WO1999033463A1 - SESQUITERPENE LACTONES SPECIFICALLY INHIBIT ACTIVATION OF NF-λB BY PREVENTING THE DEGRADATION OF IλB-α AND IλB-$g(b) - Google Patents

Abstract

Extracts from certain Mexican indian medicinal plants used in traditional indigenous medicine for the treatment of inflammations contain sesquiterpene lactones (SLs), which specifically inhibit the transcription factor NF-λB (1). Here we show that SLs prevented the activation of NF-λB by different stimuli such as phorbolesters, tumor necrosis factor (TNF)-α, ligation of the T-cell receptor and hydrogen peroxide in various cell types. Treatment of cells with SLs prevented the induced degradation of IλB-α and IλB-β by all these stimuli, suggesting that they interfere with a rather common step in the activation of NF-λB. SLs did neither interfere with DNA-binding activity of a ctivated NF-λB nor with the activity of the protein tyrosine kinases p59fyn and p60src. Micromolar amounts of SLs prevented the induced expression of the NF-λB target gene ICAM-1. Inhibition of NF-λB by SLs resulted in an enchanced cell-killing of murine fibroblast cells by TNF-α. SLs lacking an exomethylene group in conjugation with the lacone function displayed no inhibitory activity on NF-λB. The analysis of the cellular redox-state by FACS showed that the SLs had no direct or indirect anti-oxidant properties.

Description

Sesquiterpene Iactones specifically inhibit activation of NF-κB by preventing the

degradation of IκB-α and IκB-β

Field of the of the invention The present invention is concerned with the use of extracts from certain Mexican indian medicinal plants applied for the treatment of inflammations. These extracts contain

sesquiterpene Iactones (Sis), which specifically inhibit the transcription factor NF-κB.

Background of the invention

The transcription factor NF-κB is one of the key regulators of genes involved in the

immune and inflammatory response (for review see 2). In mammalian cells, NF-κB is

composed of a homo- or heterodimer of various DNA-binding subunits. Five different DNA- binding subunits share a N-terminal homology domain, which confers DNA-binding,

dimerization, nuclear translocation and interaction with the inhibitory IκB proteins (for review

see 3, 4). In most cell types these proteins sequester NF-κB, which is frequently a heterodimer

of the p50 and p65 (RelA) subunits, in the cytoplasm by masking their nuclear localization

sequence. Constitutive NF-κB activity in the cell nucleus can only be detected in certain

neurons, some cells of the monocyte/macrophage lineage and B cells (for review see 5, 6). Stimulation of cells with a variety of pathogenic agents including inflammatory cytokines, phorbol esters, UV irradiation and oxidants finally leads to the intracellular generation of reactive oxygen intermediates (ROIs) as a key event and results in the activation of NF-κB

(for review see 7, 8). The two major forms of IκB proteins, termed IκB- and IκB-β, can be

inducibly phosphorylated and ubiquitinylated (9-11). These posttranslational modifications tag the molecule for the subsequent proteolytical degradation by the ubiquitin-26S proteasome

pathway (12-14). This induced degradation of IκB proteins unmasks the nuclear localization

sequences of the DNA-binding subunits of the NF-κB dimer and allows NF-κB to enter the nucleus, to bind to its DNA sequence and to induce transcription. The target genes whose

transcription is mainly regulated by NF-κB include many cytokines, cell adhesion molecules,

such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), as well as acute-phase proteins and immunoreceptors (for review see 15).

Among its many different biological activities NF-κB seems to play an important role

in cell killing. NF-κB has been shown recently to counteract the induction of apoptosis by the

cytokine TNF-α, ionizing radiation and the cancer chemotherapeutic agent daunorubicine (for

review see 16). However, there is also evidence for apoptosis-promoting properties of NF-κB.

Glutamate was found to induce NF-κB in neuronal cells and acetylsalicylic acid (Aspirin^®)

protected these cells from NF-κB-induced cell death (17, 18). Along this line, the

overexpression of the cellular and viral anti-apoptotic proteins Bcl-2 and E1B 19 K both

negatively interfered with the activation of NF-κB under these conditions (19-21).

The role of NF-κB in the immune response is also evident from gene disruption

experiments. The targeted deletion of the p50, p65, RelB and c-Rel subunits resulted in an impaired immune response and/or in a reduced viability of the mice (22-26). These findings

and the immunological relevance of most of the NF-κB target genes make this transcription

factor an interesting therapeutical target for the identification of inhibitors. One group of NF- KB inhibitors exerts its inhibitory effects by scavenging ROIs. These inhibitors include N- acetyl-L-cysteine (27, 28), pyrrolidine dithiocarbamate (29), acetylsahcylic acid (30, 31) or curcumin (32). All these compounds are structurally unrelated, but share the property of being anti-oxidative. Another group of inhibitors interferes with the induced degradation of IκB-

family members by affecting the functioning of the 26S proteasome (14, 33, 34). Further

inhibitors of NF-κB exert their effects only in the cell nucleus by impairing the transcriptional

activity of NF-κB already bound to DNA. Examples are inhibitors of the p38 MAP kinase (35,

36) and, at least in some cell types, glucocorticoids. Here, the activated glucocorticoid

receptor directly binds to the NF-κB dimer and thereby prevents transcriptional activation

(37).

WO 96/25666 (PCT/US96/02122) describes a polypeptide IκB-β which binds to and

effects NF-κB gene activation. Also disclosed is a nucleotide sequence encoding IκB-β and

methods of identifying compositions which affect IκB-β/NF-κB complexes. WO92/20795

(PCT/US9204073) describes the purification, cloning, expression and characterization of IκB.

Summary of the invention

It has been found that Sis prevents the activation of NF-κB by different stimuli; more

precisely it has been found that treatment of cells with Sis prevented the induced

degradation of IκB-α and IκB-β by these stimuli, suggesting that they interfere with a

common step in the activation of factor NF-κB.

We recently identified SLs isolated from extracts of Mexican indian medicinal plants as

specific inhibitors of NF-κB (1). SL-containing plant extracts are frequently used in the traditional Mexican indian medicine for the treatment of infections of the skin and other organs (for review see 38). The SL parthenolide is also contained in drugs such as Feverfew^® (Tanacetum parthenium) used against migraine, an illness that has been implicated with neurogenic inflammatory processes (39). The anti-inflammatory activity of the SL-containing plant extracts was confirmed in the hen egg tests where they showed a delay in cell culture experiments (40) and the onset of capillary reactions of the allantois membrane (1).

The present invention shows that SLs prevent a common step in NF-κB activation. They did not interfere with the generation of oxygen radicals, but prevented the induced

degradation of IκB-α and IκB-β as well as the induced expression of the NF-κB target gene ICAM-1. Structural studies identified the exomethylene group in conjugation with the lactone group as the decisive structural feature for the inhibitory activity.

Numerous biological activities have been reported for SLs, including antimicrobial (48), antiviral (49) and antitumor activities (50). Furthermore SLs or SL-containing plant extracts were found to have anti-inflammatory properties (51, 52). Anti-phlogistic activities were also seen in hen eggs assays, in the reduced production of the inflammatory cytokine IL- 6 and in cell culture experiments (1, 40). It was previously reported that the anti-inflammatory effects of SLs can be assigned -at least to a certain extent- to the inhibition of transcription

factor NF-κB, a central mediator of the immune response (1). The present invention shows

that SLs do not interfere with the generation of ROIs following the stimulation of cells, but

prevent the induced degradation of IκB-α and IκB-β. The SLs do not directly act on the

DNA-binding subunits of NF-κB. Also the IκB subunits seems not to be a direct target for the

SLs. The incubation of cell extracts from unstimulated cells with SLs did not change the inducibility of NF-κB by the dissociating agent desoxycholate (M.L.S., unpublished results). Furthermore the re-synthesis of the putative target protein(s) after more than 18 h makes the

IκB proteins an unlikely candidate for the SLs, since IκB-α is completely resynthesized within one hour (53)

Various protein kinases have been implicated in the induced activation of NF-κB, but

the kinases participating in these signaling events remain poorly defined. The signal

transducing events leading to NF-κB include the small GTP-binding proteins Racl (54) and Cdc 42 (55) which then lead to the activation of mitogen-activated protein kinase/ERK kinase kinase- 1 (MEKK-1). The activation of MEKK-1, which has recently been shown to be

necessary for the induction of NF-κB (56), results in the induced activity of mitogen-activated

protein kinase kinase (MKK4/SEK) and the c-Jun N-terminal kinase (JNK) (57). The kinase

finally phosphorylating IκB-α at serines 32 and 36 is called CHUK (58) or IKK (59). It is

currently not clear which identified or so far unidentified member(s) of the signaling cascade is the target for SLs. Furthermore the number of molecules inhibited by SLs remains unknown.

The role of NF-κB in the induction of cell death is still not yielding a homogenous

picture. The inhibition of NF-κB promotes TNF-α-mediated cell death in HeLa cells,

macrophages, fibroblasts, fibrosarcoma cells and Jurkat cells (60-62), while it is ineffective in other cell lines such as human breast carcinoma cells (64). Part of the discrepancies may be explained by differences of the cell type studied and the nature of the apoptosis-inducing stimulus. This study shows that the incubation with parthenolide facilitates cell-killing by TNF-

α in mouse L929 cells, although only additional experiments using further inhibitors of NF-κB

can really prove the protecting role of this transcription factor for L929 cells. The mechanism

of this cell-protecting effect of NF-κB is still not clear. It might rely on the induced expression of anti-apoptotic genes such as the zinc finger protein AJO or manganese superoxide dismutase (for review see 16).

The SLs tested in this study display a high degree of specificity for their inhibitory activity, since they did not influence the activity of other transcription factors such as AP-1,

RBP-Jκ and Oct-1 (1; this study). Furthermore the SLs did not impair the activity of the T-cell

kinases p59fyⁿ and p60^src. These results show that the SLs do not interfere in a non-specific manner with transcription factors or signaling molecules. A potential target-specificity of SLs may well be explained by considering the fact that the combination of the reactive Michael- acceptor system together with the oxygen-substituted isoprenoide rings forms a pattern of potential non-covalent binding sites (e.g. hydrogen bonds). These binding sites would allow the SLs to interact with complementary sites on the surface of the target molecule(s). The

relative positions of SLs and other inhibitors of NF-κB in the signaling cascade are

schematically displayed in Figure 9. This allows the usage of this drug for the analysis and dissection of the diverse signal transduction pathways finally resulting in the activation of NF-

KB. Since the various classes of inhibitors interfere with NF-κB activation at different levels in

signal transduction it is tempting to speculate that a mixture of these different inhibitors might

be highly effective in NF-κB inhibition. Furthermore the individual doses required for an

optimal blocking may presumably be reduced in such a blend, thereby reducing the respective side effects.

The inhibition of NF-κB may be of therapeutic use for the treatment of chronic

diseases such as rheumatoid arthritis or for the acute situations such as septic shock. There is

emerging evidence for the role of NF-κB during rheumatoid arthritis (for review see 2) and

the importance of NF-κB during Crohn^'s disease. Here, the local administration of antisense phosphorothioate oligonucleotides to the p65 subunit of NF-κB abrogated established experimental colitis in mice (65). Also other chronic inflammatory diseases such as

Alzheimer^'s disease involve the activation of NF-κB. The amyloid β peptide, which is a major

component of the plaque, induces an increase in ROIs and activates the nuclear translocation

of the p50 and p65 subunits of NF-κB in the neurons directly surrounding the plaque (5).

Moreover the septic shock syndrome is associated with a massive activation of NF-κB. Septic shock occurs when microbial products such as LPS stimulate the expression of inflammatory cytokines. This massive production of cytokines leads to failure of circulation and general organ function. Therefore it is of therapeutical interest to develop drugs that are able to

interfere with the activity of NF-κB.

The complete inhibition of NF-κB is lost only to 50 % after 18 h post treatment,

suggesting an irreversible mechanism such as a covalent modification of proteins. Also other drugs such as Aspirin^® and omeprazole (Antra^®), lead to to an irreversible inactivation of their target molecules (for review see 66, 67). The covalent modification of a target protein by a drug reduces the required frequency of drug application.

The determination of the structural requirements of SLs for NF-κB inhibition

presented in this study provides the basis for a rational development of a new generation of

anti-inflammatory substances interfering with NF-κB.

This and other aspects of the present invention become more fully appreciated upon consideration of the invention described below.

Brief description of the drawings

Fig. 1. SLs inhibit NF-κB activation by different stimuli. Fig. 2. SLs do not interfere with DNA-binding of activated NF-κB.

Fig. 3. Effects of SLs on DNA-binding of Oct-1 and the fragmentation of IκB-α.

Fig 4. SLs inhibit the degradation of IκB-α and IκB-β induced by various stimuli.

Fig. 5. Reversibility of SL treatment.

Fig. 6. Effect of parthenolide on the cell killing by TNF-α.

Fig. 8. SLs do not change the intracellular redox-state.

Fig. 9. Distinct positions of NF-κB inhibitors in the activation cascade.

Detailed description of the preferred embodiments

Fig. 1 A, shows the inhibition of NF-κB activated by H₂O₂. Jurkat JR cells were preincubated with the indicated amounts of parthenolide for one h and stimulated with various concentrations of H₂O₂ for 90 min. Subsequently total cell extracts were prepared and tested for DNA-binding

of activated NF-κB by EMS A. B shows the inhibition of NF- B activated by CD3/CD28 ligation.

Jurkat J16 cells were preincubated with the indicated amounts of parthenolide for 1 h and

activated by immobilized α-CD3 antibodies and crosslinked α-CD28 antibodies. Ten minutes

after stimulation cells were harvested and assayed for NF-κB activity by EMSA. The SLs were

kept with the cells during both stimulations. The filled arrowhead indicates the location of the

DNA-NF-κB complex, the circle indicates the position of a constitutively DNA-binding protein

and the open arrowhead points to the unbound oligonucleotide.

Fig. 2. HeLa cells were stimulated for 20 min with PMA and total cell extracts of the stimulated cells were pooled. These extracts were incubated for 1 h with various concentrations of parthenolide as indicated. Subsequently these extracts were tested together with a protein extract from unstimulated HeLa cells as a control for DNA-binding activity of NF-κB by EMS A. Bound and free oligonucleotides were separated by electrophoresis on a native gel, dried and exposed. The symbols used are as explained in Fig.1.

Fig. 3. Mouse L929 fibroblasts were preincubated with parthenolide for 1 h prior to stimulation

with murine TNF-α. Subsequently cell extracts from these cells were prepared, together with

extracts from unstimulated and TNF-α-stimulated cells as indicated. Equal amounts of the protein

extracts were simultaneously tested for DNA-binding activity of NF-κB (A), Oct-1 (C) and for

the presence of IκB-α in Western blots (B). A, DNA-binding activity of NF-κB. Cells were

treated as indicated and total cell extracts were tested for NF-κB binding by EMSA. The symbols

used are as described for Fig. 1. B, IκB-α Western blot. Total cell extracts were separated by

SDS-PAGE and transferred to a PVDF membrane. The arrow points to the IκB-α protein which

was detected with an α-IκB-α antibody. The positions of prestained protein markers are

indicated. C, DNA-binding activity of Oct-1. The indicated cell extracts were tested for the activity of the constitutively DNA-binding protein Oct-1 by EMSA. The open arrowhead points to the position of the unbound oligonucleotide, the filled arrowhead indicates the position of the DNA-protein complex.

Fig. 4. Cells were preincubated with 10 μM of parthenolide 1 h prior to stimulation with 2000 U

TNF-α (A), 50 ng/ml PMA (B), ligation of the CD3/CD28 receptors (C) and 100 μM of H₂O₂

(D). Cells were harvested at the indicated time points and total cell extracts were prepared. Equal

amounts of protein were tested by EMSAs for DNA-binding of NF-κB and by Western blot

experiments for the occurence of IκB proteins. The shifted DNA-protein complexes are shown in the upper parts of the respective panels with the filled arrowhead pointing to the position of the

NF-KB-DNA complex. The same extracts were simultaneously tested for the occurence of IκB-α

and IκB-β in Western blots with the arrows pointing to the respective IκB proteins. Representative experiments are shown, for further details see text.

Fig. 5. HeLa cells were preincubated with 5 μM of parthenolide for 1 h. Subsequently the medium was replaced by parthenolide-free medium and the cells were grown for the indicated

time periods prior to stimulation with PMA for 15 min. NF-κB activity was then assessed by

EMSA and the amount of DNA-bound NF-κB was quantitated in a Phosphorlmager (Molecular

Dynamics). A, schematic outline of the experimental strategy. The incubation time with parthenolide is symbolized by the black columns, the stimulation period is highlighted by

hatching. B, time-dependence of NF-κB inhibition. The diagram shows the percental inhibition of

NF-κB activity in dependence of the time period between parthenolide preincubation and PMA-

stimulation. The complete inhibition of NF-κB in the experiment without incubation in

parthenolide-free medium was set as 100%. A typical experiment is shown.

Fig. 6. L929 cells were incubated with either 5 μM parthenolide (♦), 2000 U/ml TNF-α (σ), or a

combination of both (v). At the indicated time points cell viability was determined using the MTT

assay. Viable cells remaining after treatment with mTNF-α are shown as a percentage of viable

untreated cells. A representative experiment is shown.

Fig. 7. A, effect of TNF-α on the expression of ICAM-1. Jurkat JR cells were stimulated for 10 h

with 2000 U/ml of TNF-α, stained with an α-ICAM-1 antibody coupled to phycoerythrine and analyzed by flow cytometry. The profiles of untreated (white) and TNF-α-stimulated (black) cells are shown. B, effect of parthenolide on ICAM-1 expression. Jurkat cells were treated as in (A) in

the presence of 10 μM parthenolide. Profiles of unstimulated (white) and stimulated (black) cells

are displayed.

Fig. 8. A, effects of TNF-α on the concentration of intracellular ROIs. Jurkat JR cells were either

left untreated or stimulated for 15 min with human TNF-α (2000 U/ml). Subsequently ROIs were

measured by FACS analysis of DFCH- stained cells. FACS profiles of untreated (white) and TNF-

α-stimulated cells (dark) are shown. B, effect of parthenolide on the cellular redox-state. Jurkat

T-cells were incubated with 5 μM parthenolide and further treated and analyzed as described in

part (A) of this figure. FACS profiles of untreated (white) and TNF-α-stimulated cells (dark) are

shown. C, comparison of ROI amounts. The mean fluorescence of unstimulated cells was set as 1 and directly compared to the fluorescence of the TNF-stimulated cells in the presence or absence of parthenolide as indicated.

Fig. 9. Diverse stimuli of NF-κB are given on the top. The various classes of NF-κB inhibitors

are shown in boxes. Abbreviations not used in the text are: B7-1: B-cell activation antigen, PKC: protein kinase C. TCR: T-cell receptor. Further details are given in the text.

Experimental Procedures

Cell culture Jurkat T leukemia cells (subclone JR „Wurzburg") were maintained in RPMI 1640 supplemented with 10% heat-inactivated FCS and 1% (w/v) penicillin/streptomycin (all purchased from GIBCO Laboratories, Gran Island, NY). HeLa cells and L929 fibroblasts were grown in Dulbecco's modified Eagle medium (DMEM) containing 10% FCS and 1% (w/v) penicillin/streptomycin. All

cells were grown in an incubator at 37°C and 5% CO₂. TNF-α and poly (dl-dC) were obtained

from Boehringer Mannheim (Mannheim, Germany). Parthenolide, isohelenin, isophoronoxide, limonenoxide, caryophyllenoxide, sclareolide and santonin were from Sigma Inc. (St. Louis,

MO). Antibodies directed against IκB-α and NF-κB were from Santa Cruz Inc. (Santa Cruz,

CA), α-CD28 antibodies were obtained from Pharmingen Inc. (San Diego, CA) and α-CD3 antibodies were isolated from a hybridoma cell line. The phycoerythrine-conjugated antibody against ICAM-1 was obtained from Dianova (Hamburg, Germany). All other chemicals were either from Sigma, Aldrich (Steinheim, Germany) or Roth (Karslruhe, Germany).

Electrophoretic mobility shift assay (EMSA)

HeLa or L929 cells (5 x 10⁵) were grown overnight on 10 cm dishes, Jurkat cells (approximately

1 x lOVml) in cell culture flasks. One hour prior to stimulation by either TNF-α, phorbol- 12-

myristate 13 -acetate (PMA) or hydrogen peroxide, cells were preincubated with the indicated amounts of the tested substances for 60 minutes at 37°C. The tested substances were dissolved in dimethyl sulfoxide (DMSO) as a solvent. In the following cells were stimulated for 20 min with

PMA at a final concentration of 50 ng/ml, TNF-α (2000 U/ml), α-CD3/α-CD28 (1 μg/ml)

antibodies or for the indicated periods with the specified amounts of hydrogen peroxide. Cells were harvested by centrifugation and washed twice with cold TBS buffer (25 mM Tris/HCl pH 7.4, 137 mM NaCl, 5 mM KC1, 0.7 mM CaCl , 0.1 mM MgCl₂). The pellet was resuspended in TOTEX buffer (20 mM Hepes/KOH pH 7.9, 035 M NaCl, 20% (v/v) glycerol, 1% (v/v) NP-40, 1 M MgCl₂, 0.5 mM EDTA, 0J mM EGTA, 1 mM phenylmethylsulfonyl fluoride (PMSF)) and incubated on ice for 30 min. The samples were carefully vortexed every 10 min. The cell debris was pelleted upon centrifugation with 14000 rpm at 4°C for 10 min. Equal amounts of supernatant were tested for DNA binding activity as described in detail elsewhere (1). Briefly, the

extracts were incubated with 2 μg poly (dl-dC), 2 μg BSA and 10000 cpm of a ³²P labeled

oligonucleotide on ice in 5 x binding buffer (20% (w/v) Ficoll 400, 100 mM Hepes/KOH pH 7.9, 1 mM DTT and 300 mM KC1) in a final volume of 20 μl. Subsequently the free and the oligonucleotide-bound proteins were separated by electrophoresis on a native 4% polyacrylamide gel. The gel was dried after electrophoresis and exposed to an X-ray film (Amersham Hyperfilm). The following oligonucleotides (binding site underlined) were used:

NF-κB: 5 '-AGTTGAGGGGACTTTCCCAGGC-3 '

3 '-TC AACTCCCCTGAAAGGGTCCG-5 '

Oct- 1 : 5 '-TGTCGAATGCAAATC ACT AGAA-3 '

3 -ACAGCTTACGTTTAGTGATCTT 5'

Western Blotting

Cell extracts were separated on a 12 % reducing SDS polyacrylamide gel. Subsequently the proteins were transferred from the SDS gel onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA) using a semi-dry blotting apparatus (Bio-Rad laboratories, Munich, Germany). Transfer efficiency was monitored by Ponceau S staining of membranes. Prior to the

incubation with the α-IκB-α and IκB-β antibodies (Santa Cruz Inc. Santa Cruz, CA), the

membrane was blocked with 5% non-fat dry milk powder in TBST buffer (25 mM Tris/HCl pH 7.4, 137 mM NaCl, 5 mM KC1, 0.7 mM CaCl₂, 0.1 mM MgCl₂, 0.05 % (v/v) Tween-20). The

membrane was then incubated in a small volume of TBST containing an 1:1000 dilution of the α-

IκB antibodies. After an overnight-incubation the membrane was extensively washed in TBST

buffer and incubated for another hour in TBST containing a 1 :3000 dilution of α-rabbit antibody

coupled to horseradish peroxidase. After extensively washing the membrane the immunoreactive bands were visualized by enhanced chemiluminiscence according to the instructions of the manufacturer (Amersham Lifescience, Braunschweig, Germany). In vitro kinase assays

The Src family protein tyrosine kinases p60^src and p59^f^ were expressed in baculovirus-infected Sf9 cells and purified by affinity chromatography as described (41). The effect of SLs on protein kinase activity was determined using enolase as a substrate. Briefly, serial concentrations of

parthenolide and isohelenin were preincubated at 30°C with pόO^*0 or p59^fyn protein in 50 μl

kinase buffer (30 mM Hepes pH 1.2, 5 mM MgCl₂, 5 mM MnCl₂, 1 μM ATP, 10 μCi ³²P-γ-ATP)

containing 5 μg of acid-treated rabbit muscle enolase (Sigma Inc., St. Louis, MO). After 15 min,

the reaction was ended by the addition of Laemmli buffer. The samples were analyzed by SDS- PAGE and subjected to autoradiography.

Cell killing assays

The cytotoxic activity of TNF was determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide assay (MTT assay) essentially as described by Mosmann (42).

L929 cells were seeded at a density of lxl 0⁴ cells per well in 96-well microtiter plates (flat bottomed) and incubated for 16 h in 0J ml culture medium. The supernatant was then removed and replaced by fresh medium containing TNF (2000 U/ml) or/and parthenolide (5 μM). At the indicated times 20 μl of a MTT solution (5 mg/ml PBS) was added to all wells. After another 3 h incubation supematants were removed followed by addition of 100 μl of a 24:1 (v/v) isopropanol/HCl solution. After 15 min at room temperature the absorbance of each well was determined with an automated plate reader (Digiscan, Asys Hitech, Austria) at 550 nm.

FACS analysis

The intracellular formation of reactive oxygen intermediates was measured using dichlorofluorescein-diacetate (DFCH) according to Royall and Ischiropoulus (43). Briefly, a 10 mM stock solution of DFCH (Molecular Probes, Eugene, OR) was prepared in DMSO under nitrogen and stored at -20°C. Cells were incubated with 5 μM DFCH in phosphate-buffered saline (PBS) for 0.5 h at 37°C. ICAM-1 expression on Jurkat cells was determined using a FITC- conjugated anti-CD 54 (ICAM-l)-antibody (Dianova, Hamburg, Germany). In both cases measurements were performed in duplicates using a FACScan (Becton Dickinson, Heidelberg, Germany) flow cytometer. Dead cells were excluded by forward/side scatter gating and staining with propidium iodide.

Example 1 SLs inhibit a common step in NF-κB activation

From a previous study it was evident that SLs prevent the PMA-induced activation of NF-κB

(1). Therefore we studied whether SLs can inhibit NF-κB activation in response to stimuli

different from PMA. As an example for a receptor-mediated pathway mouse L929 cells were

stimulated with TNF-α with or without preincubation with various amounts of isohelenin or parthenolide. After 1 h of preincubation cells were stimulated for 20 min with murine TNF-α and total extracts were tested for DNA-binding activity by EMSA. The TNF-induced binding

of NF-κB was completely prevented by preincubation of cells with 5 μM of parthenolide and

20 μM of isohelenin, respectively (data not shown). Since it is known that most inducers of

NF-κB lead to the formation of ROIs, we tested whether SLs would interfere with H₂O₂-

induced NF-κB binding activity. Therefore Jurkat JR cells were preincubated for 1 h with

various amounts of parthenolide and isohelenin and treated with various concentrations of H₂O₂ for 90 min. The extracts prepared from these stimulated cells were subsequently

analyzed by EMSA (Fig. 1 A). Only H₂O₂ concentrations between 100 and 250 μM activated

NF-κB, whereas the addition of 1 mM H₂O₂ failed to efficiently induce NF-κB probably due

to an oxidative destruction of the protein. The H₂O₂-induced DNA-binding activity of NF-κB was completely prevented by preincubation with the two SLs in low micromolar

concentrations. Another stimulus of NF-κB with special relevance in T-cells is the activation of the CD3/CD 28 pathway, which also leads to an increased concentration of ROIs (44). T-

cell specific activation of NF-κB in Jurkat T-cells by crosslinking the CD3- and CD28-

receptors was prevented by preincubation with low micromolar concentrations of the two SLs (Fig. 1 B). All these experiments indicate that SLs interfere with one or more common steps

during NF-κB activation in different cell types rather than with one single event specific for an

individual stimulus. They are therefore likely to display their inhibitory properties after the point of integration of the different signals.

Example 2 SLs do not affect activity of Src family protein tyrosine kinases Tyrosine kinases of the Src family have been implicated in NF-κB activation in response to various stimuli including UV radiation, T-cell receptor ligation and stimulation with prooxidants. We therefore investigated the effect of parthenolide and isohelenin on kinase activity of recombinant p60^src and p59* After pretreatment with various concentrations of SLs, kinase activities were determined by incubation of the protein kinases with the substrate

rabbit muscle enolase and ³²P-γ-ATP. Tyrosine-phosphorylated proteins were then separated

by SDS-PAGE and visualized by autoradiography. Neither increasing amounts up to 20 μM of

isohelenin nor of parthenolide did affect the ability of p59^fyn to phosphorylate itself or the substrate enolase. Also the recombinant p60^src kinase was completely unchanged in its phosphorylating activity in the presence of both SLs (data not shown). These results suggest

that SLs do not prevent NF-κB activation by inhibiting Src tyrosine kinases. Furthermore these data indicate that SLs do not unspecifically alter the activity of cytoplasmatic enzymes by reacting with the sulfhydryl group of cysteine residues, because both Src family tyrosine kinases possess exposed and redox-reactive cysteines which were shown to be important for their activity (45).

Example 3

SLs do not prevent DNA-binding of NF-κB

In order to investigate the mechanism of action of SLs we first tested the potential effects of

the SLs parthenolide and isohelenin on the DNA-binding activity of NF-κB in band-shift

experiments. Therefore HeLa cells were stimulated with PMA and cell extracts were prepared

20 min after stimulation. These extracts, which contain the activated nuclear form of NF-κB,

were pooled and incubated either with various concentrations of the two SLs or with the solvent DMSO as a control. The preincubated extracts were analyzed for DNA-binding

activity of NF-κB in an EMSA. Increasing concentrations of parthenolide completely

abrogating NF-κB activation in the intact cell (see Fig. 1) did not influence DNA-binding of

activated NF-κB in vitro (Fig. 2). The same amounts of isohelenin also failed to reduce the

DNA-binding activity of NF-κB (data not shown). This experiment excludes that SLs directly

inhibit the DNA-binding activity of activated NF-κB, e.g. by modifying reactive amino acids

such as cysteine residues in the DNA-binding or dimerisation domains. The induced DNA-

binding complex was confirmed to be a p50/p65 NF-κB dimer by competition assays with

unlabeled oligonucleotides and antibody reactivity (data not shown).

Example 4

SLs prevent the induced degradation of IκB-α and IκB-β

The part of the NF-κB activation cascade that is influenced by SLs was further analyzed by

following the fate of IκB proteins in Western blots. Mouse L929 cells were preincubated for 1

h with 5 μM of either isohelenin or parthenolide. Subsequently murine TNF-α was added

without changing the medium and kept for 20 min on the cells. Total cell extracts were tested

for the presence of IκB-α in Western blots and simultaneously for DNA-binding of NF-κB

and Oct-1 by EMS As (Fig. 3). The TNF-α-induced DNA-binding of activated NF-κB in the

EMSA experiment coincided with the degradation of IκB-α as detected by Western blotting

(Fig. 3 A,B). Preincubation of cells with 5 μM parthenolide completely prevented the

induction of the DNA-binding form of NF-κB and protected IκB-α from proteolysis by the

26S-proteasome. Identical results were obtained for HeLa cells preincubated with 5 μM isohelenin (data not shown). In a control experiment the DNA-binding activity of the Oct-1 protein remained unchanged (Fig. 3 C).

In a more detailed analysis we tested the behaviour of IκB-α and IκB-β proteins in the

presence or absence of SLs with different NF-κB-inducing conditions at various time points

(Fig. 4 A-D). The rapid TNF-α-induced DNA-binding of NF-κB in L929 cells remained

unchanged up to 120 min (Fig. 4 A). Preincubation with parthenolide completely prevented

the induced degradation of IκB-α and IκB-β as monitored by Western blotting. Sixty minutes

after the addition of TNF-α only the IκB-α protein was resynthesized. The PMA-induced

DNA-binding of NF-κB in HeLa cells took place with a significantly slower kinetic when

compared to TNF-α. DNA-binding was complete thirty minutes after stimulation with PMA

and resulted in a full degradation of IκB-α, which was not apparent in the presence of

parthenolide (Fig. 4 B). The IκB-β protein was not inducibly degraded by PMA, suggesting

the importance of alternative PMA-signaling pathways for IκB-β. The effects of CD3/CD28

receptor ligation on NF-κB and IκB proteins were monitored in peripheral blood lymphocytes.

Various time points after receptor ligation total cell extracts were prepared and tested for

DNA-binding activity and IκB degradation. The DNA-binding of NF-κB was significantly

enhanced already 5 min after receptor triggering. The observed constitutive DNA-binding

activity is most likely due to the constitutively active form of NF-κB from mature B-cells,

which are contained in the peripheral blood lymphocytes. The stimulation of NF-κB by

CD3/CD28 ligation is reflected by a degradation of the IκB-α and IκB-β proteins, which again

can be prevented by preincubation with parthenolide (Fig. 4 C). The activation of NF-κB by

100 μM of H₂O₂ in Jurkat JR cells appeared only after an incubation period of 90 minutes. As

already seen for the other tested stimuli, the (in the case of H₂O₂ incomplete) degradation of IκB-α and IκB-β proteins was abrogated by preincubation with parthenolide (Fig. 4 D).

Identical results were obtained for all the stimuli described here with the SL isohelenin (data

not shown). These experiments indicate that SLs prevent the induced degradation of IκB-α

and IκB-β by diverse stimuli and therefore interfere with a common step in the signaling

cascade leading to the activation of NF-κB.

Example 5

SLs irreversibly inhibit NF-κB activation

We next tested whether SLs act as competitive or irreversible inhibitors. HeLa cells were

incubated with 5 μM of parthenoUde for 1 h. Subsequently the cells were washed with

medium void of SLs and further grown for various periods as schematically displayed in Figure 5 A. After stimulation with PMA for 20 min cells were lyzed and the extracts were

assayed for NF-κB activity by EMSA. The amount of DNA-bound NF-κB dimers was

quantitated with a phosphoimager and the results are displayed graphically (Fig. 5 B). The

total inhibition of NF-κB binding activity seen after immediate stimulation of cells following

the preincubation with parthenolide was set as 100 %. The inhibition was still almost complete after 2 h of incubation and decreased to approximately 50 % after 18 h of incubation in parthenolide-free medium (Fig. 5 B). This kinetic behaviour suggests that the SLs act by covalently and thus irreversibly modifying their target molecule(s), presumably by their

reactive Michael system in the lactone ring. The partial restauration of inducible NF-κB activation, which is occuring 18 h after SL-treatment, is most probably due to the re-synthesis of the inactivated protein(s).

Example 6 SLs promote killing of mouse L929 cells by TNF-α

The role of NF-κB during the TNF-α-induced cell death is still not clear and seems to depend on the tested cell line (for review see 16). We therefore wanted to address the question

whether cell death of L929 cells in response to TNF-α is influenced by parthenolide. Mouse

L929 cells were incubated either with 5 μM parthenolide or 2000 U/ml TNF-α alone or by a

combination of both. After various incubation times the cell viability was measured (Fig. 6).

Treatment with parthenolide alone did not influence the cell viability and TNF-α-induced cell

death was enhanced in the presence of parthenolide. This experiment shows that the NF-κB

inhibitor parthenolide is also enhancing the TNF-α-induced cell killing of mouse L929 cells.

Example7

SLs prevent the induced expression of the NF-κB target gene ICAM-1

The inducible transcription of the ICAM-1 gene in response to TNF-α, E -lβ and PMA is

controlled by a NF-κB binding site in its promoter (for review see 47). This feature makes

ICAM-1 surface expression a good read-out to test the effect of SLs on the expression of

endogenous NF-κB target genes. The induction of ICAM-1 gene expression and the

appearance of the protein on the cell surface was analyzed by FACS. Treatment of Jurkat T-

cells with TNF-α resulted in a strong induction of ICAM-1 expression, as displayed in Figure 7 A. Preincubation with either 5 μM parthenolide almost completely prevented the induction of ICAM-1 synthesis (Fig. 7 B). In a control experiment parthenolide or isohelenin did not influence the amount of the T-cell receptor CD3 protein on the surface of Jurkat cells in the

presence or absence of TNF-α (data not shown). These results indicate that the induced

transcription of NF-κB target genes is specifically inhibited by SLs.

Example 7 Structural determinants for the inhibitory activity of SLs

We next investigated the structural features of the SLs which confer inhibitory activity on NF-

KB activation pathways. Two structural hallmarks of SLs are an isoprenoide ring system and a

lactone ring. In many cases this lactone ring contains a conjugated exomethylene group. Both groups together form a reactive Michael system which is a target for nucleophilic substrates, e.g. for cysteine residues in proteins. Various isoprenoide substances lacking either the lactone

ring or the exomethylene group were tested for their effects on NF-κB activation. The structure of these tested compounds is given in Table 1. None of these substances showed

cytotoxic effects on HeLa cells at concentrations of 5 and 10 μM after 10 h of incubation

time. One hour after preincubation with 5 and 10 μM of the respective drugs HeLa cells were

stimulated for 20 min with PMA. Subsequently total extracts of these cells were tested by

EMSA on NF-κB activation. All tested isoprenoides that lacked either the lactone or the

exomethylene group in the α position to the lactone function displayed no inhibitory effect on

the pathway leading to the activation of NF-κB (see Table 1). Another interesting structural

element especially of parthenolide is its epoxide ring which is also a likely site for the addition of nucleophilic reagents. To investigate the importance of this epoxide ring we also tested substances with this structural feature, even in combination with exomethylene groups to provide a bi-reactive substrate for (the) target molecule(s). Again none of the tested

substances displayed inhibitory properties on NF-κB as assessed by EMSA (Table 1). This failure of inhibition occured irrespective of a synthetic or natural origin of the tested substances.

Example 8 SLs inhibit NF-κB activation without having anti-oxidative properties

The chemical structure of the SLs suggests that they do not have anti-oxidative properties

such as many other inhibitors of NF-κB. In order to exclude that SLs display any direct or

indirect anti-oxidative effects we measured the potential influence of parthenoUde

preincubation on the TNF-α induced change of the intracellular redox state in Jurkat T-cells.

Quantitation of the intracellular levels of ROIs by FACS analysis with the dye DFCH

demonstrated that preincubation of Jurkat T-cells for 1 h with 5 μM of parthenolide did not

reduce the amount of intracellular ROIs that were generated by TNF-α-stimulation (Fig. 8

A,B). This observation is confirmed by a direct comparison of ROI-concentrations under various conditions (Figure 8 C).

Example 9

Crystallography of the NF-κB/ IκB-α/IκB-β/parthenolide complex

Co-Crystallization of the NF-κB/IκB-α or NF-κB/IκB-β complex in the presence of

parthenolide revealed the structure of the parthenolide binding site on IκB-α and IκB-β. The structural data as resulting from mass spectroscopic studies (results are not shown) are relevant for rational drug design.

Example 10 In vivo effect of sesquiterpene Iactones

Animal experiments have been designed to investigate the in vivo effect of sesquiterpene Iactones, namely parthenolide and isohelenin, based on the above described mechanism.

Abbreviations

CHUK, conserved helix-loop-helix ubiquitous kinase; DFCH, dichlorofluorescein-diacetate; DMEM, Dulbecco's modified Eagle medium; DMSO, dimethyl sulfoxide; EMSA,

electrophoretic mobility shift assay; ICAM-1, intercellular adhesion molecule- 1; IKK, IκB kinase; JNK, c-Jun N-terminal kinase; MEKK-1, mitogen-activated protein kinase/ERK kinase kinase- 1; MKK4, mitogen-activated protein kinase kinase 4; MTT, 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl tetrazolium bromide; PBS, phosphate-buffered saline; PMA, phorbol-12- myristate 13 -acetate; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene difluoride; ROIs, reactive oxygen intermediates; SLs, sequiteφene Iactones; TNF, tumor necrosis factor; VCAM-1, vascular cell adhesion molecule- 1.

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Claims

Claims

1. Use of sesquiterpene Iactones by binding I╬║B-╬▒ and I╬║B-╬▓ thereby inhibiting the

activation of NF-╬║B.

2. Use of sesquiterpene Iactones for drug design.

3. Use of sesquiterpene Iactones as drugs.

4. Characterization of the the structure of the sesquiteφene lactone binding sites by co-

crystallization of NF-κB/IκB-α or NF-κB/IκB-β complexes with sesquiteφene Iactones.

5. Characterization of the in vivo effect by sesquiteφene Iactones using animal models.