Design And Synthesis Of Enzyme-targeted Inhibitors And Their Anti-cancer Applications

The Enzyme is the catalyst of all chemical reactions related to the life.It plays an important role in metabolism,signal transduction and regulation.Life without enzymes is no more than leaving the biological organisms.In tumor microenvironment,the activity of some enzymes are thriving and higher than that in normal cells.In the treatment of tumor,drug that effectively inhibit enzymes activity which are closely related to the tumor growth and proliferation will hopefully suppressed tumor cell growth and proliferation ultimately.DNA topoisomerase I(TOPOI),dihydrofolate reductase(DHFR)and thioredoxin reductase(Trx R)are enzymes that highly expressed under the tumor microenvironment.They play vital roles in the proliferation and growth of tumors.In this study we designed and synthesized these three enzyme inhibitors.Their application in anti-tumor were investigated.1.Camptothecin(CPT)is one of the most effective clinically used drug for the treatment of digestive tract tumors,such as gastric cancer and colorectal cancer.It can effectively inhibit DNA topoisomerase type I(TOPOI)activity of tumor cells,further deprive DNA of replication,resulted in tumor cells death.However,due to the notorious side effects,its clinical application is hindered.According to the fact,we synthesized a cancer targeted CPT prodrug: Biotin-ss-CPT based on disulfide bond.On the anti-proliferative activity of different tumor cells and normal cell,we found that it can be selectively assimilated by MGC 803 gastric cancer cells,then released CPT by biological responses,further inhibited the activity of TOPO I and eventually resulted in tumor cell death.Moreover,the response of prodrug Biotin-ss-CPT to glutathione(GSH)to liberate CPT was detected by HRMS,HPLC and fluorescence spectrum.In vivo antitumor results showed that the antitumor effect of Biotin-ss-CPT was as potent as CPT that effectively resulted in tumor cell necrosis.However,Biotin-ss-CPT did not possess the side effects as CPT,such as liver and kidney toxicity,as well as weight loss.Together,Biotin-ss-CPT solved the key problem of CPT in poor selectivity and side effects.2.Cervical cancer is currently one of the most common gynecologic malignant tumors.X-ray radiation therapy is often used in the treatment of cervical cancer,but the treatment effect of radiation therapy turns to be indistinctive,mainly because of the amplification of Dihydrofolate Reductase(DHFR)gene,which cause the increase in DHFR activity.As DHFR is an important enzyme for the synthesis of thymidine,which is the precursor of DNA.The increase in the DHFR activity promotes the DNA replication of tumor cells,resulting in the attenuation the treatment effect of radio therapy.In order to improve the radiotherapy effect,we synthesized a series of DHFR inhibitors deriving from methotrexate(MTX)analogues.Activity screening revealed that compound 2a could effectively inhibit the activity of dihydrofolate reductase(DHFR).The combination of 2a and X-ray radiotherapy(4 Gy)can exerted synergistic effects in killing cervical cancer cells.Their combination displayed better selectivity and radiosensitization than MTX.In vitro experiments showed that 2a + 4 Gy significantly induced cells into Sub-G1 phase,as well as mitochondrial membrane potential depolarization,bulk deposition of ROS,activation of Caspase 3,8 and 9,resulted in cell death.Moreover,the migration ability of tumor cells significantly inhibited.In vivo antitumor effect showed that 2a did not possess the liver and kidney toxicity and did not cause significant weight loss which DHFR inhibitors(such as MTX)typically did.And did not bring further toxicity even in the presence of radiation.Interestingly,the antitumor effect of 2a in combination of 4 Gy was obviously better than 2a or radiation alone.In conclusion,DHFR inhibitors successfully reversed the radioresistance problem which induced by radio therapy.This was a good try in tumor therapy,which deserves further research and development.3.The insensitivity to radiation therapy for malignant melanoma(A375 cells)is mainly attributed its prosperous redox system.The radiation generated reactive oxygen species(ROS)are easy to be eliminated by the thioredoxin reductase(Trx R),causing resistance to radiation and resulting in the insignificance of radiation therapy.Nucleic acids have been certified to be targets for cisplatin analogues.Recent studies have also demonstrated that Trx R is also an important enzyme for them.Therefore,we synthesized two cisplatin analogues as Trx R inhibitors,and their inhibitory effects on Trx R activity were compared due to introduce of substituent into the ligand.A375 cells were treated with the combination of cisplatin analogues with X-ray(2 Gy)radiotherapy.Results showed that the compound 3b inhibited Trx R activity more obviously than 3a.As the activity of Trx R was inhibited by synthetic cisplatin analogues,ROS-removing function of Trx R were deprived,which caused irreversible damage to tumor cell structure,so that the radiotherapy effect is greatly improved.The anti-proliferative effect of 3b + 2 Gy is about five times as potent as 3b alone.Moreover,3b + 2 Gy can effectively induce cell cycle into Sub-G1 phase,cause serious mitochondrial membrane depolarization,activate Caspase 3,8 and 9 and resulted in cell death.In this work,DNA topoisomerase type ?,dehydrofolic reductase and thiodeoxygenase reductase inhibitors were synthesized and their antitumor applications were studied.This work provides a mirror and reference for the design and synthesis of these enzyme inhibitors.It also paves the way for the synthesis of cancer-targeted antineoplastic precursors and provides a valuable solution to reverse the radiation resistance caused by X-ray radiotherapy.It is of great significance in chemical synthesis,biology or clinical application.