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Names | |
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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 | |
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Properties | |
C18H16N2O2 | |
Molar mass | 292.338 g·mol−1 |
Appearance | Yellow 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]
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]
A number of physicochemical deficiencies hamper the use of blebbistatin as a chemical tool in particular applications. [2]
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]
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]
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.
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.
species | myosin isoform or muscle type | assay type | IC50 |
---|---|---|---|
Dictyostelium discoideum | myosin II motor domain | basal ATPase | 2.96 ± 0.45 μM, [7] 4.4 ± 0.3 μM, [9] 4.9 μM [23] |
Dictyostelium discoideum | myosin II motor domain | actin activated ATPase | 3.9 ± 0.3 μM [9] |
Rabbit | skeletal muscle II | basal ATPase | 0.50 μM, [23] 0.3 ± 0.03 μM, [9] 0.41 ± 0.03 μM [7] |
Rabbit | skeletal muscle II | actin activated ATPase | 0.11 ± 0.009 μM [9] |
Porcine | b-cardiac muscle | basal ATPase | 1.2 μM [23] |
Scallop | striated muscle | basal ATPase | 2.3 μM [23] |
Human | nonmuscle IIA | basal ATPase | 5.1 μM [23] |
Chicken | nonmuscle IIB | basal ATPase | 1.8 μM [23] |
Human | nonmuscle IIA | actin activated ATPase | 3.58 μM [26] |
Human | nonmuscle IIB | actin activated ATPase | 2.30 μM [26] |
Mouse | nonmuscle IIC | actin activated ATPase | 1.57 μM [26] |
Turkey | smooth muscle | basal ATPase | 79.6 μM [23] |
Acanthamoeba | myosin II | basal ATPase | 83 μM [23] |
Rat | myosin 1B | basal ATPase | >150 μM [23] |
Acanthamoeba | myosin IC | basal ATPase | >150 μM [23] |
Mouse | myosin V | basal ATPase | >150 μM [23] |
Bovine | myosin X | basal ATPase | >150 μM [23] |
smooth muscle myosin IIA heavy-chain | actin activated ATPase | 3 μM [24] | |
smooth muscle myosin IIB heavy-chain | actin activated ATPase | 3 μM [24] | |
Rabbit | femoral, renal and saphenous artery | tonic contraction | 5 μM [24] |
Chicken | gizzard | contraction | 20 μM [24] |
Chicken | gizzard smooth muscle HMM | basal ATPase | 15 ± 0.6 μM [27] |
Chicken | gizzard smooth muscle | actin activated ATPase | 6.47 μM [26] |
Rat | bladder | contraction | 100% inhibition at 15 μM [28] |
Mouse | intact paced cardiac papillary muscle | contraction | 1.3 μM [29] |
Mouse | Ca2+-activated, permeabilized cardiac papillary muscle | contraction | 2.8 μM [29] |
Rat | skinned cardiac trabeculae | Ca2+ activated force | 0.38 ± 0.03 μM [15] |
Rat | native demembranated right ventricular trabeculae | isometric force development | 3.17 ± 0.43 μM [30] |
Drosophila | nonmuscle myosin-2 | actin activated ATPase | no inhibition [31] |
Drosophila | nonmuscle myosin-2 M466I mutation | actin activated ATPase | 36.3 ± 4.1 μM [31] |
Drosophila | cardiac tubes | heart wall motion | 100 μM resulted in full inhibition [32] |
Starfish | nonmuscle myosin-2 | oocyte cytokinesis | effective at 300 μM [33] |
C. elegans | nonmuscle myosin-2 | acto-myosin colocalization microscopy | effective at 100 μM [34] |
C. elegans | nonmuscle myosin-2 | ventral enclosure | effective at 100 μM [35] |
Podocoryna carnea (cnidarian) | nonmuscle myosin-2 | stolon tip pulsations and gastrovascular flow | effective at 255 μM [36] |
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]
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]
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.
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]
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 (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.
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.
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]
Muscle contraction is the activation of tension-generating sites within muscle cells. In physiology, muscle contraction does not necessarily mean muscle shortening because muscle tension can be produced without changes in muscle length, such as when holding something heavy in the same position. The termination of muscle contraction is followed by muscle relaxation, which is a return of the muscle fibers to their low tension-generating state.
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.
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.
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.
ROCK1 is a protein serine/threonine kinase also known as rho-associated, coiled-coil-containing protein kinase 1. Other common names are ROKβ and P160ROCK. ROCK1 is a major downstream effector of the small GTPase RhoA and is a regulator of the actomyosin cytoskeleton which promotes contractile force generation. ROCK1 plays a role in cancer and in particular cell motility, metastasis, and angiogenesis.
Cardiac muscle troponin T (cTnT) is a protein that in humans is encoded by the TNNT2 gene. Cardiac TnT is the tropomyosin-binding subunit of the troponin complex, which is located on the thin filament of striated muscles and regulates muscle contraction in response to alterations in intracellular calcium ion concentration.
Troponin C, also known as TN-C or TnC, is a protein that resides in the troponin complex on actin thin filaments of striated muscle and is responsible for binding calcium to activate muscle contraction. Troponin C is encoded by the TNNC1 gene in humans for both cardiac and slow skeletal muscle. In slow skeletal muscle. structural analysis,anlaizie;10.164.138.220 Hotspot in for phone lunch everyday. Troponin C, also known as TN-C or TnC, is a protein that resides in the troponin complex on actin thin filaments of striated muscle and is responsible for binding
Troponin I, slow skeletal muscle is a protein that in humans is encoded by the TNNI1 gene. It is a tissue-specific subtype of troponin I, which in turn is a part of the troponin complex.
Myosin-10 also known as myosin heavy chain 10 or non-muscle myosin IIB (NM-IIB) is a protein that in humans is encoded by the MYH10 gene. Non-muscle myosins are expressed in a wide variety of tissues, but NM-IIB is the only non-muscle myosin II isoform expressed in cardiac muscle, where it localizes to adherens junctions within intercalated discs. NM-IIB is essential for normal development of cardiac muscle and for integrity of intercalated discs. Mutations in MYH10 have been identified in patients with left atrial enlargement.
Troponin I, fast skeletal muscle is a protein that in humans is encoded by the TNNI2 gene.
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).
Transcriptional enhancer factor TEF-1 also known as TEA domain family member 1 (TEAD1) and transcription factor 13 (TCF-13) is a protein that in humans is encoded by the TEAD1 gene. TEAD1 was the first member of the TEAD family of transcription factors to be identified.
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.
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.
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.
Protein phosphatase 1 regulatory subunit 14A also known as CPI-17 is a protein that in humans is encoded by the PPP1R14A gene.
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.
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.
para-Nitroblebbistatin is a non-phototoxic, photostable myosin inhibitor with low fluorescence. Its myosin inhibitory properties are very similar to those of blebbistatin.
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.