Design,Synthesis,Bioactivity And Structure-Activity Relationship Of Allatostatin Analogs

Traditional pesticides have made great contributions to pest control,crops' safety and human health.However,inappropriate application of pesticides and the growing concern about their inherent disadvantages,including pest resistance,environmental pollution and toxicity to non-target organisms,led to the implementation of strict regulations around the world governing their use.Therefore,new approaches to develop novel compounds with high effectiveness,low toxic and high selectivity,are needed in pesticide discovery research.Insect neuropeptides have been a prime target in the development of novel,selective and eco-friendly insect control agents in recent decades,since they regulate most of the key functions including metabolic,homeostatic,developmental,reproductive and behavioral events during an insect's life.The FGLa-allatostatins(ASTs),a family of pleiotropic neuropeptides,which were originally identified from Diploptera punctata for their ability to inhibit juvenile hormone(JH)biosynthesis by the corpora allata(CA)rapidly and reversibly,share a common C-terminal pentapeptide sequence:Phe1/Tyr1-Xaa2-Phe3-Gly4-Leu5-NH2(where Xaa2=Ser,Gly,Asp,Ala and Asn).The FGLa-ASTs have numerous other biological actions,including modulation of myotropic activity and gut enzyme activity,and they also can act as neurotransmitters.Owing to these characteristics described above,the FGLa-ASTs have been employed in the design of new potential IGRs.However,like other natural bioactive peptides,the FGLa-ASTs have some shortcomings as potential pesticides,such as susceptibility to metabolic inactivation by peptidases in hemolymph and midgut,difficulties in absorption through the insect' cuticle in vivo and high production cost.These issues preclude their application in pest control.Therefore,much effort has been expended to overcome these problems and create better AST peptidomimetics.In our previous studies,several series of analogs of the pentapeptide were designed and synthesized employing the peptidomimetic approach,by replacing portions of the peptides with unnatural or natural structures to probe the structure-activity relationship(SAR)of the core pentapeptide region of AST.Subsequently,H17 was discovered to have a significant inhibitory effect on JH biosynthesis by cockroach CA,both in vitro and in vivo.In order to discover more potent AST analog(s)as IGRs and to further investigate the stucture-activity relationship at the N-terminus,H17 and another active AST analog,K24,were selected as the lead in present study.The results are summarized below:1.For the purpose of exploring the structure-activity relationships at the N-terminus,four series of AST analogs were designed and synthesized based on four modifiable sites in the lead K24 and H17and their bioactivities evaluated.This work was carried out as follows:i.Six AST analogs were synthesized by modifying the linker of K24.Bioassay results showed that all target analogs exhibited differing potencies in inhibiting JH biosynthesis as a consequence of varying linker length.In particular,analog L6,with the longest linker(n=5),had a very strong effect on inhibition of JH biosynthesis both in vitro and in vivo.SAR study indicated that longer linkers increased their ability to inhibit JH biosynthesis.ii.To assess the substituent effect on the benzene ring at the N-terminus(Naa1),32 substituent-modified analogs were designed and synthesized by replacing 4-NO2 of the lead compound H17 with different substituents in different position.Bioassay results indicated that M11(4-Br)exhibited improved potency in comparison with the lead H17.The SAR studies demonstrated that different positions of substituents on the benzene ring of the cinnamic acid led to different activities.The para-position on the benzene ring was the most important factor in determining inhibitory activity.In general,electron-withdrawing substituents seemed to favor higher potency.iii.Thirteen analogs with heterocyclic(naphthalene)rings replacing the benzene ring of the cinnamoyl were designed and synthesized to assess the heterocyclic(naphthalene)effect on bioactivity.The bioassay results showed the incorporation of thiophene(N5)and naphthalene(N13)displayed a positive effect on inhibiting JH biosynthesis.In particular,N13 was more active than some native ASTs,such as AST 1,AST 3 and AST 13.The reason is probably because that naphthalene had a more hydrophobic region which can form the ?-? interaction with the receptor.iv.Eleven analogs were designed and synthesized by modifying the Gly2 of H17 with hydrophilic residues and hydrophobic residues respectively.Bioassay showed that five analogs with hydrophilic residues P1,P3,P4,P5 and P6 were more potent than the lead H17.The activity of P5 was about 12 times higher than the lead.P5 also was more active than six natural ASTs(AST 1,AST 3,AST 6,AST 8,AST 11 and AST 13).SAR studies demonstrated that analogs containing hydrophilic residues exhibited a strong effect on JH inhibition in vitro,whereas hydrophobic residues exerted a negative effect.This implied that the hydrogen-bond donor in hydrophilic residues at the Xaa2 site could bind to the AST receptor.2.To study the mechanism of AST analogs in inhibiting JH biosynthesis,we first reported the activity of AST analogs in activation of Dippu-AstR.The results indicated all the selected AST analogs can activate Dippu-AstR,albeit with different potencies.The EC50 of L5,L6,M10,Mlland Dlwas in the nanomolar range.3.In order to discover AST analogs with practical value,the in vivo activity of L6,M11,N13 and P5 were evaluated following injection or topical treatment.The results indicated that all selected analogs had a significant effect on JH biosynthesis as well as basal oocyte growth.P5 showed lower IC50 value than the lead H17 following injection.Accordingly,P5 can be considered as an IGR candidate for further study.4.The potential bioactivity,acute toxicity,carcinogenic toxicity,mutagenic toxicity and log P values of all the analogs were predicted using the Prediction System developed by the Shanghai Institute of Organic Chemistry,Chinese Academy of Science.The toxicity prediction result showed that most AST analogs demonstrated no acute toxicity,carcinogenic toxicity or mutagenic toxicity.The bioactivity prediction result indicated that the analogs may have potential activity as both fungicides and growth promoter.Therefore,we evaluated the fungicidal activity of some analogs in vitro using the standard procedures.The results showed that the analogs demonstrated different in vitro fungicidal activity against seven typical agricultural pathogens.This is the first report of fungicidal activity of AST analogs which may be a class of potential insect anti-fungal peptides.5.In order to obtain the conformation-activity relationship of the AST analogs,we selected five typical Gly2 modified analogs and studied their conformation in solution by using the NMR spectroscopy and molecular dynamic methods.The results showed that five analogs formed a "linear" conformation instead of a turn.Analogs with higher activity maintained a characteristic "linear" conformation,the distance between C?(Xaa2)and C?(Leu5)was longer than 8 A.In summary,four series of AST analogs were designed and synthesized.Their structure-activity relationships were subsequently analyzed.Ten novel analogs showed higher in vitro activity than the lead K24 or H17 whereas the in vivo activity of L6,M11,N13 and P5 were identified by injection and topical treatment.Furthermore,the activity of AST analogs in activation of Dippu-AstR and in vitro fungicidal activity were reported for the first time.P5 was discovered as a new IGR which can be used for the cockroach control.