Study On The Mechanism Of Action And Target Of Neocryptolepine Derivatives Z24 Against Botrytis Cinerea

Cryptolepis sanguinolenta（Lindl.）Schlechter is a traditional plant found throughout Africa that is frequently utilized in traditional medicine.It has a wide range of pharmacological activities,including anti-inflammatory,hypolipidemic,antibacterial,antimalarial,and anti-tumor effects.In recent years,neocryptolepine,cryptolepine,isocryptolepine,and other key active ingredients of Cryptolepis sanguinolenta（Lindl.）Schlechter have undergone systematic structural optimization and antifungal activity testing.This work revealed for the first time that neocryptolepine has good antifungal activity against plant pathogenic fungi,and with this as the lead compound,structural derivation and optimization were used to create the candidate chemical Z24,which has a larger antifungal spectrum,improved antifungal activity,and decreased toxicity.As a result,the purpose of this research is to examine and clarify the target and mechanism of action of a prospective lead chemical Z24 against B.cinerea Pers.,which will serve as a basis for future biorational designs of neocryptolepine-based antifungal medicines.B.cinerea Pers.was used as the test strain in this work,and DARTS and LC-MS/MS were used to identify the direct target of Z24.The hypothesized target proteins were thoroughly investigated using enzymology,antibody preparation,molecular docking,transcriptomics,variable splicing,metabolomics,and exogenous metabolites.The primary findings and results of this study are as follows:1.Z24,a derivative of neocryptolepine,is a highly effective and low toxic antifungal agentAccording to the results of an in vitro activity study,Z24 outperformed cryptolepine in its superior antifungal efficacy against four major plant pathogenic fungi,outperforming pyrimethanil(EC₅₀=4.45 g/m L),with the highest antifungal activity against B.cinerea Pers.(EC₅₀=0.56 g/m L).In an in vivo investigation,Z24 has a high protective effect and can significantly reduce B.cinerea Pers.infection of tomato fruits when applied at a concentration of 100 g/m L.In the pot experiment,100 g/m L Z24 effectively suppressed B.cinerea Pers.infection on cucumber leaves,and its protective efficacy was comparable to pyrimethanil.Simultaneously,Z24 can limit the spore germination of B.cinerea Pers.and induce the accumulation of reactive oxygen species in the spores.Z24 also dramatically results in the loss of mycelium membrane integrity and cell microstructure disintegration,promoting apoptosis in B.cinerea Pers.mycelium cells.In addition,Z24 had lower cytotoxicity in HIEC cells,HL7702 cells,and GES-1 cells than neocryptolepine,which was less toxic in HIEC cells and GES-1cells than pyrimethanil.Acute oral toxicity of LD₅₀>2000 mg/kg and 2000 mg/kg.Z24did not elicit histological alterations in rats,nor aberrant changes in AKP/ALP,GOT,Urea Nitrogen,and GPT damage indices in rat serum.Finally,the seed germination test revealed that Z24 had comparatively low plant toxicity.2.Based on DARTS and LC-MS/MS techniques,the suspected target protein Bcthi4 of Z24 was obtainedDARTS,Coomassie bright blue staining,silver staining,and mass spectrometry were used to identify the direct target protein-thiaminothiazole synthase（Bcthi4）of Z24,and the relative abundance of Bcthi4 protein in the identification list was 37.11%.The docking approach was then utilized to confirm that the Z24 and Bcthi4 homologous proteins exhibited high bonding ability,with a docking score of-4.302 Kcal/mol.According to KEGG analysis,these Z24-enriched proteins were primarily involved in biological processes such as metabolic pathways,secondary metabolite biosynthesis,ribosomes,antibiotic biosynthesis,microbial metabolism in different environments,carbon metabolism,amino acid biosynthesis,glycolysis/glycoconeogenesis,and pyruvate metabolism.3.B.cinerea’s Bcthi4 protein was identified as a direct target protein of Z24.In the confirmation of the target protein,the primer was first designed by referring to the B.cinerea B05.10 Bcthi4 gene sequence（Gene ID:5430120）published by Gen Bank,the recombinant plasmid Bcthi4-p ET-B2M was constructed after the Bcthi4gene was amplified by PCR and connected to the p ET-B2M subclonal vector.Bcthi4-p ET-B2M plasmid was transferred into E.coli BL21（DE3）receptor cells,and high purity Bcthi4 recombinant protein was obtained after induction culture by IPTG.After animal immunization,indirect ELISA and Western blotting detection showed that the prepared rabbit polyclonal antibody against Bcthi4 gene of B.cinerea had high sensitivity and strong specificity,and could be used for further Western blotting.Then,the specific binding reaction of Z24 and Bcthi4 was detected by the Western blotting method.For this purpose,we designed and applied CETSA and DARTS technology to confirm the binding reactions of Z24 and Bcthi4 protein,and found that Z24 and Bcthi4proteins had good binding activity in a concentration dependent manner.Moreover,0.05μg/m L and 0.1μg/m L Z24 significantly inhibited B.cinerea Pers.and B.cinerea B05.10 Bcthi4 activity.In summary,thiaminothiazole synthase（Bcthi4）was indeed the direct target of Z24 through the joint verification of Western blotting and ELISA.4.Z24 regulates thiamine metabolism by affecting the binding of TPP to Bcthi4 ribose switchesBcthi4,a ribose switching site in the thiamine biosynthesis pathway,controls gene expression in fungi by variable splicing.The thiamine metabolic pathway was significantly enriched in the KEGG pathway analysis of differentially variable splicing genes,and three differentially variable splicing genes,including Bcthi4,Bcthi6,and Bcrpb8,were enriched into this pathway,indicating that they play a crucial role in thiamine metabolism.Notably,significant upregulation of AS events was detected in Bcthi4,the ribose switch responsible for thiamine thiazole ring synthesis,as well as the A5SS splicing pattern,indicating that Z24 treatment induced selective splicing and increased splicing of Bcthi4 precursor m RNA of B.cinerea Pers.gene.Furthermore,during Z24 stress development,B.cinerea Pers.produced a high level of expression of the target gene Bcthi4,showing that mycelial cells were thiamine starved,B.cinerea Pers.growth could be partially restored by boosting the expression of genes involved in thiamine synthesis,similar to how the commercially marketed fungal drug tebuconazole works.These findings suggest that Z24 regulates thiamine metabolism in B.cinerea via modulating the binding of TPP and the Bcthi4 ribose switch.5.Z24 is a thiamine biosynthesis pathway inhibitorExogenous thiamine application to PDA plates and PD water medium containing Z24 significantly reduced Z24’s inhibitory activity against B.cinerea Pers.and B.cinerea B05.10,whereas thiamine alone could not promote B.cinerea growth,indicating that Z24 is an inhibitor of the thiamine biosynthesis pathway.Furthermore,after dense growth of B.cinerea Pers.and B.cinerea B05.10 under Z24 stress,thiamine and its active form thiamine pyrophosphate in mycelial cells showed a trend of continuous accumulation with increasing compound concentration,indicating that mycelial cells were in a state of thiamine deficiency,and that only the accumulation of metabolites such as thiamine can restore B.cinerea growth.In addition,non-targeted metabolomics showed that abnormal thiamine metabolism further affected secondary metabolites biosynthesis,amino acid biosynthesis,alanine,aspartate and glutamate metabolism,ABC transporter,aminoacyl-t RNA biosynthesis and other biological processes in B.cinerea Pers..6.Based on virtual screening,potential Bcthi4 small molecule inhibitors were identifiedThe three-dimensional structure of the B.cinerea Bcthi4 protein has yet to be determined,the prior literature analysis revealed that the fraction of Bcthi4 Alpha Fold prediction three-dimensional structure score was more than 70 points,indicating that the structural reliability of Alpha Fold prediction was good（Alpha Fold ID:AF-A0A384JNK6-F1）.Therefore,computer virtual screening is performed by Bcthi4’s Alpha Fold predictive structure（The binding pocket is selected from the binding pocket of Bcthi4 protein and substrate,the key amino acids are CYS89/GLU110/GLY118/VAL183/HIS234/MET286,etc）.The Lig Prep Module of Schr?dinger software was used to process 2D formats of 78.8K compounds for hydrogenation and energy optimization,and 3D structures were output for virtual screening,yielding the Top 200 small molecule compounds with strong binding force to the target protein Bcthi4.The top 5 compounds（Z1601701772,Z2506212851,Z1738460435,HY-Q01831,HY-Q03084）and Bcthi4 protein binding patterns were mapped in 2D and 3D to further study the interaction patterns between the two and predict the biological activity of small molecule ligands,it lays the groundwork for future research into the design and synthesis of novel chemical entities based on the Bcthi4 protein,as well as the development of antifungal agents.