Study On The Structural Modification And Biological Activity Of Nitazonide By Biological Target

NTZ,as an anti-parasitic drug,has shown activity against multiple microorganisms and even novel coronavirus.Therefore,researchers’ interest in NTZ derivatives is stimulated.How to use the target or mechanism of NTZ to develop more efficient and broad-spectrum drugs,the primary task is to explore the drug target or mechanism of NTZ.Therefore,it is necessary to modify the structure of NTZ to obtain derivatives that can carry out affinity chromatography and fluorescence probe localization.In this study,biotin and fluorescent labeled NTZ probes were designed and synthesized based on the existing laboratory basis and a series of studies were carried out,laying a theoretical foundation for the study of the biological mechanism of NTZ.In order to explore the targeting location and protein of NTZ,in this study,fluorescent probe L-1 linked to rhodamine B and biotin probe B7 were designed and synthesized using NTZ as the targeting ligand.Then,different cells and Escherichia-coli were used as research objects to compare the effects of NTZ and the two probes on cell viability and antibacterial activity in vitro.Compared with NTZ,L-1 and B7 showed no significant difference in the activity of CCK-8 cells at different concentrations and at different times.The results showed that L-1,B7 and NTZ did not have the anti-Escherichia coli effect,which indirectly indicated that the modification of NTZ structure did not change its activity.Western Blot was further used to detect the effects of fluorescent probe L-1 and biotin probe B7 on autophagy marker LC3 B,and the results showed that B7 could significantly induce autophagy and had the same biological function as NTZ.Subsequently,inverted fluorescence microscope was used to observe the fluorescence uptake of L-1 and rhodamine B in mammalian cells,and the results showed that compared with the blank control,they showed stronger fluorescence,indicating that both L-1 and rhodamine B could enter cells.DAPI was further used to costain L-1,and it was found that L-1 did not enter the nucleus.According to its distribution in the cytoplasm,it was preliminarily determined that the drug-acting organelles surrounded the nucleus,and it might be endoplasmic reticulum.However,cell fluorescence intensity was significantly reduced when TIZ competed with L-1,which indicated that TIZ and L-1 might have the same target after entering cells and form a competitive relationship,thus affecting the uptake of L-1 by cells.Fluorescence localization results showed that the molecular target of NTZ was located in the cytoplasm.In order to further screen drug targets of NTZ and verify them with the results of related target methods,the experimental conditions of drug affinity reaction dependent on target stability were optimized in this study,and more than 20 possible NTZ targeting proteins were preliminarily obtained.Comparative analysis showed that these proteins were mainly concentrated in golgi apparatus and endoplasmic reticulum.The result is consistent with that of fluorescence probe.In conclusion,two NTZ target probes were successfully synthesized in this study,which proved that the two probes maintained the related biological activity of NTZ,and preliminarily confirmed that the biological target of NTZ was located in the cytoplasm,which laid a material and theoretical basis for further screening biological target of NTZ and elucidating its mechanism of action.