Antifeedant activity and high mortality in the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae) induced by biostable insect kinin analogs.

The insect kinins are multifunctional neuropeptides found in a variety of arthropod species, including the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae). A series of biostable insect kinin analogs based on the shared C-terminal pentapeptide core region were fed in solutions of artificial diet to the pea aphid over a period of 3 days and evaluated for antifeedant and aphicidal activity. The analogs contained either alpha,alpha-disubstituted or beta-amino acids in key positions to enhance resistance to tissue-bound peptidases and retain activity in a number of insect kinin bioassays and/or on expressed receptors. Three of the biostable analogs demonstrated antifeedant activity, with a marked reduction in honeydew formation observed after 1 day, and very high mortality. In contrast, an unmodified, parent insect kinin and two other analogs containing some of the same structural components that promote biostability are inactive. The most active analog, double Aib analog K-Aib-1 ([Aib]FF[Aib]WGa), featured aphicidal activity calculated at an LC(50) of 0.063 nmol/microl (0.048 microg/microl) and an LT(50) of 1.68 days, matching the potency of some commercially available aphicides. The mechanism of this activity has yet to be established. The aphicidal activity of the biostable insect kinin analogs may result from different potential mechanisms as disruption of digestive processes by interfering with gut motility patterns, digestive enzyme release, and/or with fluid cycling in the gut, and also nutrient transport across the gut itself; all processes shown to be regulated by the insect kinins in other insects. However the mechanism(s) is(are) not yet known. The active insect kinin analogs represent potential leads in the development of selective, environmentally friendly pest aphid control agents.

Bioavailability of beta-amino acid and C-terminally derived PK/PBAN analogs.

The ability of linear beta-amino acid substituted peptides (PK-betaA-1: Ac-YFT[beta(3)P]RLa; PK-betaA-2: Ac-Y[beta(3)homoF]TPRLa; PK-betaA-3: Ac-Y[beta(3)F]TPRLa; PK-betaA-4: Ac-[beta(3)F]FT[beta(3)P]RLa) and unsubstituted analogs (Ac-YFTPRLa and YFTPRLa) of the pyrokinin(PK)/pheromone biosynthesis-activating neuropeptide (PBAN) family to penetrate the insect cuticle and exert biological activity (i.e., stimulate sex pheromone biosynthesis), was tested by topical application on Heliothis peltigera moths. The present results clearly indicate that small linear synthetic peptides can penetrate the cuticle very efficiently by contact application and activate their target organ. The time responses of the peptides applied in DDW and DMSO were tested and the activities of topically applied and injected peptides were compared. The results clearly indicate that PK-betaA-4 and PK-betaA-3 exhibited high bioavailability (ability to penetrate through the cuticle and exertion of bioactivity) with the latter showing longer persistence in both solvents than any other analog in the study; indicative that incorporation of a beta-amino acid at the Phe(2) position can enhance longevity in topical PK/PBAN analogs. PK-betaA-4 was significantly more active in DMSO than in DDW, and significantly more active than the parent peptide LPK in DMSO. PK-betaA-1 and PK-betaA-2 exhibited negligible activity. Interestingly, Ac-YFTPRLa was highly potent in both solvents; its activity in DDW did not differ from that of PK-betaA-4 and PK-betaA-3, and was higher than that of LPK. Even the unacylated peptide YFTPRLa was active in both solvents, at a similar level to LPK. Topically applied PK-betaA-4 and Ac-YFTPRLa exhibited significantly higher activity than the injected peptides. PK-betaA-3 and YFTPRLa were equally potent in both routes of administration.

Synthesis, conformational analysis and immunological activity of beta3Phe-substituted Cyclolinopeptide A analogues.

CLA, a natural, highly hydrophobic cyclic nonapeptide with sequence c(Pro(1)-Pro(2)-Phe(3)-Phe(4)-Leu(5)-Ile(6)-Ile(7)-Leu(8)-Val(9)-), isolated from linseed oil, was found to possess a strong immunosuppressive activity comparable, in low doses, with that of CsA, with a mechanism that depends on the inhibition of the interleukin-1 and interleukin-2 action. Structural analysis of CLA and its related compounds has underlined that the presence of the tetrapeptide Pro-Pro-Phe-Phe sequence, the Pro-Pro cis amide bond, and the 'edge-to-face' interaction are possible important features for the immunosuppressive activity of CLA. To evaluate the role and significance of 'edge-to-face' interaction in the process of molecular recognition by receptors, we have synthesised three linear precursors and three cyclic analogues of CLA, in which one or both Phe residues have been replaced by beta(3)Phe residues. A conformational analysis by NMR in CD(3)CN/H(2)O mixture has been carried out on the CLA analogue, in which Phe(3) has been replaced by a betaPhe, to study the influence of the mutation on the three-dimensional structure. All linear and cyclic CLA analogues containing betaPhe have been tested in the humoral and cellular immune response in vivo assays in mice. The peptide activities have been compared with CsA, as a reference drug.

Biostable beta-amino acid PK/PBAN analogs: agonist and antagonist properties.

The pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family plays a significant role in a multifunctional array of important physiological processes in insects. PK/PBAN analogs incorporating beta-amino acids were synthesized and evaluated in a pheromonotropic assay in Heliothis peltigera, a melanotropic assay in Spodoptera littoralis, a pupariation assay in Neobellieria bullata, and a hindgut contractile assay in Leucophaea maderae. Two analogs (PK-betaA-1 and PK-betaA-4) demonstrate greatly enhanced resistance to the peptidases neprilysin and angiotensin converting enzyme that are shown to degrade the natural peptides. Despite the changes to the PK core, analog PK-betaA-4 represents a biostable, non-selective agonist in all four bioassays, essentially matching the potency of a natural PK in pupariation assay. Analog PK-betaA-2 is a potent agonist in the melanotropic assay, demonstrating full efficacy at 1pmol. In some cases, the structural changes imparted to the analogs modify the physiological responses. Analog PK-betaA-3 is a non-selective agonist in all four bioassays. The analog PK-betaA-1 shows greater selectivity than parent PK peptides; it is virtually inactive in the pupariation assay and represents a biostable antagonist in the pheromonotropic and melanotropic assays, without the significant agonism of the parent hexapeptide. These analogs provide new, and in some cases, biostable tools to endocrinologists studying similarities and differences in the mechanisms of the variety of PK/PBAN mediated physiological processes. They also may provide leads in the development of PK/PBAN-based, insect-specific pest management agents.

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.

Biostable agonists that match or exceed activity of native insect kinins on recombinant arthropod GPCRs.

The multifunctional arthropod 'insect kinins' share the evolutionarily conserved C-terminal pentapeptide motif Phe-X(1)-X(2)-Trp-Gly-NH(2), where X(1)=His, Asn, Ser, or Tyr and X(2)=Ser, Pro, or Ala. Insect kinins regulate diuresis in many species of insects. Compounds with similar biological activity could be exploited for the control of arthropod pest populations such as the mosquito Aedes aegypti (L.) and the southern cattle tick Rhipicephalus (Boophilus) microplus (Canestrini), vectors of human and animal pathogens, respectively. Insect kinins, however, are susceptible to fast enzymatic degradation by endogenous peptidases that severely limit their use as tools for pest control or for endocrinological studies. To enhance resistance to peptidases, analogs of the insect kinins incorporating bulky alpha,alpha-disubstituted amino acids in positions adjacent to both primary and secondary peptidase hydrolysis sites were synthesized. In comparison with a control insect kinin, several of these analogs are highly stable to hydrolysis by degradative enzymes ANCE, neprilysin and Leucine aminopeptidase. Six analogs were evaluated by calcium bioluminescence assay on recombinant receptors from mosquito and tick. Four of these analogs either matched or exceeded the potency of the control kinin peptide agonist. One of these was about 5-fold more potent than the control agonist on the tick receptor. This analog was 8-fold more potent than the control agonist on the mosquito receptor, and twice more potent than the endogenous Aedes kinin-II. The analog also demonstrated potent activity in an in vitro Aedes Malpighian tubule fluid secretion assay. Similar comparisons of analog potency cannot be made to tick kinins because no endogenous kinin has yet been identified. These potent, biostable analogs represent ideal new tools for endocrinologists studying arthropod kinin-regulated processes in vivo, particularly for ticks in which their role remains to be established.

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.

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.

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.

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.

A comparison of the pupariation acceleration activity of pyrokinin-like peptides native to the flesh fly: Which peptide represents the primary pupariation factor?

Five native pyrokinin-like peptides (Neb-PK-1, Neb-PK-2, Neb-PVK-1, [L9]Neb-PVK-2, [I9]Neb-PVK-2) identified in the neuropeptidome of the flesh fly Neobellieria bullata were compared for their quantitative and/or qualitative effects on puparium formation (pupariation). In a standard pupariation bioassay, both Neb-PVK-1 and [I9]Neb-PVK-2 proved inactive, whereas [L9]Neb-PVK-2 demonstrated only weak activity. In contrast, both Neb-PK-1 and Neb-PK-2 demonstrated potent threshold doses, with Neb-PK-2 about 10-fold more active than Neb-PK-1. Analysis of neuromuscular activity during pupariation using a tensiometric technique demonstrates that the two native Neb-PKs accelerate the onset of immobilization and cuticular shrinkage more than motor programs associated with retraction of the anterior segments and longitudinal body contraction. It was further determined that the sensitivity of various components of the pupariation process to these peptides decreases in the following order: immobilization>cuticular shrinkage>motor program for anterior retraction>motor program for longitudinal contraction congruent to tanning of cuticle of the newly formed puparium. A paradoxical situation was observed whereby the motor programs of pupariation are temporally dissociated from actual morphogenesis of the puparium. The tensiometric data suggest that the most likely candidate for a primary pupariation factor is Neb-PK-2, rather than Neb-PK-1.

Analogues of cyclolinopeptide A containing alpha-hydroxymethyl amino acid residues.

Linear and cyclic cyclolinopeptide A (CLA) analogues containing alpha-hydroxymethylleucine (HmL) in positions 1, 4, and 1&4, and alpha-hydroxymethylvaline (HmV) in position 5, were synthesized by the solid-phase peptide strategy and cyclized with the 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide/1-hydroxy-7-azabenzotriazole (EDC/HOAt) reagent. The peptides were examined for their immunosuppressive activity in the lymphocyte proliferation assays (LPA). Only HmL-containing peptides demonstrated at about 25% lower immunosuppressive activity, but they are four times more soluble in water solutions than the native CLA. It seems from the LPA results that peptide [(HmL4)CLA] is the most promising for further studies. This peptide was characterized in solution, at room temperature in CDCl3, and the conformation compared with that observed for CLA in the solid state.