Sesquiterpene lactones (SLs) have potent antiinflammatory properties. We have shown previously that they exert this effect in part by inhibiting activation of the transcription factor NF-B, a central regulator of the immune response. We have proposed a molecular mechanism for this inhibition based on computer molecular modeling data. In this model, SLs directly alkylate the p65 subunit of NF-B, thereby inhibiting DNA binding. Nevertheless, an experimental evidence for the proposed mechanism was lacking. Moreover, based on experiments using the SL parthenolide, an alternative mode of action has been proposed by other authors in which SLs inhibit IB-␣ degradation. Here we report the construction of p65/ NF-B point mutants that lack the cysteine residues alkylated by SLs in our model. In contrast to wild type p65, DNA-binding of the Cys 38 3 Ser and Cys 38,120 3 Ser mutants is no longer inhibited by SLs. In addition, we provide evidence that parthenolide uses a similar mechanism to other SLs in inhibiting NF-B. Contrary to previous reports, we show that parthenolide, like other SLs, inhibits NF-B most probably by alkylating p65 at Cys 38 . Although a slight inhibition of IB degradation was detected for all SLs, the amount of remaining IB was too low to explain the observed NF-B inhibition.The transcription factor NF-B promotes the expression of over 150 target genes in response to inflammation, viral and bacterial infections, and other stressful situations (1, 2). Both the nature of the NF-B inducers and the function of its target genes highlight its role, as a central mediator of the human immune response.NF-B is most frequently composed of a p50 and a p65 subunit retained in the cytoplasm in an inactive form by binding to IB, an inhibitory subunit. Inducers of NF-B, such as bacterial lipopolysaccharides (LPS) 1 or inflammatory cytokines, activate the IB kinase complex (IKC), which phosphorylates IB on serines 32 and 36. Phosphorylation causes IB ubiquitinylation and its subsequent degradation by the 26-S proteasome. Degradation of the inhibitor allows NF-B to translocate to the nucleus, where it stimulates transcription of its target genes.2 Genes that are regulated by NF-B include, for example, proinflammatory and inflammatory cytokines such as interleukin-1, -2, -4, and -6 or TNF-␣, as well as genes encoding immunoreceptors, cell adhesion molecules, acute phase proteins, and enzymes such as cyclooxygenase-II. Because of its central role in regulating inflammatory responses, a pharmacological inhibition of NF-B activation could be beneficial in the treatment of inflammation (4). Using helenalin as a model, we have shown that SLs inhibit neither IB degradation nor NF-B nuclear translocation. SLs interact directly with NF-B. DNA binding of NF-B is prevented by selectively alkylating cysteine sulfhydryl groups in its p65 subunit (5, 6). There are strong indications that this is a general mechanism for SLs, which possess ␣,␤-or ␣,␤,␥-unsaturated carbonyl structures such as ␣-methylene-␥-lactones or ␣,␤-unsubstituted...

Trypanosomiasis and leishmaniasis are important parasitic diseases affecting millions of people in Africa, Asia, and South America. In a previous study, we identified several flavonoid glycosides as antiprotozoal principles from a Turkish plant. Here we surveyed a large set of flavonoid aglycones and glycosides, as well as a panel of other related compounds of phenolic and phenylpropanoid nature, for their in vitro activities against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania donovani. The cytotoxicities of more than 100 compounds for mammalian L6 cells were also assessed and compared to their antiparasitic activities. Several compounds were investigated in vivo for their antileishmanial and antitrypanosomal efficacies in mouse models. Overall, the best in vitro trypanocidal activity for T. brucei rhodesiense was exerted by 7,8-dihydroxyflavone (50% inhibitory concentration [IC 50 ], 68 ng/ml), followed by 3-hydroxyflavone, rhamnetin, and 7,8,3,4-tetrahydroxyflavone (IC 50 s, 0.5 g/ml) and catechol (IC 50 , 0.8 g/ml). The activity against T. cruzi was moderate, and only chrysin dimethylether and 3-hydroxydaidzein had IC 50 s less than 5.0 g/ml. The majority of the metabolites tested possessed remarkable leishmanicidal potential. Fisetin, 3-hydroxyflavone, luteolin, and quercetin were the most potent, giving IC 50 s of 0.6, 0.7, 0.8, and 1.0 g/ml, respectively. 7,8-Dihydroxyflavone and quercetin appeared to ameliorate parasitic infections in mouse models. Generally, the test compounds lacked cytotoxicity in vitro and in vivo. By screening a large number of flavonoids and analogues, we were able to establish some general trends with respect to the structure-activity relationship, but it was not possible to draw clear and detailed quantitative structure-activity relationships for any of the bioactivities by two different approaches. However, our results can help in directing the rational design of 7,8-dihydroxyflavone and quercetin derivatives as potent and effective antiprotozoal agents.

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Alcoholic extracts prepared form Arnicae flos, the collective name for flowerheads from Arnica montana and A. chamissonis ssp. foliosa, are used therapeutically as anti-inflammatory remedies. The active ingredients mediating the pharmacological effect are mainly sesquiterpene lactones, such as helenalin, 11alpha,13-dihydrohelenalin, chamissonolid and their ester derivatives. While these compounds affect various cellular processes, current data do not fully explain how sesquiterpene lactones exert their anti-inflammatory effect. We show here that helenalin, and, to a much lesser degree, 11alpha,13-dihydrohelenalin and chamissonolid, inhibit activation of transcription factor NF-kappaB. This difference in efficacy, which correlates with the compounds' anti-inflammatory potency in vivo, may be explained by differences in structure and conformation. NF-kappaB, which resides in an inactive, cytoplasmic complex in unstimulated cells, is activated by phosphorylation and degradation of its inhibitory subunit, IkappaB. Helenalin inhibits NF-kappaB activation in response to four different stimuli in T-cells, B-cells and epithelial cells and abrogates kappaB-driven gene expression. This inhibition is selective, as the activity of four other transcription factors, Oct-1, TBP, Sp1 and STAT 5 was not affected. We show that inhibition is not due to a direct modification of the active NF-kappaB heterodimer. Rather, helenalin modifies the NF-kappaB/IkappaB complex, preventing the release of IkappaB. These data suggest a molecular mechanism for the anti-inflammatory effect of sesquiterpene lactones, which differs from that of other nonsteroidal anti-inflammatory drugs (NSAIDs), indomethacin and acetyl salicylic acid.

Prompted by results of our previous studies where we found high activity of some sesquiterpene lactones (STLs) against Trypanosoma brucei rhodesiense (which causes East African sleeping sickness), we have now conducted a structure-(in-vitro)-activity study on a set of 40 STLs against T. brucei rhodesiense, T. cruzi, Leishmania donovani and Plasmodium falciparum. Furthermore, cytotoxic activity against L6 rat skeletal myoblast cells was assessed. Some of the compounds possess high activity, especially against T. brucei (e.g. helenalin and some of its esters with IC50-values of 0.05-0.1 µM, which is about 10 times lower than their cytotoxic activity). It was found that all investigated antiprotozoal activities are significantly correlated with cytotoxicity and the major determinants for activity are α,β-unsaturated structural elements, also known to be essential for other biological activities of STLs. It was observed, however, that certain compounds are considerably more toxic against protozoa than against mammalian cells while others are more cytotoxic than active against the protozoa. A comparative QSAR analysis was therefore undertaken, in order to discern the antiparasitic activity of STLs against T. brucei and cytotoxicity. Both activities were found to depend to a large extent on the same structural elements and molecular properties. The observed variance in the biological data can be explained in terms of subtle variations in the relative influences of various molecular descriptors.