Screening Of Plant Virus Inhibitors Targeting G-Quadruplex And Target Discovery Of NK0209 Against Tobacco Mosaic Virus

Plant viruses cause many plant diseases which are seriously harmful to agricultural production.Anti-viral chemicals are important sources for preventing and controlling plant viruses.At present,the ideal plant virus inhibitors are still limited in classes and structures.The development of highly efficient and environmentally friendly plant virus inhibitors with novel structures mostly depends on the discovery of new targets.Identification of biological macromolecules that play important role in the process of virus proliferation,and exploration of potential targets of existing plant virus inhibitors,both are expected to provide new targets for the research and development of novel plant virus inhibitors.G-quadruplexes are non-classical high-level structures formed by guanine-rich nucleic acid sequences,Which have significant biological functions,This study aimed to discover novel quinoline inhibitors through targeting the G-quadruplex in the plant virus genome.NK0209 is a derivative of harmine,a natural plant secondary metabolite,and it has a significant effect on controlling plant viruses in the field.In this study,the helicase of plant virus was identified to be a potential target of NK0209with the methods of Activity-Based Protein Profiling（ABPP）and Drug Affinity Responsive Target Stability（DARTS）.The detailed conclusions are as follows:1 Design of plant virus inhibitors targeting G-quadruplex of TMVTobacco mosaic virus（TMV）,as a model plant virus,causes serious damage to the quality and the yield of agricultural products.In this study,a parallel RNA G-quadruplex structure forming sequence TMV-PQS1 was identified from the TMV genome.Then the quinoline compound BMPQ-1 was discovered to be a plant virus inhibitor through target-based designing and screening by using G-quadruplex as a novel target.The fluorescence spectroscopy assay showed that the fluorescence intensity of BMPQ-1 increased dramatically with the addition of TMV-PQS1,and fluorescence titration indicated that BMPQ-1 exhibited strong binding affinity（K_d=4.77μmol/L）with TMV-PQS1 G-quadruplex.CD melting experiments proved that BMPQ-1 stabilized TMV-PQS1 G-quadruplex with aΔT_mvalue of about 5.3℃.The above results suggested that BMPQ-1 interacted and stabilized TMV-PQS1G-quadruplex.The inhibitory activity of BMPQ-1 at the concentrations of 100μg/m L and 500μg/m L against TMV was 44.12%and 67.56%,respectively,which is equivalent to the commercial plant virus inhibitor Ningnanmycin,and the compound had no significant toxic effect on the growth of Nicotiana benthamiana.G-quadruplexes are widely distributed in organisms and have structural similarities.The interaction between BMPQ-1 and other nucleic acid structures had been evaluated and the results showed that BMPQ-1 was able to target the telomere multimeric G-quadruplexes,induce the formation of endogenous telomeric G-quadruplex structures in cells,and shift the conformational ensemble of multimeric G-quadruplexes from hybrid-1 topology to hybrid-2 topology.The bioactivity assay showed that BMPQ-1 effectively inhibited the tumor growth with IC₅₀values of 1.40μmol/L and has no significant effect on the weight of mice,which indicated its good biocompatibility.2 Target discovery of plant virus inhibitor NK0209NK0209 is a derivative of harmine,an active plant virus inhibitor that is produced by the plant.The identification and verification of the target of NK0209 are conducive to elucidating its mechanism of action,carrying out the optimization of lead compounds based on the target structure,and discovering plant virus inhibitors with novel structures.Multiple target identification methods,ABPP and DARTS,were applied to explore the target of NK0209,and 24 proteins including TMV-Replicase that were found to be potential targets of NK0209.The helicase domain of TMV-Replicase participates in the regulation of TMV replication and assembly.Recombinant TMV-Helicase was obtained by in vitro expression and purification,and then chemical biology and biophysics methods were performed to verify the interaction between NK0209 and TMV-Helicase.Fluorescence gel electrophoresis experiments showed that TMV-Helicase could cross-link with NK0209-Probe.Drug Affinity Responsive Target Stability（DARTS）assay proved that TMV-Helicase was protected by NK0209 and was resistant to degradation by protease.Microscale thermophoresis（MST）analysis revealed that the dissociation constant between of NK0209 and TMV-Helicase was 57μmol/L.Molecular docking showed that NK0209could interact with the tyrosine residue at position 297,the alanine residue at position 311 and the leucine residue at position 313 of TMV-Helicase.Gel filtration chromatography indicated that NK0209 induced aggregation of TMV-Helicase.The above results concluded that NK0209 targeted TMV-Helicase and induced aggregation of this protein to result in anti-viral activity against TMV.Formation or unwinding of G-quadruplex structure in the viral genome can regulate genome replication or gene transcription.Helicase unwinds the higher structure of nucleic acid to ensure the normal replication of the genome or transcription of viral genes.This work found that nucleic acids and protein in the process of virus replication and transcription can be used as targets for the development of plant virus inhibitors.It provides new insight for the discovery of novel plant virus inhibitors.