+ MODEL
ARTICLE IN PRESS
JPT-05965; No of Pages 15
Pharmacology & Therapeutics xx (2007) xxx – xxx
www.elsevier.com/locate/pharmthera
Associate editor: Madhani
Q11
Potential of p38-MAPK inhibitors in the treatment of ischaemic heart disease
3
James E. Clark, Negin Sarafraz, Michael S. Marber ⁎
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The Cardiovascular Division, Kings College London, The Rayne Institute, St Thomas' Hospital, London, SE1 7EH, United Kingdom
OO
F
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Abstract
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Chronic heart failure is debilitating, often fatal, expensive to treat and common. In most patients it is a late consequence of myocardial
infarction (MI). The intracellular signals following infarction that lead to diminished contractility, apoptosis, fibrosis and ultimately heart failure
are not fully understood but probably involve p38-mitogen activated protein kinases (p38), a family of serine/threonine kinases which, when
activated, cause cardiomyocyte contractile dysfunction and death. Pharmacological inhibitors of p38 suppress inflammation and are undergoing
clinical trials in rheumatoid arthritis, Chrohn's disease, psoriasis and surgery-induced tissue injury. In this review, we discuss the mechanisms,
circumstances and consequences of p38 activation in the heart. The purpose is to evaluate p38 inhibition as a potential therapy for ischaemic heart
disease.
© 2007 Elsevier Inc. All rights reserved.
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Keywords: p38; MAPK; Inhibitors; Heart failure; Ischaemia; Infarction
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Contents
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1.
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4.
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UN
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RR
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Introduction . . . . . . . . . . . . . . . . . . . . . . .
p38-Mitogen-activated protein kinase . . . . . . . . . .
Structure and function of p38-MAPK . . . . . . . . . .
Mechanisms of p38-MAPK activation. . . . . . . . . .
4.1. Mitogen-activated protein kinase kinases. . . . .
4.2. Autophosphorylation . . . . . . . . . . . . . . .
5. p38, myocardial ischaemia and ischaemic heart disease .
6. Pharmacological inhibitors of p38-MAPK. . . . . . . .
7. Clinical trials of p38-MAPK inhibition . . . . . . . . .
8. Summary and conclusions . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . .
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ED
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CT
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PR
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1. Introduction
Atherosclerotic vascular disease manifests predominantly as
heart disease and stroke, which are the most frequent causes
of death in the United Kingdom. Collectively atherosclerotic
vascular disease was responsible for 40% of total mortality in the
United Kingdom in 2004. Despite recent reductions in this high
⁎ Corresponding author. Tel.: +44 20 7188 1008.
E-mail address: mike.marber@kcl.ac.uk (M.S. Marber).
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mortality, morbidity is increasing as more patients survive 36
myocardial infarction (MI) and stroke. For example, about 37
1.3 million people in the United Kingdom have survived acute 38
MI, 2 million have angina and 0.9 million have heart failure. The 39
extraordinarily high impact of atherosclerosis on the Nation's 40
health is reflected in its economic cost. Atherosclerotic disease 41
of just the coronary arteries costs the United Kingdom healthcare 42
system £3500 million with an estimated further £4400 million 43
lost to the economy through premature death, illness and infor- 44
mal care (The United Kingdom Heart Attack Study (UKHAS) 45
Collaborative Group, 1998; Office for National Statistics, 2000). 46
0163-7258/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.pharmthera.2007.06.013
Please cite this article as: Clark, J. E. et al. Potential of p38-MAPK inhibitors in the treatment of ischaemic heart disease. Pharmacol Ther (2007), doi:10.1016/j.
pharmthera.2007.06.013
2
Study
Model
End point
Inhibitor
Outcome
(Weinbrenner
et al., 1997)
p38 phosphorylation and
cell viability (trypan blue)
SB203580 5 μM during ischaemia
Inhibition of p38 activation abolishes protection in
preconditioned hearts and cardiomyocytes
t1:4
(Wang et al., 1998a)
Ex vivo: buffer perfused rabbit heart subjected
to global ischaemia–reperfusion
In vitro: isolated rabbit cardiomyocytes
In vitro: neonatal rat cardiac myocytes
cotransfected with p38α/β, active
MKK3/MKK6 or dominant negative isoforms
Cell survival, hypertrophic
response, apoptosis
The hypertrophic response in myocytes is mediated by
p38β isoform, whereas over expression of p38α results
in increase cell death
t1:5
(Nagarkatti & Afi, 1998)
In vitro: ischaemia in rat myoblast cell
line H9C2
Cell viability (MTT)
Modulation of p38α/β using
overexpression of dominant
negative isoforms or
upstream activators
SB203580 15 μM before/during
ischaemia
t1:6
(Meldrum et al., 1998)
t1:7
LV function, coronary flow,
CK release and tissue TNF
LDH release
t1:8
(Mackay & MochlyRosen, 1999)
(Ma et al., 1999)
t1:9
(Craig et al., 2000)
t1:10
(Hoover et al., 2000)
Ex vivo: buffer perfused rat heart exposed
to H2O2
In vitro: simulated ischaemia in neonatal
rat cardiomyocytes
Ex vivo: buffer perfused rat heart subjected
to global ischaemia–reperfusion
In vitro: neonatal rat cardiac myocytes
cotransfected with MKK6, TNF-α, TAK-1
and IL-6
In vitro: cardiac myocytes (transfected
with MKK6) exposed to sorbitol
t1:11
(Saurin et al., 2000)
In vitro: simulated ischaemia in cultured
rat neonatal cardiac myocytes
t1:12
t1:13
t1:14
(Yue et al., 2000)
In vitro: simulated ischaemia–reperfusion in
rat neonatal cardiomyocytes
In vitro: simulated ischaemia–reperfusion in
rat neonatal cardiomyocytes
In vivo: regional ischaemia–reperfusion in
pig heart
t1:15
(Mackay & MochlyRosen, 2000)
(Barancik et al., 2000)
t1:16
(Marais et al., 2001)
t1:18
t1:19
(Gysembergh
et al., 2001)
(Schneider et al., 2001)
t1:20
(Martin et al., 2001)
t1:21
(Rakhit et al., 2001)
t1:22
(Sanada et al., 2001)
UN
CO
RR
Ex vivo: buffer perfused rat heart subjected
to global ischaemia–reperfusion
In vitro: neonatal cardiac myocytes
Ex vivo: buffer perfused rabbit heart subjected
to focal ischaemia–reperfusion
Ex vivo: buffer perfused rat heart subjected to
global ischaemia–reperfusion
In vitro: simulated ischaemia in neonatal/adult
rat cardiocytes overexpressing wild-type
p38α MAPK
In vitro: simulated ichaemia–reperfusion injury
in neonatal rat cardiomyocytes
In vivo: regional ischaemic preconditioning in
dog heart
SB203580 1 mmol/min prior to insult
SB203580 10 μM during ischaemia
LV Function, apoptosis and
CK release
Apoptosis and IL-1 translation
and transcription
SB203580 10 μM before/during
ischaemia
SB203580 5 μM
α-B crystallin expression
and phosphorylation;
MAPKAP-K2 and
p38 activation and apoptosis
p38 phosphorylation, CK
and LDH release and
cell viability (MTT)
SB203580 5 μM during insult
LV function and apoptosis
(TUNEL)
p38 phosphorylation, apoptosis
and LDH release
p38 phosphorylation and
ATF-2 phosphorylation
and Infarct size
p38 phosphorylation, LV
function and apoptosis
SB242719 10 μM
SB203580 10 μM
SB203580 10 μM during ischaemia
p38 activity and infarct size
SB203580 1 μM during ischaemia
LV function and necrosis
SB202190 10 μM before ischaemia
LDH release
SB203580 10 μM during ischaemia
p42/44 and p38 MAPK
phosphorylation
HSP27 phosphorylation,
arterial blood pressure,
infarct size and collateral flow
SB203580 1 μM during ischaemia
EC
TE
SB203580 10 μM during ischaemia
DP
Inhibition of p38 with SB203580 increased sorbitolmediated apoptosis
Inhibition of p38 activation during prolonged ischaemia
reduces injury and contributes to preconditioninginduced cardioprotection p38α is phosphorylated during
ischaemia whereas p38β is deactivated
Inhibition of p38/JNK leads to cardioprotection, whereas
inhibition of ERK pathway exacerbates injury
Incubation with SB203580 during ischaemia–
reperfusion attenuates cell death
Inhibition of p38 during ischaemia decrease infarct size
and delays cell death
RO
SB203580 1 μM during ischaemia
SB203580 1-10 μM during
ischaemia/reperfusion
SB203580 administered prior to ischaemia blocks
preconditioning, but is protective during prolonged
ischaemia
p38 inhibition decreases myocardial TNF production,
cardiomyocyte death and dysfunction
p38 inhibition, by SB203580, during ischaemia protects
against cardiomyocyte apoptosis
Inhibition of p38 attenuates reperfusion injury by
reducing apoptosis and improving cardiac function
Activating both p38 and TNF-α augments myocardial
survival during stress which is inhibited by SB203580
SB203580 1 μM during ischaemia/
reperfusion
p38 inhibition by SB203580 during ischaemia and
reperfusion is cardioprotective
OF
Inhibition of p38 activity during coronary artery
occlusion is cardioprotective
p38 inhibition reduces ischaemic injury and does not
block protective effect of preconditioning
SB203580 is cardioprotective through inhibition of p38
isoform and not due to inhibition or activation of other
kinases
SB203580 protected against injury, but p38β isoform
does not contribute to survival
SB203580 treatment during the preischemic and
postischaemic periods had no significant effect on
infarct size
ARTICLE IN PRESS
t1:1
t1:2
J.E. Clark et al. / Pharmacology & Therapeutics xx (2007) xxx–xxx
Please cite this article as: Clark, J. E. et al. Potential of p38-MAPK inhibitors in the treatment of ischaemic heart disease. Pharmacol Ther (2007), doi:10.1016/j.
pharmthera.2007.06.013
Table 1
Apoptosis and Fas-L cyclin
D1 expression
SB203580 10 μM during stress
LV function
SB202190 10 μM during rewarming/
reperfusion
p38α activity, infarct size and
LV function
p38α+/− mice were used to
knockdown p38 activity
Inhibition of p38 activity attenuated stress-induced
apoptosis and reversed changes in Fas-L and cyclin
D1 expression
SB202190, when present during reperfusion, improves
recovery of LV function Inhibition of p38 did not protect
against rewarming-induced injury
Activation of p38α during ichaemia–reperfusion is
detrimental; reduction in p38α expression results
in protection
The p38 activation that accompanies short-term
hibernation does not appear to contribute to the
contractile deficit
Inhibition of p38 activation attenuates ischaemia–
reperfusion injury in heart transplantation from nonheart-beating donors
RWJ-67657 treatment post-MI had beneficial effects on
LV remodelling and dysfunction
FR167653-mediated inhibition of p38 activity during
regional ichaemia–reperfusion injury reduces infarct size
t1:26
(Sharov et al., 2003)
t1:27
(Clanachan et al., 2003)
t1:28
(Otsu et al., 2003)
t1:29
(Gorog et al., 2004)
Ex vivo: buffer perfused mouse heart subjected
to low-flow global ischaemia–reperfusion
LV function, Coronary flow
and apoptosis
SB203580 1 μM during ischaemia
t1:30
(Koike et al., 2004)
In vivo: canine heart transplantation from nonheart-beating donors
Cardiac output (CO) and
LV function
FR167653 Dose not reported during
cold storage
t1:31
(See et al., 2004)
In vivo: regional ischaemia in rat hearts
t1:32
(Yada et al., 2004)
In vivo: regional ichaemia–reperfusion in
mouse hearts
RWJ-67657 50 mg/day 7 days post
MI for 21 days
FR167653 2 mg/kg i.p.
before ischaemia
t1:33
(Kaiser et al., 2004)
t1:35
(Aleshin et al., 2004)
In vitro: simulated ischaemia–reperfusion in
neonatal cardiomyocytes expressing dominantnegative p38
In vivo: regional ischaemia–reperfusion in
dominant-negative MKK6 transgenic mice or
dominant-negative p38α transgenic mice
Ex vivo: buffer perfused rat heart subjected to
global ischaemia–reperfusion
LV function and postinfarction
remodelling
Protein kinase activation and
kinase activity, Nuclear factor
κB activity, inflammatory
cytokines and infarct size
Cell death, apoptosis, DNA
fragmentation and Infarct size
TNFα mRNA expression; LV
function and CK release
FR167653 1.0 mg/kg i.p. before I/R,
and 1.0 mg/L during perfusion
t1:36
(Martindale et al., 2005)
(Kabir et al., 2005)
LV dimensions (by echo);
αB-crystallin expression;
DNA fragmentation
p38 and HSP27
phosphorylation and
infarct size
p38 activity was modified by cardiac
overexpression of MKK6
t1:37
In vivo: regional ischaemia–reperfusion in
hearts from mice overexpressing cardiacrestricted wild type MKK6
Ex vivo: buffer perfused mouse heart subjected
to global ischaemia–reperfusion in presence/
absence of antimycin A
t1:38
(Wang et al., 2005)
Ex vivo: buffer perfused rat heart subjected to
global ischaemia–reperfusion
p38, activation and cytokine
expression, activation of
caspases and LV function
SB203580 20 μM before ichaemia
t1:39
(Okada et al., 2005)
In vitro: hypoxia-reoxygenation in neonatal rat
cardiomyocyte
MAPKAP phosphorylation;
Cyt. C release from
mitochondria, caspase-3
activation and LDH release
SB203580 10 μM throughout the
experiment
UN
CO
RR
EC
TE
Reduction of endogenous p38 using
overexpression of dominant negative
p38 isoform or upstream activation
DP
p38α functions as a prodeath signalling effector in both
cultured myocytes as well as in the intact heart
RO
SB203580 1 μM at the same time as
antimycin A
FR167653 inhibited ichaemia–reperfusion-mediated
myocardial TNFα production and p38 activation and
improved functional recovery
Overexpression of MKK6 resulted in less myocardial
damage following ischaemia–reperfusion and enhanced
functional recovery
Cardioprotection initiated by antimycin A is dependant
upon p38 activation but independent of the upstream
kinase MKK3; however, during lethal ischaemia,
inhibition of p38 activity was protective
Inhibition of p38 activation during ischaemia resulted in
less MAPKAPK2, caspase-1, caspase-3 and caspase-11
activation, and TNF, IL-1beta, IL-6 production after
myocardial ichaemia as well as increasing functional
recovery of the heart
SB203580 abrogated activation of p38 MAPK,
translocation of HSP27, and F-actin reorganization,
prevented cytochrome C release, caspase-3 activation,
and DNA fragmentation
OF
3
(continued on next page)
ARTICLE IN PRESS
SB203580 sensitive ischaemic activation of p38 by
TAB1-associated autophosphorylation contributes to
myocardial injury
J.E. Clark et al. / Pharmacology & Therapeutics xx (2007) xxx–xxx
SB203580 1 μM during ischaemia
(Tanno et al., 2003)
Please cite this article as: Clark, J. E. et al. Potential of p38-MAPK inhibitors in the treatment of ischaemic heart disease. Pharmacol Ther (2007), doi:10.1016/j.
pharmthera.2007.06.013
Ex vivo: buffer perfused mouse heart subjected
to global ischaemia–reperfusion (mkk3−/−
and mkk3+/+)
In vitro: global ischaemia in H9c2 myoblasts
expressing wild-type and drug-resistant p38α
In vitro: cardiomyocytes isolated from dogs
with heart failure simulated by hypoxia,
Angiotensin-II or nor-epinephrine
Ex vivo: buffer perfused rat heart subjected to
hypothermia-rewarming and global ischaemia–
reperfusion
In vivo: regional Ichaemia–reperfusion in
p38α+/+ and p38α+/− mice
Infarction/risk volume,
p38, TAB1 and HSP27
phosphorylation
t1:24
4
t1:41
Study
Model
t1:42
(Sumida et al., 2005)
Ex vivo: buffer perfused rat heart subjected to
global ischaemia–reperfusion
t1:43
(Liu et al., 2005)
In vivo: mouse hearts subjected to regional
ischaemia–reperfusion
t1:44
(House et al., 2005)
t1:45
(Li et al., 2005)
t1:47
(Gupta et al., 2005)
Ex vivo: buffer perfused mouse heart subjected
to global low-flow ischaemia–reperfusion
In vitro: rat neonatal cardiomyocytes
overexpressing MKK6
In vivo: hearts from MKK6bE transgenic mice
subjected to ischaemia–reperfusion
In vitro: adult rat ventricular myocytes
(ARVMs) during sepsis
t1:48
(Kim et al., 2006)
In vitro:Hypoxia–re–oxygination in neonatal
rat cardiomyocyte
t1:49
(Khan et al., 2006)
Ex vivo: buffer perfused rat heart subjected to
global ischaemia–reperfusion
t1:50
(Bellahcene et al., 2006)
Ex vivo: buffer perfused mouse hearts from
mkk3−/− mice subjected to TNF-α
t1:51
(Engel et al., 2006)
In vivo: regional ischaemia in rat hearts
t1:52
(Li et al., 2006)
In vivo: angiotensin and L-NAME-mediated
cardiac hypertrophy in rats
t1:53
(Vahebi et al., 2007)
t1:54
t1:55
UN
CO
End point
Inhibitor
Outcome
p38 and JNK activities LV
contractility, CK release,
mitochondrial ATP generation
and infarct size
LV function, cardiac
remodelling
SB203580 10 μM during reperfusion
p38 inhibition exerts cardioprotection only when
contractile force-induced necrosis is prevented
SC-409 30 mg/kg/day given after MI
for 12 weeks
Infarct size and p38 and HSP27
phosphorylation
LV remodelling, cytokine
release and LV function
SB203580 2 μM during ischaemia/
reperfusion
SB239068 20 μM and 1200 ppm in
drinking water or culture medium
during exposure
Inhibition of p38 MAPK attenuates cardiac remodelling
and improves cardiac function in mice with heart failure
after infarction
Inhibition of p38 during ichaemia–reperfusion injury
protects against myocardial cell death
p38 inhibition prevents induction of inflammatory
cytokines in cardiomyocytes and extracellular
remodelling in heart
RR
EC
Cell fractional shortening, cell
viability (MTT), caspase-3
activity
Cell viability (trypan blue),
apoptosis (TUNEL), necrosis,
ROS generation
Coronary flow, LV function,
LDH release, infarct size and
apoptosis (TUNNEL)
LV function, p38 and HSP27
phosphorylation
SB203580 10 μM before insult
SB203580 1 μM during ischaemia/
TE
reperfusion
SB203580 10 μM before TNF-α
DP
SB203580 1 μM before ischaemia
Left ventricular remodelling,
fractional shortening and
neovascularisation
LV function, arterial
inflammatory cell infiltration,
and cardiomyocyte apoptosis
SB203580 2 mg/kg at time of surgery
In vitro: isolated skinned cardiac muscle fibre
bundles
Cardiac myofilament function,
Phosphorylation of α-Tm
and TNI
Reduction of endogenous p38 using
overexpression of dominant negative
p38α isoform or upstream activation
(Riad et al., 2007)
In vivo: diabetic mellitus induced by a single
injection of streptozotocin
SB239063 40 mg/kg/day for 43 days
after induction of diabetes mellitus
(Clark et al., 2007)
In vivo: regional ischaemia in hearts from
mkk3−/− and mkk3+/+ mice
LV function, p38
phosphorylation and peripheral
ICAM-1 and VCAM-1
LV function, LV remodelling,
p38 and HSP27
phosphorylation
SB203580 pretreatment followed by bigET-1
administration decreased p38 phosphorylation and downregulated ET(B) receptor expression in sepsis group
Inhibition of p38α prevented hypoxia re–oxygenation
induced apoptosis of cardiomyocytes
Inhibition of p38 MAPK improved cardiac function after
reperfusion and attenuated ischaemic reperfusioninduced myocardial apoptosis and necrosis
Activation of p38 contributes to TNF-α induced
contractile depression in intact heart and in isolated
cardiac myocytes through MKK3; inhibition of p38
abolished contractile depression caused by TNFα
SB203580 and FGF-1 induces cardiomyocyte mitosis,
reduces scarring, and rescues function after MI
RO
SD-282 60 mg/kg administered for
4 days during treatment
Knockout of MKK3
SD-282, reduces inflammatory response and apoptosis,
resulting in a reduction of myocardial damage, which, in
turn, improves cardiac function following angiotensin II
and L-NAME treatment
Activation of p38α directly depresses saromeric function
by decreased phosphorylation of α-tropomyosin, which
is reversed by inhibition of p38α activity or by over
expressing dominant negative isoform
Inhibition of p38MAPK reduced cardiac cell adhesion
molecules expression indicating both antiinflammatory
and vasculoprotective effects in the diabetic heart
Postinfarction LV remodelling continues in the absence
of MKK3 as does p38 activation
OF
ARTICLE IN PRESS
Table 1 (continued )
J.E. Clark et al. / Pharmacology & Therapeutics xx (2007) xxx–xxx
Please cite this article as: Clark, J. E. et al. Potential of p38-MAPK inhibitors in the treatment of ischaemic heart disease. Pharmacol Ther (2007), doi:10.1016/j.
pharmthera.2007.06.013
t1:40
ARTICLE IN PRESS
J.E. Clark et al. / Pharmacology & Therapeutics xx (2007) xxx–xxx
54
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61
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66
67
68
69
70
71
72
73
74
75
76
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78
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80
81
82
83
84
85
86
87
88
89
92
93
94
95
96
97
98
99
100
101
OF
53
EC
52
CO
RR
51
UN
50
RO
2. p38-Mitogen-activated protein kinase
49
surprisingly these cellular effects have clear consequence(s),
translating into involvement in complex pathophysiologies,
such as wound healing (Lim et al., 1998), inflammatory arthritis
(Badger et al., 1996), sepsis (Kotlyarov et al., 1999), acute respiratory distress syndrome (Carter et al., 1999), and malignant
hypertension (Behr et al., 2001).
Four p38 isoforms (α, β, δ and γ) exist, which have preserved
structure but variable sensitivity to pharmacological inhibition.
All 4 isoforms have a Thr180-Gly181 -Tyr182 (TGY) dual phosphorylation motif which is used by investigators to infer activation. p38α and β have high sequence homology and share
sensitivity to pharmacological inhibition by prydinyl imidazole
molecules (such as SB203580) but have only 60% homology
with p38γ and δ, which are resistant to SB203580 (SB) inhibition (Eyers et al., 1999). Of the SB-sensitive isoforms, p38α is
the predominant form in human and rodent myocardium (Lemke
et al., 2001; Rakhit et al., 2001; Sanada et al., 2001; Braz et al.,
2003). Studies with knockout mice and cells have shown that
p38α is essential for embryonic development as knockout of the
α isoform results in embryonal lethality, but mice lacking p38β,
p38γ, and p38δ are viable (Allen et al., 2000; Adams et al., 2000;
Tamura et al., 2000; Brancho et al., 2003).
DP
91
48
3. Structure and function of p38-MAPK
TE
90
Atherosclerotic plaque rupture/erosion results in MI, which is
characterised by necrosis and apoptosis of cardiomyocytes.
Although the acute condition alone may result in death due to
ventricular arrhythmias or pump failure, in the patients with
substantial infarction that survive, a chronic phase of ventricular
remodelling occurs. Remodelling, is a maladaptive process
characterised by cardiomyocyte apoptosis, fibrosis, thinning of
the ventricular wall at the site of infarction, ventricular chamber
enlargement and hypertrophy of surviving cardiomyocytes
(Pfeffer & Braunwald, 1990; Swynghedauw, 1999; Udelson
et al., 2003). These events may, eventually, lead to heart failure
which is frequently lethal despite current best care. Therefore,
intervention to minimise pathological cardiac remodelling is
highly desirable to reduce the mortality and the incidence and
severity of congestive heart failure after MI.
Various intracellular signalling pathways are thought to play
a critical role in the myocardial response to ischaemia and
consequent pathological remodelling. Multiple mitogen-activated protein kinase (MAPK) are activated during ischaemia
and may contribute to the structural and functional changes.
MAPK are highly conserved serine/threonine kinases that are
activated by a dual phosphorylation of a Thr-X-Tyr motif, in
response to wide a variety of stimuli such as cytokines, osmotic
and other environmental stresses and consequently play a role in
numerous cell functions including growth and proliferation
(English et al., 1999; Pearson et al., 2001). Three of the five
major MAPK cascades have been extensively studied in the
heart: extracellular signal-regulated kinase (ERK1 and ERK2),
c-Jun N-terminal kinases (JNK1 and JNK2) and p38 kinases. It
has been shown that JNK and p38 contribute to, whereas ERK/
ERK2 protect against, apoptotic cell death. Although the mechanisms by which p38 and JNK induce apoptosis may be cell and
stimulus specific, there is overwhelming evidence that the
activation of p38-MAPK (or p38) that occurs during prolonged
ischaemia accelerates injury since its inhibition by pharmacological or genetic means slows the rate of infarction/death
(Saurin et al., 2000; Martin et al., 2001; see Table 1). Although
this evidence is based on animal data, it seems likely similar
mechanisms operate in the human heart since p38 is identically
activated by ischaemia (Han et al., 1995; Cain et al., 1999; Cook
et al., 1999; Lee et al., 2000; Lemke et al., 2001) and early
clinical trials indicate a potential benefit (de Winter et al., 2005).
Thus, superficially at least, inhibitors of p38 have therapeutic
potential in ischemic heart disease (Force et al., 2004).
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p38-MAPK are activated by a wide range of extracellular
influences, including radiation, ultraviolet light, heat shock,
osmotic stress, proinflammatory cytokines such as interleukin
(IL)-1 and tumour necrosis factor (TNF)-α, and certain mitogens
(Sugden & Clerk, 1998) in addition to myocardial ischaemia
(Bogoyevitch et al., 1996; Saurin et al., 2000; Luss et al., 2000;
Ping & Murphy, 2000). Furthermore, the consequent activation
of p38-MAPK is intimately involved in multiple cellular responses, including growth, proliferation, differentiation, and
death (English et al., 1999; Ono & Han, 2000). Perhaps not
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The catalytic site of p38 lies in a pocket between the N- and 125
C-terminal domains. These domains are connected by a single 126
hinge and the L16 loop of the C-terminal domain which wraps 127
back around the N-terminal domain and controls the relationship 128
between the relatively rigid domains (see Fig. 2). In addition, in 129
the inhibitor bound nonphosphorylated state, there is a mis- 130
alignment between the N- and C-lobes which prevents the 131
cooperation between a lysine residue (Lys53) in the N-terminal 132
lobe and aspartic acid residue (Asp168) in the C-terminal lobe, 133
imperative to binding and stabilization of the α phosphate group 134
and adjacent ribose of ATP, respectively (Wilson et al., 1996; 135
Gum et al., 1998). Therefore, it is widely thought that the 136
nondual phospho- form of p38 is inactive as a result of steric 137
obstruction of the peptide-binding channel and low ATP affinity. 138
Thr180 and Tyr182 are located on a flexible “activation loop” 139
that guards the active site. Dual phosphorylation of these 2 140
amino acids in response to exotoxin, cytokines, physical stress 141
(such as hyperosmolarity), and chemical oxidant stress, such as 142
hydrogen peroxide (Han et al., 1994; Freshney et al., 1994; 143
Rouse et al., 1994; Raingeaud et al., 1995) is thought to cause the 144
activation loop to refold and move out of the peptide-binding 145
channel. This movement is then thought to exert a “crank- 146
handle” effect on the overall tertiary structure of the kinase 147
reorienting the N- and C-, terminal lobes so that Lys53 and 148
Asp168 move towards one another by 2.5–5 Å. This alters the 149
conformation of the catalytic site enabling the cooperation 150
necessary for ATP binding and allowing substrate access 151
(Wilson et al., 1996; Diskin et al., 2007). The docking grooves 152
used by substrates and activators consist of 2 regions (see Fig. 2), 153
the CD region and the ED region (Tanoue et al., 2000). The CD 154
region is part of a shallow groove formed by the acidic residues 155
Asp313, Asp316 , Glu81, and the aromatic residues Phe129 and 156
Please cite this article as: Clark, J. E. et al. Potential of p38-MAPK inhibitors in the treatment of ischaemic heart disease. Pharmacol Ther (2007), doi:10.1016/j.
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4. Mechanisms of p38-MAPK activation
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4.1. Mitogen-activated protein kinase kinases
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Although the intracellular activation cascade for p38 under
most physiological conditions is still unclear, several upstream
MAPK kinases (MKK) have been identified from in vitro
analysis, including MKK3 and MKK6 (Derijard et al., 1995;
Han et al., 1996). MKK4 is predominately involved in JNK
activation but is able to activate p38-MAPK, at least, in vitro
(Deacon & Blank, 1997). Using MKK-targeted mouse lines, it
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has been shown that, in response to most stress stimuli, MKK3
and MKK6 are the principal MKK activating p38α and β,
respectively (see Fig. 1). MKK3 and MKK6 are in turn activated by phosphorylation by a MKK kinase (MKKK). The
MKKK, being responsible for activation of the p38 cascade,
appears to be cell type and stimulus specific, and several have
been implicated (Yamaguchi et al., 1995; Moriguchi et al.,
1996; Ichijo et al., 1997; Hutchison et al., 1998; Gallo &
Johnson, 2002; Ge et al., 2002; Cheung et al., 2003).
However, p38 activation is not limited to this traditional
phospho-relay signalling cascade. Since SB203508 (the most
widely used p38 kinase inhibitor) occupies the catalytic site,
without inhibiting upstream MKK, it should only inhibit the
phosphorylation events downstream of p38 without inhibiting
the dual-phosphorylation of p38 itself (Young et al., 1997).
However, certain conditions, such as myocardial ischaemia,
cause a SB-sensitive form of p38 dual phosphorylation. Two
mutually exclusive explanations for these observations are
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Tyr311. The ED region is part of a deeper groove formed by
residues 159–163 at one side and residues Gln120 , His126 and
Phe129 at the opposite side (Haar et al., 2007). It is believed that
these 2 binding regions facilitate activator (MAPK kinase 3,
MKK3) and substrate (MK2 and MEF2) binding (Chang et al.,
2002; Haar et al., 2007).
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Fig. 1. Mechanisms of p38-MAPK activation. Classical activation by MKK3/MKK6 is depicted as mechanism ①. TAB1-induced autoactivation is depicted as
mechanism ②. TCR-mediated Tyr323 phorphorylation by ZAP70 is depicted as mechanism ③. TAB1 activates TAK1, which in turn activates MKK3/MKK6. In
addition, TAB1 is a p38 substrate. PhosphoTAB1 is less able to activate TAK1 (★). Pharmacological inhibition of p38-MAPK diminishes p38 dual phosphorylation
and phosphoTAB-1. Heavy lines represent an interaction; dotted lines represent a modification (phosphorylation); open arrows represent a multi-element pathway.
Please cite this article as: Clark, J. E. et al. Potential of p38-MAPK inhibitors in the treatment of ischaemic heart disease. Pharmacol Ther (2007), doi:10.1016/j.
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Ge and co-workers elegantly reported that auto-phosphorylation of p38 can occur, facilitated by an interaction with the nonenzymatic adaptor protein transforming growth factor-βactivated protein kinase-1 (TAK1) binding protein-1 (TAB1;
Ge et al., 2002). TAB1 is known to perform a similar function by
inducing the autophosphorylation of TAK1, which in turn
activates MKK3/MKK6. In vitro co-expression experiments
have shown that the interaction of TAB1 and p38α leads to
phosphorylation of the TGY activation motif. TAB1-dependent
p38α activation appears to play a role in the injury response
during myocardial ischaemia (Tanno et al., 2003; Fiedler et al.,
2006), myocyte-derived dendritic cell maturation (Matsuyama
et al., 2003), and peripheral T-cell anergy maintenance (OhkusuTsukada et al., 2005). The interpretation of the TAB1-p38
interaction was, however, complicated by Cohen's group who
demonstrated that the phosphorylation of TAB1 on Ser423 and
Tyr431 was p38-MAPK-dependent and hence prevented by
SB203580. The authors proposed a feedback control mechanism
of TAK1 activity, whereby p38 activity inhibits TAK1, through
the phosphorylation of TAB1. Inhibition of p38 activity (by
SB203580) abolishes this feedback control of TAK1, causing
unopposed activation of the parallel JNK pathway and
consequently IKK (Cheung et al., 2003). This is depicted in
Fig. 1. Although TAB1 and MKK3/MKK6 mechanisms interact
through the potential modulation of TAK1, other possibilities
also exist. For example there is some evidence to suggest that
TAB1 causes p38 redistribution to the cytoplasm and may
restrict access to downstream targets, such as MAPK-activated
protein kinase 2 (MAPKAPK2). This is in direct contrast to the
pattern seen with MKK3/MKK6 (Lu et al., 2005).
Using MKK3/MKK6 double knockout and MKK4/MKK7
double knock out mouse embryonic fibroblasts (MEF), Kang
et al. have shown that peroxynitrite-induced phosphorylation of
p38α is associated with an ∼85 kDa disulfide complex in wild
type MEF (Kang et al., 2006). This association was diminished
in MKK3/MKK6 knockout MEF (Kang et al., 2006). The
authors suggested that phosphorylation of p38 mediated by
TAB-1 can be modulated by a yet unknown binding partner(s) in
a manner dependent on a disulfide complex (Kang et al., 2006).
In addition to TAB-1-mediated activation, p38 can also
autophosphorylate through an alternative pathway in response
to T-cell antigen receptor (TCR) activation (Dong et al., 2002;
Rincon & Pedraza-Alva, 2003). In this pathway, activation of
TCR leads to recruitment of a Syk family kinase, ZAP-70,
which directly phosphorylates p38 on Tyr323 (Rouse et al.,
1994). In a recent study it was shown that the phosphorylation
of p38 on Try323 can be blocked in the presence of the DNA
damage inducible gene Gadd45a, an autoimmune suppressor.
Absence of Gadd45a has been shown to result in chronic
phosphorylation of p38, T-cell hyperproliferation and autoimmunity (Salvador et al., 2005a, 2005b). Following ZAP-70
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phosphorylation of p38, an autophosphorylation event similar
to that induced by interaction with TAB-1 occurs resulting in
dual phosphorylation, and activation of the kinase.
However, regulation of p38 kinase activity in vitro, at least is
not solely dependent on upstream kinases and binding partners.
Diskin and co-workers, using a in vitro mutation approach, made
intrinsically active p38 isoforms based on activating mutations
previously found in the yeast MAPK kinase p38/Hog1 (Bell
et al., 2001). Single and multiple point mutations of human p38α
resulted in high intrinsic activity independent of activation by
dual phosphorylation. Structural analysis of these p38 mutants
has identified a hydrophobic core stabilised by 3 aromatic
residues, Tyr69, Phe327 and Trp337, in the vicinity of the L16
Loop region. It is believed that the hydrophobic core is an inherent stabiliser that maintains the low basal activity level of
unphosphorylated p38 (Diskin et al., 2004). Upon activation,
however, a segment of the L16 Loop, including Phe327 becomes
disordered allowing ATP and substrate binding. The mutation of
these amino acids involved in the hydrophobic core results in the
conformational changes imposed naturally by dual phosphorylation, namely destabilising the hydrophobic core and locking
the kinase in a constitutively active state. In addition, in this
active state, p38 is able to autophosphorylate in an invitro kinase
assay (Diskin et al., 2004). More recently p38β, p38γ and p38δ
mutants were similarly constructed (Askari et al., 2007). In these
mutants, a highly conserved aspartic acid located in the activation loop (Asp170 in Hog-1; Asp176 in p38α, p38β and p38δ;
and Asp179 in p38) was mutated. The spontaneous kinase
activity of p38β, p38γ and p38δ appeared to be lower than the
dual phosphorylated wild-type isoforms whereas the p38α
isoform presented the highest spontaneous activity. Therefore, it
is apparent that modifications of the amino acids in the hydrophobic core along with the mutations in Asp176 are capable of
activating p38s (Askari et al., 2007), the former likely explaining
the mechanism by which ZAP-70 induces autoactivation
(Mittelstadt et al., 2005). In addition, these mutants provide a
tool to dissect isoform-specific downstream signalling (Askari
et al., 2007).
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(i) that p38 is able to autophosphorylate its activation loop or
(ii) that SB203580 inhibits a kinase upstream of p38 involved in
its activation by trans-phosphorylation during ischaemia.
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5. p38, myocardial ischaemia and ischaemic heart disease
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Ischaemic heart disease remains the leading cause of death, 284
accounting for approximately 1 quarter of all deaths in the 285
United Kingdom. Currently, the most effective method of 286
reducing mortality in such patients is to achieve rapid 287
reperfusion by lysis or mechanical disruption of the occlusive 288
coronary thrombus and plaque. The mortality from acute MI 289
under these circumstances is inversely related to the amount of 290
myocardial salvage achieved by reperfusion.
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There is increasing evidence from preclinical investigations 292
that inhibition of p38 during prolonged ischaemia slows the rate 293
of infarction/death and inhibits the production of inflammatory 294
cytokines, such as TNF-α, IL-1 and IL-8, which aggravate 295
ischaemic injury (Young et al., 1997; see Table 1 for summary). 296
It was fist demonstrated as early as 1996 that p38α and β 297
are activated in response to ischaemia and reperfusion in the 298
heart (Bogoyevitch et al., 1996). Since then, using gene transfer 299
Please cite this article as: Clark, J. E. et al. Potential of p38-MAPK inhibitors in the treatment of ischaemic heart disease. Pharmacol Ther (2007), doi:10.1016/j.
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punctate distribution (Court NW et al., 2002), and p38δ mRNA 357
is broadly expressed in a wide variety of mouse and human 358
tissues including the heart (Wang et al., 1997; Beardmore et al., 359
2005). The C-terminal tail of p38γ allows its interaction with 360
PDZ domains of its substrate protein(s) and association with 361
α1-syntrophin and SAP90/PSD95 in skeletal muscle and 362
neuronal synapses, respectively (Zhang et al., 2003; Hasegawa 363
& Cahill, 2004; Sabio et al., 2004). p38γ-catalysed phosphory- 364
lation of hDlg (the mammalian homologue of the Drosophila 365
tumour suppressor Dlg) triggers its dissociation from the 366
cytoskeleton, indicating that this may regulate the integrity of 367
intercellular-junctional complexes, cell shape, volume and cell 368
polarity in response to many kinds of external stimuli. In support 369
of these findings, Parker et al. identified a novel p38δ substrate as 370
stathmin, a cytoplasmic protein that was previously reported to be 371
a substrate of several intercellular signalling kinases which have 372
been linked to regulation of microtubule (MT) dynamics in a 373
phosphorylation-dependent manner (Belmont & Mitchison, 374
1996). This may suggest that a common theme in p38 pathway 375
activation may be the re-organisation of the cytoskeletal frame- 376
work to enhance cell survival in times of stress such as ischaemia 377
(Parker et al., 1998). Moreover, both p38γ and p38δ phos- 378
phorylate the MT-associated protein Tau in neurons in vivo. 379
Hyperphosphorylated Tau is the major component of the paired 380
helical filaments, which constitute one of the main neuropatho- 381
logical hallmarks of many neurodegenerative disorders (Sabio 382
et al., 2005). So, although there is only circumstantial evidence to 383
support a role for γ and δ isoforms in the heart, this is likely an 384
emerging area of research. In the absence of isoform-selective 385
pharmacological inhibitors, this is, to some extent, aided by the 386
availability of a number of p38 isoform-targeted mouse lines 387
(Beardmore et al., 2005; Sabio et al., 2005) and spontaneously 388
active mutants (Askari et al., 2007).
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techniques, the α isoform that has been implicated in myocyte
apoptosis, consistent with the findings that this isoform alone
contributes to cell death following ischaemia (Saurin et al.,
2000; Martin et al., 2001). p38s phosphorylate a number of
known transcription factors to alter their transactivating potential influencing gene expression. However, the immediate
downstream targets of p38 that aggravate myocardial injury
are still largely unknown. One downstream substrate of p38α
is MAPKAP2, which can, in turn, phosphorylate HSP27, a
heat shock protein, which is thought to confer a number of protective effects (Kim et al., 2005). In addition, phosphorylation of
MAPKAP2 can also result in phosphorylation of factors that
transactivate cytokine genes, such as TNF-α, a cytokine implicated in chronic heart failure. Interestingly, TNF-α also
activates p38 and thus p38 has been considered as the keystone
in an autoamplifying cytokine cascade by most investigators and
an attractive target for antiinflammatory drug development (Lee
et al., 2000; Kuma et al., 2005).
Furthermore, a proapoptotic role for p38α and/or p38β
during myocardial ischaemia is suggested by protection of cardiac myocytes from ischaemic damage using a selective p38α/
p38β isoform inhibitor, SB203580 (Wang et al., 1998a). Using
adenoviral-mediated expression of p38α and p38β in rat neonatal cardiomyocytes our group have previously shown that after
2.5 hr simulated ischaemia p38α was activated, whereas p38β
activation was significantly inhibited (Saurin et al., 2000).
Inhibition of p38α activation during prolonged ischaemia, but
not β, resulted in an increase in cell viability (Saurin et al., 2000).
This strongly supported Wang et al. who suggested that p38α
activation in cardiac myocytes is sufficient to cause apoptosis
whereas activation of the β isoforms leads to protection and
hypertrophy (Wang et al., 1998a). However, there is some evidence to suggest that p38 isoforms may have potential protective
function and suggest a possible adverse effect of prolonged p38
inhibition in the heart. Glembotski's laboratory have demonstrated chronic activation of p38 through overexpression of
MKK6 in the heart can result in improved functional recovery
from ischaemia and MI (Martindale et al., 2005). The protective
role of p38β has also been investigated in a recent study by Kim
and co-workers who have shown that activation of p38β by
carbon monoxide promotes the nuclear translocation of heat
shock factor-1 (HSF-1), which regulates the expression of
cytoprotective HSP70 in cells and tissues (Kim et al., 2005).
HSF-1 can also serve as a negative regulator of proinflammatory
genes, including IL-1β, and TNF-α (Xie et al., 2002). The role
of p38s (the α isoform predominantly) in myocardial ischaemic
injury has been studied extensively since the findings of (Wang
et al. 1998a). These studies, which in the main suggest p38
activation during ischaemia worsens injury and depresses LV
function, are too numerous to review and appear in Table 1.
There is little information in the literature regarding the roles
of either the γ and δ isoform of p38 in the myocardium during
ischaemia. Conserved cardiac expression of p38γ amongst several different species suggests that this isoform may play an
important role in the heart and therefore is unlikely to be functionally redundant (Court NW et al., 2002). p38γ is localised in
the cytoplasm of the cardiac myocyte and is reported to have a
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6. Pharmacological inhibitors of p38-MAPK
Early efforts in drug discovery of small molecule inhibitors of
kinases were met with scepticism that selectivity could ever be
accomplished, due to the high degree of structural similarity in
the adenosine binding pocket among the entire kinome. Thus, it
was somewhat of a surprise when SB203580, the first reported
p38 inhibitor, emerged showing selectivity over the closely
related JNK and ERK MAPK families (Lantos et al., 1984). The
pyridinyl imidazole antiinflammatory agents were soon shown
to be highly selective p38 inhibitors and the bi-cyclic pyridinyl
imidazole SKF-86002 was the first compound reported to inhibit
LPS-stimulated cytokine production (Lee et al., 1988, 1994). It
was not long before investigators explored dual 5-lipooxygenase/cyclooxygenase (LO/COX) and cytokine inhibition as
potential mechanisms for the potent anti-inflammatory activity
of these compounds (Lee et al., 1993), and subsequently,
SB203580 was used as a pharmacological inhibitor to study the
cascade of kinases (via p38) involved in cytokine production
(Gallagher et al., 1997). The structures of representative classes
of p38 inhibitors are shown in Fig. 3.
The crystal structures of pyridinyl imidazole-p38α complexes
have recently become available and suggest that SB203580 binds
Please cite this article as: Clark, J. E. et al. Potential of p38-MAPK inhibitors in the treatment of ischaemic heart disease. Pharmacol Ther (2007), doi:10.1016/j.
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ketones, indole amides, diamides, quinazolinones, and pyridylaino-quinazolines (Cirillo et al., 2002). Unlike the imidazolebased p38α inhibitors, the urea-containing inhibitors act in a
noncompetitive manner (Kulkarni et al., 2007). Crystallographic
studies of urea-containing p38α inhibitors, such as BIRB-796,
have revealed that these compounds bind to at a site remote from
ATP pocket, and induce a significant movement of Phe169, such
that this residue fills the ATP pocket, preventing ATP binding
(Pargellis et al., 2002). Thus, inhibitors of p38 can be divided
into 2 groups dependent upon their mode of binding to p38; these
are (i) active site or “gatekeeping” inhibitors (such as SB203580)
and (ii) those which bind remotely and interfere with ATP binding indirectly (such as BIRB-796).
Enthusiasm for “blanket” pharmacological inhibition of p38
is tempered by the fact that this kinase is involved in innumerable biological processes and therefore not surprising that
under many circumstances its activation leads to myocardial
protection rather than injury (Weinbrenner et al., 1997; Craig
et al., 2000; Hoover et al., 2000; Communal et al., 2000; Force
et al., 2004; Zheng & Zuo, 2004; Martindale et al., 2005). This
particularly seems to be the case when p38 activation occurs as a
consequence of an intervention that precedes lethal myocardial
ischaemia, such as ischaemic or pharmacological preconditioning (Nagarkatti & Afi, 1998; Marais et al., 2001; Sanada et al.,
2001). However, in these studies the same inhibitor, at the same
concentration, reduces injury if present solely during lethal
ischaemic injury (Nagarkatti & Afi, 1998; Marais et al., 2001;
Sanada et al., 2001; Tanno et al., 2003). The cause of this apparent paradoxical observation may relate to an attenuation of
p38 activation during lethal ischaemia by its prior transient
activation. Thus there is ample evidence from cardiac as well as
other research fields that p38 activation can have beneficial
consequences whilst it is also incontrovertible that restricting
p38 inhibition to the activation that accompanies lethal
myocardial ischaemia reduces infarction (Nagarkatti & Afi,
1998; Marais et al., 2001; Sanada et al., 2001; Marais et al.,
2005; Table 1).
However, if we are to consider currently available p38
inhibitors with the aim of treating chronic conditions, it is likely
greater levels of selectivity will be required to avoid the inhibition of beneficial forms of activation. As more kinases are
investigated, a better understanding of selectivity over the
kinome has followed. With expanded kinase panels, it has now
emerged that “classic” p38α inhibitors like SB203580, which
were once described as being selective over certain kinases, now
have been shown to have similar, and sometimes lower IC50s.
These actions are a result of similarities in the inhibitory binding
site or in particular the hydrophobic pocket equivalent to that
formed by Thr106 (Fabian et al., 2005). So if broad inhibition of
p38 is not the answer, what is? At present, the majority of
pharmacological inhibitors of p38 are selective for the α and β
isoforms of the kinase. It is clear from the published data that the
during prolonged ischaemia the α isoform plays an important
role in the progression of dysfunction. Perhaps a more rational
approach to inhibit p38 in a site- and condition-specific manner
might be to target the activation of the kinase pathway upstream
of p38 itself, such as TAB1 or MKK3/MKK6. Using a model of
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to the active site of both phosphorylated (active) and unphosphorylated (inactive) p38 in an ATP-competitive manner (see
Figs. 2 and 3). These inhibitors bind to an aryl-specificity pocket
behind the site, which is normally occupied by the adenine ring of
ATP. The interaction occurs between the 4-pyridinyl group
(analogous to the N-1 adenine of ATP) and the N-H of Met109. In
addition, studies have also have implicated Thr106 as a key residue
conferring selectivity (Wilson et al., 1996; Gum et al., 1998). The
2 adjacent residues, His107 and Leu108, along with Thr106 lie at the
back of the ATP pocket and are identical in p38α and p38β, but
are different in p38γ and p38δ (Met106, Pro107 and Phe108, respectively) which are insensitive to SB203580 inhibition. Using a
mutagenesis approach it has been shown that if these 3 residues in
p38α and p38β are changed to Met-Pro-Phe (as found in p38γ
and p38δ) the mutant kinase is no longer inhibited by SB203580.
By contrast, introduction of the Thr-His-Leu sequence of p38α
into p38γ or p38δ confers sensitivity to SB203580. Taken
together, these studies have identified Thr106 as the key residue
forming the aryl-specificity pocket (Saccani et al., 2002).
In addition to novel prydinyl imidazole compounds, a new
group of selective p38 inhibitors are the arly-pyridinyle-heterocylces. In these compounds, the imidazole core is replaced by
other heteroaryl scaffolding, and consequently these compounds
generally exhibit in vitro potency similar to prydinyl imidazoles
(Boehm et al., 2001). Other structurally diverse p38 inhibitors
include a subset of novel non-aryl-pyridinyls such as triazanapthalenones, N,N′-diary ureas, benzzophenones, pyrazole
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Fig. 2. Crystal structure of p38 with SB203580 occupying the ATP binding site.
The Thr106 residue (4), which is important for binding of pyridinyl imidazole
inhibitors, and the 2 residues within the activation loop that are phosphorylated
(Thr180 (2) and Try182 (1)) are highlighted. Tyr323 (3), which has been implicated
in TCR-mediated activation of p38 is also shown. SB203580 is shown in green.
The activation loop is shown in orange. ED and CD activator/substrate binding
regions are highlighted. The C-terminal extension that forms the L16 loop
bridging the domains is also indicated (created in JenaLib Jmol; PDB entry
1a9u; Wang et al., 1998b).
9
Please cite this article as: Clark, J. E. et al. Potential of p38-MAPK inhibitors in the treatment of ischaemic heart disease. Pharmacol Ther (2007), doi:10.1016/j.
pharmthera.2007.06.013
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Fig. 3. Structure of representative classes of p38 MAPK inhibitors. p38 inhibitors can be divided into 2 groups dependant upon their mode of binding to p38; active site
inhibitors, such as SB203580 and RJW-67657, bind competitively to the ATP site of the enzyme whereas others bind remotely and interfere with ATP binding
indirectly (such as BIRB-796).
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coronary artery ligation in a mkk3-targetted mouse line, we have
recently demonstrated that removing MKK3 does not alter
pathological remodelling and progression to ventricular dys-
function after MI (Clark et al., 2007). Maybe this is not sur- 499
prising considering the multitude of pathways involved in 500
ischaemia and inflammation but it does, perhaps, highlight the 501
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7. Clinical trials of p38-MAPK inhibition
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8. Summary and conclusions
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Despite the apparent dichotomy in preclinical research of the
consequences of p38 inhibition, 2 types of molecular inhibitors
of p38, aryl-prydinyl heterocycles (namely SB242235 and RWJ67657) and non-aryl-prydinyl heterocycles (VX-745, BIRB-796
and RO3201195), have so far advanced to clinical trials. Results
of phase I and early phase II trials were also promising. In one
study, orally administered SB242235 (1–500 mg), which has
been shown to have potent antiinflammatory effects in a rat
model of arthritis (Badger et al., 2000), was well tolerated and
suppressed production of TNF-α, IL-1β, IL-6, and IL-8 in a
dose-dependent manner within 3 hr (Adams et al., 2001). A
randomised clinical study to examine the efficacy of RWJ-67657
to combat the effects of endotoxin on normal healthy volunteer
was recently carried out. Development of flu-like symptoms,
which were associated with raised serum levels of TNF-α, IL-6
and IL-8, were reduced in a dose dependent manner by RWJ67657 (Fijen et al., 2001).
However, clinical studies have not been limited to healthy
controls; VX-745 (Vertex) has been given to patients with active
rheumatoid arthritis (Haddad, 2001; Weisman, 2002), and although the drug is well tolerated, it was associated with adverse
effects, such as elevation in liver transaminases. Preclinical safety
evaluations of this drug in animals have revealed that at that
concentrations VX-745 can cross the blood–brain barrier and
exert adverse neurological side effects (Weisman, 2002). For these
reasons, further investigation on VX-745 has been suspended. The
rise in the level of liver transaminases has also been observed in a
series of double-blinded, randomised, placebo-controlled studies
of BIRB-796 in healthy volunteers. This compound also suppressed neutrophil activation ex vivo, but no inhibition of LPSinduced TNF-α production was observed (Wood et al., 2002).
Currently, the most clinically advanced p38 inhibitors are the
Scios compound (now Johnson & Johnson) SCIO-323 for treatment of stroke and the Vertex compound VX-702, which has
been tested in patients with acute coronary syndromes in whom
percutaneous coronary intervention (PCI) is planned. There
were no adverse side effects and the serum level of C-reactive
protein (CRP), which is considered a risk factor in MI patients,
was suppressed (de Winter et al., 2005). However, although
many p38 inhibitors have advanced to phase I, II or III clinical
trial, many of these studies have been stopped prematurely due
to adverse side effects. One reason for this might be the crossreactivity against other kinases or other cellular signalling molecules. An alternative explanation is that p38-dependant signalling is vital to normal cell function necessitating a greater
understanding of mechanisms of activation in the hope that this
will reveal circumstance-specific targets.
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biological processes, such as cell growth, apoptosis and inflammation. In the heart p38 is activated by various pathological
conditions, such as ischaemia and pressure overload. Studies
using genetic and pharmacological inhibitors of p38 suggests
that this kinase plays a key role in left ventricular matrix remodelling (Petrich & Wang, 2004), cell survival following
ischaemia–reperfusion (Ma et al., 1999; Shao et al., 2006) and
possibly, in post MI remodelling (Ren et al., 2005). Investigators
have shown that multiple isoforms of p38 are activated in
response to specific stimuli and take part in distinct signalling
pathways, which result in activation of specific downstream
substrates. From the existing evidence, it appears that p38α and
p38β are differentially regulated during myocardial stresses and
that the consequences of activation of each isoform may differ by
cell type. This highlights the likelihood that different members
within a single kinase family can play distinct roles in the heart
during ischaemia. Despite continued interest in the p38 pathway
few studies to date have addressed the role of p38 isoforms other
than p38α during ischaemia. Furthermore, since there are no
isoform-specific pharmacological inhibitors of p38 activity, the
contribution of each isoform remains unclear.
Understanding the physiological roles of each p38 isoform
and identifying their mechanism(s) of activation and potential
substrates are important avenues that may lead to pharmacological inhibitors with greater circumstance selectivity thereby
avoiding the potential pitfalls of chronic systemic inhibition.
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potential importance of other pathways which warrant further
investigation.
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The p38 kinase pathway has been studied intensely since its
discovery in the early 1990s. It has been implicated in various
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