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| Targets |
The primary target of MLK-IN-1 is MLK-3 (mixed-lineage kinase 3), a serine/threonine protein kinase belonging to the MLK family, which is a subgroup of MAP kinase kinase kinases (MAP3Ks). MLK-3 is involved in the activation of the JNK and p38 MAPK signaling pathways, which play critical roles in cellular stress responses, inflammation, and apoptosis. MLK-IN-1 exhibits high specificity for MLK-3, with excellent brain penetration, making it a valuable tool for studying the role of MLK-3 in the central nervous system. By selectively inhibiting MLK-3, MLK-IN-1 modulates downstream JNK and p38 signaling pathways.
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| ln Vitro |
In the presence of Tat-activated microglia, MLK-IN-1 (100 nM; pre-treatment 20 mins HIV-1 Tat) stimulates ongoing axonogenesis and guards against the effects of HIV-Tat in vitro[1].
MLK-IN-1 at 100 nM supports sustained axonogenesis in cultures challenged with HIV-1 Tat-activated microglia and protects neuronal cells from Tat-induced damage. This demonstrates its neuroprotective activity in an in vitro model of neuroinflammation. The compound's high specificity for MLK-3 and excellent brain penetration make it a valuable probe for studying the role of MLK-3 in neurodegenerative diseases. Its purity is reported to be 99.74%, and it is soluble in DMSO at 50 mg/mL (115.61 mM). |
| ln Vivo |
In vivo, MLK-IN-1 is used to study the role of MLK-3 in neuroinflammation and neurodegeneration. Its excellent brain penetration allows it to effectively reach its target in the central nervous system. The compound's ability to protect neuronal cells from Tat-induced damage in vitro suggests potential in vivo applications in models of HIV-associated neurocognitive disorders and other neurodegenerative conditions. As a research tool, MLK-IN-1 enables the investigation of MLK-3-mediated signaling pathways in various disease models, including neuroinflammation and neurodegeneration.
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| Enzyme Assay |
In vitro enzyme activity assays for MLK-IN-1 typically involve measuring its ability to inhibit MLK-3 kinase activity. Recombinant MLK-3 enzyme is incubated with its substrate in the presence of ATP and increasing concentrations of MLK-IN-1. Kinase activity is measured by detecting phosphorylation of the substrate, and the IC50 is determined from the dose-response curve. The compound's high specificity for MLK-3 is confirmed by testing against a panel of other kinases. Binding assays, such as surface plasmon resonance or isothermal titration calorimetry, can be used to measure the direct binding affinity of MLK-IN-1 to MLK-3.
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| Cell Assay |
Cellular assays for MLK-IN-1 are performed using neuronal cell cultures or microglial cell lines. In HIV-1 Tat-activated microglia challenge conditions, MLK-IN-1 at 100 nM is used to pretreat cells 20 minutes before Tat treatment. The compound's ability to support sustained axonogenesis and protect neuronal cells from Tat-induced damage is then assessed. Readouts include neurite outgrowth measurements, neuronal survival assays, and analysis of downstream signaling pathways (e.g., JNK and p38 phosphorylation) to confirm MLK-3 inhibition.
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| Animal Protocol |
In vivo animal studies with MLK-IN-1 are conducted in models of neuroinflammation and neurodegenerative diseases. The compound's excellent brain penetration makes it suitable for oral or intraperitoneal administration. Efficacy endpoints include assessment of neuroinflammation, neuronal survival, and behavioral outcomes. For example, in HIV-1 Tat-induced neurotoxicity models, MLK-IN-1 could be administered to evaluate its ability to protect against Tat-induced neuronal damage and neuroinflammation. Its effects on MLK-3-mediated JNK and p38 signaling pathways would be assessed by analyzing phosphorylation levels of downstream targets in brain tissue.
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| ADME/Pharmacokinetics |
MLK-IN-1 has a molecular weight of 432.49 and a molecular formula of C23H20N4O3S. It exhibits excellent brain penetration, which is a key feature for studying central nervous system targets. The compound is supplied as a powder with a purity of 99.74% and is soluble in DMSO at 50 mg/mL (115.61 mM). For in vivo studies, MLK-IN-1 can be formulated in appropriate vehicles. The compound should be stored as a powder at -20°C for up to 3 years, and in solvent at -80°C for up to 1 year.
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| Toxicity/Toxicokinetics |
No specific toxicity data is available for MLK-IN-1 in the provided literature. As a research compound, its safety profile would be an important consideration for in vivo applications. Potential toxicities could be related to its effects on MLK-3-mediated JNK and p38 signaling pathways in various tissues. Standard preclinical safety studies would be required to evaluate its safety for potential therapeutic applications. The compound is for research use only and is not intended for human therapeutic use.
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| References | |
| Additional Infomation |
MLK-IN-1 is a potent, brain-penetrant, and highly selective MLK-3 inhibitor. It is compound 68 from patent US20140256733A1 and has a purity of 99.74%. The compound is used as a chemical probe for studying neuroinflammation and neurodegenerative diseases, particularly in the context of HIV-1 Tat-induced neurotoxicity. At 100 nM, MLK-IN-1 supports sustained axonogenesis and protects neuronal cells from Tat-induced damage in microglia challenge conditions. Its excellent brain penetration and high specificity for MLK-3 make it a valuable tool for investigating the role of MLK-3 in the central nervous system.
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| Molecular Formula |
C23H20N4O3S
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| Molecular Weight |
432.494903564453
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| Exact Mass |
432.125
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| CAS # |
1627729-62-7
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| PubChem CID |
90388209
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| Appearance |
Light yellow to green yellow solid powder
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| LogP |
4.8
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
31
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| Complexity |
601
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S1C2C(=CC=CC=2C=C1C1=CN=C2C=CC(NC3C=CC(=C(C=3)OC)OC)=NN12)OC
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| InChi Key |
NTIXYCZKSHEEOH-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H20N4O3S/c1-28-17-8-7-15(12-19(17)30-3)25-21-9-10-22-24-13-16(27(22)26-21)20-11-14-5-4-6-18(29-2)23(14)31-20/h4-13H,1-3H3,(H,25,26)
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| Chemical Name |
N-(3,4-dimethoxyphenyl)-3-(7-methoxy-1-benzothiophen-2-yl)imidazo[1,2-b]pyridazin-6-amine
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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: 62.5 mg/mL (144.51 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 | 2.3122 mL | 11.5610 mL | 23.1219 mL | |
| 5 mM | 0.4624 mL | 2.3122 mL | 4.6244 mL | |
| 10 mM | 0.2312 mL | 1.1561 mL | 2.3122 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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