Optimization Of Carbazole Alkaloid As Anti-TMV Agents And Study On Photodynamic Anti-TMV Targeting Its G-Quadruplex

Tobacco mosaic virus(TMV)is one of the most serious plant viruses,which causes hundreds of millions loss every year.Chemical protection is still the main means of control;however,the efficient chemicals are limited,and it is difficult to control plant viruses,and thus,it is highly desired to develop novel and efficient antivirals.It is beneficial to develop novel antivirals by establishing accurate and high-efficient screening method,and designing new compounds based on novel mechanism of action.Therefore,this study focuses on the establishment of screening method and photodynamic antivital therapy targeting TMV G-quadruplexe(G4),and the results are as follows:1.Establishment of a digital screening method for anti-TMV compounds,and the discovery of effective anti-TMV carbazole derivativesFacing the limitation of common screening methods of anti-virus,such as time consuming and laborious,the construction of viral expression vectors containing fluorescent proteins can intuitively evaluate the inhibition of small molecules.However,most of the reported fluorescence methods are lack of quantification and cannot achieve high-throughput screening.In this study,an automatic readout and digital fluorescence screening method was established,which could achieve the quantitative analysis of compounds to virus,and avoids subjective errors from the operators.Moreover,the fluorescence area was monitored in real time to achieve high-throughput screening and long-term continuous observation.The anti-TMV activities of commercial ningnanmycin at 50 μmol/L were 87.8%,82.3%,and 64.6% post injection for 72 h,120 h,150 h;under the same condition,the anti-TMV activities of ribavirin were 64.5%,57.6%,and 41.8%,suggesting that ningnanmycin was more efficient than ribavirin.In addition,these results were in accordant with that from half-leaf method,suggesting that this screening method is competent to screen anti-TMV compounds.Carbazole and β-carboline alkaloids exhibited various antiviral activities.To find highly active antiviral compounds,a series of carbazole and β-carboline derivatives with substitutions at positions 7 and 11 were designed,synthesized by introducing the acylhydrazone moiety.Their anti-TMV activities were evaluated using our screening method,among them,compounds 5 and 11 exhibited high anti-TMV activities(inhibition of compounds 5 and 11 at 50 μmol/L were 76.3% and 63.1% at 150 hpi),which was comparable to that of commercial ningnanmycin(inhibition: 64.6%).They delayed the multiplication of TMV in the early stage of infection from the observation at different time points,and kept high activities against TMV for a long-term.These results were in accordant with that from half-leaf method,and the correlation coefficient of inhibition rates at different time points were >0.8,which further proved the accuracy and repeatability of this method.2.Developing photodynamic anti-TMV strategy that targets TMV G-quadruplexes,and discovering photosensitive chlorin Ce6 with high anti-TMV activityPhotodynamic therapy(PDT)shows great application prospects in agriculture due to its advantages of high efficiency and fast action speed,however,the target of photosensitizers is poor,which leads to damage to normal tissues and cells.G-quadruplexes are non-canonical secondary structure of nucleic acids,and some G4 ligands exhibit good photophysical properties.Our previous studies have found that TMV G4 s influenced the replication of gene,which will be expected to be the potential drug targets.Therefore,we take TMV G4 s as biotarget to screen the bound photosensitizers,destroying nucleic acids by photodynamic pathway,aiming to providing a novel strategy for plant viruses.Based on the previously identified putative G-quadruplex sequences(TMV PQS5)in TMV genome,photosensitive chlorins Ce6 and Ce6 T were discovered to interacted with TMV PQS5 with high affinity(Kd values of Ce6 and Ce6T: 0.94 μmol/L and 1.77μmol/L)via UV-Vis method.Both two compounds produced reactive oxygen species(ROS)and cleaved TMV PQS5 upon photo-irradiation(cleavage of Ce6 and Ce6 T after irradiation for 120 min: 55% and 40%);the ROS level and photo-cleavage of TMV PQS5 were decreased after adding of ROS scavenger,suggesting that the ROS generation was contributed to the photo-cleavage of TMV PQS5 by chlorins.In addition,they were not cleave ss RNA under the same condition,suggesting that chlorins preferentially interacted with TMV G4.Considering the relatively higher ROS yield and binding affinity with TMV PQS5 of Ce6,Ce6 was selected to was deeply studied.Using the fluorescence method,Ce6 could not inhibit the fluorescence of GFP from TMV-GFP mutant vector that was mutated in PQS5 sequence,suggesting that Ce6 interacted TMV G4.Moreover,Ce6(78.1%)exhibited comparable anti-TMV activity to that of ningnanmycin(76.6%)at500 μg/m L under normal irradiation,and displayed higher activity(92.2%)upon photo-irradiation;and Ce6 reduced the expression of TMV cp gene and exhibited higher activity by q RT-PCR.In conclusion,this study established a digital fluorescence method to screen the anti-TMV activities of compounds,and discovering that carbazole derivatives 5 and 11 exhibited high activities,and developing photodynamic anti-TMV strategy by targeting its G4 and found the high activity photosensitizer Ce6.The results of this study provided a accurate and quick screening method for plant virus inhibitors,and expected to provide a molecularly targeted PDT for plant viruses.