Design,Synthesis And Biological Activity Evaluation Of AI-2 Mediated Quorum Sensing Inhibitors In V. Harveyi

The discovery and popularization of traditional antibiotic therapy have improved the treatment of bacterial infectious diseases.However,due to the abuse of antibiotic therapy for a long time,Antibiotic resistance is getting worse,the separation rate of resistant bacteria continues to rise,and the mortality rate of bacterial infectious diseases continues to increase.The World Health Organization launched the“World Antibiotics Awareness Week”in 2015,and the theme for 2018 is“Change Can't Wait.Our Time with Antibiotics is Running Out”.Therefore,the problem of antibiotic resistance should not be underestimated,and it is urgent to find a new target for the treatment of bacterial infectious diseases.The main cause of bacterial resistance is the formation of biofilm which reduces the sensitivity of bacteria to antibiotics by 10-1000 times,and the formation of biofilms is regulated by quorum sensing which is a regulation of bacterial biofilm formation and the release of virulence factors,so quorum sensing inhibitors can increase the sensitivity of bacteria to antibiotics and keep the bacteria in a non-toxic or low pathogenic state.The advantage of this strategy is that it does not affect bacterial growth and protein synthesis,thus reducing the heritable mutations of bacteria,and it's will be more effective to use with antibiotics.In addition,it dosen't destroy the cell membrane,so there won't be any released toxin which will cause the host be infected again.Quorum sensing is roughly classified into four types according to the type of signal molecules:the quorum sensing mediated by oligopeptides?AIP?in Gram-positive bacteria;the quorum sensing mediated by homoserine lactone signal molecule?AI-1?in Gram-negative bacteria;the quorum sensing mediated by AI-2;the quorum sensing mediated by AI-3.Among them,AI-2 and AI-3mediated population effects are less studied.Because AI-2 is widely present in Gram-positive and negative bacteria,there are about 70 strains in total,and the structural mechanism of AI-3 is not clear at the moment.Therefore this study is focus on AI-2 quorum sensing inhibitor.In recent years,virtual screening has played a important role in the discovery of lead compounds,predicting compound targeting by mimicking the interaction between small molecules and receptors.The molecular docking method is divided into rigid docking,semi-flexible docking and flexible docking.The accuracy of the three docking methods increases in turn,and also because the amount of calculation increases in turn,resulting in a decrease in screening speed.The whole process is completed in the computer,which greatly improves the speed of drug screening.GOLD is a molecular docking program that calculates the binding mode between macromolecules and small molecules.It is the product of collaboration between Sheffield University,GlaxoSmithKline and CCDC.It uses genetic algorithm?GA?for protein-ligand docking.When docking,the ligand is completely flexible and the protein is partially flexible.The amino acid residue and ligand of the active site of the receptor are flexible,and the calculation amount is more flexible.The docking has been reduced a lot,so the screening speed has also been improved while ensuring the accuracy of the calculation,which provides a lot of convenience for this research.In 2002,Chen X et al.reported the crystal structure of LuxP,which contains the original AI-2signal molecule,provides the receptor structure and the location of the active site,and provides support for the structure-based virtual screening to find small molecule inhibitors.Therefore,this study uses GOLD to perform virtual screening and compound design of AI-2 mediated quorum sensing inhibitor based on LuxP crystal structure.10 potential inhibitors were obtained by virtual screening of the wizard module in GOLD,and the IC50 of the compound was determined by the bioluminescence experiment of Vibrio harveyi.The strains for biological activity screening were selected from Vibrio harveyi BB170 strain,because the wild-type Vibrio harveyi contained both AI-1and AI-2 signaling molecules and their receptors,while the BB170 strain was a mutation of Vibrio harveyi strain,which lacks the receptor LuxN of AI-1,so it does not respond to AI-1 signal changes,so it does not interfere with the determination of AI-2 type population effects.The activity was determined by the bioluminescence intensity of Vibrio harveyi,and when the bioluminescence intensity was reduced by half,the concentration of the compound at this time was the IC50 of the test compound.According to the activity data and structural characteristics,Str5015 was used as the lead compound,and PyMOL and LigPlot+analysis were used to design the scaffold and the structure of 20 target compounds.After 20 target compounds were synthesized,the biological activity was evaluated again to obtain the compound YB-1 whose activity was comparable to the positive control,its IC₅₀0 reached 7.1±2.89?M.In summary,a novel 3-bromophenyl-3,5-dibromo-2-hydroxybenzoate?Str5015?with IC50 value of 60?M was obtained by in silico screening and in vitro activity test.20 compounds were synthesized by structure optimization and were confirmed by ESI-MS,¹H-NMR.The studies revealed that compound3,5-dibromo-N-?2-bromo-4-nitrophenyl?-4-hydroxybenzamide?YB-1?has an IC50value of 7.1±2.89?M against AI-2 mediated quorum sensing,the structure-activity relationship was obtained which was based on the activity results of all target compounds:?1?The activity of linking the two benzene rings with amide bond is better than that with ester bond;?2?The introduction of nitro group at R4 can significantly increase the activity,and the stronger the electron withdrawing ability of the substituent at this position,the better the activity of the compound;?3?When there is a strong electron withdrawing group at R₄ and a strong electron donating group at R₃,the compound's activity is better;?4?When R₅ is bromine substituted and the R6 is unsubstituted,the activity is better;?5?The activity will be improved when R7 is replaced by the carbonate chain.This study has a guiding role in further transformation of YB-1 to find a more active small molecule inhibitor.