Rational Design, Synthesis, and Aphicidal Activity of Novel Insect Short Neuropeptide Analogs as Potential Aphid Control Agents.

Short neuropeptide F (sNPF) is an insect-specific neuropeptide named for its C-terminal phenylalanine. It consists of 6-19 amino acids with a conserved RLRFa structure, regulating feeding, growth, circadian rhythms, and water-salt balance in insects. Its receptor belongs to GPCR-As and binds sNPF to regulate the insect nervous system. Many research groups are evaluating sNPF for plant protection and pest control. In this study, the natural sNPF from the pea aphid (Acyrthosiphon pisum) was used as a lead compound. Five novel sNPF analogs were designed and synthesized through molecular docking and peptidomimetics, altering the N-terminal amino acid to Ser, Thr, Tyr, Leu, or Gln. Aphid bioassays showed that the analog I-3 (YLRLRFa, LC50 = 1.820 mg/L) was more active than the natural Acypi-sNPF-1 and pymetrozine. The structure-activity relationship analysis indicated that N-terminal tyrosine incorporation, combined with increased ClogP and TPSA, enhanced aphidicidal activity. Furthermore, Toxtree's toxicity predictions suggest a low risk for all compounds, and a toxicity assay conducted on the honeybee (Apis mellifera) for I-3, which exhibits high aphidicidal activity, indicates that I-3 does not pose a toxicity risk to non-target organisms. Thus, I-3 can be utilized as a selective and environmentally friendly insecticide to manage pea aphids.

Discovery of piperonyl-tethered sulfoximines as novel low bee-toxicity aphicides targeting Amelα1/ratβ2 complex

Targeting the insect nicotinic acetylcholine receptor (nAChR) is recognized as a validated strategy for developing novel insecticides. This study employed the Mpα1/ratβ2 complex to rationally design sulfoximine derivatives with enhanced efficacy against Myzus persicae. Based on the binding cavity and the interaction of the low bee-toxicity insecticide flupyradifurone with Amelα1/ratβ2, lead compound 7g was optimized. Two novel series (I-01∼30 and II-01∼16) were synthesized via bridge-chain shortening or fluorine atom introduction. Thereinto, compound II-08 exhibited better aphicidal activity (LC50 = 53.4 mg/L) and Mpα1/ratβ2 sensibility (EC50 = 0.111 μM) versus I-14 (LC50 = 77.6 mg/L, EC50 = 0.220 μM), which indicated that aphicidal efficiency could be related to the two-electrode voltage clamp responses on Mpα1/ratβ2. Moreover, representative sulfoximines I-09, I-14, 1-15, I-23, and II-08 showed essential nontoxicity (IV, LD50 > 100 μg/bee at 48 h) toward Apis mellifera and lower agonist responses on Amelα1/ratβ2 than sulfoxaflor. Critical hydrogen-bond donors (Lys144 and Ile134) in Mpα1 were identified for structural optimization to improve aphicidal potency. This work provides a solid rational design strategy for developing novel insecticides targeting the Mpα1/ratβ2 complex.

GW182 Controls Drosophila Circadian Behavior and PDF-Receptor Signaling

BackgroundHuo Luo Xiao Ling Dan (HLXLD), a famous Traditional Chinese Medicine (TCM) classical formula, possesses anti-atherosclerosis (AS) activity. However, the underlying molecular mechanisms remain obscure.AimThe network pharmacology approach, molecular docking strategy, and in vitro validation experiment were performed to explore the potential active compounds, key targets, main signaling pathways, and underlying molecular mechanisms of HLXLD in treating AS.MethodsSeveral public databases were used to search for active components and targets of HLXLD, as well as AS-related targets. Crucial bioactive ingredients, potential targets, and signaling pathways were acquired through bioinformatics analysis. Subsequently, the molecular docking strategy and molecular dynamics simulation were carried out to predict the affinity and stability of active compounds and key targets. In vitro cell experiment was performed to verify the findings from bioinformatics analysis.ResultsA total of 108 candidate compounds and 321 predicted target genes were screened. Bioinformatics analysis suggested that quercetin, dihydrotanshinone I, pelargonidin, luteolin, guggulsterone, and β-sitosterol may be the main ingredients. STAT3, HSP90AA1, TP53, and AKT1 could be the key targets. MAPK signaling pathway might play an important role in HLXLD against AS. Molecular docking and molecular dynamics simulation results suggested that the active compounds bound well and stably to their targets. Cell experiments showed that the intracellular accumulation of lipid and increased secretory of TNF-α, IL-1β, and MCP-1 in ox-LDL treated RAW264.7 cells, which can be significantly suppressed by pretreating with dihydrotanshinone I. The up-regulation of STAT3, ERK, JNK, and p38 phosphorylation induced by ox-LDL can be inhibited by pretreating with dihydrotanshinone I.ConclusionOur findings comprehensively demonstrated the active compounds, key targets, main signaling pathways, and underlying molecular mechanisms of HLXLD in treating AS. These findings would provide a scientific basis for the study of the complex mechanisms underlying disease and drug action.

Background Aquilaria sinensis (Lour.) Gilg (ASG) has been used as traditional medicine for centuries. However, the active ingredients from leaves and their anti-inflammatory mechanism are rarely reported. The network pharmacology and molecular docking strategies were applied to explore the potential mechanisms of Benzophenone compounds from the leaves of ASG (BLASG) against inflammation. Methods BLASG-related targets were obtained from the SwissTargetPrediction and PharmMapper databases. Inflammation-associated targets were retrieved from GeneGards, DisGeNET, and CTD databases. Cytoscape software was used to draw a network diagram of BLASG and its corresponding targets. DAVID database was applied for enrichment analyses. A protein-protein interaction (PPI) network was constructed to identify the hub targets of BLASG. Molecular docking analyses were performed by AutoDockTools 1.5.6. Moreover, we used ELISA and qRT-PCR assays to validate the anti-inflammatory effects of BLASG by cell experiments. Results Four BLASG were extracted from ASG, and corresponding 225 potential targets were identified. PPI network analysis indicated that SRC, PIK3R1, AKT1, and other targets were the core therapeutic targets. Enrichment analyses revealed that the effects of BLASG are regulated by targets associated with apoptosis and inflammation-related pathways. In addition, molecular docking revealed that BLASG combined well with PI3K and AKT1. Furthermore, BLASG significantly decreased the inflammatory cytokines levels and down-regulated PIK3R1 and AKT1 gene expression in RAW264.7 cells. Conclusion Our study predicted the potential targets and pathways of BLASG against inflammation, which offered a promising strategy to reveal the therapeutic mechanism of natural active components in the treatment of diseases.

ChuShiWeiLing Decoction (CSWLD) is a famous classical Chinese prescription for the treatment of eczema with desirable effect in clinical practice. It has gradually exerted good curative effects on perianal eczema (PE) in recent years, but its specific mechanism is not elucidated yet. This research explores the underlying pharmacological mechanism of CSWLD in addressing PE through network pharmacology combined with molecular docking strategy. The key chemical compounds and potential target genes of CSWLD were screened by bioinformatics. The major targets of CSWLD were discovered using network modules. Functional annotation of Gene Ontology (GO) was undertaken, as well as pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG). Molecular docking of core protein-ligand interactions was modeled using AutoDock software. Pymol software was used to perform a molecular dynamics simulation for the ideal core protein-ligand that was discovered by molecular docking. A total of 2,853 active compounds and 922 targets of CSWLD were collected. The target with a higher degree was identified through the PPI network, namely TNF, IL6, ALB, STAT3, EGFR, TLR4, CXCL8 and PTPRC. GO and KEGG analyses suggested that CSWLD treatment of PE mainly involves cellular activation, activation of leukocytes, and adhesion among leukocytes. The molecular docking results showed that wogonin, hederagenin and quercetin of CSWLD could bind to IL-6 and TNF, respectively. Our results indicated that the bioactives, potential targets, and molecular mechanism of CSWLD against PE.

This study aimed to investigate the therapeutic effect of black ginseng (BG) on non-alcoholic fatty liver disease (NAFLD) using network pharmacology combined with the molecular docking strategy. The saponin composition of BG was analyzed by liquid chromatography-mass spectrometry (LC/MS) instrument. Then the network pharmacology was applied to explore the potential targets and related mechanisms of BG in the treatment of NAFLD. After screening out key targets, molecular docking was used to predict the binding modes between ginsenoside and target. Finally, a methionine and choline deficiency (MCD) diet-induced NAFLD mice model was established to further confirm the therapeutic effect of BG on NAFLD. Twenty-four ginsenosides were annotated based on the MS and tandem MS information. Ten proteins were screened out as key targets closely related to BG treatment of NAFLD. The molecular docking showed that most of the ginsenosides had good binding affinities with AKT1. The validation experiment revealed that BG administration could reduce serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and improve the MCD diet-induced histological changes in liver tissue. Moreover, BG could upregulate the phosphorylation level of AKT in the liver of NAFLD mice, thereby exerting the therapeutic effect on NAFLD. Further studies on the active ginsenosides as well as their synergistic action on NAFLD will be required to reveal the underlying mechanisms in-depth. This study demonstrates that network pharmacological prediction in conjunction with molecular docking is a viable technique for screening the active chemicals and related targets of BG, which can be applied to other herbal medicines.

Neuropeptides and their receptors play vital roles in controlling the physiology and behavior of animals. Short neuropeptide F (sNPF) signaling regulates several physiological processes in insects such as feeding, locomotion, circadian rhythm and reproduction, among others. Previously, the red imported fire ant (Solenopsis invicta) sNPF receptor (S. invicta sNPFR), a G protein-coupled receptor, was immunolocalized in queen and worker brain and queen ovaries. Differential distribution patterns of S. invicta sNPFR protein in fire ant worker brain were associated both with worker subcastes and with presence or absence of brood in the colony. However, the cognate ligand for this sNPFR has not been characterized and attempts to deorphanize the receptor with sNPF peptides from other insect species which ended in the canonical sequence LRLRFamide, failed. Receptor deorphanization is an important step to understand the neuropeptide receptor downstream signaling cascade. We cloned the full length cDNA of the putative S. invicta sNPF prepropeptide and identified the putative “sNPF” ligand within its sequence. The peptide ends with an amidated Tyr residue whereas in other insect species sNPFs have an amidated Phe or Trp residue at the C-terminus. We stably expressed the HA-tagged S. invicta sNPFR in CHO-K1 cells. Two S. invicta sNPFs differing at their N-terminus were synthesized that equally activated the sNPFR, SLRSALAAGHLRYa (EC50 = 3.2 nM) and SALAAGHLRYa (EC50 = 8.6 nM). Both peptides decreased the intracellular cAMP concentration, indicating signaling through the Gαi-subunit. The receptor was not activated by sNPF peptides from other insect species, honey bee long NPF (NPY) or mammalian PYY. Further, a synthesized peptide otherwise identical to the fire ant sequence but in which the C-terminal amidated amino acid residue ‘Y’ was switched to ‘F’, failed to activate the sNPFR. This discovery will now allow us to investigate the function of sNPY and its cognate receptor in fire ant biology.

Chronic oral lethal and sub‐lethal toxicities of different binary mixtures of pesticides and contaminants in bees (Apis mellifera, Osmia bicornis and Bombus terrestris)

Motivation can critically influence learning and memory. Multiple neural mechanisms regulate motivational states, among which signalling via specific neuropeptides, such as NPY in vertebrates and NPF and its short variant sNPF in invertebrates, plays an essential role. The honey bee (Apis mellifera) is a privileged model for the study of appetitive learning and memory. Bees learn and memorize sensory cues associated with nectar reward while foraging, and their learning is affected by their feeding state. However, the neural underpinnings of their motivational states remain poorly known. Here we focused on the short neuropeptide F (sNPF) and studied if it modulates the acquisition and formation of colour memories. Artificially increasing sNPF levels in partially fed foragers with a reduced motivation to learn colours resulted in significant colour learning and memory above the levels exhibited by starved foragers. Our results thus identify sNPF as a critical component of motivational processes involved in foraging and in the cognitive processes associated with this activity in honey bees.

Honey bee (Apis mellifera) workers emerge from the pupae with no circadian rhythms in behavior or brain clock gene expression but show strong rhythms later in life. This postembryonic development of circadian rhythms is reminiscent of that of infants of humans and other primates but contrasts with most insects, which typically emerge from the pupae with strong circadian rhythms. Very little is known about the internal and external factors regulating the ontogeny of circadian rhythms in bees or in other animals. We tested the hypothesis that the environment during early life influences the later expression of circadian rhythms in locomotor activity in young honey bees. We reared newly emerged bees in various social environments, transferred them to individual cages in constant laboratory conditions, and monitored their locomotor activity. We found that the percentage of rhythmic individuals among bees that experienced the colony environment for their first 48 h of adult life was similar to that of older sister foragers, but their rhythms were weaker. Sister bees isolated individually in the laboratory for the same period were significantly less likely to show circadian rhythms in locomotor activity. Bees experiencing the colony environment for only 24 h, or staying for 48 h with 30 same-age sister bees in the laboratory, were similar to bees individually isolated in the laboratory. By contrast, bees that were caged individually or in groups in single- or double-mesh enclosures inside a field colony were as likely to exhibit circadian rhythms as their sisters that were freely moving in the same colony. These findings suggest that the development of the circadian system in young adult honey bees is faster in the colony than in isolation. Direct contact with the queen, workers, or the brood, contact pheromones, and trophallaxis, which are all important means of communication in honey bees, cannot account for the influence of the colony environment, since they were all withheld from the bees in the double-mesh enclosures. Our results suggest that volatile pheromones, the colony microenvironment, or both influence the ontogeny of circadian rhythms in honey bees.

Effects of carboxyl and acylamino linkers in synthetic derivatives of aphid alarm pheromone (E)-β-farnesene on repellent, binding and aphicidal activity

Summary Bees, like many other organisms, evolved an endogenous circadian clock, which enables them to foresee daily environmental changes and exactly time foraging flights to periods of floral resource availability. The social lifestyle of a honey bee colony has been shown to influence circadian behavior in nurse bees, which do not exhibit rhythmic behavior when they are nursing. On the other hand, forager bees display strong circadian rhythms. Solitary bees, like the mason bee, do not nurse their offspring and do not live in hive communities, but face the same daily environmental changes as honey bees. Besides their lifestyle mason and honey bees differ in their development and life history, because mason bees overwinter after eclosion as adults in their cocoons until they emerge in spring. Honey bees do not undergo diapause and have a relatively short development of a few weeks until they emerge. In my thesis, I present a comparison of the circadian clock of social honey bees (Apis mellifera) and solitary mason bees (Osmia bicornis and Osmia cornuta) on the neuroanatomical level and behavioral output level. I firstly characterized in detail the localization of the circadian clock in the bee brain via the expression pattern of two clock components, namely the clock protein PERIOD (PER) and the neuropeptide Pigment Dispersing Factor (PDF), in the brain of honey bee and mason bee. PER is localized in lateral neuron clusters (which we called lateral neurons 1 and 2: LN1 and LN2) and dorsal neuron clusters (we called dorsal lateral neurons and dorsal neurons: DLN, DN), many glia cells and photoreceptor cells. This expression pattern is similar to the one in other insect species and indicates a common ground plan of clock cells among insects. In the LN2 neuron cluster with cell bodies located in the lateral brain, PER is co-expressed with PDF. These cells build a complex arborization network throughout the brain and provide the perfect structure to convey time information to brain centers, where complex behavior, e.g. sun-compass orientation and time memory, is controlled. The PDF arborizations centralize in a dense network (we named it anterio-lobular PDF hub: ALO) which is located in front of the lobula. In other insects, this fiber center is associated with the medulla (accessory medulla: AME). Few PDF cells build the ALO already in very early larval development and the cell number and complexity of the network grows throughout honey bee development. Thereby, dorsal regions are innervated first by PDF fibers and, in late larval development, the fibers grow laterally to the optic lobe and central brain. The overall expression pattern of PER and PDF are similar in adult social and solitary bees, but I found a few differences in the PDF network density in the posterior protocerebrum and the lamina, which may be associated with evolution of sociality in bees. Secondly, I monitored activity rhythms, for which I developed and established a device to monitor locomotor activity rhythms of individual honey bees with contact to a mini colony in the laboratory. This revealed new aspects of social synchronization and survival of young bees with indirect social contact to the mini colony (no trophalaxis was possible). For mason bees, I established a method to monitor emergence and locomotor activity rhythms and I could show that circadian emergence rhythms are entrainable by daily temperature cycles. Furthermore, I present the first locomotor activity rhythms of solitary bees, which show strong circadian rhythms in their behavior right after emergence. Honey bees needed several days to develop circadian locomotor rhythms in my experiments. I hypothesized that honey bees do not emerge with a fully matured circadian system in the hive, while solitary bees, without the protection of a colony, would need a fully matured circadian clock right away after emergence. Several indices in published work and preliminary studies support my hypothesis and future studies on PDF expression in different developmental stages in solitary bees may provide hard evidence.

Background:Clozapine is superior to all other antipsychotics in treating schizophrenia in terms of its curative efficacy; however, this drug is prescribed only as a last resort in the treatment of schizophrenia, given its potential to induce cardiac arrest. The mechanism of clozapine-induced cardiac arrest remains unclear, so we aimed to elucidate the potential mechanisms of clozapine-induced cardiac arrest using network pharmacology and molecular docking.Methods:We identified and analyzed the overlap between potential cardiac arrest–related target genes and clozapine target genes. We conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. We then constructed a protein–protein interaction (PPI) network and screened the core targets. We used molecular docking to evaluate the binding energy between clozapine and core targets.Results:We identified a total of 2405 target genes related to cardiac arrest and 107 target genes for clozapine. Among these, we found 41 overlapping target genes. The main enriched GO biological processes included the upregulation of the mitogen-activated protein kinase (MAPK) cascade and the adenylate cyclase–activating adrenergic receptor signalling pathway. The KEGG enrichment analysis showed that the neuroactive ligand–receptor interaction and the forkhead box O (FoxO) signalling pathway seemed to be the key signalling pathways involved in clozapine-induced cardiac arrest. The 7 core targets identified in the established PPI network were G-protein–coupled receptor kinase 2, 5-hydroxytryptamine 2A receptor, dopamine D2 receptor, glycogen synthase kinase 3β, cyclin-dependent kinase 2, CREB-binding protein, and signal transducer and activator of transcription 3. The molecular docking results indicated a high affinity between clozapine and all of these core targets.Limitations:The relatively small scope of the predictive and modelling methods, which predominantly comprised network pharmacology and molecular docking strategies, is a limitation of this study.Conclusion:Network pharmacology and molecular docking approaches unveiled target genes for clozapine and potential mechanisms by which it may cause cardiac arrest, including the MAPK cascade, neuroactive ligand–receptor interactions, and the FoxO signalling pathway.