Para-nitroblebbistatin, the non-cytotoxic and photostable myosin II inhibitor

Article abstract of DOI:10.1002/anie.201403540

Blebbistatin, the best characterized myosin II-inhibitor, is commonly used to study the biological roles of various myosin II isoforms. Despite its popularity, the use of blebbistatin is greatly hindered by its blue-light sensitivity, resulting in phototo

Full text of DOI:10.1002/anie.201403540

Angewandte
Chemie
DOI: 10.1002/anie.201403540
Enzyme Inhibition
para-Nitroblebbistatin, the Non-Cytotoxic and Photostable Myosin II
Inhibitor**
Miklꢀs Kꢁpirꢀ, Boglꢂrka H. Vꢂrkuti, Lꢂszlꢀ Vꢁgner, Gergely Vçrçs, Gyçrgy Hegyi, Mꢂtꢁ Varga,
and Andrꢂs Mꢂlnꢂsi-Csizmadia*
Abstract: Blebbistatin, the best characterized myosinII-inhib-
itor, is commonly used to study the biological roles of various
myosinII isoforms. Despite its popularity, the use of blebbi-
statin is greatly hindered by its blue-light sensitivity, resulting in
phototoxicity and photoconversion of the molecule. Addition-
ally, blebbistatin has serious cytotoxic side effects even in the
absence of irradiation, which may easily lead to the misinter-
pretation of experimental results since the cytotoxicity-derived
phenotype could be attributed to the inhibition of the myosinII
function. Here we report the synthesis as well as the in vitro and
in vivo characterization of a photostable, C15 nitro derivative
of blebbistatin with unaffected myosinII inhibitory properties.
Importantly, para-nitroblebbistatin is neither phototoxic nor
cytotoxic, as shown by cellular and animal tests; therefore it
can serve as an unrestricted and complete replacement of
blebbistatin both in vitro and in vivo.
types, e.g. in cancer research and developmental biology, and
in the field of cell motility.[2] A well-known limitation of
blebbistatin treatment is the phototoxicity of the inhibitor
upon blue-light illumination,[3] which causes structural
changes in the molecule accompanied by the generation of
reactive oxygen species responsible for the phototoxic
effect.[3c]
The blue-light susceptibility of blebbistatin greatly hin-
ders in vivo imaging, because excitation wavelengths below
500 nm seriously damage the blebbistatin-treated samples.[4]
Additionally, incubation with blebbistatin exerts a significant
cytotoxic effect even without irradiation,[3c] which causes
serious problems in several in vivo systems. Further difficul-
ties may arise from blebbistatinꢀs own fluorescence, which
interferes with fluorescent signals such as that from the green
fluorescent protein (GFP) and with Fçrster resonance energy
transfer (FRET) experiments.[5] For in vivo applications of
blebbistatin, optimization of its chemical structure is essen-
tial. Introduction of a nitro group at the C7 position
(Scheme 1) of the tricyclic core of blebbistatin (7) led to
B
lebbistatin is a cell-permeable, specific inhibitor of class II
myosins, a group of actin-based ATP-driven motor proteins
responsible for various biological processes including muscle
contraction, cell migration, differentiation, and cytokinesis.[1]
Since blebbistatin is the best characterized myosinII-specific
inhibitor, it rapidly became the compound of choice to inhibit
myosinII-dependent processes in different species and cell
[*] G. Hegyi, A. Mꢀlnꢀsi-Csizmadia
MTA-ELTE Molecular Biophysics Research Group (Hungary)
E-mail: malnalab@yahoo.com
Homepage: http://www.malnalab.hu
M. Kꢁpirꢂ,[+] B. H. Vꢀrkuti,[+] L. Vꢁgner, G. Vçrçs,
A. Mꢀlnꢀsi-Csizmadia
Department of Biochemistry, Eçtvçs Lorꢀnd University
Pꢀzmꢀny Pꢁter sꢁtꢀny 1/c, 1117 Budapest (Hungary)
Scheme 1. Synthesis of para-nitroblebbistatin (6) and para-chlorobleb-
bistatin (8). Reagents and conditions: a) H2SO4, HNO3, 08C, 15 min;
b) POCl3, CH2Cl2, 508C, 18 h; c) LiHMDS, À788C to 08C, 3 h; d) oxa-
ziridine, À108C, 16 h; e) BF3·2H2O, CH3OH, N-chlorosuccinimide,
1008C microwave 30 min. The substituted positions applied in Ref. [6]
(C7) and the present study (C15) are indicated in blebbistatin (7) (for
detailed synthesis protocols see the Supporting Information).
M. Varga
Department of Genetics, Eçtvçs Lorꢀnd University
Pꢀzmꢀny Pꢁter sꢁtꢀny 1/c, 1117 Budapest (Hungary)
A. Mꢀlnꢀsi-Csizmadia
Drugmotif Ltd., Szt Erszebet krt 11, 2112 Veresegyhaz (Hungary)
[+] These authors contributed equally to this work.
[**] We are grateful to Mihaly Kovacs for his valuable comments. This
work was funded by the European Research Council (European
Community’s Seventh Framework Programme (FP7/2007-2013)/
ERC grant agreement no. 208319), ERC-PoC (grant agreement no.
620315), and Hungarian Research and Innovation Fund
slightly decreased fluorescence and increased photostability.[6]
However, the biological application of this blebbistatin
derivative was hampered by its strongly decreased binding
constant and specificity to myosinII. Recently, we reported
a highly myosinII-specific, photoinducible, C15-substituted
azido derivative of blebbistatin, called para-azidoblebbista-
tin,[7] which forms covalent cross-links with its target proteins
upon UV and bluelight irradiation. We observed that para-
azidoblebbistatin—unlike blebbistatin—is nonfluorescent
(KTIA_AIK_12-1-2013-0005). M.V. is a Jꢀnos Bolyai Fellow of the
Hungarian Academy of Sciences. B.H.V. was supported by the
European Union and the State of Hungary, cofinanced by the
European Social Fund in the framework of TꢃMOP 4.2.4. A/1-11-1-
2012-0001 “National Excellence Program”.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201403540.
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and non-cytotoxic. These findings and
the electrochemical properties of the
azido group initiated the idea that
a
C15 nitro- or chloro-substituted
blebbistatin derivative (para-nitro-
blebbistatin or para-chloroblebbista-
tin) could be a non-photoreactive,
and similarly non-cytotoxic and non-
fluorescent myosinII-specific inhibi-
tor. The replacement of blebbistatin
by such a derivative in biological
experiments would be advantageous.
Below we demonstrate that by means
of the C15 nitro substitution
(Scheme 1) we managed to eliminate
the fluorescence, photosensitivity, and
photo- and cytotoxicity of blebbistatin
without affecting its in vitro and
in vivo inhibitory properties. We also
present the structural model of para-
nitroblebbistatin in complex with myo-
sinII in which blebbistatin is extended
by a nitro group at the C15 position in
the atomic structural model of the
blebbistatin–myosin complex (PDB:
1YV3).[8]
We synthesized para-nitroblebbi-
statin (6) by nitration of the pyrrolidi-
none building block of blebbistatin
and subsequent application of Davisꢀ
oxaziridine protocol[6] (Scheme 1).
para-Chloroblebbistatin (8) was syn-
thesized by the direct chlorination of
blebbistatin (7).
The strong fluorescence of blebbi-
statin is a serious limitation in fluores-
cent microscopy. In contrast, signifi-
cantly reduced fluorescence character-
izes para-nitroblebbistatin and para-
chloroblebbistatin, as their emission
intensity is less than 1% of that of
blebbistatin (Figure 1A and Fig-
ure S1A).
Figure 1. In vitro characterization of para-nitroblebbistatin (NBleb) and blebbistatin (Bleb). A) The
fluorescence emission spectra of NBleb and Bleb were recorded using an excitation wavelength of
430 nm. B) The inhibition of the relative basal ATPase activities of DdMD and SkS1 (inset)
myosinII isoforms were recorded at increasing concentrations of NBleb and Bleb. The inhibition
of actin-activated ATPase activities of C) DdMD and D) SkS1 were measured at an inhibitor
concentration of 20 mm in the presence of increasing concentrations of actin. Data represents the
mean valuesÆstandard deviation of three independent experiments. Photoconversion of E) NBleb
and F) Bleb were followed by measuring their absorbance spectra after irradiation at 480Æ10 nm
at the indicated times.
The in vitro inhibitory effect of the
derivatives was evaluated on the Dictyostelium discoideum
myosinII motor domain (DdMD) and the rabbit skeletal
muscle myosin S1 (SkS1). The half maximal inhibitory con-
centration (IC50) of the basal ATPase activity of para-
nitroblebbistatin was the same as that of blebbistatin on
DdMD (2.33 Æ 0.13 mm and 2.96 Æ 0.45 mm, respectively) as
well as on SkS1 (0.4 Æ 0.05 mm and 0.41 Æ 0.03 mm, respec-
tively; Figure 1B). Both inhibitors completely suppressed
actin activation of the myosin ATPase activity even at high
concentrations of actin (80 mm) on both types of myosin
isoforms (Figure 1C,D). Since the inhibitory properties of
para-chloroblebbistatin and para-nitroblebbistatin are very
similar, these results confirm that C15 substitution of
blebbistatin does not substantially alter its inhibitory charac-
teristics (Figure S1B,C).
To characterize the photostability of para-nitroblebbista-
tin, para-chloroblebbistatin, and blebbistatin, the effect of
blue-light irradiation on their absorption spectra was com-
pared (Figure 1E,F and Figure S1D). Upon irradiation, the
absorption spectrum of blebbistatin changed markedly, which
is consistent with previously published studies[3b,c,6] and
indicates photoinduced changes in the molecular structure.
The absorption spectra of the C15 chloro- and nitro-substi-
tuted derivatives of blebbistatin remained essentially
unchanged upon blue-light irradiation.
We presumed that the improved photostability could be
accompanied by a reduced phototoxic effect of the inhibitors.
To test our hypothesis, we measured the blue-light-induced
phototoxicity of the compounds on HeLa cells. The cells were
treated with either para-nitroblebbistatin, para-chlorobleb-
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bistatin, or blebbistatin and then irradiated with blue light.
The irradiation wavelength was 480 Æ 10 nm, which is the
most commonly used excitation range in fluorescence mi-
croscopy for GFP detection. The energy density was
3.3 mJmmÀ2, which is an average energy input in confocal
microscopy applications (1–1400 mJmmÀ2).[3a] Following irra-
diation, the inhibitors were removed by washing and after
18 h of incubation the mortality rates were determined by
trypan blue staining and subsequent counting of the cells. We
observed no difference in the mortality and cell morphology
in the nontreated control and para-nitroblebbistatin-treated
cells, while blebbistatin treatment caused extensive cell death
and led to a number of morphologically defective cells that
were not stained by trypan blue (Figure 2A). para-Chloro-
blebbistatin treatment resulted in even more pronounced cell
death than blebbistatin treatment (Figure S2). This result
indicates that improved photostability is not necessarily
accompanied by reduced phototoxicity.
To test the suitability of para-nitroblebbistatin for live-cell
imaging, we performed a series of confocal microscopy
experiments employing blue-light excitation. EGFP-a-tubu-
lin mCherry-H2B-labeled HeLa Kyoto cells were imaged
repeatedly over 12 h in a confocal microscope; three z-
sections were applied every 10 min in the presence of 50 mm
blebbistatin or para-nitroblebbistatin, which is a commonly
applied concentration of blebbistatin. After blebbistatin
treatment, the cells became defective as they did not enter
into mitosis and most of them died or became morphologi-
cally seriously defective by the end of the experiment
(Figure 2B, Movies S1–S3). Cells that were already in the
mitotic phase before blebbistatin treatment, did not complete
cytokinesis and formed multinuclear cells as a consequence of
myosinII inhibition.[9] Additionally, fluorescent precipitates
of blebbistatin hampered microscopic imaging. Cells treated
with para-nitroblebbistatin did not show any sign of photo-
toxic damage and none of them died by the end of the
experiment. The ratios of cells entering into the mitotic phase
were the same in the para-nitroblebbistatin-treated cells as in
the control experiment. Due to the myosinII inhibitory effect
of para-nitroblebbistatin, all of the mitotic cells failed to
perform cytokinesis and thus became multinuclear. Since
para-nitroblebbistatin is not fluorescent, microscopic imaging
was not perturbed by fluorescent aggregates.
Figure 2. Phototoxicity of para-nitroblebbistatin and blebbistatin.
A) HeLa cells were treated with 10 mm of NBleb or Bleb then irradiated
at 480Æ10 nm for 15 min along with untreated control cells. After the
inhibitors had been removed by washing and the cells had been
incubated for 18 h, the dead cells were stained with trypan blue and
visualized by light microscopy (right panel). Live (unstained), dead
(blue), and morphologically defective cells (red arrowhead) were
counted and their ratios are presented in the column diagram (left
panel, N=350–400, mean valuesÆstandard deviation). B) HeLa Kyoto
cells were treated with DMSO (control), 50 mm NBleb, or Bleb and
imaged repeatedly in a confocal microscope for 12 h. The excitation
wavelengths were 488 and 543 nm for the GFP and mCherry, respec-
tively. Most of the Bleb-treated cells became highly autofluorescent,
which is an indication of cell death or serious defectiveness of cellular
functions (Movies S1–S3). C) Zebrafish larvae (3 dpf in age) were
treated with 10 mm NBleb or Bleb and irradiated at 470Æ20 nm for
10 min along with untreated control larvae. Lifespans of the larvae
were monitored for 36 h after treatment (N=10, mean valuesÆstan-
dard deviation). All data represent at least three independent experi-
ments.
In addition to experiments using HeLa cells, the photo-
toxicity of blebbistatin and para-nitroblebbistatin was also
compared using Danio rerio (zebrafish) as a vertebrate
model. Embryos 3 dpf (days post fertilization) in age were
treated with the inhibitors at a concentration of 10 mm and
irradiated with blue light (wavelength: 470 Æ 20 nm, energy
density: 0.4 mJmmÀ2). The mortality rate of the embryos was
monitored for the following 36 h (Figure 2C). After 36 h the
majority of the embryos that had received blebbistatin
treatment were dead (86 Æ 5%), whereas the mortality in
the para-nitroblebbistatin-treated samples was significantly
lower (18 Æ 10%).
cells. The cells were treated with the inhibitors at a concen-
tration of 20 mm and the change in cell number, viability, and
the ratio of multinuclearity was monitored for 3 days (Fig-
ure 3A,B). Both inhibitors efficiently suppressed cytokinesis
and therefore the initial cell number did not rise during the
experiment. Consistently, the ratio of multinuclear cells
continuously increased and by the third day, practically all
cells were multinuclear. Blebbistatin treatment resulted in
After elucidating that para-nitroblebbistatin is not photo-
toxic, we further evaluated its myosinII inhibition on differ-
ent cellular systems. First, we compared the effect of para-
nitroblebbistatin and blebbistatin on the cytokinesis of HeLa
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a significant cell mortality, leading to 90% death ratio of
HeLa cells in 3 days. Since the entire experiment was
performed in the dark, this result reflects the cytotoxicity of
blebbistatin in itself, without irradiation. In contrast, para-
nitroblebbistatin did not affect the death rate of the cells.
Additionally, the live blebbistatin-treated cells displayed
severely defective cellular morphologies while para-nitro-
blebbistatin treatment did not damage the cells (Figure 3C).
We further confirmed that para-nitroblebbistatin also induces
multinuclearity of Dictyostelium discoideum (Dd) cells,
similarly to blebbistatin (Figure S3). Neither of the inhibitors
exerted cytotoxic effects on Dd cells. As blebbistatin was
named after its characteristic effect of suppressing the
blebbing of cells, we compared para-nitroblebbistatin and
blebbistatin inhibition on this process as well by using the
continuously blebbing M2 human melanoma cell line. Solu-
tions of either compound at a concentration of 10 mm
completely inhibited the blebbing within 10 min, and during
this time no morphological difference was detected between
cells treated with the inhibitors (Figure S4, Movies S4 and
S5). These experiments demonstrate that para-nitroblebbi-
statin has the same in vivo specificity to nonmuscle myosinII
as blebbistatin.
To explore the functional effects of para-nitroblebbistatin
on vertebrates, we studied the development of the lateral line
organ in zebrafish embryos, which involves nonmuscle
myosinII function.[10] The lateral line is formed by pLLp
(posterior lateral line primordia) cells migrating from the ear
to the tip of the tail, meanwhile a series of mechanosensory
receptors (neuromasts) are deposited regularly (Figure 4).
Transgenic cldnb:gfp zebrafish embryos[11] with GFP-labeled
pLLp and neuromasts were treated with increasing concen-
trations of para-nitroblebbistatin and blebbistatin starting at
1 dpf. After 24 h of incubation (at 2 dpf) the positions of the
pLLp were studied. The treatment resulted in similar effects
Figure 4. Comparison of the in vivo effects of para-nitroblebbistatin
and blebbistatin on zebrafish embryos. A) The schematic representa-
tion depicts the migration of the lateral line primordium (pLLp)
starting from the head region at 1 dpf heading to the tip of the tail
while neuromasts are deposited regularly. The primordium cells are
fluorescent due to the specific expression of claudin-GFP. B) 1 dpf
embryos were treated with increasing concentrations of NBleb or Bleb
and fluorescence stereomicroscope images (excitation wavelength
470Æ20 nm) were recorded after 24 h of incubation. The migration
fronts of pLLps are indicated by white arrowheads. C) Lifespans of
10 mm NBleb- or Bleb-treated and untreated embryos were monitored
for 36 h (N=10, mean valuesÆstandard deviation from three inde-
pendent experiments).
Figure 3. In vivo effects of para-nitroblebbistatin and blebbistatin on
HeLa cells. A) HeLa cells were treated with 20 mm NBleb or Bleb and
incubated for 3 days along with DMSO-treated control cells, while the
changes in cell number and the rates of mortality were followed.
B) HeLa cells were treated with 20 mm NBleb, Bleb, or DMSO and
incubated for 3 days, while the ratio of their multinuclearity was
followed. Data represent the mean valuesÆstandard deviation of at
least three independent experiments. C) Representative images of
Hoechst-stained HeLa cells were taken on the third day of the
experiment by confocal microscopy, excited at 405 nm wavelength.
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for both inhibitors, including the halt of the pLLp and
a curved body shape (Figure 4B). However, animal mortality
associated with blebbistatin administration was higher than
that after para-nitroblebbistatin treatment. In 36 h the
mortality rate of the para-nitroblebbistatin-treated embryos
was similar to that of the nontreated control, whereas all fish
died in the presence of 10 mm of blebbistatin. This reflects
blebbistatinꢀs high cytotoxicity in the dark which is a clearly
different effect than its blue-light-induced phototoxicity
(Figure 4C).
Here, we have demonstrated that the nitro substitution of
blebbistatin at the C15 position decreases the fluorescence of
the compound by a hundred times, increases the chemical
stability upon blue-light irradiation, and greatly reduces its
phototoxicity and cytotoxicity without affecting specific
myosinII inhibitory properties. The analysis of the molecular
structure of the myosin–ADP–vanadate–blebbistatin com-
plex (PDB: 1YV3)[8] indicates that the nitro substituent at
C15 does not significantly affect the interaction of the
inhibitor with myosin, because the nitro substituent causes
no steric hindrance for any of the side chains in the
blebbistatin binding site of myosin (Figure 5).
ation, is a source of constant nuisance for experimental
biologists. Furthermore, although the solubility of blebbista-
tin is around 20 mm (Figure S5), it is usually applied in vivo at
concentrations of 50–100 mm. As the in vivo solubility of
blebbistatin in the more hydrophobic cellular environment
can be much higher than that measured in vitro, such high
concentrations of the inhibitor induce nonspecific enzymatic
interactions[7] and more pronounced cytotoxicity in in vivo
experiments. Cytotoxicity and other nonspecific effects may
easily lead to the misinterpretation of experimental results
since myosin inhibition is interspersed with the side effects.
Therefore, the non-cytotoxic, non-phototoxic, and nonfluor-
escent para-nitroblebbistatin is an ideal and complete
replacement of blebbistatin for the study of the specific role
of myosinII in physiological, developmental, and cell biolog-
ical studies.
Received: March 20, 2014
Revised: May 2, 2014
Published online: && &&, &&&&
Keywords: asymmetric synthesis · cytotoxicity · enzymes ·
.
inhibitors · proteins
The high cytotoxicity of blebbistatin, which irreversibly
damages the in vivo systems even without blue-light irradi-
[1] a) A. F. Straight, A. Cheung, J. Limouze, I. Chen, N. J. West-
wood, J. R. Sellers, T. J. Mitchison, Science 2003, 299, 1743 –
1747; b) M. Kovacs, J. Toth, C. Hetenyi, A. Malnasi-Csizmadia,
J. R. Sellers, J. Biol. Chem. 2004, 279, 35557 – 35563.
[2] a) S. G. Turney, P. C. Bridgman, Nat. Neurosci. 2005, 8, 717 – 719;
b) A. J. Engler, S. Sen, H. L. Sweeney, D. E. Discher, Cell 2006,
126, 677 – 689; c) M. Krieg, Y. Arboleda-Estudillo, P. H. Puech, J.
Kꢁfer, F. Graner, D. J. Mꢂller, C. P. Heisenberg, Nat. Cell Biol.
2008, 10, 429 – 436.
[3] a) T. Sakamoto, J. Limouze, C. A. Combs, A. F. Straight, J. R.
Sellers, Biochemistry 2005, 44, 584 – 588; b) J. Kolega, Biochem.
Biophys. Res. Commun. 2004, 320, 1020 – 1025; c) A. Mikulich, S.
Kavaliauskiene, P. Juzenas, Biochim. Biophys. Acta Gen. Subj.
2012, 1820, 870 – 877.
[4] a) T. Kondo, K. Hamao, K. Kamijo, H. Kimura, M. Morita, M.
Takahashi, H. Hosoya, Biochem. J. 2011, 435, 569 – 576; b) C. S.
Rex, C. F. Gavin, M. D. Rubio, E. A. Kramar, L. Y. Chen, Y. Jia,
R. L. Huganir, N. Muzyczka, C. M. Gall, C. A. Miller, G. Lynch,
G. Rumbaugh, Neuron 2010, 67, 603 – 617.
[5] K. Wong, A. Van Keymeulen, H. R. Bourne, J. Cell Biol. 2007,
179, 1141 – 1148.
[6] C. Lucas-Lopez, S. Patterson, T. Blum, A. F. Straight, J. Toth,
A. M. Z. Slawin, T. J. Mitchison, J. R. Sellers, N. J. Westwood,
Eur. J. Org. Chem. 2005, 1736 – 1740.
[7] M. Kepiro, B. H. Varkuti, A. Bodor, G. Hegyi, L. Drahos, M.
Kovacs, A. Malnasi-Csizmadia, Proc. Natl. Acad. Sci. USA 2012,
109, 9402 – 9407.
[8] J. S. Allingham, R. Smith, I. Rayment, Nat. Struct. Mol. Biol.
2005, 12, 378 – 379.
[9] S. Shu, X. Liu, E. D. Korn, Proc. Natl. Acad. Sci. USA 2005, 102,
1472 – 1477.
[10] a) S. Ernst, K. Liu, S. Agarwala, N. Moratscheck, M. E. Avci, D.
Dalle Nogare, A. B. Chitnis, O. Ronneberger, V. Lecaudey,
Development 2012, 139, 4571 – 4581; b) M. J. Harding, A. V.
Nechiporuk, Development 2012, 139, 3130 – 3135.
Figure 5. Structure of the myosin–ADP–vanadate–para-nitroblebbista-
tin complex. The model was created by the extension of Bleb with
a nitro group at the C15 position in the crystal structure model of
myosin–ADP–vanadate–blebbistatin (PDB: 1YV3). Myosin residues
form a cavity halfway between the nucleotide-binding pocket and the
actin-binding cleft, where the binding site of Bleb is situated (lower
panel). Bleb and ADP–vanadate are shown by stick and space-filling
representations, respectively. The possible attractive (green) and
repulsive (red) interactions of the partially negatively charged nitro
group of para-nitroblebbistatin are highlighted (upper left panel). The
distance between the side chains of amino acids is indicated in ꢄ.
There is no steric hindrance between the additional nitro group and
the myosin residues (upper right panel).
[11] P. Haas, D. Gilmour, Dev. Cell 2006, 10, 673 – 680.
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Communications
Enzyme Inhibition
No side effects: Blebbistatin, the most
popular myosinII inhibitor, is phototoxic,
cytotoxic, and light sensitive. However, its
C15 nitro analogue displays none of these
side effects and maintains the specificity
and inhibitory properties of the parent.
Thus, para-nitroblebbistatin can replace
blebbistatin both in vitro and in vivo.
M. Kꢁpirꢂ, B. H. Vꢃrkuti, L. Vꢁgner,
G. Vçrçs, G. Hegyi, M. Varga,
A. Mꢃlnꢃsi-Csizmadia*
&&&& — &&&&
para-Nitroblebbistatin, the Non-Cytotoxic
and Photostable Myosin II Inhibitor
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