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Blebbistatin

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)
  • CC1=CC2=C(C=C1)N=C3C(C2=O)(CCN3C4=CC=CC=C4)O
Properties
C18H16N2O2
Molar mass 292.338 g/mol
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).

Introduction

Blebbistatin [1] is a myosin inhibitor mostly specific for myosin II. It is widely used in research to inhibit heart muscle, non-muscle myosin II, skeletal muscle. However, its adverse characteristics e.g. its cytotoxicity and blue-light instability or low solubility in water often make its application challenging or impossible [2] [3]. Recently its applicability was improved by chemical design and its derivatives overcome the limitations of blebbistatin. E.g. para-nitroblebbistatin and para-aminoblebbistatin are photostable, and they are neither cytotoxic nor fluorescent [3] [4].

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[5]. 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[6], hence its name. At cellular level blebbistatin also inhibits cytokinesis [6] may also disrupt mitotic spindle formation [7]. Migration of cells can be either enhanced or inhibited depending on other conditions [8]. At the organ level blebbistatin stops the contraction of skeletal muscle [9] or heart muscle [10]. 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 [11] [12] [13].

Adverse characteristics

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 [14] [15] [16].

Fluorescence

Blebbistatin is a relatively strong fluorophore, it absorbs around 430 nm and emits around 560 nm [17], therefore its fluorescence interferes with GFP imaging or FRET experiments [3].

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[18][19][20]. 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[3], 4.4 ± 0.3 μM[4], 4.9 μM[18]
Dictyostelium discoideum myosin II motor domain actin activated ATPase 3.9 ± 0.3 μM[4]
Rabbit skeletal muscle II  basal ATPase 0.50 μM[18], 0.3 ± 0.03 μM[4], 0.41 ± 0.03 μM [3]
Rabbit skeletal muscle II  actin activated ATPase 0.11 ± 0.009 μM[4]
Porcine b-cardiac muscle basal ATPase 1.2 μM[18]
Scallop striated muscle basal ATPase 2.3 μM[18]
Human nonmuscle IIA basal ATPase 5.1 μM[18]
Chicken nonmuscle IIB basal ATPase 1.8 μM[18]
Turkey smooth muscle basal ATPase 79.6 μM[18]
Acanthamoeba myosin II basal ATPase 83 μM[18]
Rat myosin 1B basal ATPase >150 μM[18]
Acanthamoeba myosin IC basal ATPase >150 μM[18]
Mouse myosin V basal ATPase >150 μM[18]
Bovine myosin X basal ATPase >150 μM[18]
smooth muscle myosin IIA heavy-chain actin activated ATPase 3 μM[19]
smooth muscle myosin IIB heavy-chain actin activated ATPase 3 μM[19]
Rabbit femoral, renal and saphenous artery  tonic contraction 5 μM[19]
Chicken gizzard contraction 20 μM[19]
Mouse intact paced cardiac papillary muscle contraction 1.3 μM[21]
Mouse  Ca2+-activated, permeabilized cardiac papillary muscle contraction 2.8 μM[21]
Drosophila nonmuscle myosin-2  actin activated ATPase no inhibition[22]

Derivatives

para-nitroblebbistatin

2D structure of para-nitroblebbistatin

A non-fluorescent, non-phototoxic, non-cytotoxic derivative developed in 2014 [3]. 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[23], for heavy meromyosin fragment of chicken skeletal muscle myosin IC50=0.4 μM[23]). It has been successfully used in fluorescent imaging experiments involving myosin IIA-GFP expressing live dendritic cells[24].

para-aminoblebbistatin

2D structure of para-aminoblebbistatin

A water-soluble blebbistatin derivative developed in 2016 [4], 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 myosinII 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[25]. 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. [26] 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[27]. 

(S)-nitro-blebbistatin

This derivative was developed in 2005 to increase the photostability and decrease the fluorescence of blebbistatin[28]. (S)-nitro-blebbistatin is indeed stable to prolonged irradiation at 450-490 nm and has been successfully used in fluorescent live cell imaging[29]. However its affinity to myosin significantly decreased with the nitro-substitution (for nonmuscle myosin IIA, the IC50=27 μM)[28]. In many cases due to the low solubility, it is not possible to achieve full inhibition of myosin with (S)-nitro-blebbistatin.

(+) blebbistatin or (R) blebbistatin

The inactive enantiomer of blebbistatin [1], which inhibits the ATPase activity by maximum 10% [30]. 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[31] and the works of Verhasselt et al from 2017 [32].[33] [34].

para-chloroblebbistatin

A photostable, non-fluorescent, phototoxic derivative. Its fluorescence is <1% of that of blebbistatin myosin inhibitory properties are similar to those of blebbistatin. It is even more phototoxic than blebbistatin. [3] 

Category:Myosin inhibitor Category:Organic compounds

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