Study On The Anti-PVY Activity And Preliminary Mechanism Of Action Of Novel Pyrido[1,2-α]pyrimidine Interionic Compound

Potato virus Y（Potato virus Y,PVY）belongs to the genus Potyvirus in the family Potyridae,causing billions of dollars in economic losses in the world annually.In this study,a series of novel pyrido[1,2-α]pyrimidinone mesoionic compounds with a dithioacetal moiety was applied for anti-PVY inactivated activity screening by the half-leaf method and the mechanism was studied preliminarily.The research results are as follows:1.The anti-PVY activity of a series of novel pyrido[1,2-α]pyrimidinone mesoionic compounds with a ringy dithioacetal moiety was screened by the half-leaf method.The results showed that some of those compounds showed good anti-PVY inactivating activity,of which compounds S34,S35 and S36 have excellent inactivation activity against PVY,with the EC₅₀ values of 117.1μg/m L,104.1μg/m L and 111.0μg/m L,respectively,which are better than Ningnanmycin（124.9μg/m L）.Compound S35 showed the best anti-PVY activity which indicate as a potential anti-PVY agent.2.,In order to analysis the relationship between the structure of compounds and its anti-PVY activity,31 novel pyrido[1,2-α]pyrimidinone mesoionic compounds were used to establish the model of 3D-QSAR with the template molecular of compound S35,based on the anti-PVY inactivation activity.The 3D-QSAR model was used to analyze the structure-activiry relationship between the structure of compounds and its anti-PVY inactivation activity.The 3D-QSAR results indicate that the anti-PVY activity of pyrimidinone mesoionic compounds can be enhanced by introducing of hydrogen bond donor and reducing of steric hindrance on R₁ group.Besides,the R₂ and R₃ moiety with large hindrance and electropositive groups would favore anti-PVY inactivating activity.3.To evaluate the anti-PVY mechanism of novel pyrido[1,2-α]pyrimidinone mesoionic compounds,wild type PVY coat protein(PVY CP^WT)was obtained by prokaryotic expression and purification.And then,the protein was applied in Microscale Thermophoresis（MST）assay to detect the interaction between compounds and PVY CP protein.MST detection results showed compounds that showed good anti-PVY inactivation activity,such as compounds S34,S35 and S36,have high binding affinity with PVY CP^WT protein,with the dissociation constant（K_d）values of 23.8μM,6.9μM and 20.6μM,respecitivily.Meanwhile,compounds that with lower anti-PVY inactivation activity,such as compounds S14,S15,S21 and S36,have poor binding affinity with PVY CP^WT protein,with the K_d values of 269.8μM,302.7μM,171.9μM and 197.5μM.The anti-PVY inactivation activities of compounds correlate with the capability of interaction between compounds and PVY CP^WT protein,which indicates that PVY CP can be treated as the target protein of pyrimidinone mesoionic derivatives.4.In order to find out the binding site of compound S35,molecular docking was applied to find poteintial binding sites.The binding sites were confirmed by site-directed mutagenesis experiment and MST assay.The results showed that the binding affinities between compound S35 and mutant PVY CP dramtically decreased with the mutation of Ser52,Glu204 or Arg208 on PVY CP,with the K_d values increased from 6.9μM to 219.3μM,231.1μM and 188.6μM,respectively.Further more,the binding capacity between compound S35 and mutant PVY CP protein was further decreased,with K_d value of 309.5μM,when mutate all the three amino acid of PVY CP protein.The results indicated that the amino acid Ser52,Glu204 and Arg208of PVY CP protein are the binding sites of compound S35.