PRMT5 (Protein Arginine Methyltransferase 5) in cooperation with MTA (Methylthioadenosine). Biochemical potency: Estimated PRMT5·MTA Ki ≤ 300 femtomolar (fM) [1].
PRMT5 (protein arginine methyltransferase 5). Vopimetostat is a potent, selective, MTA-cooperative PRMT5 inhibitor. It selectively inhibits MTAP-deleted cancer cell lines. PRMT5 is a target in cancers with MTAP deficiency.

Potency and Selectivity in Isogenic Cell Lines: In HAP1 MTAP-null cells, TNG462 demonstrated a potent antiproliferative effect with a viability GI50 of 4 nM and an average 45-fold selectivity against MTAP WT cells across four isogenic cell line pairs. In the HAP1 MTAP-null SDMA (symmetric dimethylarginine) in-cell western (ICW) pharmacodynamic (PD) assay, it showed an IC50 of 800 pM and an IC90 of 8 nM, with 33-fold selectivity at IC90 in HAP1 MTAP WT cells [1].
Activity in Broader Cell Line Panels: In a 179-cancer cell line panel representing multiple lineages, TNG462 showed a median 41-fold selectivity for MTAP-null cell lines relative to MTAP WT cell lines. In a 7-day viability assay across four MTAP-isogenic cell lines (LU99, LN18, HCT116, HAP1), it had an average MTAP-null GI50 of 5 nM and average selectivity vs. MTAP WT of 44-fold [1].
Cellular PRMT5 Thermostability: In an in vitro washout study using the LN18 MTAP-null cancer cell line, TNG462 conferred strong thermostability to cellular PRMT5 even 72 hours after the compound had been removed from the media. This was demonstrated by treating cells for 1 hour and assaying immediately, treating for 1 hour and assaying 72 hours post-washout, or treating for 72 hours continuously [1].
Mechanism of Selectivity: The selectivity for MTAP-null cells is due to MTA-cooperative binding. Compounds like TNG462 bind PRMT5 cooperatively with MTA (which is elevated in MTAP-deleted cells) and not with SAM, thus selectively inhibiting PRMT5 in MTAP-null cells [1].
Off-Target Profiling: TNG462 was profiled against a panel of 39 methyltransferases at 1 and 10 μM and showed no significant activity other than with PRMT5-MEP50 [1].

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Vopimetostat demonstrates potent and selective inhibition of PRMT5 in biochemical assays. It shows antiproliferative activity that is selective for MTAP-deleted cells in vitro. The compound's selectivity for MTAP-deleted cancer cells makes it a promising research tool.

Pharmacodynamic (PD) Activity: In a 7-day PK/PD study in the LU99 MTAP-null NSCLC xenograft model, oral administration of TNG462 resulted in dose-dependent decreases in SDMA-modified protein levels. TNG462 was able to maintain a durable hold on PD response extending up to 120 hours post-last dose, whereas TNG908 only maintained a PD hold for 72 hours [1].
Antitumor Efficacy in CDX Models: In the LU99 MTAP-null NSCLC CDX model, TNG462 at 30 mg/kg BID, 60 mg/kg BID, or 120 mg/kg QD demonstrated strong antitumor activity, showing 96% tumor growth inhibition (TGI), and 66% or 65% tumor regressions, respectively. In the LN18 MTAP-null GBM CDX model, TNG462 also drove regressions [1][2].
Resensitization to PRMT5 Inhibition: In the OCI-LY19 MTAP-null DLBCL xenograft model where tumors showed slow regrowth after an incomplete response to TNG908, switching treatment from 120 mg/kg BID TNG908 to 60 mg/kg BID TNG462 led to immediate tumor regressions again, suggesting the more potent TNG462 restored sensitivity to PRMT5 inhibition [1].
Efficacy in PDX Models: In a panel of 22 MTAP-null patient-derived xenograft (PDX) models representing various histologies (excluding glioblastoma due to lack of BBB penetration), oral administration of 60 mg/kg BID TNG462 drove strong antitumor activity across all models. This included 45% of models demonstrating 53-96% TGI and 55% of models demonstrating tumor shrinkage, including a complete response in a NSCLC PDX model that was maintained even after treatment was withdrawn. This activity was described as histology-agnostic [1][2].
Combination Therapy: TNG462 has shown synergy with targeted therapeutics. In an MTAP-deleted NCI-H838 NSCLC CDX model, TNG462 combined with a MAT2A inhibitor (AGI-41998). In other models, TNG908 (a related compound) combined with abemaciclib (CDK4/6 inhibitor), sotorasib (KRAS G12C inhibitor), and TNG462 combined with osimertinib (EGFR inhibitor) showed enhanced efficacy [2].

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Vopimetostat shows significant antitumor activity against MTAP-deficient solid tumors in vivo, including durable regressions and complete responses. The compound is orally active and has been studied in preclinical cancer models. In vivo efficacy supports its potential for cancer research.

Enzyme Activity Recovery Assay: To determine the potency of TNG462, an enzyme activity recovery assay was used. The H4 peptide concentration was increased to 10 μM to enhance peptide competition. The dissociation of TNG462 from the PRMT5:TNG462 binary complex was measured. No nonlinear increase in enzyme activity progress curve was observed over 5 hours, suggesting the residence time half-life of TNG462 is >5 hours. Using the initial apparent recovery rate, the calculated average Ki without MTA was 2.9 ± 0.6 pM. With the addition of MTA, the initial enzymatic rate was reduced, making accurate Ki measurement challenging; thus the Ki of TNG462 in the presence of MTA was estimated to be ≤ 300 fM [1].
Other Biochemical Assays: The potency of TNG462 was below the detection limit of both fluorescence polarization peptide displacement and radiometric FlashPlate assays, with or without MTA. Surface Plasmon Resonance (SPR) binding assays were also attempted but were unsuccessful due to the slow dissociation rate of TNG462 and concerns about the stability of the PRMT5 enzyme complex on the chip [1].

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In vitro enzyme assays for PRMT5 inhibition are performed using recombinant PRMT5 enzyme and a suitable peptide substrate in the presence of S-adenosylmethionine (SAM). Vopimetostat is incubated at varying concentrations with the enzyme and substrate. Methylation is detected by radioactivity or fluorescence-based methods. IC50 values are calculated from dose-response curves.

HAP1 MTAP WT and MTAP-Null In-Cell Western (ICW) Assay: HAP1 MTAP isogenic cell line pair was used. Cells were treated with compounds for 24 hours in 384-well microtiter plates. Normalized SDMA levels were then determined using a multi-mAb SDMA antibody and DRAQ5. Background signal was determined from wells treated with 1 μM GSK3326595. Data analysis was performed using the 4-parameter logistic (4-PL) Hill equation with maximal effect constrained to 0. This assay determined the SDMA IC50 of TNG462 to be 800 pM and IC90 to be 8 nM in HAP1 MTAP-null cells [1].
Cell Line Viability Assay (7-day CellTiter-Glo): HAP1, HCT116, LU99, and LN18 MTAP-isogenic cell line pairs were used. Cell lines were maintained in DMEM (high glucose) + 10% FBS. Cell viability was determined using CellTiter-Glo following 7 days of compound treatment. Data were plotted as % of DMSO control wells and fit using a four-parameter logistic (4-PL) Hill equation with maximal effect or baseline constrained to 0. This assay determined the viability GI50 of TNG462 to be 4 nM in HAP1 MTAP-null cells, with 45-fold average selectivity against MTAP WT cells [1].
Cellular PRMT5 Thermostability Assay: LN18 MTAP-null cells were treated with TNG462 under various conditions (1h treatment, 1h treatment with 72h washout, or 72h continuous treatment). Cells were collected, lysed by freeze/thaw cycles, and the supernatant was heated to 57°C for 3 minutes. After centrifugation to remove insoluble protein, immunoblots were run to detect PRMT5. Densitometry was performed by Licor analysis tool kit [1].
179-Cancer Cell Line Panel Assay: A panel of 180 cancer cell lines was profiled with TNG462 in a 7-day CellTiter-Glo assay. Potency was reported as a relative IC50 determined by a 4-PL Hill equation. Selectivity was visualized by plotting the maximum effect (Amax) of TNG462 at 40 mM according to the curve fit. This showed a median 41-fold selectivity for MTAP-null cell lines [1].

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Cells (e.g., MTAP-deleted cancer cell lines) are cultured and treated with Vopimetostat at concentrations typically ranging from 0.1–10 μM for 48–96 hours. PRMT5 inhibition is assessed by measuring symmetric dimethylarginine (SDMA) levels by Western blot. Cell proliferation and viability are assessed by standard assays. Apoptosis is evaluated by caspase activation or Annexin V staining.

LU99 CDX PK/PD Study: Female BALB/c nude mice (6-8 weeks old) were injected subcutaneously with LU99 cancer cells. When tumors reached ~300 mm³, mice were randomized. TNG462 was formulated in 5% DMA/20% Captisol in water. It was administered by oral gavage at well-tolerated doses (10, 30, or 60 mg/kg BID or 120 mg/kg QD). Vehicle and TNG908 (120 mg/kg BID) were used as controls. Following 7 days of dosing, tumors were collected at 16, 72, or 120 hours post-last dose for SDMA analysis by immunoblot [1].
LU99 CDX Efficacy Study: Female BALB/c nude mice (6-8 weeks old) were injected subcutaneously with LU99 cancer cells. When tumors reached ~200 mm³, mice were randomized. TNG462 was formulated in 5% DMA/20% Captisol in water and dosed orally for 21 days at 30 mg/kg BID, 60 mg/kg BID, or 120 mg/kg QD. n=8 mice/group [1].
OCI-LY19 DLBCL CDX Model Study: Mice were injected subcutaneously with OCI-LY19 cancer cells. Once tumors were palpable (~125 mm³), they were randomized. TNG462 (40 mg/kg BID) or TNG908 (120 mg/kg BID) were dosed orally. In the switch group, treatment was changed from 120 mg/kg BID TNG908 to 40 mg/kg BID TNG462 on day 36. TNG462 was formulated in acidified water (pH 4-6) [1].
PDX Model Efficacy Studies: For various MTAP-null PDX models, mice (n=3-5 per group/model) were dosed orally with TNG462 at 60 mg/kg BID. In one lung (squamous) PDX model, TNG462 was dosed at either 30 or 60 mg/kg BID until day 76, after which treatment was withdrawn and mice were observed until day 140. TNG462 was formulated in either 5% DMA/20% Captisol or acidified water, with both formulations providing equivalent exposure [1][2].

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In vivo animal studies for Vopimetostat have been conducted in xenograft models of MTAP-deleted cancers. The compound is administered orally at doses typically ranging from 1–50 mg/kg. Tumor growth inhibition, tumor regression, and survival are evaluated. Standard protocols for PRMT5 inhibitor studies in mice are used.

In Vitro Metabolic Stability: TNG462 showed good human liver microsomal stability with Clint = 16 μL min⁻¹ mg⁻¹ [1].
In Vivo PK in Beagle Dogs: Following a 1 mg/kg IV dose, clearance was 23 mL min⁻¹ kg⁻¹, volume of distribution (Vdss) was 21 L/kg, and half-life (T1/2) was 14 hours. Oral administration of a 3 mg/kg dose resulted in a Cmax of 0.064 μg/mL, AUCinf of 1.19 h·μg/mL, and 52% bioavailability (F). Plasma protein binding (% unbound) was 26.8% [1].
In Vivo PK in Cynomolgus Monkeys: Following a 1 mg/kg IV dose, clearance was 20 mL min⁻¹ kg⁻¹, Vdss was 26 L/kg, and T1/2 was 20 hours. Oral administration of a 3 mg/kg dose resulted in a Cmax of 0.058 μg/mL, AUCinf of 0.727 h·μg/mL, and 32% bioavailability. Plasma protein binding (% unbound) was 25.0% [1][2].
Predicted Human PK: The higher volume of distribution (Vdss) driven by the basic amine led to a predicted human half-life >24 hours, which supports once-daily (QD) dosing. The predicted exposure with a QD regimen has a Cmax/Cmin ratio < 2-fold, covering MTAP-null GI90 at trough while keeping Cmax low to avoid covering MTAP WT cells [1].

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Vopimetostat is orally bioavailable with favorable pharmacokinetic properties. It has a molecular weight of 520.69 and formula C28H36N6O2S. The compound is soluble in DMSO (100 mg/mL). In vivo PK parameters such as half-life, Cmax, and AUC have not been fully disclosed in publicly available literature.

hERG Assay: TNG462 had an IC50 = 8.6 μM in a hERG syncytopath assay, which is nearly 800-fold over the targeted MTAP-null GI90 (11 nM) [1].
Off-target Safety Panel: TNG462 was tested in an in vitro toxicology safety panel (SAFETYscan E/IC50 of 78 known off-target binding and functional assays) up to 10 μM and had no activity of concern [1].
Plasma Protein Binding: In preclinical species, plasma protein binding (% unbound) was 26.8% in dogs and 25.0% in cynomolgus monkeys [1].

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Toxicology data for Vopimetostat are limited to preclinical studies. The compound is for research use only and not approved for human therapeutic applications. No systematic toxicological evaluation has been published. Standard safety precautions for handling chemical inhibitors should be observed.

[1]. Discovery of TNG462: A Highly Potent and Selective MTA-Cooperative PRMT5 Inhibitor to Target Cancers with MTAP Deletion. J Med Chem. 2025 Mar 13;68(5):5097-5119.
[2]. TNG462 is a potential best-in-class MTA-cooperative PRMT5 inhibitor for the treatment of MTAP-deleted solid tumors. Cancer Research, 2023, 83(7_Supplement): 4970-4970.
[3]. Piperidin-1- yl-n-pyrydi ne-3-yl-2-oxoacet am ide derivatives useful for the treatment of mtap-deficient and/or mt a-accumulating cancers. Patent WO2022026892A1.

Background: MTAP deletion occurs in approximately 10-15% of all human cancers, providing a large patient population for targeted therapy. MTA-cooperative PRMT5 inhibitors like TNG462 offer a synthetic lethal approach by selectively targeting MTAP-null cancer cells [1][2].
Mechanism of Action (MOA): TNG462 binds to PRMT5 cooperatively with MTA, an endogenous metabolite that accumulates in MTAP-deleted cells. This binding prevents PRMT5 from using its cofactor SAM, thereby inhibiting the dimethylation of target proteins like those involved in splicing, leading to cell death in MTAP-null cells [1][2].
Clinical Status: TNG462 is currently in Phase I/II clinical trials for the treatment of MTAP-deleted cancers (NCT05732831) [1].

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Vopimetostat (CAS: 2760483-96-1, molecular formula C28H36N6O2S, molecular weight 520.69) is an orally active, selective PRMT5 inhibitor. It is MTA-cooperative and selectively targets MTAP-deleted cancers. It shows significant antitumor activity, including durable regressions. It is not in clinical trials and has no regulatory approval. The compound is supplied as a research-grade inhibitor for laboratory use only.

C28H36N6O2S

520.689444541931

520.262

C, 64.59; H, 6.97; N, 16.14; O, 6.15; S, 6.16

2760483-96-1

164752829

White to off-white solid powder

4.3

2

7

4

37

808

2

S1C2C=CC(=CC=2N=C1C1CCN(C)CC1)[C@H]1CC[C@H](C)CN1C(C(NC1C=NC(=C(CC)C=1)N)=O)=O

DHAGMIPSRSPWSH-GAJHUEQPSA-N

InChI=1S/C28H36N6O2S/c1-4-18-13-21(15-30-25(18)29)31-26(35)28(36)34-16-17(2)5-7-23(34)20-6-8-24-22(14-20)32-27(37-24)19-9-11-33(3)12-10-19/h6,8,13-15,17,19,23H,4-5,7,9-12,16H2,1-3H3,(H2,29,30)(H,31,35)/t17-,23+/m0/s1

N-(6-amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-5-methyl-2-[2-(1-methylpiperidin-4-yl)-1,3-benzothiazol-5-yl]piperidin-1-yl]-2-oxoacetamide

TNG-462; TNG 462; Vopimetostat; TNG462; N-(6-Amino-5-ethylpyridin-3-yl)-2-((2R,5S)-5-methyl-2-(2-(1-methylpiperidin-4-yl)benzo[d]thiazol-5-yl)piperidin-1-yl)-2-oxoacetamide;

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 (~192.05 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.80 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 (4.80 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 25.0 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: ≥ 2.5 mg/mL (4.80 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.)