Study On The Interaction Of TNF-α And Sesquiterpene Lactones From I. Japonica And Anti-inflammatory Effects Of The Compounds

【Background】TNF-α is a pleiotropic cytokine with strong proinflammatory and immunomodulatoryproperties. It exerts its biological functions by interacting with two TNF receptors (TNFR),namely TNFR1and TNFR2. Signaling through TNFR1appears to play a more importantrole in the proinflammatory and cytotoxic effects of TNF-α. In turn, TNFR2-mediatedsignals have been shown to be involved in particular anti-infective defence. Directinhibition of TNF-α by protein-based biological agents such as anti-TNF antibodies andsoluble receptor has achieved a remarkable success in treatment of various inflammatorydiseases and validated the extracellular inhibition of this cytokine as an effective therapy.Unfortunately, the currently available TNF blockers inhibit both TNFR1and TNFR2signaling. Therefore, long-term use of TNF blockers can cause serious side effects such asan increased risk of infection.Medicinal plant-derived compounds have been a major source of pharmacologicallyactive substances from which drugs can be developed. Currently, many natural productshave been found to regulate TNF-α production or activity and being advanced for thetreatment of inflammatory disorders. These small-molecule drugs might provide acost-effective alternative to protein-based therapeutics. However, most of thesesmall-molecule TNF-α antagonists found thus far have typically been limited to inhibitorsof TNF-α production and/or release, uncharacterized inhibitors of TNF-α cell-based assays,and other inhibitors of intracellular signaling pathways that are involved in TNF-αexpression or activity. Despite considerable incentives, natural leads for analogoussmall-molecule inhibitors of TNF-α that can directly disrupt the interaction between TNF-αand its receptor have not been reported.In our previous study, we have identified a dimeric sesquiterpene lactone, XFH-31,isolated from an herb Inula japonica Thunb, as a novel TNF-α antagonist, which directlybound to TNF-α in Biacore assay, inhibited its interaction with TNFR1, and blocked theTNFR1-mediated proinflammatory activities. In this study, we continued our research onXFH-31and the structural analogues isolated from the same fraction of the plants. 【Aims】To discover a new class of drug candidates from Inula-derived sesquiterpene lactonefor anti-inflammatory therapy that can derictly target TNF-α and provide basis for furtherstructural optimization, molecular design, and drug development of novel TNF-αantagonists.【Methods】1. Surface plasmon resonance (SPR) analysis was performed on a BIAcore T100instrument to assay the direct binding of compounds to TNF-α;2. Gel filtration, Native PAGE, and Circular dichroism (CD) spectroscopy were usedto analyze the effects of XFH-31on the three-dimensional conformation of TNF-α;3. The binding of TNF-α to TNFR1or TNFR2was detected with a ELISA assay, insome experiment, the binding of125I-labeled TNF-α to L929cell surface was assayed tostudy the effects of compounds on the binding of TNF-α to membrane receptors;4. MTT assay was used to analyze the TNF-α-mediated L929cytotoxicity;5. NF-κB reporter gene assay and Western blotting were used for assay of TNF-αsignaling in293cells and bEnd.3mouse endothelial cells;6. Real-time PCR was performed to assay for TNF-α-induced expression ofchemokine and adhesional molecules in bEnd.3mouse endothelial cells;7. The in vivo anti-inflammatory effect of XFH-31was evaluated in a mouse model ofacute hepatitis induced by TNF-α plus D-galactosamine.【Results】1. XFH-31directly binds to TNF-α, selectively inhibits the interaction between TNF-αand TNFR1, blocks TNFR1-mediated signaling, and antagonizes the pro-inflammatoryactivities of TNF-α both in vitro and in vivo.(1) SPR analysis performed on BIAcore T100confirmed that XFH-31was able to bindto TNF-α with a KDof7.02μM;(2) XFH-31did not promote subunit disassembly of trimeric TNF-α. However, themigrating behavior of trimeric TNF-α during Native PAGE changed remarkably afterincubation with XFH-31, suggesting the binding of XFH-31to TNF-α may cause analteration in conformational state of the protein. The result of measurement of CD spectraof TNF-α in the absence or presence of XFH-31further confirmed that XFH-31did cause aconformational change at the level of the secondary structure of TNF-α; (3) XFH-31(2.5–10μM) effectively inhibits the binding of TNF-α to TNFR1in adose-dependent manner, while displaying only marginal inhibitory effects on that toTNFR2;(4) XFH-31inhibited TNF-α-mediated cytotoxicity on L929cells dose-dependentlywithin the range of2.5–10μM and inhibited the binding of125I-labeled TNF-α to L929cellsurface;(5) The result of dual-luciferase reporter assay showed that XFH-31inhibited theNF-κB activation induced by TNF-α, rather than by IL-1, in293cells dose-dependently inthe range of2.5–10μM; Western blotting assay further showed that XFH-31dose-dependently inhibited IκB phosphorylation and degradation induced by TNF-α, buthad no significant effect on that induced by IL-1;(6) XFH-31(2.5–10μM) dose-dependently suppressed the expression of MCP-1,ICAM-1, and VCAM-1and blocked all of the TNF-α-activated signaling cascadesincluding NF-κB, p38, JNK and ERK MAPkinase activation in bEnd.3mouse endothelialcells;(7) In TNF-α/D-GalN-induced murine hepatitis model, treatment with XFH-31(8mg/kg, i.p.) significantly suppressed TNF-α/D-GalN-induced elevation of serum ALTlevels and reduced the lethal toxicity. XFH-31administration not only markedly reducedapoptosis of hepatocytes but also suppressed the elevation of intrahepatic expression ofchemokines and adhesion molecules including MCP-1, MCP-2, ICAM-1, and VACM-1,suggesting XFH-31exerts its therapeutic effect by effectively inhibiting theTNFR1-mediated hepatocytotoxic and inflammatory responses, thus blocking the liverfailure in the experimental animals.2. Several Inula-derived dimeric sesquiterpene lactones structurally analogous toXFH-31were identified to bind directly to TNF-α, selectively inhibit the interactionbetween TNF-α and TNFR1, and antagonize the cytotoxic activities of TNF-α in vitro.(1) From37Inula-derived sesquiterpene lactones,13sesquiterpene lactones werescreened out which could effectively antagonize the cytotoxic activity of TNF-α on L929cells, while showing no impact on the survival and proliferation of L929cells alone;(2) From above13sesquiterpene lactones that could inhibit TNF-α-mediatedcytotoxicity on L929cells and had no nonspecific cytotoxicity on L929cells,10compounds, including XFH-31, TX-79, TX-109, XY-32, TX-69, XY-56, TX-65, TX-91,XY-34, XY-48, were found to be able to effectively inhibit the interaction between TNF-α and TNFR1, among them,6compounds showed stronger inhibitory effect on theinteraction between TNF-α and TNFR1than on that between TNF-α and TNFR2;(3)6sesquiterpene lactones were further discovered to be able to bind directly toTNF-α in Biacore assay, they are TX-79, TX-91, TX-109, XY-15, XY-27and XY-32. Fivecompounds among them are dimeric sesquiterpene lactones.【Conclusion】XFH-31, a new dimeric sesquiterpene lactones derived from Japonica Thunb, candirectly bind to TNF-α, selectively inhibit its binding to TNFR1, effectively blockTNFR1-mediated signaling in TNF-α-stimulated cells, and antagonize its proinflammatoryactivities both in vitro and in vivo. Besides XFH-31, there are other conpounds,structurally analogous to XFH-31, that can also directly target TNF-α, selectively inhibit itsbinding to TNFR1and antagonize its activity. These new findings not only extend ourunderstanding on the molecular mechanisms underlying the anti-inflammatory activity ofthis herb, but also emphasize the potential of these compounds as interesting leads for thedevelopment of novel, small-molecule TNF-α antagonist that can be pharmaceutically usedin anti-inflammatory remedies.