Study On The Sulfur-containing Dimer Metabolites Of Natural α,β-unsaturated Sesquiterpene Lactones As H₂S Donor

The α,β-unsaturated sesquiterpene lactones are a class of natural products with extensive biological activities,which have been the focus of drug research,especially in anti-cancer,anti-inflammatory,anti-oxidant and neuroprotective aspects.Some compounds are considered to have the potential for further development or be used as lead compounds.In our previous studies,sesquiterpenoids withα-methylene-γ-lactone structural unit,such as parthenolide（PA）,alantolactone（AL）and isoalantolactone（IAL）,have been proved to produce sulfur-containing dimer metabolites（R-S_n-R or R-S_n-R’,n=1-6）after gastrointestinal metabolism in rats or incubation by rat intestinal bacteria in vitro.However,whether these metabolites are biologically active and their possible mechanism of action remain unclear.In recent years,PA,AL and IAL have been proved to have significant anti-colon cancer or anti-colitis activities.Since the in vivo metabolites of drugs may be the material basis for their true efficacy,and the R-S_n-R skeleton structure of these metabolites is similar to that of known thioalkyl hydrogen sulfide（H₂S）donors,this study aims to demonstrate the possibility of sulfur-containing dimer metabolites of naturalα,β-unsaturated sesquiterpene lactones as H₂S donors and their potential anti-colon cancer activities and mechanisms.These results provide evidence and new idea for the drug development of naturalα,β-unsaturated sesquiterpene lactones.Objective:By studying three sesquiterpenoids withα-methylene-γ-lactone units（PA,AL and IAL）and their sulfur-containing dimer metabolites,to investigate the potential of sulfur-containing dimer metabolites of these sesquiterpene lactones as H₂S donors,and to explore the possible pharmacological mechanisms of naturalα,β-unsaturated sesquiterpene lactones through the H₂S/sulfide pathway,especially in intestinal cancer and inflammation.Methods:1.The corresponding sulfur-containing dimer metabolites（sulfide dimers,disulfide dimers and trisulfide dimers）of PA,AL and IAL were prepared by chemical synthesis or microbial incubation,and purified by column chromatography and preparative high performance liquid chromatography.The structures were identified by HR-MS and NMR.2.The release trend of H₂S from sulfur-containing dimer metabolites was plotted by fluorescence spectrophotometry.The reaction products of sulfur-containing dimer metabolites and bioactive thiol cysteine（Cys）were detected by ultra performance liquid chromatography and ultrahigh performance liquid chromatography combined with mass spectrometry（UHPLC-MS）.The reaction intermediates between disulfide dimer/trisulfide dimer of PA and Cys were captured and identified by chemical derivatization and liquid chromatography combined with mass spectrometry（LC-MS）,and the reaction mechanism of their H₂S release was speculated.3.In vitro cell and enzyme activity experiments:The cytotoxicity of PA,AL,IAL and their sulfur-containing dimer metabolites on colon cancer HT-29cells and HCT116 cells was investigated by CCK-8 method.Flow cytometry was used to detect the apoptotic effect of PA and its three sulfur-containing dimer metabolites on HT-29 cells.ELISA was used to investigate the inhibitory effect of sulfur-containing dimer metabolites on the increase of interleukin-6 and tumor necrosis factor-αsecreted by lipopolysaccharide-induced in RAW264.7 macrophages.The inhibitory effects of these compounds on the R132H mutant and wild type of isocitrate dehydrogenase 1（IDH1）and fatty acid synthase thioesterase domain（FASN-TE）were investigated by enzyme activity inhibition experiments.4.The potential anti-colon cancer targets of disulfide dimer and trisulfide dimer of PA were searched by network pharmacology,and their core targets were analyzed by protein-protein interaction.Additionally,their mechanisms were analyzed by GO enrichment analysis and KEGG pathway analysis.Molecular docking technique was used to predict the theoretical binding ability of compounds to target protein.Results:The corresponding sulfur-containing dimer metabolites（sulfide dimers,disulfide dimers and trisulfide dimers）of PA,AL and IAL were obtained（R-S_n-R,R=PA/AL/IAL,n=1-3）.The results of fluorescence spectrophotometry showed that the disulfide dimers and trisulfide dimers of these three drugs could spontaneously release H₂S in varying degrees in the absence of Cys,and the ability to release H₂S could be stimulated with the addition of Cys,while the release of H₂S could not be detected in the monosulfide dimers regardless of the presence of Cys.The reaction products of disulfide dimers and trisulfide dimers with Cys were idenified by LC-MS,while no reaction products of monosulfide dimers were detected.Taking PA as an example,the highly reactive intermediates between PA-S₂-PA/PA-S₃-PA and Cys were successfully captured,and the relatively complete reaction mechanism was deduced.The inhibition of PA-S₂-PA and PA-S₃-PA on the proliferation of HT-29cells and HCT116 cells was stronger than that of PA.Meanwhile PA and its sulfur-containing dimer metabolites（PA-S_n-PA,n=1-3）showed proapoptotic effect on HT-29 cells.AL,IAL and their corresponding disulfide dimers and trisulfide dimers,showed weaker inhibition to HT-29 cells and HCT116 cells proliferation.PA,AL and their corresponding disulfide dimers and trisulfide dimers showed concentration-dependent inhibition of interleukin-6.In vitro inhibition of enzyme activity showed that,compared with wild-type IDH1,PA-S₃-PA,AL,AL-S₃-Al and IAL showed selective inhibitory activity against IDH1 R132H mutant,with IC₅₀values of 0.41±0.02μM、23.00±4.37μM、1.14±0.31μM and 6.85±0.45μM,respectively.PA-S₂-PA、PA-S₃-PA、AL-S₂-AL and AL-S₃-AL showed about 50%inhibition ratio against FASN-TE with the concetration of 75μM。The potential anti-colon cancer targets of PA-S₂-PA,PA-S₃-PA and PA sulfides（PA-SH,PA-SSH）were integrated by network pharmacology,and seven core targets were obtained by topological analysis.The theory binding ability of these four structures with nine target proteins（EGFR,HIF1A,ESR1,SRC,ERBB2,CASP3,PIK3CA,NFKB,IKBKB）were predicted by molecular docking technology.Based on the screening results of enzyme activity,the theory binding of IDH R132H mutant with PA-S₃-PA was calculated by molecular docking.Conclusions:Based the advantages of liquid chromatography and mass spectrometry,combined with the preliminary screening of cell and enzyme activity,and the predictive ability of network pharmacology,we proved that the sulfur-containing dimer metabolites of sesquiterpenoids withα-methylene-γ-lactone structural unit could release H₂S and has the potential to be H₂S slow-release donor.The structure-activity relationship analysis showed that both the parent R structure and the number of sulfur atoms of dimer would affect the H₂S release performance and bioactivity.These results provide a new idea for the study of H₂S donor.Additionally,this study also demonstrated the possibility that natural sesquiterpenoids withα-methylene-γ-lactone structural unit may be involved in the regulation of H₂S in the intestinal tract and exert pharmacological activity on this basis,which provided a new perspective for the druggability ofα,β-unsaturated sesquiterpene lactones,and the pharmacodynamic substance basis and mechanism of Chinese herbal medicine containing these compounds.