Comparison of rates of penetration through insect cuticle of amphiphylic analogs of insect pyrokinin neuropeptides

Rates of penetration through the cuticle of amphiphylic analogs, synthesized by addition of 6-phenylhexanoic acid or 9-fluoreneacetic acid or 1-pyrenebutyric acid to the amino terminus of the pentapeptide Phe-Thr-Pro-Arg-Leu-amide, were assessed by quantitative analysis using reversed phase liquid chromatography. The analogs effectively penetrated the cuticle of both the adult American cockroach and tobacco budworm moth. However, the amounts of analogs that penetrated the cuticle of the cockroach were significantly lower and the rates of penetration were slower than for moth cuticle. Penetration of the analogs through the cuticle was dependent upon the size of the lipidic attachment to the pentapeptide. The 6-phenylhexanoic acid analog penetrated most rapidly followed by the 9-fluoreneacetic acid analog and the 1-pyrenebutyric acid analog penetrated slowest. All of the analogs exhibited an initial rapid period of penetration lasting 2-3 h followed by the establishment of a steady slow release state which lasted between 9-24 h and was dependent upon both the size and surface area of the aromatic lipidic portion of the analog and species of insect to which the analog was applied. The results confirmed the hypothesis that the insect cuticle could be employed as a slow release device for delivery of analogs of insect neuropeptides.     

An amphiphilic, PK/PBAN analog is a selective pheromonotropic antagonist that penetrates the cuticle of a heliothine insect

A linear pyrokinin (PK)/pheromone biosynthesis activating neuropeptide (PBAN) antagonist lead (RYF[dF]PRLa) was structurally modified to impart amphiphilic properties to enhance its ability to transmigrate the hydrophobic cuticle of noctuid moth species and yet retain aqueous solubility in the hemolymph to reach target PK/PBAN receptors within the internal insect environment. The resulting novel PK/PBAN analog, Hex-Suc-A[dF]PRLa (PPK-AA), was synthesized and evaluated as an antagonist in a pheromonotropic assay in Heliothis peltigera against 4 natural PK/PBAN peptide elicitors (PBAN; pheromonotropin, PT; myotropin, MT; leucopyrokinin, LPK) and in a melanotropic assay in Spodoptera littoralis against 3 natural PK/PBAN peptide elicitors (PBAN, PT, LPK). The analog proved to be a potent and efficacious inhibitor of sex pheromone biosynthesis elicited by PBAN (84% at 100 pmol) and PT (54% at 100 pmol), but not by MT and LPK. PPK-AA is a selective pure antagonist (i.e., does not exhibit any agonistic activity) as it failed to inhibit melanization elicited by any of the natural PK/PBAN peptides. The analog was shown to transmigrate isolated cuticle dissected from adult female Heliothis virescens moths to a high extent of 25-30% (130-150 pmol), representing physiologically significant quantities. PPK-AA represents a significant addition to the arsenal of tools available to arthropod endocrinologists studying the endogenous mechanisms of PK/PBAN regulated processes, and a prototype for the development of environmentally friendly pest management agents capable of disrupting the critical process of reproduction.     

Production and regulation of extracellular proteases from the entomopathogenic fungus Isaria fumosoroseus (Cordycipitaceae; Hypocreales) in the presence of diamondback moth,Plutella xylostella cuticle

Extracellular proteases of entomopathogenic fungi have been implicated as components of the insect infection process. To elucidate their role in the infection process, it is essential to characterize these enzymes. During the current studies synthesis and regulation of extracellular proteases (Pr1 and Pr2) by Isaria fumosoroseus (Cordycipitaceae; Hypocreales) isolate IF28.2 were investigated as a function of carbon source (with special reference to diamondback moth cuticle), temperature and pH. The highest level of Pr1 and Pr2 activity were found in the supernatants from 1% glucose plus 1% diamondback moth cuticle (18.83±1.25 and 12.44±1.36 U/mg per h for Pr1 and Pr2, respectively). Maximum Pr1 production by the depressed mycelia was observed from the supernatants having diamondback moth cuticle as a nutrient source, whereas depressed mycelia showed maximum Pr2 activity from the cultures having chitin as the basic nutrient. The optimum pH for Pr1 and Pr2 activity was 8 while 35°C was the best temperature for protease production.     

Synthesis of (S)-3-(1-hydroxy-p-carboran-12-yl)alanine, a novel hydrophobic tyrosine-mimetic for peptides

A new, p -carborane containing analog of tyrosine, 3-[1-hydroxy-1,12-dicarba-closo-dodecaboran (12)-12-yl]alanine, was prepared from protected 3-[1-hydroxy-1,12-dicarba-closo-dodecaboran (12)-12-yl]propionic acid in five steps using Oppolzer's sultam methodology for asymmetric hydroxyamination as the key step. The tyrosine mimetic can function as a hydrophobic surrogate for tyrosine residues in insect and mammalian neuropeptides to enhance the lipophilicity, and therefore, the cuticle and/or tissue permeability properties of mimetic analogs. As an amino acid, insertion of the mimic is not limited to the N-terminus but can replace a tyrosine residue at any position within a peptide sequence.     

斑蝥素对小菜蛾体壁组织结构的影响

为探究昆虫天然产物斑蝥素(cantharitin,CTD)对鳞翅目昆虫小菜蛾Plutella xylostella(L.)的触杀机制,利用透射电子显微镜观察了该物质处理小菜蛾4龄幼虫体壁组织结构的变化。斑蝥素亚致死剂量0.15μg/头和半致死剂量0.3μg/头点滴处理试虫前胸背板,中毒试虫反映结果类似。处理12h后中毒试虫虫体发黑,陆续死亡。处理12h后,体壁细胞细胞核固缩并且边极化,线粒体扭曲,内脊模糊出现空泡化,线粒体和粗面内质网数量下降;处理24h后,体壁细胞出现的类似处理12h后的症状,且症状更为明显。结果表明CTD对小菜蛾体壁表皮层组织结构无显著破坏作用,但对体壁细胞有明显的致毒作用。     

An active insect kinin analog with 4-aminopyroglutamate, a novel cis-peptide bond, type VI beta-turn motif

The insect kinins are potent diuretic peptides that preferentially form a cis-Pro, type VI beta-turn. An insect kinin analog containing (2S,4S)-4-aminopyroglutamate, a novel cis-peptide bond, type VI beta-turn motif, demonstrates significant activity in the physiological range in a cricket diuretic assay. This is the first instance of a 4-aminopyroglutamate analog of a peptide with a preference for a type VI turn that demonstrates significant bioactivity. The results provide further confirmatory evidence for the active conformation of the insect kinins, and a new scaffold with which to design biostable, peptidomimetic analogs capable of disrupting critical insect kinin-regulated processes in insects.     

cis-peptide bond mimetic tetrazole analogs of the insect kinins identify the active conformation

The insect kinin neuropeptides have been implicated in the regulation of water balance, digestive organ contraction, and energy mobilization in a number of insect species. A previous solution conformation study of an active, restricted-conformation cyclic analog, identified two possible turn conformations as the likely active conformation adopted by the insect kinins at the receptor site. These were a cisPro type VI beta-turn over C-terminal pentapeptide core residues 1-4 and a transPro type I-like beta-turn over core residues 2-5, present in a ratio of 60:40. Synthesis and evaluation of the diuretic activity of insect kinin analogs incorporating a tetrazole moiety, which mimics a cis peptide bond, identifies the active conformation as the former. The discovery of a receptor interaction model can lead to the development of potent agonist and antagonist analogs of the insect kinins. Indeed, in this study a tetrazole analog with D stereochemistry has been shown to demonstrate partial antagonism of the diuretic activity of natural insect kinins, providing a lead for more potent and effective antagonists of this critical neuropeptide family. The future development of mimetic agonists and antagonists of insect kinin neuropeptides will provide important tools to neuroendocrinologists studying the mechanisms by which they operate and to researchers developing new, environmentally friendly pest insect control strategies.     

Fungal infection dynamics in response to temperature in the lepidopteran insect Galleria mellonella

This study examines how the dynamics of fungus–insect interactions can be modulated by temperature. The wax moth, Galleria mellonella, is a well‐studied and important model insect whose larvae in the wild develop optimally at around 34 °C in beehives. However, surprisingly little research on wax moths has been conducted at relevant temperatures. In this study, the entomopathogenic fungus Metarhizium robertsii inflicted rapid and substantial mortality on wax moth larvae maintained at a constant temperature of 24 °C, but at 34 °C a 10 fold higher dose was required to achieve an equivalent mortality. The cooler temperature favored fungal pathogenicity, with condial adhesion to the cuticle, germination and hemocoel invasion all significantly enhanced at 24 °C, compared with 34 °C. The wax moth larvae immune responses altered with the temperature, and with the infective dose of the fungus. Enzyme‐based immune defenses (lysozyme and phenoloxidase) exhibited enhanced activity at the warmer temperature. A dramatic upregulation in the basal expression of galiomicin and gallerimycin was triggered by cooling, and this was augmented in the presence of the fungus. Profiling of the predominant insect epicuticular fatty acids revealed a 4–7 fold increase in palmetic, oleic and linoleic acids in larvae maintained at 24 °C compared with those at 34 °C, but these failed to exert fungistatic effects on topically applied fungus. This study demonstrates the importance of choosing environmental conditions relevant to the habitat of the insect host when determining the dynamics and outcome of insect/fungus interactions, and has particular significance for the application of entomopathogens as biocontrol agents.     

Enhanced in vivo activity of peptidase-resistant analogs of the insect kinin neuropeptide family

The diuretic/myotropic insect kinin neuropeptides, which share the common C-terminal pentapeptide core FX(1)X(2)WG-NH(2), reveal primary (X(2)-W) and secondary (N-terminal to F) sites of susceptibility to peptidases bound to corn earworm (H. zea) Malpighian tubule tissue. Analogs designed to enhance resistance to tissue-bound peptidases, and pure insect neprilysin and ACE, demonstrate markedly enhanced in vivo activity in a weight gain inhibition assay in H. zea, and strong in vivo diuretic activity in the housefly (M. domestica). The peptidase-resistant insect kinin analog pQK(pQ)FF[Aib]WG-NH(2) demonstrates a longer internal residence time in the housefly than the native muscakinin (MK), and despite a difference of over 4 orders of magnitude in an in vitro Malpighian tubule fluid secretion assay, is equipotent with MK in an in vivo housefly diuretic assay. Aminohexanoic acid (Ahx) is shown to function as a surrogate for N-terminal Lys, while at the same time providing enhanced resistance to aminopeptidase attack. Peptidaese-resistant insect kinin analogs demonstrate enhanced inhibition of weight gain in larvae of the agriculturally destructive corn earworm moth. Potent peptidase resistant analogs of the insect kinins, coupled with an increased understanding of related regulatory factors, offer promise in the development of new, environmentally friendly pest insect control measures.     

Role of extracellular domains in PBAN/pyrokinin GPCRs from insects using chimera receptors

Pheromone biosynthesis-activating neuropeptide (PBAN) is a peptide used by a variety of moths to regulate pheromone production. Pyrokinins are peptides that activate muscle contraction in a variety of insects. These peptides have a common FXPRLamide C-terminal ending that is required for activity. Receptors have been identified from a moth and Drosophila as belonging to the rhodopsin family of G-protein coupled receptors (GPCRs) with sequence similarity to neuromedin U receptors from vertebrates. No insect GPCR has been characterized with regard to role of extracellular domains required for peptide binding and receptor activation. To begin characterizing these GPCRs we created chimera receptors using a PBAN-receptor from a moth and pyrokinin-receptors from Drosophila where extracellular domains were swapped. The N-terminal of the moth GPCR has two N-glycosylation sites that when replaced with glutamines, activity was reduced but not absent, indicating these sites contribute to receptor stability. Activity was greatly reduced by replacing the 2nd extracellular loop that has an N-glycosylation site and a cysteine that can form a disulfide bridge with a cysteine at the beginning of the 3rd transmembrane domain. Exchange of the 3rd extracellular loop between the moth and Drosophila receptor resulted in differential activation by PBAN or a diapause hormone peptide. This result indicates that the 3rd extracellular loop is directly involved in peptide ligand recognition. Results are discussed in context of the structural features of insect GPCRs that are required for receptor activation as compared to vertebrate receptors.     

Hemolymph and tissue-bound peptidase-resistant analogs of the insect allatostatins

While neuropeptides of the allatostatin family inhibit in vitro production of juvenile hormone, which modulates aspects of development and reproduction in the cockroach, Diploptera punctata, they are susceptible to inactivation by peptidases in the hemolymph, gut, and bound to internal tissues. Patterns of peptidase cleavage were investigated in two allatostatin analogs in which sterically bulky components were incorporated into the active core region to block peptidase attack. The results were used to design and synthesize the first pseudopeptide analog of an insect neuropeptide resistant to degradation by both hemolymph and tissue-bound peptidases. This pseudotetrapeptide allatostatin mimetic analog represents a valuable tool to neuroendocrinologists studying mechanisms by which the natural peptides operate and the physiological consequences of challenging an insect with an allatostatin that is not readily degraded via peptidase enzymes. Disruption of critical physiological processes modulated by neuropeptides such as the allatostatins via peptidase-resistant mimetic analogs could form the basis for novel pest insect management strategies in the future.     

Synthesis, cardiostimulatory, and cardioinhibitory effects of selected insect peptides on Tenebrio molitor

The subject of these studies was a search for proctolin antagonists among peptides originating from insect species because the proctolin antagonists constantly pose a problem. During these studies we performed the synthesis of the following peptides: a native decapeptide from Manduca sexta Mas-MT-I and its 11 analogs with shortened sequences at the N-end as well as a growth suppressor, a pentapeptide isolated from Antheraea yamamai, Any-GS and its 10 analogs, modified at position 1 and with a shortened peptide chain.Biological effects were evaluated by the cardiotropic test on the semi-isolated heart of the insect species Tenebrio molitor. Mas-MT-I and six analogs stimulate the heartbeat frequency, especially [6-10]-Mas-MT-I, whereas the [4-10]-Mas-MT-I analog shows a strong inhibition of the heartbeat frequency, if insect. The Any-GS and the analogs [Gln(1)]- and [Gly(1)]-Any-GS also show a strong cardioinhibitory effect.     

Sphingomyelin analogs with branched N-acyl chains: The position of branching dramatically affects acyl chain order and sterol interactions in bilayer membranes

Sphingolipids have been found to have single methyl branchings both in their long-chain base and in their N-linked acyl chains. In this study we determined how methyl-branching in the N-linked acyl chain of sphingomyelin (SM) affected their membrane properties. SM analogs with a single methyl-branching at carbon 15 (of a 17:0 acyl chain; anteiso) had a lower gel-liquid transition temperature as compared to an iso-branched SM analog. Phytanoyl SM (methyls at carbons 3, 7, 11 and 15) as well as a SM analog with a methyl on carbon 10 in a hexadecanoyl chain failed to show a gel-liquid transition above 10 °C. Only the two distally branched SM analogs (iso and anteiso) formed ordered domains with cholesterol in a 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer. However, domains formed by the branched SM analogs appeared to contain less sterol when compared to palmitoyl SM (PSM) as the saturated phospholipid. Sterol-enriched domains formed by the anteiso SM analog were also less stable against temperature than domains formed by PSM. Both the 10-methyl and phytanoyl SM analogs failed to form sterol-enriched domains in the POPC bilayer. Acyl chain branching weakened SM/sterol interactions markedly when compared to PSM, as also evidenced from the decreased affinity of cholestatrienol to bilayers containing branched SM analogs. Our results show that methyl-branching weakened intermolecular interactions in a position-dependent manner.     

A C-terminal aldehyde insect kinin analog enhances inhibition of weight gain and induces significant mortality in Helicoverpa zea larvae

The first reported examples of C-terminal aldehyde analogs of an insect neuropeptide are described. They are hexapeptide insect kinin analogs Boc-VFFPWG-H and Fmoc-RFFPWG-H. Activity observed for these modified analogs in an in vitro insect diuretic assay confirms that the C-terminal aldehyde group is tolerated by an insect kinin receptor. The two analogs demonstrate greatly enhanced activity over standard C-terminal amide insect kinins in a larval weight gain inhibition assay in the corn earworm Helicoverpa zea. Treatment with Boc-VFFPWG-H led to significant increases in larval mortality at doses of 500pm (45%) and 5nm (67%). Boc-VFFPWG-H represents a lead analog in the development of novel, environmentally friendly pest insect management agents based on the insect kinin class of neuropeptides.     

Conformational aspects and hyperpotent agonists of diapause hormone for termination of pupal diapause in the corn earworm

Diapause hormone (DH) is a peptide well known to induce embryonic diapause in the commercial silkmoth Bombyx mori. More recently, this same neuropeptide was reported to break diapause in pupae of the agriculturally important Heliothis/Helicoverpa complex. In this study we examine the efficacy and potency of a select group of structural analogs of the native hormone in Helicoverpa zea and report the structures of several analogs that are considerably more potent than DH in breaking diapause. Among the most potent analogs (PK-Etz, PK-2Abf, 901) were those with structural components that enhance resistance to peptidases that degrade and inactivate the native peptide in vivo, which may account, at least in part, for the observed increase in potency for these analogs. Analog 901 was previously demonstrated to both enhance biostablility and bioavailability properties in adult heliothines and thus may be a potential candidate for topical application as a diapause-terminating agent. The significant activity observed for two restricted conformation analogs is consistent with an active conformation for diapause hormone that features a transPro within a type I beta-turn in the C-terminal region. DH is also known to successfully break diapause only within a fairly narrow temperature range. While DH is effective at 21 degrees C, it is not effective at 18 degrees C. Likewise, the analogs were effective at 21 degrees C but not at 18 degrees C. By contrast, 20-hydroxyecdysone, a steroid hormone that is also capable of breaking diapause is effective at both temperatures, thus suggesting that DH and the ecdysteroids act through different mechanisms to terminate diapause.     

The MrCYP52 cytochrome P450 monoxygenase gene of Metarhizium robertsii is important for utilizing insect epicuticular hydrocarbons

Fungal pathogens of plants and insects infect their hosts by direct penetration of the cuticle. Plant and insect cuticles are covered by a hydrocarbon-rich waxy outer layer that represents the first barrier against infection. However, the fungal genes that underlie insect waxy layer degradation have received little attention. Here we characterize the single cytochrome P450 monoxygenase family 52 (MrCYP52) gene of the insect pathogen Metarhizium robertsii, and demonstrate that it encodes an enzyme required for efficient utilization of host hydrocarbons. Expressing a green florescent protein gene under control of the MrCYP52 promoter confirmed that MrCYP52 is up regulated on insect cuticle as well as by artificial media containing decane (C10), extracted cuticle hydrocarbons, and to a lesser extent long chain alkanes. Disrupting MrCYP52 resulted in reduced growth on epicuticular hydrocarbons and delayed developmental processes on insect cuticle, including germination and production of appressoria (infection structures). Extraction of alkanes from cuticle prevented induction of MrCYP52 and reduced growth. Insect bioassays against caterpillars (Galleria mellonella) confirmed that disruption of MrCYP52 significantly reduces virulence. However, MrCYP52 was dispensable for normal germination and appressorial formation in vitro when the fungus was supplied with nitrogenous nutrients. We conclude therefore that MrCYP52 mediates degradation of epicuticular hydrocarbons and these are an important nutrient source, but not a source of chemical signals that trigger infection processes.     

Side chain to side chain cyclization of an enkephalin analog results in loss of opioid receptor selectivity

The cyclic enkephalin analog H-Tyr-D-Lys-Gly-Phe-Glu-NH2 (I) and the structurally related open chain analogs H-Tyr-D-Nle-Gly-Phe-Gln-NH2 (II) and H-Tyr-D-Lys(For)-Gly-Phe-Abu-NH2 (III) were tested in mu and delta opioid receptor-representative binding assays and bioassays. Whereas both linear analogs showed a pronounced preference for mu receptors over delta receptors, the conformationally restricted cyclic peptide I was found to be unselective. This finding represents the first reported example of a peptide cyclization resulting in a loss of receptor selectivity. From this and earlier studies, it was concluded that the receptor selectivity of cyclized peptide analogs relative to that of their linear correlates may depend on the size and relative rigidity of their ring structures.     

Ethyl (E)-3,5-ethano-7,11-dimethyl-2,4-dodecadienoate,A new insect growth regulator with potent juvenile hormone activity

A number of analogs of ethyl (2E,4E)-3,7,11-trimethyl-2,4-dodecadienoate were prepared and bioassayed for juvenile hormone activity on the yellow-fever mosquito (Aedes aegypti), the greater wax moth (Galleria mellonella), the yellow mealworm (Tenebrio molitor), the house fly (Musca domestica), and the tobacco budworm (Heliothis virescens). The analog ethyl (E)-3,5-ethanol-7,11-dimethyl-2,4-dodecadienoate (VI), containing a cyclopentene ring, showed remarkable potency on the above insect species. Since this compound possesses a fixed 3-s-trans-diene conformation it may provide some insight into the active conformation of bound 2,4-dienoate analogs.     

(+)-2,3-dehydro-10-oxo-alpha-isosparteine in Uresiphita reversalis larvae fed on Cytisus monspessulanus leaves

Quinolizidine alkaloids, found in the leaves of Cytisus monspessulanus L. (Leguminosae), were characterized in the cuticle of larvae of the pyralid moth Uresiphita reversalis (Lepidoptera: Pyralidae) when the latter were fed on this weed. By GC-MS analysis of the methanolic extracts of the cuticle, four quinolizidine alkaloids, N-methylcytisine, cytisine, aphylline and anagyrine, were identified as possible defense substances. In addition, the quinolizidine alkaloid, (+)-2,3-dehydro-10-oxo-alpha-isosparteine was characterized in both the insect and host plant.