Blebbistatin

Last updated
Blebbistatin
Blebbistatin.png
Names
IUPAC name
3a-Hydroxy-6-methyl-1-phenyl-2,3-dihydropyrrolo[2,3-b]quinolin-4-one
Other names
(S)-Blebbistatin, (-)-Blebbistatin
Identifiers
3D model (JSmol)
PubChem CID
UNII
  • CC1=CC2=C(C=C1)N=C3C(C2=O)(CCN3C4=CC=CC=C4)O
Properties
C18H16N2O2
Molar mass 292.338 g·mol−1
AppearanceYellow solid
10 μM
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).

Blebbistatin is a myosin inhibitor mostly specific for myosin II. [1] [2] It is widely used in research to inhibit heart muscle myosin, non-muscle myosin II, and skeletal muscle myosin. Blebbistatin has been especially useful in optical mapping of the heart, [3] and its recent use in cardiac muscle cell cultures has improved cell survival time. [4] [5] However, its adverse characteristics e.g. its cytotoxicity and blue-light instability or low solubility in water often make its application challenging. [6] [7] Recently its applicability was improved by chemical design and its derivatives overcome the limitations of blebbistatin. [2] [8] E.g. para-nitroblebbistatin and para-aminoblebbistatin are photostable, and they are neither cytotoxic nor fluorescent. [7] [9]

Contents

Mode of action and biological effects

Blebbistatin inhibits myosin ATPase activity and this way acto-myosin based motility. It binds halfway between the nucleotide binding pocket and the actin binding cleft of myosin, predominantly in an actin detached conformation. [10] This type of inhibition relaxes the acto-myosin myofilaments and leads to several biological effects.

Blebbistatin inhibits the formation of blebs in melanoma cell culture, [11] hence its name. At a cellular level, blebbistatin also inhibits cytokinesis [11] and may also disrupt mitotic spindle formation. [12] Migration of cells can be either enhanced or inhibited depending on other conditions. [13] In neurons, blebbistatin was found to promote neurite outgrowth. [14] At the organ level blebbistatin stops the contraction of skeletal muscle [15] or heart muscle. [16] Blebbistatin has also been found to stabilize the super relaxed state in the myofilaments, where myosin heads are in a helical order and interact with each other but not with actin. [17] [18] [19]

Adverse characteristics

A number of physicochemical deficiencies hamper the use of blebbistatin as a chemical tool in particular applications. [2]

Photo-instability

Upon blue light illumination, blebbistatin becomes inactive and phototoxic due to changes in the structure of the compound accompanied by the generation of ROS [20] [6] [21]

Fluorescence

Blebbistatin is a relatively strong fluorophore. When dissolved in water, it absorbs at 420 nm and emits at 490 nm however in DMSO or when perfused through cardiac tissue, it absorbs around 430 nm and emits around 560 nm, [22] therefore at high concentrations its fluorescence interferes with GFP imaging or FRET experiments. [7] Reduction of the concentration of blebbistatin to 6.25 uM allows for FRET imaging in isolated adult mouse cardiac muscle cells. [5]

Cytotoxicity

Long-term incubation with blebbistatin results in cell damage and cytotoxicity, which are independent of the myosin inhibitory effect.

This photo-instability, phototoxicity and fluorescence makes in-vivo imaging of blebbistatin-treated samples impossible.

Myosin specificity

Blebbistatin is a potent inhibitor of nonmuscle myosin IIA and IIB, cardiac myosin, skeletal myosin and smooth muscle but does not inhibit myosin I, V and X. [23] [24] [25] The table below summarizes IC50 data of blebbistatin on different myosin isoforms.

speciesmyosin isoform or muscle typeassay typeIC50
Dictyostelium discoideum myosin II motor domainbasal ATPase2.96 ± 0.45 μM, [7] 4.4 ± 0.3 μM, [9] 4.9 μM [23]
Dictyostelium discoideummyosin II motor domainactin activated ATPase3.9 ± 0.3 μM [9]
Rabbitskeletal muscle IIbasal ATPase0.50 μM, [23] 0.3 ± 0.03 μM, [9] 0.41 ± 0.03 μM [7]
Rabbitskeletal muscle IIactin activated ATPase0.11 ± 0.009 μM [9]
Porcineb-cardiac musclebasal ATPase1.2 μM [23]
Scallop striated musclebasal ATPase2.3 μM [23]
Humannonmuscle IIAbasal ATPase5.1 μM [23]
Chickennonmuscle IIBbasal ATPase1.8 μM [23]
Humannonmuscle IIAactin activated ATPase3.58 μM [26]
Humannonmuscle IIBactin activated ATPase2.30 μM [26]
Mousenonmuscle IICactin activated ATPase1.57 μM [26]
Turkeysmooth musclebasal ATPase79.6 μM [23]
Acanthamoeba myosin IIbasal ATPase83 μM [23]
Ratmyosin 1Bbasal ATPase>150 μM [23]
Acanthamoebamyosin ICbasal ATPase>150 μM [23]
Mousemyosin Vbasal ATPase>150 μM [23]
Bovinemyosin Xbasal ATPase>150 μM [23]
smooth muscle myosin IIA heavy-chainactin activated ATPase3 μM [24]
smooth muscle myosin IIB heavy-chainactin activated ATPase3 μM [24]
Rabbitfemoral, renal and saphenous arterytonic contraction5 μM [24]
Chicken gizzard contraction20 μM [24]
Chickengizzard smooth muscle HMMbasal ATPase15 ± 0.6 μM [27]
Chickengizzard smooth muscleactin activated ATPase6.47 μM [26]
Ratbladdercontraction100% inhibition at 15 μM [28]
Mouseintact paced cardiac papillary musclecontraction1.3 μM [29]
MouseCa2+-activated, permeabilized cardiac papillary musclecontraction2.8 μM [29]
Ratskinned cardiac trabeculaeCa2+ activated force0.38 ± 0.03 μM [15]
Ratnative demembranated right ventricular trabeculaeisometric force development3.17 ± 0.43 μM [30]
Drosophila nonmuscle myosin-2actin activated ATPaseno inhibition [31]
Drosophila nonmuscle myosin-2 M466I mutationactin activated ATPase36.3 ± 4.1 μM [31]
Drosophila cardiac tubesheart wall motion100 μM resulted in full inhibition [32]
Starfishnonmuscle myosin-2oocyte cytokinesiseffective at 300 μM [33]
C. elegans nonmuscle myosin-2acto-myosin colocalization microscopyeffective at 100 μM [34]
C. elegans nonmuscle myosin-2ventral enclosureeffective at 100 μM [35]
Podocoryna carnea (cnidarian)nonmuscle myosin-2stolon tip pulsations and gastrovascular floweffective at 255 μM [36]

Derivatives

The main aims of the structure-activity relationship work on the blebbistatin scaffold are the improvement of the physicochemical properties and the ATPase inhibitory potency, for use as chemical or pharmacological tools. Several analogs with superior properties have been developed, and guidelines for their optimal use have been described. [2] [8]

para-Nitroblebbistatin

2D structure of para-nitroblebbistatin Para-nitroblebbistatin 2D structure.jpg
2D structure of para-nitroblebbistatin

A non-fluorescent, non-phototoxic, non-cytotoxic derivative developed in 2014. [7] Its myosin inhibitory properties are similar to those of blebbistatin (for rabbit skeletal muscle myosin S1 IC50=0.4 μM, for Dictyostelium discoideum myosin II motor domain IC50=2.3 μM, for human β-cardiac myosin subfragment 1 IC50=13 μM, [37] for heavy meromyosin fragment of chicken skeletal muscle myosin IC50=0.4 μM [37] ). It has been successfully used in fluorescent imaging experiments involving myosin IIA-GFP expressing live dendritic cells [38]

para-Aminoblebbistatin

2D structure of para-aminoblebbistatin Para-aminoblebbistatin 2D structure.png
2D structure of para-aminoblebbistatin

A water-soluble blebbistatin derivative developed in 2016, [9] its high water solubility (~400 uM) enables in vivo research applications. Para-aminoblebbistatin is a slightly weaker myosin inhibitor than blebbistatin (for rabbit skeletal muscle myosin S1 IC50=1.3 μM, for Dictyostelium discoideum myosin II motor domain IC50=6.6 μM with only 90% maximal inhibition), it is non-fluorescent, photostable, neither cytotoxic nor phototoxic.

Azidoblebbistatin

A photoreactive myosin inhibitor developed in 2012. [39] A permanent inhibition of myosin may be achieved by covalently crosslinking the inhibitor azidoblebbistatin to its target by photoaffinity labeling (PAL). Briefly, upon UV illumination, the aryl-azide moiety in azidoblebbistatin forms a reactive nitrene. [40] This reaction is utilized to form covalent link between the inhibitor and myosin.

Azidoblebbistatin is also sensitive to two-photon irradiation, i.e. the covalent crosslink may also be generated by two-photon excitation microscope, therefore azidoblebbistatin is suitable for molecular tattooing. [41]

(S)-Nitroblebbistatin

This derivative was developed in 2005 to increase the photostability and decrease the fluorescence of blebbistatin. [42] (S)-nitro-blebbistatin is indeed stable to prolonged irradiation at 450-490 nm and has been successfully used in fluorescent live cell imaging. [43] However its affinity to myosin significantly decreased with the nitro-substitution (for nonmuscle myosin IIA, the IC50 = 27 μM). [42] In many cases due to the low solubility, it is not possible to achieve full inhibition of myosin with (S)-nitro-blebbistatin. It is effective for FRET imaging of isolated adult mouse cardiac muscle cells. [5]

(+)-Blebbistatin

(+)-Blebbistatin (or (R)-blebbistatin) is the inactive enantiomer of blebbistatin [1] which inhibits the ATPase activity by maximum 10%. [44] In research, it is useful compound for control treatment, to check the non-myosin related toxic effects of blebbistatin.

Other derivatives

The blebbistatin scaffold has been modified in several ways to optimize myosin isoform specificity or to improve the inhibitory properties and to map the structure-activity relationship. Major steps in the optimization include the work of Lucas-Lopez et al. from 2008 [45] and the works of Verhasselt et al. from 2017. [46] [47] [48] [49] The latter studies also include modifications of the A- and C-rings of the scaffold.

para-Chloroblebbistatin

A photostable, non-fluorescent, phototoxic derivative. Its fluorescence is less than 1% of that of blebbistatin myosin inhibitory properties are similar to those of blebbistatin. It is even more phototoxic than blebbistatin. [7]

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<span class="mw-page-title-main">GSK-3</span> Class of enzymes

Glycogen synthase kinase 3 (GSK-3) is a serine/threonine protein kinase that mediates the addition of phosphate molecules onto serine and threonine amino acid residues. First discovered in 1980 as a regulatory kinase for its namesake, glycogen synthase (GS), GSK-3 has since been identified as a protein kinase for over 100 different proteins in a variety of different pathways. In mammals, including humans, GSK-3 exists in two isozymes encoded by two homologous genes GSK-3α (GSK3A) and GSK-3β (GSK3B). GSK-3 has been the subject of much research since it has been implicated in a number of diseases, including type 2 diabetes, Alzheimer's disease, inflammation, cancer, addiction and bipolar disorder.

<span class="mw-page-title-main">Myosin light-chain kinase</span> Class of kinase enzymes

Myosin light-chain kinase also known as MYLK or MLCK is a serine/threonine-specific protein kinase that phosphorylates a specific myosin light chain, namely, the regulatory light chain of myosin II.

<span class="mw-page-title-main">Bleb (cell biology)</span> Bulge in the plasma membrane of a cell

In cell biology, a bleb is a bulge of the plasma membrane of a cell, characterized by a spherical, "blister-like", bulky morphology. It is characterized by the decoupling of the cytoskeleton from the plasma membrane, degrading the internal structure of the cell, allowing the flexibility required for the cell to separate into individual bulges or pockets of the intercellular matrix. Most commonly, blebs are seen in apoptosis but are also seen in other non-apoptotic functions, including apocrine secretion. Blebbing, or zeiosis, is the formation of blebs.

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<span class="mw-page-title-main">TNNI1</span> Protein-coding gene in the species Homo sapiens

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<span class="mw-page-title-main">MYH10</span> Protein-coding gene in the species Homo sapiens

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<span class="mw-page-title-main">TNNI2</span> Protein-coding gene in the species Homo sapiens

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<span class="mw-page-title-main">MYL2</span> Protein-coding gene in the species Homo sapiens

Myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC-2) also known as the regulatory light chain of myosin (RLC) is a protein that in humans is encoded by the MYL2 gene. This cardiac ventricular RLC isoform is distinct from that expressed in skeletal muscle (MYLPF), smooth muscle (MYL12B) and cardiac atrial muscle (MYL7).

<span class="mw-page-title-main">TEAD1</span> Protein-coding gene in the species Homo sapiens

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<span class="mw-page-title-main">MYL3</span> Protein-coding gene in the species Homo sapiens

Myosin essential light chain (ELC), ventricular/cardiac isoform is a protein that in humans is encoded by the MYL3 gene. This cardiac ventricular/slow skeletal ELC isoform is distinct from that expressed in fast skeletal muscle (MYL1) and cardiac atrial muscle (MYL4). Ventricular ELC is part of the myosin molecule and is important in modulating cardiac muscle contraction.

<span class="mw-page-title-main">MYL4</span> Protein-coding gene in the species Homo sapiens

Atrial Light Chain-1 (ALC-1), also known as Essential Light Chain, Atrial is a protein that in humans is encoded by the MYL4 gene. ALC-1 is expressed in fetal cardiac ventricular and fetal skeletal muscle, as well as fetal and adult cardiac atrial tissue. ALC-1 expression is reactivated in human ventricular myocardium in various cardiac muscle diseases, including hypertrophic cardiomyopathy, dilated cardiomyopathy, ischemic cardiomyopathy and congenital heart diseases.

<span class="mw-page-title-main">MYH1</span> Protein-coding gene in the species Homo sapiens

Myosin-1, also known as 'striated muscle myosin heavy chain 1', is a protein that in humans is encoded by the MYH1 gene. This gene is most highly expressed in fast type IIX/D muscle fibres of vertebrates and encodes a protein found uniquely in striated muscle; it is a class II myosin with a long coiled coil tail that dimerizes and should not be confused with 'Myosin 1' encoded by the MYO1 family of genes (MYO1A-MYO1H). Class I MYO1 genes function in many cell types throughout biology and are single-headed membrane-binding myosins that lack a long coiled coil tail.

<span class="mw-page-title-main">PPP1R14A</span> Protein found in humans

Protein phosphatase 1 regulatory subunit 14A also known as CPI-17 is a protein that in humans is encoded by the PPP1R14A gene.

<span class="mw-page-title-main">MYL7</span> Protein-coding gene in the species Homo sapiens

Atrial Light Chain-2 (ALC-2) also known as Myosin regulatory light chain 2, atrial isoform (MLC2a) is a protein that in humans is encoded by the MYL7 gene. ALC-2 expression is restricted to cardiac muscle atria in healthy individuals, where it functions to modulate cardiac development and contractility. In human diseases, including hypertrophic cardiomyopathy, dilated cardiomyopathy, ischemic cardiomyopathy and others, ALC-2 expression is altered.

<span class="mw-page-title-main">Rho kinase inhibitor</span>

Rho-kinase inhibitors are a series of compounds that target rho kinase (ROCK) and inhibit the ROCK pathway. Clinical trials have found that inhibition of the ROCK pathway contributes to the cardiovascular benefits of statin therapy. Furthermore, ROCK inhibitors may have clinical applications for anti-erectile dysfunction, antihypertension, and tumor metastasis inhibition. More recently they have been studied for the treatment of glaucoma and as a therapeutic target for the treatment of cardiovascular diseases, including ischemic stroke. While statin therapy has been demonstrated to reduce the risk of major cardiovascular events, including ischemic stroke, the interplay between the ROCK pathway and statin therapy to treat and prevent strokes in older adults has not yet been proven.

<i>para</i>-Nitroblebbistatin Chemical compound

para-Nitroblebbistatin is a non-phototoxic, photostable myosin inhibitor with low fluorescence. Its myosin inhibitory properties are very similar to those of blebbistatin.

<i>para</i>-Aminoblebbistatin Chemical compound

para-Aminoblebbistatin is a water-soluble, non-fluorescent, photostable myosin II inhibitor, developed from blebbistatin. Among the several blebbistatin derivatives it is one of the most promising for research applications. Furthermore, it has a favourable overall profile considering inhibitory properties and ADME calculations.

References

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