p38 MAP Kinase Inhibition in Cardiovascular Disease
MAPKs are serine/threonine protein kinases that process and regulate cellular behavior in response to a range of extracellular stimuli. These enzymes phosphorylate the OH group of serine or threonine, and play a role in the regulation of cell proliferation, apoptosis, and embryonic development.
In mammalian cells, several distinct MAPKs have been identified, including four main subgroups (i) p38, (ii) c-jun N-terminal kinases (JNKs), (iii) extracellular signal-regulated kinases (ERKs 1/2), and (iv) ERK 5/BMK-1. The p38 and JNKs are involved in the regulation of cellular responses to environmental stressors such as tobacco smoke, osmotic stress, and pro-inflammatory cytokines, whereas the ERKs are generally activated by mitogenic stimuli.
The MAPKs constitute intracellular signaling pathways, which convey signals by phosphorylation events; thus, MAPKs are activated by dual phosphorylation of two residues (threonine and tyrosine) in their activation loop by upstream MAPK kinases (mitogen-activated protein kinase kinase [MKK]: MAP2K). These upstream kinases are activated in turn by MAP3K (mitogen-activated protein kinase kinase kinase [MAPKKK]: MAP3K) forming classical (canonical) activation pathways (Fig. 1). The p38 MAPKs have the amino acid glycine between the threonine and tyrosine residues (known as the Thr-Gly-Tyr motif) in their activation loop, in contrast to ERKs and JNKs, which have glutamine or proline instead.
(Enlarge Image)
Figure 1.
Schematic of MAPK signaling pathway. MAPK mitogenactivated protein kinase, MAPKK(K) mitogen-activated protein kinase kinase (kinase)
The major upstream activators (MKKs) of p38 are MKK3 and MKK6, with MKK4 (upstream kinase of JNK) reported as also making some contribution to the activation of p38. In addition, an alternative pathway involving TAB1 (transforming growth factor-β-activated protein kinase1 binding protein) has been described, which results in autophosphorylation of p38α, after interaction with TAB1. Downstream substrates of p38 MAPKs include transcription factors, other protein kinases, and proteins (Fig. 2).
(Enlarge Image)
Figure 2.
Substrates of p38α mitogen-activated protein kinase. HSP heat shock protein, MAPKAPK mitogen-activated protein kinase activated protein kinase
The p38 family comprises four isoforms: α, β, γ, and δ, with differences in function and tissue expression reported between isoforms. p38α and β are approximately 70 % identical, whereas p38γ and p38δ share approximately 60 % sequence identity with p38α. p38α and β are ubiquitously expressed, while the p38γ isoform is expressed in only skeletal and cardiac tissue and p38δ is found in certain adult tissue types such as lung and spleen.
p38α (often known as just 'p38') was the first isoform to be identified and was recognised as a 38-kDa protein activated by lipopolysaccharide (LPS) and inflammatory cytokines. It was also distinguished as the molecular target of pyridinyl imidazole compounds, known to inhibit tumor necrosis factor (TNF)-α and IL-1 in LPS-stimulated human monocytes. Subsequent to these discoveries, p38α is the best described of the isoforms and its relevance in inflammatory pathways, including within cardiovascular disease, is established by several preclinical studies. This review hence focuses on the p38α isoform.
p38α MAPK is a key component in the regulation of inflammatory mediators at the level of transcription and translation. Inflammatory mediators such as TNF-α, IL-1, IL-6, and IL-8 are commonly implicated in the detrimental inflammatory pathogenesis of different disease models. For example, rheumatoid arthritis (RA) is characterized by progressive destruction of articular cartilage, and inflammatory mediators (TNF-α and IL-1) found with the synovium are believed to contribute to the process of articular destruction. Likewise, in chronic obstructive pulmonary disease (COPD), inflammatory cytokines are implicated in its complex pathology. Preclinical studies have shown increased levels of p38 MAPKs in alveolar walls of COPD subjects, and reduced cytokine production in different lung cells of COPD patients following p38 MAPK inhibition.
In cardiovascular disease, the downstream substrates of p38α MAPKs, including TNF-α, IL-1, and heat shock proteins (HSPs) are understood to be major factors in the different stages of atherosclerosis. It is known that upstream activators of p38 include oxidized LDLs, which play a central role in foam cell formation in atherosclerotic plaque development. p38 signaling is also pivotal to the vascular inflammatory response. The generation of reactive oxygen species (ROS) is a main feature of vascular inflammation and leads to reduced nitric oxide (NO) bioavailability and smooth muscle contraction. p38 MAPK is considered to function in an amplification circuit both upstream and downstream of ROS generation.
In addition, it is understood p38α is activated during both myocardial ischemia and reperfusion injury, and animal models studies have indicated pharmacological inhibition of p38 can attenuate infarction size.
p38α is understood to be activated in harmful myocardial remodeling following infarction injury. In a preclinical animal model, it was shown that p38α increased the expression of genes associated with fibrosis, whilst p38β stimulated B-type natriuretic peptide transcription, thereby also indicating differences in function between isoforms.
Furthermore, p38α is thought to mediate a negative inotropic effect in cardiac myocytes, by reducing myofilament response to calcium ions. Pharmacological inhibition of p38 has also been shown to negate the negative inotropic effect of TNF-α, post-myocardial infarction.
The role of p38 MAPK in diseases where inflammation is detrimental has led to the evaluation of p38 pharmacological inhibition in a range of different diseases where systemic inflammation is a common finding.
Most p38 MAPK inhibitors are adenosine triphosphate (ATP)-competitive and target the highly conserved ATP-binding site of the kinase. Type 1 inhibitors are ATP-competitive, which bind to active and inactive forms of p38 and target the enzyme's hydrophobic pocket. The pyridinyl imidazole molecules (cytokine-suppressive anti-inflammatory drugs such as SB203580) represent type 1 inhibitors. Type 2 inhibitors do not directly compete with ATP for the binding pocket but induce a conformational change in p38 (allosteric inhibitors), which thus prevents ATP binding and subsequent activation by dual phosphorylation. The BIRB-796 compound is an example and inhibits all four isoforms of p38.
Over the last 2 decades, many different p38 MAPK inhibitors have been developed with variations in compound structures, with the aim to improve selectivity of target isoform(s) to improve efficacy and reduce systemic toxicity associated with this drug class. Adverse side effects associated with some of the earlier p38 MAPK inhibitor compounds tested were partly attributed to poor drug target selectivity resulting in adverse 'off-target' actions of inhibitors affecting other kinases.
Mitogen-Activated Protein Kinases (MAPKs)
MAPKs are serine/threonine protein kinases that process and regulate cellular behavior in response to a range of extracellular stimuli. These enzymes phosphorylate the OH group of serine or threonine, and play a role in the regulation of cell proliferation, apoptosis, and embryonic development.
In mammalian cells, several distinct MAPKs have been identified, including four main subgroups (i) p38, (ii) c-jun N-terminal kinases (JNKs), (iii) extracellular signal-regulated kinases (ERKs 1/2), and (iv) ERK 5/BMK-1. The p38 and JNKs are involved in the regulation of cellular responses to environmental stressors such as tobacco smoke, osmotic stress, and pro-inflammatory cytokines, whereas the ERKs are generally activated by mitogenic stimuli.
The MAPKs constitute intracellular signaling pathways, which convey signals by phosphorylation events; thus, MAPKs are activated by dual phosphorylation of two residues (threonine and tyrosine) in their activation loop by upstream MAPK kinases (mitogen-activated protein kinase kinase [MKK]: MAP2K). These upstream kinases are activated in turn by MAP3K (mitogen-activated protein kinase kinase kinase [MAPKKK]: MAP3K) forming classical (canonical) activation pathways (Fig. 1). The p38 MAPKs have the amino acid glycine between the threonine and tyrosine residues (known as the Thr-Gly-Tyr motif) in their activation loop, in contrast to ERKs and JNKs, which have glutamine or proline instead.
(Enlarge Image)
Figure 1.
Schematic of MAPK signaling pathway. MAPK mitogenactivated protein kinase, MAPKK(K) mitogen-activated protein kinase kinase (kinase)
The major upstream activators (MKKs) of p38 are MKK3 and MKK6, with MKK4 (upstream kinase of JNK) reported as also making some contribution to the activation of p38. In addition, an alternative pathway involving TAB1 (transforming growth factor-β-activated protein kinase1 binding protein) has been described, which results in autophosphorylation of p38α, after interaction with TAB1. Downstream substrates of p38 MAPKs include transcription factors, other protein kinases, and proteins (Fig. 2).
(Enlarge Image)
Figure 2.
Substrates of p38α mitogen-activated protein kinase. HSP heat shock protein, MAPKAPK mitogen-activated protein kinase activated protein kinase
p38 Isoforms
The p38 family comprises four isoforms: α, β, γ, and δ, with differences in function and tissue expression reported between isoforms. p38α and β are approximately 70 % identical, whereas p38γ and p38δ share approximately 60 % sequence identity with p38α. p38α and β are ubiquitously expressed, while the p38γ isoform is expressed in only skeletal and cardiac tissue and p38δ is found in certain adult tissue types such as lung and spleen.
p38α (often known as just 'p38') was the first isoform to be identified and was recognised as a 38-kDa protein activated by lipopolysaccharide (LPS) and inflammatory cytokines. It was also distinguished as the molecular target of pyridinyl imidazole compounds, known to inhibit tumor necrosis factor (TNF)-α and IL-1 in LPS-stimulated human monocytes. Subsequent to these discoveries, p38α is the best described of the isoforms and its relevance in inflammatory pathways, including within cardiovascular disease, is established by several preclinical studies. This review hence focuses on the p38α isoform.
p38 MAPK in Disease
p38α MAPK is a key component in the regulation of inflammatory mediators at the level of transcription and translation. Inflammatory mediators such as TNF-α, IL-1, IL-6, and IL-8 are commonly implicated in the detrimental inflammatory pathogenesis of different disease models. For example, rheumatoid arthritis (RA) is characterized by progressive destruction of articular cartilage, and inflammatory mediators (TNF-α and IL-1) found with the synovium are believed to contribute to the process of articular destruction. Likewise, in chronic obstructive pulmonary disease (COPD), inflammatory cytokines are implicated in its complex pathology. Preclinical studies have shown increased levels of p38 MAPKs in alveolar walls of COPD subjects, and reduced cytokine production in different lung cells of COPD patients following p38 MAPK inhibition.
In cardiovascular disease, the downstream substrates of p38α MAPKs, including TNF-α, IL-1, and heat shock proteins (HSPs) are understood to be major factors in the different stages of atherosclerosis. It is known that upstream activators of p38 include oxidized LDLs, which play a central role in foam cell formation in atherosclerotic plaque development. p38 signaling is also pivotal to the vascular inflammatory response. The generation of reactive oxygen species (ROS) is a main feature of vascular inflammation and leads to reduced nitric oxide (NO) bioavailability and smooth muscle contraction. p38 MAPK is considered to function in an amplification circuit both upstream and downstream of ROS generation.
In addition, it is understood p38α is activated during both myocardial ischemia and reperfusion injury, and animal models studies have indicated pharmacological inhibition of p38 can attenuate infarction size.
p38α is understood to be activated in harmful myocardial remodeling following infarction injury. In a preclinical animal model, it was shown that p38α increased the expression of genes associated with fibrosis, whilst p38β stimulated B-type natriuretic peptide transcription, thereby also indicating differences in function between isoforms.
Furthermore, p38α is thought to mediate a negative inotropic effect in cardiac myocytes, by reducing myofilament response to calcium ions. Pharmacological inhibition of p38 has also been shown to negate the negative inotropic effect of TNF-α, post-myocardial infarction.
Pharmacological Inhibition of p38
The role of p38 MAPK in diseases where inflammation is detrimental has led to the evaluation of p38 pharmacological inhibition in a range of different diseases where systemic inflammation is a common finding.
Most p38 MAPK inhibitors are adenosine triphosphate (ATP)-competitive and target the highly conserved ATP-binding site of the kinase. Type 1 inhibitors are ATP-competitive, which bind to active and inactive forms of p38 and target the enzyme's hydrophobic pocket. The pyridinyl imidazole molecules (cytokine-suppressive anti-inflammatory drugs such as SB203580) represent type 1 inhibitors. Type 2 inhibitors do not directly compete with ATP for the binding pocket but induce a conformational change in p38 (allosteric inhibitors), which thus prevents ATP binding and subsequent activation by dual phosphorylation. The BIRB-796 compound is an example and inhibits all four isoforms of p38.
Over the last 2 decades, many different p38 MAPK inhibitors have been developed with variations in compound structures, with the aim to improve selectivity of target isoform(s) to improve efficacy and reduce systemic toxicity associated with this drug class. Adverse side effects associated with some of the earlier p38 MAPK inhibitor compounds tested were partly attributed to poor drug target selectivity resulting in adverse 'off-target' actions of inhibitors affecting other kinases.