Discovery And Mechanism Study Of Novel Hydroxypyranone Derivatives As Pseudomonas Aeruginosa Biofilm Inhibitors

Objective:Nowadays,bacterial infections lead to approximately 16 million people death every year all over the world,and more than 70% of them are associated with drug resistance caused by bacterial biofilm formation.Biofilm is a membrane-like structure formed by a multi-layer matrix secreted by microorganisms to adhere to the surface of the medium for wrapping itself.Once bacteria form biofilm,they will decrease antimicrobial effects through various mechanism of actions,for example,restricting drugs penetrance and affecting drugs dissociation by changing pH values in biofilms,which will enhance drug resistance and results in bacterial resistance and chronic infection diseases.Therefore,inhibition of bacterial biofilm formation,reduction of bacterial resistance are expected to alleviate bacterial resistance and refractory chronic infection.However,until now,no efficient biofilm formation inhibitor has been successfully applied in clinic.Therefore,it is urgent to develop new biofilm inhibitors.In this thesis,the compounds of different skeleton types designed and synthesized by our group were screened to find new and effective biofilm inhibitors,and further explored the anti-biofilm mechanism of active compounds,and finally obtain novel biofilm inhibits lead compounds that with clear mechanism,safety and effectiveness.Methods: 1.Experimental strain:Pseudomonas aeruginosa is the most common conditional pathogen that is easily inducible to acquire biofilm resistance in clinic practice.Thus,standard strain PAO1(ATCC 15692)of Pseudomonas aeruginosa were selected as a model strain for screening biofilm inhibitors.2.Activity screening and evaluation:(1)Crystal violet staining assay(determining compound concentration): the biofilm inhibitory activities of berberine derivatives,cajaninstilbene acid derivatives and hydroxypyrones derivatives that designed and synthesized in our laboratory were evaluated using 96-well plate crystal violet staining method.(2)Minimum inhibitory concentration(MIC): the MICs of the compounds that with biofilm inhibitory activity were determined by half dilution method to investigate whether these compounds affect the growth of bacteria within the effective concentration range.(3)Laser scanning confocal microscopy: confocal photographs were used to further confirm the inhibitory effects of candidate compounds on biofilm formation and morphology of Pseudomonas aeruginosa biofilm.(4)Concentration dependent experiment:in order to further verify the biofilm inhibitory effects of the potential compounds,concentration-dependent crystal violet staining and laser confocal microscopy were used to detect the suppressive effects of the potential compound at different dosages on the biofilm formation of Pseudomonas aeruginosa.3.Mechanism research(1)Detection of virulence factors: the virulence factors such as elastase,rhamnolipid and pyocyanin which were under the regulation of Las,Rhl,and PQS quorum sensing systems(QS),respectively to explore the possible QS pathways that interrupted by the potentional compound.(2)Fluorescent reporter strains: the molecular pathway of the potential compound was further validated by PAO1-lasB-gfp,PAO1-rhlA-gfp and PAO1-pqsA-gfp fluorescent reporter strains.(3)Computer molecular docking: using computer molecular docking to simulate the interaction of potential compounds with the corresponding pathway regulatory proteins to further explore the possible target and mechanism of actions of the potential compound.4.Safety evaluation:Finally,the toxicity of the potential compounds in murine macrophage RAW264.7 cells were evaluated by an MTT(3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide)assay.Results: 1.Biofilm inhibitory activity preliminary screening results:(1)The novel hydroxypyrones derivatives that with good biofilm inhibitory activities were identified.Among them,compound 6a showed the best activity,whose biofilm inhibition rate at screening concentration(20 ?M)was 50.98.±1.97%.(2)The minimum inhibitory concentration of the the novel hydroxypyrones derivatives were all larger than 512 ?M,which indecated that this kind of compounds had no suppressive effect on the growth of bacteria within the effective concentration range of biofilm inhibition activity.(3)The laser confocal images further confirmed the significant biofilm inhibitory activity of 6a.(4)Biofilm inhibition concentration gradient experiments demonstrated that the inhibitory effects of different concentrations of 6a on the biofilm formation of Pseudomonas aeruginosa were concentration-dependent,which suggests that 6a had a targeted specific inhibitory effects on bacterial biofilms.2.Mechanism study results:(1)Virulence factors(elastase,rhamnolipid and pyocyanin)detection resutls revealed that compound 6a specifically inhibited the secretion of pyocyanin,indicating that compound 6a may exert biofilm inhibition by specifically acingt on the PQS pathway.(2)Further fluorescent reporter strains(PAO1-lasB-gfp,PAO1-rhlA-gfp,and PAO1-pqsA-gfp)analysis showed that compound 6a also had speicific effect on the GFP fluorescent protein expression of the PAO1-pqsA-gfp reporter strain.This result further confirmed that the mechanism of compound 6a was specific to the PQS pathway at the molecular level.(3)Computer docking analysis showed that compound 6a had a good affinity to PqsR and the binding score was 6.7014,which was even higher than that of the endogenous signal molecule PQS.This result revealed that compound 6a may compete with the PQS signaling molecule to specifically bind to the PqsR receptor protein,thus inhibiting PQS quorum sensing pathway and ultimately inhibiting biofilm production.3.Safety evaluation:MTT assay showed that 6a as a biofilm inhibitor shown no cytotoxic to mammalian cells within effective concentration range of biofilm inhibition.Conclusion:A new class of low-toxic and high-efficiency pyrone-based biofilm inhibitors was discovered by screening the biofilm inhibitory activities of a series of compounds designed and synthesized in my group.Furthermore,through the mechanism study of the most active compound 6a,it is clarified that the mechanism of 6a is likely to compete with the PQS signaling molecule to specifically bind to the PqsR receptor protein and thus specificly inhibit PQS quorum sensing pathway,and ultimately decrese the virulence factor secretion and biofilm formation of P.aeruginosa.The discovery of such PQS-specific targeting biofilm inhibitor provides a new candidate for the development of targeted biofilm inhibitors for the treatment of clinical chronic infectious diseases.