Pyridinyl Imidazole Research Articles

Acquisition of Sensitivity of Stress-activated Protein Kinases to the p38 Inhibitor, SB 203580, by Alteration of One or More Amino Acids within the ATP Binding Pocket

Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase compete with ATP for binding. Mutation of 23 residues in the ATP pocket indicated that several residues which affected binding of pyridinyl imidazole photoaffinity cross-linker 125I-SB 206718 did not affect kinase activity, and vice versa, suggesting that pyridinyl imidazoles bind p38 differently than ATP. Two close homologues of p38, SAPK3 and SAPK4, are not inhibited by SB 203580 and differ from p38 by three amino acids near the hinge of the ATP pocket. Substitution of the three amino acids in p38 by those in SAPK3/4 (Thr-106, His-107, and Leu-108 to Met, Pro, and Phe) resulted in decreased 125I-SB 206718 cross-linking and loss of inhibition by SB 203580. Substitution of just Thr-106 by Met resulted in incomplete loss of inhibition. Conversely, substitution of the three amino acids of p38 into SAPK3, SAPK4, or the more distantly related JNK1 resulted in inhibition by SB 203580, whereas mutation of just Met-106 to Thr resulted in weaker inhibition. These results indicate that these three amino acids can confer specificity and sensitivity to SB 203580 for at least two different classes of MAPKs.

Induction of Apoptosis by SB202190 through Inhibition of p38β Mitogen-activated Protein Kinase

p38, a subfamily of the mitogen-activated protein kinase, regulates gene expression in response to various extracellular stimuli. The pyridinyl imidazoles like SB202190 are specific inhibitors of p38alpha and p38beta and have been widely used in investigation of the biological functions of p38. Here we show that SB202190 by itself was sufficient to induce cell death, with typical apoptotic features such as nucleus condensation and intranucleosomal DNA fragmentation. SB202190 stimulated the activity of CPP32-like caspases, and its apoptotic effect was completely blocked by the protease inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and expression of bcl-2. In addition, SB202190 was able to potentiate apoptosis induced by Fas(APO-1) ligation or UV irradiation. Expression of p38beta attenuated the apoptotic effect of SB202190 and the cell death induced by Fas ligation and UV irradiation. In contrast, expression of p38alpha induced cell death mildly. These results indicate that SB202190 induces apoptosis through activation of CPP32-like caspases and suggest that distinct members of the p38 subfamily of mitogen-activated protein kinase have different functions in apoptosis.

Conversion of SB 203580-insensitive MAP kinase family members to drug-sensitive forms by a single amino-acid substitution.

Specific inhibitors of protein kinases have great therapeutic potential, but the molecular basis underlying their specificity is only poorly understood. We have investigated the drug SB 203580 which belongs to a class of pyridinyl imidazoles that inhibits the stress-activated protein (SAP) kinases SAPK2a/p38 and SAPK2b/p38 beta 2 but not other mitogen-activated protein kinase family members. Like inhibitors of other protein kinases, SB 203580 binds in the ATP-binding pocket of SAPK2a/p38. The SAP kinases SAPK1 gamma/JNK1, SAPK3 and SAPK4 are not inhibited by SB 203580, because they have methionine in the position equivalent to Thr106 in the ATP-binding region of SAPK2a/p38 and SAPK2b/p38 beta 2. Using site-directed mutagenesis of five SAP kinases and the type I and type II TGF beta receptors, we have established that for a protein kinase to be inhibited by SB 203580, the sidechain of this residue must be no larger than that of threonine. Sensitivity to inhibition by SB 203580 is greatly enhanced when the sidechain is even smaller, as in serine, alanine or glycine. Thus, the type I TGF beta receptor, which has serine at the position equivalent to Thr106 of SAPK2a/p38 and SAPK2b/p38 beta 2, is inhibited by SB 203580. These findings explain how drugs that target the ATP-binding site can inhibit protein kinases specifically, and show that the presence of threonine or a smaller amino acid at the position equivalent to Thr106 of SAPK2a/p38 and SAPK2b/p38 beta 2 is diagnostic of whether a protein kinase is sensitive to the pyridinyl imidazole class of inhibitor.

Osmolyte Strategy in Human Monocytes and Macrophages: Involvement of p38MAPKin Hyperosmotic Induction of Betaine and Myoinositol Transporters

Betaine and myoinositol are compatible organic osmolytes which are specifically accumulated by cells exposed to hyperosmotic medium. A role for compatible organic osmolytes in the regulation of immune function for rat liver macrophages has been described recently. This report describes an osmolyte strategy in human peripheral blood monocytes and human peripheral blood-derived macrophages. Hyperosmotic (405 mOsm) exposure of monocytes and macrophages led to an upregulation of betaine/γ-amino-n-butyric acid (GABA) transporter BGT-1 and sodium-dependent myoinositol transporter SMIT in mRNA levels within 6 to 12 h. Induction of BGT-1 and SMIT mRNA occurred regardless of whether hyperosmolarity was induced by addition of NaCl (50 mM) or raffinose (100 mM). Betaine (5 mM) inhibited upregulation of BGT-1 as well as SMIT mRNA. After hyperosmotic (405 mOsm) exposure uptake of betaine and myoinositol was increased up to 10-fold compared to normoosmotic conditions. Hypoosmotic exposure led to a rapid efflux of betaine and myoinositol. Treatment of cells with the pyridinyl imidazole SB 203580 (10 μM), a specific inhibitor of p38 MAP kinase, inhibited the hyperosmolarity-induced increase in BGT-1 and SMIT mRNA as well as betaine and myoinositol uptake by 45–70%. The data show that human peripheral blood monocytes and human peripheral blood-derived macrophages use betaine and myoinositol are compatible organic osmolytes when exposed to osmotic stress and that p38MAPKis involved in hyperosmolarity-induced upregulation of osmolyte transporters BGT-1 and SMIT.

Inhibitors of p38 mitogen-activated protein kinase promote neuronal survival in vitro.

Mammalian mitogen-activated protein kinases include the extracellular signal-regulated protein kinase, the c-Jun amino-terminal kinase, and the p38 subgroups. Sustained activation of Jun kinase and p38 have been shown to precede apoptosis of PC12 pheochromocytoma cells induced by withdrawal of trophic factors. To investigate the possible role of p38 in neuronal apoptosis, we tested the effect of two selective p38 inhibitors, the pyridinyl imidazole compounds SB203580 and SB202190, on different populations of chick embryonic neurons in vitro. Both substances promoted the in vitro survival of sensory, sympathetic, ciliary and motor neurons in a dose-dependent fashion. When assayed in nerve growth factor-stimulated PC12 cells, SB203580 pretreatment inhibited the activation of both ribosomal S6 kinases-1 and -2 with the same IC50 (approximately 30 microM) that inhibited apoptosis in primary neurons. Thus, p38 inhibitor-sensitive pathways may be involved in apoptosis of neurotrophic factor-deprived primary neurons, and in activation of ribosomal S6 kinases.

Interferon-gamma expression by Th1 effector T cells mediated by the p38 MAP kinase signaling pathway

Signal transduction via MAP kinase pathways plays a key role in a variety of cellular responses, including growth factor-induced proliferation, differentiation and cell death. In mammalian cells, p38 MAP kinase can be activated by multiple stimuli, such as pro-inflammatory cytokines and environmental stress. Although p38 MAP kinase is implicated in the control of inflammatory responses, the molecular mechanisms remain unclear. Upon activation, CD4+ T cells differentiate into Th2 cells, which potentiate the humoral immune response or pro-inflammatory Th1 cells. Here, we show that pyridinyl imidazole compounds (specific inhibitors of p38 MAP kinase) block the production of interferon-gamma (IFNgamma) by Th1 cells without affecting IL-4 production by Th2 cells. These drugs also inhibit transcription driven by the IFNgamma promoter. In transgenic mice, inhibition of the p38 MAP kinase pathway by the expression of dominant-negative p38 MAP kinase results in selective impairment of Th1 responses. In contrast, activation of the p38 MAP kinase pathway by the expression of constitutivelyactivated MAP kinase kinase 6 in transgenic mice caused increased production of IFNgamma during the differentiation and activation of Th1 cells. Together, these data demonstrate that the p38 MAP kinase is relevant for Th1 cells, not Th2 cells, and that inhibition of p38 MAP kinase represents a possible site of therapeutic intervention in diseases where a predominant Th1 immune response leads to a pathological outcome. Moreover, our study provides an additional mechanism by which the p38 MAP kinase pathway controls inflammatory responses.

Inhibitors of p38 mitogen‐activated protein kinase promote neuronal survival in vitro

Mammalian mitogen-activated protein kinases include the extracellular signal-regulated protein kinase, the c-Jun amino-terminal kinase, and the p38 subgroups. Sustained activation of Jun kinase and p38 have been shown to precede apoptosis of PC12 pheochromocytoma cells induced by withdrawal of trophic factors. To investigate the possible role of p38 in neuronal apoptosis, we tested the effect of two selective p38 inhibitors, the pyridinyl imidazole compounds SB203580 and SB202190, on different populations of chick embryonic neurons in vitro. Both substances promoted the in vitro survival of sensory, sympathetic, ciliary and motor neurons in a dose-dependent fashion. When assayed in nerve growth factor-stimulated PC12 cells, SB203580 pretreatment inhibited the activation of both ribosomal S6 kinases-1 and -2 with the same IC50 (approximately 30 μM) that inhibited apoptosis in primary neurons. Thus, p38 inhibitor-sensitive pathways may be involved in apoptosis of neurotrophic factor-deprived primary neurons, and in activation of ribosomal S6 kinases. J. Neurosci. Res. 52:483–490, 1998. © 1998 Wiley-Liss, Inc.

Selective Activation of p38 Mitogen-activated Protein (MAP) Kinase Isoforms by the MAP Kinase Kinases MKK3 and MKK6

The cellular response to treatment with proinflammatory cytokines or exposure to environmental stress is mediated, in part, by the p38 group of mitogen-activated protein (MAP) kinases. We report the molecular cloning of a novel isoform of p38 MAP kinase, p38 beta 2. This p38 MAP kinase, like p38 alpha, is inhibited by the pyridinyl imidazole drug SB203580. The p38 MAP kinase kinase MKK6 is identified as a common activator of p38 alpha, p38 beta 2, and p38 gamma MAP kinase isoforms, while MKK3 activates only p38 alpha and p38 gamma MAP kinase isoforms. The MKK3 and MKK6 signal transduction pathways are therefore coupled to distinct, but overlapping, groups of p38 MAP kinases.

Coordinate regulation of stress- and mitogen-activated protein kinases in the apoptotic actions of ceramide and sphingosine.

We characterized participation of the stress-activated protein kinase (SAPK) cascade in the lethal actions of the cytotoxic lipid messengers ceramide and sphingosine in U937 human monoblastic leukemia cells. Acute exposure of U937 cells to either lipid resulted in loss of proliferative capacity, degradation of genomic DNA, and manifestation of apoptotic cytoarchitecture. Ceramide robustly stimulated p46-JNK1/p54-JNK2 activity and increased expression of c-jun mRNA and c-Jun protein; in contrast, sphingosine moderately stimulated p46-JNK1/p54-JNK2 and failed to modify c-jun/c-Jun expression. Dominant-negative blockade of normal c-Jun activity by transfection with the TAM-67 c-Jun NH2-terminal deletion mutant abolished the lethal actions of ceramide but was without effect on those of sphingosine, indicating that ceramide-related apoptosis is directly dependent on activation of c-Jun, whereas sphingosine-induced cell death proceeds via an unrelated downstream mechanism. Characterization of the mitogen-activated protein kinase (MAPK) cascade in these responses revealed a further functional disparity between the two lipids: basal p42-ERK1/ p44-ERK2 activity was gradually reduced by ceramide but immediately and completely suppressed by sphingosine. Moreover, blockade of the MAPK cascade by the aminomethoxyflavone MEK1 inhibitor PD-98059 unexpectedly activated p46-JNK1/p54-JNK2 and induced apoptosis in a manner qualitatively resembling that of sphingosine. Both lipids sharply increased p38-RK activity; selective pharmacological inhibition of p38-RK by the pyridinyl imidazole SB-203580 failed to mitigate the cytotoxicity associated with either ceramide or sphingosine, suggesting that p38-RK is not essential for lipid-induced apoptosis. These findings demonstrate that reciprocal alterations in the SAPK and MAPK cascades are associated with the apoptotic influence of either lipid inasmuch as (i) ceramide-mediated lethality is primarily associated with strong stimulation of SAPK and weak inhibition of MAPK, whereas (ii) sphingosine-mediated lethality is primarily associated with weak stimulation of SAPK and strong inhibition of MAPK. We therefore propose that leukemic cell survival depends on the maintenance of an imbalance of the outputs from the MAPK and SAPK systems such that the dominant basal influence of the MAPK cascade allows sustained proliferation, whereas acute redirection of this balance toward the SAPK cascade initiates apoptotic cell death.

Characterization of the Structure and Function of the Fourth Member of p38 Group Mitogen-activated Protein Kinases, p38δ

We have cloned and characterized a new member of the p38 group of mitogen-activated protein kinases here termed p38delta. Sequence comparisons revealed that p38delta is approximately 60% identical to the other three p38 isoforms but only 40-45% to the other mitogen-activated protein kinase family members. It contains the TGY dual phosphorylation site present in all p38 group members and is activated by a group of extracellular stimuli including cytokines and environmental stresses that also activate the other three known p38 isoforms. However, unlike the other p38 isoforms, the kinase activity of p38delta is not blocked by the pyridinyl imidazole, 4-(4-fluorophenyl)-2-2(4-hydroxyphenyl)-5-(4-pyridyl)-imidazole (identicalto SB202190). p38delta can be activated by MKK3 and MKK6, known activators of the other isoforms. Nonetheless, in-gel kinase assays provide evidence for additional activators. The data presented herein show that p38delta has many properties that are similar to those of other p38 group members. Nonetheless important differences exist among the four members of the p38 group of enzymes, and thus each may have highly specific, individual contributions to biologic events involving activation of the p38 pathways.

P38-2, a Novel Mitogen-activated Protein Kinase with Distinct Properties

Mitogen-activated protein (MAP) kinases are involved in many cellular processes. Here we describe the cloning and characterization of a new MAP kinase, p38-2. p38-2 belongs to the p38 subfamily of MAP kinases and shares with it the TGY phosphorylation motif. The complete p38-2 cDNA was isolated by polymerase chain reaction. It encodes a 364-amino acid protein with 73% identity to p38. Two shorter isoforms missing the phosphorylation motif were identified. Analysis of various tissues demonstrated that p38-2 is differently expressed from p38. Highest expression levels were found in heart and skeletal muscle. Like p38, p38-2 is activated by stress-inducing signals and proinflammatory cytokines. The preferred upstream kinase is MEK6. Although p38-2 and p38 phosphorylate the same substrates, the site specificity of phosphorylation can differ as shown by two-dimensional phosphopeptide analysis of Sap-1a. Additionally, kinetic studies showed that p38-2 appears to be about 180 times more active than p38 on certain substrates such as ATF2. Both kinases are inhibited by a class of pyridinyl imidazoles. p38-2 phosphorylation of ATF2 and Sap-1a but not Elk1 results in increased transcriptional activity of these factors. A sequential kinetic mechanism of p38-2 is suggested by steady state kinetic analysis. In conclusion, p38-2 may be an important component of the stress response required for the homeostasis of a cell.

Apoptosis Induced by Withdrawal of Trophic Factors Is Mediated by p38 Mitogen-activated Protein Kinase

p38 is a member of the mitogen-activated protein (MAP) kinase superfamily activated by stress signals and implicated in cellular processes involving inflammation and apoptosis. Unlike the extracellular signal-regulated kinases (p42 and p44 MAP kinases), which are stimulated by insulin in many cell types, p38 activity is inhibited by insulin in postmitotic fetal neurons for which insulin is a potent survival factor (Heidenreich, K. A., and Kummer, J. L. (1996) J. Biol. Chem. 271, 9891-9894). These data suggested that insulin's effects on neuronal survival are mediated by inhibition of a p38-mediated apoptotic pathway. To better understand the relationship between p38 activity and cell survival, we induced apoptosis in two cell lines and examined the ability of insulin or a specific p38 inhibitor (a pyridinyl imidazole compound PD169316) to block p38 activity and cell death. In Rat-1 fibroblasts grown in the presence of serum, p38 activity was undetectable by immune complex assays, and the number of apoptotic cells was very low (<0.5%). After the removal of serum for 16 h, p38 activity was markedly elevated, and apoptosis increased by 14-15-fold. Insulin (50 ng/ml) inhibited p38 activity by approximately 70% and blocked apoptosis by at least 80%. PD169316 also blocked p38 enzyme activity and apoptosis by approximately 80%. Similar results were obtained in differentiated PC12 cells that were deprived of nerve growth factor (NGF) for 16 h. In the presence of NGF, p38 activity and the number of apoptotic cells was very low (approximately 1.0%). After NGF withdrawal, p38 activity was selectively elevated and apoptosis increased to 15%. Both insulin and PD169316 markedly blocked the increase in p38 activity and apoptosis. The MAP kinase kinase inhibitor, PD98059, had no effect on apoptosis in Rat-1 fibroblasts and only partially blocked apoptosis in PC12 cells. PD98059 did not influence insulin's ability to block apoptosis, indicating that the extracellular signal-regulated kinase pathway does not mediate insulin's survival effects. These data further support the role of p38 in cellular apoptosis and support the hypothesis that insulin promotes cell survival, at least in part, by inhibiting the p38 pathway.

Novel Homologues of CSBP/p38 MAP Kinase: Activation, Substrate Specificity and Sensitivity to Inhibition by Pyridinyl Imidazoles

A novel homologue of p38 MAP kinase, called SAPK4, has been cloned which shares 61% amino acid identity with p38 and is expressed predominantly in testes, pancreas and small intestine. We also cloned an alternative form of p38β, termed p38β2, which lacks the additional 8 amino acid insertion unique to p38β. p38, p38β, p38β2, ERK6/p38γ/SAPK3, and SAPK4 were characterized with respect to stimulus-dependent activation in transfected cells, substrate specificity, and sensitivity to inhibition by pyridinyl imidazoles. All homologues were stimulated, although to differing extents, by IL-1β, TNF, sorbitol, and UV. Only SAPK3 and SAPK4 were stimulated significantly by PMA. p38β showed the weakest activation overall. MBP, ATF-2, and both MAPKAP kinase-2 and kinase-3 were good substrates of p38 and p38βin vitro.In contrast, only MBP, ATF2, and MAPKAP kinase-3 proved to be significant substrates of SAPK3 and SAPK4, and of these three, MAPKAP kinase-3 was by far the weakest. p38β had very poor kinase activity for all substrates except MBP. While both p38 and p38β2 were comparably inhibited by SB 203580 and SB 202190, neither SAPK3 nor SAPK4 were inhibited. p38β was partially inhibited by both inhibitors. These data suggest that SAPK3 and SAPK4 form a distinct subset of the p38 MAP kinases with different expression pattern, response to stimuli, substrate specificity, and inhibitor sensitivity.

Pyridinyl Imidazole Inhibitors of p38 Mitogen-activated Protein Kinase Bind in the ATP Site

The site of action of a series of pyridinyl imidazole compounds that are selective inhibitors of p38 mitogen-activated protein kinase in vitro and block proinflammatory cytokine production in vivo has been determined. Using Edman sequencing, 125I-SB206718 was shown to cross-link to the nonphosphorylated Escherichia coli-expressed p38 kinase at Thr175, which is proximal to the ATP binding site. Titration calorimetric studies with E. coli-expressed p38 kinase showed that SB203580 bound with a stoichiometry of 1:1 and that binding was blocked by preincubation of p38 kinase with the ATP analogue, FSBA (5'-[p-(fluorosulfonyl)benzoyl]adenosine), which covalently modifies the ATP binding site. The intrinsic ATPase activity of the nonphosphorylated enzyme was inhibited by SB203580 with a Km of 9.6 mM. Kinetic studies of active, phosphorylated yeast-expressed p38 kinase using a peptide substrate showed that SB203580 was competitive with ATP with a Ki of 21 nM and that kinase inhibition correlated with binding and biological activity. Mutagenesis indicated that binding of 125I-SB206718 was dependent on the catalytic residues K53 and D168 in the ATP pocket. These findings indicate that the pyridinyl imidazoles act in vivo by inhibiting p38 kinase activity through competition with ATP and that their selectivity is probably determined by differences in nonconserved regions within or near the ATP binding pocket.

Actions of IL-1 are selectively controlled by p38 mitogen-activated protein kinase: regulation of prostaglandin H synthase-2, metalloproteinases, and IL-6 at different levels.

The role of p38 mitogen-activated protein kinase (MAPK) in responses of human fibroblasts and vascular endothelial cells to IL-1 was investigated by use of a pyridinyl imidazole compound (SB 203580), which specifically inhibits the enzyme. SB 203580 inhibited (50% inhibitory concentration approximately 0.5 microM) IL-1-induced phosphorylation of heat shock protein 27 (an indicator of p38 MAPK activity) in fibroblasts without affecting the other known IL-1-activated protein kinase pathways (p42/p44 MAPK, p54 MAPK/c-Jun N-terminal kinase and beta-casein kinase). SB 203580 significantly inhibited IL-1-stimulated IL-6, (30 to 50% at 1 microM) but not IL-8 production from human fibroblasts (gingival and dermal) and umbilical vein endothelial cells. IL-1 induction of steady state level of IL-6 mRNA was not significantly inhibited, which is consistent with p38 MAPK regulating IL-6 production at the translational level. SB 203580 strongly inhibited IL-1-stimulated PG production by fibroblasts and human umbilical vein endothelial cells. This was associated with the inhibition of the induction of PGH synthase-2 protein and mRNA. SB 203580 also inhibited the stimulation of collagenase-1 and stromelysin-1 production by IL-1 without affecting synthesis of the tissue inhibitor of metalloproteinases (TIMP)-1. SB 203580 prevented the increase in collagenase-1 and stromelysin-1 mRNA stimulated by IL-1. In a model of cartilage breakdown, short-term IL-1-stimulated proteoglycan resorption and inhibition of proteoglycan synthesis were unaffected by SB 203580, while longer term collagen breakdown was prevented. It is concluded that 1) p38 MAPK plays an important role in the regulation of some, but not all, responses to IL-1, and 2) it is involved in the regulation of mRNA levels of some IL-1-responsive genes.

Synthesis of 4-[2-(4-Azidophenyl)-5-(3-iodo-125I-phenyl)-1H-imidazol-4-yl]pyridine (SB 206718-[125I]), a pyridinyl imidazole cytokine inhibitor

The pyridinyl imidazole cytokine-suppressing anti-inflammatory drug (CSAID), SB 206718 (1) was required in 125 I-labeled form for photoaffinity ligand studies. The target compound (SB 206718-[ 125 I], [ 125 I]1) was obtained via conversion of the highly functionalized 1 to a tributylstannyl derivative. Radioiododestannylation using Na 125 I in the presence of chloramine-T gave good radiochemical yields of the title compound (42-69%, four radiosyntheses) at high radiochemical purity (>98%) after HPLC purification at specific activities of 1670-1736 Ci/mmol.

Role for p38 Mitogen-activated Protein Kinase in Platelet Aggregation Caused by Collagen or a Thromboxane Analogue

p38 mitogen-activated protein kinase (MAPK) was identified in platelets on the basis of (a) its reactivity with antibodies to C-terminal and N-terminal peptides, and (b) its ability to activate MAPK-activated protein kinase-2, which phosphorylates the small heat shock protein, hsp27. p38 MAPK was activated in platelets by collagen fibers, a collagen-related cross-linked peptide, thrombin, or the thromboxane analogue U46619. A highly specific inhibitor of p38 MAPK, a pyridinyl imidazole known as SB203580, inhibited the platelet enzyme in vitro (IC50 approximately 0.5 microM). At similar concentrations it also inhibited agonist-stimulated phosphorylation of hsp27 in platelets, and platelet aggregation and secretion induced by minimal aggregatory concentrations of collagen or U46619, but not thrombin. Inhibition of aggregation was overcome by increasing agonist dose. SB203580 might act by inhibiting thromboxane generation, but this was only inhibited by 10-20% at low agonist concentrations. p38 MAPK provides a crucial signal, which is necessary for aggregation caused by minimal concentrations of collagen fibers or U46619. Thrombin or high doses of these agonists generate signals that bypass the enzyme, or render the enzyme no longer rate-limiting.

Dissection of Protein Kinase Cascades That Mediate Cellular Response to Cytokines and Cellular Stress

Publisher Summary To control the biological functions of proteins, it is therefore critical to be able to regulate the activities of protein kinases. Some protein kinases are controlled by allosteric effectors, such as the second messengers, cyclic AMP, calcium ions, and diacylglycerol, but others are themselves regulated by phosphorylation catalyzed by “kinase kinases” that operate in protein kinase “cascades.” The first protein kinase cascade to be identified was that involved in the adrenergic regulation of glycogenolysis in skeletal muscle. Over the past few years, work in a number of laboratories has largely elucidated the MAP kinase (MAPK) cascade that plays a critical role in mediating several of the intracellular actions of growth factors. In this pathway, the interaction of growth factors with their receptors triggers a series of protein-protein interactions at the plasma membrane that culminate in activation of the GTP-binding protein Ras. The MAPK cascade plays a critical role in amplifying the initial stimulus in a situation in which classical second messengers such as cyclic AMP and calcium ions may not exist. The pharmaceutical company SmithKline Beecham (SB) has developed a novel class of pyridinyl imidazoles called CSAIDS that inhibit the production of the cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF-α) in monocytes by bacterial lipopolysaccharide (LPS). They found that this drug binds specifically to one protein in monocytes, termed CSBP (CSAID-binding protein), which they isolated, cloned, and sequenced and showed was a novel MAPK homolog.

Localization of the Human Stress Responsive MAP Kinase-Like CSAIDs Binding Protein (CSBP) Gene to Chromosome 6p21.3/21.2

The proinflammatory cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) play a pivotal role in the initiation of inflammatory responses. Soluble protein antagonists of IL-1 and TNF, such as IL-1ra, sTNFR-Fc fusion, and monoclonal antibodies to TNF have proven to be effective at blocking acute and chronic responses in a number of animal models of inflammatory diseases such as rheumatoid arthritis, septic shock, and inflammatory bowel disease. Consequently, there has been considerable interest in discovering compounds that could inhibit the production of these cytokines and might therefore become treatments. Recently, a structurally related series of pyridinyl imidazoles was found to block IL-1 and TNF production from LPS-stimulated human monocytes and to ameliorate inflammatory diseases significantly in vivo, leading to their being named CSAIDs (cytokine suppressive anti-inflammatory drugs). The protein target of these compounds, termed CSBP (CSAID binding protein), was discovered to be a new member of the MAP kinase family of serine-threonine protein kinases whose kinase activity is activated by LPS in human monocytes. Independently, the same kinase, or its rodent homologues, was found to respond also to chemical, thermal, and osmotic stress and IL-1 treatment. Inhibition of this kinase correlated with reduction in inflammatory cytokine production from LPS-activatedmore » monocytes. 15 refs., 1 fig.« less

SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1

A class of pyridinyl imidazoles inhibit the MAP kinase homologue, termed here reactivating kinase (RK) [Lee et al. (1994) Nature 372, 739–746]. We now show that one of these compounds (SB 203580) inhibits RK in vitro (IC 50 = 0.6 μM), suppresses the activation of MAPKAP kinase-2 and prevents the phosphorylation of heat shock protein (HSP) 27 in response to interleukin-1, cellular stresses and bacterial endotoxin in vivo. These results establish that MAPKAP kinase-2 is a physiological RK substrate, and that HSP27 is phosphorylated by MAPKAP kinase-2 in vivo. The specificity of SB 203580 was indicated by its failure to inhibit 12 other protein kinases in vitro, and by its lack of effect on the activation of RK kinase and other MAP kinase cascades in vivo. We suggest that SB 203580 will be useful for identifying other physiological roles and targets of RK and MAPKAP kinase-2.