Sesquiterpene lactones as inhibitors of human neutrophil elastase

Human neutrophil elastase (HNE) is a serine protease that has been implicated in the abnormal turnover of connective tissue proteins and has been described as an important pathogenic factor in several inflammatory diseases such as rheumatoid arthritis or cystic fibrosis. Here we investigated 17 sesquiterpene lactones (SLs) for their ability to inhibit human neutrophil elastase in an in vitro assay. Podachaenin was the most active compound with an IC(50) value of 7 microM. SLs do not covalently bind to the amino acids of the catalytic triad, thus differing from other elastase inhibitors with a lactone moiety. In contrast to most other biological activities of SLs HNE inhibition is not mediated by alpha,beta-unsaturated carbonyl functions. Ligand binding calculations using the X-ray structure of HNE and the program FlexX revealed structural elements which are a prerequisite for their inhibitory activity.     

Sesquiterpene lactones are potent and irreversible inhibitors of the antibacterial target enzyme MurA

We report the identification of the sesquiterpene lactones cnicin and cynaropicrin as potent, irreversible inhibitors of the bacterial enzyme MurA. They covalently bind to the thiol group of Cys115. Judging from the structure-activity relationships, we conclude that the unsaturated ester side chain of cynaropicrin and cnicin is of particular importance for the inhibition of MurA. These results provide evidence that MurA is a target protein of SLs with a probably high relevance for their known antibacterial effect.     

Sesquiterpene lactones inhibit inducible nitric oxide synthase gene expression in cultured rat aortic smooth muscle cells.

Nitric oxide (NO) is an important regulator and effector molecule in various inflammatory disease states. High output of NO during inflammation is generated by the inducible isoform of nitric oxide synthase (iNOS). Sesquiterpene lactones are derived from Mexican-Indian medicinal plants and are known to have potent anti-inflammatory properties. The mechanisms by which sesquiterpene lactones exert their anti-inflammatory effects are not fully understood. In the current studies we determined if the sesquiterpene lactones, parthenolide and isohelenin, modulate iNOS gene expression in cultured rat aortic smooth muscle cells (RASMC) treated with lipopolysaccharide and interferon-gamma. Treatment with parthenolide or isohelenin inhibited NO production and iNOS mRNA expression in a concentration-dependent manner. Transient transfection studies with an iNOS promoter-luciferase reporter plasmid demonstrated that parthenolide and isohelenin also inhibited activation of the iNOS promoter. Inhibition of iNOS promoter activation was associated with inhibition of both I-kappaBalpha degradation and nuclear translocation of NF-kappaB. Neither parthenolide nor isohelenin induced the heat shock response in RASMC. We conclude that sesquiterpene lactones inhibit iNOS gene expression by a mechanism involving stabilization of the I-kappaBalpha/NF-kappaB complex. This effect is not related to induction of the heat shock response. The ability of sesquiterpene lactones to inhibit iNOS gene expression may account, in part, for their anti-inflammatory effects.     

Estrogen effects in the myocardium: inhibition of NF-kappaB DNA binding by estrogen receptor-alpha and -beta

We have previously shown that estrogen effects in the heart include direct hormone effects on the myocardium. In a recent study we found that one beneficial effect of estradiol on the myocardium is the inhibition of apoptosis in cardiac myocytes. This effect was associated with a reduction of NF-kappaB activity. In the present study we have analyzed the functional mechanism of NF-kappaB inhibition in the myocardium by estrogen receptors-alpha and -beta. Despite the previous finding that 17-beta-estradiol (10 nM) inhibited the staurosporine-induced binding of p65/p50 NF-kappaB complexes to their cognate DNA elements in cultured rat cardiac myocytes, myocyte extracts showed no change in expression or cellular localization of p65, p50, and IkappaB upon staurosporine or estradiol treatment. Addition of either estrogen receptor-alpha or estrogen receptor-beta as recombinant protein was sufficient to inhibit staurosporine-dependent p65/p50 DNA binding in cardiac myocytes. 17-beta-Estradiol inhibits staurosporine-induced p65/p50 DNA binding associated with apoptotic cell death of cardiac myocytes via estrogen receptors-alpha and -beta. This is not associated with changes in p65, p50 and IkappaB expression or subcellular localization. Thus, inhibition of NF-kappaB activity by estrogenic compounds might inhibit NF-kappaB dependent gene expression such as pro-inflammatory cytokines in the myocardium.     

Targeting nuclear factor-kappa B activation pathway by thymoquinone: role in suppression of antiapoptotic gene products and enhancement of apoptosis

Thymoquinone (TQ), derived from the medicinal plant Nigella sativa, exhibits antiinflammatory and anticancer activities through mechanism(s) that is not fully understood. Because numerous effects modulated by TQ can be linked to interference with the nuclear factor-kappaB (NF-kappa B) signaling, we investigated in detail the effect of this quinone on NF-kappa B pathway. As examined by DNA binding, we found that TQ suppressed tumor necrosis factor-induced NF-kappa B activation in a dose- and time-dependent manner and inhibited NF-kappaB activation induced by various carcinogens and inflammatory stimuli. The suppression of NF-kappaB activation correlated with sequential inhibition of the activation of I kappa B alpha kinase, I kappa B alpha phosphorylation, I kappa B alpha degradation, p65 phosphorylation, p65 nuclear translocation, and the NF-kappa B-dependent reporter gene expression. TQ specifically suppressed the direct binding of nuclear p65 and recombinant p65 to the DNA, and this binding was reversed by DTT. However, TQ did not inhibit p65 binding to DNA when cells were transfected with the p65 plasmid containing cysteine residue 38 mutated to serine. TQ also down-regulated the expression of NF-kappa B-regulated antiapoptotic (IAP1, IAP2, XIAP Bcl-2, Bcl-xL, and survivin), proliferative (cyclin D1, cyclooxygenase-2, and c-Myc), and angiogenic (matrix metalloproteinase-9 and vascular endothelial growth factor) gene products. This led to potentiation of apoptosis induced by tumor necrosis factor and chemotherapeutic agents. Overall, our results indicate that the anticancer and antiinflammatory activities previously assigned to TQ may be mediated in part through the suppression of the NF-kappa B activation pathway, as shown here, and thus may have potential in treatment of myeloid leukemia and other cancers.     

Glutathionylation of the p50 subunit of NF-kappaB: a mechanism for redox-induced inhibition of DNA binding.

The cellular redox status can modify the function of NF-kappaB, whose DNA-binding activity can be inhibited by oxidative, nitrosative, and nonphysiological agents such as diamide, iodoacetamide, or N-ethylmaleimide. This inhibitory effect has been proposed to be mediated by the oxidation of a conserved cysteine in its DNA-binding domain (Cys62) through unknown biochemical mechanisms. The aim of this work was to identify new oxidative modifications in Cys62 involved in the redox regulation of the NF-kappaB subunit p50. To address this problem, we exposed p50, both the native form (p50WT) and its corresponding mutant in Cys62 (C62S), to changes in the redox pair glutathione/glutathione disulfide (GSH/GSSG) ratio ranging from 100 to 0.1, which may correspond to intracellular (patho)physiological states. A ratio between 1 and 0.1 resulted in a 40-70% inhibition of the DNA binding of p50WT, having no effect on the C62S mutant. Mass spectrometry studies, molecular modeling, and incorporation of (3)H-glutathione assays were consistent with an S-glutathionylation of p50WT in Cys62. Maximal incorporation of (3)H-glutathione to the p50WT and C62S was of 0.4 and 0.1 mol of (3)H-GSH/mol of protein, respectively. Because this covalent glutathione incorporation did not show a perfect correlation with the observed inhibition in the DNA-binding activity of p50WT, we searched for other modifications contributing to the maximal inhibition. MALDI-TOF and nanospray-QIT studies revealed the formation of sulfenic acid as an alternative or concomitant oxidative modification of p50. In summary, these data are consistent with new oxidative modifications in p50 that could be involved in redox regulatory mechanisms for NF-kappaB. These postranslational modifications could represent a molecular basis for the coupling of pro-oxidative stimuli to gene expression.     

Antifungal activity of sesquiterpene lactones

Forty-five sesquiterpene lactones were screened for their antifungal activities against Microsporum cookei, Trichophyton mentagrophytes and Fusarium sp. The screening tests showed that a majority of sesquiterpene examined possess at least weak antifungal activity, the eudesmanolides being the most active. The antifungal activity of sesquiterpene lactones cannot be explained by the presence or absence of two potential active sites (the exocyclic methylene and, in pseudoguaianolides, a β-unsubstituted cyclopentenonel) but other functions must play a role in enhancing or reducing this activity.