Myosin II[1]

In vitro and in vivo inhibitory properties of para-aminoblebbistatin[1]
After the physico-chemical characterization of para-aminoblebbistatin, we compared its in vitro and in vivo inhibitory properties to those of blebbistatin and para-nitroblebbistatin. We measured the basal and actin activated ATPase activites of rabbit skeletal muscle myosin S1 (SkS1) and Dictyostelium discoideum myosin II motor domain (DdMD) at increasing concentrations of para-aminoblebbistatin, para-nitroblebbistatin or blebbistatin (Fig. 3a,b). All three myosin II inhibitors decreased the basal as well as actin activated ATPase activity of SkS1 to a maximal extent (~98–100%) yielding half-maximal inhibitory concentration values of IC50,AmBleb = 1.3 ± 0.1 μM, IC50,NBleb = 0.3 ± 0.04 μM and IC50,Bleb = 0.3 ± 0.03 μM for the basal ATPase of SkS1 and IC50,AmBleb = 0.47 ± 0.06 μM, IC50,NBleb = 0.1 ± 0.004 μM and IC50,Bleb = 0.11 ± 0.009 μM for the actin activated ATPase activity of SkS1. Para-nitroblebbistatin and blebbistatin also exerted maximal inhibition on DdMD (~100%), while AmBleb reached 90% and 80% inhibition on basal and actin activated ATPase activities of DdMD yielding half-maximal inhibitory concentration values of IC50,AmBleb = 6.6 ± 2 μM, IC50,NBleb = 5.3 ± 1.6 μM and IC50,Bleb = 4.4 ± 0.3 μM for the basal ATPase and IC50,AmBleb = 6.7 ± 1.9 μM, IC50,NBleb = 3.4 ± 0.3 μM and IC50,Bleb = 3.9 ± 0.3 μM for the actin activated ATPase activity of DdMD. It should be noted that IC50 of AmBleb is higher by 4.3 and 1.7 times on SkS1 and DdMD, respectively.[1]

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Next, we compared the non-muscle myosin II inhibition by para-aminoblebbistatin, para-nitroblebbistatin and blebbistatin in human melanoma (M2) and HeLa cells. As blebbistatin acquired its name from its ability to block blebbing of M2 cells1, we compared blebbing indices17 of M2 cells in time, incubated in the three different myosin II inhibitors at different concentrations (Fig. 3c–e). Even 5 μM concentrations of the myosin II inhibitors achieved significant decrease in the blebbing indices of the cells within an hour, whereas higher concentrations of the inhibitors completely stopped blebbing of the cells within >40–60 minutes (10 μM) or >20 minutes (25 and 50 μM). The rate constants for the inhibition of blebbing of M2 cells at 5 μM inhibitor concentrations were similar, whereas at 10 and 25 μM concentration of para-aminoblebbistatin the rate constants were slightly slower than those of para-nitroblebbistatin or blebbistatin (Table 1).[1]

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Enhanced imaging by the non-phototoxic and non-cytotoxic para-aminoblebbistatin.

In live zebrafish embryo tests, myosin II inhibition by blebbistatin proved to be highly toxic after 24 hours of incubation, whereas using para-nitroblebbistatin the cytotoxic effect was negligible8. In order to compare the cyototoxicity of para-aminoblebbistatin to that of blebbistatin, we treated zebrafish embryos with the inhibitors at different concentrations and followed their development for 72 hours and determined their lifespan (Fig. 4b). After 40 hours, blebbistatin-treated zebrafish embryos started to die, and at 69 hours all of them died at all blebbistatin concentrations. In contrast, embryos treated with para-aminoblebbistatin did not show any sign of cytotoxicity even at high concentrations and their fitness was comparable to the untreated control embryos[1].

Steady state ATPase measurements[1]
MgATPase activities of 500 nM (for basal ATPase) or 50 nM (for actin activated ATPase) SkS1 (rabbit skeletal S1 fragment) and 1 μM (for basal ATPase) or 300 nM (for actin activated ATPase) DdMD were measured at increasing concentrations of blebbistatin, para-nitroblebbistatin or para-aminoblebbistatin in the absence or in the presence of 45 μM actin for SkS1 or 15 μM actin for DdMD. Measurements were carried out using a pyruvate kinase/lactate dehydrogenase coupled assay (NADH-coupled assay) at 25 °C or 20 °C in case of SkS1 or DdMD, respectively. Myosin and actin together with the indicated concentration of the inhibitor were pre-incubated for 5 minutes at room temperature and the measurements were initiated with 1 mM ATP. Data were corrected for background ATPase activity of actin. G-actin was prepared accordingly from rabbit skeletal muscle24 and polymerized by 2 mM MgCl2 for 1 hour at room temperature. Measurements were performed in low salt buffer containing 5 mM HEPES, 2 mM MgCl2, 0.1 mM EGTA and 2 mM DTT at pH 7.2 or assay buffer containing 20 mM HEPES, 40 mM NaCl, 4 mM MgCl2 at pH 7.3 for basal ATPase activity measurements with DdMD.

Conditions of cellular assays[1]
For blebbing index determination, M2 cells were incubated for 30 minutes in PBS in every case prior to the addition of inhibitors, in order to initiate extensive blebbing. Blebbing of the cells was monitored before as well as after inhibitor addition to the cells. DMSO concentration was 0.1 vol/vol% in all experiments. Blebbing index was calculated by summing the initiated blebs in a 5-minute interval (starting at the indicated time points) on a given standard area17 (whole cells with similar size, diameter = 12 ± 1 μm) and normalizing it to the blebbing index prior to inhibitor treatment.[1]
For wound healing assays, 200 μl of HeLa cells were plated on 35 mm borosilicate glass imaging dishes at 300,000 cells/ml concentrations and incubated for 24 hours. 1 hour prior to wounding, cells were incubated in 20 μM of the different myosin II inhibitors. After one hour, scratches were made with pipette tips in monolayer cultures and the motility of cells for occupying the wound was monitored for 24 hours with a Foculus Digital Camera (IEEE1394). Analysis of cell motility was carried out by ImageJ, by measuring the distance between two lines fitted on the two sides of the approaching cell edges created by the pipette tips.[1]
For cell growth assays, HeLa Kyoto H2B-mCherry eGFP-α-tubulin cells were grown on an eight-chambered Lab-Tek borosilicate cover glass system, with the initial cell concentration of 100,000 cells/ml. As soon as the cells attached to the surface, they were treated with 0, 2, 5, 10, 25 or 50 μM concentrations of para-aminoblebbistatin, para-nitro- or blebbistatin. Cells were grown for three days in the presence of the indicated inhibitor concentrations and cell numbers were quantified each day. The final vol/vol% of DMSO in all experiments was 0.1. Data analysis was carried out by Fiji software. On the third day of the experiment, multinuclear cells were visualized by using a Zeiss LSM 710 with a Plan Apo 20x/0.8 objective. For phototoxicity experiments, confocal time-lapse imaging of HeLa Kyoto H2B-mCherry eGFP-α-tubulin cells for 12 hours was performed in DMEM further 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, with the following parameters: 2% laser intensity at λexc = 488 nm, 3% laser intensity at λexc = 543 nm, 1.2× zoom, average 4, line step 1, 1000 × 1000 pixels, 0.35 μm pixel size, 0.65 μs dwell time, 3 slice/z-stack with 7 μm between slices at every 10 minutes[1].

Conditions of assays with zebrafish embryos For the fast escape response experiments, 6 dpf embryos were placed in 50 μl drops of E3 medium on a plastic plate. They were treated with 0, 2, 5, 10 or 20 μM of the inhibitors. 10 taps were carried out with 1 minute intervals every hour for 4 hours. The tapping force was standardized. The videos were recorded by a Ximea Camera (MQ003MG-CM) at 500 fps for 2 s. Data were analyzed by Flote software version 2.1 and maximal angular velocity parameters of C-start reflex were quantified. For the experiments investigating the effect of the inhibitors on the heart muscle of zebrafish, 6 dpf larvae were embedded in 1% agarose and kept in E3 in the presence of 20 μM concentrations of the inhibitors. Videos were taken at 15.1 fps before and after inhibitor treatment every hour for 3 hours. For the cytotoxicity assay, zebrafish embryos were incubated in E3 medium containing 0 μM (Control), 5 μM, 10 μM or 20 μM of para-aminoblebbistatin, para-nitroblebbistatin or blebbistatin in 0.1 vol/vol% final concentration of DMSO and monitored for 70 hours. Embryos were considered to be dead when the whole body became necrotic and the heart stopped beating. Dead embryos were removed from the experiment to prevent contamination. Images of zebrafish were captured with a Zeiss Stereo Lumar V12 microscope using a NeoLumar S 0.8x FWD 80mm objective at 50× zoom.

[1]. A highly soluble, non-phototoxic, non-fluorescent blebbistatin derivative. Sci Rep. 2016 May 31;6:26141.

[2]. para-Nitroblebbistatin, the non-cytotoxic and photostable myosin II inhibitor. Angew Chem Int Ed Engl. 2014 Jul 28;53(31):8211-5.

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.[1]

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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.[2]

C18H17N3O2

307.35

307.132

C, 70.34; H, 5.58; N, 13.67; O, 10.41

2097734-03-5

129626534

Orange to red solid powder

1.4±0.1 g/cm3

571.3±60.0 °C at 760 mmHg

299.3±32.9 °C

0.0±1.7 mmHg at 25°C

1.715

0.34

2

4

1

23

526

1

CC1=CC2=C(C=C1)N=C3[C@](C2=O)(CCN3C4=CC=C(C=C4)N)O

LYWLZINJPRNWFF-GOSISDBHSA-N

InChI=1S/C18H17N3O2/c1-11-2-7-15-14(10-11)16(22)18(23)8-9-21(17(18)20-15)13-5-3-12(19)4-6-13/h2-7,10,23H,8-9,19H2,1H3/t18-/m1/s1

(3aS)-1-(4-aminophenyl)-3a-hydroxy-6-methyl-2,3-dihydropyrrolo[2,3-b]quinolin-4-one

para-amino-Blebbistatin; para-aminoblebbistatin; 2097734-03-5; (3aS)-1-(4-aminophenyl)-3a-hydroxy-6-methyl-2,3-dihydropyrrolo[2,3-b]quinolin-4-one; p-aminoblebbistatin; CHEMBL4164044; SCHEMBL22512479; TQR0309;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMF: ~20 mg/mL (65.1 mM)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)