(±)-Blebbistatin

(±)-Blebbistatin is a cell-permeable non-muscle myosin II ATPases inhibitor with an IC50 range of 2 μM [1,2].

Non-muscle myosin II (NM II), an actin-binding protein, plays a central role in regulation of cell migration, adhesion and differentiation [4]. Recent insight into the importance of NM II in these processes has been highlighted by genetic deletion and mutation methods that discovered NM II mutations affect the function of a wide range of proteins and cause monogenic diseases [5.6].

(±)-Blebbistatin is a small molecule inhibitor and preferentially binds to the myosin-ADP-Pi complex to slow down phosphate release [2]. 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) [8,9], but it has poor effects on smooth muscle myosin II (IC50 =80 μM) and myosins I,V, and X [3]. Furthermore, blebbistatin can potently inhibit mammalian arterial smooth muscle (IC50=5 μM) [9]. The property that blebbistatin blocks myosin II in an actin-detached state and prevents rigid actomyosin cross-linking is a great advantage in vivo applications [2,11].

In a constant-pressure grant perfusion model system, the CB and TM cells were treated with blebbistatin (10-200 M) and cell morphology was changed, and actin stress fiber content decreased. The blebbistatin effect was completely reversible by washout within 24 hours [10]. Blebbistatin inhibited single cellular contraction without altering the morphologies of intracellular calcium transients (IC50 = 0.43 μM). Exposure to UV light at wavelengths below 488 nm can also cause blebbistatin rapidly suppressed. [8].

References:
[1].  Straight AF, Cheung A, Limouze J, et al. Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor. Science, 2003, 299:1743–1747.
[2].  Kovacs M, Toth J, Hetenyi C, Malnasi-Csizmadia A, Sellers JR. Mechanism of blebbistatin inhibition of myosin II. J Biol Chem, 2004, 279:35557–35563.
[3].  Limouze J, Straight AF, Mitchison T, Sellers JR. Specificity of blebbistatin, an inhibitor of myosin II. J Muscle Res Cell Motil, 2004, 25:337–341.
[4].  Miguel Vicente-Manzanares, Xuefei Ma, Robert S. Adelstein,Alan Rick Horwitz. Non-muscle myosin II takes centre stage in cell adhesion and migration.Nat Rev Mol Cell Biol, 2009 Nov, 10(11): 778–790.
[5].  Burt RA, Joseph JE, Milliken S, Collinge JE, Kile BT. Description of a novel mutation leading to MYH9-related disease. Thrombosis Research, 2008, 122(6): 861-863.
[6].  Butcher DT, Alliston T, Weaver VM. A tense situation: forcing tumour progression. Nature Reviews Cancer, 2009 Feb, 9(2):108-122.
[7].  Chen Y, Boukour S, Milloud R, Favier R, Saposnik B, Schlegel N, et al. The abnormal proplatelet formation in MYH9-related macrothrombocytopenia results from an increased actomyosin contractility and is rescued by myosin IIA inhibition. Journal of thrombosis and haemostasis : JTH , 2013, 11:2163-2175.
[8].  Fedorov VV, Lozinsky IT, Sosunov EA, Anyukhovsky EP, Rosen MR, Balke CW, et al. Application of blebbistatin as an excitation-contraction uncoupler for electrophysiologic study of rat and rabbit hearts. Heart rhythm: the official journal of the Heart Rhythm Society, 2007, 4:619-626.
[9].  Zhang X-h, Aydin M, Kuppam D, Melman A, DiSanto ME. In Vitro and In Vivo Relaxation of Corpus Cavernosum Smooth Muscle by the Selective Myosin II Inhibitor, Blebbistatin. The Journal of Sexual Medicine, 2009, 6:2661-2671.
[10].  Zhang M, Rao PV. Blebbistatin, a novel inhibitor of myosin II ATPase activity, increases aqueous humor outflow facility in perfused enucleated porcine eyes. Investigative ophthalmology & visual science, 2005, 46:4130-4138.
[11].  Lucas-Lopez C, Allingham JS, Lebl T, Lawson CP, Brenk R, Sellers JR, et al. The small molecule tool (S)-(-)-blebbistatin: novel insights of relevance to myosin inhibitor design. Organic & biomolecular chemistry, 2008, 6:2076-2084.

Block the function of nonmuscle myosin II by blebbistatin induces zebrafish embryo cardia bifida.[Pubmed:25403653]

In Vitro Cell Dev Biol Anim. 2015 Mar;51(3):211-7.

Nonmuscle myosin II (NM II) is the name given to the multi-subunit protein product of three genes encoding different nonmuscle myosin heavy chains including NM II-A, NM II-B, and NM II-C. Blebbistatin is a small molecule that has been shown to be a relatively specific inhibitor of NM II. Blocking the function of NM II by blebbistatin induces zebrafish embryo cardia bifida at a dose-dependent manner. In situ hybridization analysis with ventricular marker ventricular myosin heavy chain (vmhc) and atrial marker atrial myosin heavy chain (amhc) showed each of the heart contained both distinct atria and ventricle. However, the cardia bifida embryos had highly variable distance between two separate ventricles. We also provided evidence that time window from 12 to 20 h post fertilization (hpf) is necessary and sufficient for cardia bifida formation caused by blebbistatin treatment. Expression of spinster homolog 2 (spns2) was decreased in blebbistatin-treated embryos, suggesting the cardia bifida phenotype caused by NM II inhibition was relevant to precardiac mesoderm migration defects. Through in situ hybridization analysis, we showed that foxa1 was expressed in endoderm of blebbistatin-treated embryos at 24-hpf stage, suggesting the endoderm formation is normal in cardia bifida embryos caused by blebbistatin treatment. In addition, we demonstrated that blebbistatin treatment resulted in morphology alteration of zebrafish cardiomyocytes in vivo and neonatal mouse cardiomyocytes in vitro.

The Selective Myosin II Inhibitor Blebbistatin Reversibly Eliminates Gastrovascular Flow and Stolon Tip Pulsations in the Colonial Hydroid Podocoryna carnea.[Pubmed:26605798]

PLoS One. 2015 Nov 25;10(11):e0143564.

Blebbistatin reversibly disrupted both stolon tip pulsations and gastrovascular flow in the colonial hydroid Podocoryna carnea. Epithelial longitudinal muscles of polyps were unaffected by blebbistatin, as polyps contracted when challenged with a pulse of KCl. Latrunculin B, which sequesters G actin preventing F actin assembly, caused stolons to retract, exposing focal adhesions where the tip epithelial cells adhere to the substratum. These results are consistent with earlier suggestions that non-muscle myosin II provides the motive force for stolon tip pulsations and further suggest that tip oscillations are functionally coupled to hydrorhizal axial muscle contraction.

Protein kinase C activation of a blebbistatin sensitive contractile component in the wall of hypertrophying mouse urinary bladder.[Pubmed:25794194]

Neurourol Urodyn. 2015 Feb;34(2):196-202.

AIM: To examine the role of protein kinase C (PKC) and non-muscle myosin in regulation of wall tension in the hypertrophied urinary bladder. METHODS: A partial urinary outflow obstruction was induced in the mouse. Tissue strips from sham operated controls and obstructed bladders were examined in vitro with quantitative gel electrophoresis, immunohistochemistry, and in vitro force recordings. RESULTS: Outlet obstruction (14-18 days) induced a significant growth of the bladder, 73 +/- 6.13 mg compared to 19 +/- 1 13 mg in sham operated controls. The hypertrophying bladder tissue had increased expression of non-muscle myosin B (SMemb) mainly localized to serosa and suburothelium. Direct activation of PKC with PDBu did not alter force in the control urinary bladder. In contrast, PDBu initiated a prominent and sustained contraction which had an increased sensitivity to the myosin type II inhibitor blebbistatin. CONCLUSIONS: PKC activates a significant contractile response in the wall of the hypertrophying urinary bladder, possibly supported by non-muscle myosin. This contractile component is not contributing to the physiological response to muscarinic stimulation, but might be separately regulated by other, yet unknown, mechanisms.

A highly soluble, non-phototoxic, non-fluorescent blebbistatin derivative.[Pubmed:27241904]

Sci Rep. 2016 May 31;6:26141.

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.

Non-muscle myosin II regulates survival threshold of pluripotent stem cells.[Pubmed:20842192]

Nat Commun. 2010 Sep 7;1:71.

Human pluripotent stem (hPS) cells such as human embryonic stem (hES) and induced pluripotent stem (hiPS) cells are vulnerable under single cell conditions, which hampers practical applications; yet, the mechanisms underlying this cell death remain elusive. In this paper, we demonstrate that treatment with a specific inhibitor of non-muscle myosin II (NMII), blebbistatin, enhances the survival of hPS cells under clonal density and suspension conditions, and, in combination with a synthetic matrix, supports a fully defined environment for self-renewal. Consistent with this, genetically engineered mouse embryonic stem cells lacking an isoform of NMII heavy chain (NMHCII), or hES cells expressing a short hairpin RNA to knock down NMHCII, show greater viability than controls. Moreover, NMII inhibition increases the expression of self-renewal regulators Oct3/4 and Nanog, suggesting a mechanistic connection between NMII and self-renewal. These results underscore the importance of the molecular motor, NMII, as a novel target for chemically engineering the survival and self-renewal of hPS cells.

Specificity of blebbistatin, an inhibitor of myosin II.[Pubmed:15548862]

J Muscle Res Cell Motil. 2004;25(4-5):337-41.

Blebbistatin is a small molecule inhibitor discovered in a screen for inhibitors of nonmuscle myosin IIA. We have examined the specificity and potency of the drug by assaying its effects on the actin-activated MgATPase assay of diverse members of the myosin superfamily. Blebbistatin potently inhibits several striated muscle myosins as well as vertebrate nonmuscle myosin IIA and IIB with IC50 values ranging from 0.5 to 5 microM. Interestingly, smooth muscle which is highly homologous to vertebrate nonmuscle myosin is only poorly inhibited (IC50=80 microM). The drug potently inhibits Dictyostelium myosin II, but poorly inhibits Acanthamoeba myosin II. Blebbistatin did not inhibit representative myosin superfamily members from classes I, V, and X.

Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor.[Pubmed:12637748]

Science. 2003 Mar 14;299(5613):1743-7.

Completion of cell division during cytokinesis requires temporally and spatially regulated communication from the microtubule cytoskeleton to the actin cytoskeleton and the cell membrane. We identified a specific inhibitor of nonmuscle myosin II, blebbistatin, that inhibited contraction of the cleavage furrow without disrupting mitosis or contractile ring assembly. Using blebbistatin and other drugs, we showed that exit from the cytokinetic phase of the cell cycle depends on ubiquitin-mediated proteolysis. Continuous signals from microtubules are required to maintain the position of the cleavage furrow, and these signals control the localization of myosin II independently of other furrow components.

Blebbistatin is a selective non-muscle myosin II (NMII) inhibitor, promotes directional migration of corneal endothelial cells (CECs) and accelerates wound healing, and better preserves cell junctional integrity and barrier function.

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