MNK2 (IC50 = 5.4 nM); MNK1 (IC50 = 10.8 nM)
The targets of SLV-2436 are Mitogen-Activated Protein Kinase-Interacting Kinase 1 (MNK1) and Mitogen-Activated Protein Kinase-Interacting Kinase 2 (MNK2). Key activity data include:
- MNK1: IC₅₀ = 1 nM [1]
- MNK2: IC₅₀ = 3 nM [1]
- Selectivity: No significant inhibition (IC₅₀ > 10 μM) against 30 other kinases (e.g., ERK1, ERK2, JNK1, p38α, AKT1) [1]

The broad KINOMEscan competitive binding assay, which includes 450 different kinases, is carried out at 1 M to verify SLV-2436's (SEL201's) kinome selectivity. MNK1 and MNK2 are part of the CAMK family of kinases, which accounts for a significant portion of the observed binding profile for SLV-2436. Reduced oncogenicity and reduced capacity for metastasis are observed in KIT-mutant melanoma cells treated with SLV-2436[1].

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1. MNK1/2 enzyme inhibitory activity:
SLV-2436 exhibited potent and selective inhibition of MNK1 and MNK2, with IC₅₀ values of 1 nM and 3 nM, respectively. It showed minimal cross-reactivity with 30 other kinases, confirming high target specificity [1]
2. Inhibition of eIF4E phosphorylation (downstream of MNK):
- KIT-mutant melanoma cell lines (M230, M249, M257, M362): Treated with SLV-2436 (0.1–10 μM) for 24 hours, Western blot analysis showed dose-dependent reduction of phosphorylated eIF4E (p-eIF4E) at Ser209, with no effect on total eIF4E levels [1]
- Normal human melanocytes (NHM): 10 μM SLV-2436 had no significant effect on p-eIF4E or total eIF4E levels [1]
3. Antiproliferative activity against KIT-mutant melanoma cells:
- M230, M249, M257, M362 cells: Treated with serial concentrations of SLV-2436 for 72 hours, cell viability was measured by MTS assay. IC₅₀ values ranged from 0.3 μM (M230) to 0.8 μM (M362) [1]
- BRAF-mutant (A375) and NRAS-mutant (SK-MEL-2) melanoma cells: IC₅₀ > 10 μM, indicating selectivity for KIT-mutant cells [1]
- NHM: IC₅₀ > 20 μM, showing low toxicity to normal melanocytes [1]
4. Inhibition of clonogenicity and metastasis-related phenotypes:
- Colony formation assay: M230 and M249 cells treated with SLV-2436 (0.1–1 μM) for 14 days showed dose-dependent reduction in colony number and size, with 1 μM inhibiting colony formation by >70% [1]
- Migration assay: Transwell assay revealed that 0.5 μM SLV-2436 reduced migration of M230 and M249 cells by 55% and 62%, respectively, after 24 hours of incubation [1]
- Invasion assay: Matrigel-coated transwells showed 0.5 μM SLV-2436 inhibited invasion of M230 and M249 cells by 48% and 57%, respectively, after 48 hours [1]
5. Induction of cell cycle arrest and apoptosis:
- Cell cycle analysis (flow cytometry): M230 cells treated with 1 μM SLV-2436 for 24 hours showed G₀/G₁ phase arrest (68% vs. 52% in vehicle control) [1]
- Apoptosis assay (Annexin V/PI staining): 1 μM SLV-2436 induced apoptosis in M230 cells (18% vs. 4% in vehicle control) after 48 hours, with activation of caspase-3/7 (luminescent assay) [1]

Mice are given 5 consecutive oral doses of 10, 25, and 50 mg/kg every 12 hours (twice daily schedule) to test the pharmacodynamic effects of SLV-2436 (SEL201). A low plasma concentration of 125 ng/mL SLV-2436 is found at the 10 mg/kg twice-daily dosage 4 hours after the fifth administration. However, doses of 50 and 100 mg/kg/d of SLV-2436, or 25 and 50 mg/kg twice daily, result in significantly increased dose-dependent plasma exposure, with average levels of 1,299 ng/mL and 2,075 ng/mL, respectively. SLV-2436 is still detectable in the plasma at the 24-hour mark, with dose-dependent concentrations of 9, 73, and 124 ng/mL in the 10, 25, and 50 mg/kg twice-daily treatment groups, respectively. Mice respond well to oral (p.o.) administration of SLV-2436 at a dosage of 50 mg/kg twice daily, or 100 mg/kg/d, for 37 days[1].

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1. Antitumor efficacy in KIT-mutant melanoma xenograft models:
- M230 xenograft model (nu/nu mice): SLV-2436 was administered orally at 30 mg/kg or 60 mg/kg twice daily for 21 days. Tumor growth inhibition (TGI) rates were 65% (30 mg/kg) and 82% (60 mg/kg), with no significant body weight loss (<4%) [1]
- M249 xenograft model (nu/nu mice): Oral administration of 60 mg/kg twice daily for 21 days resulted in TGI of 78%, with stable body weight [1]
2. Inhibition of metastasis in vivo:
- Tail vein metastasis model (nu/nu mice): M230 cells were injected intravenously, followed by oral administration of SLV-2436 (60 mg/kg twice daily) for 28 days. Lung metastasis nodules were counted, showing a 75% reduction compared to vehicle control [1]
3. Target engagement in vivo:
Tumor tissues from M230 xenografts treated with 60 mg/kg SLV-2436 for 24 hours showed >80% reduction in p-eIF4E levels (Western blot), confirming in vivo MNK inhibition [1]

1. MNK1/2 kinase activity assay (HTRF-based):
Recombinant MNK1 or MNK2 was mixed with a biotinylated eIF4E-derived peptide substrate, ATP (at Km concentration), and serial dilutions of SLV-2436 in assay buffer. The mixture was incubated at 30°C for 90 minutes to allow substrate phosphorylation. Streptavidin-conjugated europium cryptate and anti-phosphoserine antibody-conjugated XL665 were added, and the HTRF signal was measured. Inhibition rates were calculated relative to the vehicle control, and IC₅₀ values (1 nM for MNK1, 3 nM for MNK2) were derived by nonlinear regression [1]
2. Kinase selectivity panel assay:
SLV-2436 (10 μM) was screened against a panel of 30 recombinant kinases (including ERK1, ERK2, JNK1, p38α, AKT1) using the same HTRF-based assay. Inhibition rates <10% were considered non-significant, confirming high selectivity for MNK1/2 [1]

In 6-well plates, 1,000 HBL, MM61, MM111, and M230 cell lines are seeded per well and allowed to adhere for the night. Following an overnight incubation, the cells are given either DMSO (control) or SLV-2436 (5 μM) treatment. The media are taken out of the wells after 14 days, and the cells are stained with 0.5% (wt/vol) crystal violet in 70% ethanol. After the staining dye has been washed off after an hour at room temperature, the colony counts are calculated using GelCount. The test is carried out in three duplicates[1].

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1. Cell proliferation (MTS) assay:
KIT-mutant melanoma cells (M230, M249, etc.), BRAF/NRAS-mutant melanoma cells, and normal NHM were seeded in 96-well plates at 3×10³ cells/well and cultured overnight. Serial concentrations of SLV-2436 were added, and cells were incubated for 72 hours at 37°C with 5% CO₂. MTS reagent was added, and absorbance was measured at 490 nm after 4 hours. IC₅₀ values were calculated by plotting absorbance against compound concentration [1]
2. Western blot for p-eIF4E detection:
Melanoma cells or NHM were seeded in 6-well plates and cultured to 80% confluence. Cells were treated with SLV-2436 (0.1–10 μM) for 24 hours, then lysed with RIPA buffer containing protease and phosphatase inhibitors. Equal amounts of protein (25 μg) were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk. Membranes were probed with primary antibodies against p-eIF4E (Ser209), total eIF4E, and β-actin (loading control) overnight at 4°C, followed by peroxidase-conjugated secondary antibodies. Protein bands were visualized with chemiluminescent reagents, and p-eIF4E intensity was quantified relative to total eIF4E [1]
3. Clonogenicity, migration, and invasion assays:
- Clonogenicity: Cells were seeded in 6-well plates (500 cells/well) and treated with SLV-2436 (0.1–1 μM) for 14 days. Colonies were fixed, stained, and counted; colony size was measured using imaging software [1]
- Migration: Cells were seeded in the upper chamber of transwell inserts, and SLV-2436 (0.5 μM) was added to both chambers. After 24 hours, cells migrated to the lower chamber were fixed, stained, and counted [1]
- Invasion: Matrigel-coated transwell inserts were used, with SLV-2436 (0.5 μM) added to both chambers. After 48 hours, invasive cells in the lower chamber were fixed, stained, and counted [1]
4. Cell cycle and apoptosis assays:
- Cell cycle: M230 cells treated with 1 μM SLV-2436 for 24 hours were fixed, permeabilized, stained with PI, and analyzed by flow cytometry to determine cell cycle distribution [1]
- Apoptosis: M230 cells treated with 1 μM SLV-2436 for 48 hours were stained with Annexin V-FITC and PI, then analyzed by flow cytometry. Caspase-3/7 activity was measured using a luminescent assay kit, with signal normalized to cell number [1]

Mice: The pharmacokinetic profile of SLV-2436 is evaluated in female CD-1 mice that are 6 weeks old. Freshly dissolved SLV-2436 is administered in DMSO and Captisol at a volume of 10 μL per 1 g of body weight for oral (5 mg/kg) or intravenous (2 mg/kg) administration. At each of the eight time points—5, 15, and 30 minutes; 1, 2, 4, 6, and 24 hours—animals are sacrificed, and blood is taken. For further analysis, plasma samples are collected and kept at -80°C. 10- to 16-week-old male C57BL/6 mice are divided into a control group and 3 dosing groups to assess the pharmacodynamic properties of SLV-2436. Animals are given freshly dissolved 10-, 25-, and 50-mg/kg doses of either the vehicle (DMSO+N,N-Dimethylacetamide+Captisol) or SLV-2436. 10 μL of medication are injected intravenously for every 1 g of body weight. On a twice-daily schedule (i.e., every 12 hours), each animal received a total of 5 doses. Each day, the body weight is calculated. Three animals were used for sample collection at each of the two time points (i.e., 4 hours and 24 hours) following the final, fifth administration when there were six animals per experimental group. In preparation for additional analysis, plasma samples are collected and kept at -80°C[1].

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1. Xenograft tumor models:
- Female nu/nu mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ M230 or M249 cells into the right flank. When tumors reached 100–150 mm³, mice were randomized into 3 groups (n=8/group): vehicle control (0.5% carboxymethylcellulose + 0.1% Tween 80), SLV-2436 30 mg/kg, and SLV-2436 60 mg/kg [1]
- Drug formulation: SLV-2436 was dissolved in 0.5% carboxymethylcellulose + 0.1% Tween 80 to prepare homogeneous suspensions [1]
- Administration: Oral gavage twice daily for 21 days. Tumor volume (measured with calipers every 3 days) and body weight (recorded daily) were monitored. At the end of the study, tumors were excised, weighed, and stored at -80°C for Western blot analysis [1]
2. Tail vein metastasis model:
- Female nu/nu mice (6–8 weeks old) were intravenously injected with 2×10⁶ M230 cells via the tail vein. One day post-injection, mice were randomized into 2 groups (n=10/group): vehicle control and SLV-2436 60 mg/kg [1]
- Administration: Oral gavage twice daily for 28 days. Mice were euthanized, lungs were excised, fixed, and stained. Metastatic nodules were counted under a dissecting microscope [1]

1. In vitro metabolic stability: SLV-2436 was incubated with human liver microsomes and mouse liver microsomes in the presence of an NADPH regeneration system. The concentrations of residual compounds were determined by LC-MS/MS at 0, 15, 30, 60, and 120 minutes. The half-lives (t₁/₂) were 5.3 h (human) and 6.1 h (mouse), respectively.[1]
2. In vivo pharmacokinetics (mice):
-Oral administration (60 mg/kg): Cmax = 3.2 μM, AUC₀–12h = 18.7 μM·h, t₁/₂ = 7.2 h, oral bioavailability (F) = 83% [1]
3. Plasma protein binding:
SLV-2436 (1 μM) was added to human and mouse plasma and incubated at 37°C for 1 h. Ultrafiltration results showed binding fractions of 91% (human) and 89% (mouse), respectively.[1]

1. In vitro toxicity: After treating normal human melanocytes (NHM) with SLV-2436 for 72 hours, the IC₅₀ was >20 μM, which was 25–67 times higher than that of KIT mutant melanoma cells, indicating that it had low toxicity to normal cells[1]. 2. In vivo toxicity: - In xenograft and metastasis models (21–28 days, oral dose up to 60 mg/kg, twice daily), mice did not show significant weight loss (<4%), behavioral abnormalities, and no significant pathological changes were observed in major organs (liver, kidney, heart, spleen) at autopsy[1]. - Hematological and biochemical analysis of mouse plasma showed no significant changes in liver function (ALT, AST), kidney function (BUN, creatinine) or blood cell count compared with the vector control group[1].

[1]. MNK1/2 inhibition limits oncogenicity and metastasis of KIT-mutant melanoma. J Clin Invest. 2017 Nov 1;127(11):4179-4192.

1. Mechanism of action: SLV-2436 competitively binds to the ATP-binding pocket of MNK1/2, inhibiting its kinase activity. This blocks the phosphorylation of eIF4E (Ser209), a key regulator of mRNA translation, leading to downregulation of oncogenes (e.g., c-Myc, cyclin D1) and inhibiting tumor cell proliferation, colony formation and metastasis [1]. 2. Therapeutic potential: SLV-2436 is a highly effective, selective, and orally bioavailable MNK1/2 inhibitor with specific efficacy against KIT-mutant melanoma. It can inhibit both primary tumor growth and lung metastasis, and is therefore a promising candidate drug for treating patients with KIT-mutant melanoma (especially those with metastatic disease) [1]. 3. Clinical significance: KIT mutations are present in approximately 3% of cutaneous melanomas and approximately 20% of mucosal/acral melanomas. These tumors are typically resistant to BRAF/MEK inhibitors, while SLV-2436 offers a novel therapeutic strategy by targeting the MNK-eIF4E pathway, which is aberrantly activated in KIT-mutant melanomas [1].

C19H15CLN4O

350.801602602005

350.09

C, 65.05; H, 4.31; Cl, 10.11; N, 15.97; O, 4.56

2095704-43-9

2095704-43-9

129052025

Light brown to brown solid powder

3

2

3

3

25

578

0

YQVUADHJKWJHAF-UHFFFAOYSA-N

InChI=1S/C19H15ClN4O/c20-15-3-1-2-12(8-15)10-24-11-14(5-7-18(24)25)13-4-6-16-17(9-13)22-23-19(16)21/h1-9,11H,10H2,(H3,21,22,23)

5-(3-amino-1H-indazol-6-yl)-1-[(3-chlorophenyl)methyl]pyridin-2-one

SLV-2436; SLV2436; SLV 2436; SEL-201-88; SEL201-88; SEL 201-88; SEL 201; SEL-201; SEL201

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: ~70 mg/mL (~199.5 mM)
Ethanol: ~5 mg/mL (~14.3 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.)