Structural And Small Molecule Intervention Studies On Pathogen Proteins

In recent years,the wide use of pesticides with single varieties and other reasons lead to the emergence of plant pathogen resistance,so it`s of great scientific significance and potential application value to strengthen the research of novo pesticides targeting new targets and novo mechanisms.Dihydrolipoic acid succinyltransferase（DLST）of Xanthomonas oryzae pv.oryzae（Xoo）is a potential target of plant pathogens.Methanulfonazole has been reported as a fungicides with broad spectrum activity,while it is unclear whether it acts as an inhibitor of Xoo DLST,and the concrete mechanism is also ambiguous.Therefore,structural optimization and the discovery of its derivatives progress slowly.In addition,small-molecule fungicides inhibiting cell division protein Fts Z of Xoo have attracts increasing interests,which preliminarily verified the feasibility of targeting Fts Z against plant pathogens.These are beneficial attempts to explore new antimicrobial agents against plant pathogens based on new targets and mechanisms.The analysis of protein structure can not only deepen the understanding of the mechanism,but also promote the structure-based design and optimization of lead compounds.We firstly resolved the crystal structure of Xoo DLST.After protein expression in prokaryotic system,protein purification,crystalization and X-ray diffraction,we obtained the Xoo DLST crystal structure.Inspections of the structure revealed that 171 amino acids at N-terminal of full length Xoo DLST was cleavage.This provided guidance for the study of full-length structure of Xoo DLST and also indicated the structural basis for the optimization of methanesulfonazole.Casein hydrolase ClpP,highly conserved in bacteria,is less reported in plant pathogen Xoo.Inspired by antibacterial effects caused by the non-selective degradation of Fts Z proteins through small-molecule activators of Staphylococcus aureus ClpP（Sa ClpP）,this paper intends to study the structure of Xoo ClpP and the small-molecule intervention on its function,so as to provide new target validation for pesticides research based on new targets and new mechanisms against Xoo.Mutagenesis studies based on the structure of Xoo ClpP indicated that S68 and Y70play an important role in the function of Xoo ClpP,which provides a potential binding site for target-based drug design.The broad-spectrum ClpP activators ADEP4 and ONC212 could activate Xoo ClpP and degrade the substrate protein Xoo Fts Z.The co-crystal structure of the Xoo ClpP_S68Y bond with ADEP4 reveals E59,Y70 and Y90as its binding sites.Notably,the activity of ONC212 against Xoo was better than that of the commercial drug bismerthiazol.These evidence indicates the feasibility of achieving anti-Xoo infections through non-selective degradation of Fts Z by a activator of Xoo ClpP.And it also provide the basis for further screening and evaluation of small molecule activators as well as structure-based design and optimization of Xoo ClpP.These crystallographic studies and small-molecule intervention on DSLT and ClpP lay a foundation for the discovery of novel anti-plant pathogenic fungicides.One of the mechanisms of resistance caused by antimicrobial agents is the bacteriocidal or bacteriostatic effects,and the occurrence of drug resistance of plant pathogens is similar.Therefore,one of the possible ways to alleviate antimicrobial resistance is to explore new strategies of antimicrobial infections utilizing non-conventional antibiotics,such as antivirulence.Herein,we take Staphylococcus aureus transpeptidase Sortase A（SrtA）as an example to explore its inhibitors against methicillin-resistant Staphylococcus aureus infection through non-conventional antibiotics（non-bactericidal or non-bacteriostatic）,so as to provide reference for non-antibiotic strategies against plant pathogen infections.SrtA has been proved to be an ideal target for the development of antivirulence drugs in biology.It reduces the virulence of pathogens by interfering with the anchoring of bacterial surface proteins.However,the discovery of SrtA inhibitors is still in its infancy.Herein,2400（candidate）drugs on the market or in clinical stage were screened by fluorescence resonance energy transfer technique（FRET）,and identified the Tideglusib（TD）as an inhibitor of Sa SrtA.Further structural optimization was also conducted by cooperation.The activity and non-covalent mechanism of small molecule inhibitor were identified at the biochemical level,and the direct interaction with target protein was revealed by biophysical methods.The active compounds TD and TD32 were characterized to interfere with viruelnce-related phenotypes at the bacterial level,such as interfering with the anchoring of surface proteins on the cell wall and inhibiting the formation of biofilms.Finally,the mouse model experiment based on drug-resistant bacteria infection showed that TD could significantly inhibit the infection of drug-resistant strain USA300.This part of the study further verified the feasibility of targeting virulence regulators against bacterial infections,and provided ideas and approches for exploring anti-plant pathogen infections.In summary,focusing on the study of the crystal structures of potential targets of pathogens as well as screening and evaluation of corresponding active molecules,we resolved the crystal structures of Xoo DLST and Xoo ClpP,and verifies the activity of small-molecule activators ONC212 of Xoo ClpP against Xoo,which provides a potential candidate target for the development of new pesticides.In addition,we also found that TD and its derivatives as Sa SrtA inhibitors have great potential in the development of new antivirulence drugs,and the strategy of antiinfection based on small-molecule intervention provides a reference for the development of anti-plant pathogen infection drugs.