-Blebbistatin (S-Blebbistatin) Chemical Structure.png)
(-)-Blebbistatin is a potent, selective, reversible and cell-permeable small molecule inhibitor for non muscle myosin II ATPase with IC50 of around 2 μM in cell-free assays. It has been extensively used in research community to inhibit heart muscle myosin, non-muscle myosin II, and skeletal muscle myosin. As a cell-permeable molecule with high affinity and selectivity toward the myosin II contractile molecule, (-)-Blebbistatin preferentially binds to the myosin-ADP-Pi complex to slow down phosphate release. The inhibitor completely eliminate contraction of activity of actin-activated Mg-ATPase and motility of myosins II for several species in vitro (IC50 = 0.5-5.0 μM), but it has poor effects on smooth muscle myosin II (IC50 =80 μM) and myosins I,V, and X.
Physicochemical Properties
| Molecular Formula | C18H16N2O2 |
| Molecular Weight | 292.33 |
| Exact Mass | 292.121 |
| Elemental Analysis | C, 73.95; H, 5.52; N, 9.58; O, 10.95 |
| CAS # | 856925-71-8 |
| Related CAS # | Blebbistatin;674289-55-5 (racemic); 856925-71-8 (S-isomer); 1177356-70-5 (R-isomer) |
| PubChem CID | 5287792 |
| Appearance | Light yellow to yellow solid powder |
| Density | 1.3±0.1 g/cm3 |
| Boiling Point | 486.7±55.0 °C at 760 mmHg |
| Melting Point | 210-212ºC |
| Flash Point | 248.1±31.5 °C |
| Vapour Pressure | 0.0±1.3 mmHg at 25°C |
| Index of Refraction | 1.681 |
| LogP | 0.93 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 3 |
| Rotatable Bond Count | 1 |
| Heavy Atom Count | 22 |
| Complexity | 497 |
| Defined Atom Stereocenter Count | 1 |
| SMILES | CC1=CC2=C(C=C1)N=C3[C@](C2=O)(CCN3C4=CC=CC=C4)O |
| InChi Key | LZAXPYOBKSJSEX-GOSISDBHSA-N |
| InChi Code | 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 |
| Chemical Name | 1,2,3,3a-tetrahydro-3aS-hydroxy-6-methyl-1-phenyl-4H-Pyrrolo[2,3-b]quinolin-4-one |
| Synonyms | (S)-Blebbistatin; (-)-Blebbistatin; 856925-71-8; (S)-(-)-Blebbistatin; (S)-blebbistatin; Blebbistatin, (-)-; (-)Blebbistatin; Blebbistatin (S)-form [MI]; CHEBI:75388; Blebbistatin. |
| HS Tariff Code | 2934.99.9001 |
| Storage |
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. |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
Myosin II (IC50: 0.5 to 5 μM) The primary target of (-)-Blebbistatin (S-Blebbistatin) is the ATPase activity of myosin II. It competitively binds to the ATP-binding site of myosin II, inhibiting ATP hydrolysis and subsequent myosin II-mediated contraction. For rabbit skeletal muscle myosin II ATPase, the IC₅₀ value of (-)-Blebbistatin is approximately 8-10 μM [2,4] - In HEI-OC-1 hair cell-like cells and cochlear hair cells, (-)-Blebbistatin targets myosin II to block neomycin-induced apoptosis, with an effective concentration range of 5-20 μM (no explicit Ki/EC₅₀ reported) [4] - In platelets, (-)-Blebbistatin inhibits myosin II ATPase activity to suppress platelet aggregation and thrombosis, with an IC₅₀ of approximately 8 μM for platelet myosin II ATPase [3] |
| ln Vitro |
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]. Corneal endothelial cell intercellular calcium wave regulation: Treatment of primary rabbit corneal endothelial cells with thrombin (1 U/mL for 30 min) inhibited intercellular calcium wave propagation—propagation distance decreased from 200 μm (control) to 50 μm. Pretreatment with 10 μM (-)-Blebbistatin for 1 h restored calcium wave propagation to 180 μm and maintained the integrity of tight junctions (detected by ZO-1 immunofluorescence). This effect was attributed to inhibited myosin II-mediated cytoskeletal rearrangement, which preserved intercellular communication [2] - HEI-OC-1 cell apoptosis inhibition: Neomycin (1 mM for 24 h) increased the apoptosis rate of HEI-OC-1 cells to 45% (vs. 5% in control). Pretreatment with 5 μM, 10 μM, or 20 μM (-)-Blebbistatin for 1 h reduced the apoptosis rate to 30%, 18%, and 12%, respectively. Western blot analysis showed that neomycin-induced cleaved caspase-3 expression (3.5-fold vs. control) was downregulated to 1.8-fold vs. control after 10 μM drug treatment. Additionally, neomycin decreased Bcl-2/Bax ratio (0.3-fold vs. control), while 10 μM (-)-Blebbistatin restored the ratio to 0.8-fold vs. control [4] - Cochlear hair cell survival promotion: In vitro-cultured mouse cochlear hair cells (P3-P5) treated with neomycin (0.5 mM for 24 h) had a survival rate of 40%. Pretreatment with 10 μM (-)-Blebbistatin for 1 h increased the survival rate to 75%. Phalloidin staining showed that the drug preserved the integrity of hair cell stereocilia, which are critical for auditory function [4] - Platelet aggregation suppression: In vitro platelet aggregation assays showed that ADP (10 μM)-induced platelet aggregation rate was 70% (control), while 10 μM (-)-Blebbistatin reduced it to 35%. Thrombin (0.1 U/mL)-induced platelet adhesion rate decreased from 65% (control) to 28% after treatment with 10 μM (-)-Blebbistatin, without affecting platelet viability (trypan blue exclusion >95%) [3] |
| ln Vivo |
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. 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]. Mouse carotid artery thrombosis inhibition: C57BL/6 mice (male, 8-10 weeks old) were used to establish a FeCl₃-induced carotid thrombosis model. The saline control group had a thrombosis formation time of 15±3 min and a thrombus weight of 8.5±1.2 mg. Mice treated with 1 mg/kg (-)-Blebbistatin (intravenous injection) showed prolonged thrombosis formation time (28±4 min) and reduced thrombus weight (5.2±0.8 mg). The 5 mg/kg dose further extended formation time to 35±5 min and reduced thrombus weight to 3.1±0.5 mg. No significant prolongation of tail bleeding time (a marker of bleeding risk) was observed in drug-treated groups [3] - Mouse cochlear hair cell protection: ICR mice (female, 6-8 weeks old) received intraperitoneal neomycin (100 mg/kg/day for 7 days), resulting in a cochlear outer hair cell survival rate of 35% (vs. 90% in control) and a 40±5 dB SPL increase in auditory brainstem response (ABR) threshold (4-16 kHz). Concurrent intracochlear injection of 10 μM (-)-Blebbistatin (0.5 μL/ear, once every 2 days for 4 times) increased outer hair cell survival rate to 68% and limited ABR threshold elevation to 18±3 dB SPL. Immunofluorescence staining confirmed preserved outer hair cell morphology in drug-treated mice [4] |
| Enzyme Assay |
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. Myosin II ATPase activity assay: Myosin II was purified from rabbit skeletal muscle and resuspended in a buffer containing 20 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, and 1 mM DTT (final concentration: 0.1 mg/mL). Different concentrations of (-)-Blebbistatin (0.1 μM-50 μM) were mixed with myosin II and preincubated at 37°C for 20 min. The reaction was initiated by adding 2 mM ATP and incubated at 37°C for 30 min. A molybdate-ascorbic acid chromogenic reagent was added to detect inorganic phosphate (a product of ATP hydrolysis) by measuring absorbance at 660 nm. The inhibition rate of ATPase activity was calculated relative to the vehicle control, and the IC₅₀ was determined by fitting the dose-response curve [2,4] |
| Cell Assay |
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. 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. 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. Corneal endothelial cell calcium wave assay: Primary rabbit corneal endothelial cells were cultured to confluence and divided into three groups: control, thrombin-treated (1 U/mL for 30 min), and drug-pretreated (10 μM (-)-Blebbistatin for 1 h followed by thrombin). Cells were loaded with 5 μM Fura-2 AM (37°C for 45 min), and calcium wave propagation was monitored using a laser confocal microscope (excitation wavelengths: 340 nm/380 nm). ImageJ software was used to analyze the time and distance of calcium wave propagation from the stimulation point to the edge of the wave, and propagation speed was calculated [2] - HEI-OC-1 cell apoptosis assay: HEI-OC-1 cells were seeded in 6-well plates (5×10⁵ cells/well) and divided into control, neomycin-treated (1 mM for 24 h), and drug+neomycin groups (5/10/20 μM (-)-Blebbistatin for 1 h followed by neomycin). Cells were collected, stained with Annexin V-FITC/PI, and analyzed by flow cytometry to determine apoptosis rate. Total cellular protein was extracted, and Western blot was performed to detect cleaved caspase-3, Bcl-2, and Bax (primary antibodies incubated at 4°C overnight, secondary antibodies at room temperature for 1 h, ECL chemiluminescence for visualization) [4] - Cochlear hair cell survival assay: Cochleae were isolated from neonatal C57BL/6 mice (P3-P5) and cultured in DMEM/F12 medium. Samples were divided into control, neomycin-treated (0.5 mM for 24 h), and drug-pretreated groups (10 μM (-)-Blebbistatin for 1 h followed by neomycin). After fixation, hair cell cilia were stained with Alexa Fluor 488-conjugated phalloidin, and nuclei with DAPI. Viable hair cells (intact cilia and normal nuclei) were counted under a fluorescence microscope, and survival rate was calculated [4] |
| Animal Protocol |
5-25 μM Zebrafish embryos model
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] Mouse carotid thrombosis model protocol: C57BL/6 mice (male, 8-10 weeks old) were randomly divided into three groups (n=6/group): saline control, 1 mg/kg (-)-Blebbistatin, and 5 mg/kg (-)-Blebbistatin. The drug was dissolved in DMSO and diluted with saline (final DMSO concentration <5%), then administered via tail vein injection (10 μL/g body weight). Thirty minutes after administration, the left carotid artery was exposed, and a 5 mm×5 mm filter paper soaked in 10% FeCl₃ was applied for 10 min to induce thrombosis. Blood flow was monitored by Doppler ultrasound to record thrombosis formation time (time to complete blood flow obstruction). Mice were euthanized 24 h later, the carotid artery was dissected, and thrombus weight was measured [3] - Mouse cochlear hair cell protection protocol: ICR mice (female, 6-8 weeks old) were divided into three groups (n=6/group): control, neomycin-only, and drug+neomycin. The neomycin-only group received intraperitoneal injections of 100 mg/kg/day neomycin for 7 days. The drug+neomycin group received intracochlear injections of 10 μM (-)-Blebbistatin (0.5 μL/ear) via the round window membrane on the same day as neomycin administration, once every 2 days for a total of 4 injections. On day 8, ABR thresholds (4 kHz, 8 kHz, 16 kHz) were measured. Mice were then euthanized, cochleae were harvested and fixed, and immunofluorescence staining (anti-actin antibody to label hair cells) was performed. The number of outer hair cells in the basal, middle, and apical turns of the cochlea was counted to calculate survival rate [4] |
| Toxicity/Toxicokinetics |
In vitro toxicity: Treatment of HEI-OC-1 cells with 20 μM (-)-Blebbistatin for 24 h resulted in a cell survival rate of 92%, which was not significantly different from the control group (95%). Incubation of corneal endothelial cells with 10 μM (-)-Blebbistatin for 48 h did not alter cell morphology or proliferation rate (vs. control) [2,4] - In vivo toxicity: Intravenous injection of 5 mg/kg (-)-Blebbistatin into mice for 7 consecutive days did not cause significant changes in body weight (control: +5%; drug group: +4%) or serum levels of ALT (alanine transaminase), AST (aspartate transaminase), or Scr (serum creatinine) (vs. control). Intracochlear injection of 10 μM (-)-Blebbistatin did not induce inflammatory responses in cochlear tissues (no neutrophil infiltration observed by HE staining) [3,4] - No data on half-lethal dose (LD₅₀), drug-drug interactions, or plasma protein binding rate of (-)-Blebbistatin were reported in the four literatures [1,2,3,4] |
| References |
[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. |
| Additional Infomation |
(S)-blebbistatin is the (S)-enantiomer of blebbistatin. It is a blebbistatin and a tertiary alpha-hydroxy ketone. \n(–)-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] \n\nPurpose: 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.\n\nMethods: 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.\n\nResults: 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.\n\nConclusions: 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] \n\nArterial 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] \n\nAging, 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] Stereochemistry and fluorescence properties: Literature [1] confirmed that the absolute configuration of (-)-Blebbistatin is the S-configuration, with a specific optical rotation of [α]D²⁵ = -123° (c=0.1, methanol). This compound emits blue fluorescence (emission wavelength: 450 nm) when excited by 365 nm ultraviolet light, and its fluorescence intensity increases with increasing solvent polarity, enabling intracellular drug localization imaging [1] - Mechanism of action: (-)-Blebbistatin binds to the ATP-binding site of myosin II, competitively inhibiting ATP hydrolysis. This blocks the interaction between the myosin II head and actin, thereby suppressing myosin II-mediated cell contraction, adhesion, and cytoskeletal rearrangement. In thrombosis models, it inhibits platelet myosin II to reduce platelet aggregation and thrombus contraction; in hair cells, it blocks neomycin-induced apoptosis by inhibiting myosin II-mediated apoptotic signaling (e.g., reducing Bax translocation to mitochondria) [2,3,4] - Tool compound characteristics: Literature [1] noted that (-)-Blebbistatin is a key small-molecule tool for studying myosin II function. Compared with the racemic mixture, the S-configuration ((-)-Blebbistatin) has 100-fold higher inhibitory activity against myosin II and greater specificity, making it suitable for selectively targeting myosin II in biological studies [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
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. 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. 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.4208 mL | 17.1040 mL | 34.2079 mL | |
| 5 mM | 0.6842 mL | 3.4208 mL | 6.8416 mL | |
| 10 mM | 0.3421 mL | 1.7104 mL | 3.4208 mL |