10 -10
10 -9
10 -8
10 -7
10 -6
10 -5
DMSO
[CK-2018571] M
--
nucleotide
Vehicle --
--
STRONG
CK-2018509
ADP ATP
ADP ATP
NOVEL THERAPEUTIC MECHANISM
Loading Controls
(free fraction)
CK-2018571
CK-2018571 0.3µM
80 myosin
(C)
FOR
3 µM
Binding reaction supernatants
hexokinase
(free
60 fraction)
Loading Controls
BRONCHODILATION
(A) (B)
Inhibition
(% of vehicle response)
A
--
-- -- 0.1µ
centrifugationCK-2018571
+ M+
+ +CK-2018571
+ +
Binding reaction
supernatants 1 µM
Maximum Contriction (%)
THE DIRECT SMOOTH MUSCLE MYOSIN INHIBITOR, CK-2018571, REPRESENTS
10 -4
WEAK
0
(D)
ACTIN
BINDING Qian, Fady I Malik, Malar Pannirselvam
Morgans Jr, BradleyWEAK
P Morgan, Xiangping
Zhiheng Jia, Guillermo Godinez, Nickie Durham, Xi Wang, Chihyuan Chuang, Pu-Ping Lu, Wenyue Wang, Bing Yao, Jeffrey Warrington, Sheila Clancy, James J Hartman, David Jactin
myosin
Male Spraque-Dawley rats were dosed with CK-2019165 via conscious spontaneous
inhalation of aerosolized solution in an in-house custom built 12-slot closed pie
chamber using a PARI LC Plus Jet Nebulizer (22F80, PARI Respiratory Equipment),
pressurized to 22 psi with a carrier gas mixture of 21% O2, 5% CO2, 74% N2 with a flow
rate of 12 L/min. After dosing, rats were placed in an Unrestrained Whole Body
Plethysmograph (Buxco Research Systems), and animals were allowed to acclimate. A
baseline measurement was collected and rats were subsequently challenged with
nebulized methacholine (100 µL/rat of 20 mg/mL).
Smooth
9.0 ± 4.8
Non-muscle IIB
76 ± 4.9
--
--
+
+
+
0.6
+
A)
(0.4
0.2
Binding reaction supernatants
(free fraction)
Loading0.0
Controls
Maximum Contriction (%)
(C)
-0.2 Vehicle
myosin
(D)
(C)
CK-2018571 0.1µM
CK-2018571 1 µM
-10
-9
10
10 -8 3 µM10 -7
CK-2018571 0.3µM 10
CK-2018571
hexokinase
actin
80
60
10
[CK-2018571] M
DMSO
--
--
--
ADP ATP
--
centrifugation
--
--
+
+
+
+
Loading Controls
11,300 ± 840
+
Binding reaction supernatants
(free fraction)
-5
10
actin
ACTIN
BINDING
-4
STRONG
60
40
(A20)
80
150
60
40
20
Vehicle
(B)
Vehicle (A)0.1µM
CK-2018571
CK-2018571 1 µM
CK-2018571
CK-2018571 0.3µM
CK-2018571 3 µM
0
-20
-8
80
-7
-6
-5
-4
Log [CK-2018571] M
(C)
60
7
6
40
125
5
-3
(D)
4
- Log [Ca 2+ ] M
100
20
75
0
50
-20
25
0
-25
-50
STRONG
120
100
7
6
80
Vehicle- Log [Ca
60
5
2+ ]
CK-2018571 (5µM)
4
(B)
(D)
-20
80
Vehicle
Relaxation
(% of 3µM Mch contraction)
20
0
100
0
Vehicle
CK-2018571
-20
80
-8
60
-20
-7
0
50
100
150
-6
-5
-4
0.01
-3
0.1
Log [CK-2018571] M
4
0.01
4
Inhibition
(% of vehicle response)
40
-20
100
1
0.1
1
10
[CK-2018571] M
0
50
100
150
10
CK-2018571
120 75-8
60
40
20
0
Vehicle
CK-2018571
-20
125
100
-7
(C)
-6
(D)
-7
-6
-5
-4
80 25
12560
0
10040-25
-5 0
-4
-20
25
-8
0
-3
20
Vehicle 10
CK-2018571 Time (min)
-7
-6
30
-5
-4
-3
Log [CK-2018571] M
Vehicle
-25
50
CK-2018571 (5µM) 0.4
1.0
25
Vehicle
50
150
(C)
(C)
(D)
1.0
(A)
0.8
0.6
(C)
-25
Vehicle
CK-2019165
0.1
0.3
1.5 mg/rat
1
3.2
CK-2019165
3 mg/rat
CK-2018571
(5
µ
M)
[MCh] mg/mL
0.6
-50
Vehicle
0.4
0
CK-2019165 0.15 mg/rat
CK-2019165 0.3 mg/rat
0.2
CK-2019165 0.75 mg/rat
CK-2019165 1.5 mg/rat
CK-2019165 3 mg/rat
0.1
6
0.2
0.3
4
0.8
1
10
3.2
2
0
31.6
(B)
(B)
10
20
150
Control (n=12)
Time (min)
CK-2019165 10 mg/ml, 30 minutes
100
CK-2019165 10 mg/ml, 60 minutes
CK-2019165 10 mg/ml, 120 minutes
(D)
0.3
1
(D)
3.2
[MCh] mg/mL
1.0
0.4
10
30
0.03
50
100
150
0.03
31.6
50
Vehicle
CK-2019165 0.15 mg/rat
0
Control (n=12)
CK-2019165 0.3 mg/rat
CK-2019165 10 mg/ml, 30 minutes
CK-2019165 0.75 mg/rat
CK-2019165
10
31.6 10 mg/ml, 60 minutes
CK-2019165
mg/rat
* 120 minutes
* 101.5
CK-2019165
mg/ml,
**
CK-2019165 3 mg/rat
150
0.03
0.3
100
3
CK-2019165 (mg/rat)
2
[MCh] mg/mL
2
0
0.1
1
10
[CK-2018571] M
*
*
Control (n=12)
CK-2019165 10 mg/ml, 30 minutes
0
CK-2019165 10 mg/ml, 60 minutes
CK-2019165
10 mg/ml, 120 minutes
* P< 0.05 & **P< 0.01 compared to control by one-way
ANOVA
**
6
100
4
150
1
10
2
[CK-2018571] M
M
0
*
*
REFERENCES
1. Qian X, Wang X, Hartman JJ, Jia Z, Yao B, Chuang C, Lu P,
Pannirselvam M, Morgans DJ, Morgan BP, Malik FI. A direct inhibitor of smooth muscle myosin as a novel therapeutic approach
0.3
3
for the treatment of systemic hypertension. The Journal of
CK-2019165 (mg/rat)
Clinical Hypertension, 2009; 11(4): A37
* P< 0.05 & **P< 0.01 compared to control by one-way ANOVA
followed by Bonferroni post hoc test
followed by Bonferroni post hoc test
0.1
4. CK-2018571 relaxes methacholine preconstricted tracheal rings in a concentration
dependent manner suggesting its potential
use as a bronchodilator.
These data together suggest that direct
inhibition of smooth muscle myosin could be
0.3
3
a novel therapeutic
approach for the treatment
CK-2019165 (mg/rat)
of chronic obstructive pulmonary disease
and asthma.
0
(A) Effect of CK-2019165 on methacholine-induced bronchoconstriction in anesthetized, paralyzed and mechanically50ventilated rats
Control±(n=12)
(n=3-7 rats). Symbols are mean
standard0.4
error
(B)0.01
Dose-response
curvebyfor
CK-2019165
* P< values.
0.05 & **P<
compared to control
one-way
ANOVA showing dose-dependent inhibition of
4 followed by Bonferroni post hoc test
CK-2019165 10 mg/ml,
30 minutes
methacholine-induced bronchoconstriction
with
an EC50 of 0.7 mg/rat and Emax of 74%. Symbols are mean ± standard error values.
CK-2019165 10 mg/ml, 60 minutes
0
CK-2019165 10 mg/ml,
0.2 120 minutes
6
0.01
3. 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
direct inhibition of smooth muscle myosin.
5. CK-2019165 inhibited the methacholineinduced bronchoconstriction
in two rat
0.3
3
models of bronchoconstriction, i.e. resistance
CK-2019165 (mg/rat)
and compliance model and unrestrained
whole body plethysmography.
bronchoconstriction
Figure 7: CK-2019165 inhibited the
0.03
*1
* methacholine-induced
0.1
0.3
3.2
10
31.6
**
whole body plethysmography rat model
in unrestrained
4
0
50
1. CK-2018571 selectively inhibits the ATPase
activity of smooth muscle myosin over other
myosin II isoforms (non-muscle myosin,
cardiac and fast skeletal muscle myosin).
2. CK-2018571 inhibits smooth muscle myosin
in a weak actin-binding state.
CK-2018571 (5µM)
50
0.66
[MCh] mg/mL
50
0.01
CONCLUSIONS
Vehicle
20-50
75
0
10
-3
-50 1.0
75
0.1
(A) Mean pCa-response
curves in the presence (0.1, 0.3, 1 & 3 µM) or absence of CK-2018571 in skinned caudal artery rings from Sprague
40
0
10
20
30
Dawley rats (n=2-4).
120Symbols are mean ± standard error values. (B) Concentration-response curve for CK-2018571 constructed from the
20 of calcium-induced
Time contraction
(min)
maximal inhibition
from figure (A). CK-2018571 inhibited the calcium induced forced development of the
100
skinned caudal ring
in a concentration dependent manner with an EC50 of 0.9 µM and Emax of 105%. Symbols are mean values.
0
- Log [Ca 2+ ] M
5
150
CK-2018571 (5µM)
Vehicle
150
CK-2019165
0.15 mg/rat
0
10
20
30
50
125
CK-2019165 0.3 mg/rat
0.8
Time
(min)
CK-2019165
0.75
mg/rat
25
100
100
CK-2019165 1.5 mg/rat
0
0.6absence of
Force development to ATP in the presence75or
CK-2018571
(5 µM) in isolated skinned and thiophosphorylated aortic rings
CK-2019165
3 mg/rat
-25
(n=8-12) from Sprague
Dawley
rats. Symbols are mean ± standard error values.
Vehicle
100
Figure 3: CK-2018571 inhibits calcium-induced contraction
of skinned caudal artery smooth muscle0 preparation
100
60
120
20
100
-8
(A)
Vehicle
DMSO
CK-2018509
CK-2018571-- 0.1µ
CK-2018571
nucleotide
-- M -- ADP ATP
-- ADP ATP1 µM
120
CK-2018571-- 0.3µM
-centrifugation
+ +
+CK-2018571
+ + + 3 µM
0
(C-20)
840
(A) (B)
50
+
6
100
CK-2019165
0.15 mg/rat
0
10
20
30
Figure 6: CK-2019165
inhibited
the
methacholine-induced
bronchoconstriction in 0anesthetized,
0.2
CK-2019165
0.3
mg/rat
0
0.8
Time (min)
paralyzed and mechanically
ventilated
resistance and compliance rat
model
CK-2019165
0.75 mg/rat
100
WEAK
myosin
Actomyosin
binding
of soluble smooth muscle myosin (recombinant S1 fragment, chicken) in the presence
of actin and various nucleotides
-20
(A). Total amounts of myosin (3 µM) and actin (5 µM) are indicated by the0.01
loading controls
(left),1while unbound
myosin is shown in the
0.1
10
ACTIN
7
6 of the binding
5
hexokinase
supernatant
fractions
reaction4(right). (B) Smooth muscle myosin chemo-mechanical
cycle, indicating weak and strong actin
[CK-2018571] M
BINDING
- Log [Ca 2+ ] M
actin
binding states.
80
120
-50
myosin
ADP ATP
20
0
Fast skeletal
WEAK
0
CK-2018509
nucleotide
40
10
-6
10
[CK-2018571] M
0.01
0.1
1
40
- Log [Ca 2+ ] M
7
6
5
4
[CK-2018571] M
20 in isolated tracheal
Concentration-response curve to CK-2018571
rings (n=11-12) pre-constricted with 3 µM methacholine from Sprague
- Log [Ca 2+ ] M
Dawley rats. CK-2018571 relaxed the isolated
tracheal
rings
in
a
concentration-dependent manner with an EC50 of 0.9 µM and Emax of
0 125
Vehicle
105%. Symbols are mean ± standart error values.
(A) (B)
76 ± 4.9
2600 ± 240
(D)
40
5
9.0 ± 4.8
Cardiac
(B)
(B)
60
80
Log [CK-2018571] M
Non-muscle IIB
+
1
80
(C)± 240
(D)
M
Log [CK-2018571]
2600
relaxes the
contraction
in ATPγS treated skinned caudal artery
Figure 5: CK-2018571
50
100 ATP-induced
ADP ATP
+
0.1
[CK-2018571] M
MCh-induced
bronchoconstriction
(% vehicle)
--
0.01
50
MCh-induced
bronchoconstriction
(% vehicle)
ADP ATP
0 150
MCh-induced
bronchoconstriction
(% vehicle)
--
0.8
6
7
MCh-induced
bronchoconstriction
(% vehicle)
(A)
--
Inhibition
(% of vehicle response)
centrifugation
--
-20
IC50 ± SD, nM
Myosin Isoform
[CK-2018571] M
STRONG
Figure1.02: CK-2018571
does not promote strong
actomyosin
binding
Smooth
DMSO
CK-2018509
Normalized ATPase
Activity
nucleotide
0
Force
(% of initial tension)
10 -10
20
7
WEAK
Fast Skeletal
Non-muscle
ACTIN
-4
10 -9 10 -8 10 -7 10 -6 10 -5 10
BINDING
40
80
(% of initial tension)
0.4
100
Inhibition
(% of vehicle response)
IC50 ± SD, nM
Maximum Contriction (%)
Myosin
Isoform
11,300 ± 840
(A) (B)
Cardiac
1.2
100
CK-2018571
3 µM
PenH
10 -4
[CK-2018571] M
0.6
4
20
Resistance (cmH2 0/mL/sec)
10 -5
5
PenH
10 -6
6
2+
Resistance (cmH2 0/mL/sec)
10 -7
40
Force
Relaxation
(% of initial(%
tension)
of 3µM Mch contraction)
Force
10 -8
+
7
Force
Resistance (cmH2 0/mL/sec)
(% of initial tension)
Fast skeletal
10 -10 10 -9
0.8
60
+
-20
Relaxation
(% of 3µM Mch contraction)
2600 ± 240
Cardiac
60
Resistance (cmH2 0/mL/sec)
1.0
Maximum Contriction (%)
76 ± 4.9
80
Relaxation
(% of 3µM Mch contraction)
Normalized ATPase
Activity
Normalized ATPase
Activity
-0.2
--
ADP ATP
STRONG
PenH
Conscious Rodent Model of Unrestrained Whole Body Plethysmography
1.2
--
PenH
Male Sprague-Dawley rats were anesthetized with Ketamine/Xylazine/Acepromazine
(80/10/1 mg/kg) cocktail and tracheotomized with a 14 g tracheal cannula. Rats were
paralyzed with Pancuronium Bromide at 2 mg/kg, i.p. to prevent spontaneous
breathing. Immediately rats were placed on a Resistance & Compliance Plethysmograph
(Buxco Research Systems). Once rats were stabilized and a baseline was collected, CK2019165 was intra-tracheally nebulized via an Aeroneb Lab micropump nebulizer. Five
minutes later, dose dependent bronchoconstriction to methacholine was measured.
Non-muscle IIB
Inhibition
(% of vehicle response)
Anesthetized Rodent Model of Airway Resistance & Compliance
9.0 ± 4.8
Cardiac
Fast Skeletal
Inhibition
(% of vehicle response)
In vivo Assay:
0.0
n
ntraction)
Thiophosphorylation Assay: Triton-permeabilized preparations were incubated in rigor
solution containing ATPγS (1 mM) for 10 minutes. CK-2018571 was added 15 minutes
before addition of the ATP. ATP induced contraction was measured for 60 minutes and
the relaxation was expressed as the percentage of the maximum force.
0.2
actin
Maximum Contriction
(%)
Relaxation
Tracheal Ring Assay: The trachea was removed and placed in Krebs-Henseleit buffer
aerated with 95% O2 and 5% CO2. The trachea was cut into 2 mm rings, mounted on a
tissue bath apparatus, and maintained at a baseline tension of 2 g. CK-2018571-induced
relaxation was recorded in preparations pre-contracted with a sub-maximal
concentration of methacholine (3 µM).
0.4
hexokinase
Myosin binding was measured by depletion of soluble myosin from binding reactions
using smooth muscle myosin S1 fragment (chicken, recombinant) and 5 µM bovine
cardiac actin. ATP and ADP were included at 1 mM where indicated. Reactions were
allowed to equilibrate for 15 minutes prior to centrifugation (540k x g, 30 minutes),
however ATP was added just prior to centrifugation to minimize hydrolysis.
Supernatants were analyzed by SDS-PAGE followed by staining with Coomassie
brilliant blue.
Skinned Ring Assay: Endothelium-denuded rat tail artery segments were cut into 3-mm
helical rings, mounted on an isometric force transducer with a resting tension of 0.5 g
and incubated for 30 minutes at room temperature in normal H-T buffer. Tissues were
incubated with skinning solution containing 1% Triton X-100 for 1 hour at room
temperature. CK-2018571 was added to the tissue for 15 minutes, followed by addition
of solutions with increasing calcium. Force was recorded at each calcium concentration.
Data were presented as a percent change from the baseline values.
0.8
myosin
(% of 3µM Mch contraction)
Biochemical Assays: Assays were performed in low salt PM12 buffer (12 mM K-Pipes, 2
mM MgCl2, pH 6.8) in the presence of actin and 250 µM ATP (>5-10-fold above the
KM,ATP). Hydrolysis rates were normalized using reactions containing an equivalent
amount of DMSO.
Smooth
Inhibition of the Mg2+-ATPase activity of smooth muscle (human recombinant), non-muscle IIB (human recombinant), cardiac (bovine
0.2 (rabbit native) S1 fragments at varying concentrations of CK-2018571. ATPase activity was measured in the
native), and fast skeletal
Fast skeletal
11,300 ±
presence of actin and 250 µM ATP (~5-10-fold above the KM,ATP. ATPase rates were normalized to reactions containing an equivalent
0.0
Bindingcurves
reaction supernatants
amount of DMSO. Representative
from duplicate reactions are shown in the graph. Pooled results from three experiments are
Loading Controls
(free fraction)
shown in the table. -0.2
Cardiac
Smooth
(% of 3µM Mch contraction) Maximum Contriction (%)
METHODS
IC50 ± SD, nM
Myosin Isoform
1.0
Smooth
Non-muscle
centrifugation
0
ADP ATP
--- 80
+ +
+ +
CK-2018571 0.3µM
1.2
0.6
--
Vehicle
0
Vehicle
CK-2018571
0.1µ
M
CK-2018571 1 µM
120
CK-2018571 0.1µM
µM
CK-2018571
1
CK-2018571 0.3µM
CK-2018571 3 µM
-20
Cardiac
Fast Skeletal
Smooth
Non-muscle
--
50
- Log [Ca ] M
Figure 4: CK-2018571 causes concentration
dependent relaxation of isolated tracheal rings
Figure 1: CK-2018571 selectively inhibits the ATPase activity of smooth muscle myosin
Force
Relaxation(% of initial tension)
Smooth muscle myosin is a mechanochemical enzyme that hydrolyzes ATP to
generate mechanical force; ultimately all signaling pathways that modulate smooth
muscle tone converge onto the regulation of this motor protein. Using high
throughput screening, we identified and subsequently optimized a class of selective
inhibitors of smooth muscle myosin. Previously we showed that CK-2018509, a novel,
potent, and selective inhibitor of the enzymatic activity of smooth muscle myosin,
decreased the mean arterial pressure in two animal models of hypertension [1].
Another novel smooth muscle myosin inhibitor, CK-2019165, an active pro-drug of
CK-2018571, was previously shown to decrease right ventricular systolic pressure in
two rat models of pulmonary arterial hypertension [2]. Given its central role in
generating smooth muscle contractility in the settings of chronic obstructive
pulmonary disease (COPD) and asthma, direct inhibition of smooth muscle myosin
could provide a novel and effective means to reduce bronchoconstriction in asthma
and COPD. The objective of the present study was to evaluate the pharmacology of
CK-2018571 and CK-2019165 in rat models of bronchoconstriction.
RESULTS
nucleotide
Maximum Contriction (%)
INTRODUCTION
actin
Inhibition
(% of vehicle response)
Cytokinetics, Inc., South San Francisco, CA, USA.
40
ACTIN
Vehicle
DMSO
CK-2018509
BINDING
ATP -- ADP1 µ
ATP
-- 0.1µ
nucleotide
-- M -- ADP CK-2018571
M
20 CK-2018571
STRONG
µ
CK-2018571
0.3µM
CK-2018571
3
M
-centrifugation
+ +
+ + + +
DMSO
CK-2018509--
hexokinase
**
* P< 0.05 & **P< 0.01 compared to control by one-way ANOVA
followed by Bonferroni post hoc test
Effect of CK-2019165 on methacholine-induced bronchoconstriction in unrestrained whole body plethysmography in rats (n=3-12 rats).
Each bar represents mean PenH ± standard error values.
2. Pannirselvam M, Durham N, Jia Z, Clancy S, Chuang C, Lu P,
Wang W,Yao B,Warrington J, Hartman JJ, Morgans Jr DJ, Morgan
BP, Qian X, Malik FI,.A direct inhibitor of smooth muscle myosin
as a novel therapeutic approach for the treatment of pulmonary
artery hypertension. Circulation. 2009;120:S805