| Traditional insecticides have made great contributions to crop protection and pest control.However,the irrational use of pesticides and their inherent shortcomings such as the pest resistance,toxicity to non-target organisms,food safety,environmental pollution and ecology interference,have made it urgent to develop novel effective and eco-friendly insecticides.It is clear that peptides and proteins constitute the major classes of bioactive ligands for drug design and development.The intrinsic properties of natural peptides,such as poor bioavailability,short biological half-lives and low receptor selectivity have limited their use as drugs,and much effort has been expended to find ways to replace portions of peptides with nonpeptide structures(termed as peptidomimetics).Recently,the rational design and development of drugs via peptidomimetics approach has proven to be a powerful and successful pathway for drug discovery,which made it a hotspot.We believe that the discovery of peptide-based environmentally acceptable and toxicologically innocuous pesticide using peptidomimetics strategy is a powerful pathway for pesticide discovery,even though there is no peptide or peptidomimetic that has been registered for use as commercial chemical pesticide.The insect kinin neuropeptides are pleiotropic peptides that are involved in the regulation of hindgut contraction,diuresis and digestive enzyme release.This family of neuropeptides have been identified/isolated from many insects,and they share a common C-terminal pentapeptide sequence Phe1-Xaa2-Yaa3-Trp4-Gly5-NH2(where Xaa2 = His,Asn,Phe,Ser or Tyr;Yaa3 = Pro,Ser or Ala).Recently,the multiple bioactivities of insect kinin analogs,especially their aphicidal activity attracted the attention of researchers.And the bioactive and eco-friendly insect kinins(IKs)are regarded as the leads for the discovery of new eco-insecticide.However,IKs and their analogs also have some shortcomings such as lack of contact toxicity,rapid degradation in vivo,low bioavailability and high production cost.These issues preclude their application in pest control.To discover potential eco-insecticide with novel structure as the leads and new pesticide development strategy,we chose the insect kinas and/or analogs as the hit.and the peptidomimetic approach as the strategy in our present study.The results are summarized as follows:1.The natural insect kinins were not suitable as the lead for the discovery of insecticide due to its long sequences.In this dissertation,the hexapeptide analog Aib-Phe-Phe-Aib-Trp-Gly-NH2 was selected as the lead compound which showed enhanced peptidases resistance,antifeedant and aphicidal activity against pea aphid.10 analogs(Series Ⅰ)were designed and synthesized by removing or replacing the N-terminal Aib group of the enzymatic fragment Aib-Phe.The aphicidal bioassay results showed that the pentapeptide analog 1-10 without the Aib exhibited higher activity than the lead and other hexapeptide analogs.This indicated for the first time that the N-terminal Aib did not play an important role in the activity and could be removed to simplify its structure and improve the activity.And the core pentapeptide analog can be corroborated as the new ideal lead in the further discovery of novel eco-insecticide.2.Aim to assess the role of the other site Phe of the enzymatic fragment Aib-Phe,21 new analogs(Series Ⅱ)were designed and synthesized by replacing Phe with H,Ph(CH2)nCO(n=0,1,2,3,4),cinnamic acid and its benzene substituted derivatives.The aphicidal bioassay results showed that analogs of series Ⅱ exhibited aphicidal activity.Of particular interest was the analog Ⅱ-B-1(LC50=0.019mmol/L)which exhibited improved aphicidal activity compared with 1-10(LC50=0.045mmol/L)or the commercial insecticide pymetrozine(LC50=0.034mmol/L).The relationships between structure and aphicidal activity were studied and analyzed with the interaction model of insect kinin receptor and ligands proposed by Nachman et al.3.With the purpose of discovery of analogs with higher activity and exploration of their structure-activity relationship,three other series analogs were designed and synthesized based on the Series Ⅱ.There were three modifiable sites in the third lead compound Ⅱ-B-1 using the peptidomimetics approach.All these work were specialized as follows:Ⅰ.Nine analogs(series Ⅲ)with heterocyclic(naphthalene)rings replacing the benzene ring of the cinnamoyl were designed and synthesized to assess the heterocyclic(naphthalene)effect to the bioactivity.The bioassay results showed that their aphicidal acitivity were generally lower than the third lead compound,indicating that the incorporation of heterocyclic(naphthalene)rings played a negative effect.Ⅱ.The Aib3 site was located in the other enzymatic fragment Aib-Trp and played an important role in the formation of the typical conformation including β-turn,γ-turn or line conformation.Modification of this site could lead to the discovery of analogs with higher activity.Therefore,analogs(Series IV)incorporated of line natural/unnatural amino acids,sterically hindered unnatural amino acids and hydrophobic/hydrophilic amino acids in Aib3 site were designed,synthesized.The aphicidal bioassay results indicated that analog Ⅳ-3.Ⅳ-5.Ⅳ-6.Ⅳ-9 and Ⅳ-10 all showed higher aphicidal activity than Ⅱ-B-1 and commercial insecticide pymetrozine.The incorporation of line natural/unnatural amino acids,sterically hindered unnatural amino acids and hydrophobic/hydrophilic amino acids in Aib3 site might not only improve the enzyme-resistance of the analogs,but also form the potential active conformation.Ⅲ.Ten analogs(series Ⅴ)were synthesized by using natural/unnatural amino acids to replace Gly5 of the Ⅱ-B-1.Bioassay results showed that the aphicidal activity of analogs analog decreased compared with the third lead Ⅱ-B-1.Analog V-9 containing Inp(piperidine-4-carboxylic acid)demonstrated significant activity,but was still less active than Ⅱ-B-1.The result indicated that Inp was a simple mimicry for Gly5 which showed conservatism.However,in consideration of its easy-modification we can modify this site in the design of nonpeptide/pseudopeptide analogs.4.In order to obtain the conformation and aphicidal activity relationship of the analogs,we selected 8 typical analogs and study their conformation in solution using the NMR and molecular dynamic techniques.Afterwards,the conformation and activity relationship were discussed.The results showed that analogs with good aphicidal activity formed a conformation like type Ⅳ β-turn and the side aromatic group of residue Phe2 and Trp4 located close to each other in the same side of the peptide chain.5.The log P value,acute toxicity,carcinogenic toxicity,mutagenic toxicity and potential bioactivity of all the analogs were predicted using the Prediction System developed by Shanghai Institute of Organic Chemistry,Chinese Academy of Science.The toxicity prediction result showed that most analogs demonstrated no acute toxicity,carcinogenic toxicity and mutagenic toxicity except those compounds containing nitro group,halogen atoms and aromatic heterocyclic.The bioactivity prediction result indicated that the analogs were also potential growth promoter and fungicide.Hence,we evaluated the in vitro fungicidal activity of all the analogs using the standard procedure.The results showed that the analogs demonstrated good in vitro fungicidal activity against 12 typical agricultural pathogens.This is the first discovery of fungicidal activity of insect kinin analogs which may be a class of potential insect antifungus peptides.In summary,five series of novel insect kinin analogs were designed and synthesized using the peptidomimetics approach to develop new eco-insecticide.Some of these analogs showed higher aphicidal activity than the commercial insecticide Pymetrozine.Their structure-activity relationships were studied based on the aphicidal and in vtro fungicidal activity evaluation. |
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