Screening Of Doxorubicin Target CMPK1 And The Mechanism Study

Anthracycline is an effective tumor inhibitor,adriamycin(ADM)is the most wide ly used anthracyline in clinical use,and its role in tumor chemotherapy has been conf irmed.Inhibit the replication of cancer cells through inserting into the segment of DN A base,and unstabilize the tumor DNA by associating with topoisomerase II(TOPO I I)have been confirmed as the mechanism of ADM.ADM has been clinically used fo r nearly 50 years,besides many side effects,ADM caused multidrug resistance occurr ed frequently.The corresponding mechanism is unknown,it is necessary to unveil the mechanism of adriamycin for further pharmacological understanding.This study begin s with screening and confirmation of the new adriamycin target,uses human protein microarray as material,and systematically analyzes the relationship between mechanism of adriamycin and resistance.So that to supplement and improve the understanding o f pharmacological actions of adriamycin.The main results are as follows:1.Design and preparation of the visual ADM probe.Based on the association of the drug and biomacromolecules,and the steric effect as well as the retaining of the drug,biotinylated aminocaproic acid was used as an i ntermediate;by coordinating with Cy5 conjugated streptavidin,the visible drug probe f ormed.Except for anthracycline,most drugs without auto-fluorescence,based on auto-f luorescence of adriamycin,designed and synthesized biotin adriamycin double fluoresce nce system,and verified the validity of biotin labeled probe compared to its auto-fluor escence,so that to assist the establishment of microarray based screening platform.d U sing DMAP and DCC as catalyst,the biotinylated ADM with long chain was synthesi zed by one step,and the physical and chemical analysis was consistent with rational design.Biological experiments found that the biotinylated ADM can effectively combin e with streptavidin-magnetic beads(? 93.7);the IC50 of biotinylated ADM and ADM had no significant difference to the ductal epithelial carcinoma cells MCF7/W and mul tidrug resistance MCF7/ADM cells,and the position of biotinylated ADM in the cells was consistent with ADM detected by laser scanning confocal microscope;the tracing effect of biotinylated ADM probe was good when used human derived microarray as material,and yielded 14 hits: TNFAIP6,APOA2,DYNLL1,TRMT112,MGAT1,S LC22A6,COASY,CMPK1,COX6B2,CLIC2,ZNF684,TRDMT1,H3F3 B,DVL2.2.The verification of ADM target CMPK1.Based on the screened 14 hits,competive binding method was used on the huma n derived protein microarray with biotinylated ADM and ADM,identified 3 hits: SLC22A6,CMPK1 and COX6B2;14 hits were expressed by HEK293 cells,biolayer interf erometry(BLI)was used,and identified 4 hits: SLC22A6,CMPK1,TRMT112 and D YNLL1.Full consideration of the hits identified by the above two methods,CMPK1 was choosed for further analysis,and detected the affinity of CMPK1 with ADM(Kd =1.2±0.5 ?M)by microscale thermophoresis(MST);the docking analysis by computer showed that the association between CMPK1 and ADM was by hydrogen bonds and ionic bonds.3.Study on the association between ADM and CMPK1.CMPK1 is a phosphorylation kinase of the precursor of DNA as well as activated kinase of prodrug.Based on the CMPK1 enzymatic properties,HPLC method was es tablised by quantitative analysis of the CMPK1 catalysate,the phosphorylation activity of CMPK1 to CMP,d CMP and UMP was found to be up-regulated by ADM.When the ADM concentration was 30 ?M,the enzyme activity of CMPK1 increased the m ost.ADM can improve the activity of enzyme without interference from CMPK1 regu lators such as DTT,Na F and EDTA.ADM could effectively improve the inhibitory ef fect of gemcitabine which was a substrate of CMPK1 on pancreatic cancer cells,and its mechanism was caused by ADM intervention to CMPK1 intracellularly.4.Study on the relationship between CMPK1 low expression and ADM resistant breast cancer cells.Based on the 14 hits above,the coresponding 14 genes transfected HEK293 cells were used as material,the sensitivity of cells transfected with CMPK1 changed the most by drug intervention in vitro,RT-PCR was used to analyse the change of the 14 genes in MCF7/W and MCF7/ADM cells,the transcription level of CMPK1 genes in drug resistant cells were significantly lower than in the drug sensitive cells,western blot semi-quantitative method verified the CMPK1 protein phenotype was consistent wi th transcription.When the protein level of CMPK1 in MCF7/W was partially silenced by the si RNA,the cells also showed a decrease in sensitivity to ADM,indicating th at the expression of CMPK1 was positively correlated with the drug susceptibility of MCF7 cells to ADM.This study confirmed the CMPK1 as the target of ADM,demonstrated that the in fluence of ADM to CMPK1 upgraded the effect of gemcitabine to cancer,and elaborated that the interaction between ADM and CMPK1 avoided ductal epithelial carcinoma resistant to ADM.