Design,Synthesis And Mechanism Of Naphthalimides And Metal Hybrids Based On The Regulation Of Glycometabolism

BackgroundThe primary traditional treatments for advanced cancer are chemotherapy and radiation therapy.However,each of them has inherent defects and shortcomings.The combination of immunotherapy and traditional therapies presents a new therapeutic idea.Unfortunately,only a few chemotherapeutic drugs can achieve chemoimmunotherapy for tumors.Therefore,finding new ways to lift tumor immunosuppression and developing new chemotherapeutic drugs based on this approach is a critical project to achieve chemoimmunotherapy for tumors.During the progression of most cancers,such as prostate,glioma,liver,breast,and colon cancers,the abnormal glycosylation of tumor cells can induce tumor progression,cancer metastasis,and immunosuppression.The dysregulation of related glycosyltransferases is also closely associated with tumor progression and immunosuppression.DNA-damaging agents are among the classical antitumor agents.Platinum drugs（cisplatin and oxaliplatin）and 1,8-naphthalimides have a broad antitumor spectrum and excellent antitumor properties,which make them widely used in the treatment of prostate cancer,glioma,hepatocellular carcinoma,and lung cancer.However,chemotherapy resistance and immunosuppression have limited their clinical use.Therefore,by exploring the mechanism of aberrant glycosylation in tumor progression,a new immunotherapeutic strategy for synergistic drug development has been developed.In this approach,platinum drugs and 1,8-naphthylimine were structurally designed and optimized to become superior immune-responsive antitumor agents.This strategy provides a new direction to exploit aberrant tumor glycosylation to develop chemotherapeutic drug candidates and improve tumor diagnosis and treatment.ObjectiveThe objective of this study is to investigate the effects of aberrant glycosylation and glycosyltransferase dysregulation on tumorigenesis,progression,migration,invasion,and the immune environment in prostate cancer,glioma,and liver cancer.We aim to explore novel therapeutic markers and targets and use these therapeutic markers and glycosylation targets to develop immune-responsive antitumor drugs.MethodsMedicinal chemistry:A series of excellent natural and non-natural sugars,such as acetylglucosamine（GN）,4-fluoroacetylglucosamine（FGN）,2-deoxy-D-glucose,have been searched for through the screening of chemical libraries of small-molecule monosaccharides.Natural or non-natural sugars were prepared by substitution modification and structural optimization of Pt（Ⅳ）heterodimers and1,8-naphthylimide-substituted compounds.Pharmacology（cellular level）:cytotoxicity testing（MTT）was performed to evaluate the activity of the target compounds and to screen for compounds FGNPt,DG2 and H4 targeting prostate cancer,glioma and hepatocellular carcinoma.transwell and wound healing assays evaluated the anti-migration ability of the drugs.Drug uptake assays were evaluated（ICP-MS）to detect the targeting ability of the drugs at the tumor site.Flow cytometry and protein immunoblotting techniques were used to probe the effect of lead compounds on apoptosis.Real-time fluorescence quantitative PCR（q PCR）,immunoprecipitation（Co-IP）and protein immunoblotting（Western Blot）probed the molecular mechanism of their inhibition of tumor cell proliferation and relief of immunosuppression.q PCR and enzyme-linked immunosorbent assay（ELSA）assays detected cytokine levels.Pharmacology（animal level）:In vivo antitumor efficacy of the lead compounds was evaluated by in situ,xenograft and lung metastasis animal models,and immunohistochemistry and flow cytometry were used to detect the effect of the lead compounds on tumor immune infiltration in mice.Results1.GNPt hybrids:In this part,four platinum hybrids were designed and synthesized by natural aminohexose（GN）and 4-fluoro-aminohexose（FGN）ligated with cisplatin and oxaliplatin.The cytotoxicity test（MTT）screened that FGNPt was significantly cytotoxic to prostate cancer,especially to castration-resistant prostate cancer（CRPC）cells PC3(IC₅₀0.23±0.39μmol/L)and DU145(IC₅₀0.77±0.15μmol/L).And it significantly inhibited the growth of mice CRPC subcutaneous tumor（RM-1）（inhibition rate:85.1%）.In addition,FGNPt effectively inhibited the recruitment of myeloid-derived immunosuppressive cells（MDSC）in tumors and blood,thereby activating tumor-killing CD4⁺and CD8⁺T lymphocytes.Proteomic sequencing revealed that CRPC progression was correlated with GALNT3,a key glycosyltransferase in Mucin-type O-glycosylation biosynthesis,and that GALNT3 could be used as a biomarker of CRPC to detect CRPC development and progression.mi R-26a was downregulated by CRPC,inhibiting its binding to the transcriptional region of GALNT3,promoting GALNT3 expression that promotes the expression of O-glycosylated MUC-C and TNFR,thereby causing chemoresistance of CRPC and immunosuppression induced by MDSCs.In contrast,FGNPt exerts resistance to platinum-based drug chemoresistance and immunosuppression in CRPC by inhibiting this pathway.2.DGPt hybrids:In this section,the DGPt was found to have excellent anti-glioma efficacy through anti-glioma screening of 12 excellent anti-tumor compounds from the laboratory’s anti-tumor compound library.Further anti-glioma screening of six 2-deoxyglucose substituted platinum hybrids（DGPt）revealed that DG1,DG2 and DG6 had excellent efficacy against glioma cell lines U251,U87,U118-MG and C6.However,acute toxicity testing revealed that DG1 was more toxic to mice,so DG2 was selected for experimental studies.In a mouse model of glioma in situ（GL261）,compound DG2（inhibition rate:77%）showed more potent inhibition than the medicines temozolomide（inhibition rate:34%）,oxaliplatin（inhibition rate:39%）and cisplatin（inhibition rate:56%）.Drug uptake assay（ICP-MS）assessment demonstrated that DG2 had significantly greater blood-brain barrier penetration efficiency than cisplatin and oxaliplatin,with high accumulation in glioma in situ.In addition,DG2 effectively activated tumor CD4⁺,CD8⁺T lymphocytes and M1-type macrophages and inhibited TAM-type macrophage expression.Transcriptomic and TCGA database studies revealed that DG2 activates tumor-killing T lymphocytes and M1-type macrophages and suppresses activation of TAM-type macrophages,possibly by inhibiting the expression of fucosyltransferase 8（FUT8）,thereby downregulating fucosylation of PD1 and CD155.3.GN-NI:In this part,sixteen glycosylated naphthalimide derivatives（GN-NI）,including S1-S4,H1-H4,N1-N4 and X1-X4,were designed by combining natural aminohexose（GN）and4-fluoro-aminohexose（FGN）with naphthalimide,and all compounds were structurally confirmed(¹H NMR,¹³C NMR and ESI-MS).Cytotoxicity test（MTT）was performed to evaluate the in vitro activity,and H4 showed significant cytotoxicity against hepatocellular carcinoma（HCC）cells in comparison with the medicine Amfefite.Moreover,in a mice hepatocellular carcinoma model（H22）,H4（inhibition rate:70%）showed higher inhibition compared to the medicine Amfefite（inhibition rate:57%）.Flow cytometry assays showed that H4 could effectively activate tumor apoptosis.transwell and wound healing assays demonstrated that H4 significantly inhibited hepatocellular carcinoma migration.In addition,by combining H4 with gemcitabine,it was found that H4 significantly improved the anti-hepatocellular carcinoma efficacy in combination with gemcitabine,showing a strong synergistic effect（combination index:0.14-0.16）.Transcriptomic sequencing revealed that the combination of H4 with gemcitabine inhibited fucosyltransferase 1（FUT1）-induced chemoresistance to gemcitabine,a clinical hepatocellular carcinoma drug,and enhanced hepatocarcinoma lethality.ConclusionOverall,in tumors,aberrantly expressed glycosyltransferases regulate the glycosylation of various proteins,thereby mediating cancer metastasis,chemoresistance and immunosuppression that promote tumor progression.However,the use of natural（acetylglucosamine）or unnatural glycosyl donors（4-fluoroacetylglucosamine and 2-deoxyglucose）to modify DNA intercalators（platinum heterodimer and 1,8-naphthylimine）can enhance their lethality and targeting effects on certain tumors such as prostate cancer,glioma and hepatocellular carcinoma,by targeting specific glycosyltransferases in tumors,thereby relieving tumor aberrant glycosylation-induced chemotherapy resistance and immunosuppression.Therefore,modification of chemotherapeutic agents by glycosyl donors to target aberrant glycosyltransferases provides a new direction for the development of novel and efficient chemotherapeutic drug candidates using aberrant tumor glycosylation to improve the diagnosis and treatment of tumors.