9.0 ± 4.8
0.6
Non-muscle IIB
76 ± 4.9
0.4
Cardiac
2600 ± 240
Skeletal
11300 ± 840
10
Myosin (30 µM)
±Compound
DMSO
CK-2018571
0.6
ATP
(10 µM)
CK-2018571 (0.5µM, n=2)
-log [Ca2+ ], mol/L
Force
(% of maximum tensio
Smooth
0.8
Phosphate Prod
(µM)
Normalized ATPas
Activity
1.0
60
0.2
0.0
20
IN VITRO
1.2
57
1
tin
200
300
CK-2018571 (1µM, n=2)
CK-2018571
Amplitude
Rate(0.5µM, n=2)
DMSO
-1)
15
15 myosin)
(mol Pi /mol
(s15
CK-2018571
0.6
ATP
±Compound
(10 µM)
Myosin
Isoform
IC
±
SD,
nM60
50 ± SD,
Myosin Isoform
IC 50
nM
AcidIsoform
Myosin
IC
SD, nM 16
DMSO
50 ±
10
Smooth
9.0 ± 4.80.4
t=5s
DMSO
CK-2018571
40 CK-2018571
4
4.8
Smooth
9.0
±
Smooth
9.0 ± 4.8
Non-muscle
IIB
76 ± 4.9
1.2
1.0
-20
9
RESULTS
ATP
(150
µM)
Myosin (30 µM)
ATP
0.53
7
6
5
-log [Ca2+ ], mol/L
125( n=8)
DMSO
CK-2018571 (1µM, n=2)
100
CK-2018571 (0.5µM, n=2)
100
Myosin (15 µM)
±Compound
8
80
75
100
80
4
( n=8)
DMSO 100
DMSO ( n=8)
CK-2018571 (1µM,CK-2018571
n=2)
(1µM, n=2)
CK-2018571
n=2)
80 (0.5µM,
CK-2018571
(0.5µM, n=2)
Force
(% of maximum tension)
actin
80
100
Force
Force
(% of maximum
tension)
(% of maximum tension)
myosin
Bl
eb
b i
st
a
100
0
Cytokinetics,
Inc., South San Francisco, CA, USA
DMSO ( n=8)Time, msec
Force
(% of initial tension)
1.2
40
Total phosphate determination
using malachite green
Total phosphate determination
using malachite green
Force
(% of maximum tension)
76 ± 4.9
(free fraction)
18
-4
SmoothSmooth Cardiac Cardiac
Smooth
Cardiac
hexokinase
Non-muscle
Skeletal
Non-muscleFast
Fast Skeletal
Non-muscle
Fast
Skeletal
Myosin
(30 µM)
10
0.2
CK
-2
0
-5
(% of maximum tension)
9.0 ± 4.8
Non-muscle IIB
0.6
-6
Phosphate Produced
Phosphate Produced
(µM)
Force
(mol / mol
myosin)
0.8
10 -7
Normalized ATPase
Activity
INTRODUCTION
Smooth
1.0
IC 50 ± SD, nM
Phosphate Produced
Phosphate
Normalized
ATPase Produced
(mol / mol myosin)
(µM)
Normalized ATPase
Activity
Activity
Normalized
ATPase
Normalized
ATPase
D
Activity
Activity
Normalized ATPase
15
Myosin Isoform
1.2
Acid
0
10
10
10
Sheila
Clancy,
Zhiheng
Jia,
Malar
Pannirselvam,
Xiangping
Qian,
Bradley
Morgan,
Fady Malik, Jim Hartman
0.0
[ CK-2018571], mol/L
Binding reaction supernatants
10 -8
Loading Controls
Cardiac
Fast Skeletal
Normalized ATPase
Activity
Normalized ATPase
Activity
Smooth
Non-muscle
10 -9
0
0.4
Phosphate Produced
(mol / mol
myosin)
Produced
D
PhosphateM
Produced (µM)
D
(mol / mol myosin)
M
Phosphate Produced
Phosphate Produced
SO
(µM)SBinding
Phosphate Produced
Phosphate Produced
/ mols myosin)
O(molRate,
(mol / mol myosin)
Actin-Stimulated
(µM)
(µM)
ADP Release Rate (s-1)
CK Binding Rate, s
Actin-Stimulated D M
D
DPhosphate CM
ADP Release Rate (s ) SO
Produced
-2
Phosphate
Produced
D
Phosphate ProducedMS
01
KSM
D Phosphate Produced
O / mol myosin)
O2
85
(mol / mol myosin)(mol
SO
M
(µM)
0
71
(µM)
SO
Phosphate
Produced
Phosphate Produced 1
D
C
Phosphate
Produced
Phosphate
Produced
M
CK
K- myosin)
(mol
myosin) 8
SO
(mol / mol
CK/ molProduced
57
C
Bl
20 Phosphate
(µM)
Phosphate
Produced
K Binding Rate, s Produced
Phosphate
C
(µM)-201
-2
eb
myosin)
K
1 8(mol / mol
1
01
D
20
D (µM)
85
b i
85
M -20
Phosphate
Produced
Binding Rate,
s (µM)
57
Produced
71
71
Phosphate
st
1
D Phosphate M
S
(mol
/ molProduced
myosin)
Phosphate
Produced
1
1
Phosphate
Produced
M
S
Phosphate
Produced
at
O
8
8
B
(µM)
Phosphate
Produced
(mol
/
mol
myosin)
O
57 Phosphate
(mol
/molmol
myosin)
SO
Binding Rate, s
5
Produced
in
l
D
B
D
(mol
/
myosin)
e
D Binding Rate, s
7
(µM)
C
Bl
le
M1 MS (µM)
M
K
(µM)
b b
-2
eb
CK b 1b
SO
01
O
DCK S
i
st
CK
M O
b i
85
-2 iBsle
at
71
st
01 tab DM CK -S2DO0M1
in
CK
at Ble
2
b
8
85 C tiCsSO -2 S5O7
0
in
-2
b
D 18
01
7 K nKt-a 01 1
b i
M 5
85
CK 1 -2 2C0tB1in 857
s
0
SO 7 1
K
71
tB a
-2
1 8-l8e25b07 1
01
t
b
1
l
85 B 5 1i8s
eb in
Bl
l
eb
Bl 71 eb 715ta7t B
b i
in l
1C
b
e
b i
stB
b
st
aletB Bb ist istat K- eb
at
b ib l
in
nies leb atin in 20 b is
b i
tb a
1 8 ta
tb in
is stat
57 tin
ta in
1
t
10 -10
5
Produced
D Phosphate
M (mol / mol myosin)
SO
THE SMALL MOLECULE SMOOTH MUSCLE MYOSIN INHIBITOR,
CK-2018571, SELECTIVELY INHIBITS ATP HYDROLYSIS AND RELAXES SMOOTH MUSCLE
Acid
ATP
(150 µM)
Myosin (15 µM)
±Compound
t=5s
80
80
(A) (B)
-1
-1
-1
-1
(B)
(C)
(A)
(B)
(C)
(D)
(A)
(A) (B) (C) (D)
-1
-1
SO
ADP Release Rate (s-1)
Actin-Stimulated
ADP Release Rate (s-1)
-1
-1
-1
-1
-1
-1
-1
10
10
0
15
-115 -1
5
(D)
-1
-1
5
5 0
C
Actin-Stimulated
K
-2 -1
- 1 ADP Release Rate01(s )
Actin-Stimulated
85
D
Actin-Stimulated
-1
M
71
ADPS Release Rate (s )
ADP Release Rate (s-1)
O
0
-1
20
0
0
-1
10
-1
15
-1
40
20
R
57
1
57
1
Nucleotide binding
monitored by
fluorescence
C
K
C
-2
01
8
K
-2
01
8
57
1
-2
01
8
K
C
SO
M
57
1
SO
20
Force
(% of initial tension)
57
1
C
K
C
K
D
-2
01
8
M
-2
01
8
SO
57
1
C
K
C
K
Force
(% of initial tension)
-2
01
8
K
57
1
C
-2
01
8
D
57
1
-2
01
8
57
1
SO
SO
M
M
D
SO
M
D
71
D
-2
01
8
4 µM -1 s0-1
2.8 µM -1 s -1
M
Figure 4:
15
40
K
mantATP,
µM
40
60
C
M
10
-1
Binding Rate, s - 1
SO
Rosenfeld SS and Taylor EW. The ATPase mechanism of skeletal and smooth muscle acto-sufbragment
1. J. Biol. Chem. 259:11908-19 (1984).
Nucleotide binding
monitored by
fluorescence
Myosin (2 uM) Wilson
-2
01
8
DP, Sutherland C, Walsh MP. Ca2+ activation of smooth muscle contraction: evidence for the
0
5
10
15
binding
and (B) actin-stimulated mantADPActin (12 uM) involvement of calmodulin that is bound to the triton insoluble fraction even in the absence of Ca2+. J.
Effects of CK-2018571
on (A)
mantATP
mantATP, µM
(2 uM)
Biol.Mant-ATP
Chem.
277:2186-92 (2002).
+/-CK-2018571Myosin
release. CK-20187571 was present at saturating (6 µM) concentrations.
Myosin
(2
uM)
Actin
(12
uM)
Myosin
(2
uM)
20
Mant-ATP
Actin (12 uM)
Actin (12 uM)
+/-CK-2018571
Mant-ATP
Mant-ATP
D
0
K
30
C
40
-Stimulated
ease Rate (s-1)
timulated
ase Rate (s-1)
s-1)
5
D
0
-1
M
D
-1
-1
D SO
C
M
57 Actin-Stimulated
K
1
SO
-1 -201
ADP Release Rate (s ) 8 5
-1
0
30
Actin-Stimulated
ADP Release Rate (s-1)
SO
Actin-Stimulated
Actin-Stimulated
ADP Release
(s-1) -1
D Rate
ADP
Release
M Rate (s )
Actin-Stimulated
ADP Release Rate (s-1)
Binding Rate, s - 1
Binding Rate, s - 1
Binding Rate, s - 1
K
C
Actin-Stimulated
ADP Release Rate (s-1)
Binding Rate, s - 1
-2
01
8
K
C
-2
01
8
M
D
(B) The myosin chemomechanical cycle, indicating weak and strong actin
STRONG
binding states.
-1
-1
-1
57
1
SO
BINDING
and various nucletoides.
Total amounts of myosin (3 µM) and actin (5 µM) are
indicated by the loading controls (left), while unboundWEAK
myosin is shown in the
STRONG
binding reaction supernatant fractions (right). Hexokinase is included in the no
ACTIN
nucleotide and ADP reactions to deplete residual ATP. BINDING
Binding Rate, s
57
1
SO
M
D
Binding Rate, s - 1
-1
-1
-1
(% of
maximum tension)
Force
(% of maximum tension)
Force
(% of maximum tension)
(% of maximum tension)
Force
Force
(% of
maximum tension)
Force
tin
Bl
eb
b i
st
a
(D)
-1
-1
40
0
M
-4
-1
-1
20
D
-5
60
40
20
Phosphate Produced
1
(mol
/ molActin-Stimulated
Binding
Rate,
s -myosin)
-6
(C)
80
60
60
-1
BindingBinding
Rate, s - 1 Rate, s
-7
-1
Force
(% of initial tension)
-1
-1
-1
Actin-Stimulated
ADP Release Rate (s-1)
Thiophosphorylation Assay: Triton-permeabilized preparations were
incubated in rigor solution containing ATP-γ-S (1 mM) for 10 minutes
CK-2018571 was added 15 minutes before addition of ATP. ATP-induced
contraction was measured for 60 minutes and the relaxation was
expressed as percentage of the maximum force.
-1
Force Force
(% of initial
tension)
(% of
initial tension)
-1
(B) (B)(C) (C)(D)(D)
(A) (A)
-1
0
-1
Force
(% ofForce
initial tension)
(% of initial tension)
-1
Binding Rate, s - 1
-8
Phosphate Produced
(mol / mol myosin)
-9
-1
-1
40
-10
Actin-Stimulated
ADP Release Rate (s-1)
-1
Actin-Stimulated
ADP Release Rate (s-1)Phosphate
Phosphate
(mol / mol myosin)
-1
Force
(% of initial tension)
-1
Normalized ATPase
Activity
-1
in
-1
Force
Force
(% of maximum tension)
-4
Force
(% of initial tension)
-5
Force
(% of initial tension)
-6
-1
(% of initial tension)
-7
-1
(%(%ofForce
tension)
ofmaximum
maximum
tension)
Force
(% of maximum tension)
-4
Force
(% Force
ofForce
initial tension)
tension)
(% of initial
tension)
-5
Force
(% of initial
(% of maximum tension)
-6
Bl
eb
b i
st
a
Binding Rate, s - 1
-1
-4
Force
(% of initial
Force tension)
-8
-5
Force
(% of maximum tension)
-7
-4 -4
Force
(% of initial tension)
-9
-6
Force
(% of initial tension)
-8
-5 -5
tin
57
1
-10
-7
Force
Force
(% of maximum tension) (% of initial tension)
-9
-6 -6
Binding Rate, s - 1
-8
-1
-7 -7
-1
-9
-1
-8-8
-1
-9-9
-10
Phosphate Produced
Force
(µM) Binding
Rate, s
Phosphate Produced
(% of initial tension)
Normalized ATPase
Activity
-4
Force
(%
of
maximum
tension)
Produced
-5
Normalized ATPase
Activity
-6
-1
tin
-7
Normalized ATPase
Activity
CK
-2
0
Normalized ATPase
ATPase
Normalized
Activity
Activity
Bl
eb
b i
st
a
57
1
18
M
SO
Activity
-8
-10
CK
-2
0
D
M
-9
Phosphate Produced
(mol / mol myosin)
Normalized ATPase
Activity
M
-10
-10
-10
18
M
SO
Phosphate Produced
(µM)
Normalized ATPase
Activity
Normalized ATPase
Activity
1.0
±Compound
(10 µM)
0.8
Smooth
muscle myosin is a mechanochemical enzyme that hydrolyzes
60
Selectively Inhibits The ATPase
51.0 CK-2018571
0.4
Acid
ATP
0.10
10
AcidMyosin (30 µM) Myosin
Cardiac
ATP
50(30 µM)
2600 ± 240
10
CK-2018571
Slows
ATP
Hydrolysis
…
CK-2018571
Relaxes
Smooth Muscle Tissue in vitro …
0.6
ATP0.2to generate mechanical force; ultimately all signaling pathways
-±Compound
(10
µM)
0.8
myosin
60
+
+
+
+
+
+
+
±Compound
(10 µM)
t=5s
0.8
Activity
of
Smooth
Muscle
Myosin
60
40
5
0.4
Acid
Smooth
Cardiac
25
Smooth
Cardiac
0.2 20
that0.0modulate smooth muscle tone converge
on the regulation
Cardiac
Skeletal
11300 ± of
840this
±
2600
240
Acid
100
100
Non-muscle
IIB
0.6
DMSODMSO
( n=8)( n=8)
+
+Fast+Skeletal
+Total+phosphate
+ Non-muscle
+determination 76
actin
0.2 -IIB± 4.9 76 ± 4.9
00.6
Non-muscle
motor protein. We used a high throughput screen
to identify Fast Skeletal
Non-muscle
0
t=5s
15
15
20
Skeletal
11300
±
840
CK-2018571
(1µM,
n=2)
using malachite
green
0.0
Total phosphate determination
40
t=5s
5
0.4
CK-2018571
(1µM,
n=2)
Smooth
Cardiac
-- Cardiac
hexokinase
Total phosphate determination
compounds
0 SD, nM 0
-- Cardiac
-- Smooth
-9
-8
Smooth
40
100
+
+ supernatants
+ Cardiac
+Cardiac
5
0.4 1.2
±
2600
240
0.0
DMSO
(
n=8)
Myosin
Isoform
IC
±
Smooth -- Binding
10inhibit
10 -7 the
10 -6 ATPase
10 -5 10 -4 activity of smooth muscle myosin;
10 -10 10that
using
malachite
green
-25
100
100
DMSO
(n=12)
reaction
( n=8) DMSO
( n=8) 80
50 2600 ± 240
100 DMSODMSO
Non-muscle
FastSkeletal
Skeletal
Myosin IsoformNon-muscle
ICNon-muscle
nM
using malachite green
CK-2018571 (0.5µM, n=2)
1.2resulted in compounds
(80
n=8)(1µM,
Smooth 0.2
Cardiac
Fast
FastCardiac
Skeletal
200 15 15
0
100
30015 (D)
(B)
(C)
(A)
50 ± SD,
Loading Non-muscle
Controls 10
optimization of
the initial
hit compounds has
0
CK-2018571
n=2)
[ CK-2018571],
mol/L
CK-2018571
(0.5µM, n=2)
Fast
Skeletal
100
10
10 (free
10 fraction)
10
10
10
CK-2018571
(1µM,
n=2)
CK-2018571
(1µM,
n=2)
DMSO
( n=8)
15
1.0
nucleotideSmooth
ADP ATP
ADP ATP Cardiac Myosin
CK-2018571
(5
µM, n=8)
0.2 Fast
Myosin
Isoform
IC±50SD,
± IC
SD,
nM
Cardiac
-50
----1.2
msec
Time,
CK-2018571
(1µM,
n=2)
Smooth
Isoform
IC
nM
80
Myosin
Isoform
±
SD,
nM
1.2
1.2
Myosin
(30
µM)
ATP
CK-2018571
(0.5µM,
n=2)
Non-muscle
Skeletal
[
CK-2018571],
mol/L
50
80
100
100
-20
50
20 ( n=8)
80 ( n=8)
with nanomolar potency. A potent representative
of this chemical
Skeletal
±9.0
840± 4.8
1.0
CK-2018571
(0.5µM,
n=2)
DMSO
10
DMSO
Smooth
CK-2018571
(0.5µM,
n=2)
15 ± 840
0.0
Isoform
Myosin
IC 5011300
± SD, nM
1.2
Non-muscle
Fast Skeletal
1.0
80
ATP
Myosin
(30
µM)
Non-muscle
Fast Skeletal
15
CK-2018571
(1µM,
n=2)
1.0
±Compound
(10determination
µM) CK-2018571
20
0.8
Skeletal
11300
CK-2018571
(0.5µM,
n=2)
-20
1.0
myosin
60
Myosin
(30
µM)
ATP
15
10
0
4.8
Smooth
9.0
±
(1µM,
n=2)
Total
phosphate
Myosin
(30
µM)
ATP
centrifugation
10
0.0
4.8
Smooth
9.0
±
series, CK-2018571, inhibits the steady-state ATPase activity of human
ATP
Myosin
(30
µM)
-- -- Myosin
+0.8 + Isoform
+1.2 + +IC
+ ± SD,Cardiac
10
Smooth
9.0 ± 4.8
CK-2018571
(1µM,
n=2)
10 0
Smooth
9.0
± 4.8
0
10
20
30
Myosin
Isoform
IC
±
nM
SD,
Acid
±Compound
(10
µM)
1.0
Smooth
nM
60
(10
µM)
±Compound
±Compound
(10
µM)
50
0.8
0.8
80
1.2
9
8
7
6
5
4
60Total
(10
µM)malachite
CK-2018571
(0.5µM,
n=2)
phosphate
determination
60
0.8
100green
60
50
80
ATP
Myosin (30 µM) ±Compound
n=8)
Myosin
Isoform
SD,± nM
Non-muscle
IIB
Acid
0.6 1.2
(0.5µM, n=2)
10
4.8
Smooth
9.0
±IC
Acidusing
9Acid
8
7 DMSO ( 6
5 CK-2018571
4 CK-2018571
50 ±76
smooth muscle myosin at approximately 10-fold lower concentrations
80
Acid
Non-muscle
FastNon-muscle
Skeletal
1.0
(0.5µM, n=2)
Non-muscle
IIB 4.9
Amplitude
Rate
15
0.6
76
± 4.9
Time,
min
hexokinase
IIB
(2.5 µM)
Myosin
125
76
± 4.9
(10
µM)
±Compound
0
ATP
Myosin
(30 µM)
0.8-9
2+
Non-muscle
IIB
-10
-8
-7
-6
-50.6
-4
CK-2018571
(1µM,
n=2)
0.6
60
76
4.9
±
10
4.8
Smooth
9.0
±
using
malachite
green
t=5s
10
10
10
10
10
10
10
2+
t=5s
mol/L
],
-log
[Ca
1.0
Non-muscle
IIB
t=5s
(10Acid
±Compound
µM)
0.6
76
0.8
Myosin Isoform
± SD, nM
1.2 ± 4.9
(mol
Pi /mol
Actin(8
µM)
than are required to inhibit non-muscle myosin,
the most closely
(s-1) IC 50DMSO
-log [Ca
DMSO
0.4 1.0
40 6080 40
0.4-6
0
5 55myosin)
0.4
CK-2018571
(0.5µM, n=2) 40
-9
-8
-7
-5
-4
ATP
Myosin
(30(30µM)
Myosin
µM)t=5s
40 ], mol/L
AcidATP
5
0.4
Non-muscle
IIB
Cardiac
0.6-10
10
76Cardiac
4.99.0
±Cardiac
0.6 actin
4.8
Smooth
±
10
±2600
2600
240
10
10
10
10
10
10
10
4.8
Smooth
9.0
±
± 0.6
2600
240
±
2600
240
100
Cardiac
mantADP(2
µM)
[
CK-2018571],
mol/L
Non-muscle
IIB
1.0
0.6
±
240
76
4.9
±
t=5s
CK-2018571
±Compound
(10
µM)
CK-2018571
0.8
related myosin II and has greater selectivity
versus
striated
muscle
ATP
Myosin
(30
µM)
0.2
0.2
t=5s
60
(10 µM)
±Compound
10
0.8
Smooth 16
± 4.8 0.53
t=5s
40 60
0.2
0.2
5Cardiac
0.4
DMSO
ATP (1(10
mM)
40
µM)
5 ±9.0
0.4
0.8 µM)
5
0.4 (15
[ CK-2018571],
Myosin
Myosin (15 µM)+/-CK-2018571
Acid
ATP ±Compound
ATP
20 4060 20
Skeletal
11300
± 840
Skeletal
11300
±2600
840
Cardiacmol/L
-20
Cardiac
75
240
Acid
0.0
±
2600
240
0.0
±
2600
240
Acid
20
Skeletal
11300
±
840
myosin IIs. Transient kinetic studies demonstrate that CK-2018571
Non-muscle
IIB
0.6
76
4.9
±
0.0 ±Compound
±Compound
80
20
(150
µM)
0 0
Skeletal
11300
± 840IIB
Non-muscle
Total
phosphate
determination
0.6
76
± 4.9
Total phosphate
determination
0.2 IIB
(150
µM)
DMSO
Non-muscle
DMSO
CK-2018571
0.0
0
0.6
76
4.9
±
0.2
Total
phosphate
determination
0.4
411300 ± 840
-20
CK-2018571
Smooth
Cardiac
t=5s
t=5s green
0.2 γ-phosphate group of
0.4
using
malachite
malachite
green
0
2040
inhibits the myosin-catalyzed hydrolysis of the
Skeletal
Total
phosphate
CK-2018571
5
50
0.4
40
0.0
Acidusing
using
malachite
green 1000 determination
5 0.10
0.4
( n=8)
t=5s
Cardiac 60
0 DMSO
20
Skeletal
11300
±
840
±
2600
240
Acid
0
10
10
10
10
10
10
10
Total
phosphate
determination
Cardiac
10
10
10
10
10
10
10
-0.0
0
Non-muscle
Fast
Skeletal
±
2600
240
9
8
7
6
5
4
myosin +
+ +
+ + 10[+
+ 10 ±
40 n=2)
10 0.2
10
515 0
20
Skeletal
11300
84010 10 10
using malachite green
with no effect
on nucleotide binding or 0.0
release from0.4
the enzyme.
ATP,
binding
Total Nucleotide
phosphate
determination
Smooth
Cardiac
using
malachite green
CK-2018571],
mol/L
CK-2018571
(1µM,
[
CK-2018571],
mol/L
Cardiac
0.2 -10
25
100
±
2600
240
0
020
Skeletal
11300 ±4840
-9
-8
-7
-4[ CK-2018571],
( n=8)
0.2
monitored
by greendetermination
10 0.0
10
10mol/L
10IC 10± SD,
10 nM
µM s
8
6
9
7
10
10 -6 10 -5 Myosin
40
0DMSO
using
malachite
Total
phosphate
-20
0
Non-muscle
Fast Skeletalassays indicate that CK-2018571
WEAK
50
-20 CK-2018571
Actin
co-sedimentation
Total phosphate
determination
80
-- 10
+ -810+ -7 10
+ -6 +
+ -4 + 10
+ 10 [Isoform
0.2 1.2 actin
(0.5µM, n=2)
20
-log [Ca2+ ], mol/L
Skeletal
11300
±
840
fluorescence
0.2stabilizes a15
-20
CK-2018571],
mol/L
0
-10
-9
-5
0.0
CK-2018571
(1µM,
n=2)
10
10
10
10
10 mol/L
10
10
DMSO
using malachite green 0
[
CK-2018571],
1.0
using
malachite
Total
phosphategreen
determination
-2090
Total phosphate
weak actin-binding conformation
of myosin
the
20
0.6 0
10
10
10
10
10
Isoformin IC
Myosin
±presence
SD, nM of ATP.
7
5
4
1.2
Myosin
(30 µM) determination
ATP
9 8
76
Skeletal
11300
± 840
50
[-CK-2018571],
mol/L--log [Ca2+ ], mol/L
8010
100.0
Smooth
9.0
± 104.8
-CK-2018571
(0.5µM, n=2) CK-2018571
0
2.8 µM s
9 8
820
7 6
6 5
5 4
4
-10
-9
-8
-7
-6 hexokinase
-5
-4 -0.0
+
+
+
+
using
malachite
green
-20
Binding
reaction
supernatants
ACTIN
DMSO
[
CK-2018571],
mol/L
using
malachite
green
Total
phosphate
determination
-25
10
10
10rat tail
10
10
100.8 10
±Compound
(10 µM)
2+
(n=12)
60DMSO
Consistent with this mechanism, CK-2018571 relaxes
skinned
0
100
200 0.6 300
], [Ca
mol/L
0 (B)
Loading
Controls
8 -log
7 -log
6 2+ mol/L
5
4
1.0
Total
phosphate
determination
-20
0 [Ca
-7
-6
2+
0
(C)
(D)
(A)
(free
fraction) green
DMSO
0.0
Myosin
-20 9
Acid (15 µM)
10 -10 10 -9 10 -8 BINDING
10
10(30
10 -5 10 -4using
malachite
ATP
Myosin
µM)
ATP
], mol/L
-log],[Ca
BindingSmooth
reaction supernatants
DMSO DMSO
10
0.6
Time,
msec
4.8
9.0
±
0
5
10
15
CK-2018571
2+
Non-muscle
IIB
0.6
76
4.9
±
[
CK-2018571],
mol/L
0.6
nucleotide
artery
muscle
tissue.
Importantly,
this
relaxation
occurs
regardless
of
0
100
200
300
ADP
ATP
ADP
ATP
CK-2018571
CK-2018571
(5 µ-log
M,[Ca
n=8)
-50 green
-- -- [ CK-2018571],
-], mol/L
Loading Controls
±Compound
(10 µM)
using
malachite
0.8
±Compound
CK-2018571
60
Figure 1:
DMSO
mol/L-mantATP, µM0.6
(150
µM)
(free fraction)
6
5
4
CK-2018571
t=5s(15 µM)
7
569
4 5 8
Myosin
myosin
0.6
Myosin
(1599 µM) 8 8 ATP
ATP
7 6
4 7
Acid
0
Myosin
(15
µM)
-10or by
-9
-8
-70.4
-6 Time,-5msec
-4
40
0.4
CK-2018571
5
ATP
whether
the
skinned
muscle
has
been
activated
by
calcium
Myosin
(15
µM)
10
10
10 centrifugation
10
10
10
10
-20
±Compound
Non-muscle IIB
ATP
2+
(150
µM)
0.6
76 ± 4.9
Cardiac
-20
STRONG
2600 ±of240
±Compound
[Ca
-log20
], mol/L
Myosin
(15
µM)
-- -- +
+ the
+ steady-state
+ t=5s
+ +Mg2+-ATPase activity
(150
±Compound
ATP µM)
smooth
CK-2018571 selectively
inhibits
±Compound
DMSO
Acid
(150
µM)
0
10
(150 µM)
myosin
-log [Ca2+ ], mol/L
Rate0.4 0.4 Amplitude
hexokinase
thiophosphorylation
of the myosin regulatory light chain, supporting [ CK-2018571],
0.6
-log
[Ca2+ ],30mol/L
0.2 mol/L
±Compound
(150
µM) 125
Acid
0.4
DMSO
40
5
DMSO
0.4
CK-2018571
0.4
muscle myosin. S1 fragments of recombinant human myosin IIs were assayed in
Acid
Pi /mol myosin)
(s-1) 0.6(mol0.4
0.6
± 240 by direct
2600tissue
Time, min
Myosin (2.5 µM)
Myosin (15 µM)
Skeletal
11300 ± 840
ATPAcid100 Acid 20
9 Acid
8
67
5 6
4
evidence that CK-2018571 relaxes Cardiac
smooth muscle
actin
0.0salt buffer in the presence
CK-2018571
CK-2018571
87
5
4
of
actin
and
250
µM
ATP.
low
-20 9
±Compound
0.2
Amplitude
Rate
hexokinase
(150 µM)
125
Actin(8
µM)
0DMSO 0.216 0.2 0.2
Total
phosphate
determination
0.53
0.4
Myosin
(15
µM)
2+
ATP
Myosin
(15
µM)
inhibition of activated smooth Skeletal
muscle myosin.
The
ability
of
-1
ATP
75
0.2
mantADP(2
µM)
(mol Pi /mol myosin)
20
(s )
11300 ± 840
0.2
-log [Ca ], mol/L 2+
Acid
using malachite
green
0.0
±Compound
40
DMSO
(150
µM)
DMSO
CK-2018571
100 CK-2018571
±Compound
0 µM)
4
ATPphosphate
(1 mM)
+/-CK-2018571 Total
determination
(150
0.10
], mol/L
CK-2018571actin
to relax intact tracheal smooth muscle and aortic ring 0
0.6
10 -6 10 -5 Total
10 -10 10 -9 10 -8 10 -7
10 -4phosphate determination
0.4
50
Total
phosphate
determination
0.2
DMSO
9
8 -log [Ca
7
6
5
4
0.0
0.4 CK-2018571
using
malachite
green
DMSO
0.2
Total phosphate
determination
Total
phosphate
determination
reaction supernatants 80
0.53
16 [ CK-2018571],
-- +
using
malachite
green+ Binding
myosin
Acid
+
+
+
+
+
Total
phosphate
determination
mol/L
0.0
DMSO
0
100
200
300
0.6
using
malachite
green
preparations
suggests
this
mechanism
may
prove
useful
in
diseases
of
75
Loading Controls
Binding
reaction
supernatants
0.0
0.0
30
Acid
0
using
malachite
green
Myosin
(15
µM)
-10
-9
-4
-8
-7
-6
-5
using
malachite
green
Binding
reaction
supernatants
(free
fraction)
25
Binding reaction supernatants
ATP
10
10
10
10
10
10
10
0 0 0 Time,
100100
CK-2018571
Loading Controls
300 300
msec
CK-2018571 0.0
100200200 200300
DMSO
CK-2018571
Controls
-20
using
malachite
green
Total phosphate determination
(free
fraction)
4
supernatants
-+ reaction
+ + Loading
+Loading
+ Controls
+ + (free fraction)
actin Binding
±Compound
CK-2018571
60
(free
fraction)
0.10
smooth
muscle hypercontractility,
such as hypertension
and asthma.
-log [Ca2+ ], mol/L
(150
µM)
Time,
msec
msec
Time,
[ CK-2018571],
mol/L
0.0
50
Promotes
Weak
Actin-Binding
SmoothCK-2018571
Cardiac
Time,
msec
0
using
malachite
green
DMSO
0.2
0
100
200
300
Binding
reaction
supernatants
Myosin
(15
µM)
myosin
0.4
Total
phosphate
determination
Loading Controls
ATP100
Nucleotide
binding
20
0
100
200
300
-- + +
DMSO ( n=8)
Controls
(free
fraction)
myosin +
0.6 4 µM
+ + + +
0.2
hexokinase --20 (free fraction)
monitored
by
--myosin
-- +Loading
Acid
Non-muscle
Fast
Skeletal
+ myosin
+myosin
+ -s
40
9 using 8
7
6
5
4
Time, msec
0.0
±Compound
Time,
msec
-25
15
CK-2018571
(n=12)
DMSO
WEAK
(150
µM)
malachite
25
Binding reaction supernatants
Rate(D) Amplitude
hexokinase
fluorescence
CK-2018571
(1µM,
n=2) green
125
(B)
(C)
(A)
Total
phosphate
determination
myosin ± SD, nM
0.4 20
Amplitude
200
0 RateRate
100
300
hexokinase
-1 Rate
125 125 (5 µ M, n=8)
10
+
+ +
+ + + +
Amplitude
actin -hexokinase
myosin)
P /mol
Myosin (15
µM)
Loading
Controls
nucleotide
ADP (free
ATP IC-1.2
-50
-- Myosin
-- actin
--Isoform
ATP CK-2018571
Amplitude
-log
[Ca2+ ], mol/L
hexokinase
50 ADP ATP
125
80
9
5(s ) (s-1)(s(mol
-1)4 i (mol P /mol
fraction)
myosin
0.0 7DMSO0.2 6
CK-2018571
(0.5µM,
n=2)
Figure
5:
myosin)
100determination
08
using malachite green
Binding
reaction
supernatants
/mol
myosin)
(mol
P
i
Total
phosphate
Acid
-1
2.8
s
µM
i (mol
myosin)±Compound
Pi /mol msec
(s ) 0.53
100
Time,
ACTINControls
0.6
Rate
Amplitude
hexokinase
0 CK-2018571
200
300
125100
actin actin actin
(150 µM)
1.0
DMSO
Loading
16
2+ 100
centrifugation
100 20
hexokinase ATERIALS
-- -- Smooth
+(free+fraction)
+ +9.0
+
+
0.0DMSODMSO
--(300.53
ATP
2+
0[Ca ], mol/L
75 10green
+ AND
+ -- +ETHODS
+ -16 Myosin
using
0-25
/mol myosin)
(mol
PµM)
0malachite
30
-log
(s-1)
10
Binding
reaction
supernatants
4.8
±
0.4
i
DMSO
(n=12)
Time,
msec
0.53
16
BINDING
DMSO
75
actin
0
5
10
15 DMSO 100 16
100 75 CK-2018571 inhibits Ca -dependent force production in skinned caudal artery
Myosin
µM)
DMSO
CK-2018571
0.53 (15
±Compound
(10
µM)
0
200
300
ATP
4
60
CK-2018571
(B)
(C)
(D)
(A)
Amplitude
Rate
0.6 0.8 hexokinase
Loading
Controls
0.10
myosin
125
DMSO
CK-2018571
Total phosphate determination
75minfrom Sprague Dawley
Myosin (2.5 µM)
Acid50 50 Time,
4 16
(free fraction)
CK-2018571
DMSO
mantATP, µM
0.10
rings
rats.
0.53
CK-2018571
Acid
±Compound
(150 µM)
myosin
nucleotide -CK-2018571
-- 76
75
(5 µ M,
n=8)
CK-2018571
0.2
0.10
-- -- ADP ATP -- ADP ATP
myosin
-1) 4
Actin(8
µM)
Time,
msec
+reaction
++ ±DMSO
+
++DMSO+
+ CK-2018571
+
+ +-50 +0.4 CK-2018571
0.0
4
Non-muscle
IIB
CK-2018571
(mol
P
/mol
myosin)
0.6
4.9
using
malachite
green
-0.10
50
(s
myosin
Binding
supernatants
+
+
Myosin
(15
µM)
DMSO
CK-2018571
ATP
50
i
STRONG
25
4
mantADP(2
µM)
CK-2018571
0.10
Biochemical Assays:
myosinmyosin
0
100
200
300
+ -++ ++ ++ ++ +
t=5s
2550
actin
Loading Controls
100
Acid
+ +--+-- +++++ -±Compound
--(free
+fraction)
+
+
+
+ 5+ ++ +
µM)
40
actin(150
+/-CK-2018571Amplitude
ATP (1 mM)
0.4
myosin
Rate
25
centrifugation -myosin
hexokinase
+
+
+
+
+
+
actin
Rate
Amplitude
125
-- + +
+ + + +
hexokinase
125
25
Cardiac
0
Time,
msec
0.4
0
10
20
30
±
2600
240
0.2
0
25
DMSO
80
-+
+
+
+
+
+
+
0.53
16
actin actin Acid
-1
DMSO
-1
-+
+
+
+
+
+
+
/mol
myosin)
(mol
P
)
phosphate
myosin) determination
(molTotal
Pi /mol
0.2
hexokinase hexokinase
(s )
--- actin
-+
+ +
+0.2+ Figure CK-2018571
-0
3: (s Time,
+
+
+
75
---- -+ +
+ +
+
+ -0(n=12)
-25 -25
Assays were performed in low salt PM12 buffer (12 mM K-Pipes, 2 mM
min i
Myosin (2.5 µM)
actin
myosin
100
0 DMSO
DMSO
(n=12)
60
actin
(B)
(C)
(D)
(A)
Amplitude
Rate
100
hexokinase
0.0
hexokinase
-20
125
Skeletal
11300
±
840
(B)
(C)
(D)
(A)
---+
+
+
+
DMSO
CK-2018571
using
malachite
green
0.0
-hexokinase
Actin(8
µM)
Binding
reaction
supernatants
---hexokinase
--25
(n=12)
DMSO
++ ATP
-- +ATP
-- + ADP
-4chemical
Nucleotide
binding
DMSO
+ ADP
+ +
+
CK-2018571
nucleotide nucleotide
0.53
16slows
ADP
ATP
µM,
(5
0.10
MgCl2, pH 6.8).
-50 -50
--- -- -ATP
ADP
-25 (n=12)
CK-2018571
(5
µ
M,n=8)
n=8)
DMSO
(n=12)
-25 CK-2018571
----by
smooth
muscle
myosin.
CK-2018571
the
cleavage
of
ATP
DMSO
-1)
40
0
Total
phosphate
determination
Total
phosphate
determination
(B)
(C)
(D)
(A)
(mol
P
/mol
myosin)
0.2
(s
0
100
200
300
monitored
by
75
50
µM)
4 µM s
DMSO
40
(B)Amplitude
(C)
(A) (B)
(C)
(D)
(A)
0.53
16
Loading Controls
Independent
of Calcium Activation
i (D)
Total
phosphate
determination
WEAK mantADP(2
Rate
hexokinase
nucleotide
125
ADP -ATP
ADPATP
ATP
n=8)
µM,100
(free
fraction)
fluorescence
---- +
--CK-2018571
---actin
nucleotide
using malachite
green
-ADP
ADP
ATP
CK-2018571
(5 µ M,(5n=8)
DMSO+
nucleotide
-50-50
-- --- 0.0
-ADP+
ATP
ADP
ATP
75
CK-2018571
(5
µ
M,
n=8)
centrifugation
myosin
Myosin
(2 uM)
-50CK-2018571
centrifugation
--ATP
(1
mM)
+/-CK-2018571
+
+
+
+
+
--+
+
+
using
malachite
green
--+
+
+
+
+
4
0.0
CK-2018571
Binding
reaction
supernatants
0.10
0
10
20
30
using
malachite
green
0
10
20
30
-1
Cardiac
Time,
msec
(A)
Measurement
of) phosphate
burst
magnitude under single turnover
0
Binding Smooth
reaction supernatants
-10
-9
-8
-7
-6
-5
-4
/mol
myosin)
(mol P
20
(s
50
10
10
10
10
10
10
10
100
30
i
Actin
(12
uM)
DMSO
(
n=8)
DMSO
0
100
200
300
0
100
200
300
0.53
16
DMSO
25
Steady-state
ATPase
activity
was
measured
using
a Loading Controls
80
phosphate
2.8 µM s
centrifugation
Loading
actinControls
--determination
----+ Skeletal
+--CK-2018571
100
Time,min
min
DMSO
Myosin
(2.5 µM)
4
Time,
Non-muscle
Myosin
(2.5 µM)
myosin
centrifugation
(free
fraction)
+ -+TotalACTIN
+
+ centrifugation
+ Fast
+ --++ --++ + ++ + +++ +++ ++ + conditions.
0 0
20
2075 3020 30
0.10
fraction)
15CK-2018571
Mant-ATP
[ CK-2018571],
mol/L (free
010 10 (1µM,
10 n=2)
30
+/-CK-2018571
CK-2018571
0
Time,
msec
using+malachite
green
50
Actin(8
µM)
Actin(8
µM)
Binding that
reactioncouples
supernatants
-+
+
+
+
+
+
actin
CK-2018571
myosin to the0.0 0
25
BINDING
Time,
msec
spectrophotometric assay
ADP production
5
10
0.53
16 15 4
Time,Time,
min min
Myosin
(2.5
µM)
DMSO
CK-2018571Myosin Isoform 20
Myosin
(2.5
µM)
IC 50 ± SD,0 nM DMSO
60
100
200myosin
300
1.2
µM)
mantADP(2
mantADP(2
µM)
-Loading Controls
80
Time,
min
-20
Myosin
(2.5
µM)
CK-2018571
CK-2018571
(0.5µM,
n=2)
75
0
0.10
mantATP,
µM
+
+
+
+
+
+
+
Actin(8
(B)
Determination
of
the
ATP
hydrolysis
rate
using
rapid
mixing
quench
flow.
µM)
(free fraction)
-+
+
+
+
+
+
+
actin
myosin
Actin(8 µM)
+/-CK-2018571
ATP
(1 mM)
binding
50
ATP
mM)
+/-CK-2018571
oxidation of NADH using pyruvate kinase and lactate dehydrogenase
Actin(8
Time, msec
1.0
µM)(1 µM)
mantADP(2
0
DMSO Nucleotide
CK-2018571
myosin
mantADP(2
µM)
25
Myosin (30
µM)
ATP ATP
STRONG
4
monitored
by+
-CK-201857110 DMSO
0.10
s
4
µM
40
4.8
Smooth
9.0
±
myosin
hexokinase
-mantADP(2
µM)
DMSO
+
+
+
+
+
+
+/-CK-2018571
(1
mM)
---+
+
+
+
WEAK rates were measured
50125
10
±Compound
(10 µM)
860
6
9binding
7
5 -25
4 DMSO (n=12)
while
regenerating ATP. ATPase
in the presence
ATP (1 mM)
+/-CK-2018571
0.8+
Amplitude
Rate
hexokinase
fluorescence
hexokinase
-myosin
Nucleotide
---CK-2018571
ATP
(1
mM)
+/-CK-2018571
+
+
+
Amplitude
Rate
25
CK-2018571 DMSO
+
hexokinaseactin
125 -25
Acid
myosin-- + +-- +
DMSO (n=12)
+ ++ + + + ++ ++ Non-muscle
80
monitored
by
IIB
DMSO
20
-1) (s -1)(mol
2+
0.6
76 ±(s
4.9
80
Nucleotide binding
(B)
(C)
(D)
(A)
CK-2018571
of actin and 250 µM ATP (>5-10-fold above the KM,ATP). Hydrolysis
rates
/mol
myosin)
P
0
DMSO
(mol
P
/mol
myosin)
Nucleotide
binding
+
+
+
+
+
+
+
-25
actin
],
mol/L
-log
[Ca
fluorescence
Amplitude
Rate
i
monitored by
i
2.8 µM -s
hexokinase
t=5s
CK-2018571
125
4 µM s
0
nucleotide
DMSO
ADPADP
ATP
ATPWEAK
ACTIN
CK-2018571
(5 µ M, n=8)
nucleotide
actin
-50
--monitored
by
ATP ---- ADP
ADP
ATP
CK-2018571
CK-2018571
(5
µ
M,
n=8)
Amplitude
100
Rate
hexokinase
Nucleotide
binding
4
µM
s
-50
--40
-60
125
actin
5
0.4
0
100
fluorescence
0.6
WEAK
+
+
+
+
+
+
60
were normalized using reactions containing an equivalent amount of
actin-- -- --+
Cardiac
monitored
by
fluorescence
40
±
2600
240
-1
0-1
Without
Significantly
Slowing
4
µM
s
hexokinase
-Nucleotide
binding
-CK-2018571
(mol
P
/mol
myosin)
)
(s
+ -+ +WEAK
(mol5 Pi /mol myosin)
BINDING
(s 0)
0.53i4 µM s
Nucleotide
binding
0
10
fluorescence
-25
monitored
by
DMSO (n=12)
-- 100DMSO DMSO16 16
hexokinase
40
µM s
--15 --+ 0.2
--++ +
75
actin
0.53
DMSO.
monitored
by
+
+
+
+
ACTIN
actin
centrifugation
WEAK
4 µM2.8
100 -25
40
ATP
-+
+
+
+
+
2.8 Myosin
µM
s s (15 µM)
mantATP,
µM
centrifugation
fluorescence
DMSO
(n=12)
WEAK
ACTIN
--+
+
+
+
20
0
10
20
30
Skeletal
11300
±
840
hexokinase
-30
fluorescence
ATP
Binding
or
ADP
Release
---75
0.0
DMSO
CK-2018571
2.8
µM
s
+
+
+
+
0
10
20
30
BINDING
ACTIN
±Compound
4
DMSO
(150 µM) Total phosphate determination
CK-2018571
0.53
16
-25
0.10
DMSO (n=12)
0
20
BINDING
DMSO
0.53
16
20
50
STRONG
nucleotide
ADP
ATP
ADP
ATP
75
mantATP,
µM s
0.4
CK-2018571
(5 µ M, n=8)
-50
----Time,
min
2.8
µM
BINDING
Myosin
(2.5
µM)
DMSO
CK-2018571
ACTIN
nucleotide
ATP
ADP ATP CK-2018571
using malachite green
2.8 µM s
CK-2018571 (5Time,
µM, n=8)
---- + -- + -- + ADP
-50
-- ADP
mantATP, µM
4 20
75CK-2018571
ACTIN
myosin
+
+
+
+
0.10
min
Myosin binding was measured
by CK-2018571
depletion of soluble myosin from
Acid
Myosin
(2.5
µM)
DMSO
mantATP, µM
0
STRONG
0
nucleotide
Actin(8
µM)
4
ADP
ATP
ATP
(5
µ
M,
n=8)
CK-2018571
-50
----0.10
10
10
10
10
10
10
10
50
BINDING
0
0
5
10
15
25
STRONG
BINDING
DMSO
CK-2018571
0
5
10
15
STRONG 50
mantADP(2 µM)
Actin(8 µM)
4
CK-2018571 10
binding
S1 fragment
(chicken,
[ CK-2018571],
mol/L
mantATP,
µM
0.10
myosin
myosinreactions
+ -- +centrifugation
+
+
+
+
+
-+
+
+
+
+
+
+
mantATP,
µM
+ -- using
+ smooth
+
+ muscle
+ +myosin
+
actin
--+
+
+
+
+
+
centrifugation
50
+/-CK-2018571
ATP (1mantADP(2
mM)
0
1030
20
centrifugation ---- -µM)0 -20
-- +
+ ++ ++ STRONG
++ STRONG
+ + + + 25 0.2
recombinant) and 5 µM bovine cardiac actin. ATP and ADP were
0
100
2010
30 20
25
-myosin
+
+
+
+
+
+
+
80
40 hexokinase
0
ATP
(1
mM)
+/-CK-2018571
DMSO
40
-+
+
+
+
+
+
+
actin
Myosin (2 uM)
Time, min
(2.5 µM)
Time, min
(2.5(n=12)
included at 1 mM where indicated. Reactions were actin
allowed --to
-25 Myosin
+
+ +
+ --+ -- + + ++ -- + + -9
8µM)
725
6
5Time, 4
DMSO
min
Myosin (2.5 Myosin
µM)
0
40
40
CK-2018571
Total phosphate determination
Actin (12 uM)
(B)
(C)
(D)
(A)
80
60
Actin(8
µM)
0
30
2+
DMSO
Actin(8
µM)
Actin(8
µM)
0.0
equilibrate --for -15 minutes
(540k x g, 30actin -hexokinase
-+ +-- Binding
+ --reaction
+ ADP+supernatants
30+nucleotide
-- +to +centrifugation
using malachite green
[Ca
], mol/L
+ + prior
+/-CK-2018571
ATP +-- ADP ATP
CK-2018571-log
(5 µ
M, n=8)
Nucleotide
binding Mant-ATP
-50
-DMSO 30
-25
mantADP(2
µM)
30 CK-2018571
40
mantADP(2
µM)
200 4 µM
0DMSO (n=12)
100
300
0.6
Loading
Controls
0
mantADP(2
µM)
40
actin
monitored
by
(B)
(C)
(D)
(A)
s
40
(free
minutes), however ATP was added just prior tohexokinase
centrifugation --to
--fraction)
20
CK-2018571
-60
+ centrifugation
+ -- --+ -- + WEAK
+/-CK-2018571
ATP (1 mM) ATP (1 mM)
+/-CK-2018571
Time,
-25ATP (1
20
nucleotide -DMSO
(n=12)
ADP ATP
ADP ATP
20
CK-2018571
(5msec
µ
M, n=8) 20 30
-50
-Myosinfluorescence
(15 myosin
µM)
--+/-CK-2018571
+
+
+
+
+
+
ATP
-hexokinase
Nucleotide binding
0
10
20 mM)
30
---minimize hydrolysis. Supernatants were analyzed by SDS-PAGE
+
+
+
+
80
80 DMSO 10 30 -1 -1
±Compound
20
(150 µM)
-25
DMSO
(n=12)
myosin
DMSO
monitored by
10
s DMSO
0.4
4 80
µM
40
2.8 µM
s
Time,
min
centrifugation
followed by--staining
with+Coomassie
Myosin (2.5 µM) Nucleotide binding
10
-- +
+ + brilliant
+ + blue.nucleotide
ADPACTIN
ATP
CK-2018571
CK-2018571
(5 µ M, n=8)
20
-50
-- -- -- 10 ADP ATP -- WEAK
CK-2018571
Acid
monitored by
0
10 0
20
30 0 20
fluorescence
60
60
Actin(8 µM) Myosin fluorescence
(2 uM)
nucleotide hexokinase
0
ATP
CK-2018571
(5 µ M, n=8)
0
5
10
15 CK-2018571
Amplitude
Rate
-50
-- ADP
-- -- -- ADP ATP BINDING
125Nucleotide binding
Time, min
20
0
Myosin (2.5 µM)
mantADP(2 µM)
Actin (12 uM)Nucleotide binding
10µM 60-1 -1
mantATP,
-1)
-1
-1
0.2
0
/mol
myosin)
(mol
P
10
(s
monitored
by
-1
4 µM s4 µM2.8
Stopped-flow (Hi-Tech SF61DX2) and quench-flow
(Biologic SFM/400)
40i
centrifugation
+/-CK-2018571
Actin(8 µM)
ATP (1 mM)Mant-ATP
+/-CK-2018571
s -1µM s
-- -- +actin +
+ + ACTIN
+STRONG
+WEAK
40
WEAK
100 monitored by Nucleotide binding
fluorescence
0
10
20
30
mantADP(2
µM)
centrifugation
-1
-1
fluorescence
-- -- + + BINDING
+ + + +
80
0
experiments were performed at 25°C using smooth muscle myosin S1
monitored by
s
4
µM
Total phosphate determination
DMSO 200 0.53
DMSO
40 0
16
0
10
20
30
WEAK
ATP (1 mM)
+/-CK-2018571
0
5
10
15
20
using(2.5
malachite
green75
fluorescence
CK-2018571 0.0
Binding reaction supernatants
Time,
min
2.8 µM -1 s -1
Myosin
µM)
fragment (recombinant, chicken). Mant-ATP binding and Mant-ADP 80
-1
-1
ACTIN
60
0
100
200
300 s
Nucleotide binding
mantATP,
µM
DMSO
CK-2018571
2.8 µM
Loading Controls
4
DMSO
ACTIN
Time, min
(free fraction)CK-2018571
Myosin
monitored
by µM)
0 0.10
20 Time, msec
Nucleotide
binding
50
Actin(8
µM)(2.5
release were monitored using fluorescence (λex=360 nm and
BINDING
actin
0
5
10
15
CK-2018571
-1
-1
0
fluorescence Myosin (2 uM)
-monitored
by
STRONG
Actin(8
µM)Myosin (2 uM)
4 µM s
myosin +
2.8
s
40
60
+ WEAK
+
+BINDING
+myosin
+ +
0
5
10
15 µM
ACTIN
mantADP(2
µM)
40
mantATP,
µM
Actin (12 uM)
λem=400 nm). ATP hydrolysis was monitored using malachite green to
fluorescence
Figure 6:
25(12 uM) Mant-ATP
Nucleotide binding
Actin
mantADP(2
µM)
mantATP,
µM
+/-CK-2018571
Myosin
(2 uM)
0
Mant-ATP
myosin
+/-CK-2018571
monitored
by
+/-CK-2018571
ATP
(1
mM)
20
s
4
µM
40
STRONG
-+
+
+
+
+
+
+
actin
BINDING
quantify total acid-labile
Actin (12 uM)
0
5
10
15
Amplitude
WEAK phosphate.
hexokinasefluorescence
125
+/-CK-2018571
(1 mM)
2.8 µM s Rate
30
0 ATP
CK-2018571 relaxes skinned, thiophosphorylated caudal artery rings from
ACTINSTRONG
Mant-ATP
+/-CK-2018571
myosin)
(mol Pi /mol
(s-1)
80
mantATP,
µM
Myosin
(2
uM)
0
20
DMSO
actin
100
Myosin
(2
uM)
-hexokinase2.8 µM
Sprague Dawley rats. Force development is expressed as a percentage of the
80
-- s -Nucleotide
binding
+ BINDING
+ -- + +
Myosin
(2 uM)
0
5
10
15
DMSO
Nucleotide
binding
-25
DMSO
(n=12)
Actin (12 uM)
monitored
by
Actin
(12
uM) (12
ACTIN
DMSO
0.53
16
20
Tissue Assays:
monitored
by
Actin
uM)
mantATP,
µM
initial force (n=8-12, mean +/- sem).
(B) CK-2018571
(C)
(A) CK-2018571
STRONG
75Mant-ATP
fluorescence Mant-ATP
Mant-ATP
40(D)
+/-CK-2018571
0
+/-CK-2018571
fluorescence
60
+/-CK-2018571
Nucleotide
binding
BINDING
nucleotide
DMSO
CK-2018571
0
5
10 -ADP ATP
CK-2018571
(5 µ M, n=8)
-50
-- ADP ATP
--15 -- STRONG
60
4
CK-2018571
0.10
monitored by
40
50
Nucleotide
binding
mantATP, µM
10
fluorescence
-- + +
Nucleotide
binding
myosin +
Skinned Ring Assay: Endothelium-denuded rat tail artery segments
+ + + +
actin
monitored
byNucleotide
centrifugation --1
-1 40
+ +
4 µM -1 s4-1µM30
40
STRONG
monitored
by
0 Nucleotide
10
20
30
binding25
s
40
WEAK WEAK -- + + + + actin
binding
were cut into 3-mm helical rings, mounted on an isometric force
myosin
30
-+
+ +
+Nucleotide
+ + binding
+
fluorescence
monitored
by
0
monitored
by
fluorescence
0
monitored by
Time, min
fluorescence
Myosin (2.5 µM)
fluorescence
transducer with a resting tension of 0.5 g, and incubated for
EFERENCES
30
hexokinase -20
-Actin(8
µM)
40
20
+ + -- fluorescence
+ + 20-40
-1
-1
-25
20
DMSO (n=12)
-1
-1
2.8
µM
s
30 minutes at room temperature in normal H-T buffer. Tissues were
(B)
(C)
(D)
(A)
mantADP(2
µM)
2.8 µM s
ACTIN ACTIN
nucleotide -CK-2018571 (5 µ M, n=8)
-50
-- -- ADP ATP -- ADP ATP
40
ATP (1 mM)
+/-CK-2018571
20
skinned by incubation with skinning solution containing 1% Triton
30
0
0
10
10
BINDINGBINDING
30
0
5
10
15
80
0
5
10
15
DMSO
Kovacs
M, Toth
J, Hetenyi
C, Malnasi-Csizmadia
A, Sellers J. Mechanism of blebbistatin Inhibition of
centrifugation --- + +
+ + + +
X-100 for 1 hour at room temperature. CK-2018571 was added to the
Figure 2:
0
10
20
30
30
mantATP,
µM 10µM
CK-2018571
mantATP,
20
myosin
II.
J.
Biol.
Chem.
279:35557
(2004).
60
Time, min
tissue for 15 minutes, followed by addition of solutions with increasing
Myosin (2.5 µM)
00
CK-2018571 inhibits smooth muscle myosin in a weak actin-binding state.
Nucleotide binding Actin(8 µM)
20
STRONG
STRONG
20
calcium. The force generated at the plateau of each corresponding pCa
monitored by
10 s
4 µM
mantADP(2 µM)
40
Ramamurthy B, Yengo C, Straight A, Mitchison T, Sweeney HL. Kinetic mechanism of blebbistatin
Myosin (2 uM)
WEAK
0
fluorescence
(A) Characterization
of myosin affinity by actin cosedimentation. Strong
+/-CK-2018571
ATP (1 mM)
condition was recorded, and data were presented as a percent change
Actin (12 uM)
inhibition of nonmuscle myosin IIB. Biochemistry 43:14832 (2004).
10
80
actomyosin binding is revealed by depletion of soluble smooth muscle myosin 20
10
DMSO
0
Mant-ATP
+/-CK-2018571
from the baseline values (Wilson et al., 2002).
2.8
µM
s
ACTIN chicken) upon centrifugation in the presence of actin
CK-2018571
(recombinant S1 fragment,
5
4
CONCLUSIONS
1. CK-2018571 selectively inhibits the
ATPase activity of smooth muscle
myosin as compared to other myosin II
isoforms (non-muscle myosin, cardiac
and skeletal muscle myosins).
2. CK-2018571 inhibits smooth muscle
myosin in a weak actin-binding state,
consistent with its ability to relax
smooth muscle tissue in vitro.
3. CK2018571 inhibits the chemical
cleavage of ATP by smooth muscle
myosin, a mechanism distinct from
previously identified myosin inhibitors
such as blebbistatin and BTS.
4. CK-2018571 inhibits calcium-induced
force development in skinned caudal
artery and relaxes skinned rings
activated by thiophosphorylation,
consistent with relaxation occurring as
a consequence of the direct inhibition
of smooth muscle myosin.
30