Study On The Synthesis And Biological Activity Of A New Fungicide Targeting The FtsZ Protein Of Xanthomonas Oryzae

At present,the commonly applied bactericides in China were bismerthiazol?BT?and thiodiazole copper?TC?dating from 1970s and 2002s,respectively.That long-term usage of common bactericides has led to BT-resistant and TC-resistant Xoo strains to be isolated,thus putting the toughest challenge for treatment of Xoo infections.Given the above-mentioned situation,highly efficient antibacterial agents must be energetically pursued and developed.Distinctly,designing novel antibacterial compounds based on potential targets from Xoo can efficiently promote the discovery of highly bioactive and innovative anti-Xoo agents.Luckly,Filamentous temperature-sensitive protein Z?FtsZ?,a eukaryotic tubulin homologue,is an important functional and highly conserved guanosine triphosphatase that occupies an important status in process of cell division.Therefore,FtsZ has been widely studied and exploited as an identified target for creating antibacterial drugs in medicine field.To date,a number of small-molecule FtsZ inhibitors were reported to have the ability to perturb FtsZ's polymerization and eventually inhibit bacterial cell division.This finding has made certain natural products to be regarded as promising leading compounds in the novel anti-FtsZ drug discovery through structural optimizations.In this dissertation,computer aided drug design?CADD?was employed to verificated the feasibility of target compounds design,subsequently,the target compounds were elaborately designed and screened the antibacterial activity and other targeting assay.The work presented here is the first report of FtsZ inhibitors targeting plant bacterial diseases.Meanwhile,the screening platform of XooFtsZ inhibitors was establish through antibacterial assay,morphology change and GTPase activity in vitro,the targeting XooFtsZ ability of compounds was systematic assessed to find the lead compounds,which can efficiently promote the discovery of highly bioactive anti-Xoo agents.This work is summarized as follows:?1?The 75 compounds were designed and synthesized,including 22 compounds in ? series,29 compounds in ? series,and 24 compounds in ? series.The structures of all the compounds were confirmed by ¹H NMR,¹³C NMR and HRMS.?2?The XooFtsZ was expressed and purificated by cloning technologies,and furtherly optimizated enzyme activity conditions:50 mM HEPES-KOH,50 mM KCl,1 mM EDTA,pH=8.0,6?M FtsZ,1.25 mM GTP,2.5 mM MgCl₂;?3?An array of novel N-?cinnamoyl?-N'-?substituted?acryloyl hydrazide derivatives containing both the cinnamoyl and pyridinium moieties were prepared to explore novel antibacterial compounds against Xoo-induced infections directed by targeting FtsZ from Xoo.Bioassay results showed that ?₇ had excellent anti-Xoo activity(EC₅₀=0.99 mg/L)in vitro and distinct curative activity?62.3%at 200 mg/L?in vivo.Elaborate investigations found that ?₇ could reduce the GTPase activity of recombinant XooFtsZ with the IC₅₀ value of 171.3?M?90.31 mg/L?,and subsequently disturb the self-assembly and polymerization of XooFtsZ from TEM images.The bonding constant between ?₇ and XooFtsZ was calculated as 10^3.46 M^-1by the fluorescence titration experiment.Docking study displayed that ?₇ had strong interactions with ASN34,GLN193 and GLN197 residues that located in?-helix regions of XooFtsZ.Moreover,prolonged and filamentous bacteria were observed from TEM and FM images after incubation with ?₇,indicating that this kind of target compounds could block the binary division and normal reproduction of Xoo.These above-mentioned evidences preliminary validated that,the designed compounds were potential Fts Z inhibitors in phytopathogen Xoo.?4?The ?-series compounds and ?-series compounds has the mordate antibacterial activity,but some compounds exhibited good anti-cancer activity by anti-tumor activity assay,and furtherly targeting the tubulin through immunofluorescence assays.Docking study displayed that ?₂₃ had strong hydrogen bond interactions with ARG374 residues,the corresponding hydrogen bond distances of 2.2?that located in M regions of tubulin,which was targeting taxol binding domains.Meanwhile,the ?₂₀had strong hydrogen bond interactions with SER369 residues,the corresponding hydrogen bond distances of 2.5?that located in M regions of tubulin.Finally,the target compounds could stabilize microtubules and promote protofilament assembly.?5?The screening platform of XooFtsZ inhibitors was establish through antibacterial assay,morphology change and GTPase activity in vitro.It's worth noting that sanguinarine and berbine are also potential XooFtsZ inhibitors.At the same time,the 4'-demethylepipodophyllotoxin may be a potential inhibitor of XooFtsZ protein as thefirstreport.Dockingstudyfurtherlydisplayedthat4'-Demethylepipodophyllotoxin had strong interactions with ARG205 and ASP38residues of XooFtsZ.Moreover,prolonged and filamentous bacteria were observed from TEM and FM images after incubation with 4'-Demethylepipodophyllotoxin,indicating that this kind of target compounds could block the binary division and normal reproduction of Xoo.Finally,bioassay results showed that4'-Demethylepipodophyllotoxin had moderate curative activity?47.9%at 200 mg/L?in vivo than thiodiazole copper?32.2%at 200 mg/L?.