Myosin II
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].

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].

---

para-Nitroblebbistatin inhibited the basal ATPase activity of Dictyostelium discoideum myosin II motor domain (DdMD) with an IC50 of 2.33 ± 0.13 µM, which was similar to blebbistatin (IC50 = 2.96 ± 0.45 µM). [1]
para-Nitroblebbistatin inhibited the basal ATPase activity of rabbit skeletal muscle myosin S1 (SkS1) with an IC50 of 0.4 ± 0.05 µM, comparable to blebbistatin (IC50 = 0.41 ± 0.03 µM). [1]
At a concentration of 20 µM, para-Nitroblebbistatin completely suppressed the actin-activated ATPase activity of both DdMD and SkS1 myosin isoforms, even at high actin concentrations (up to 80 µM). [1]
The fluorescence emission intensity of para-Nitroblebbistatin was less than 1% of that of blebbistatin when excited at 430 nm. [1]
The absorption spectrum of para-Nitroblebbistatin remained essentially unchanged upon irradiation with blue light (480 ± 10 nm), indicating high photostability, in contrast to blebbistatin which underwent significant photoconversion. [1]
Molecular modeling based on the crystal structure of the myosin-ADP-vanadate-blebbistatin complex (PDB: 1YV3) indicated that the addition of a nitro group at the C15 position of blebbistatin (to form para-Nitroblebbistatin) does not cause steric hindrance with any side chains in the myosin binding pocket. [1]

In HeLa cells, treatment with 20 µM para-Nitroblebbistatin for 3 days in the dark efficiently suppressed cytokinesis, leading to a continuous increase in multinuclear cells, with practically all cells becoming multinuclear by day 3. This effect was comparable to blebbistatin treatment. [1]
In the continuously blebbing M2 human melanoma cell line, a 10 µM solution of para-Nitroblebbistatin completely inhibited cell blebbing within 10 minutes, an effect identical to blebbistatin. [1]
In transgenic zebrafish embryos (1 dpf) expressing GFP in the posterior lateral line primordium (pLLp), treatment with increasing concentrations of para-Nitroblebbistatin for 24 hours halted the migration of the pLLp and caused a curved body shape, phenocopying the effects of blebbistatin treatment. [1]
In Dictyostelium discoideum cells, para-Nitroblebbistatin induced multinuclearity, similar to blebbistatin. [1]

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).

---

The inhibitory effect on myosin II ATPase activity was evaluated in vitro. The basal ATPase activity of purified myosin isoforms (Dictyostelium discoideum myosin II motor domain, DdMD, or rabbit skeletal muscle myosin S1, SkS1) was measured in the presence of increasing concentrations of para-Nitroblebbistatin or blebbistatin. The assay measured the release of inorganic phosphate from ATP. IC50 values were determined from dose-response curves. To test the effect on actin-activated ATPase activity, the ATPase activity was measured in the presence of a fixed concentration of inhibitor (20 µM) and increasing concentrations of actin (up to 80 µM). All experiments were performed in triplicate. [1]

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.

---

Phototoxicity Assay in HeLa Cells: HeLa cells were treated with 10 µM para-Nitroblebbistatin, blebbistatin, or left untreated. Cells were then irradiated with blue light (480 ± 10 nm, energy density 3.3 µJ µm⁻²) for 15 minutes. After irradiation, the inhibitors were removed by washing. Cells were incubated for an additional 18 hours, then stained with trypan blue. Live (unstained), dead (blue), and morphologically defective cells were counted under a light microscope. [1]
Long-term Live-cell Imaging: HeLa Kyoto cells expressing EGFP-α-tubulin and mCherry-H2B were treated with 50 µM para-Nitroblebbistatin or blebbistatin. Cells were repeatedly imaged over 12 hours in a confocal microscope (excitations at 488 nm and 561 nm), acquiring three z-sections every 10 minutes. Cell fate, division, and morphology were analyzed. [1]
Cytokinesis Inhibition and Cytotoxicity Assay in HeLa Cells: HeLa cells were treated with 20 µM para-Nitroblebbistatin or blebbistatin and incubated in the dark for 3 days. Cell number, viability (by trypan blue exclusion), and the ratio of multinuclear cells (visualized by Hoechst staining and confocal microscopy) were monitored daily. [1]
Blebbing Inhibition Assay in M2 Melanoma Cells: M2 human melanoma cells were treated with 10 µM para-Nitroblebbistatin or blebbistatin. Cell blebbing was observed by microscopy over 10 minutes. [1]
Effect on Dictyostelium discoideum: Dictyostelium cells were treated with para-Nitroblebbistatin or blebbistatin, and the induction of multinuclearity was assessed. [1]

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).

---

Zebrafish Phototoxicity Assay: Zebrafish larvae at 3 days post-fertilization (dpf) were treated with 10 µM para-Nitroblebbistatin or blebbistatin. They were then irradiated with blue light (470 ± 20 nm, energy density 0.4 µJ µm⁻²) for 10 minutes. Mortality was monitored for 36 hours post-treatment. [1]
Zebrafish Developmental Phenotyping: Transgenic zebrafish embryos (1 dpf) expressing GFP in the posterior lateral line primordium (cldnb:gfp) were treated with increasing concentrations of para-Nitroblebbistatin or blebbistatin. After 24 hours of incubation (at 2 dpf), embryos were anesthetized and visualized under a fluorescence stereomicroscope (excitation 470 ± 20 nm) to assess the migration front of the pLLp and body morphology. Mortality was monitored over 36 hours. The specific vehicle or solvent used for drug dissolution is not detailed in the provided text. [1]

Para-Nitroblebbistatin has no phototoxic effect on HeLa cells. After 18 hours of blue light irradiation and incubation, the cell mortality and morphology of cells treated with para-nitroblebbistatin were not significantly different from those of the untreated control group, while bubiusstatin caused a large number of cell deaths and morphological defects. [1] Para-nitroblebbistatin has no cytotoxic effect on HeLa cells. After 3 days of incubation with 20 µM para-nitroblebbistatin in the dark, the cell mortality rate was similar to that of the DMSO control group, and the cell morphology was not damaged. In contrast, under the same conditions, bubiusstatin treatment caused 90% of the cells to die within 3 days, and the surviving cells showed severe morphological defects. [1] In zebrafish larvae, after treatment with 10 µM para-nitroblebbistatin and subsequent blue light irradiation, the mortality rate after 36 hours was 18 ± 10%, which was significantly lower than the mortality rate of 86 ± 5% caused by bubiusstatin under the same conditions. [1]
Under dark conditions (no irradiation), zebrafish embryos treated with 10 µM p-nitrobrabisstatin for 36 hours showed similar mortality rates to the untreated control group. In contrast, all embryos treated with 10 µM brabisstatin died under the same dark conditions, highlighting the inherent cytotoxicity of brabisstatin. [1]
P-nitrobrabisstatin had no cytotoxic effect on Dendrobium distichum cells. [1]

[1]. 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 in vitro and in vivo. Despite its wide applicability, blebbistatin's poor water solubility (below 10 μM in aqueous buffer) and sensitivity to blue light limit its applications. Blue light irradiation causes photoconversion of the molecule, resulting in severe phototoxicity in addition to its cytotoxicity. Furthermore, blebbistatin forms insoluble aggregates in aqueous media at concentrations above 10 μM and exhibits extremely high fluorescence intensity and high adhesion to various surface types, limiting its experimental applications. This paper reports a highly soluble (440 μM in aqueous buffer), non-fluorescent, and photostable C15 amino-substituted blebbistatin derivative, termed para-aminoblebbistatin. Importantly, experiments on HeLa cells and zebrafish embryos demonstrated that this derivative is neither phototoxic nor cytotoxic. Furthermore, p-aminoblebbistatin, possessing myosin II inhibitory properties similar to blebbistatin or p-nitroblebbistatin (not to be confused with C7-substituted nitroblebbistatin), has been tested on rabbit skeletal muscle myosin S1 as well as M2 and HeLa cells. Due to its significantly improved solubility and photochemical properties, and the absence of phototoxicity or cytotoxicity, p-aminoblebbistatin may be a viable alternative to blebbistatin, especially in applications requiring high concentrations of inhibitors or blue light irradiation. [Sci Rep. 2016 May 31;6:26141.]

---

p-Nitroblebbistatin is a C15 nitro derivative of blebbistatin, synthesized to overcome the major limitations of the parent compound (phototoxicity, cytotoxicity, fluorescence, photosensitivity) while maintaining its specificity and potency in inhibiting myosin II. [1]
Compared to blebbistatin, the main advantages of p-nitroblebbistatin are: 1) significantly reduced fluorescence intensity (emission intensity less than 1% of blebbistatin), thus avoiding interference with commonly used fluorescent probes such as GFP; 2) high photostability under blue light irradiation; 3) no phototoxicity; 4) no inherent cytotoxicity in the dark. [1]
This study concludes that p-nitroblebbistatin is an ideal and complete alternative to blebbistatin, and can be used to study the specific roles of myosin II in physiological, developmental and cell biological research, both in vitro and in vivo. [1]

C18H15N3O4

337.329404115677

337.106

C, 64.09; H, 4.48; N, 12.46; O, 18.97

1621326-32-6

102361739

Yellow to orange solid powder

2.2

1

5

1

25

608

1

O[C@]12C(C3C=C(C)C=CC=3N=C1N(C1C=CC(=CC=1)[N+](=O)[O-])CC2)=O

KAUXNLHXGQGFOS-GOSISDBHSA-N

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

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

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;

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)

DMSO : ~22 mg/mL (~65.22 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.

---

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).

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)]
*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)

---

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
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

---

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.

---

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