Comparative topical pheromonotropic activity of insect pyrokinin/PBAN amphiphilic analogs incorporating different fatty and/or cholic acid components

This study presents a comparison of the topical pheromonotropic activity in the tobacco budworm moth of a series of amphiphilic pseudopeptide analogs of the insect pyrokinin/PBAN peptide class incorporating fatty acids of varying chain lengths. While the C16 analog fails to penetrate the moth cuticle, and the C12 only moderately so, shorter chain analogs transmigrate the moth cuticle readily with decreasing cuticle-retention properties. A cholic acid analog topically induces twice the maximal pheromone titer of injected native hormone. From a pest management perspective, these non-aromatic hydrophobic components are expected to be more environmentally benign than benzenoid components previously used in topical insect peptide analogs.     

Design, synthesis and biological activity of peptidomimetic analogs of insect allatostatins

Allatostatins (ASTs) comprise a family of insect neuropeptides isolated from cockroaches and found to inhibit the production of juvenile hormone (JH) by the corpora allata (CA). For this reason, the ASTs can be regarded as possible IGR candidates for pest control. Six peptidomimetic analogs according to the C-terminal pentapeptide of ASTs were prepared by solid-phase organic synthetic methods in an attempt to obtain new simple substitution agents. Assays of inhibition of JH biosynthesis in vitro by corpora allata from the cockroach Diploptera punctata showed that the activity of analog I (IC₅₀: 0.09 μM) was more active than that of the C-terminal pentapeptide (Tyr-Xaa-Phe-Gly-Leu-NH₂, IC₅₀: 0.13 μM) it mimicked and the activity of the analog II (IC₅₀: 0.13 μM) proved roughly equivalent to the C-terminal pentapeptide. The results indicate that a new simple mimicry for Tyr-Xaa-Phe-Gly has been discovered; analog I may be a novel compound candidate for potential IGRs. This study will be useful for the design of new AST analogs for insect management.     

Identification of selective and non-selective, biostable beta-amino acid agonists of recombinant insect kinin receptors from the southern cattle tick Boophilus microplus and mosquito Aedes aegypti

The multifunctional arthropod 'insect kinins' share the evolutionarily conserved C-terminal pentapeptide motif Phe-X1-X2-Trp-Gly-NH2, where X1=His, Asn, Ser, or Tyr and X2=Ser, Pro, or Ala. Eight different analogs of the insect kinin C-terminal pentapeptide active core in which the critical residues Phe 1, Pro3 and Trp 4 are replaced with beta 3-amino acid and/or their beta2-amino acid counterparts were evaluated on recombinant insect kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini) and the dengue vector, the mosquito Aedes aegypti (L.). A number of these analogs previously demonstrated enhanced resistance to degradation by peptidases. Single-replacement analog beta 2 Trp 4 and double-replacement analog [beta 3 Phe 2, beta 3 Pro 3] of the insect kinins proved to be selective agonists for the tick receptor, whereas single-replacement analog beta 3 Pro 3 and double-replacement analog [beta 3 Phe, beta 3 Pro 3] were strong agonists on both mosquito and tick receptors. These biostable analogs represent new tools for arthropod endocrinologists and potential leads in the development of selective, environmentally friendly arthropod pest control agents capable of disrupting insect kinin-regulated processes.     

Active fragments and analogs of the insect neuropeptide leucopyrokinin: structure-function studies

Evaluation of analogs of the blocked insect myotropic neuropeptide leucopyrokinin (LPK) has demonstrated its relative insensitivity to amino acid substitution in the N-terminal in contrast to the C-terminal region. Truncated analogs of LPK without the first, second, and third N-terminal amino acids retain a significant 144%, 59% and 30% of the activity of the parent octapeptide, respectively. The [2-8]LPK analog is the first fragment of an insect neuropeptide to exhibit greater activity than the parent hormone. In contrast, truncated analogs of the insect myotropic, proctolin, exhibit little or no activity. The pentapeptide fragment Phe-Thr-Pro-Arg-Leu-NH2 has been identified as the active core of LPK.     

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.     

Design,synthesis and aphicidal activity of N-terminal modified insect kinin analogs

The insect kinins are a class of multifunctional insect neuropeptides present in a diverse variety of insects. Insect kinin analogs showed multiple bioactivities, especially, the aphicidal activity. To find a biostable and bioactive insecticide candidate with simplified structure, a series of N-terminal modified insect kinin analogs was designed and synthesized based on the lead compound [Aib]-Phe-Phe-[Aib]-Trp-Gly-NH₂. Their aphicidal activity against the soybean aphid Aphis glycines was evaluated. The results showed that all the analogs maintained the aphicidal activity. In particular, the aphicidal activity of the pentapeptide analog X Phe-Phe-[Aib]-Trp-Gly-NH₂ (LC₅₀ = 0.045 mmol/L) was similar to the lead compound (LC₅₀ = 0.048 mmol/L). This indicated that the N-terminal protective group may not play an important role in the activity and the analogs structure could be simplified to pentapeptide analogs while retaining good aphicidal activity. The core pentapeptide analog X can be used as the lead compound for further chemical modifications to discover potential insecticides.     

Chlorinated tyrosine derivatives in insect cuticle

A method for quantitative measurement of 3-monochlorotyrosine and 3,5-dichlorotyrosine in insect cuticles is described, and it is used for determination of their distribution in various cuticular regions in nymphs and adults of the desert locust, Schistocerca gregaria. The two chlorinated tyrosine derivatives were present in all analyzed regions in mature adult locusts, the highest concentrations were found in the sclerotized cuticle of femur and tibia, but significant amounts were also present in the unsclerotized arthrodial membranes. Small amounts of the two amino acids were obtained from pharate, not-yet sclerotized cuticle of adult femur and tibia, the amounts increased rapidly during the first 24 h after ecdysis and more slowly during the next two weeks. Control analyses using stable isotope dilution mass spectrometry have confirmed that the chlorinated tyrosines are not artifacts formed during sample hydrolysis. Mono- and dichlorotyrosine are also present in cuticular samples from other insect species, such as the beetle, Tenebrio molitor, the moth Hyalophora cecropia, the cockroach Blaberus craniifer, and the bug Rhodnius prolixus, but not in the sclerotized puparial cuticle of the blowfly, Calliphora vicina, or in sclerotized ootheca from the cockroach, Periplaneta americana. Cuticular sclerotization and formation of chlorotyrosines occur simultaneously in locust legs; sclerotized cuticles tend to have a higher content of chlorotyrosines than unsclerotized cuticles, but it is concluded that the chlorotyrosines are not just a by-product from the sclerotization process.     

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.     

A brominated-fluorene insect neuropeptide analog exhibits pyrokinin/PBAN-specific toxicity for adult females of the tobacco budworm moth

An analog of the insect pyrokinin/PBAN class of neuropeptides, which features a 2-amino7-bromofluorene attached to the carboxy-terminal bioactive core of the insect pyrokinin/PBAN class of neuropeptides (Phe-Thr-Pro-Arg-Leu-NH(2)), via a succinnic acid linker, was tested in adult H. virescens moths. This analog was found to induce pheromone production when injected into or applied topically to moths. Topical application of as much as 1 nmol of the analog to moths induced production of significant amounts of pheromone for only 1-2 h, whereas injection of 500 pmol induced pheromone production for up to 20 h. All insects died within 24 h after injection of 500 pmol of the analog. Mortality studies indicated that the LD(50) for the analog was 0.7 pmol when injected. A non-pyrokinin/PBAN peptide analog formed by attachment of 2-amino-7-bromofluorene to Ala-Ala-Arg-Ala-Ala-NH(2) (via the succinnic acid linker) did not induce mortality when injected at 1 nmol. Similarly no mortality was found when up to 2 nmol of an analog containing a non-brominated fluorene ring, formed by attachment of 9-fluoreneacetic acid to Phe-Thr-Pro-Arg-Leu-NH(2,) was injected into moths. The data indicated that both the bromine and active core of the pyrokinin neuropeptides (Phe-Thr-Pro-Arg-Leu-NH(2)) were critical for a specific toxic action and suggested that the brominated analog poisoned the moths by interacting with pyrokinin receptors.     

A photoswitchable thioxopeptide bond facilitates the conformation-activity correlation study of insect kinin

Thioxopeptide bond psi[CS-N], a nearly isosteric modification of the native peptide bond, was introduced into insect kinin active core pentapeptide to evaluate the impact of backbone cis/trans photoswitching on bioactivity. The thioxo analog Phe(1)-Tyr(2)-psi[CS-N]-Pro(3)-Trp(4)-Gly(5)-NH(2) (psi[CS-N](2)-kinin), was synthesized by Fmoc solid-phase peptide strategy. The reversible photoswitching property was characterized via spectroscopic methods and HPLC, which showed that the cis conformer increased from 15.7 to 47.7% after 254 nm UV irradiation. A slow thermal reisomerization (t(1/2) = 40 min) permitted us to determine the cockroach hindgut myotropic activity of the thioxopeptide in the photostationary state. The results indicated that the activity increased significantly after UV irradiation and recovered to the ground level after thermal re-equilibration. In the present study, by utilizing the phototriggered isomerization in a specific position of peptide backbone, we revealed that the cis psi[CS-N](2)-kinin conformer is the active conformation when interacting with kinin receptor on cockroach hindgut. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Beta-amino acid analogs of an insect neuropeptide feature potent bioactivity and resistance to peptidase hydrolysis

Insect neuropeptides of the insect kinin class share a common C-terminal pentapeptide sequence F(1)X(1)(2)X(2)(3)W(4)G(5)-NH(2) (X(2)(3) = P, S, A) and regulate such critical physiological processes as water balance and digestive enzyme release. Analogs of the insect kinin class, in which the critical residues of F(1), P(3), and W(4) were replaced with beta(3)-amino acid or their beta(2)-homo-amino acid variants, have been synthesized by the solid phase peptide strategy. The resulting single- and double-replacement analogs were evaluated in an insect diuretic assay and enzyme digestion trials. Analogs modified in the core P(3) position produce a potent and efficacious diuretic response that is not significantly different from that obtained with the endogenous achetakinin peptides. The analogs also demonstrate enhanced resistance to hydrolysis by ACE and NEP, endopeptidases that inactivate the natural insect neuropeptides. This paper describes the first instance of beta-amino acids analogs of an arthropod peptide that demonstrate significant bioactivity and resistance to peptidase degradation.     

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.     

Cell culture techniques for studying insect cuticle

Evidence that biosynthetic pathways critical to the formation of insect cuticle are retained in continuous insect cell lines opens new possibilities for research on the cuticle system. Recent findings indicate that chitin, molting hormone, and catecholamines are all produced by a vesicle cell line derived from embryos of the cockroach Blattella germanica. The chitin that is formed by this cell line is particulate and does not show the characteristic featherlike crystalline structure found in mature cuticle. The molting hormone is produced as ecdysone and is released into the culture medium. The addition of 20-hydroxyecdysone to the cultures increases the production of chitin fourfold. These responses are similar to those found in insect organ cultures.     

Comparative structure-activity analysis of insect kinin core analogs on recombinant kinin receptors from Southern cattle tick Boophilus microplus (Acari: Ixodidae) and mosquito Aedes aegypti (Diptera: Culicidae)

The systematic analysis of structure-activity relationships of insect kinins on two heterologous receptor-expressing systems is described. Previously, kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the dengue vector, the mosquito Aedes aegypti (L.), were functionally and stably expressed in CHO-K1 cells. In order to determine which kinin residues are critical for the peptide-receptor interaction, kinin core analogs were synthesized as an Ala-replacement series of the peptide FFSWGa and tested by a calcium bioluminescence plate assay. The amino acids Phe(1) and Trp(4) were essential for activity of the insect kinins in both receptors. It was confirmed that the pentapeptide kinin core is the minimum sequence required for activity and that the C-terminal amide is also essential. In contrast to the tick receptor, a large increase in efficacy is observed in the mosquito receptor when the C-terminal pentapeptide is N-terminally extended to a hexapeptide. The aminoisobutyric acid (Aib)-containing analog, FF[Aib]WGa, was as active as superagonist FFFSWGa on the mosquito receptor in contrast to the tick receptor where it was statistically more active than FFFSWGa by an order of magnitude. This restricted conformation Aib analog provides information on the conformation associated with the interaction of the insect kinins with these two receptors. Furthermore, the analog FF[Aib]WGa has been previously shown to resist degradation by the peptidases ACE and nephrilysin and represents an important lead in the development of biostable insect kinin analogs that ticks and mosquitoes cannot readily deactivate.     

Aliphatic amino diacid Asu functions as an effective mimic of Tyr(SO3H) in sulfakinins for myotropic and food intake-inhibition activity in insects

The aliphatic amino diacid alpha-aminosuberic acid can function as an effective, stable mimic of the hydrolysis-susceptible Tyr(SO3H) group in sulfakinin neuropeptide analogs for both hindgut contractile activity in cockroach and food intake-inhibition activity in the desert locust. In the analog, the acidic sulfate group is replaced with an acidic carboxyl group. The degree of activity of sulfakinin analogs is correlated with the carboxyl/alpha-carbon distance in the cockroach hindgut contractile assay. The results represent an important step in the design and synthesis of biostable, sulfakinin analogs that could potentially suppress the feeding behavior of destructive insect pests of agricultural importance.     

The life cycle of the insect pathogenic fungus Metarhizium anisopliae in the termite Nasutitermes exitiosus

In in vitro and in vivo studies the mode of penetration from Metarhizium anisopliae through the termite integument is elucidated. Serial sections and haemolymph studies elucidate the infective cycle within the host.Conidia germinate and penetrate the cuticle after a not-obligate formation of one or more appressoria. A penetration plate or hyphal bodies between cuticle layers form the base for the invasion of the body cavity, where the haemolymph distributes the multiplicating hyphal bodies. After the death of the insect due to various fungal toxins, organs and tissues are penetrated. Before the gut is invaded, the fungus breaks through to the outside and grows with a special air mycelium, which forms the conidiophores. From these structures the conidia, which stick together in bunches of chains, develope. These conidia can infect the nest mates.     

Leucokinins, a new family of ion transport stimulators and inhibitors in insect Malpighian tubules

Leucokinins are octapeptides isolated from heads of the cockroach Leucophaea maderae. In the cockroach they increase motility of the isolated hindgut. Surprisingly, synthetic leucokinins have biological activity in a different insect and in a different tissue. In isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti, leucokinins depolarize the transepithelial voltage. This effect on voltage is dependent on extracellular Cl. One leucokinin, LK-8, the effects of which were studied further in isolated Malpighian tubules, was found to inhibit transepithelial fluid secretion at low concentrations (10(-11) M threshold), and to stimulate fluid secretion at high concentrations (3.5 x 10(-9) M threshold). Together, the depolarizing effects on voltage and the stimulation of fluid secretion suggest that leucokinins increase the Cl permeability of the tubule wall thereby increasing the availability of Cl for secretion with Na, K and water. Structure-function comparisons of the seven leucokinins studied suggest that the active region of the octapeptide is segregated to the C-terminal pentapeptide. In view of the known effects of leucokinins on hindgut motility in the cockroach, our finding of effects in mosquito Malpighian tubules suggests that leucokinins may be widely distributed in insects where they may have diverse functions in a variety of organs.     

Effect of pH on Penetration of Naphthaleneacetic Acid and Naphthaleneacetamide Through Isolated Pear Leaf Cuticle

Penetration of naphthaleneacetic acid through enzymatically isolated upper pear (Pyrus communis L. cv. Bartlett) leaf cuticle increased as the donor pH was decreased. Naphthaleneacetamide penetration was not influenced by donor pH. The effect of pH on naphthaleneacetic acid penetration was reversible. Higher receiver (simulated leaf interior) pH favored penetration of naphthaleneacetic acid. Changes in the degree of dissociation, and hence polarity, as controlled by hydrogen ion concentration was the prime factor in the response of naphthaleneacetic acid to pH. At pH values lower than the pK (4.2 for naphthaleneacetic acid), the molecule was primarily undissociated, lipophilic, and penetrated into the cuticle; whereas, at pH values above the pK naphthaleneacetic acid was ionized, hydrophilic, and penetrated the cuticle with difficulty or not at all. Data presented are consistent with the hypothesis that naphthaleneacetic acid and naphthaleneacetamide penetration through the cuticle takes place by diffusion.     

N-terminal modified analogs of HVFLRFamide with inhibitory activity on the locust oviduct

New N-terminal analogs of the peptide HVFLRFamide, the minimum sequence of the insect myosuppressins capable of inhibiting spontaneous and induced contractions of the locust oviduct, were synthesized and tested for biologic activity on locust oviduct. Most active, as judged by the ability to inhibit proctolin-induced contractions of locust oviduct, was (N(alpha)-acetyl)-HVFLRFamide. D-Pro-HVFLRFamide was also highly inhibitory. Interestingly, low doses of the pentapeptide analog (N(alpha)-imidazoleacrylyl)-VFLRFamide inhibited oviduct contractions. This is the first pentapeptide analog shown to inhibit contractions of locust oviduct, and this result indicates that the alpha-amino group of His is not absolutely required for inhibitory activity. In all cases when His was replaced by a D-amino acid, the analogs were stimulatory, resulting in an increase in basal tonus of the locust oviduct. The results provide further insight into the structural features of the HVFLRFamide molecule that are required for inhibitory activity on locust oviduct muscle.