PRMT5

TNG908 is a potent inhibitor that binds the PRMT5·MTA complex, leading to 15-fold-selective killing of MTAP-deleted (MTAP-null) cells compared to MTAPintact (MTAP WT) cells. In summary, a series of compounds was discovered that inhibits PRMT5 cooperatively with MTA and includes compounds that are potent and selective in MTAP-null cells vs MTAP WT cells. The series was initiated from a high-throughput biochemical screening hit and was guided by both ligand- and structure-based drug design, leading to TNG908, which has a Tanimoto coefficient of 0.3 relative to the original hit. Potency was increased by nearly 2000-fold with the addition of only 83 Da, no additional rotatable bonds, and a reduction in lipophilicity as measured in LogD (2.4 vs 2.8) while maintaining high permeability, low efflux, and a high Kp,uu,CSF = 0.9. [1]

TNG908 shows selective antitumor activity when dosed orally in mouse xenograft models, and its physicochemical properties are amenable for crossing the blood-brain barrier (BBB), supporting clinical study for the treatment of both CNS and non-CNS tumors with MTAP loss. TNG908 has low to moderate clearance and moderate to high bioavailability across species, strong efficacy across a panel of xenograft models including those representing glioblastoma and nonsmall cell lung cancer, and selective efficacy in vivo in a colorectal cancer model. TNG908 was nominated as a development candidate and is currently in a Phase I/II clinical study (NCT05275478).[1]

Biochemical Fluorescence Anisotropy Peptide Displacement Assay[1]
A fluorescence anisotropy (FA) assay was established to measure binding of C-terminal 5′-TAMRA-labeled histone H4 peptide (1–21) with PRMT5/MEP50. The test compound competes with the peptide to bind to PRMT5/MEP50 protein. Assay buffer: 30 mM Bicine (pH 8.0), 150 mM NaCl, 1.5 mM DTT, 0.003% Tween-20. The two peptides utilized for these studies were Me0: Ac-SGRGKGGKGLGKGGAKRHRKV-K(5-TAMRA)-NH2 and Me2: Ac-SGR(Sym Me2)GKGGKGLGKGGAKRHRKV-K(5-TAMRA)-NH2. Me0 peptide is not methylated and was used to determine the compound potency in the absence of cofactor and in the presence of 50 μM 5′-methylthioadenosine (MTA). Me2 peptide is symmetrically methylated at Arginine 3 and was used to determine the compound potency in the presence of 50 μM S-adenosyl-l-methionine (SAM). Inhibitor potency was assessed at equilibrium by measuring the dose-dependent displacement of a fixed concentration of the peptide from PRMT5/MEP50. Following incubation at RT for 30 min, the plate was read on an Envision plate reader. For data analysis, fluorescence anisotropy (FA) detected equals 1000 × (S – G × P)/(S + G × 2 × P), where S = detector 2 or channel 2 signal, P = detector 1 or channel 1 signal, G = G factor. Fluorescence anisotropy is normalized to percent inhibition using percent inhibition = (signal – MinAVG)/(MaxAVG – MinAVG) × 100, where MinAVG = the average value of the min value and MaxAVG = the average value of the max value. Curves are fit by XL-Fit as percent inhibition vs log [compound concentration] using a 4-parameter logistic model 205 y = A + ((B – A)/(1 + ((C/x)∧D))) with fixed 0% and 100% inhibition limits to calculate the IC50. A: bottom = 0%. B: top = 100%. C: relative IC50. D: Hill slope. The apparent Ki values of TNG908 were calculated using the Cheng–Prusoff equation for a competitive inhibitor.

HAP1 MTAP WT and MTAP-null In-Cell Western Assay[1]
A HAP1 MTAP-isogenic cell line pair was acquired from Horizon Discovery (HZGHC004894c005) and maintained in DMEM (high glucose) + 10% FBS in a humidified, 10% CO2 tissue culture incubator. The SAM-cooperative PRMT5 inhibitor, GSK3326595, was maintained as a 10 mM DMSO stock. All test compounds were maintained as 10 mM DMSO stocks.[1]
On day 0, MTAP WT or MTAP-null cells are seeded in a 384-well plate and incubated in a humidified, 5% CO2 tissue culture incubator for 16–24 h. On day 1, the test compounds are dispensed to wells at defined concentrations using a Tecan D300e digital dispenser (n = 4), and the volume of DMSO is normalized to the highest class volume. Each plate includes wells dosed with defined concentrations of GSK3326595 as a plate control. The compounds are incubated with cells for 24 h in a humidified, 5% CO2 tissue culture incubator. On day 2, the compound-treated cells are fixed with a final concentration of 4% formaldehyde. The cells are then washed/permeabilized with 1 × PBS + 0.1% Triton X-100 and then blocked with 5% goat serum/1 × TBS. The fixed cells are then incubated overnight at 4 °C with a primary SDMA antibody cocktail.[1]
On day 3, the cells are washed with 1 × PBS + 0.1% Triton X-100 and then incubated at room temperature for 1 h with a NIR fluorescent secondary antibody cocktail that also contains DRAQ5. The cells are washed with 1 × PBS + 0.1% Triton X-100 and then washed again with ddH2O. The plates are then imaged using a NIR fluorescent imager.[1]
For data analysis, the SDMA signal is normalized to the DRAQ5 signal. Assay background is determined by the signal from wells treated with 1 μM GSK3326595 and subtracted from every well. The data are plotted as percent of the DMSO control wells for the MTAP WT and the MTAP-null cell lines independently and fitted to the 4-parameter logistic (4-PL) Hill equation with maximal effect constrained to 0. The fit was performed using GraphPad Prism or the default IC50 fitting procedure in Dotmatics Studies 5.4 as part of a customized data analysis protocol.

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HAP1 MTAP WT and MTAP-null Viability Assay[1]
A HAP1 MTAP-isogenic cell line pair was acquired from Horizon Discovery (HZGHC004894c005) and maintained in DMEM (high glucose) + 10% FBS in a humidified 5% or 10% CO2 tissue culture incubator. All test compounds are maintained as 10 mM DMSO stocks.[1]
On day 0, MTAP WT and MTAP-null cells are seeded in a 96-well plate and incubated in a humidified 5% or 10% CO2 tissue culture incubator for 16–24 h. On day 1, the test compounds are dispensed to wells at defined concentrations using a Tecan D300e digital dispenser (n = 3), and the volume of DMSO is normalized to the highest class volume (0.2%). The compound-treated plates are incubated for 7 days in a humidified 5% or 10% CO2 tissue culture incubator.[1]
On day 7, the plates are removed from the tissue culture incubator and allowed to equilibrate to room temperature. Then, either a 1/2 volume CellTiter-Glo Luminescent Cell Viability Assay reagent is added to each well or the media is removed from every well and a 1:3 dilution of CellTiter-Glo 2.0 Cell Viability Assay reagent in 1 × PBS is added. Ten minutes after addition, the luminescent signal is detected by an Envision plate reader. The data are plotted as percent of the DMSO control wells for the MTAP WT and the MTAP-null cell lines independently and fitted to the 4-parameter logistic (4-PL) Hill equation with maximal effect constrained to 0. The fit was performed using GraphPad Prism or the default IC50 fitting procedure in Dotmatics Studies 5.4 as part of a customized data analysis protocol.

In Vivo Pharmacology[1]
All protocols were approved by the Institutional Animal Care and Use Committee at Pharmaron (Beijing, China) following the guidance of the Association of Assessment and Accreditation of Laboratory Animal Care.
After 7 days of acclimatization, 10 million LN-18, LU99, HCT116 MTAP WT, or HCT116 MTAP-null tumor cells were injected subcutaneously into the right flank of each animal. Once the tumors reached 200–300 mm3 in size, animals were randomized to treatment groups. TNG908 was administered by oral gavage twice daily in a 5% DMA/20% Captisol solution. Tumor volume was measured using calipers and calculated as (length × width × width)/2.
For data analysis, tumor growth inhibition was calculated using the following equation: percent TGI = [1 – (mean treated TVfinal – mean treated TVinitial)/(mean vehicle TVfinal – mean vehicle TVinitial)] × 100. Tumor regression was calculated as follows: percent tumor regression= [mean TVfinal – mean TVinitial] × 100.

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Western Blotting[1]
Protein lysates were generated by lysis of frozen tumor tissue using RIPA buffer. Samples were normalized by protein concentration using a Pierce Rapid Gold BCA Protein Assay Kit. SDS-PAGE was run using Invitrogen NuPAGE 4–12% Bis-Tris Midi Protein Gels. Antibodies SDMA, ACTB were used at 1:1000 dilution.

[1]. Discovery of TNG908: A Selective, Brain Penetrant, MTA-Cooperative PRMT5 Inhibitor That Is Synthetically Lethal with MTAP-Deleted Cancers. J. Med. Chem. 2024, 67, 8, 6064–6080.
[2]. K. Briggs, et al. TNG908 is a brain-penetrant, MTA-cooperative PRMT5 inhibitor for the treatment of MTAP-deleted cancer.MOLECULAR TARGETED AGENTS 2| VOLUME 174, SUPPLEMENT 1, S84, OCTOBER 2022.

TNG908 is a small molecule inhibitor of protein arginine methyl transferase 5 (PRMT5).

C21H23N5O2S

409.16

C, 61.59; H, 5.66; N, 17.10; O, 7.81; S, 7.83

2760481-53-4

164753116

Typically exists as white to off-white solids at room temperature

3.2

2

6

2

29

620

2

C[C@H]1CC[C@@H](N(C1)C(=O)C(=O)NC2=CN=C(C(=C2)C)N)C3=CC4=C(C=C3)SC=N4

NXXBDYHMHHINFC-YVEFUNNKSA-N

InChI=1S/C21H23N5O2S/c1-12-3-5-17(14-4-6-18-16(8-14)24-11-29-18)26(10-12)21(28)20(27)25-15-7-13(2)19(22)23-9-15/h4,6-9,11-12,17H,3,5,10H2,1-2H3,(H2,22,23)(H,25,27)/t12-,17+/m0/s1

N-(6-amino-5-methylpyridin-3-yl)-2-((2R,5S)-2-(benzo[d]thiazol-5-yl)-5-methylpiperidin-1-yl)-2-oxoacetamide

TNG908; A1AAV; SCHEMBL24365689; TNG-908; EX-A7872

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 : ~100 mg/mL (~244.20 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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