Pawel Zubrzak

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.

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.

Biostable β-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.

Bioavailability of β-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 β3Phe‐substituted Cyclolinopeptide A analogues

CLA, a natural, highly hydrophobic cyclic nonapeptide with sequence c(Pro1‐Pro2‐Phe3‐Phe4‐Leu5‐Ile6‐Ile7‐Leu8‐Val9‐), 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 β3Phe residues. A conformational analysis by NMR in CD3CN/H2O mixture has been carried out on the CLA analogue, in which Phe3 has been replaced by a βPhe, to study the influence of the mutation on the three‐dimensional structure. All linear and cyclic CLA analogues containing βPhe 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. Copyright

Analogues of cyclolinopeptide a containing α-hydroxymethyl amino acid residues†

Linear and cyclic cyclolinopeptide A (CLA) analogues containing α‐hydroxymethylleucine (HmL) in positions 1, 4, and 1&4, and α‐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.