This work was supported by The Austrian Science Fund (FWF) projectDNTI ‐ Drugs from Nature Targeting Inflammation (S10704, S10711,S10703, and S10709/10713).

Plumericin isolated from Himatanthus sucuuba is a novel potent NF‐κB inhibitor directly targeting IKK‐β.

Introduction

Results

Conclusions

Atanas G. Atanasov,†,* Nanang Fakhrudin,†,Birgit Waltenberger,‡ Muris Cabaravdic,┴ Elke H. Heiss,† Stefan M. Noha,§ Anna M. Grzywacz,† Judit Mihaly‐Bison,┴ Daniela Schuster,§ Johannes M. Breuss,┴ Judith M. Rollinger,‡ Valery Bochkov,┴ Hermann Stuppner,‡ Verena M. Dirsch†

†Department of Pharmacognosy, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria; ‡,§Institute of Pharmacy (Pharmacognosy‡/Pharmaceutical Chemistry§) andCenter for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80‐82, 6020 Innsbruck, Austria; ┴Center for Physiology and Pharmacology, Institute for VascularBiology and Thrombosis Research, Medical University of Vienna, Schwarzspanierstrasse 17, 1090 Vienna, Austria; Faculty of Pharmacy, Gadjah Mada University, Sekip Utara,55281 Yogyakarta, Indonesia; *Correspondence: atanas.atanasov@univie.ac.at

Inflammation is a prominent part of various and at first glance diverse pathologicalconditions, for many of which no optimal treatment is currently available. Thetranscription factor NF‐κB is a well‐established key regulator of the inflammatoryresponse. In search for new NF‐κB inhibitors from natural sources, we investigatedextracts from the stem bark of Himatanthus sucuuba, a medicinal plant traditionallyused in the Amazon region of South America to treat different inflammation‐relateddiseases, such as arthritis, cough, ulcer, pain, gastritis, and tumors. Bioactivity‐guidedfractionation led to the identification of the spirolactone iridoid plumericin as a potentinhibitor of the NF‐κB signaling pathway exerting a new chemical scaffold compared tothe other known NF‐κB inhibitors.

Plumericin effectively abolishes NF‐κB activation in a luciferase reporter cell model, inhibits TNF‐α‐inducedexpression of the pro‐inflammatory adhesion molecules VCAM‐1 and ICAM‐1 in endothelial cells, and suppressesneutrophil recruitment to the peritoneum in response to thioglycollate in mice. The molecular mechanism ofaction of plumericin reveals to be a direct inhibition of IKK‐β, an upstream kinase that phosphorylates the NF‐κBinhibitor IκB. Consequently, plumericin blocks the phosphorylation and following degradation of IκB and thusthe activation of the pro‐inflammatory NF‐κB signaling pathway.

The anti‐inflammatory action of plumericin provides a scientific rationale for the traditionalethnopharmacological use of H. sucuuba, and suggests this compound as a promising natural product that couldbe further explored as new pharmaceutical lead.

Geographical distribution of H. sucuuba

http://www.tropicos.org/

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Structurally similar compounds isolated from H. sucuubaand their NF‐κB inhibitory potential:

Chemical structure of the known IKK‐β inhibitor parthenolide:

Inactiveup 30 µM

Inactiveup 30 µM

Inactiveup 30 µM

ActiveIC50 = 1.1 µM

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Plumericin inhibits TNF‐α‐induced cell surface expression of the endothelial adhesion molecules VCAM‐1 (a) and ICAM‐1 (b).

HUVECtert cells were pretreated with the indicated concentrations of plumericin orsolvent vehicle (DMSO 0.1%) for 30 min prior to stimulation with 10 ng/mL TNF‐αfor 14 h. The protein expression levels were analyzed by flow cytometry. Data shownare mean ± SEM (n=3; *p<0.05, **p<0.01, n.s. not significant, ANOVA/Dunnett).

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Plumericin from H. sucuuba inhibits TNF‐α‐induced NF‐κB activation. 

HEK293/NF‐κB‐luc cells were pretreated for 30min with the indicated concentrations ofplumericin, solvent vehicle (DMSO 0.1%), or 5μM parthenolide as positive control, prior tostimulation with 2 ng/mL TNF‐α for 4 h. Thedata represent mean ± SEM (n=3; **p<0.01,***p<0.001, n.s. not significant,ANOVA/Dunnett).

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Plumericin inhibits the phosphorylation and degradation of the IKK‐β  substrate IκB‐α.

HUVECtert cells were preincubated with plumericin (5μΜ) or solvent vehicle (DMSO, 0.1%) for 30 min prior tostimulation with TNF‐α for 0, 4, 8, or 12 min. Western blotanalysis was performed for α‐tubulin, IκB‐α, andphospho(Ser32/Ser36)‐IκB‐α (p‐IκB‐α). Representativeblots out of three independent experiments are shown.

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Plumericin inhibits the enzyme activity of purified human IKK‐β.The enzyme activity of human recombinantIKK‐β was determined for 30 min at 30°C inthe presence of solvent vehicle (DMSO) ordifferent concentrations of plumericin. Thephosphorylation of the Ser32/Ser36‐IκB‐αsubstrate peptide was detected by ELISA.The data shown are mean ± SEM (n=4;*p<0.05, **p<0.01, ***p<0.001,ANOVA/Dunnett).

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Binding of plumericin and parthenolideas suggested by docking studies. 

(A) Plumericin (green) and parthenolide (blue)overlay well in their predicted binding occupying thearea of a hydrophobic groove on the IKK‐β proteinsurface. (B, C) Covalent binding of parthenolide (B)and predicted binding of plumericin (C) to Cys179(ball‐and‐stick style) and binding into thehydrophobic groove of IKK‐β.

Plumericin inhibits neutrophil recruitment in a thioglycollate‐induced peritonitis in mice. C57BL/6 male mice (5‐7 animals per group) werepretreated i.p. with 2 μL plumericin (final concentration250 μM) or DMSO dissolved in 1 mL of saline. Thirty minlater the animals were injected i.p. with 1 mL of solventvehicle or 4% sterile thioglycollate containing again 2 μLDMSO or plumericin. Five h after the second injection micewere sacrificed, the lavage fluid was collected and theneutrophil number was quantified by flow cytometry. Thegraph shows box and whisker plots representing median,upper and lower quartile, and lowest and highest detectedvalues (***p<0.001, ANOVA/Dunnett).

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EMBO Conference on Cellular Signaling & Molecular Medicine25‐29 May 2012, Cavtat‐Dubrovnik, Croatia