Design Synthesis And Biological Activity Study Of MCR-1 Inhibitors

In recent years,drug-resistant bacteria widely spread around the world due to the abuse of antibiotics,and more than 700,000 people die of drug-resistant bacteria infection worldwide each year,and seriously threatening human health.Additionally,the emergence of multi-drug-resistant gram-negative bacteria poses a great challenge to clinical treatment,and the drugs used to treat multi-drug-resistant gram-negative bacteria infection are very limited.To solve this issue,polymyxins,which are old antibiotics,have been reused in clinic,and they are considered to be the last line of defense against multi-drug-resistant gram-negative bacteria infection.However,at the end of 2015,the plasmid-mediated colistin resistance gene,mcr-1?mobile colistin resistance?,was reported for the first time in China.It could not only be transferred vertically,but also be transferred horizontally among different bacteria species,and it has attracted widespread attention globally.This also led to the breakdown of the final defense,and it also increases the risk of pan-drug-resistant bacteria spreading.Currently,the combination of non-antibiotics and antibiotics is becoming a very valuable therapy,which could restore the efficacy of antibiotics against drug-resistant bacteria without exerting selective pressure on bacteria.Therefore,MCR-1,a protein encoded by mcr-1 gene,could be used as a target for drug design.The mcr-1 positive bacteria might restore the sensitivity to polymyxins by designing and synthesizing compounds for inhibiting the biological activity of MCR-1 protein,and it may provide a solution for clinical polymyxins resistance.At the beginning of the research,there were no reports on MCR-1 small molecule inhibitors.This paper describes the activity evaluation of MCR-1 inhibitors in vitro and in vivo,and this is the first time that MCR-1 inhibitors have been discovered by targeting MCR-1 protein using computer-aided drug design.The major work of this paper are mainly divided into the following parts:1.The finding of small molecule inhibitors targeting MCR-1 protein In this paper,the MCR-1 protein?PDB code:5GRR?was used as the target,and the virtual screening of 8000+small molecules in our in-house compound library was carried out by computer-aided drug design,and a total of 30 compounds were screened out as candidate molecules.Subsequently,E.coli BL21?DE3?expressing full-length mcr-1gene was constructed as the activity evaluation system for testing the bioactivity of30 candidate compounds.Among them,racemate compound str5329 could restore the sensitivity of BL21?DE3?expressing mcr-1 gene to colistin,and it had no influence on the growth of tested strains.Therefore,str5329 was used as a lead compound for structural optimization in this study,and 35 new compounds were designed and synthesized.After the cell-based activity evaluation,nine compounds display better biological activity than that of str5329.Among them,compound 23 and 25 could restore the sensitivity of BL21?DE3?expressing mcr-1 gene to colistin at 25??,and they exhibit optimal bioactivity.According to the results of activity evaluation,structure-activity relationship of the compounds was analyzed:the biological activity of compounds was mainly formed by the combination of R¹,R⁴ and R⁵ groups,that is,when the R¹ was aliphatic alkyl substitution?cyclohexyl,hexyl or octyl?,the R⁴ was carboxyl substitution and the R⁵ was alkoxyl substitution,the compounds could exhibit biological activity,and the biological activity of compounds increased with the extension of the carbon chain at the R¹ group.Wherein R¹ was substituted with hexyl or octyl,the introduction of methyl group at R² position could increase the biological activity of compounds,while the introduction of methyl group at R³ position could decrease the biological activity of compounds.This results may provide a basis for further rational optimization of small molecule compounds.2.The activity evaluation of active compounds on MCR-1 proteinIn order to validate the inhibitory effect of active compounds on MCR-1 protein,the lead compound str5329,active compound 23 and 25,and inactive compound 18were selected for the experiment at the enzyme assay.The results showed that str5329,compound 23 or 25 could inhibit the PEA transfer reaction mediated by MCR-1protein,and compound 25 displays the best inhibitory activity on MCR-1 protein,while compound 23 or 25 exhibit much better inhibitory activity on MCR-1 protein than that of str5329.A dditionally,compound 18,which showed no biological activity at the cell-based assay,also showed no significant inhibitory effect on PEA transfer reaction mediated by MCR-1 protein,indicating that the results of bioactivity evaluation at the cell-based level were consistent with those at the protein-based level.The results further confirmed that the active compounds restored the sensitivity of BL21?DE3?expressing mcr-1 gene to colistin by inhibiting the bioactivity of MCR-1protein.In order to further explore the relationship between the structure of compounds and the inhibitory activity of MCR-1 protein,the molecular docking analysis was carried out between compounds and the constructed full-length MCR-1 protein.Among them,active compounds such as compound 23 or 25 could occupy the cavity of MCR-1 protein,and they have a good match with the active site of MCR-1 protein,and they interact with key amino acids of Glu246 and Thr285 by hydrogen bonds.Moreover,there was no significant difference in the interaction mode between the two configurations of compounds and MCR-1 protein,this result suggested that the configurations of compounds may have no effect on inhibitory activity of MCR-1protein.This result laid a foundation for further study of MCR-1 small molecule inhibitors.3.The activity evaluation of compound 25 in vivo After testing the bioactivity of compounds in vitro,compound 25 was selected to perform the activity evaluation in vivo.We established an acute peritonitis infection model in mice,and the survival rate of mice,the bacteria amounts in liver and liver pathology of mice treated with compound 25 or colistin alone or compound 25 combined with colistin were investigated.Compared to the mice treated with compound 25(10 mg·kg^-1)or colistin(7.5 mg·kg^-1)alone,the survival rate of the mice treated with compound 25(10mg·kg^-1)combined with colistin(7.5 mg·kg^-1)was significantly improved,and the bacteria amounts in liver was decreased,and the inflammatory effect in liver was significantly alleviated.The results showed that the therapeutic effect of compound 25combined with colistin is better than that of colistin alone on infected mice.4.The discovery of Nebivolol as a MCR-1 protein inhibitor The discovery of new uses from existing drugs could greatly shorten the time of drug development,and the pace of clinical application would be accelerated,and this could provide solutions for the treatment of clinical diseases.In this paper,the drugs of FDA library were also docked with MCR-1 protein,and nine drugs were screened out as candidate compounds.After the cell-based activity evaluation,we found that nebivolol could restore the sensitivity of BL21?DE3?expressing mcr-1 gene to colistin for the first time.Furthermore,nebivolol could inhibit PEA transfer reaction mediated by MCR-1in protein-based assay,that is,nebivolol had inhibitory activity on MCR-1 protein.We will further study the combined application of nebivolol and colistin,and we hope that this finding could provide new ideas for the clinical treatment of mcr-1 positive bacteria infection.In conclusion,we identified str5329 as a MCR-1 protein inhibitor by targeting MCR-1 protein using computer-aided drug design,and str5329 was used as a lead compound for further optimization.After the cell-based activity evaluation,nine compounds display better biological activity than that of str5329,among them,compound 23 and 25 exhibit optimal biological activity.Subsequently,the mechanism study was carried out in protein level,and compound 23 or 25 exhibit inhibitory activity for the PEA transfer reaction mediated by MCR-1 protein.In animal level,the therapeutic effect of compound 25 combined with colistin is better than that of compound 25 or colistin alone on infected mice.These results laid a foundation for further study on MCR-1 protein inhibitors.Additionally,nebivolol was screened out from FDA library,and we find and validate that nebivolol has inhibitory activity against MCR-1 protein for the first time,and the therapy of nebivolol combined with colistin is expected to provide new ideas for clinical treatment of mcr-1 positive bacteria infection.