| In nature,pests evolve the resistance to insecticides to survive.Resistance mutations on target proteins are the most severe challenge of pest control worldwide.In this study,we focus on the structural and functional study of major insecticides targets,ryanodine receptor（RyR）and voltage-gated sodium channel（Na V）,which provides insight on how to identify new binding sites on traditional targets and how to develop novel insecticides to overcome the resistance crisis.The first project is to develop insecticidal compounds targeting insect RyR N-terminal domain（NTD）.Insect RyR mainly expresses in the muscle and central nervous system,controlling insect motion and vision.Diamide insecticides are widely used to control the lepidopteran and coleopteran pests,and the molecular target is RyR.Due to the extensive use of diamide insecticides,the pests such as diamondback moth（DBM）have generated more than thousand times resistance,reducing the effect of diamide dramatically.The exact binding sites of diamide on RyR and the resistance mechanism still remain elusive.The locations of the resistance mutations imply that the transmembrane domain is the potential binding site.RyR NTD,containing two subdomains（NTD-A,NTD-B）,plays an important role in regulating the channel gating.Comparing the DBM RyR NTD-A（PDB ID:5Y9V）and mammalian RyR NTD-A（PDB ID:2XOA）,β1-β2 chain and loop 4,both located in the interface between NTD-A and NTD-B of insect RyR,showed distinct structural differences and could become insect-specific pocket to develop potential insecticides.The structural model for DBM RyR NTD in open state for virtual screening was constructed by combining DBM NTD-A crystal structure and a homology model constructed for DBM NTD-B.Totally about 3.5 million compounds were screened comparing the results of docking and binding free energies against NTD from DBM and mammalian and eight insect-specific hits were obtained.Among them,ortho-phthalaldehyde（OPA）can activate DBM RyR on the cell-based and insect-based bioassays,and shows good insect-specific properties.The molecular dynamics simulation of NTD-A/OPA complex showed the major interactions and the stability of the complex.OPA was also evaluated by in silico ADME（absorption,distribution,metabolism,and excretion）toxicity assessment and showed good scores in the predicted pharmacokinetic relevant properties.OPA derivatives could become potential green insecticides targeting insect RyR NTD,a distinct site different from the traditional diamide-binding region.It has laid the foundation for further structure-based pesticide design with the aim to overcome the current resistance to diamide insecticides.The second project is to study the regulation of insect voltage-gated sodium channel（Na V）by calmodulin（CaM）with the aim to develop potential green insecticides targeting this interaction.Na Vplays an essential role in the rapid depolarization of excitable cells.Insect para-sodium channel shares similar overall structural organization with mammalian Na V.Na Vαsubunit is composed of four domains（DI-DIV）each containing six segments（S1-S6）.Many insects have evolved knockdown resistance（kdr）or super-kdr mutations to dichlorodiphenyl-trichloroethane（DDT）and pyrethoids,the insecticides targeting Na Vchannels.These mutations are clustered on the S5 or S6 helices of DI-III and have been found in many insect species such as German cockroach（Blattella germanica）,Codling moth（Cydia pomonella）,house fly（Musca domestica）,and African malaria mosquito（Anopheles gambiae）.The proximal portion of Na Vcytosolic C-terminal domain（CTD）is composed of sixαhelices（αI-αVI）withαVI containing an IQ motif that is known to interact with CaM.Interaction of CaM and Na VCTD is essential for controlling fast and long term inactivation.Although the mechanism of regulation of Na Vby CaM in mammals has been investigated extensively,the details of this interaction are still controversial and the counterpart in insect is little known.We expressed and purified insect CaM,Na VCTD,and Na VCTD-CaM proteins.The crystals of Na VCTD-CaM and CaM-IQ complex were obtained and the resolution of Na VCTD-CaM complex is 10（?）in the primary X-ray experiment.The optimization of crystallization condition is undergoing.We also characterized the interaction of CaM and CTD IQ motif using isothermal titration calorimetry（ITC）.In the absence of Ca2+,CaM binds to IQ motif at the ratio of 1:1.The binding of insect CTD IQ motif and CaM is weaker than those in human Na V1.2 and human Na V1.5.In the presence of Ca2+,CaM binds to IQ motif at the ratio of 1:2.The binding in insect is weaker than that in human Na V1.2 but stronger than that in human Na V1.5.These results demonstrate that insect Na Vhas special CaM regulation and can be targeted by potential novel insecticides.The high-resolution crystal structure and binding data will lay a foundation for future green insecticides screening and design. |
