Discovering allatostatin type-C receptor specific agonists

Abstract

Currently, there is no pesticide available for the selective control of the pine processionary moth (Thaumetopoea pityocampa-specific), and conventional methods typically rely on mechanical techniques such as pheromone traps or broad-spectrum larvicidal chemicals. As climate change increases the range and dispersion capacity of crop and forest pests, outbreaks of the pine processionary occur with greater frequency and significantly impact forestry and public health. Our study is carried out to provide a T. pityocampa-specific pesticide targeting the Allatostatin Type-C Receptor (AlstR-C). We use a combination of computational biology methods, a cell-based screening assay, and in vivo toxicity and side effect assays to identify, for the first time, a series of AlstR-C ligands suitable for use as T. pityocampa-specific insecticides. We further demonstrate that the novel AlstR-C targeted agonists are specific to lepidopteran larvae, with no harmful effects on coleopteran larvae or adults. Overall, our study represents an important initial advance toward an insect GPCR-targeted next-generation pesticide design. Our approach may apply to other invertebrate GPCRs involved in vital metabolic pathways.

Fig. 1. Workflow of the study.

Schema indicates the research approaches for the next-generation pesticide design. It combines in silico approaches (i.e., virtual screening, MD simulations, and post-MD analyses), in vitro biological assays (i.e., TGFα shedding assay, and Glo-Sensor cAMP assay), and in vivo approaches (i.e., toxicity and side effect analysis).

Fig. 2. Virtual screening studies provide ten molecules for further applications.

Compounds shown in (a–d) and (f–j) are from the GPCR-Targeted library. e is the only compound from the Peptidomimetic library. The Glide docking score (kcal/mol) of each molecule is given below the compound name. Black: carbon atoms, red: oxygen atoms, blue: nitrogen atoms, green: chlorine atoms, gray: sulfur atoms, dark brown: bromine atoms, and light brown: fluorine atoms. Source data are provided as a Source Data file.

Fig. 3. MD simulations show robust contacts between AlstR-C and V029-3547.

a Ribbon and surface representation of V029-3547:AlstR-C complex. V029-3547 is at the orthosteric pocket, located at the receptor’s extracellular site. Ligand is colored blue and represented in ball and stick format. Loop regions in contact with the compound are colored yellow (ECL1), green (ECL2), and purple (ECL3). The orange-colored surface shows the binding pocket of AlstR-C. b Protein-ligand interactions plot shows the contacts throughout the simulations. Stacked bar charts are normalized according to simulation time. Purple: hydrophobic interactions, green: hydrogen bonds, blue: water bridges, and pink: ionic interactions. c Detailed representation of interactions of active site residues and ligand atoms. Interactions that occur more than 15.0% of the simulation time, are shown. Lime color shows hydrophobic contacts, and blue illustrates the water bridges. Green is an indicator of π-π stacking interactions. d Illustration of the interactions and contacts throughout the simulation time. The top panel depicts the total number of contacts, while the bottom panel displays which residues interact with the ligand in each trajectory frame. A deeper shade of orange denotes specific residues that make more than one contact with the ligand. Source data are provided as a Source Data file.

Fig. 4. TGF-α shedding assay reveals agonist properties of the predicted ligands.

a–j AP-TGFα release responses of ligand-treated AlstR-C on the left. (Statistical significance was assessed by a one-tailed Student’s t-test for each molecule P < 0.05 (n = 4)). Real-time molecule treatment effects on cAMP production on the right. Concentration-dependent decreases in RLUs were observed at all treatments (Two-way ANOVA, P < 0.05, Dunnett’s Test, D074-0013: Adjusted P < 0.0001, D074-0034: Adjusted P value 0.0042, J100-0311: Adjusted P < 0.0001, and V029-3547 Adjusted P value 0.0403). Error bars, SEM for four biologically independent replicates from one experiment (n = 4). k AP-TGFα release after Serotonin treatment on 5-HTR4 expressing cells. l The hit molecules were tested on the 5-HTR4 receptor. Molecules did not show a significant difference among each other compared to Serotonin, the natural ligand of the 5-HTR4 (One-way ANOVA, P < 000.1, F = 30.97, n = 4 biologically independent replicates). Source data are provided as a Source Data file.

Fig. 5. The in vivo experiments validate insecticidal properties of candidate molecules and specificity towards T. pityocampa AlstR-C.

a–c Results of one-time application of different doses of compounds on T. pityocampa larvae on days 3, 7, and 14. Each dose application has three independent replicas (n = 10), and error bars represent the standard deviation (SD). d Stack bar chart illustrates the side effects of 1000 ppm of molecules on C. sychophanta larvae (n = 50), adults (n = 50), and L. decemlineata larvae (n = 30). Mortality rates of applied molecules are not significantly different from negative controls (Two-way ANOVA and Tukey test, P > 0.05). Data are representative of three independent experiments. Source data are provided as a Source Data file.

Fig. 6. Probit analysis revealed the lethality concentrations of pesticide candidates compared to the natural ligand.

a Natural ligand of the AlstR-C receptor of T. pityocampa (b–e) Pesticide candidates. f Lethal dose of diflubenzuron on L. decemlineata as a positive control. Lethal concentration values of LC_50-90 with 95% CI (P > 0.05). Slopes were obtained over 14. day mortality values (n = 180). Source data are provided as a Source Data file.

Fig. 7. Effects of the four identified hit compounds, the native peptide AST-C, untreated application medium (PBS with 0.1% BSA), and a non-AST-C targeting control ligand (7119812967) on T. pityocampa larval length and head capsule width.

Left: Changes in larva body length are demonstrated following the 14th day after being treated with pesticide candidates, AST-C, and control ligand 7119812967. P values are given above the bars (n = 20). Right: Effects of treatment with hit compounds, native peptide, and 7119812967 on head capsule width. P values are given above the bars (n = 20). Each n corresponds to an individual larva. Numbers above bars denote P values as determined by one-way ANOVA. Tukey’s test was used for multiple comparisons. Error bars are presented in ±SEM. Source data are provided as a Source Data file.