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| Targets |
MLS000544460 targets Eya2 phosphatase. Eya2 is a member of the Eyes Absent (Eya) family of proteins, which function as transcriptional co-activators and tyrosine phosphatases. Eya2 plays a critical role in various developmental processes, including organogenesis, and is also involved in cancer. Eya2 phosphatase activity is required for cell migration, and its overexpression has been linked to tumor metastasis. MLS000544460 is a highly selective and reversible inhibitor of Eya2 phosphatase. It binds to the Eya2 phosphatase domain with a Kd of 2.0 μM and inhibits its enzymatic activity with an IC₅₀ of 4 μM. The compound does not bind to Eya3 phosphatase, demonstrating its selectivity for Eya2. It is also ineffective against other tyrosine phosphatases, including Eya3 and PTP1B. The compound's binding to Eya2 is enhanced in the absence of Mg²⁺, indicating that this class of compounds does not require Mg²⁺ for its interaction with the Eya2 ED. By inhibiting Eya2 phosphatase, MLS000544460 can modulate cell migration and other Eya2-dependent processes.
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| ln Vitro |
MLS000544460 does not bind to ED, or Eya3 phosphatase [1]. In MCF10A cells expressing human Eya2, Eya2 phosphatase-dead (D274N), and YFP (as a negative control), MLS000544460 (10 μM) suppresses Eya2-mediated cell migration [1]. This class of chemicals does not require Mg2+ to interact with Eya2 ED, as shown by the increased affinity with which it binds MLS000544460 (Kd=0.80 μM) when dialyzed with 10 μM EDTA to remove Mg2+ [1].
In vitro, MLS000544460 inhibits Eya2 phosphatase activity with an IC₅₀ of 4 μM and a Kd of 2.0 μM. It does not bind to Eya3 phosphatase, demonstrating its selectivity for Eya2. The compound (10 μM) inhibits Eya2-mediated cell migration in human MCF10A cells expressing Eya2. This effect is specific to Eya2-mediated migration, as the compound does not inhibit migration in cells expressing a phosphatase-dead mutant of Eya2 (D274N) or in control cells expressing YFP. The compound's ability to inhibit Eya2-mediated cell migration is consistent with its role as an Eya2 phosphatase inhibitor. The compound also has anticancer activity, likely due to its inhibition of Eya2, which is involved in tumor progression and metastasis. The compound's selectivity for Eya2 over other phosphatases makes it a valuable tool for studying the specific role of Eya2 in cell migration and cancer. |
| ln Vivo |
Detailed in vivo activity data for MLS000544460 is not extensively reported in the available literature. However, its in vitro anticancer activity and its ability to inhibit Eya2-mediated cell migration suggest that it may have potential for treating cancer in vivo. Eya2 is overexpressed in various cancers and is associated with poor prognosis. Inhibiting Eya2 phosphatase activity could potentially reduce tumor growth and metastasis. The compound's selectivity for Eya2 over other phosphatases suggests that it may have a favorable safety profile. However, in vivo studies are needed to evaluate its efficacy and safety in animal models of cancer. The compound's pharmacokinetic properties, such as oral bioavailability and tissue distribution, would also need to be assessed. Currently, MLS000544460 is primarily a research tool for in vitro studies.
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| Enzyme Assay |
The inhibition of Eya2 phosphatase by MLS000544460 is typically measured using a phosphatase assay. The assay is performed in a 96-well plate format. The reaction mixture contains 50 mM Tris-HCl buffer (pH 7.5), 50 mM NaCl, 1 mM DTT, 0.1 mg/mL BSA, and a fluorogenic substrate, such as 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP). The substrate is dephosphorylated by the phosphatase to release the fluorescent product, which is measured at excitation 358 nm and emission 450 nm. The reaction is initiated by the addition of the Eya2 enzyme. The increase in fluorescence is monitored over time. The percentage of inhibition at each compound concentration is calculated relative to a control without inhibitor. The IC₅₀ is determined by fitting the data to a four-parameter logistic model. To determine the Kd, a binding assay such as isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) is used. The selectivity of the compound for Eya2 over other phosphatases, such as Eya3 and PTP1B, is assessed using the same phosphatase assay with the respective enzymes.
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| Cell Assay |
For in vitro cellular assays, the effect of MLS000544460 on cell migration is typically assessed using a wound healing or transwell migration assay. In a wound healing assay, MCF10A cells expressing Eya2 are grown to confluence in a 6-well plate. A scratch wound is created in the cell monolayer using a pipette tip. The cells are then treated with MLS000544460 at various concentrations (e.g., 1-10 μM). The migration of cells into the wound area is monitored over time using a microscope. The percentage of wound closure is calculated at various time points. In a transwell migration assay, cells are seeded in the upper chamber of a transwell insert containing a porous membrane. The lower chamber contains a chemoattractant. The cells are treated with MLS000544460 in the upper chamber. After incubation, cells that have migrated to the lower side of the membrane are fixed, stained, and counted. The percentage of inhibition of cell migration is calculated relative to untreated control cells. These assays provide a measure of the compound's ability to inhibit Eya2-mediated cell migration.
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| Animal Protocol |
Detailed in vivo animal experiment protocols for MLS000544460 are not extensively reported in the available literature. Typically, to assess the in vivo efficacy of a compound that inhibits cell migration and has anticancer activity, a xenograft mouse model would be employed. In such a study, immunodeficient mice are injected with cancer cells that overexpress Eya2. When tumors reach a certain size, MLS000544460 is administered orally or intraperitoneally at various doses. Tumor volume is measured regularly, and final tumor weights are recorded. The number and size of metastases would also be assessed. The compound's ability to inhibit tumor growth and metastasis would be evaluated by comparing tumor volumes, weights, and metastatic burden between treated and control groups. The compound's effects on Eya2 phosphatase activity in vivo could be assessed by measuring the phosphorylation status of Eya2 substrates in tumor tissues. These studies would provide critical data on the compound's in vivo efficacy and mechanism of action. Currently, no such detailed in vivo protocols for MLS000544460 have been published.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties for MLS000544460 are not extensively reported in the available literature. The compound has a molecular weight of 341.36 and is soluble in DMSO (125 mg/mL). It is formulated for in vivo administration using a combination of DMSO, PEG300, Tween-80, and saline, or DMSO and corn oil. These formulations suggest that the compound may have limited aqueous solubility. Specific pharmacokinetic parameters such as half-life, volume of distribution, clearance, and oral bioavailability have not been determined experimentally. The compound's metabolism is likely to involve hepatic cytochrome P450 enzymes. The compound's elimination route is unknown. Further studies are needed to fully characterize the pharmacokinetic profile of this compound. The compound's in vivo activity would depend on its ability to reach target tissues in sufficient concentrations.
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| Toxicity/Toxicokinetics |
Toxicological data for MLS000544460 is limited, as it is a research chemical. Standard safety precautions should be observed when handling this compound. No specific toxicity studies, such as acute or chronic toxicity in animal models, have been detailed in the public domain. The compound's selectivity for Eya2 over other phosphatases suggests that it may have a reduced risk of off-target effects. However, as with all research chemicals, it should be handled with care, using appropriate personal protective equipment and following institutional safety guidelines. The compound is not approved for clinical use and should only be used in preclinical research settings.
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| References | |
| Additional Infomation |
MLS000544460 is a research tool for studying the role of Eya2 phosphatase in cell migration and cancer. Its mechanism of action involves selective inhibition of Eya2 phosphatase. It has a Kd of 2.0 μM and an IC₅₀ of 4 μM for Eya2. It is not approved for clinical use.
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| Molecular Formula |
C17H12FN3O2S
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|---|---|
| Molecular Weight |
341.3595
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| Exact Mass |
341.063
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| CAS # |
352336-36-8
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| PubChem CID |
7285259
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| Appearance |
White to off-white solid powder
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| LogP |
4
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
24
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| Complexity |
452
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S(C1=C([H])C([H])=C([H])C([H])=N1)C1=C([H])C([H])=C(/C(/[H])=N/N([H])C(C2C([H])=C([H])C([H])=C(C=2[H])F)=O)O1
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| InChi Key |
VDKNIVFIPSLUFD-RGVLZGJSSA-N
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| InChi Code |
InChI=1S/C17H12FN3O2S/c18-13-5-3-4-12(10-13)17(22)21-20-11-14-7-8-16(23-14)24-15-6-1-2-9-19-15/h1-11H,(H,21,22)/b20-11+
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| Chemical Name |
3-fluoro-N-[(E)-(5-pyridin-2-ylsulfanylfuran-2-yl)methylideneamino]benzamide
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| Synonyms |
MLS 000544460; MLS-000544460
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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 : ~125 mg/mL (~366.18 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (6.09 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 20.8 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (6.09 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9295 mL | 14.6473 mL | 29.2946 mL | |
| 5 mM | 0.5859 mL | 2.9295 mL | 5.8589 mL | |
| 10 mM | 0.2929 mL | 1.4647 mL | 2.9295 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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