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para-Nitroblebbistatin is a novel, photostable, non-cytotoxic, fluorescent and specific Myosin II inhibitor, used in the study of the specific role of myosin II in physiological, developmental, and cell biological studies.
| Targets |
Myosin II
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|---|---|
| ln Vitro |
Blebbistatin, the best characterized myosin II-inhibitor, is commonly used to study the biological roles of various myosin II isoforms. Despite its popularity, the use of blebbistatin is greatly hindered by its blue-light sensitivity, resulting in phototoxicity and photoconversion of the molecule. Additionally, blebbistatin has serious cytotoxic side effects even in the absence of irradiation, which may easily lead to the misinterpretation of experimental results since the cytotoxicity-derived phenotype could be attributed to the inhibition of the myosin II function. Here we report the synthesis as well as the in vitro and in vivo characterization of a photostable, C15 nitro derivative of blebbistatin with unaffected myosin II inhibitory properties. Importantly, para-nitroblebbistatin is neither phototoxic nor cytotoxic, as shown by cellular and animal tests; therefore it can serve as an unrestricted and complete replacement of blebbistatin both in vitro and in vivo[1].
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| Enzyme Assay |
Basal and actin activated ATPase measurements [1]
Relative basal or actin activated ATPase activities of 3 µM or 0.5 µM DdMD W501+ and 3 µM or 0.2 µM SkS1 constructs were measured at increasing concentrations of blebbistatin/para-nitroBlebbistatin/parachloroblebbistatin or actin using a pyruvate kinase/lactate dehydrogenase coupled assay (NADH-coupled assay) at 20⁰C. In the actin activated ATPase measurements, 20 µM inhibitor was pre-incubated with myosin for 15 minutes on ice. G-actin was prepared accordingly from rabbit skeletal muscle and polymerized by 2mM MgCl2 for 1 hour at room temperature. Basal ATPase activity measurements were carried out in assay buffer and actin activated ATPase activity measurements were performed in low salt buffer (2 mM MgCl2, 1 mM HEPES, 2 mM 2- mercaptoethanol, pH 7.3). |
| Cell Assay |
Cellular assays and microscopic imaging [1]
For phototoxicity assays, HeLa cells were grown into 96-well plates achieving monolayer cultures. Cells were incubated in PBS containing 10 µM blebbistatin or para-nitroBlebbistatin for 30 minutes then irradiated with 480±10 nm light for 15 minutes. The applied energy was 3.3 µJ/µm2 . After irradiation the inhibitors were washed out and cells were maintained in cell culture medium. After 18 hours of incubation, the mortality rate was determined by direct trypan blue staining and subsequent counting of the cells in the illuminated area. Widefield images of HeLa cells were acquired on a modified light microscope (Motic AE31) equipped with a Sony cyber-shot camera using an LWD PH 20x/0.40 objective (Figure 2A and Figure S2). Confocal imaging of HeLa cells was performed using Plan Apo 63x/1.40 Oil DIC objective (Figure 3C). For acquisition and image analysis Zeiss LSM 710 Zen 2011 software was used. For live cell confocal time-lapse imaging of HeLa Kyoto cells, DMEM medium was supplemented with 25 mM HEPES in order to achieve CO2 independent media. Confocal time-lapse imaging was performed on a Zeiss LSM 710 using a Plan Apo 20x/0.8 objective (Figure 2B and Movies S1, S2 and S3). Inhibitor treated Dd cells were cultured in 50-ml Falcon tubes for three days at 21 °C with shaking at 200 rpm. Two photon imaging of Dd and M2 cells was performed by a Femtonics two photon microscope (equipped with a Ti-sapphire laser, Spectraphysics) using a Plan Apo 63×/1.4 oil DIC II objective (Figures S3, S4 and Movies S4 and S5). Acquisition and image analysis were performed using Femtonics MES software. |
| Animal Protocol |
Phototoxicity assays and microscopic imaging of zebrafish embryos [1]
Zebrafish embryos were incubated with 5 µM blebbistatin and para-nitroBlebbistatin and irradiated with 470 ± 20 nm light for 10 minutes. The applied energy was 0.4 µJ/µm2 . The embryos were monitored for 36 hours and were considered to be dead when became necrotic. Images 8 of zebrafishes were captured by a Zeiss Stereo Lumar.V12 microscope using a NeoLumar S 0.8x FWD 80mm objective. For acquisition AxioVision 4.8 software was used (Figure 4B). Fish husbandry and embryo treatments [1] Transgenic Tg(−8.0cldnb:lynEGFP)zf106 (cldn:gfp) fish stocks were used. For blebbistatin and para-nitroBlebbistatin treatments 1 dpf embryos were dechorionated and placed in the indicated reagent concentrations, diluted in standard E3 embryo medium in a 20-well plate. All protocols used in this study were approved by the Hungarian National Food Chain Safety Office (Permit Number: XIV-I-001/515-4/2012). |
| References | |
| Additional Infomation |
Blebbistatin is a commonly used molecular tool for the specific inhibition of various myosin II isoforms both in vitro and in vivo. Despite its popularity, the use of blebbistatin is hindered by its poor water-solubility (below 10 micromolar in aqueous buffer) and blue-light sensitivity, resulting in the photoconversion of the molecule, causing severe cellular phototoxicity in addition to its cytotoxicity. Furthermore, blebbistatin forms insoluble aggregates in water-based media above 10 micromolar with extremely high fluorescence and also high adherence to different types of surfaces, which biases its experimental usage. Here, we report a highly soluble (440 micromolar in aqueous buffer), non-fluorescent and photostable C15 amino-substituted derivative of blebbistatin, called para-aminoblebbistatin. Importantly, it is neither photo- nor cytotoxic, as demonstrated on HeLa cells and zebrafish embryos. Additionally, para-aminoblebbistatin bears similar myosin II inhibitory properties to blebbistatin or para-nitroblebbistatin (not to be confused with the C7 substituted nitroblebbistatin), tested on rabbit skeletal muscle myosin S1 and on M2 and HeLa cells. Due to its drastically improved solubility and photochemical feature, as well as lack of photo- or cytotoxicity, para-aminoblebbistatin may become a feasible replacement for blebbistatin, especially at applications when high concentrations of the inhibitor or blue light irradiation is required.[ Sci Rep. 2016 May 31;6:26141.]
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| Molecular Formula |
C18H15N3O4
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|---|---|
| Molecular Weight |
337.329404115677
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| Exact Mass |
337.106
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| Elemental Analysis |
C, 64.09; H, 4.48; N, 12.46; O, 18.97
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| CAS # |
1621326-32-6
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| PubChem CID |
102361739
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| Appearance |
Yellow to orange solid powder
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| LogP |
2.2
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
25
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| Complexity |
608
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| Defined Atom Stereocenter Count |
1
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| SMILES |
O[C@]12C(C3C=C(C)C=CC=3N=C1N(C1C=CC(=CC=1)[N+](=O)[O-])CC2)=O
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| InChi Key |
KAUXNLHXGQGFOS-GOSISDBHSA-N
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| InChi Code |
InChI=1S/C18H15N3O4/c1-11-2-7-15-14(10-11)16(22)18(23)8-9-20(17(18)19-15)12-3-5-13(6-4-12)21(24)25/h2-7,10,23H,8-9H2,1H3/t18-/m1/s1
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| Chemical Name |
(3aS)-3a-hydroxy-6-methyl-1-(4-nitrophenyl)-2,3-dihydropyrrolo[2,3-b]quinolin-4-one
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| Synonyms |
para-nitroblebbistatin; p-Nitroblebbistatin; 1621326-32-6; CHEMBL4162111; (3aS)-3a-hydroxy-6-methyl-1-(4-nitrophenyl)-2,3-dihydropyrrolo[2,3-b]quinolin-4-one; (3aS)-1,2,3,3a-tetrahydro-3a-hydroxy-6-methyl-1-(4-nitrophenyl)-4H-pyrrolo[2,3-b]quinolin-4-one; SCHEMBL23917202; TQR0306;
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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 : ~22 mg/mL (~65.22 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 | 2.9645 mL | 14.8223 mL | 29.6446 mL | |
| 5 mM | 0.5929 mL | 2.9645 mL | 5.9289 mL | |
| 10 mM | 0.2964 mL | 1.4822 mL | 2.9645 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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