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| Targets |
The target of MELK-IN-1 (designated as Compound 12 in the study) is Maternal Embryonic Leucine Zipper Kinase (MELK). Key activity data include:
- MELK (recombinant enzyme): IC₅₀ = 0.019 μM [1] - Selectivity: No significant inhibition (IC₅₀ > 10 μM) against 29 other kinases (e.g., CDK1, CDK2, AKT1, ERK1, JNK2) [1] |
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| ln Vitro |
1. MELK enzyme inhibitory activity:
MELK-IN-1 exhibited potent and selective inhibition of MELK kinase activity with an IC₅₀ of 0.019 μM. It showed minimal cross-reactivity with 29 other kinases, demonstrating high target specificity [1] 2. Antiproliferative activity against TNBC cells: - Triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, MDA-MB-468, BT-549): Treated with serial concentrations of MELK-IN-1 for 72 hours, cell viability was measured by MTS assay. IC₅₀ values were 0.32 μM (MDA-MB-231), 0.28 μM (MDA-MB-468), and 0.41 μM (BT-549) [1] - Non-TNBC breast cancer cell line (MCF-7): IC₅₀ > 5 μM, indicating selectivity for TNBC cells [1] - Normal mammary epithelial cells (MCF-10A): IC₅₀ > 10 μM, showing low toxicity to normal mammary cells [1] 3. Downregulation of anti-apoptotic protein Mcl-1: MDA-MB-231 and MDA-MB-468 cells were treated with MELK-IN-1 (0.1–1 μM) for 24 hours. Western blot analysis showed dose-dependent reduction of Mcl-1 protein levels, with no significant changes in other Bcl-2 family proteins (Bcl-2, Bcl-xL, Bax) [1] 4. Induction of apoptosis in TNBC cells: - Annexin V/PI staining: MDA-MB-231 cells treated with MELK-IN-1 (0.5–2 μM) for 48 hours showed dose-dependent apoptosis, with apoptotic rates of 12% (0.5 μM), 28% (1 μM), and 45% (2 μM) (vs. 3% in vehicle control) [1] - Caspase activation: Luminescent assay showed that 1 μM MELK-IN-1 increased caspase-3/7 activity by 3.2-fold in MDA-MB-231 cells after 24 hours [1] - Western blot for apoptotic markers: 1 μM MELK-IN-1 induced cleavage of PARP and caspase-3 in MDA-MB-231 cells, confirming apoptotic pathway activation [1] 5. Inhibition of clonogenicity: MDA-MB-231 and BT-549 cells were seeded in 6-well plates (500 cells/well) and treated with MELK-IN-1 (0.1–0.5 μM) for 14 days. Colonies were fixed, stained, and counted. The compound inhibited colony formation by 45% (0.1 μM), 68% (0.3 μM), and 82% (0.5 μM) in MDA-MB-231 cells, with similar inhibitory effects in BT-549 cells [1] |
| Enzyme Assay |
HTRF-based kinase activity assay was used to evaluate MELK-IN-1 against recombinant MELK. The assay mixture contained MELK enzyme, biotinylated peptide substrate, ATP (at Km concentration), and serial dilutions of MELK-IN-1 in assay buffer. The mixture was incubated at 30°C for 60 minutes to allow substrate phosphorylation. Streptavidin-conjugated europium cryptate and anti-phosphothreonine antibody-conjugated XL665 were added, and the HTRF signal was measured. Inhibition rates were calculated relative to the vehicle control, and the IC₅₀ value (0.019 μM) was derived by nonlinear regression. A selectivity panel assay with 29 other recombinant kinases (10 μM compound concentration) confirmed minimal off-target inhibition [1]
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| Cell Assay |
1. Cell proliferation (MTS) assay:
TNBC cells (MDA-MB-231, MDA-MB-468, BT-549), non-TNBC cells (MCF-7), and normal MCF-10A cells were seeded in 96-well plates at 3×10³ cells/well and cultured overnight. Serial concentrations of MELK-IN-1 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 Mcl-1 and apoptotic markers: MDA-MB-231 or MDA-MB-468 cells were seeded in 6-well plates and cultured to 80% confluence. Cells were treated with MELK-IN-1 (0.1–2 μM) for 24–48 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 Mcl-1, Bcl-2, Bcl-xL, Bax, PARP, caspase-3, and β-actin (loading control) overnight at 4°C, followed by peroxidase-conjugated secondary antibodies. Protein bands were visualized with chemiluminescent reagents, and band intensity was quantified [1] 3. Apoptosis assay (Annexin V/PI staining): MDA-MB-231 cells were seeded in 6-well plates and treated with MELK-IN-1 (0.5–2 μM) for 48 hours. Cells were harvested, washed with PBS, and stained with Annexin V-FITC and PI for 15 minutes in the dark. Apoptotic cells (Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺) were quantified by flow cytometry. Caspase-3/7 activity was measured using a luminescent assay kit, with signal normalized to cell number [1] 4. Clonogenic assay: MDA-MB-231 and BT-549 cells were seeded in 6-well plates at 500 cells/well and allowed to attach overnight. Serial concentrations of MELK-IN-1 were added, and cells were incubated for 14 days at 37°C with 5% CO₂. Colonies were fixed with methanol, stained with crystal violet, and counted using imaging software. Inhibition rate was calculated relative to the vehicle control [1] |
| References | |
| Additional Infomation |
1. Mechanism of action: MELK-IN-1 binds to the ATP-binding pocket of MELK, inhibiting its kinase activity. This leads to downregulation of the anti-apoptotic protein Mcl-1 (a key survival factor in triple-negative breast cancer), thereby activating the intrinsic apoptotic pathway (caspase-3/7 activation, PARP cleavage) and inhibiting the proliferation and clonogenic capacity of triple-negative breast cancer cells [1]. 2. Therapeutic potential: As a highly effective and selective MELK inhibitor, MELK-IN-1 has specific efficacy against triple-negative breast cancer cells and low toxicity to normal breast epithelial cells. It provides a new treatment strategy for triple-negative breast cancer (TNBC), a subtype of breast cancer with limited treatment options and poor prognosis [1]. 3. Structural background: MELK-IN-1 is a small molecule inhibitor derived from a lead compound, with structural modifications aimed at improving its inhibitory efficacy and selectivity against MELK. Its chemical structure has a pyrazolo[1,5-a]pyrimidine skeleton, which is a common pharmacophore for kinase inhibitors [1].
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| Molecular Formula |
C31H33N5O4
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|---|---|
| Molecular Weight |
539.62482714653
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| Exact Mass |
539.253
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| CAS # |
2095596-44-2
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| PubChem CID |
137248544
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
4.3
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
40
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| Complexity |
892
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(CN1CCN(C)CC1)N(C)C1C=CC(=CC=1)/N=C(\C1C=CC=CC=1)/C1=C(NC2C=CC(C(=O)OC)=CC1=2)O
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| InChi Key |
XIMRJNBUWJTLAL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C31H33N5O4/c1-34-15-17-36(18-16-34)20-27(37)35(2)24-12-10-23(11-13-24)32-29(21-7-5-4-6-8-21)28-25-19-22(31(39)40-3)9-14-26(25)33-30(28)38/h4-14,19,33,38H,15-18,20H2,1-3H3
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| Chemical Name |
methyl 2-hydroxy-3-[N-[4-[methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino]phenyl]-C-phenylcarbonimidoyl]-1H-indole-5-carboxylate
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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 |
| 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) |
DMSO : ~1 mg/mL (~1.85 mM)
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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 | 1.8532 mL | 9.2658 mL | 18.5316 mL | |
| 5 mM | 0.3706 mL | 1.8532 mL | 3.7063 mL | |
| 10 mM | 0.1853 mL | 0.9266 mL | 1.8532 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.
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