Identification of a Novel Inhibitor of Cimex lectularius Acetylcholinesterase.

Acetylcholinesterase (AChE) stands as a primary target of commercial insecticides, notably organophosphates and carbamates. Despite their widespread use in agricultural and indoor pest control, concerns over their high toxicity and the emergence of resistance have restricted their efficacy. In this study, we conducted high-throughput virtual screening against both wild-type (WT) and resistant Cimex lectularius AChE utilizing a library encompassing 1 270 000 compounds. From this screening, we identified 100 candidate compounds and subsequently assessed their inhibitory effects on purified AChE enzymes. Among these candidates, AE027 emerged as a potent inhibitor against both WT and resistant AChE, exhibiting IC50 values of 10 and 43 µM, respectively. Moreover, the binding of AE027 significantly stabilized AChE, elevating its melting temperature by approximately 7 °C. Through molecular docking and molecular dynamics simulation, we delineated the binding mode of AE027, revealing its interaction with a site adjacent to the catalytic center, which is distinct from known inhibitors, with differing poses observed between WT and resistant AChE. Notably, the resistance mutation F348Y, positioned at a site directly interfacing with AE027, impedes ligand binding through steric hindrance. Furthermore, we evaluated the toxicity and pharmacokinetic properties of AE027 utilizing bioinformatics tools. These findings lay a crucial foundation for the development of a novel generation of insecticides that can combat both WT and resistant pest populations effectively and safely.

Functional Characterization of Double Mutations T929I/K1774N in the Voltage-Gated Sodium Channel of Megalurothrips usitatus (Bagnall) Related to Pyrethroid Resistance.

Megalurothrips usitatus (Bagnall), the main pest on legume vegetables, is controlled by pyrethroids in the field. Field strains of M. usitatus resistant to pyrethroids were collected from three areas in Hainan Province (Haikou, Ledong, and Sanya City), and two mutations, T929I and K1774N, were detected in the voltage-gated sodium channel. In this study, the sodium channel in M. usitatus was first subcloned and successfully expressed in Xenopus oocytes. The single mutation (T929I or K1774N) and double mutation (T929I/K1774N) shifted the voltage dependence of activation in the hyperpolarization direction. The three mutants all reduced the amplitude of tail currents induced by type I (permethrin and bifenthrin) and type II (deltamethrin and λ-cyhalothrin) pyrethroids. Homology modeling analysis of these two mutations shows that they may change the local hydrophobicity and positive charge of the sodium channel. Our data can be used to reveal the causes of the resistance of M. usitatus to pyrethroids and provide guidance for the comprehensive control of M. usitatus in the future.

CRISPR/Cas9-Mediated Knockout of the PxJHBP Gene Resulted in Increased Susceptibility to Bt Cry1Ac Protoxin and Reduced Lifespan and Spawning Rates in Plutella xylostella.

Juvenile hormone binding protein (JHBP) is a key regulator of JH signaling, and crosstalk between JH and 20-hydroxyecdysone (20E) can activate and fine-tune the mitogen-activated protein kinase cascade, leading to resistance to insecticidal proteins from Bacillis thuringiensis (Bt). However, the involvement of JHBP in the Bt Cry1Ac resistance of Plutella xylostella remains unclear. Here, we cloned a full-length cDNA encoding JHBP, and quantitative real-time PCR (qPCR) analysis showed that the expression of the PxJHBP gene in the midgut of the Cry1Ac-susceptible strain was significantly higher than that of the Cry1Ac-resistant strain. Furthermore, CRISPR/Cas9-mediated knockout of the PxJHBP gene significantly increased Cry1Ac susceptibility, resulting in a significantly shorter lifespan and reduced fertility. These results demonstrate that PxJHBP plays a critical role in the resistance to Cry1Ac protoxin and in the regulation of physiological metabolic processes associated with reproduction in adult females, providing valuable insights to improve management strategies of P. xylostella.

Natural product guvermectin inhibits guanosine 5'-monophosphate synthetase and confers broad-spectrum antibacterial activity.

Bacterial diseases caused substantial yield losses worldwide, with the rise of antibiotic resistance, there is a critical need for alternative antibacterial compounds. Natural products (NPs) from microorganisms have emerged as promising candidates due to their potential as cost-effective and environmentally friendly bactericides. However, the precise mechanisms underlying the antibacterial activity of many NPs, including Guvermectin (GV), remain poorly understood. Here, we sought to explore how GV interacts with Guanosine 5'-monophosphate synthetase (GMPs), an enzyme crucial in bacterial guanine synthesis. We employed a combination of biochemical and genetic approaches, enzyme activity assays, site-directed mutagenesis, bio-layer interferometry, and molecular docking assays to assess GV's antibacterial activity and its mechanism targeting GMPs. The results showed that GV effectively inhibits GMPs, disrupting bacterial guanine synthesis. This was confirmed through drug-resistant assays and direct enzyme inhibition studies. Bio-layer interferometry assays demonstrated specific binding of GV to GMPs, with dependency on Xanthosine 5'-monophosphate. Site-directed mutagenesis identified key residues crucial for the GV-GMP interaction. This study elucidates the antibacterial mechanism of GV, highlighting its potential as a biocontrol agent in agriculture. These findings contribute to the development of novel antibacterial agents and underscore the importance of exploring natural products for agricultural disease management.

Novel Nitrophenyl Substituted Anthranilic Diamide Derivatives: Design, Synthesis, Selectivity, and Antiresistance.

Diamide insecticides have gained popularity due to their high efficacy and low toxicity to nontarget organisms. However, diamide-associated resistance has emerged recently, causing a significant reduction in their potency, thereby hindering sustainable agricultural development. Here, we explored novel diamide insecticide analogs and, using a structure-based approach, rationally designed and synthesized 28 nitrophenyl substituted anthranilic diamides. Most of the compounds showed moderate to good activity against Mythimna separata, Plutella xylostella, and Spodoptera frugiperda. Among them, compounds Ia and Im showed extraordinarily high activity and their mode of action was verified on isolated neurons. Additionally, Im exhibited over 10-fold greater potency than chlorantraniliprole in a HEK293 cell line stably expressing S. frugiperda ryanodine receptors (SfRyRs) containing the resistance mutations, G4891E and I4734M. The binding modes of Im in the SfRyRs were predicted using in silico molecular docking analysis. Our novel nitrophenyl substituted anthranilic diamide derivatives provide valuable insights for the design of insecticidal RyR-targeting compounds to effectively control both wild type and diamide insecticide-resistant lepidopteran pests.

A detoxification pathway initiated by a nuclear receptor TcHR96h in Tetranychus cinnabarinus (Boisduval).

Understanding the mechanism of detoxification initiation in arthropods after pesticide exposure is crucial. Although the identity of transcription factors that induce and regulate the expression of detoxification genes in response to pesticides is beginning to emerge, whether transcription factors directly interact with xenobiotics is unclear. The findings of this study revealed that a nuclear hormone receptor, Tetranychus cinnabarinus hormone receptor (HR) TcHR96h, regulates the overexpression of the detoxification gene TcGSTm02, which is involved in cyflumetofen resistance. The nuclear translocation of TcHR96h increased after cyflumetofen exposure, suggesting direct binding with cyflumetofen. The direct binding of TcHR96h and cyflumetofen was supported by several independent proteomic assays that quantify interactions with small molecules. Together, this study proposes a model for the initiation of xenobiotic detoxification in a polyphagous agricultural pest. These insights not only provide a better understanding of the mechanisms of xenobiotic detoxification and metabolism in arthropods, but also are crucial in understanding adaptation in polyphagous herbivores.

Characterization of Six Diamide Insecticides on Ryanodine Receptor: Resistance and Species Selectivity.

Ryanodine receptor (RyR) has been used as an insecticide target to control many destructive agricultural pests. The effectiveness of these insecticides has been limited by the spread of resistance mutations identified in pest RyRs, but the detailed molecular impacts of the individual mutations on the activity of different diamide compounds have not been fully explored. We created five HEK293 cell lines stably expressing wild type rabbit RyR1, wild type Spodoptera frugiperda RyR (Sf RyR), or Sf RyR carrying different resistance mutations, including G4891E, G4891E/I4734M, and Y4867F, respectively. R-CEPIA1er, a genetically encoded fluorescent protein, was also introduced in these cell lines to report the Ca2+ concentration in the endoplasmic reticulum. We systematically characterized the activities of six commercial diamide insecticides against different RyRs using the time-lapse fluorescence assay. Among them, cyantraniliprole (CYAN) displayed the highest activity against all three resistant Sf RyRs. The good performance of CYAN was confirmed by the toxicity assay using gene-edited Drosophila expressing the mutant RyRs, in which CYAN showed the lowest LD50 value for the double resistant mutant. In addition, we compared their acitivty between mammalian and insect RyRs and found that flubendiamide has the best insect-selectivity. The mechanism of the anti-resistance property and selectivity of the compounds was proposed based on the structural models generated by homology modeling and molecular docking. Our findings provide insights into the mechanism of insect resistance and guidance for developing effective RyR agonists that can selectively target resistant pests.

Development of an efficient insecticide substrate and inhibitor screening system of insect P450s using fission yeast.

Metabolic resistance is one of the most frequent mechanisms of insecticide resistance, characterized by an increased expression of several important enzymes and transporters, especially cytochrome P450s (CYPs). Due to the large number of P450s in pests, determining the precise relationship between these enzymes and the insecticide substrates is a challenge. Herein, we developed a luminescence-based screening system for efficient identification of insecticide substrates and insect P450 inhibitors. We recombinantly expressed Bemisia tabaci CYP6CM1vQ (Bt CYP6CM1vQ) in the fission yeast Schizosaccharomyces pombe and subsequently permeabilized the yeast cells to convert them into "enzyme bags". We exploited these enzyme bags to screen the activity of twelve luciferin substrates and identified Luciferin-FEE as the optimal competing probe that was further used to characterize the metabolism of eight candidate commercial insecticides. Among them, Bt CYP6CM1vQ exhibited notable activity against pymetrozine and imidacloprid. Their binding modes were predicted by homology modeling and molecular docking, revealing the mechanisms of the metabolism. We also tested the inhibitory effect of eight known P450 inhibitors using our system and identified letrozole and 1-benzylimidazole as showing significant activity against Bt CYP6CM1vQ, with IC50 values of 23.74 µM and 1.30 µM, respectively. Their potential to be developed as an insecticide synergist was further proven by an in vitro toxicity assay using imidacloprid-resistant Bemisia tabaci. Overall, our luciferin-based enzyme bag method is capable of providing a robust and efficient screening of insect P450 substrates and, more importantly, inhibitors to overcome the resistance.

Clip-SP1 cleavage activates downstream prophenoloxidase activating protease (PAP) in Plutella xylostella.

Melanization is a component of the humoral immune defense of insects and is induced by serine protease-mediated phenoloxidase (PO) catalysis. Prophenoloxidase (PPO) in the midgut of Plutella xylostella is activated by the CLIP domain serine protease (clip-SP) in response to Bacillus thuringiensis (Bt) infection, but the detailed signaling cascade following this activation is unknown. Here, we report that activation of clip-SP enhances PO activity in the P. xylostella midgut by cleaving three downstream PPO-activating proteases (PAPs). First, the expression level of clip-SP1 was increased in the midgut after Bt8010 infection of P. xylostella. Then, purified recombinant clip-SP1 was able to activate three PAPs - PAPa, PAPb and PAP3 - which in turn enhanced their PO activity in the hemolymph. Furthermore, clip-SP1 showed a dominant effect on PO activity compared to the individual PAPs. Our results indicate that Bt infection induces the expression of clip-SP1, which is upstream of a signaling cascade, to efficiently activate PO catalysis and mediate melanization in the midgut of P. xylostella. And it provides a basis for studying the complex PPO regulatory system in the midgut during Bt infection.

Enhanced resistance to Bacillus thuringiensis Cry1Ac toxin mediated by the activation of prophenoloxidase in a cosmopolitan pest.

Plutella xylostella has evolved resistance to Bacillus thuringiensis Cry1Ac toxin over a long evolutionary period. Enhanced immune response is an important factor in insect resistance to a variety of insecticides, and whether phenoloxidase (PO), an immune protein, is involved in resistance to Cry1Ac toxin in P. xylostella remains unclear. Here, spatial and temporal expression patterns showed that prophenoloxidase (PxPPO1 and PxPPO2) in the Cry1S1000-resistant strain was more highly expressed in eggs, 4th instar, head, and hemolymph than those in G88-susceptible strain. The results of PO activity analysis showed that after treatment with Cry1Ac toxin PO activity was about 3 times higher than that before treatment. Furthermore, knockout of PxPPO1 and PxPPO2 significantly increased the susceptibility to Cry1Ac toxin. These findings were further supported by the knockdown of Clip-SPH2, a negative regulator of PO, which resulted in increased PxPPO1 and PxPPO2 expression and Cry1Ac susceptibility in the Cry1S1000-resistant strain. Finally, the synergistic effect of quercetin showed that larval survival decreased from 100 % to <20 % compared to the control group. This study will provide a theoretical basis for the analysis of immune-related genes (PO) genes involved in the resistance mechanism and pest control of P. xylostella.

Analysis of the Effect of Plutella xylostella Polycalin and ABCC2 Transporter on Cry1Ac Susceptibility by CRISPR/Cas9-Mediated Knockout.

Many insects, including the Plutella xylostella (L.), have developed varying degrees of resistance to many insecticides, including Bacillus thuringiensis (Bt) toxins, the bioinsecticides derived from Bt. The polycalin protein is one of the potential receptors for Bt toxins, and previous studies have confirmed that the Cry1Ac toxin can bind to the polycalin protein of P. xylostella, but whether polycalin is associated with the resistance of Bt toxins remains controversial. In this study, we compared the midgut of larvae from Cry1Ac-susceptible and -resistant strains, and found that the expression of the Pxpolycalin gene was largely reduced in the midgut of the resistant strains. Moreover, the spatial and temporal expression patterns of Pxpolycalin showed that it was mainly expressed in the larval stage and midgut tissue. However, genetic linkage experiments showed that the Pxpolycalin gene and its transcript level were not linked to Cry1Ac resistance, whereas both the PxABCC2 gene and its transcript levels were linked to Cry1Ac resistance. The larvae fed on a diet containing the Cry1Ac toxin showed no significant change in the expression of the Pxpolycalin gene in a short term. Furthermore, the knockout of polycalin and ATP-binding cassette transporter subfamily C2 (ABCC2) genes separately by CRISPR/Cas9 technology resulted in resistance to decreased susceptibility to Cry1Ac toxin. Our results provide new insights into the potential role of polycalin and ABCC2 proteins in Cry1Ac resistance and the mechanism underlying the resistance of insects to Bt toxins.

Expression and purification of snustorr snarlik protein from Plutella xylostella.

Snustorr snarlik (Snsl) is a type of extracellular protein essential for insect cuticle formation and insect survival, but is absent in mammals, making it a potential selective target for pest control. Here, we successfully expressed and purified the Snsl protein of Plutella xylostella in Escherichia coli. Two truncated forms of Snsl protein, Snsl 16-119 and Snsl 16-159, were expressed as a maltose-binding protein (MBP) fusion protein and purified to a purity above 90% after a five-step purification protocol. Snsl 16-119, forming stable monomer in solution, was crystallized, and the crystal was diffracted to a resolution of ∼10 Å. Snsl 16-159, forming an equilibrium between monomer and octamer in solution, was shown to form rod-shaped particles on negative staining electron-microscopy images. Our results lay a foundation for the determination of the structure of Snsl, which would improve our understanding of the molecular mechanism of cuticle formation and related pesticide resistance and provide a template for structure-based insecticide design.

Molecular Basis for the Selectivity of the Succinate Dehydrogenase Inhibitor Cyflumetofen between Pest and Predatory Mites.

Acaricides that act as inhibitors of the mitochondrial succinate dehydrogenase (SDHIs) provide excellent control of phytophagous mites but display limited toxicity to predatory mites and other beneficial organisms. However, the molecular mechanism of selectivity is not fully understood. Here, we first confirm that SDHI acaricides are over 10,000-fold more toxic to spider mites than predatory mites. Next, we show that differential penetration, pro-acaricide activation, or metabolism are most likely not the main reason for this selectivity. In contrast, the inhibition of AB-1 on the SDH target is approximately 200-fold more potent in spider mites compared to predatory mites, revealing strong target-site selectivity. Strikingly, a key motif associated with differential binding was identified and validated by gene editing in Drosophila. Our findings contribute to understanding the selectivity of SDHIs, which can be used for the rational design of selective acaricides in support of an integrated pest management.

Functional Characterization of Knockdown Resistance Mutation L1014S in the German Cockroach, Blattella germanica (Linnaeus).

The effectiveness of pyrethroid insecticides is seriously threatened by knockdown resistance (kdr), which is induced in insects by inherited single-nucleotide polymorphisms in the voltage-gated sodium channel (VGSC) gene. VGSC's L1014F substitution results in the classic kdr mutation, which is found in many pest species. Other substitutions of the L1014 locus, such as L1014S, L1014C, L1014W, and L1014H, were also reported. In 2022, a new amino acid substitute L1014S of Blattella germanica was first discovered in China. We modified the BgNav1-1 sodium channel from cockroaches with the L1014S mutation to study how pyrethroid sensitivity and channel gating were affected in Xenopus oocytes. The L1014S mutation reduced the half-maximal activation voltage (V1/2,act) from -19.0 (wild type) to -15.5 mV while maintaining the voltage dependency of activation. Moreover, the voltage dependence of inactivation in the hyperpolarizing shifts from -48.3 (wild type) to -50.9 mV. However, compared with wild type, the mutation L1014S did not cause a significant shift in the half activation voltage (V1/2,act). Notably, the voltage dependency of activation was unaffected greatly by the L1014S mutation. Tail currents are induced by two types of pyrethroids (1 µM): type I (permethrin, bifenthrin) and type II (deltamethrin, λ-cyhalothrin). All four pyrethroids produced tail currents, and significant differences were found in the percentages of channel modifications between variants and wild types. Further computer modeling showed that the L1014S mutation allosterically modifies pyrethroid binding and action on B. germanica VGSC, with some residues playing a critical role in pyrethroid binding. This study elucidated the pyrethroid resistance mechanism of B. germanica and predicted the residues that may confer the risk of pyrethroid resistance, providing a molecular basis for understanding the resistance mechanisms conferred by mutations at the 1014 site in VGSC.

Calmodulin Modulation of Insect Ryanodine Receptors.

Ryanodine receptor (RyR) is a giant calcium release channel located on the membrane of the endoplasmic reticulum (ER). Here, we report the regulation of RyRs from two major agricultural pests, diamondback moth and fall armyworm, by insect calmodulin (CaM). The recombinantly expressed full-length insect RyR could be pulled down by insect CaM in the presence of Ca2+, but the efficiency is lower compared to rabbit RyR1 and insect RyR with the CaM-binding domain (CaMBD) replaced by rabbit RyR1 sequence. Interestingly, the enhanced binding of CaM in the mutant insect RyR resulted in an increased sensitivity to the diamide insecticide chlorantraniliprole (CHL), suggesting that this CaM-CaMBD interface could be targeted by potential synergists acting as molecular glue. The thermodynamics of the binding between insect CaM and CaMBD was characterized by isothermal titration calorimetry, and the key residues responsible for the insect-specific regulation were identified through mutagenesis studies.

The Potential Role of the Methionine Aminopeptidase Gene PxMetAP1 in a Cosmopolitan Pest for Bacillus thuringiensis Toxin Tolerance.

Methionine aminopeptidases (MetAPs) catalyze the cleavage of the N-terminal initiator methionine (iMet) in new peptide chains and arylamides, which is essential for protein and peptide synthesis. MetAP is differentially expressed in two diamondback moth (DBM; Plutella xylostella) strains: the G88 susceptible strain and the Cry1S1000 strain, which are resistant to the Bt toxin Cry1Ac, implicating that MetAP expression might be associated with Bt resistance. In this study, we identified and cloned a MetAP gene from DBMs, named PxMetAP1, which has a CDS of 1140 bp and encodes a 379 amino acid protein. The relative expression of PxMetAP1 was found to be ~2.2-fold lower in the Cry1S1000 strain compared to that in the G88 strain. PxMetAP1 presents a stage- and tissue-specific expression pattern, with higher levels in the eggs, adults, integument, and fatbody of DBMs. The linkage between PxMetAP1 and Cry1Ac resistance is verified by genetic linkage analysis. The knockout of PxMetAP1 in G88 by CRISPR/Cas9 leads to a ~5.6-fold decrease in sensitivity to the Cry1Ac toxin, further supporting the association between the PxMetAP1 gene and Bt tolerance. Our research sheds light on the role of MetAP genes in the development of Bt tolerance in P. xylostella and enriches the knowledge for the management of such a cosmopolitan pest.

Structural basis for directional chitin biosynthesis.

Chitin, the most abundant aminopolysaccharide in nature, is an extracellular polymer consisting of N-acetylglucosamine (GlcNAc) units1. The key reactions of chitin biosynthesis are catalysed by chitin synthase2-4, a membrane-integrated glycosyltransferase that transfers GlcNAc from UDP-GlcNAc to a growing chitin chain. However, the precise mechanism of this process has yet to be elucidated. Here we report five cryo-electron microscopy structures of a chitin synthase from the devastating soybean root rot pathogenic oomycete Phytophthora sojae (PsChs1). They represent the apo, GlcNAc-bound, nascent chitin oligomer-bound, UDP-bound (post-synthesis) and chitin synthase inhibitor nikkomycin Z-bound states of the enzyme, providing detailed views into the multiple steps of chitin biosynthesis and its competitive inhibition. The structures reveal the chitin synthesis reaction chamber that has the substrate-binding site, the catalytic centre and the entrance to the polymer-translocating channel that allows the product polymer to be discharged. This arrangement reflects consecutive key events in chitin biosynthesis from UDP-GlcNAc binding and polymer elongation to the release of the product. We identified a swinging loop within the chitin-translocating channel, which acts as a 'gate lock' that prevents the substrate from leaving while directing the product polymer into the translocating channel for discharge to the extracellular side of the cell membrane. This work reveals the directional multistep mechanism of chitin biosynthesis and provides a structural basis for inhibition of chitin synthesis.

Structural Insight Into Ryanodine Receptor Channelopathies.

The ryanodine receptors (RyRs) are large cation-selective ligand-gated channels that are expressed in the sarcoplasmic reticulum (SR) membrane. They mediate the controlled release of Ca2+ from SR and play an important role in many cellular processes. The mutations in RyRs are associated with several skeletal muscle and cardiac conditions, including malignant hyperthermia (MH), central core disease (CCD), catecholaminergic polymorphic ventricular tachycardia (CPVT), and arrhythmogenic right ventricular dysplasia (ARVD). Recent breakthroughs in structural biology including cryo-electron microscopy (EM) and X-ray crystallography allowed the determination of a number of near-atomic structures of RyRs, including wildtype and mutant structures as well as the structures in complex with different modulating molecules. This allows us to comprehend the physiological gating and regulatory mechanisms of RyRs and the underlying pathological mechanisms of the disease-causing mutations. In this review, based on the insights gained from the available high-resolution structures of RyRs, we address several questions: 1) what are the gating mechanisms of different RyR isoforms; 2) how RyRs are regulated by multiple channel modulators, including ions, small molecules, and regulatory proteins; 3) how do disease-causing mutations affect the structure and function of RyRs; 4) how can these structural information aid in the diagnosis of the related diseases and the development of pharmacological therapies.