The Interaction Of Anticancer Drug Cisplatin And ATPase

Cisplatin is a widely used anticancer drug in clinic.The metal binding domain(MBD)of copper efflux protein ATPase interacts with cisplatin,which is involved in drug resistance of tumor cells.Although all MBDs share high sequence homology and similar protein folding,the various MBDs possess distinct functions in the cisplatin transporting process.Tetrathiomolybdate(TM)is a clinically used drug for the treatment of Wilson’s disease.Both TM and ATP7B are related to the function of cisplatin.TM can enhance drug efficacy and reduce cisplatin resistance,while ATP7B is involved in cisplatin resistance.However,the molecular mechanism and correlation among these agents are unknown.In this dissertation,we investigate the effect of ATPases on the platinum based drugs,and study how to overcome the drug resistance and improve cell sensitivity by small molecular drugs.The interaction between N-terminal metal binding domain of ATP7A/7B and cisplatin is determined.And the effect of TM on the platination of MBDs is studied.The research contains the following aspects:In the first part,we prepare nine MBDs of the two ATPases,and analyze their reactivity towards cisplatin.In addition,the effect of copper loading on the reaction between protein and cisplatin is also investigated.The results show that the various MBDs have different reactivity towards cisplatin.Cisplatin binds to cysteine in the copper-binding region,and copper binding generally enhances the platination rate of the MBDs,but the degree of promotion is very different.Furthermore,platinum binding does not cause copper ion ejection from the MBDs,but only weakens the Cu(I)-MBD interaction.Therefore,the copper dye BCA can compete for copper binding from platinum Cu-MBDs,but cannot remove copper from natural Cu-MBDs.In the second part,we investigate the reaction of TM with the metal binding domain of ATP7B(WLN4),and study the effect of TM on the platination of WLN4.Crystal structure indicates that TM induces the dimerization of Cu-WLN4 through a unique sulfur-bridged Mo2S6O2 cluster.The Mo atoms directly bind to the thiol groups of WLN4 at copper binding residues.The Mo-coordination expels copper ions from Cu-WLN4 and forms a copper free protein dimer.An identical protein dimer can be formed by the reaction of apo-WLN4 with TM.As the copper coordination residues are also the cisplatin binding site of ATP7B,the occupation of Mo atom at these residues inhibits the platinum binding to the protein.In order to identify the two domains linked by TM,the conserved sequence CxxC is mutated to SxxS.The results from mass spectra,UV-vis and SEC-MALS measurements confirm the presence of Mo2S2O2 mediated intramolecular crosslinking in the multi-domain proteins,which effectively inhibites the reaction of protein with cisplatin.These results provide the structural and chemical insights into the effect of TM on the function of ATPase,and elucidate the molecular mechanism how TM attenuate the cisplatin resistance arisen from copper efflux proteins.In the third part,the reaction between the N-terminal metal binding domain of ATPases and cisplatin or Pt/Atoxl is studied.The efficiency of reaction with cisplatin is compared with that induced by Pt/Atoxl.According to the fluorescence results,the efficiency of platinated MBD induced by cisplatin on WLN1,WLN4,MNK5 and MNK6 proteins is consistent with that induced by Pt/Atox1.The efficiency of obtaining platinum from Pt/Atoxl by MNK3,MNK4 and WLN2 is higher than that of direct reaction with cisplatin.The results show that MBDs could react with platinum from Pt/Atox1.The transfer of Pt from Atoxl to ATPase has also been determined by ion exchange chromatography.Before cisplatin targeting DNA,the protein ATPase in cytoplasm affects drug distribution and transport,which can help in understanding the mechanism of resistance.Tetrathiomolybdate inhibits the reaction of ATPases with cisplatin.This result explains the synergy of TM and cisplatin in cancer chemotherapy,and provide molecular basis in the sensitization of cellular responses to cisplatin.