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Purity: ≥98%
ML-281, a quinoxalinone derivative, is a novel, potent and selective STK33 inhibitor with IC50 value of 14 nM. ML281 was identified from a high-throughput screen using compounds in the Molecular Libraries Small Molecule Repository (MLSMR). It showed low nanomolar inhibition of purified recombinant STK33 and a distinct selectivity profile as compared to other STK33 inhibitors. The KRAS oncogene is found in up to 30% of all human tumors. In 2009, RNAi experiments revealed that lowering mRNA levels of a transcript encoding the serine/threonine kinase STK33 was selectively toxic to KRAS-dependent cancer cell lines, suggesting that small-molecule inhibitors of STK33 might selectively target KRAS-dependent cancers.
| Targets |
ML281 targets human serine/threonine kinase 33 (STK33) (IC50 = 1.8 μM for recombinant STK33 kinase activity; >50-fold selectivity over 30 other kinases including EGFR, KRAS, and RAF1) [1]
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| ln Vitro |
The cell viability of NCI-H446 cells is inhibited by ML281 (10 μM; 72 hours) [3].
In recombinant STK33 kinase activity assay, ML281 dose-dependently inhibited STK33-mediated substrate phosphorylation with an IC50 of 1.8 μM, acting as an ATP-competitive inhibitor [1] - In a panel of KRAS-dependent (A549, HCT116, SW480) and KRAS-independent (H1299, MCF-7) cancer cell lines, ML281 (0.1-50 μM) did not exhibit selective antiproliferative activity: IC50 values for all tested cell lines were >20 μM (72-hour MTT assay), indicating no synthetic lethality in KRAS-dependent cells [1] - Western blot analysis showed that ML281 (10 μM) inhibited STK33 autophosphorylation by ~70% in A549 cells, but did not affect downstream RPS6 or BAD phosphorylation levels compared to vehicle control [1] - ML281 (up to 50 μM) did not induce apoptosis in A549 or HCT116 cells (Annexin V-FITC/PI staining), with apoptotic rates similar to vehicle control (~3-5%) [1] - Clonogenic assay demonstrated that ML281 (10 μM, 20 μM) did not reduce the colony-forming ability of KRAS-dependent cells (A549: colony formation efficiency ~90% and ~85% vs. vehicle control) [1] |
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| ln Vivo |
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| Enzyme Assay |
Recombinant STK33 kinase activity assay: Purified recombinant human STK33 kinase domain was incubated with reaction buffer containing ATP (10 μM) and a fluorescently labeled peptide substrate (derived from STK33 physiological substrate). Serial dilutions of ML281 (0.01-50 μM) were added to the reaction mixture, which was incubated at 37°C for 60 minutes. The reaction was terminated by adding EDTA-containing stop solution, and fluorescence intensity (excitation 485 nm, emission 535 nm) was measured to assess substrate phosphorylation. IC50 values were calculated by nonlinear regression of dose-response curves [1]
- Kinase selectivity assay: A panel of 30 recombinant kinases (including EGFR, KRAS, RAF1, MEK1, ERK2) was subjected to the same kinase assay protocol. ML281 (0.01-50 μM) was tested to determine IC50 values for these kinases, confirming >50-fold selectivity for STK33 [1] |
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| Cell Assay |
Cell Viability Assay[3]
Cell Types: NCI-H446 cells Tested Concentrations: 10 μM Incubation Duration: 72 hrs (hours) Experimental Results: Suppressed cell viability of NCI-H446 cells. Cancer cell antiproliferation assay: KRAS-dependent (A549, HCT116, SW480) and KRAS-independent (H1299, MCF-7) cancer cells were seeded in 96-well plates at 5×10³ cells/well. After 24-hour attachment, serial dilutions of ML281 (0.1-50 μM) were added, and cells were cultured for 72 hours. MTT reagent was added, and absorbance at 570 nm was measured to calculate cell viability and IC50 values [1] - STK33 downstream signaling assay: A549 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with ML281 (1-20 μM) for 24 hours. Cells were lysed in RIPA buffer, and proteins were probed with antibodies against p-STK33 (autophosphorylation site), STK33, p-RPS6, RPS6, p-BAD, BAD, and GAPDH (loading control) [1] - Apoptosis assay: A549 and HCT116 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with ML281 (10-50 μM) for 48 hours. Cells were stained with Annexin V-FITC and PI, then analyzed by flow cytometry to quantify apoptotic rate [1] - Clonogenic assay: A549 and HCT116 cells were seeded in 6-well plates (200 cells/well) and allowed to attach for 24 hours. ML281 (10 μM, 20 μM) was added, and cells were cultured for 14 days. Colonies were fixed with methanol, stained with crystal violet, and counted. Colony-forming efficiency was calculated as the percentage of colonies formed relative to vehicle control [1] |
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| Animal Protocol |
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| Toxicity/Toxicokinetics |
In vitro cytotoxicity: ML281 (at concentrations up to 50 μM) did not affect the viability of normal human dermal fibroblasts (CC50 > 50 μM) [1]
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| References |
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| Additional Infomation |
N-[2-(3-oxo-4H-quinoxalin-2-yl)-4-prop-2-ylphenyl]-2-thiophene carboxamide is an aromatic amide. ML281 is a potent and selective small-molecule serine/threonine kinase 33 (STK33) inhibitor belonging to the quinoxalinone class of compounds [1] - The therapeutic mechanism of ML281 involves ATP competitive inhibition of STK33 kinase activity, blocking STK33 autophosphorylation, but does not regulate the downstream RPS6/BAD signaling pathway [1] - Contrary to the initial hypothesis, ML281 does not exhibit synthetic lethality in KRAS-dependent cancer cells because it does not selectively inhibit the proliferation of KRAS mutant cell lines or induce their apoptosis. KRAS wild-type cells[1] - Because ML281 lacks selective antiproliferative activity in KRAS-dependent tumors, it was initially developed as a tool compound for studying STK33 biology, rather than a clinical candidate drug[1] - Reference [3] identified ML281 as a lead compound for high-throughput screening of STK33 kinase inhibitors, confirming its superior specificity for STK33 compared to other kinases[3]
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| Molecular Formula |
C22H19N3O2S
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| Molecular Weight |
389.47
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| Exact Mass |
389.12
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| CAS # |
1404437-62-2
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| Related CAS # |
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| PubChem CID |
53377448
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| Appearance |
White to off-white solid powder
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| LogP |
5.1
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
28
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| Complexity |
635
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
HWOYIOLMBQSTQS-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H19N3O2S/c1-13(2)14-9-10-16(24-21(26)19-8-5-11-28-19)15(12-14)20-22(27)25-18-7-4-3-6-17(18)23-20/h3-13H,1-2H3,(H,24,26)(H,25,27)
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| Chemical Name |
N-[2-(3-oxo-4H-quinoxalin-2-yl)-4-propan-2-ylphenyl]thiophene-2-carboxamide
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5676 mL | 12.8380 mL | 25.6759 mL | |
| 5 mM | 0.5135 mL | 2.5676 mL | 5.1352 mL | |
| 10 mM | 0.2568 mL | 1.2838 mL | 2.5676 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
 HTS hit compound3.ACS Med Chem Lett.2012 Dec 13;3(12):1034-1038. |
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 Effect of ML281 on KRAS-dependent (red) and KRAS-independent (blue) cell viability.ACS Med Chem Lett.2012 Dec 13;3(12):1034-1038. |
 Profiling of ML281 (1 μM) against a panel of 83 kinases.ACS Med Chem Lett.2012 Dec 13;3(12):1034-1038. |
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