Blebbistatin
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 | |
| |
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
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
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.
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] |
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
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
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,
References
- ^ S2CID 38625401.
- ^ PMID 29878759.
- PMID 22990759.
- PMID 18296569.
- ^ PMID 30242036.
- ^ PMID 15240150.
- ^ PMID 24954740.
- ^ PMID 30057142.
- ^ PMID 27241904.
- PMID 15205456.
- ^ S2CID 32475661.
- PMID 15109496.
- S2CID 22216223.
- PMID 22191382.
- ^ S2CID 20503064.
- PMID 17467631.
- PMID 18599626.
- PMID 19413972.
- PMID 25296316.
- PMID 15641783.
- PMID 22507270.
- PMID 22990759.
- ^ S2CID 22355306.
- ^ S2CID 28556955.
- PMID 24662474.
- ^ PMID 28714309.
- S2CID 7063637.
- PMID 22016792.
- ^ PMID 17615158.
- PMID 29052230.
- ^ PMID 25636739.
- PMID 24221941.
- ^ Bischof J. "The Molecular Mechanism of Surface Contraction Waves in the Starfish Oocyte" (PDF). The Heidelberg Document Repository.
- PMID 18190904.
- ^ Wernike D. "Studying Ventral Enclosure during C. elegans Epidermal Morphogenesis" (PDF). Concordia University Spectrum Research Repository. Retrieved 31 July 2018.
- PMID 26605798.
- ^ PMID 28119616.
- PMID 26109323.
- PMID 22647605.
- ^ "Photoreactive Crosslinker Chemistry". Thermo Fisher Scientific.
- PMID 25892202.
- ^ .
- PMID 18614806.
- ^ Lucas-Lopez C. "Supportin information for the article "Absolute Stereochemical Assignment and Fluorescence Tuning of the Small Molecule Tool, (–)-Blebbistatin"" (PDF).
- PMID 18528569.
- PMID 28220174.
- PMID 28486210.
- PMID 28512027.
- PMID 29807794.