Inhibition of farnesyltransferase prevents collagen-induced arthritis by down-regulation of inflammatory gene expression through suppression of p21(ras)-dependent NF-kappaB activation

Farnesylation of p21(ras) is an important step in the intracellular signaling pathway of growth factors, hormones, and immune stimulants. We synthesized a potent and selective farnesyltransferase inhibitor (LB42708) with IC(50) values of 0.8 nM in vitro and 8 nM in cultured cells against p21(ras) farnesylation and examined the effects of this inhibitor in the settings of inflammation and arthritis. LB42708 suppressed NF-kappaB activation and iNOS promoter activity by suppressing the I-kappaB kinase activity and I-kappaBalpha degradation. The inhibitor suppressed the expression of inducible NO synthase, cyclooxygenase-2, TNF-alpha, and IL-1beta and the production of NO and PGE(2) in immune-activated macrophages and osteoblasts as well as LPS-administrated mice. Furthermore, in vivo administration of LB42708 significantly decreased the incidence and severity of arthritis as well as mRNA expression of inducible NO synthase, cyclooxygenase-2, TNF-alpha, and IL-1beta in the paws of collagen-induced arthritic mice compared with controls. These observations indicate that the anti-inflammatory and antiarthritic effects of the farnesyltransferase inhibitor may be ascribed to the inhibition of I-kappaB kinase activity and subsequent suppression of NF-kappaB-dependent inflammatory gene expression through the suppression of p21(ras) farnesylation. Together, these findings reveal that the inhibitory effect of LB42708 on p21(ras)-dependent NF-kappaB activation may have potential therapeutic value for arthritis and other inflammatory diseases.     

IL-17 reduces TNF-induced Rantes and VCAM-1 expression

Functional diversity of the memory T-cell-derived cytokine IL-17 was explored at the receptor level. IL-17 inhibited TNF-induced chemokine Rantes expression in human synovial fibroblasts and mouse lung fibroblasts. This inhibitory activity of IL-17 (IC50=0.2 ng/ml) was 6-fold more potent than its stimulatory activity on TNF-alpha-induced IL-6 secretion (ED50=1.2 ng/ml), measured in the same cells. IL-17 also inhibited the TNF-mediated expression of adhesion molecule VCAM-1, and the NF-kappaB binding to the VCAM-1 promoter-specific site, along with the inhibitor of NF-kappaB, IkappaB-beta. Neutralization of the human IL-17 receptor (IL-17R) by M202 antibody competitively reverses the IL-17-induced IL-6 upregulation. However, M202 only partially affected the inhibitions by IL-17. Yet, IL-17R was essential for the Rantes inhibition, as assessed in lung-derived fibroblasts from IL-17R gene deficient mice. Therefore, inhibitory and stimulatory functions of IL-17 involve receptor IL-17R but show distinct dose-responses and in turn different sensitivities to an IL-17R antagonizing antibody.     

Lignans from Saururus chinensis inhibiting the transcription factor NF-kappaB

The sesquineolignans, saucerneol D and saucerneol E were isolated from the roots of Saururus chinensis together with four known lignans, manassantin A, manassantin B, (-)-saucerneol methyl ether, and (+)-saucernetin. Structure elucidation was based on the analysis of spectroscopic data and anti-inflammatory activity was studied using HeLa cells transfected with NF-kappaB reporter construct. All compounds except for (+)-saucernetin inhibited NF-kappaB dependent reporter gene expression with IC50 values of 2.5-16.9 microM.     

Saururus cernuus lignans--potent small molecule inhibitors of hypoxia-inducible factor-1

Hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective therapeutic target for solid tumors. In search of novel small molecule HIF-1 inhibitors, 5400 natural product-rich extracts from plants, marine organisms, and microbes were examined for HIF-1 inhibitory activities using a cell-based reporter assay. Bioassay-guided fractionation and isolation, followed by structure elucidation, yielded three potent natural product-derived HIF-1 inhibitors and two structurally related inactive compounds. In a T47D cell-based reporter assay, manassantin B1, manassantin A, and 4-O-methylsaucerneol inhibited hypoxia-induced HIF-1 activation with IC50 values of 3, 3, and 20 nM, respectively. All three compounds are relatively hypoxia-specific inhibitors of HIF-1 activation, in comparison to other stimuli. The hypoxic induction of HIF-1 target genes CDKN1A, VEGF, and GLUT-1 were also inhibited. These compounds inhibit HIF-1 by blocking hypoxia-induced nuclear HIF-1alpha protein accumulation without affecting HIF-1alpha mRNA levels. In addition, preliminary structure-activity studies suggest specific structural requirements for this class of HIF-1 inhibitors.     

Studies of the inhibitory activity of MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-indol- 2-yl]-2,2-dimethyl propanoic acid) on arachidonic acid metabolism in human phagocytes.

We have investigated the inhibitory activity of compound MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-i ndol-2- yl]-2,2-dimethyl propanoic acid) on 5-lipoxygenase (5-LO) product synthesis in various human phagocytes stimulated with either the ionophore A23187, opsonized zymosan (OPZ), platelet-activating factor (PAF), or formyl-methionyl-leucyl-phenylalanine (fMLP). The lipoxygenase products were analyzed by reversed-phase HPLC. MK-0591 inhibited the formation of 5-hydroxyeicosatetraenoic acid, leukotriene (LT) B4, its omega-oxidation products, and 6-trans-isomers with IC50 values of 2.8-4.8 nM in A23187-stimulated neutrophils. In these conditions, arachidonic acid at a concentration of 10 microM had no effect on MK-0591 inhibitory activity. In neutrophils stimulated with OPZ, the synthesis of LTB4, its omega-oxidation products, and 6-trans-isomers was inhibited with IC50 values of 9.5-11.0 nM. MK-0591 inhibited 5-LO product synthesis in A23187-stimulated blood monocytes, eosinophils, and alveolar macrophages with IC50 values of 0.3-0.9, 3.7-5.3, and 8.5-17.3 nM, respectively. In neutrophils primed with granulocyte--macrophage colony-stimulating factor and stimulated with PAF, lipoxygenase product synthesis was inhibited with IC50 values of 7.7-8.7 nM. At the concentration of 1 microM, MK-0591 had no inhibitory effect on 15-lipoxygenase activity in human polymorphonuclear leukocytes, nor on human platelet 12-lipoxygenase and cyclooxygenase. In conclusion, MK-0591 is a very potent and specific inhibitor of 5-LO product synthesis in various types of human phagocytes.     

Structural and functional characterization of EMF10, a heterodimeric disintegrin from Eristocophis macmahoni venom that selectively inhibits alpha 5 beta 1 integrin.

Alpha5beta1, a major fibronectin receptor, is a widely distributed integrin that is essential for cell growth and organ development. Here, we describe a novel heterodimeric disintegrin named EMF10, isolated from the Eristocophis macmahoni venom, that is an extremely potent and selective inhibitor of alpha5beta1. EMF10 inhibited adhesion of cells expressing alpha5beta1 to fibronectin (IC(50) = 1-4 nM) and caused expression of a ligand-induced binding site (LIBS) on the beta1 subunit of alpha5beta1 integrin. It partially inhibited adhesion of cells expressing alphaIIbbeta3, alphavbeta3, and alpha4beta1 to appropriate ligands only at concentration higher than 500 nM. Guinea pig megakaryocytes expressing alpha5beta1 adhered to immobilized EMF10 and showed extensive spreading and cytoskeletal mobilization. As determined by electrospray mass spectrometry, EMF10 is composed of two species with molecular masses of 14 575 and 14 949 Da, respectively. EMF10 is a heterodimer containing two subunits: EMF10A (Mr 7544 Da) and EMF10B (Mr 7405 and 7032 Da) linked covalently by S-S bonds. Subunit B showed heterogeneity and may be present as EMF10B1 (Mr 7032) and EMF10B2 (Mr 7405). In putative hairpin loops, EMF10A and EMF10B contained CKKGRGDNLNDYC and CWPAMGDWNDDYC motifs, respectively. The reduced and alkylated subunit B of EMF10 inhibited adhesion of K562 cells to fibronectin in a dose-dependent, saturable manner with IC(50) of 3 microM. The synthetic, cyclic CKKGRGDNLNDYC and CWPAMGDWNDDYC peptides expressed their inhibitory activity in the same system with IC(50) of 100 microM. We propose that alpha5beta1 recognition of EMF10 is associated with the MGDW motif located in a putative hairpin loop of the B subunit and that the expression of activity may also depend on the RGDN motif in the subunit A and on the C-termini of both subunits.     

Calyculin A and okadaic acid: inhibitors of protein phosphatase activity

Calyculin A and okadaic acid induce contraction in smooth muscle fibers. Okadaic acid is an inhibitor of phosphatase activity and the aims of this study were to determine if calyculin A also inhibits phosphatase and to screen effects of both compounds on various phosphatases. Neither compound inhibited acid or alkaline phosphatases, nor the phosphotyrosine protein phosphatase. Both compounds were potent inhibitors of the catalytic subunit of type-2A phosphatase, with IC50 values of 0.5 to 1 nM. With the catalytic subunit of protein phosphatase type-1, calyculin A was a more effective inhibitor than okadaic acid, IC50 values for calyculin A were about 2 nM and for okadaic acid between 60 and 500 nM. The endogenous phosphatase of smooth muscle myosin B was inhibited by both compounds with IC50 values of 0.3 to 0.7 nM and 15 to 70 nM, for calyculin A and okadaic acid, respectively. The partially purified catalytic subunit from myosin B had IC50 values of 0.7 and 200 nM for calyculin A and okadaic acid, respectively. The pattern of inhibition for the phosphatase in myosin B therefore is similar to that of the type-1 enzyme.     

Synthesis, biological evaluation and molecular docking studies of novel 2-(1,3,4-oxadiazol-2-ylthio)-1-phenylethanone derivatives

In present study, a series of new 2-(1,3,4-oxadiazol-2-ylthio)-1-phenylethanone derivatives (6a-6x) as potential focal adhesion kinase (FAK) inhibitors were synthesized. The bioassay assays demonstrated that compound 6i showed the most potent activity, which inhibited the growth of MCF-7 and A431 cell lines with IC(50) values of 140 ± 10 nM and 10 ± 1 nM, respectively. Compound 6i also exhibited significant FAK inhibitory activity (IC(50)=20 ± 1 nM). Docking simulation was performed to position compound 6i into the active site of FAK to determine the probable binding model.     

Adiponectin enhances IL-6 production in human synovial fibroblast via an AdipoR1 receptor, AMPK, p38, and NF-kappa B pathway

Articular adipose tissue is a ubiquitous component of human joints, and adiponectin is a protein hormone secreted predominantly by differentiated adipocytes and involved in energy homeostasis. We investigated the signaling pathway involved in IL-6 production caused by adiponectin in both rheumatoid arthritis synovial fibroblasts and osteoarthritis synovial fibroblasts. Rheumatoid arthritis synovial fibroblasts and osteoarthritis synovial fibroblasts expressed the AdipoR1 and AdipoR2 isoforms of the adiponectin receptor. Adiponectin caused concentration- and time-dependent increases in IL-6 production. Adiponectin-mediated IL-6 production was attenuated by AdipoR1 and 5'-AMP-activated protein kinase (AMPK)alpha1 small interference RNA. Pretreatment with AMPK inhibitor (araA and compound C), p38 inhibitor (SB203580), NF-kappaB inhibitor, IkappaB protease inhibitor, and NF-kappaB inhibitor peptide also inhibited the potentiating action of adiponectin. Adiponectin increased the kinase activity and phosphorylation of AMPK and p38. Stimulation of synovial fibroblasts with adiponectin activated IkappaB kinase alpha/beta (IKK alpha/beta), IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation at Ser (276), p65 and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. Adiponectin-mediated an increase of IKK alpha/beta activity, kappaB-luciferase activity, and p65 and p50 binding to the NF-kappaB element and was inhibited by compound C, SB203580 and AdipoR1 small interference RNA. Our results suggest that adiponectin increased IL-6 production in synovial fibroblasts via the AdipoR1 receptor/AMPK/p38/IKKalphabeta and NF-kappaB signaling pathway.     

Biomechanical stress induces IL-6 expression in smooth muscle cells via Ras/Rac1-p38 MAPK-NF-kappaB signaling pathways

IL-6, a proinflammatory cytokine, has been implicated in the development of vascular diseases. We previously demonstrated that mechanical stress can initiate signaling pathways leading to smooth muscle cell (SMC) proliferation and apoptosis, but little is known concerning cyclic stress-induced inflammatory response. To explore the role of stretch in the upregulation of cytokine expression in SMCs we performed RNase protection assay for a panel of cytokines and found that mechanical stress resulted in a time-dependent induction of IL-6 mRNA but not other cytokines, e.g., IL-1alpha, IL-1beta, IL-6, IL-10, IL-12p35, IL-12p40, IL-18, IFN-gamma, and macrophage migration inhibitory factor (MIF). This induction also correlated with elevated IL-6 protein levels in the supernatant. Pretreatment of the cells with NF-kappaB inhibitors inhibited NF-kappaB activity and resulted in marked inhibition (50%) of IL-6 protein. Moreover, SMC lines stably expressing dominant-negative Ras (RasN17) or Rac (RacN17) exhibited a remarkable decrease in p38 MAPK activity and IL-6 mRNA induction by mechanical stress. Furthermore, a significant inhibition of 30 and 40% in IL-6 protein was observed in SMCs pretreated with inhibitors of p38 MAPK and ERK1/2, respectively, but not JNK. Interestingly, SMCs isolated from PKC-delta-deficient mice exhibited higher levels of IL-6 compared with wild-type cells. Finally, high levels of IL-6 expression were observed in atherosclerotic lesions of vein bypass grafts, which are related to altered biomechanical stress. Our findings demonstrate that biomechanical stress-induced IL-6 expression occurs via a mechanism that involves Ras/Rac/p38 MAPK/NF-kappaB/NF-IL6 signaling pathways, which is downregulated by PKC-delta, and suggest that modulation of this event contributes to the pathogenesis of atherosclerosis.     

Leptin-induced IL-6 production is mediated by leptin receptor, insulin receptor substrate-1, phosphatidylinositol 3-kinase, Akt, NF-kappaB, and p300 pathway in microglia

Leptin, the adipocyte-secreted hormone that centrally regulates weight control, is known to function as an immunomodulatory regulator. We investigated the signaling pathway involved in IL-6 production caused by leptin in microglia. Microglia expressed the long (OBRl) and short (OBRs) isoforms of the leptin receptor. Leptin caused concentration- and time-dependent increases in IL-6 production. Leptin-mediated IL-6 production was attenuated by OBRl receptor antisense oligonucleotide, PI3K inhibitor (Ly294002 and wortmannin), Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate)), NF-kappaB inhibitor (pyrrolidine dithiocarbamate), IkappaB protease inhibitor (L-1-tosylamido-2-phenylenylethyl chloromethyl ketone), IkappaBalpha phosphorylation inhibitor (Bay 117082), or NF-kappaB inhibitor peptide. Transfection with insulin receptor substrate (IRS)-1 small-interference RNA or the dominant-negative mutant of p85 and Akt also inhibited the potentiating action of leptin. Stimulation of microglia with leptin activated IkappaB kinase alpha/IkappaB kinase beta, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation at Ser(276), p65 and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. Leptin-mediated an increase of IkappaB kinase alpha/IkappaB kinase beta activity, kappaB-luciferase activity, and p65 and p50 binding to the NF-kappaB element was inhibited by wortmannin, Akt inhibitor, and IRS-1 small-interference RNA. The binding of p65 and p50 to the NF-kappaB elements, as well as the recruitment of p300 and the enhancement of histone H3 and H4 acetylation on the IL-6 promoter was enhanced by leptin. Our results suggest that leptin increased IL-6 production in microglia via the leptin receptor/IRS-1/PI3K/Akt/NF-kappaB and p300 signaling pathway.     

Design and synthesis of 6-fluoro-2-naphthyl derivatives as novel CCR3 antagonists with reduced CYP2D6 inhibition

In our previous study on discovering novel types of CCR3 antagonists, we found a fluoronaphthalene derivative (1) that exhibited potent CCR3 inhibitory activity with an IC(50) value of 20 nM. However, compound 1 also inhibited human cytochrome P450 2D6 (CYP2D6) with an IC(50) value of 400 nM. In order to reduce its CYP2D6 inhibitory activity, we performed further systematic structural modifications on 1. In particular, we focused on reducing the number of lipophilic moieties in the biphenyl part of 1, using ClogD(7.4) values as the reference index of lipophilicity. This research led to the identification of N-{(3-exo)-8-[(6-fluoro-2-naphthyl)methyl]-8-azabicyclo[3.2.1]oct-3-yl}-3-(piperidin-1-ylcarbonyl)isonicotinamide 1-oxide (30) which showed comparable CCR3 inhibitory activity (IC(50)=23 nM) with much reduced CYP2D6 inhibitory activity (IC(50)=29,000 nM) compared with 1.     

Differential regulation of matrix metalloproteinase-2 and -9 expression and activity in adult rat cardiac fibroblasts in response to interleukin-1beta

Matrix metalloproteinases (MMPs), a family of endoproteinases, are implicated in cardiac remodeling. Interleukin-1beta (IL-1beta), which is increased in the heart following myocardial infarction, increases expression and activity of MMP-2 (gelatinase A) and -9 (gelatinase B) in cardiac fibroblasts. Previously, we have shown that IL-1beta activates ERK1/2, JNKs, and protein kinase C (PKC). However, signaling pathways involved in the regulation of MMP-2 and -9 expression and activity are not yet well understood. Using adult rat cardiac fibroblasts, we show that inhibition of ERK1/2 and JNKs inhibits IL-1beta-stimulated increases in MMP-9, not MMP-2, expression and activity. Chelerythrine, an inhibitor of PKC, inhibited activation of ERK1/2 and JNKs and expression and activity of both MMPs. Selective inhibition of PKC-alpha/beta1 using G?6976 inhibited JNKs activation and the expression and activity of MMP-9, not MMP-2. Inhibition of PKC-theta and PKC-zeta using pseudosubstrates inhibited IL-1beta-stimulated activation of ERK1/2 and JNKs and the expression and activity of MMP-2 and -9. Inhibition of PKC-epsilon had no effect. IL-1beta activated NF-kappaB pathway as measured by increased phosphorylation of IKKalpha/beta and IkappaB-alpha. Inhibition of ERK1/2, JNKs, and PKC-alpha/beta1 had no effect on NF-kappaB activation, whereas inhibition of PKC-theta and PKC-zeta inhibited IL-1beta-stimulated activation of NF-kappaB. SN50, NF-kappaB inhibitor peptide, inhibited IL-1beta-stimulated increases in MMP-2 and -9 expression and activity. These observations suggest that 1) activation of ERK1/2 and JNKs plays a critical role in the regulation of MMP-9, not MMP-2, expression and activity; 2) PKC-alpha/beta1 act upstream of JNKs, not ERK1/2; 3) PKC-zeta and -theta, not PKC-epsilon, act upstream of JNKs, ERK1/2, and NF-kappaB; and 4) activation of NF-kappaB stimulates expression and activity of MMP-2 and -9.     

EC3, a novel heterodimeric disintegrin from Echis carinatus venom, inhibits alpha4 and alpha5 integrins in an RGD-independent manner

EC3, a heterodimeric disintegrin (Mr = 14,762) isolated from Echis carinatus venom is a potent antagonist of alpha4 integrins. Two subunits called EC3A and EC3B were isolated from reduced and alkylated EC3 by reverse-phase high performance liquid chromatography. Each subunit contained 67 residues, including 10 cysteines, and displayed a high degree of homology to each other and to other disintegrins. EC3 inhibited adhesion of cells expressing alpha4beta1 and alpha4beta7 integrins to natural ligands vascular cell adhesion molecule 1 (VCAM-1) and mucosal addressin cell adhesion molecule 1 (MadCAM-1) with IC50 = 6-30 nM, adhesion of K562 cells (alpha5beta1) to fibronectin with IC50 = 150 nM, and adhesion of alphaIIbbeta3 Chinese hamster ovary cells to fibrinogen with IC50 = 500 nM; it did not inhibit adhesion of alphavbeta3 Chinese hamster ovary cells to vitronectin. Ethylpyridylethylated EC3B inhibited adhesion of Jurkat cells to immobilized VCAM-1 (IC50 = 6 microM), whereas EC3A was inactive in this system. The MLDG motif appeared to be essential for activity of EC3B. Linear MLDG peptide inhibited the adhesion of Jurkat to VCAM-1 in a dose-dependent manner (IC50 = 4 mM), whereas RGDS peptide was not active at the same concentration. MLDG partially inhibited adhesion of K562 cells to fibronectin (5-10 mM) in contrast to RGDS peptide (IC50 = 3 mM), inhibiting completely at 10 mM.     

Diphenyleneiodonium inhibits NF-kappaB activation and iNOS expression induced by IL-1beta: involvement of reactive oxygen species

AIMS: In this work, we studied the mechanisms by which diphenyleneiodonium chloride (DPI) inhibits nitric oxide (NO) synthesis induced by the proinflammatory cytokine interleukin-1beta (IL-1) in bovine articular chondrocytes. To achieve this, we evaluated the ability of DPI to inhibit the expression and activity of the inducible isoform of the NO synthase (iNOS) induced by IL-1. We also studied the ability of DPI to prevent IL-1-induced NF-kappaB activation and reactive oxygen species (ROS) production. RESULTS: Northern and Western blot analysis, respectively, showed that DPI dose-dependently inhibited IL-1-induced iNOS mRNA and protein synthesis in primary cultures of bovine articular chondrocytes. DPI effectively inhibited NO production (IC50=0.03+/-0.004 microM), as evaluated by the method of Griess. Nuclear factor-kappa B (NF-kappaB) activation, as evaluated by electrophoretic mobility shift assay, was inhibited by DPI (1-10 microM) in a dose-dependent manner. IL-1-induced ROS production, as evaluated by measurement of dichlorofluorescein fluorescence, was inhibited by DPI at concentrations that also prevented NF-kappaB activation and iNOS expression. CONCLUSIONS: DPI inhibits IL-1-induced NO production in chondrocytes by two distinct mechanisms: (i) by inhibiting NOS activity, and (ii) by preventing iNOS expression through the blockade of NF-kappaB activation. These results also support the involvement of reactive oxygen species in IL-1-induced NF-kappaB activation and expression of NF-kappaB-dependent genes, such as iNOS.     

Effects of sphondin,isolated from Heracleum laciniatum,on IL-1beta-induced cyclooxygenase-2 expression in human pulmonary epithelial cells

Recently, under large-scale screening experiments, we found that sphondin, a furanocoumarin derivative isolated from Heracleum laciniatum, possessed an inhibitory effect on IL-1beta-induced increase in the level of COX-2 protein and PGE(2) release in A549 cells. Accordingly, we examined in the present study the action mechanism of sphondin on the inhibition of IL-1beta-induced COX-2 protein expression and PGE(2) release in a human pulmonary epithelial cell line (A549). Pretreatment of cells with sphondin (10-50 microM) concentration-dependently attenuated IL-1beta-induced COX-2 protein expression and PGE(2) release. The IL-1beta-induced increase in COX-2 mRNA expression was also attenuated by sphondin (50 microM). The selective COX-2 inhibitor, NS-398 (0.01-1 microM), inhibited the activity of the COX-2 enzyme in a concentration-dependent manner, while sphondin (10-50 microM) had no effect. Sphondin (50 microM) did not affect the IL-1beta-induced activations of p44/42 MAPK, p38 MAPK, and JNK. Treatment of cells with sphondin (50 microM) or the NF-kappaB inhibitor, PDTC (50 microM) partially inhibited IL-1beta-induced degradation of IkappaB-alpha in the cytosol and translocation of p65 NF-kappaB from the cytosol to the nucleus. Furthermore, IL-1beta-induced NF-kappaB-specific DNA-protein complex formation in the nucleus was partially inhibited by sphondin (50 microM) or PDTC (50 microM). Taken together, we demonstrate that sphondin inhibits IL-1beta-induced PGE(2) release in A549 cells; this inhibition is mediated by suppressing of COX-2 expression, rather than by inhibiting COX-2 enzyme activity. The inhibitory mechanism of sphondin on IL-1beta-induced COX-2 expression may be, at least in part, through suppression of NF-kappaB activity. We conclude that sphondin may have the therapeutic potential as an anti-inflammatory drug on airway inflammation.     

Identification of a novel competitive inhibitor of p38alpha MAPK by a human PBMC screen

The pro-inflammatory cytokines TNF-alpha and IL-1beta are two of the important mediators involved in the several chronic inflammatory diseases. We used the release of TNF-alpha and IL-1beta from lipopolysaccharide-stimulated human PBMC as inflammatory indexes to discover the potential anti-inflammatory candidates. Among near 500 chemical compounds, MT4 had the suppressive action on the release of TNF-alpha and IL-1beta in PBMC with IC50 values of 22 and 44 nM, respectively. After verified the MT4 inhibitory mechanism, the results revealed that p38alpha and p38beta MAPK activity was inhibited by MT4 with an IC50 value of 0.13 and 0.55 microM, respectively. Further characterization of enzyme kinetics showed the binding mode of MT4 was competitive with the ATP substrate-binding site of p38alpha MAPK.