| Background and ObjectivesHepatitis B virus(HBV)infection is a serious global public health problem.Currently,there are about 240 million chronic HBV infections worldwide,of which about 86 million are in China.Chronic hepatitis B(CHB),HBV infection-related cirrhosis,and HBV infection-related hepatocellular carcinoma(HCC)causes more than 800,000 deaths worldwide each year,placing a huge burden on society.At present,the first-line drug nucleot(s)ide analogues used to treat HBV infection are still difficult to achieve clinical cure of CHB,because they cannot clear cccDNA in the nucleus of infected hepatocyte.New anti-HBV drugs are need to be developed.Osalmid is a kind of ribonucleotide reductase(RR)inhibitor that inhibits intracellular ribonucleotide production,thereby reducing the raw material for HBV replication.It has been found that osalmid has a strong inhibitory effect on HBV replication and cccDNA formation,but its metabolic process is still unclear.In view of the biological transformation and metabolic process in vivo,metabolites with stronger pharmacological activity or toxicity can be generated by changing the molecular structure of drugs.Therefore,this dissertation intends to identify the metabolites of osalmid and further analyze the anti-HBV activity and related mechanisms of osalmid's metabolitesMethods1.Ultra-performance liquid chromatography combined with time-of-flight mass spectrometry(UPLC-QTOF/MS)was used to identify metabolites of osalmid in vivo generated by Sprague Dawley rat and metabolites of osalmid in vitro by incubation with human liver microsomes.The metabolic pathways of osalmid were analyzed using UNIFI software.Recombinant human liver drug metabolizing enzymes were further used to clarify the liver drug metabolizing enzyme subtypes involved in osalmid metabolism2.The binding affinity between the identified metabolites of osalmid and the RRM2 active site was calculated using computer virtual molecular computational docking system.The metabolites with higher molecular binding affinity were selected for further research.Based on the stable HBV replication cell model HepG2.2.15 cell line and the nucleoside analogues cross-resistant HBV cell model HepG2.A64 cell line,inhibitory activity of the selected metabolites on HBVDNA,HBsAg,HBeAg in cell culture medium supernatant,as well as intracellular HBVDNA and cccDNA was verified.Cytotoxicity of osalmid's hydroxylation metabolite LAF-OH was determined by cell proliferation assay and cell apoptosis assay.ComboSyn software was used to calculate the drug combination index of osalmid's hydroxylation metabolite LAF-OH and nucleoside(acid)analogues through the drug combination experiment3.The inhibitory effect of osalmid's hydroxylation metabolite LAF-OH on RR activity and the effects on mRNA and protein expression levels of RR subunits RRM1,RRM2 and RRM2B were analyzed by ELISA,qPCR and Western Blot,respectively Proteomics was used to compare the differences in protein expression among cells treated by LAF-OH,osalmid and lamivudine.The biological processes in which the differential proteins participated was further analyzed to identify the possible anti-HBV mechanism of osalmid in addition to the inhibition of RR activityResults1.After oral administration of osalmid,the bile,feces,plasma and urine was collected at 0-3h,3-8h,8-12h,12-24h,separately.Six metabolites of osalmid(M1-M6)were identified in bile samples collected at 0-3 h and 3-8 h;two metabolites of osalmid(M1 and M2)were identified in bile samples collected at 8-12 h;three metabolites of osalmid(M1,M3 and M6)were identified in fecal samples collected at 0-3 h and 3-8 h;no metabolite of osalmid was identified in bile,feces samples collected at other time point and all plasma and urine samples.Five metabolites of osalmid(M1,M7-M10)were identified in samples incubated with human liver microsomes.The main metabolic processes of osalmid include hydroxylation,glucuronidation,sulfonation,acetylation and metabolic degradation.The results of recombinant hepatic drug-metabolizing enzymes screening indicate that CYP1A2 and CYP2C9 participated in phase I metabolic reactions of osalmid;UGT1A1,UGT1A6,UGT1A9,UGT2B7 and UGT2B15 participated in phase II metabolic reactions of osalmid2.The results of virtual docking demonstrate that the binding affinity of the metabolites M8,M10 and LAF-OH with RRM2 active site was stronger than that of osalmid.The HBV inhibiting assays based on HepG2.2.15 cell line and HepG2.A64 cell line revealed that HBV inhibitory activity of LAF-OH was stronger than that of osalmid in a time-and dose-dependent manner,which could significantly reduce the levels of HBV-DNA,HBsAg in culture supernatants and intracellular HBV-DNA,cccDNA levels of both wild-type HBV and drug-resistant mutant HBV model cells.The results of cell proliferation toxicity experiments and cell apoptosis experiments indicate that the half maximal proliferation inhibitory concentration(IC50)of LAF-OH in HepG2.2.15 cells,HepG2.A64 cells,HepG2 cells and HEK293 cells was 173.4?M,366.7?M,222.3?M and 191.8?M,respectively.The concentration of LAF-OH at 150?M showed no significant effect on cell apoptosis.The combination of LAF-OH with lamivudine and entecavir showed synergistic effect against HBV,while the combination of LAF-OH with adefovir and tenofovir showed additive effect against HBV3.iTRAQ screening revealed that the cell cycle,IL-17 signaling pathway and PPARsignaling pathway-related protein expression levels were significantly decreased after LAF-OH treatment.Western Blot analysis showed that LAF-OH could reduce cyclin D1 expression level by inhibiting Akt phosphorylation levels.The results of cell cycle analysis showed that the proportion of cells in S phase significantly decreased after LAF-OH treatment.Further qPCR and Western Blot analysis showed that LAF-OH could inhibit the expression of RRM2 without causing compensatory upregulation of RRM2B.RR activity analysis showed that LAF-OH could inhibit RR activity by inhibiting RRM2 activity and decreasing RRM2 expression levels simultaneouslyConclusions1.Osalmid is mainly metabolized by hydroxylation,glucuronidation,sulfonation,acetylation and metabolic degradation pathways in vivo.CYP1A2,CYP2C9,UGT1A1,UGT1A6,UGT1A9,UGT2B7 and UGT2B15 are the main hepatic metabolic enzyme subtypes of osalmid2.The binding affinity with RRM2 active site with LAF-OH is higher than osalmid,and the anti-HBV activity of LAF-OH is also higher than that of osalmid.By conducting HBV inhibition experiments,LAF-OH shows good safety as it did not produce obvious cytotoxicity while reducing HBV-DNA,HBsAg levels in cell medium supernatant and intracellular HBV-DNA,cccDNA levels.LAF-OH still has strong inhibitory activity against nucleoside analogue-resistant HBV LAF-OH exhibits additive or synergistic effects when used in combination with nucleot(s)ide analogues3.LAF-OH down-regulates the expression levels of cyclin D1 by decreasing the phosphorylation level of Akt,ameliorates the cell cycle disorder caused by HBV infection,and down-regulates the expression level of RRM2 by reducing the proportion of cells in S phase without compensatory increase in the expression level of RRM2B.LAF-OH competitively binds to the RRM2 active site while downregulating the expression level of RRM2,thus greatly inhibiting RR activity and exerting anti-HBV effect.LAF-OH could bring more potential benefits for HBV treatment by regulating IL-17-mediated immunological response and PPAR,Akt and other signaling pathways. |
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