Structure-activity relationships of p38 mitogen-activated protein kinase inhibitors

Rheumatoid arthritis and other chronic inflammatory diseases constitute a major therapeutic challenge, usually not sufficiently met by the classical antiinflammatory medications. Recent research efforts provided new insights into the molecular basis of these pathologies and disclosed new opportunities for developing improved drugs directed to the chemical mediators of the disease. The enzyme p38 MAP kinase plays a central role in the signal transduction cascade that leads to the production of both the proinflammatory cytokines, TNF-alpha and IL-1 beta, thus representing an attractive therapeutic target for novel antiinflammatory therapies. A number of p38 inhibitors belonging to different structural families have been developed as potential antiinflammatory drugs, and some of them progressed into clinical trials. The initial pyridinyl imidazole inhibitors contributed to the identification and characterization of p38 MAP kinase as the molecular target of these new drugs, and were found to act as competitive inhibitors at the ATP binding site of the enzyme. A number of variations in the pyridine and imidazole rings were subsequently introduced. Other inhibitors structurally unrelated to the pyridinylimidazoles have also been developed, such as the pyridopyridazinones, diaryl ureas, aminobenzophenones and aromatic amides. One of these structural classes, the N,N'-diarylureas, has been found to interact with a distinct allosteric site of p38 MAP kinase and requires a deep conformational change prior to binding.     

Inhibitors of p38 mitogen-activated protein kinase for the treatment of rheumatoid arthritis

The p38 mitogen-activated protein kinase pathway is involved in a number of cellular processes critical to the development of rheumatoid arthritis. The activation and infiltration of leukocytes as well as the production of inflammatory cytokines are p38-dependent processes. In addition, p38 regulates the differentiation of osteoclasts, which are directly involved in bone loss. Numerous inhibitors of p38 have demonstrated efficacy in animal models of arthritic disease and at least two p38 inhibitors are currently in phase II clinical trials for rheumatoid arthritis. Several other p38 inhibitors are currently undergoing phase I clinical trials.     

Therapeutic potential of inhaled p38 mitogen-activated protein kinase inhibitors for inflammatory pulmonary diseases

Background: Over the past two decades, p38 MAPK (mitogen-activated protein kinase) has been the subject of intense multidisciplinary research. p38 MAPK inhibitors have been shown to be efficacious in several disease models, including rheumatoid arthritis, psoriasis, Crohn's disease, and stroke. Recent studies support a role for p38 MAPK in the development, maintenance, and/or exacerbation of a number of pulmonary diseases, such as asthma, cystic fibrosis, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease (COPD). Objective: Many previous attempts to develop p38 MAPK inhibitors have failed as a result of unacceptable safety profiles. These toxicities have been varied and are believed to derive from different off-target effects. Method: The above concerns can be overcome by delivering the compound locally to minimize whole-body burden, resulting in low exposure to the gastrointestinal, liver, and CNS. This review discusses the role of p38 MAPK in various inflammatory diseases, followed by the toxicity concerns associated with p38 MAPK inhibition. It also highlights the possible beneficial effect of delivering drugs via the inhalation route. Conclusion: We present proof-of-principle confirming the therapeutic potential of inhaled p38 inhibitors for asthma and other inflammatory pulmonary diseases.     

Investigational p38 inhibitors for the treatment of chronic obstructive pulmonary disease

Introduction: The p38 protein kinases, in particular p38α and p38β, regulate the production of multiple inflammatory mediators. Consequentially, considerable effort has been focused on trying to develop p38 inhibitors for the treatment of inflammatory diseases. Some 20 p38 inhibitors have progressed to clinical development, mostly for the treatment of rheumatoid arthritis, but with little success. Increasingly, interest has turned to their use in other indications and notably chronic obstructive pulmonary disease (COPD).

Areas covered: In this review, the author discusses the eight p38 inhibitors that have been clinically evaluated. Acumapimod is the only one of four orally delivered inhibitors that remains in active development while Phase II results of PH-797804 and losmapimod are compared. The activity of two inhibitors designed for inhaled delivery, RV-568 and PF-03715455, is compared but little is known about AZD-7624 or the discontinued GSK-610677.

Expert opinion: Results from animal models provide a clear rationale for developing p38 inhibitors for COPD, and appear to be (partially) validated by the efficacy seen with PH-797804 and losmapimod. Inhaled delivery provides the opportunity to enhance p38 inhibition in the lung while reducing unwanted systemic effects of p38 inhibition. Validation of this hypothesis should come from the results of the recently completed Phase II study with RV-568.     

Inhibitory effect of p38 mitogen-activated protein kinase inhibitors on cytokine release from human macrophages

BACKGROUND AND PURPOSE: Macrophages release cytokines that may contribute to pulmonary inflammation in conditions such as chronic obstructive pulmonary disease. Thus, inhibition of macrophage cytokine production may have therapeutic benefit. p38 MAPK may regulate cytokine production, therefore, the effect of two p38 MAPK inhibitors, SB239063 and SD-282, on the release of TNF-alpha, GM-CSF and IL-8 from human macrophages was investigated. EXPERIMENTAL APPROACH: Cytokine release was measured by ELISA. Immunoblots and mRNA expression studies were performed to confirm p38 MAPK isoform expression and activity. Macrophages were isolated from lung tissue of current smokers, ex-smokers and emphysema patients and exposed to lipopolysaccharide. These cells then released cytokines in a concentration-dependent manner. KEY RESULTS: SB239063 only inhibited TNF-alpha release (EC50 0.3 +/- 0.1 microM). Disease status had no effect on the efficacy of SB239063. SD-282 inhibited both TNF-alpha and GM-CSF release from macrophages (EC50 6.1 +/- 1.4 nM and 1.8 +/- 0.6 microM respectively) but had no effect on IL-8 release. In contrast, both inhibitors suppressed cytokine production in monocytes. CONCLUSIONS AND IMPLICATIONS: The differential effects of p38 MAPK inhibitors between macrophages and monocytes could not be explained by differences in p38 MAPK isoform expression or activity. However, the stability of TNF-alpha mRNA was significantly increased in macrophages compared to monocytes. These data suggest a differential involvement for p38 MAPK in macrophage cytokine production compared with monocytes. These effects are not due to lack of p38 activation or p38alpha expression in macrophages but may reflect differential effects on the stability of cytokine mRNA.     

Role of p38 mitogen-activated protein kinase in cardiac remodelling

BACKGROUND AND PURPOSE: Mitogen-activated protein kinases (MAPK) are centrally involved in several mechanisms important for heart failure such as apoptosis, activation of inflammatory responses and cell proliferation. We therefore evaluated the effect of the selective p38 MAPK inhibitor SB 239063 on progression of left ventricular remodelling after myocardial infarction (MI) in rats. EXPERIMENTAL APPROACH: Rats were treated for 9 weeks with placebo or SB 239063 by gavage (15 mg kg(-1)) twice daily starting 7 days after ligation of the left anterior descending artery. Serial transthoracic echocardiography was performed at days 7, 36 and 70. KEY RESULTS: Over the 9 weeks, mortality was not different between the groups. On echocardiography, animals after myocardial infarction exhibited significant left ventricular dilatation as expected (week 10, end-systolic diameter, placebo sham 5.21+/- 0.34 vs. placebo MI 8.44+/- 0.57 mm). However, there was no difference between placebo and SB 239063-treated rats (week 10, end-systolic diameter, SB MI 7.76+/- 0.74 mm, not significantly different from placebo MI). Haemodynamics changed accordingly. Moreover, SB 239063 had no effect on left ventricular hypertrophy. Treatment with SB 239063 significantly reduced cytokine expression of tumour necrosis factor and interleukin-1beta after myocardial infarction. However, collagen content was not influenced by the treatment. CONCLUSION: Despite a reduction of inflammation, treatment with the p38 inhibitor SB 239063 does not affect cardiac remodelling and cardiac function when treatment is started 7 days after myocardial infarction.     

Mercury induces multidrug resistance-associated protein gene through p38 mitogen-activated protein kinase

The multidrug resistance-associated protein (MRP1) belongs to a drug efflux membrane pump that confers multidrug resistance to the cells. The MRP1 mediates the cellular efflux of various xenobiotics including heavy metals and mediates cellular resistance to heavy metals. Mercury is a well-known health hazard and an environmental contaminant. Recently, information about the uptake of the heavy metals such as mercury has been suggested. However, little is known regarding molecular mechanisms of exporting mercury. This study was designed to determine if mercury could be extruded by MRP1 in acute myeloid leukemia cells (AML-2). The MRP-1-overexpressing AML-2/DX100 cells showed a higher resistance to mercury than AML-2/WT. Probenecid, which is a specific MRP1 inhibitor, decreased the resistance to mercury. Exposing the AML-2 cells to mercury-induced MRP1 gene expression and production without altering the MRP1 activity. Mercury activated p38 mitogen-activated protein kinase (MAPK) and SB 203580, a specific p38 MAPK inhibitor, blocked the mercury-induced MRP1 production. These results suggest that MRP1 can control mercury and p38 MAPK mediates the mercury-induced MRP1 gene expression.     

Novel mitogen-activated protein kinase kinase inhibitors

INTRODUCTION: the development of new drugs over the last few decades has targeted specific proteins thought to be a key to the disease state. MAPK kinases 1 and 2 (commonly known as MEK1-2) represent such proteins as they lie downstream of important drug targets for oncology, such as EGFR, RAS and RAF. Several MEK1-2 inhibitors are currently in Phase I and II clinical trials in oncology. AREAS COVERED: this review of current literature and recent conferences provides a background on the RAS-RAF-MEK-ERK signaling pathway and a discussion of early MEK inhibitors. The potential of MEK1-2 inhibitors for the treatment of inflammation is briefly presented. Preclinical and early clinical results are discussed for MEK inhibitors currently in development. Completed clinical trials of MEK inhibitors in oncology include some disappointments as well as some promising signs of the value of these compounds and we discuss the potential for MEK inhibitors as monotherapy and their use in drug combinations. EXPERT OPINION: the utility of MEK inhibitors as anticancer agents will depend on careful patient selection based on the presence of mutations in genes such as KRAS and BRAF, the identification of additional predictive biomarkers, and an improved understanding of the benefit of drug combinations utilizing both established and emerging therapeutics.     

Enzyme fragment complementation binding assay for p38alpha mitogen-activated protein kinase to study the binding kinetics of enzyme inhibitors

The majority of protein kinase assays used in drug discovery research are enzyme activity assays. These assays are based on the measurement of phosphorylated protein or peptide substrate, which is the end product of the enzyme reaction. Since most kinase inhibitors are ATP competitive, prediction of the activity of compounds in cellular systems based on potency values in enzyme activity assays is complex, as this should take into account the affinity of the enzyme for ATP and the cellular ATP concentration. The fact that some of the most successful kinase inhibitors, such as STI 571 (imatinib mesylate, Gleevec, Novartis Pharmaceuticals, East Hanover, NJ), act through binding to the inactive isoform of the kinase provides another limitation of enzyme activity assays. Binding assays allow separate measurement of compound affinity to active and inactive kinase and do not require ATP or substrate in the reaction. Recently, a non-radioactive kinase binding assay for p38 mitogen-activated protein kinase has become available from DiscoveRx (Fremont, CA). The assay method, called HitHunter, utilizes enzyme fragment complementation of Escherichia coli beta-galactosidase to generate an assay signal by chemiluminescence. We have reconfigured the commercial assay kit to study the binding kinetics of two known reference inhibitors of the alpha-isoform of p38, the pyridinyl imidazole SB 203580 and the diaryl urea BIRB 796. Our data confirm the slow association kinetics of BIRB 796 as compared to SB 203580, which corresponded with the requirement of a relatively long preincubation time to obtain maximal effect in a cellular assay. Although neither of the two compounds showed preference for either active or inactive p38alpha, our data demonstrate that the HitHunter kinase binding assay can be used to select compounds that specifically target inactive kinase.     

p38 mitogen-activated protein kinase (MAPK) in rheumatoid arthritis

The importance of p38 MAPK inhibitors as new drug for rheumatoid arthritis is reflected by the large number of compounds that has been developed over the last years. In this review new insights such as non-stressful activation of p38 MAPK, and the role of p38 MAPK in regulation of NF-kappaB recruitment are also discussed.     

p38 mitogen-activated protein kinase mediates sidestream cigarette smoke-induced endothelial permeability

Second-hand smoke is associated with increased risk of cardiovascular diseases. So far, little is known about the signaling mechanisms of second-hand smoke-induced vascular dysfunction. Endothelial junctions are fundamental structures important for maintaining endothelial barrier function. Our study showed that sidestream cigarette smoke (SCS), a major component of second-hand smoke, was able to disrupt endothelial junctions and increase endothelial permeability. Sidestream cigarette smoke stimulated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and myosin light chain (MLC). A selective inhibitor of p38 MAPK (SB203580) prevented SCS-induced loss of endothelial barrier integrity as evidenced by transendothelial resistance measurements. Resveratrol, an antioxidant that was able to inhibit SCS-induced p38 MAPK and MLC phosphorylation, also protected endothelial cells from the damage. Thus, p38 MAPK mediates SCS-induced endothelial permeability. Inhibition of p38 MAPK may have therapeutic potential for second-hand smoke-induced vascular injury.     

Allosteric p38 kinase inhibitors

Two applications claim N,N´-bisaryl urea derivatives as allosteric inhibitors of p38 MAPK and their use as anti-inflammatory agents. The compounds have been designed to stabilise a specific conformation of the kinase and to optimise selectivity by avoiding interactions with the ATP-binding domain of the kinase. They are the first application to describe the applicant’s p38 inhibitor programme, which is close to investigational new drug filing.     

Involvement of p38 mitogen-activated protein kinase in PLL-AGE-induced cyclooxgenase-2 expression

In the present study, murine RAW 264.7 macrophages were incubated with poly-L-lysine-derived advanced glycosylation end products (PLL-AGEs) to examine cyclooxygenase-2 protein expression. Treatment of RAW 264.7 cells with PLL-AGEs caused the dose-dependent expression of cylooxygenase-2 but not cylooxygenase-1 and an increase in cylooxygenase activity. Increased cylooxygenase-2 expression was seen at 6 h and reached a maximum at 24 h. The tyrosine kinase inhibitor, genistein, and the p38 mitogen-activated protein kinase (MAPK) inhibitor, [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] (SB 203580), inhibited PLL-AGE-induced cylooxygenase-2 expression, while the Ras inhibitor, FPT inhibitor II, and the MAP kinase kinase inhibitor, (2'-amino-3'-methoxyflavone) (PD 98059), had no effect on PLL-AGE-induced cylooxygenase-2 expression. Incubation of RAW 264.7 cells with PLL-AGEs resulted in activation of p38 MAPK, and this activation was suppressed by genistein and SB 203580. Taken together, our results suggest that activation of protein tyrosine kinase and p38 MAPK is involved in AGE-induced cyclooxygenase-2 expression in RAW 264.7 macrophages.     

Activation of p38 mitogen-activated protein kinase by formyl-methionyl-leucyl-phenylalanine in rat neutrophils

The signaling pathways leading to p38 mitogen-activated protein kinase (MAPK) activation in formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated rat neutrophils were examined. Immunoblot analysis with antibodies against a phosphorylated form of p38 MAPK showed that fMLP-stimulated p38 MAPK activation was dependent on a pertussis toxin-sensitive G protein. Two phosphatidylinositol 3-kinase inhibitors, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), did not affect the p38 MAPK activation. Phosphorylation of p38 MAPK was concentration dependently attenuated by a tyrosine kinase inhibitor, genistein, and by a Ca(2+)-dependent protein kinase C inhibitor, 13-cyanoethyl-12-methyl-6,7,12,13-tetrahydroindolo[2,3-a]pyrrolo[3 , 4-c]carbazole-7-one (G?6976). However, the protein kinase C inhibitors with a broader spectrum, 2-[1-(3-dimethylaminopropyl)-5-methoxy-1H-indol-3-yl]-3-(1H-indol-3-y l)-maleimide (G?6983) and 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimi de (GF109203X), had no inhibitory effect. fMLP-stimulated p38 MAPK phosphorylation was also reduced in cells pretreated with a phospholipase C inhibitor, 1-[6-((17beta-3-methoxyestra-1,3, 5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U73122), or preloaded with an intracellular Ca(2+) chelator, 1, 2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA). We conclude that phosphorylation of p38 MAPK by fMLP stimulation in rat neutrophils is dependent on G(i/o) protein, nonreceptor tyrosine kinase, phospholipase C/Ca(2+), and probably Ca(2+)-dependent protein kinase C pathways.     

Potentiation of cyclic AMP and cyclic GMP accumulation by p38 mitogen-activated protein kinase (p38MAPK) inhibitors in rat pinealocytes.

The effects of p38 mitogen-activated protein kinase (p38MAPK) inhibitors on the adrenergic-stimulated cyclic nucleotide production in rat pinealocytes were investigated. Treatment with SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)IH-imidazole] and SB203580 [4-(4-fluoropheny)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)IH-imidazole] (1-100 microM), two pyridinyl imidazole compounds that inhibit p38MAPK, as well as SB202474 [4-(ethyl)-2-(4-methoxyphenyl)-5-(4-pyridyl)IH-imidazole], an inactive analog, was effective in potentiating norepinephrine- and isoproterenol-stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) accumulation in a concentration-dependent manner. All three compounds caused a greater increase in the cGMP than the cAMP response, with SB202474 being substantially more potent than the two active analogs. At 100 microM, SB202474 potentiated the isoproterenol-stimulated cAMP and cGMP accumulation by 65 and 500%, respectively. Pharmacological studies indicated that the potentiating effect of SB202474 was independent of protein kinase C activation, intracellular calcium elevation, or serine/threonine phosphatase inhibition, three pathways known to potentiate the beta-adrenergic-stimulated cyclic nucleotide responses in rat pinealocytes. In contrast, the potentiating effect of SB202474 was abolished in the presence of a phosphodiesterase inhibitor, isobutylmethylxanthine. At 100 microM, all three compounds inhibited cAMP- and cGMP-phosphodiesterase activities by 50 and 80%, respectively. These results suggest that the commonly used p38MAPK inhibitors can modulate cyclic nucleotide responses through phosphodiesterase inhibition, a mechanism that appears to be independent of p38MAPK inhibition.     

New inhibitors of p38 kinase

In the short time since the 1994 report that the molecular target for the 4-aryl-5-pyridin-4-yl imidazole class of cytokine suppressive anti-inflammatory agents (exemplified by SB-203580) was the human homologue of the stress-induced kinase p38, there has been an explosion of patent literature disclosing related inhibitor analogues. These compounds fall into the general structural class of vicinal aryl/pyridin-4-yl heterocycles. Compounds of this class have been claimed as p38 inhibitors or inhibitors of cytokine biosynthesis. Since cytokines mediate a variety of disease processes, inhibition of cytokine biosynthesis has potential as a therapeutic target. The SAR of binding to p38 for this intensively studied class of compounds is now understandable on the basis of recent x-ray crystallographic and mutagenesis studies. The inhibitors can be sub-classified based upon a variety of structural characteristics. Recently new inhibitor structural classes have been disclosed. Although distinctly different structurally, several of these new compounds appear to bind with the same key interactions as the vicinal aryl/pyridin-4-yl heterocycles.     

Design and synthesis of inhaled p38 inhibitors for the treatment of chronic obstructive pulmonary disease

This paper describes the identification and optimization of a novel series of DFG-out binding p38 inhibitors as inhaled agents for the treatment of chronic obstructive pulmonary disease. Structure based drug design and "inhalation by design" principles have been applied to the optimization of the lead series exemplied by compound 1a. Analogues have been designed to be potent and selective for p38, with an emphasis on slow enzyme dissociation kinetics to deliver prolonged lung p38 inhibition. Pharmacokinetic properties were tuned with high intrinsic clearance and low oral bioavailability in mind, to minimize systemic exposure and reduce systemically driven adverse events. High CYP mediated clearance and glucuronidation were targeted to achieve high intrinsic clearance coupled with multiple routes of clearance to minimize drug-drug interactions. Furthermore, pharmaceutical properties such as stability, crystallinity, and solubility were considered to ensure compatibility with a dry powder inhaler. 1ab (PF-03715455) was subsequently identified as a clinical candidate from this series with efficacy and safety profiles confirming its potential as an inhaled agent for the treatment of COPD.     

New small-molecule inhibitors of mitogen-activated protein kinase kinase

Background: Overexpression of the Ras/Raf/MEK/ERK (extracellular-signal-regulated kinase) pathway is associated with the formation, progression and survival of tumours and has also been implicated in a diverse range of therapeutic areas such as arthritis, organ transplant rejection, asthma and developmental disorders. One approach to down regulation of this pathway is through the inhibition of mitogen-activated protein kinase kinase 1/2 (MEK1/2). Objective: The importance of the mitogen-activated protein kinase (MAPK) pathway, MEK1/2 as a therapeutic target and early MEK1/2 inhibitors is discussed, followed by an overview of recent patent activity in the area. Methods: The patent literature was searched for inhibitors of MEK1/2 published within the last three years; these results are described. Other relevant publications that provide further insight into the discovery and development of these compounds are also discussed. Conclusion: The determination of a crystal structure with inhibitor bound has allowed the design of exquisitely selective and potent inhibitors of MEK1/2. Several allosteric inhibitors have advanced to clinical trial and shown some efficacy in cancer as single agents, but the future application of MEK1/2 inhibitors is likely to be either in combination with other therapies or in disorders which are genetically defined as being dependent on the MAPK pathway.