Study On The Resistance And Action Mechanisms Of Riminophenazine And Identification Of Regimens Containing TBI-166

Clofazimine(CFZ)is playing an increasingly important role in anti-tuberculosis treatment.Its structural modification,TBI-166,has been successfully synthesized in recent years.Preliminary evaluation results show that TBI-166 has stronger activity and lesser adverse reaction than CFZ,which has a good application prospect.Currently,TBI-166 has entered phase I clinical study in China,more detailed data from in vitro and in vivo studies are needed as the basis and reference for phase II clinical study.In addition,the anti-tuberculosis mechanism and resistance mechanism of CFZ are not completely clear at present,and the study on such mechanism of TBI-166 has not been carried out yet.In this study,the resistance and acting mechanism of CFZ and TBI-166 were studied by whole-genome DNA resequencing technology and DIA proteomics technology,and the application of regimen containing TBI-166 was identified in mice models.In the first part,we conducted a preliminary study on the resistance mechanism of CFZ and TBI-166.By measuring the MIC of clinical isolates of 200 MDR-TB patients,it was found that the drug resistance rate of TBI-166 in clinical strains was lower than that of CFZ.TBI-166 had relatively better bacteriostasis effect on over 95%of clinical isolates of MDR-TB and its activity is higher than that of CFZ in vitro.However,it was also found that TBI-166 was prone to high drug resistance,and there may be cross-resistance between TBI-166,CFZ and BDQ.The whole genome of 13 CFZ drug-resistant strains and 9 TBI-166 drug-resistant strains that screened were sequenced and compared with the sequences of CFZ and TBI-166 sensitive strains and H37Rv standard strains.Many high frequency mutation sites were screened and obtained,among which Rv1319c,Rv1945 gene mutation and Rv1453 promoter region C-17T mutation had the highest mutation frequency in the drug-resistant strain,while those were not detected in the sensitive strains and H37Rv standard strain,suggesting that these gene mutations may be related to CFZ drug resistance.At the same time,we made bio informatics prediction of the C-17T mutation in the promoter region of Rv1453 and verified the clinical strain by expanding the sample size,and the results all support the conclusion that this mutation is related to CFZ drug resistance.Besides,we found that the mutation rate of Rv0678 mutation in CFZ drug-resistant isolates was low.Rv0678 mutation may be related to the cross resistance of CFZ and TBI-166 or BDQ,but there is no direct correlation with TBI-166 resistance.In the second part,we discuss the mechanism of CFZ and TBI-166.Through the determination of ROS content and catalase activity in tuberculosis bacteria before and after treatment with CFZ and TBI-166,we found that both CFZ and TBI-166 can induce the accumulation of ROS in mycobacterium tuberculosis.TBI-166 may have a similar mechanism of action with CFZ,and play an anti-tuberculosis role through ROS.By comparing the changes of tuberculous protein expression before and after treatment with CFZ and TBI-166,we found that the effects of CFZ and TBI-166 on M.tuberculous mainly caused the abnormalities of RNA anabolism and signal transduction in bacterial cells.Besides,cellular oxidative phosphorylation pathway,p53 signal transduction pathway,and differential proteins RpoA,SDHA,SDhC,NuoL,Qor,and FadB4 may play an important role in the anti-tuberculosis process of the two drugs.In the third part,we evaluated the interactions profiles of TBI-166 in vivo and in vitro,and screened the combined regiments containing TBI-166.The in vivo and in vitro interaction between TBI-166 and various anti-tuberculosis drugs revealed that TBI-166 can improve the anti-tuberculosis activity of BDQ and PZA.At the same time,through the screening of the combined drug regimen containing TBI-166,we determined five high-potency chemotherapy regiments containing TBI-166,among which TBI-166+BDQ+LZD regimen had the best efficacy,which was recommended to be evaluated in the TBI-166 ?b phase clinical trial.Through this study,we obtained 5 high-potency chemotherapy schemes containing TBI-166,and evaluated the CFZ Rv0678 drug-resistant genes mutation in the clinical drug resistant strains,verified the hypothesis that CFZ exerts anti-tuberculosis effect through reactive oxygen species,and obtained many high frequency mutated genes(loci)that may be related to the resistance mechanisms of CFZ and TBI-166 and high-multiple differentially expressed proteins related to the action mechanisms of CFZ and TBI-166.All these findings provide the reference for further studies on the mechanism of action and resistance of these two drugs.