Myosin II (IC50: 0.5 to 5 μM)

Blebbistatin, with IC50 values ranging from 0.5 to 5 μM, potently inhibits both vertebrate non-muscle myosins IIA and IIB and numerous striated muscle myosins. There is only a slight inhibition of smooth muscle myosin (IC50=80 μM)[1]. The nucleotide binding of blebbistatin to skeletal muscle myosin subfragment-1 is not competitive. The inhibitor inhibits the release of phosphate by preferentially binding to ATPase intermediates, ADP, and phosphate in the active site. It inhibits the myosin head group in complexes that have a low affinity for actin [2]. Blebbistatin was shown to modify the appearance and function of activated hepatic stellate cells in vitro. Star cells undergo dendritic morphology, shrink, and lose focal adhesions and stress fibers that contain vinculin and myosin IIA. Blebbistatin inhibits endothelin-1-induced intracellular Ca2+ release, decreases collagen gel contraction, and messes with the creation of silicone wrinkles. Wound-induced cell migration is facilitated by it [3].

In a dose-dependent manner, blebbistatin fully relaxes the rat detrusor triggered by KCl and carbachol as well as the human bladder contractions caused by endothelin-1. When 10 μM blebbistatin was preincubated, it reduced carbachol reactivity by 65% and inhibited bladder contraction induced by electric field stimulation, with 50% inhibition occurring at 32 Hz.

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Blebbistatin (1 mg/kg) inhibited development of carotid AT, reduced infiltration of inflammatory cells, and prevented vascular-tissue damage, relative to the model group. Furthermore, blebbistatin also reduced the procoagulant activity of TF. Immunohistochemical and immunofluorescence data demonstrated that, compared with the model group, blebbistatin intervention reduced expression of NMMHCIIA, TF, GSK3β, p65, and p-p65 in carotid-artery endothelia in the CAL-induced AT model, but it increased levels of p-GSK3β. Blebbistatin could inhibit expression of NMMHCIIA mRNA in the CAL model[3].

Measurement of [Ca2+]i[2]
The effect of blebbistatin on thrombin and ATP-induced Ca2+ transients were analyzed with a multimode benchtop microplate reader. Cells (8000 per well) were seeded onto 96-well plates and allowed to reach confluence over 2 to 3 days. The cells were then loaded with Fura-2AM (at a final concentration of 1.25 μg/mL) for 30 minutes at room temperature. Ratiometric [Ca2+]i measurement was obtained by acquiring emission at 510 nm to excitation at 340 and 380 nm, respectively. ATP dose–response curves were fitted using the Michaelis-Menten model using the DRC-package (version 1.2.0) for R programming.

Whole Organ Explant Culture[4]
Cochlear sensory epithelium was dissected from postnatal day (P)3 wild-type FVB mice and cultured in DMEM/F12 supplemented with 2% B27, 1% N-2, and 50 μg/ml ampicillin. In the experimental group, the cochleae were treated with 0.5 mM neomycin and 1 μM blebbistatin (dissolved in DMSO) for 12 h and allowed to recover for another 12 h. Equivalent amounts of DMS were added to the control and neomycin-only groups. The tissues were cultured at 37°C with 5% CO2.

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Cell Culture[4]
HEI-OC-1 cells were divided into three groups and cultured in DMEM supplemented with 10% FBS (Pansera, P30-2602) and 50 μg/ml ampicillin for 12 h. After this initial incubation, the experimental group was treated with 2 mM neomycin and 0.01 μM to 5 μM blebbistatin in 6-well plates, while the neomycin-only group was treated with 2 mM neomycin and an equivalent volume of DMSO in place of the blebbistatin. After another 24 h of culture, the cells were thoroughly washed with PBS and cultured in DMEM with ampicillin for an additional 12 h recovery. Control cells without neomycin or blebbistatin were treated with an equivalent volume of DMSO and incubated under identical conditions. Finally, the cells were imaged with an inverted phase-contrast microscope.

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CCK-8 Assay[4]
Cell death was measured using the Cell Counting CCK-8 Kit (Protein Biotechnology, CC201-01). Briefly, HEI-OC-1 cells were exposed to 2 mM neomycin in 96-well plates for 12 h. After removing the neomycin, the tissues were allowed to recover for another 12 h. blebbistatin was added throughout the entire process in the experimental group, and an equivalent volume of DMSO was added in the neomycin-only group. All cells were then incubated with 10 μl of CCK-8 in each well for 30 min at 37°C, and a microtiter plate reader was used to measure the optical densities at 450 nm.

Model of carotid-artery ligation (CAL)[3]
A mouse model of CAL was generated using a modified method based on previous reports. Briefly, C57BL/6 J mice were anesthetized, as determined by assessment of the righting reflex. Neck hair was removed and a 1-cm midline incision made in the neck to expose the right side of the carotid artery. Two knots were tied in the upper end of the isolated carotid artery (external carotid artery) and internal carotid artery using 6.0 non-absorbable sutures. During carotid surgery, a length of ∼1 cm, between the upper-artery bifurcation and the carotid artery from the first lower ligation point, was tied using two 6.0 non-absorbable silk knots. The wound was rinsed with physiologic (0.9 %) saline, after closing muscle and skin (model group). Sham-operated mice underwent carotid-artery surgery after anesthesia without silk ligation (sham group). The blebbistatin was dissolved in absolute ethanol to the concentration of 1 × 10−2 M, and suspended at 0.5 % CMC-Na before use. In the blebbistatin group, mice were injected with blebbistatin (1 mg/kg, i.v.) to inhibit thrombosis. The blebbistatin was injected to the mice at the 0,4.7 day from the ligation. Six mice were included in each group. After 7 days, blood vessels from all groups were collected.[3]

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5-25 μM

Zebrafish embryos model

[1]. Absolute Stereochemical Assignment and Fluorescence Tuning of the Small Molecule Tool, (–)‐Blebbistatin. Eur J org Chem. 2005, 2005 (9), 1736-1740. doi.org/10.1002/ejoc.200500103
[2]. The myosin II ATPase inhibitor blebbistatin prevents thrombin-induced inhibition of intercellularcalcium wave propagation in corneal endothelial cells. Invest Ophthalmol Vis Sci. 2008 Nov;49(11):4816-27.
[3]. An inhibitor of myosin II, blebbistatin, suppresses development of arterial thrombosis. Bomed Pharmacother . 2020 Feb:122:109775.
[4]. Blebbistatin Inhibits Neomycin-Induced Apoptosis in Hair Cell-Like HEI-OC-1 Cells and in Cochlear Hair Cells. Front Cell Neurosci. 2020 Feb 5;13:590.

(S)-blebbistatin is the (S)-enantiomer of blebbistatin. It is a blebbistatin and a tertiary alpha-hydroxy ketone.

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(–)-Blebbistatin (1), a recently discovered small molecule inhibitor of the ATPase activity of non-muscle myosin II has been prepared from methyl 5-methylanthranilate (6) in three steps. This flexible synthetic route has also been used to prepare a nitro group-containing analogue 12 that has modified fluorescence properties and improved stability under microscope illumination. The key step in the synthesis of 1 and its analogues was the asymmetric hydroxylation of the quinolone intermediate 3 using the Davis oxaziridine methodology. The absolute stereochemistry of (–)-blebbistatin (1) was shown to be S by X-ray crystal structure analysis of a heavy atom (bromine) containing analogue 11, which was subsequently reduced and shown to be identical to 1.[1]

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Purpose: Thrombin inhibits intercellular Ca(2+) wave propagation in bovine corneal endothelial cells (BCECs) through a mechanism dependent on myosin light chain (MLC) phosphorylation. In this study, blebbistatin, a selective myosin II ATPase inhibitor, was used to investigate whether the effect of thrombin is mediated by enhanced actomyosin contractility. Methods: BCECs were exposed to thrombin (2 U/mL) for 5 minutes. MLC phosphorylation was assayed by immunocytochemistry. Ca(2+) waves were visualized by confocal microscopy with Fluo-4AM. Fluorescence recovery after photobleaching (FRAP) was used to investigate intercellular communication (IC) via gap junctions. ATP release was measured by luciferin-luciferase assay. Lucifer yellow (LY) uptake was used to investigate hemichannel activity, and Fura-2 was used to assay thrombin- and ATP-mediated Ca(2+) responses. Results: Pretreatment with blebbistatin (5 microM for 20 minutes) or its nitro derivative prevented the thrombin-induced inhibition of the Ca(2+) wave. Neither photo-inactivated blebbistatin nor the inactive enantiomers prevented the thrombin effect. Blebbistatin also prevented thrombin-induced inhibition of LY uptake, ATP release and FRAP, indicating that it prevented the thrombin effect on paracrine and gap junctional IC. In the absence of thrombin, blebbistatin had no significant effect on paracrine or gap junctional IC. The drug had no influence on MLC phosphorylation or on [Ca(2+)](i) transients in response to thrombin or ATP. Conclusions: Blebbistatin prevents the inhibitory effects of thrombin on intercellular Ca(2+) wave propagation. The findings demonstrate that myosin II-mediated actomyosin contractility plays a central role in thrombin-induced inhibition of gap junctional IC and of hemichannel-mediated paracrine IC.[2]

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Arterial thrombosis (AT) causes various ischemia-related diseases, which impose a serious medical burden worldwide. As an inhibitor of myosin II, blebbistatin has an important role in thrombosis development. We investigated the effect of blebbistatin on carotid artery ligation (CAL)-induced carotid AT and its potential underlying mechanism. A model of carotid AT in mice was generated by CAL. Mice were divided into three groups: CAL model, blebbistatin-treated, and sham-operation. After 7 days, blood vessels were harvested from mice in each group. The procoagulant activity of tissue factor (TF) was tested by a chromogenic assay, and thrombus severity assessed by histopathology scores. Expression of non-muscle myosin heavy chain II A (NMMHCIIA), TF, glycogen synthase kinase 3β (GSK3β), and nuclear factor-kappa B (NF-κB) was detected by immunohistochemical and immunofluorescence staining. mRNA expression was measured by quantitative polymerase chain reaction. Blebbistatin (1 mg/kg) inhibited development of carotid AT, reduced infiltration of inflammatory cells, and prevented vascular-tissue damage, relative to the model group. Furthermore, blebbistatin also reduced the procoagulant activity of TF. Immunohistochemical and immunofluorescence data demonstrated that, compared with the model group, blebbistatin intervention reduced expression of NMMHCIIA, TF, GSK3β, p65, and p-p65 in carotid-artery endothelia in the CAL-induced AT model, but it increased levels of p-GSK3β. Blebbistatin could inhibit expression of NMMHCIIA mRNA in the CAL model. Overall, our data demonstrated that blebbistatin could inhibit TF expression and AT development in arterial endothelia (at least in part) via GSK3β/NF-κB signaling.[3]

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Aging, noise, and ototoxic drug-induced hair cell (HC) loss are the major causes of sensorineural hearing loss. Aminoglycoside antibiotics are commonly used in the clinic, but these often have ototoxic side effects due to the accumulation of oxygen-free radicals and the subsequent induction of HC apoptosis. Blebbistatin is a myosin II inhibitor that regulates microtubule assembly and myosin-actin interactions, and most research has focused on its ability to modulate cardiac or urinary bladder contractility. By regulating the cytoskeletal structure and reducing the accumulation of reactive oxygen species (ROS), blebbistatin can prevent apoptosis in many different types of cells. However, there are no reports on the effect of blebbistatin in HC apoptosis. In this study, we found that the presence of blebbistatin significantly inhibited neomycin-induced apoptosis in HC-like HEI-OC-1 cells. We also found that blebbistatin treatment significantly increased the mitochondrial membrane potential (MMP), decreased ROS accumulation, and inhibited pro-apoptotic gene expression in both HC-like HEI-OC-1 cells and explant-cultured cochlear HCs after neomycin exposure. Meanwhile, blebbistatin can protect the synaptic connections between HCs and cochlear spiral ganglion neurons. This study showed that blebbistatin could maintain mitochondrial function and reduce the ROS level and thus could maintain the viability of HCs after neomycin exposure and the neural function in the inner ear, suggesting that blebbistatin has potential clinic application in protecting against ototoxic drug-induced HC loss.[4]

C18H16N2O2

292.33

292.121

C, 73.95; H, 5.52; N, 9.58; O, 10.95

856925-71-8

Blebbistatin;674289-55-5 (racemic); 856925-71-8 (S-isomer); 1177356-70-5 (R-isomer)

5287792

Light yellow to yellow solid powder

1.3±0.1 g/cm3

486.7±55.0 °C at 760 mmHg

210-212ºC

248.1±31.5 °C

0.0±1.3 mmHg at 25°C

1.681

0.93

1

3

1

22

497

1

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

LZAXPYOBKSJSEX-GOSISDBHSA-N

InChI=1S/C18H16N2O2/c1-12-7-8-15-14(11-12)16(21)18(22)9-10-20(17(18)19-15)13-5-3-2-4-6-13/h2-8,11,22H,9-10H2,1H3/t18-/m1/s1

1,2,3,3a-tetrahydro-3aS-hydroxy-6-methyl-1-phenyl-4H-Pyrrolo[2,3-b]quinolin-4-one

(S)-Blebbistatin; (-)-Blebbistatin; 856925-71-8; (S)-(-)-Blebbistatin; (S)-blebbistatin; Blebbistatin, (-)-; (-)Blebbistatin; Blebbistatin (S)-form [MI]; CHEBI:75388; Blebbistatin.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.  (2). This product is not stable in solution, please use freshly prepared working solution for optimal results.

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

Solubility in Formulation 1: ≥ 1 mg/mL (3.42 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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.

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Solubility in Formulation 2: 1 mg/mL (3.42 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 1 mg/mL (3.42 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 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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