Design,synthesis, and biological activity of substituted 2-amino-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic acid derivatives as inhibitors of the inflammatory kinases TBK1 and IKKε for the treatment of obesity

The non-canonical IκB kinases TANK-binding kinase 1 (TBK1) and inhibitor of nuclear factor kappa-B kinase ε (IKKε) play a key role in insulin-independent pathways that promote energy storage and block adaptive energy expenditure during obesity. Utilizing docking calculations and the x-ray structure of TBK1 bound to amlexanox, an inhibitor of these kinases with modest potency, a series of analogues was synthesized to develop a structure activity relationship (SAR) around the A- and C-rings of the core scaffold. A strategy was developed wherein R₇ and R₈ A-ring substituents were incorporated late in the synthetic sequence by utilizing palladium-catalyzed cross-coupling reactions on appropriate bromo precursors. Analogues display IC₅₀ values as low as 210?nM and reveal A-ring substituents that enhance selectivity toward either kinase. In cell assays, selected analogues display enhanced phosphorylation of p38 or TBK1 and elicited IL-6 secretion in 3T3-L1 adipocytes better than amlexanox. An analogue bearing a R₇ cyclohexyl modification demonstrated robust IL-6 production in 3T3-L1 cells as well as a phosphorylation marker of efficacy and was tested in obese mice where it promoted serum IL-6 response, weight loss, and insulin sensitizing effects comparable to amlexanox. These studies provide impetus to expand the SAR around the amlexanox core toward uncovering analogues with development potential.     

IκB kinase epsilon expression in adipocytes is upregulated by interaction with macrophages

Macrophage infiltration in the adipose tissue, and the interaction with adipocytes, is well documented to be involved in fat inflammation and obesity-associated complications. In this study, we isolated IκB kinase ε (IKKε) as a key adipocyte factor that is potentially affected by interaction with macrophages in adipose tissue in vivo. We showed that IKKε mRNA expression levels in white adipose tissue were increased in both genetic and diet-induced obese mouse. Furthermore, IKKε mRNA expression was decreased by the administration of vitamin B6, an anti-inflammatory vitamin, and that IKKε expression levels in adipose tissue were closely correlated with the numbers of infiltrating macrophages. In a co-culture system, we showed that IKKε expression in adipocytes was upregulated by interaction with activated macrophages. This study provides novel insight into IKKε, which is involved in adipose tissue inflammation during the development of obesity.     

Transient blockade of TBK1/IKKε allows efficient transduction of primary human natural killer cells with vesicular stomatitis virus G-pseudotyped lentiviral vectors

Background aimsVesicular stomatitis virus G (VSV-G)-pseudotyped lentiviral vectors (LVs) are widely used to reliably generate genetically modified, clinical-grade T-cell products. However, the results of genetically modifying natural killer (NK) cells with VSV-G LVs have been variable. The authors explored whether inhibition of the IKK-related protein kinases TBK1 and IKKε, key signaling molecules of the endosomal TLR4 pathway, which is activated by VSV-G, would enable the reliable transduction of NK cells by VSV-G LVs.MethodsThe authors activated NK cells from peripheral blood mononuclear cells using standard procedures and transduced them with VSV-G LVs encoding a marker gene (yellow fluorescent protein [YFP]) or functional genes (chimeric antigen receptors [CARs], co-stimulatory molecules) in the presence of three TBK1/IKKε inhibitors (MRT67307, BX-795, amlexanox). NK cell transduction was evaluated by flow cytometry and/or western blot and the functionality of expressed CARs was evaluated in vitro.ResultsBlocking TBK1/IKKε during transduction of NK cells enabled their efficient transduction by VSV-G LVs as judged by YFPexpression of 40–50%, with half maximal effective concentrations of 1.1 µM (MRT67307), 5 µM (BX-795) and 24.8 µM (amlexanox). Focusing on MRT67307, the authors successfully generated NK cells expressing CD19-CARs or HER2-CARs with an inducible co-stimulatory molecule. CAR NK cells exhibited increased cytolytic activity and ability to produce cytokines in comparison to untreated controls, confirming CAR functionality.ConclusionsThe authors demonstrate that inhibition of TBK1/IKKε enables the reliable generation of genetically modified NK cells using VSV-G LVs. The authors’ protocol can be readily adapted to generate clinical-grade NK cells and thus has the potential to facilitate the clinical evaluation of genetically modified NK cell-based therapeutics in the future.     

Discovery of azabenzimidazole derivatives as potent, selective inhibitors of TBK1/IKKε kinases

The design, synthesis and biological evaluation of a series of azabenzimidazole derivatives as TBK1/IKKε kinase inhibitors are described. Starting from a lead compound 1a, iterative design and SAR exploitation of the scaffold led to analogues with nM enzyme potencies against TBK1/IKKε. These compounds also exhibited excellent cellular activity against TBK1. Further structure-based design to improve selectivity over CDK2 and Aurora B resulted in compounds such as 5b-e. These probe compounds will facilitate study of the complex cancer biology of TBK1 and IKKε.     

Development of a High-Throughput Assay for Identifying Inhibitors of TBK1 and IKKε

IKKε and TBK1 are noncanonical IKK family members which regulate inflammatory signaling pathways and also play important roles in oncogenesis. However, few inhibitors of these kinases have been identified. While the substrate specificity of IKKε has recently been described, the substrate specificity of TBK1 is unknown, hindering the development of high-throughput screening technologies for inhibitor identification. Here, we describe the optimal substrate phosphorylation motif for TBK1, and show that it is identical to the phosphorylation motif previously described for IKKε. This information enabled the design of an optimal TBK1/IKKε substrate peptide amenable to high-throughput screening and we assayed a 6,006 compound library that included 4,727 kinase-focused compounds to discover in vitro inhibitors of TBK1 and IKKε. 227 compounds in this library inhibited TBK1 at a concentration of 10 μM, while 57 compounds inhibited IKKε. Together, these data describe a new high-throughput screening assay which will facilitate the discovery of small molecule TBK1/IKKε inhibitors possessing therapeutic potential for both inflammatory diseases and cancer.     

Osthol attenuates neutrophilic oxidative stress and hemorrhagic shock-induced lung injury via inhibition of phosphodiesterase 4

Oxidative stress caused by neutrophils is an important pathogenic factor in trauma/hemorrhagic (T/H)-induced acute lung injury (ALI). Osthol, a natural coumarin found in traditional medicinal plants, has therapeutic potential in various diseases. However, the pharmacological effects of osthol in human neutrophils and its molecular mechanism of action remain elusive. In this study, our data showed that osthol potently inhibited the production of superoxide anion (O₂^•−) and reactive oxidants derived therefrom as well as expression of CD11b in N-formylmethionylleucylphenylalanine (FMLP)-activated human neutrophils. However, osthol inhibited neutrophil degranulation only slightly and it failed to inhibit the activity of subcellular NADPH oxidase. FMLP-induced phosphorylation of extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) was inhibited by osthol. Notably, osthol increased the cAMP concentration and protein kinase A (PKA) activity in activated neutrophils. PKA inhibitors reversed the inhibitory effects of osthol, suggesting that these are mediated through cAMP/PKA-dependent inhibition of ERK and Akt activation. Furthermore, the activity of cAMP-specific phosphodiesterase (PDE) 4, but not PDE3 or PDE7, was significantly reduced by osthol. In addition, osthol reduced myeloperoxidase activity and pulmonary edema in rats subjected to T/H shock. In conclusion, our data suggest that osthol has effective anti-inflammatory activity in human neutrophils through the suppression of PDE4 and protects significantly against T/H shock-induced ALI in rats. Osthol may have potential for future clinical application as a novel adjunct therapy to treat lung inflammation caused by adverse circulatory conditions.     

Inducible SUMO modification of TANK alleviates its repression of TLR7 signalling

Adaptor proteins allow temporal and spatial coordination of signalling. In this study, we show SUMOylation of the adaptor protein TANK and its interacting kinase TANK‐binding kinase 1 (TBK1). Modification of TANK by the small ubiquitin‐related modifier (SUMO) at the evolutionarily conserved Lys 282 is triggered by the kinase activities of IκB kinase ε (IKKε) and TBK1. Stimulation of TLR7 leads to inducible SUMOylation of TANK, which in turn weakens the interaction with IKKε and thus relieves the negative function of TANK on signal propagation. Reconstitution experiments show that an absence of TANK SUMOylation impairs inducible expression of distinct TLR7‐dependent target genes, providing a molecular mechanism that allows the control of TANK function.     

Crystal structure of the ubiquitin-like domain of human TBK1

TANK-binding kinase 1 (TBK1) is an important enzyme in the regulation of cellular antiviral effects. TBK1 regulates the activity of the interferon regulatory factors IRF3 and IRF7, thereby playing a key role in type I interferon (IFN) signaling pathways. The structure of TBK1 consists of an N-terminal kinase domain, a middle ubiquitin-like domain (ULD), and a C-terminal elongated helical domain. It has been reported that the ULD of TBK1 regulates kinase activity, playing an important role in signaling and mediating interactions with other molecules in the IFN pathway. In this study, we present the crystal structure of the ULD of human TBK1 and identify several conserved residues by multiple sequence alignment. We found that a hydrophobic patch in TBK1, containing residues Leu316, Ile353, and Val382, corresponding to the “Ile44 hydrophobic patch” observed in ubiquitin, was conserved in TBK1, IκB kinase epsilon (IKK?/IKKi), IκB kinase alpha (IKKα), and IκB kinase beta (IKKβ). In comparison with the structure of the IKKβ ULD domain of Xenopus laevis, we speculate that the Ile44 hydrophobic patch of TBK1 is present in an intramolecular binding surface between ULD and the C-terminal elongated helices. The varying surface charge distributions in the ULD domains of IKK and IKK-related kinases may be relevant to their specificity for specific partners.     

Pellino-1 Positively Regulates Toll-like Receptor (TLR) 2 and TLR4 Signaling and Is Suppressed upon Induction of Endotoxin Tolerance

Endotoxin tolerance reprograms Toll-like receptor (TLR) 4-mediated macrophage responses by attenuating induction of proinflammatory cytokines while retaining expression of anti-inflammatory and antimicrobial mediators. We previously demonstrated deficient TLR4-induced activation of IL-1 receptor-associated kinase (IRAK) 4, IRAK1, and TANK-binding kinase (TBK) 1 as critical hallmarks of endotoxin tolerance, but mechanisms remain unclear. In this study, we examined the role of the E3 ubiquitin ligase Pellino-1 in endotoxin tolerance and TLR signaling. LPS stimulation increased Pellino-1 mRNA and protein expression in macrophages from mice injected with saline and in medium-pretreated human monocytes, THP-1, and MonoMac-6 cells, whereas endotoxin tolerization abrogated LPS inducibility of Pellino-1. Overexpression of Pellino-1 in 293/TLR2 and 293/TLR4/MD2 cells enhanced TLR2- and TLR4-induced nuclear factor κB (NF-κB) and expression of IL-8 mRNA, whereas Pellino-1 knockdown reduced these responses. Pellino-1 ablation in THP-1 cells impaired induction of myeloid differentiation primary response protein (MyD88), and Toll-IL-1R domain-containing adapter inducing IFN-β (TRIF)-dependent cytokine genes in response to TLR4 and TLR2 agonists and heat-killed Escherichia coli and Staphylococcus aureus, whereas only weakly affecting phagocytosis of heat-killed bacteria. Co-expressed Pellino-1 potentiated NF-κB activation driven by transfected MyD88, TRIF, IRAK1, TBK1, TGF-β-activated kinase (TAK) 1, and TNFR-associated factor 6, whereas not affecting p65-induced responses. Mechanistically, Pellino-1 increased LPS-driven K63-linked polyubiquitination of IRAK1, TBK1, TAK1, and phosphorylation of TBK1 and IFN regulatory factor 3. These results reveal a novel mechanism by which endotoxin tolerance re-programs TLR4 signaling via suppression of Pellino-1, a positive regulator of MyD88- and TRIF-dependent signaling that promotes K63-linked polyubiquitination of IRAK1, TBK1, and TAK1.     

Phosphorylation of cAMP-specific PDE4A5 (phosphodiesterase-4A5) by MK2 (MAPKAPK2) attenuates its activation through protein kinase A phosphorylation

cAMP-specific PDE (phosphodiesterase) 4 isoforms underpin compartmentalized cAMP signalling in mammalian cells through targeting to specific signalling complexes. Their importance is apparent as PDE4 selective inhibitors exert profound anti-inflammatory effects and act as cognitive enhancers. The p38 MAPK (mitogen-activated protein kinase) signalling cascade is a key signal transduction pathway involved in the control of cellular immune, inflammatory and stress responses. In the present study, we show that PDE4A5 is phosphorylated at Ser147, within the regulatory UCR1 (ultraconserved region 1) domain conserved among PDE4 long isoforms, by MK2 (MAPK-activated protein kinase 2, also called MAPKAPK2). Phosphorylation by MK2, although not altering PDE4A5 activity, markedly attenuates PDE4A5 activation through phosphorylation by protein kinase A. This modification confers the amplification of intracellular cAMP accumulation in response to adenylate cyclase activation by attenuating a major desensitization system to cAMP. Such reprogramming of cAMP accumulation is recapitulated in wild-type primary macrophages, but not MK2/3-null macrophages. Phosphorylation by MK2 also triggers a conformational change in PDE4A5 that attenuates PDE4A5 interaction with proteins whose binding involves UCR2, such as DISC1 (disrupted in schizophrenia 1) and AIP (aryl hydrocarbon receptor-interacting protein), but not the UCR2-independent interacting scaffold protein β-arrestin. Long PDE4 isoforms thus provide a novel node for cross-talk between the cAMP and p38 MAPK signalling systems at the level of MK2.     

Association of the adaptor TANK with the I kappa B kinase (IKK) regulator NEMO connects IKK complexes with IKK epsilon and TBK1 kinases

Canonical activation of NF-kappa B is mediated via phosphorylation of the inhibitory I kappa B proteins by the I kappa B kinase complex (IKK). IKK is composed of a heterodimer of the catalytic IKK alpha and IKK beta subunits and a presumed regulatory protein termed NEMO (NF-kappa B essential modulator) or IKK gamma. NEMO/IKK gamma is indispensable for activation of the IKKs in response to many signals, but its mechanism of action remains unclear. Here we identify TANK (TRAF family member-associated NF-kappa B activator) as a NEMO/IKK gamma-interacting protein via yeast two-hybrid analyses. This interaction is confirmed in mammalian cells, and the domains required are mapped. TANK was previously shown to assist NF-kappa B activation in a complex with TANK-binding kinase 1 (TBK1) or IKK epsilon, two kinases distantly related to IKK alpha/beta, but the underlying mechanisms remained unknown. Here we show that TBK1 and IKK epsilon synergize with TANK to promote interaction with the IKKs. The TANK binding domain within NEMO/IKK gamma is required for proper functioning of this IKK subunit. These results indicate that TANK can synergize with IKK epsilon or TBK1 to link them to IKK complexes, where the two kinases may modulate aspects of NF-kappa B activation.     

TBK1 has a new Akt

TANK-binding kinase 1 (TBK1) is a noncanonical IκB kinase that plays an essential role in the innate immune response to foreign pathogens. Recent studies have highlighted additional roles for TBK1 in the regulation of metabolism, although the mechanisms of this regulation have not been well characterized. In a recent issue, Tooley et al. demonstrated that TBK1-dependent activation of downstream kinase Akt is mediated via mechanistic target of rapamycin complex 2. This novel action of TBK1 reveals a key role for this kinase in the regulation of cellular metabolism and growth by diverse environmental inputs.

TANK-binding kinase 1 (TBK1), a serine/threonine kinase that belongs to the noncanonical IκB kinase family, plays an essential role in the innate immune response to viral and bacterial pathogens by regulating the type I interferon–mediated T cell response (1). Although TBK1 has been most widely studied in this context, more recent investigations using tissue-specific KO mice and drugs that inhibit kinase activity have revealed novel roles for this kinase in nonimmune cells, particularly at the intersection of immunity and metabolism. For example, TBK1 expression and activity are induced in adipose tissue in obesity by elevated expression of proinflammatory cytokines such as tumor necrosis factor α (2). TBK1 contributes to obesity by repressing energy expenditure and increasing anabolic functions as determined from analysis of mice with conditional adipose cell KO of TBK1 (3). TBK1 has also been reported to promote activation of Akt, a central kinase involved in metabolic regulation (4). However, the mechanism by which TBK1 regulates Akt has remained unclear.Akt is an essential regulator of glucose metabolism and plays an important role in controlling cellular glucose uptake and utilization through both positive and negative regulatory actions (4). Phosphorylation of Akt on T308 in its activation loop stimulates kinase activity, and phosphorylation on S473 further enhances activity and determines substrate specificity (4). Although it had been previously reported that TBK1 can directly phosphorylate Akt at S473 and T308 in in vitro kinase assays, the ability of TBK1 to mediate these phosphorylation events under physiological conditions was not known (5). In a recent study, Tooley et al. (6) contributed to the mechanistic understanding of TBK1 function in metabolic regulation by demonstrating a role for TBK1 in mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) activation and subsequent phosphorylation of Akt.To investigate how TBK1 regulates Akt activation, mouse embryonic fibroblasts (MEFs) were stimulated with epidermal growth factor (EGF) and evaluated for Akt-S473 and Akt-T308 phosphorylation (6). The intensity and duration of Akt phosphorylation at both sites was diminished significantly, both in the absence of TBK1 and in the presence of the TBK1 inhibitor amlexanox. Restoration of endogenous levels of TBK1, but not kinase-dead TBK1, rescued EGF-stimulated Akt-S473 phosphorylation. The stimulation of Akt-S473 phosphorylation by EGF, as well as by other growth factors and the hormone insulin, was found to be dependent upon mTOR activity. Together, these results validate the ability of TBK1 to regulate Akt-S473 phosphorylation and show that in response to normal growth regulatory signaling, this regulation is mediated through mTOR kinase.The kinase mTOR is the core catalytic kinase of two multisubunit complexes, mTOR complex 1 (mTORC1) and mTORC2, which are distinguished by the scaffolding proteins Raptor and Rictor, respectively (7). mTORC1 is regulated by the combination of growth factor/hormone signaling and nutrient availability to drive anabolic metabolism. mTORC2, on the other hand, is regulated by growth factor/hormone signaling to activate Akt. Together, mTORC1 and mTORC2 are key signaling nodes in the regulation of cell growth and proliferation, and dysregulation of these signaling pathways contributes to metabolic disease and cancer. In previous investigations, the authors had demonstrated that phosphorylation of mTOR on S2159 by TBK1 enhanced mTORC1 activation and downstream signaling to promote cell growth and proliferation (8). To investigate if TBK1 acts upstream of mTORC2 to regulate Akt-S473 phosphorylation through a similar mechanism, MEFs derived from mice with an alanine knock-in at S2159 (Mtor^A/A) were stimulated with EGF. A marked reduction of Akt-S473 phosphorylation was observed in Mtor^A/A MEFs compared with WT MEFs (Mtor^+/+). Using immunoprecipitation of Rictor to isolate the mTORC2 complex, TBK1 was observed to interact with mTORC2 and directly phosphorylate mTOR-S2159 to activate mTORC2 intrinsic kinase activity toward Akt-S473. TBK1 activity is increased by phosphorylation of S172 in its activation loop in response to pathogens in the innate immunity pathway. In contrast, Tooley et al. (6) found that EGF stimulation did not enhance S172 phosphorylation, supporting that it is the basal activity of TBK1 that is important for mTORC2 signaling downstream of growth factors. However, when RAW264.7 macrophages and primary bone marrow–derived macrophages were stimulated with the dsRNA mimetic poly(I:C), which induces TBK1-S172 phosphorylation, TBK1 and mTOR-S2159 were also found to be required for mTORC2-dependent phosphorylation of Akt-S473. Finally, the physiological regulation of mTORC2 activity by TBK1 was assessed by injection of Mtor^A/A and Mtor^+/+ mice with poly(I:C). Spleen tissue isolated from Mtor^A/A mice showed diminished Akt-S473 phosphorylation. Therefore, the authors conclude that under both basal and activated states, the activation of Akt by TBK1 is mediated through mTORC2 (Fig. 1) (6).Open in a separate windowFigure 1TBK1 promotes AKT activation through mTORC2. TBK1 interacts with and phosphorylates mTORC2 on S2159 of mTOR in response to either growth factor stimulation or innate immune agonists to promote AKT activation. Created using BioRender.com. mTORC2, mTOR complex 2; SGK, serum/glucocorticoid-regulated kinase; TBK1, TANK-binding kinase 1.TBK1 regulation of mTORC2-dependent phosphorylation of Akt shown in this study adds to the growing role of TBK1 as a signaling node in the regulation of cellular metabolism and growth by diverse environmental inputs. In response to foreign pathogens or inflammatory cytokines that stimulate TBK1 activation, or growth factor/hormone signaling that requires basal TBK1 activity, mTORC2 is activated to promote Akt-S473 phosphorylation and its downstream functions. Given that TBK1 expression and activity are enhanced in metabolic diseases and cancer, and the important role that Akt plays in these pathological conditions, identifying TBK1 as an upstream regulator of Akt reveals a potential novel approach to disrupt this signaling axis for therapeutic benefit (4, 9). In this regard, drugs such as amlexanox and other compounds are under investigation for their potential clinical use (10). Of note, the study by Tooley et al. (6) only examined the TBK1-dependent phosphorylation of Akt-S473 by mTORC2; mTORC2 also has additional substrates, including serum/glucocorticoid-regulated kinase and members of the PKC family (Fig. 1) (4). These kinases regulate unique cellular functions, such as regulation of the actin cytoskeleton. It will be important to determine if TBK1 regulates the activation of these kinases through mTORC2 as well, to understand the full impact of inhibiting TBK1 function therapeutically.The mechanism by which TBK1 regulates mTORC2 function has not been established. Although the kinase activity of TBK1 is required for Akt-S473 phosphorylation, neither phosphorylation of S172 in the activation loop of TBK1 nor phosphorylation of mTOR-S2159 was increased by growth factor stimulation in this study. Phosphorylation of S172 stabilizes the active confirmation of TBK1 and it is possible that additional uncharacterized phosphorylation sites could serve a similar function. Alternatively, the interaction of TBK1 with mTORC2 could impact TBK1 conformation, or multimerization, to enhance activity. Intracellular localization of mTORC2 could also be determined by TBK1 interaction, which could affect substrate availability. As little is known about the upstream regulation of mTORC2, the next acts should be elucidating further the mechanism of its activation by TBK1 to reveal novel approaches for targeting the mTORC2-Akt signaling pathway.     

Mefunidone ameliorates renal inflammation and tubulointerstitial fibrosis via suppression of IKKβ phosphorylation

Mefunidone is a new pyridone agent that attenuates renal tubulointerstitial fibrosis. However, the signaling pathways involved in the effect of mefunidone on renal tubulointerstitial fibrosis have not been well explained. Inflammatory response initiates and promotes renal tubulointerstitial fibrosis, and the inhibitor of nuclear factor kappa-B kinase beta (IKKβ) is a master regulator of inflammation. This study is determined to clarify the influence of mefunidone on renal inflammation and the phosphorylation of IKKβ. Experimental renal tubulointerstitial fibrosis was induced by unilateral ureteral obstruction (UUO) for 3, 7 and 14 days in sprague dawley rat. Treatment with mefunidone was conducted simultaneously. Obstructed kidneys were harvested for the assessment. Our results showed that treatment with mefunidone ameliorated renal inflammatory injury, renal tubular lesions and interstitial fibrosis. Further studies indicated that treatment with mefunidone mitigated the expressions of tumor necrosis factorα (TNFα) and interleukin-1β (IL-1β) in the kidney. The phosphorylation of IKKβ and inhibitor of kappa-B (IκB) and the expression of NOD-like receptor family, pyrin domain containing 3 (NALP3) were also reduced in vivo after treatment with mefunidone. In vitro, peritoneal macrophages were incubated with necrotic cells or lipopolysaccharide in the presence or absence of mefunidone. Mefunidone markedly decreased necrotic cell or LPS induced IL-1β production and LPS induced TNFα production in primary peritoneal macrophages. Furthermore, mefunidone significantly inhibited the phosphorylation of IKKβ/IκB and nuclear transition of NF-κB p65 in peritoneal macrophages stimulated by necrotic cell or lipopolysaccharide. In conclusion, mefunidone serves as a novel anti-inflammatory agent that attenuates UUO-induced renal interstitial inflammation and fibrosis, possibly through suppressing IKKβ phosphorylation.     

Combined anti-inflammatory effects of β2-adrenergic agonists and PDE4 inhibitors on astrocytes by upregulation of intracellular cAMP

Inflammation is an important hallmark of all neurodegenerative diseases and activation of different glial populations may be involved in the progression of some of these disorders. Especially, the activation of astroglia can lead to long-term detrimental morphological changes, such as scar formation. Therefore, improved strategies to modulate inflammation in these cells are currently being investigated. We investigated the interaction of phosphodiesterase (PDE) 4 inhibitors, such as rolipram, with other agents raising cellular cAMP levels. When used alone, none of the PDE4 inhibitors increased cAMP levels. The adenylate cyclase activator forskolin, the β₂-adrenergic agonist clenbuterol and the mixed β₁/β₂-adrenergic agonist isoproterenol increased intracellular cAMP levels of cortical murine astrocytes. This increase was synergistically elevated by rolipram or the PDE4 inhibitor RO-201724, but not by inhibition of PDE3. Inflammatory stimulation of the cells with the cytokines TNF-α, IL-1β and IFN-γ strongly induced PDE4B and augmented overall PDE4 activity, while PDE3 activity was low. Clenbuterol and forskolin caused downregulation of cytokines and chemokines such as IL-6 and MCP-1. This effect was further enhanced by rolipram, but not by the PDE3 inhibitor milrinone. The cAMP-raising drug combinations attenuated the upregulation of TNF-α and IL-6 mRNA and the secretion of IL-6, but did not affect initial NF-κB signalling triggered by the stimulating cytokines. These results indicate that PDE4 may be a valuable anti-inflammatory target in brain diseases, especially under conditions associated with stimulation of cAMP-augmenting astrocyte receptors as is observed by clenbuterol treatment.