Study On The Target Of Phenyloxadiazole Sulfone Compounds

As a main product for the control of diseases,pests and weeds,agrochemicals play a vital role in the development of national economy.In the process of developing agrochemicals,the discovery of new targets is significant for discovering new agrochemicals.Once discovered,it will become a breakthrough for the creation of new drugs.According to its social and economic benefits,the discovery of new targets has become an important direction for designing new agrochemicals.Phenyl oxadiazole sulfone compounds represent a novel structure and showed potent efficacy against those significant bacterial diseases such as Xanthomonas oryzae pv.oryzae（Xoo）,Xanthomonas axonopodis pv.citri（Xac）and Ralstonia solanacearum,therefore the further optimization and bioactivity study of this structure has appeared extensive concerns.However,no detailed target analysis of this bactericide has been conducted so far in living systems and the further utilization of this structure is facing great challenges.To unravel JHXJZ’s protein targets in bacteria and elucidate its mechanism of action at the molecular level,we designed a series of alkyne-functionalized probes based on the parent structure jiahuangxianjunzuo（JHXJZ）.For the first time,we unraveled dihydrolipoamide S-succinyltransferase（DLST）as a major target both in Xoo and Xac by using chemical proteomic methods.Quantitative proteomic analysis further revealed that DLST affected energy metabolism by regulating lysine succinylation in cells.We also discovered that JHXJZ interacted with multiple targets in Ralstonia solanacearum.In this study,we established a target research platform based on activity-based protein profiling and revealed DLST as a new target in pesticide field.The discovery of DLST is expected to provide theoretical support for the development of novel,highly effective and green bactericides.This work is summarized as follows:（1）Based on the parent structure JHXJZ,we designed a series of alkyne-functionalized probes 1-6 and negative probes 1-2,which were further characterized by ¹H NMR,¹³C NMR and HRMS.（2）The antibacterial activity of probes 1-6 were evaluated against Xoo,Xac and Ralstonia solanacearum by a turbidimeter test in vitro.The antibacterial activity of Probe 1 against Xoo(EC₅₀=3.58μM)was similar to JHXJZ(EC₅₀=3.51μM),and the antibacterial activity of Probe 2 against Xac(EC₅₀=7.06μM)was better than JHXJZ(EC₅₀=8.59μM).The antibacterial activity of Probe 3 against Ralstonia solanacearum(EC₅₀=84.67μM)was slightly lower than JHXJZ(EC₅₀=65.06μM).Probe 1,2 and 3can be used as activity-based probes for target identification.（3）We used Probe 1 for the target idenfication in Xoo cells.To analyze its targets,we used Probe 1 to conduct in vitro and in situ studies,followed by click chemistry,SDS-PAGE and streptavidin blot.As a result,we discovered protein p45 as a major target and Probe 1 selectivily labeled protein p45 both in vitro and in situ experiments.We further confirmed p45 as a specific target by competitive experiments.The negative probe NP 1 was used to confirm that the sulfonyl group is critical for the interaction between Probe 1 and p45.The p45 was enriched by pull down experiments and identified as dihydrolipoamide S-succinyltransferase（DLST）by LC-MS/MS analysis.The recombinantly expressed DLST was further used to confirm the interaction between Probe 1 and p45 in vitro.Finally,immunoblot analysis using an antibody confirmed the protein p45 pulled down by probe 1 was DLST.（4）We used Probe 2 for the target idenfication in Xac cells.To analyze its targets,we used Probe 2 to conduct in vitro and in situ studies,followed by click chemistry,SDS-PAGE and streptavidin blot.As a result,we discovered protein p45 as a major target of Probe 2 and p45 was selectivily labeled by Probe 2 both in vitro and in situ experiments.We further confirmed p45 as a specific target by competitive experiments.The p45 was enriched by pull down experiments and identified as by LC-MS/MS analysis.Immunoblot analysis using an antibody was further performed to confirm the protein p45 pulled down by probe 2 was DLST.（5）We used Probe 3 for the target idenfication in Ralstonia solanacearum cells.To analyze its targets,Probe 3 was used to conduct in vitro and in situ studies.As a result,Probe 3 labeld multiple targets,which were further confirmed as specific targets by competitive experiments.Pull down experiments were performed to enrich targets,followed by on-bead digestion and LC-MS/MS,and a total of 65 specific targets was identified.We further performed immunoblot analysis by using antibodies to further confirm the highly enriched DLST,GCSH（glycine cleavage system H protein）and TPX（thiol peroxidase）were the targets of Probe 3.GO analysis showed the targets were mainly involved in metabolic processes（43%）and celluar processes（37%）.KEGG enrichment analysis showed targets were mainly associated with ribosome and other pathways.（6）We used a label-free quantitative proteomic approach to identify the lysine succinylome in Xoo cells and its regulation by JHXJZ.Succinylated peptides were enriched by affinity purification with succinyl-lysine antibody,analyzed by nano LC-MS/MS.A total of 2860 succinylation sites from 765 proteins were identified with a FDR less than 1%.Quantitative data generated on 2052 sites from 597 proteins revealed 65%of succinylation sites（1332）across 69%of the identified proteins（412）were reduced by more than 2-fold（p-value<0.05）.This result showed the inhibition of DLST by JHXJZ significantly reduced succinylation levels of DLST targets,indicating the crucial role of DLST for lysine succinylation.Function classification by Gene Ontology annotation showed the succinylated proteins are mainly involved in metabolic processes（34%）,followed by cellular processes（30%）.In the molecular function classification,approximately half of the succinylated proteins（45%）were categorized as binding of various targets and 43%succinylated proteins were associated with catalytic activity-related proteins.These result indicated the essential regulatory roles of theses lysine succinylation substrates in cells.Further GO enrichment analysis also showed the metabolic process was significantly enriched,indicating that these succinylated proteins are associated with metabolism.We further performed KEGG pathway enrichment analysis of succinylated proteins and found that glycolysis/gluconeogenesis was significantly enriched,indicating the important role of lysine succinylation in regulation of energy metabolism.To test this hypothesis,we treated Xoo cells with various concentrations of JHXJZ and detected a more than40%decrease in ATP content,which further confirmed the energy production was interrupted.We revealed that DLST may affect energy metabolism by regulating lysine succinylation.Our results also indicated JHXJZ was a useful tool for the function study of DLST.