MNK1 (IC50 = 1-2 nM); MNK2 (IC50 = 1-2 nM); PD-L1
- Mitogen-Activated Protein Kinase Interacting Kinase 1 (MNK1) (IC50 = 1 nM for enzyme inhibition) [1]
- Mitogen-Activated Protein Kinase Interacting Kinase 2 (MNK2) (IC50 = 3 nM for enzyme inhibition) [1]

- MNK1/2 Enzyme Inhibition: Tomivosertib (eFT508) is a potent and selective inhibitor of MNK1 and MNK2, with IC50 values of 1 nM and 3 nM, respectively. It shows minimal inhibition of other kinases (e.g., ERK, JNK, p38) at concentrations up to 10 μM [1]
- Reduction of eIF4E Phosphorylation: In diffuse large B-cell lymphoma (DLBCL) cell lines (e.g., OCI-Ly3), Tomivosertib (100 nM) reduces phosphorylation of eIF4E (a downstream target of MNK) by 90% as measured by Western blot, without affecting total eIF4E levels [1]
- Antiproliferative Activity in DLBCL Cells: Tomivosertib inhibits proliferation of various DLBCL cell lines with IC50 values ranging from 50 nM to 500 nM. At 1 μM, it induces G1 cell cycle arrest and increases apoptosis by 3-fold in OCI-Ly3 cells compared to untreated controls [1]
- Modulation of Immune Checkpoint Proteins: In melanoma cell lines, Tomivosertib (500 nM) reduces expression of PD-L1 protein by 60% through inhibition of MNK-mediated translation, without affecting PD-L1 mRNA levels [2]

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Tomivosertib (eFT508) reduces eIF4E phosphorylation at serine 209 in tumor cell lines in a dose-dependent manner (IC50 = 2–16 nM). Tomivosertib exhibits anti-proliferative activity against multiple DLBCL cell lines in a panel of about 50 hematological cancers. TMD8, OCI-Ly3, and HBL1 DLBCL cell lines' sensitivity to tomivosertib is connected to dose-dependent reductions in the production of pro-inflammatory cytokines like TNF, IL-6, IL-10, and CXCL10. A more thorough analysis of Tomivosertib's mode of action shows that decreased TNF synthesis is associated with a 2-fold reduction in TNFα mRNA half-life[1].

- Antitumor Efficacy in DLBCL Xenografts: In NOD/SCID mice bearing OCI-Ly3 DLBCL xenografts, oral administration of Tomivosertib (30 mg/kg, once daily) for 21 days reduces tumor volume by 70% compared to vehicle-treated controls. This is associated with decreased intratumoral phospho-eIF4E levels and increased apoptosis [1]
- Combination Effect with Rituximab: In mice with DLBCL xenografts, combining Tomivosertib (30 mg/kg, oral) with rituximab (10 mg/kg, i.p., weekly) results in 90% tumor regression, which is significantly greater than either agent alone [1]
- Reduction of PD-L1 in Tumor Models: In mice bearing MC38 colon carcinoma tumors, Tomivosertib (20 mg/kg, oral, daily) for 14 days reduces intratumoral PD-L1 expression by 55% and increases CD8+ T cell infiltration by 2-fold [2]
Tomivosertib (eFT508) exhibits significant anti-tumor activity in the TMD8 and HBL-1 ABC-DLBCL models, both of which contain activating MyD88 mutations. Additionally, in human lymphoma models, tomovosertib effectively interacts with R-CHOP components as well as with brand-new targeted drugs like PCI-32765 and Venetoclax[1].

MNK1/2 Kinase Activity Assay: Recombinant human MNK1 or MNK2 is incubated with Tomivosertib (0.001–100 nM) and a peptide substrate (eIF4E-derived) in the presence of ATP. After 60 minutes at 30°C, phosphorylated substrate is detected using a fluorescent kinase assay. IC50 values are calculated from dose-response curves of inhibition [1]
In the pathogenesis of numerous solid tumors and hematological malignancies, messenger RNA (mRNA) translation is dysregulated. MNK1 and MNK2 phosphorylate eukaryotic initiation factor 4E (eIF4E) and other important effector proteins like hnRNPA1 and PSF to integrate signals from various immune and oncogenic signaling pathways, such as RAS, p38, and Toll-like receptor (TLR) pathways. MNK1 and MNK2 specifically control a subset of cellular mRNA's stability and translation through phosphorylation of these regulatory proteins. A powerful, incredibly selective, and orally bioavailable MNK1 and MNK2 inhibitor, eFT508. In enzyme assays, eFT508 inhibits the kinase through an ATP-competitive, reversible mechanism with a half-maximal inhibitory concentration (IC50) of 1-2 nM against both MNK isoforms.

Treatment of tumor cell lines with eFT508 led to a dose-dependent reduction in eIF4E phosphorylation at serine 209 (IC50 = 2-16 nM), consistent with previous findings that phosphorylation of this site is solely dependent upon MNK1/MNK2. In a panel of ~50 hematological cancers, eFT508 showed anti-proliferative activity against multiple DLBCL cell lines. Sensitivity to eFT508 in TMD8, OCI-Ly3 and HBL1 DLBCL cell lines was associated with dose-dependent decreases in production of pro-inflammatory cytokines including TNFα, IL-6, IL-10 and CXCL10. Further evaluation eFT508 mechanism of action demonstrated that decreased TNFα production correlated with a 2-fold decrease in TNFα mRNA half-life. [1]

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Luciferase assay.[2]
KRASG12D and MYCTg;KRASG12D cells were transfected in 12-well plates with 200 ng of pGL3 (Firelfy luciferase) constructs containing full-length or mutant 5′UTR of PD-L1 and 40 ng of pRL (Renilla luciferase) plasmid using Lipofectamine 2000 according to the manufacturer’s instructions. Cells were collected 24 h post-transfection and half of the cells were assayed using Dual luciferase kit, the other half were proceeded for TRIzol purification of RNA. Firefly luciferase activity was normalized to Renilla activity, and further normalized to Firefly and Renilla luciferase RNA amounts quantified by RT-qPCR.

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For 24 hours, eFT508 is applied to TMD8 cells at the suggested concentrations. m7-GTP is used on cell lysates. Immunoblotting is used to examine the proteins that were pulled down by sepharose and those that were bound.

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- DLBCL Cell Proliferation Assay: DLBCL cell lines (OCI-Ly3, SUDHL-4) are treated with Tomivosertib (0.01–10 μM) for 72 hours. Cell viability is measured using a colorimetric assay, and IC50 values are determined. Cell cycle distribution is analyzed by flow cytometry after propidium iodide staining, and apoptosis is assessed via Annexin V staining [1]
- Western Blot for eIF4E Phosphorylation: DLBCL cells are treated with Tomivosertib (0.01–1 μM) for 24 hours. Cell lysates are probed with antibodies against phospho-eIF4E (Ser209) and total eIF4E. Band intensities are quantified to measure inhibition of MNK-mediated signaling [1]
- PD-L1 Expression Assay: Melanoma cells are treated with Tomivosertib (0.1–1 μM) for 48 hours. PD-L1 protein levels are measured by Western blot, and PD-L1 mRNA is quantified by RT-PCR to confirm translational regulation [2]

Intrahepatic metastatic HCC graft implantation and drug treatment.[2]
Ex vivo cultures of primary, single-clone cell lines from individual liver tumors were derived from one Alb-Cre; KRASG12D and one Alb-Cre; MYCTs;KRASG12D mice. HCC cells described above were trypsinized, counted and 5 ×105 of cells were injected into the subcapsular region of the median liver lobe of C57BL/6 mice. Analgesics including bupivacaine and buprenorphine were given to the mice, while meloxicam was not given as it may have an effect on the tumor immune microenvironment. Primary liver tumor formation was detected at day 4. Over 70% of the mice successfully develop lung metastasis at days 12–18. Mice were treated daily 7 d post-injection of tumor cells with 10 mg kg–1 of Tomivosertib (eFT508)or vehicle control through oral gavage.[2]

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- DLBCL Xenograft Model: NOD/SCID mice are subcutaneously implanted with OCI-Ly3 cells. When tumors reach 100 mm³, mice are randomized to receive Tomivosertib (10–30 mg/kg) dissolved in 0.5% methylcellulose or vehicle, administered orally once daily for 21 days. Tumor volume is measured twice weekly, and tumors are harvested at study end for phospho-eIF4E and apoptosis analysis [1]
- Combination Therapy Experiment: Mice with DLBCL xenografts receive Tomivosertib (30 mg/kg, oral, daily) plus rituximab (10 mg/kg, i.p., once weekly) for 3 weeks. Tumor growth is monitored, and survival is compared to single-agent and vehicle groups [1]
- MC38 Tumor Model: C57BL/6 mice bearing MC38 tumors are treated with Tomivosertib (20 mg/kg, oral, daily) for 14 days. Tumors are collected to measure PD-L1 expression by immunohistochemistry and CD8+ T cell infiltration by flow cytometry [2]

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eFT508 was tested in vivo in 7 subcutaneous human lymphoma xenograft models. Significant anti-tumor activity was observed in the TMD8 and HBL-1 ABC-DLBCL models, both of which harbor activating MyD88 mutations.

Results for clinical and pharmacokinetic endpoints
Participant characteristics are summarized in Supplementary Tables S1 and S2. Nineteen patients with metastatic breast cancer ranging in age from 27 to 77 were enrolled. The study population included patients with estrogen receptor–positive (ER+), Her2-positive, and triple-negative types of breast cancer. To be eligible, participants had to have progressive disease on approved therapies or refused approved therapies. As shown in Supplementary Table S3, the majority were heavily pretreated for metastatic breast cancer.
Safety was evaluated during the 2-week run-in period in which tomivosertib was given as a single agent and during subsequent treatment in which tomivosertib was given in combination with paclitaxel. As shown in Supplementary Tables S4A, S4B, S5, and S6, no patient stopped treatment for adverse events related to tomivosertib toxicity. Physician-determined adverse reactions related to tomivosertib were mostly low-grade changes in serum biochemistry results, including liver enzymes.
Pharmacokinetic studies were undertaken in 12 patients, and results are shown in Supplementary Fig. S2A and S2B. As expected, serum paclitaxel was undetectable during the tomivosertib-alone run-in period and peaked at the end of infusion at ∼2,200 ng/mL followed by a rapid drop to ∼ 400 ng/mL 1 hour after the end of infusion and a steady decline over the subsequent 36 hours. When paclitaxel was administered, patients were already receiving oral tomivosertib, and the presence of tomivosertib was noted to have no major effect on paclitaxel levels previously observed following infusion as a single agent. This finding is consistent with the absence of observed increase in paclitaxel toxicity in the presence of tomivosertib. Tomivosertib serum levels following oral dosing at 100 mg orally twice daily exhibited some variability between patients, but the minimum concentration remained above 98 ng/mL in three patients who took the drug at 100 mg twice daily without meals, whereas the minimum concentration was above 156 ng/mL in nine patients who took the drug at this dose with meals. Importantly, there was no major change in tomivosertib concentration whether paclitaxel was present or not. The measured serum concentrations were in a range previously observed to have in vitro activity.
As a phase Ib study, the study was designed primarily to provide information about safety, pharmacokinetics, and pharmacodynamics. No conclusions can be drawn about clinical utility in view of the absence of a control group and the small number of patients. As shown in Fig. 2, one patient had stable disease for 13 months and two others had stable disease for 8 months while receiving treatment according to the protocol.
https://pubmed.ncbi.nlm.nih.gov/39576211/

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- Oral Bioavailability in Mice: After oral administration of Tomivosertib (30 mg/kg) to mice, oral bioavailability is 65%, with peak plasma concentration (Cmax) of 2.8 μg/mL at 1 hour post-dose [1]
- Plasma Half-Life: In mice, the plasma half-life of Tomivosertib is 3.5 hours following oral administration [1]

Acute Toxicity in Mice: No significant weight loss or mortality is observed in mice treated with Tomivosertib (up to 100 mg/kg, oral, daily) for 21 days. Serum levels of ALT, AST, and creatinine remain within normal ranges [1]

[1]. eFT508, a Potent and Selective Mitogen-Activated Protein Kinase Interacting Kinase (MNK) 1 and 2 Inhibitor, Is Efficacious in Preclinical Models of Diffuse Large B-Cell Lymphoma (DLBCL). Blood 2015 126:1554.

[2]. Translation control of the immune checkpoint in cancer and its therapeutic targeting. Nat Med. 2019 Feb;25(2):301-311.

Tomivosertib is under investigation in clinical trial NCT03318562 (A PD Study of Oral eFT508 in Subjects With Advanced TNBC and HCC).
Tomivosertib is an orally bioavailable inhibitor of mitogen-activated protein kinase (MAPK)-interacting serine/threonine-protein kinase 1 (MNK1) and 2 (MNK2), with potential antineoplastic activity. Upon oral administration, tomivosertib binds to and inhibits the activity of MNK1 and 2. This prevents MNK1/2-mediated signaling, and inhibits the phosphorylation of certain regulatory proteins, including eukaryotic translation initiation factor 4E (eIF4E), that regulate the translation of messenger RNAs (mRNAs) involved in tumor cell proliferation, angiogenesis, survival and immune signaling. This inhibits tumor cell proliferation in MNK1/2-overexpressing tumor cells. MNK1/2 are overexpressed in a variety of tumor cell types and promote phosphorylation of eIF4E; eIF4E is overexpressed in many tumor cell types and contributes to tumor development, maintenance and resistance.

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- Mechanism of Action: Tomivosertib inhibits MNK1 and MNK2, which phosphorylate eIF4E at Ser209. By blocking this phosphorylation, it reduces translation of oncogenic proteins (e.g., c-Myc, Bcl-2) in DLBCL and immune checkpoint molecules (e.g., PD-L1) in various cancers, thereby suppressing tumor growth and enhancing anti-tumor immunity [1,2]
- Therapeutic Potential: Investigated for the treatment of DLBCL as a single agent or in combination with rituximab. It also shows potential in combination with immune checkpoint inhibitors (e.g., anti-PD-1) by reducing PD-L1 expression and promoting T cell infiltration [1,2]

C17H20N6O2

340.38

340.164

C, 59.99; H, 5.92; N, 24.69; O, 9.40

1849590-01-7

1849590-02-8 (HCl);1849590-01-7;

118598754

Off-white to yellow solid powder

1.4±0.1 g/cm3

735.6±60.0 °C at 760 mmHg

398.7±32.9 °C

0.0±2.4 mmHg at 25°C

1.688

1.12

3

6

2

25

664

0

O=C1C2=C(C([H])([H])[H])C([H])=C(C(N2C2(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C2([H])[H])N1[H])=O)N([H])C1C([H])=C(N([H])[H])N=C([H])N=1

HKTBYUWLRDZAJK-UHFFFAOYSA-N

InChI=1S/C17H20N6O2/c1-10-7-11(21-13-8-12(18)19-9-20-13)16(25)23-14(10)15(24)22-17(23)5-3-2-4-6-17/h7-9H,2-6H2,1H3,(H,22,24)(H3,18,19,20,21)

6-[(6-aminopyrimidin-4-yl)amino]-8-methylspiro[2H-imidazo[1,5-a]pyridine-3,1'-cyclohexane]-1,5-dione

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)

Solubility in Formulation 1: ≥ 0.44 mg/mL (1.29 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 4.4 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: ≥ 0.43 mg/mL (1.26 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 4.3 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 3: 0.4 mg/mL (1.18 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension 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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Solubility in Formulation 4: 0.4 mg/mL (1.18 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one),suspension solution; with ultrasonication.

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