| Ubiquitin-conjugating enzyme UbcH5c-mediated protein ubiquitination play a critical role in TNF-a-triggered NF-κB pathway activation,suggesting that UbcH5c could serve as a potential anti-inflammatory target.We have previously found a natural sesquiterpene lactone compound IJ-5 that could inactivate UbcH5c and inhibit TNF-a-induced NF-κB activation and cytokine expression in immune cells,thereby exerting anti-inflammatory effects in vivo.Further study on molecular mechanism revealed that IJ-5 could bind covalently to Cys85 of UbcH5c through itsα,β-unsaturated carbonyl moiety by Michael addition and thereby inactivating UbcH5c.Therefore,IJ-5 is a promising lead compound for developing new UbcH5c inhibitors as anti-inflammatory agents.However,the abundance of IJ-5 in plants is very low and total synthesis of this compound is very difficult.In order to overcome the limitation of IJ-5,we used UbcH5c as target to screen for active compounds from semi-synthetic and natural sesquiterpene lactones which all containedα,β-unsaturated carbonyl moiety,and further studied the anti-inflammatory effects of active compounds,aiming to find new lead compounds which is easy to obtain and thus provide candidate compounds for developing novel anti-inflammatory agent eventually.Method:1.Screening for active compounds which possess inhibitory effect on UbcH5c from natural and semi-sythetic sesquiterpene lactones with the in vitro ubiquitin-conjugating enzyme activity measuring system.The semi-synthetic compounds which containedα,β-unsaturated carbonyl moiety were synthesized fromα-santonin.The candidate natural sesquiterpene lactones were mainly those with high abundance in plant such as alantolactone,isoalantolactone and total alantolactone.2.The luciferase reporter assay and Western blot were used to test the effects of active compounds on TNF-a-triggered NF-κB activation in cells.3.Biacore was used to analyze the interaction between active compounds and UbcH5c.4.Molecular docking was used to determine the binding mode of target compound with UbcH5c.5.Immunoprecipitation was used to study the effects of target compound on linear polyubiquitination of NEMO.6.Quantatitive real-time PCR(qPCR)was used to analyze the effects of target compound on TNF-a stimulated mRNA expression ininflammation-related cells.7.DNCB-induced dermatitis in mice were used as the animal model to invetigate the in vivo anti-inflammatory effect of the active compound.Results:1.A series of natural sesquiterpene lactones with different structure types selected from our compound library were compared for their effects on UbcH5c.It was found that,excepting IJ-5,which belongs to eudesmane,several natural sesquiterpene lactones belonging to other structural types,such as guaiane and germacrane,also possessed inhibitory activity on UbcH5c.Based on this finding,the candidate compounds for the screening of UbcH5c inhibitors should not be limited to eudesmane but could extend to other type of sesquiterpene lactones.2.Usedα-santonin as starting material,a series of derivatives were synthesized through oxidation reduction and rearrangement.Measure of their effects on UbcH5c showed that the derivative 6,10,20 possessed weak inhibitory effect at 10μM concentration.Thus,we further used 6,10,20 as precursor to synthesize 3 types of derivatives through further structural modification.From total 73 sesquiterpene lactone derivatives,14 compounds were detected to possess inhibitory effects on UbcH5c at 5pM concentration,which were 1a,1b,1e,2f,4c,5a,5g,5f,6i,6d,6g,8a,8b,9a.3.The effects of these 14 active compounds on NF-κB activation were analyzed by NF-κB luciferase reporter assay and Western blot,the result revealed that 6 compounds(1a,1b,5g,6d,8a,8b)could dose-dependently suppress TNF-a-induced NF-κB pathway activation in the range of 1-10μM.4.It was further confirmed that the compound 1a,1b,5g,6d,8a and 8b could dose-dependently inactivate UbcH5c in the range of 2.5-10μM,excepting compound 5g,other 5 compounds showed no inhibitory effects on another ubiquitin conjugating enzyme-Ubc13.This result not only confirmed the inhibitory effects of the active compounds on UbcH5c but also preliminary suggested that the effects of these 5 compounds on UbcH5c were selective.5.Biacore analysis revealed that,among above 5 active compounds,the compound 6d possess the strongest affinity to UbcH5c with a KD value of 0.873μM.Thus,we mainly focused on 6d in follow-up research.6.Covalent docking simulation showed that the steric conformation of 6d match well to the active pocket of UbcH5c.More importantly,the carbonyl and oxygen groups of 6d could form additional hydrogen bonds with the amino groups of Arg90 and Gln92 of UbcH5c,which could help 6d covenlent conjugate to Cys85 but not other Cys.8.Immunopreciptation assay demonstrated that 6d(2.5-10μM)could dose-dependently inhibit TNF-a-induced linear ployubiquitination of NEMO.This result indicated 6d could inhibit UbcH5c activity,further to exert inhibitory effect on NF-κB pathway through suppressing linear polyubiquitination of NEMO.7.qPCR assays showed that 6d(1-5μM)could dose-dependently inhibit the expressions of MMP-3,MCP-1 and IL-1 in TNF-a stimulated synovial fibroblasts.This result indicated that 6d may have therapeutic potential for rheumatoid arthritis.8.Through screening natural sesquiterpene lactones with high abundance,we found that alantolactone could inactivated UbcH5c,whereas its isomer isoalantolactone showed only slight inhibitory effect,and total-alantolactone(contain maily alantolactone and isoalantolactone)also possessed remarkable UbcH5c inhibitory activity.Both alantolactone and isoalantolactone have no inhibitory effects on Ubc13.9.Biacore analysis the interaction between alantolactone,isoalantolactone and UbcH5c revealed that alantolactone have stronger affinity to UbcH5c with a KD value of 32.7μM,whereas isoalantolactone have weaker affinity with a KD of 159.7μM.10.Covalent docking of alantolactone to UbcH5c demonstrated that the steric conformation of alantolactone can fit well with the binding site around Cys85,and the ester oxygen and carbonyl groups of alantolactone could form additional hydrogen bonds with Arg90 of the side chain of UbcH5c.In contrast,the structure of isoalantolactone can not fit with the active pocket of UbcH5c,so that itsα,β-unsaturated carbonyl moiety can not approach to Cys85,resulting in isoalantolactone unable to bind to UbcH5c.11.Alantolactone,isoalantolactone and total-alantolactone could dose-dependently supresss TNF-a-stimulated NF-κB activation in b.End3 at the range of 2.5-10μM.Among them,alantolactone was the strongest,followed by total-alantolactone,and isoalantolactone was the weakest.Meanwhile,these three compounds could inhibit MAPK pathway activation.12.Alantolactone,isoalantolactone and total-alantolactone could also dose-dependently supresss TNF-a-stimulated NF-κB activation and expressions of IL-1,IL-4,TNF-a in HaCat cells at the range of 2.5-10 μM.Among them,alantolactone was the strongest,followed by total-alantolactone,and isoalantolactone was the weakest.Those results were consistant with the result 12 and suggested that all those three components may possess strong anti-inflammatory property and therapeutic potential for dermatitis.13.We used DNCB-induced dermatitis mice as animal model to evaluate the anti-inflammatory effect of total-alantolactone in vivo.The results showed that the DNCB-induced skin lesion was alleviated significantly by topical application of total-alantolactone.Dermatitis cilinical score in total-alantolactone treatment group was significantly decreased compared with model group and the swelling degree of right ear was also significantly relieved by total-alantolactone treatment.HE stain demonstrated that the thickness of the epidermis,hyperkeratosis,and inflammatory infiltration in dermis was also significantly relieved by total-alantolactone treatment.The level of IgE,IFN-y,TNF-a in serum of dermatitic mice was significantly reduced by total-alantolactone treatment.The in vivo data indicated that total-alantolactone had prominent therapeutic effect on DNCB-induced dermatitis in mice.Conclusion:In this study two active compounds,6d and alantolactone,were detected to possess strong inhibitory activity on UbcH5c through multiple screen of semi-synthetic and natural sesquiterpene lactones.Both two compounds could bind to UbcH5c directly,their binding mode with UbcH5c was predicted by docking simulation,and these two compounds could inhibit TNF-a-induced NF-κB pathway activation and cytokine expression in inflammation related cells.The in vivo data indicated that topical application of total-alantolactone had prominent therapeutic effect on DNCB-induced dermatitis in mice,suggesting this component possess the potential to be developed into novel agent for treatment of dermatitis.The present study provided candidate compounds for developing novel anti-inflammatory agents targeting UbcH5c. |
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