murine and PDX models[2]
Generation of AML PDX models and lentiviral transduction for transgenic expression of enhanced firefly luciferase was performed as described in detail by Vick, et al. 38. For primary and secondary transplantation experiments using primary murine MLL-AF9/Flt3ITD/+, 6- to 10-week-old NSG female mice were injected with 106 AML cells by intravenous injection. Indicated doses of STM2457 or vehicle were delivered to the mice via intraperitoneal injection (IP) on day 10 post-transplant, once daily for total two weeks (14 treatments). STM2457 was dissolved in 20%(w/v) 2-hydroxyproply beta-cyclodextrin vehicle. At day 10 post-transplant, the tumor burdens of the animals were detected using IVIS Lumina II with Living Image version 4.3.1 software. Briefly, 100 μl of 30 mg/ml D-luciferin was injected into the animals intraperitoneally. Ten min after injection, the animals were maintained in general anesthesia by isoflurane and put into the IVIS chamber for imaging. The detected tumor burdens were measured and quantified by the same software. Diseased mice were assessed blindly by qualified animal technicians from the Sanger mouse facility. Mice were housed in specific pathogen-free conditions in the Wellcome Sanger Institute animal facilities. All cages were on a 12:12-h light:dark cycle (lights on, 07:30) in a temperature-controlled and humidity-controlled room. Room temperature was maintained at 72 ± 2 °F (22.2 ± 1.1 °C), and room humidity was maintained at 30–70%. The animals were culled when the tumor burden was 109 photons per second or higher. All animal studies were carried out in accordance with the Animals Act 1986, UK and approved by the Ethics Committee at the Sanger Institute. Randomization and blinding were not applied. All data in this section were plotted using GraphPad Prism (Version 9).[2]

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STM2457 pharmacokinetic analysis[2]
Three C57BL6/J mice were given IP injections of 30 mg/kg STM2457 and sampled serially up to 24 hours after dosing. Blood was collected from the tail vein at the indicated timepoints. Plasma was isolated by centrifugation, and 20 μL of plasma or blood was mixed with a precipitant solution of 120 μL acetonitrile and internal standard. Supernatant from this precipitation was diluted 1:1 v/v in water and 2.5 uL injections were characterised by LC-MS on a TSQ triple quadrupole mass spectrometer attached to an Accela pump and an HTS-CTC PAL autosampler. STM2457 was resolved on Hypersil Gold C18 solid phase (30 X 2.1 mm, 1.9 μm particles) with an increasing gradient of 5-95% B over 30 seconds. Mobile phases consisted of 0.1% formic acid in water (A) and acetonitrile (B) and the flow was held at 1.0 ml/min. Blood to plasma ratio was determined using results from the appropriate samples, and the LLOQ was set at 10 ng/ml.

[1]. Mettl3 inhibitory compounds. WO2020201773A1.

[2]. Small molecule inhibition of METTL3 as a strategy against myeloid leukaemia. Nature. 2021 Apr 26.

STM2457 is a secondary carboxamide resulting from the formal condensation of the carboxy group of 4-oxo-4H-pyrido[1,2-a]pyrimidine-2-carboxylic acid with the primary amino group of 1-[2-(aminomethyl)imidazo[1,2-a]pyridin-6-yl]-N-(cyclohexylmethyl)methanamine. It is a highly potent and selective first-in-class small molecule inhibitor of METTL3 (N6-adenosine-methyltransferase 70 kDa subunit) enzyme activity and exhibits anti-leukaemic activity. It has a role as an EC 2.1.1.348 (mRNA m6A methyltransferase) inhibitor, an antineoplastic agent and an apoptosis inducer. It is an imidazopyridine, a secondary carboxamide, a secondary amino compound and a pyridopyrimidine.

C25H28N6O2

444.5288

444.23

C, 67.55; H, 6.35; N, 18.91; O, 7.20

2499663-01-1

155167581

White to yellow solid powder

2.7

2

5

7

33

872

0

OBERVORNENYOLE-UHFFFAOYSA-N

InChI=1S/C25H28N6O2/c32-24-12-21(29-23-8-4-5-11-31(23)24)25(33)27-15-20-17-30-16-19(9-10-22(30)28-20)14-26-13-18-6-2-1-3-7-18/h4-5,8-12,16-18,26H,1-3,6-7,13-15H2,(H,27,33)

N-((6-(((cyclohexylmethyl)amino)methyl)imidazo[1,2-a]pyridin-2-yl)methyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-2-carboxamide

STM-2457; STM2457; CHEMBL5291234; STM-2457; N-((6-(((cyclohexylmethyl)amino)methyl)imidazo[1,2-a]pyridin-2-yl)methyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-2-carboxamide; N-[(6-{[(cyclohexylmethyl)amino]methyl}imidazo[1,2-a]pyridin-2-yl)methyl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-2-carboxamide; SCHEMBL22499068; GTPL11529; STM 2457

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 : ~50 mg/mL (~112.48 mM)
Ethanol : ~89 mg/mL (200.2 mM)
Water : Insoluble (<1 mg/mL)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.68 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 (4.68 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 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 (4.68 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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Solubility in Formulation 4: 5 mg/mL (11.25 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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