Hubert Dyker

Effect of helicokinins and ACE inhibitors on water balance and development of Heliothis virescens larvae

The diuretic activity of the helicokinins I (YFSPWG-amide), II (VRFSPWG-amide) and III (KVKFSAWG-amide) was tested on Heliothis virescens larvae. All three kinins increased fluid secretion in isolated Malpighian tubules in a dose-dependent manner. Injections into the haemolymph caused a significant reduction in weight gain after 24 h and, in the case of helicokinin I, led to an increased mortality of 43% within 6 days. When truncated analogues of helicokinin I were tested in vitro, only the pentapeptide (FSPWG-amide) stimulated fluid secretion. Tested in vivo the pentapeptide did not influence normal development of the larvae. An alanine scan of helicokinin I showed that the substitution of phenylalanine, tryptophan and glycine led to a massive decrease or even loss of diuretic activity. The substitution of the other amino acids had no effect in vitro. The ACE inhibitors captopril, enalapril-maleate and lisinopril were tested for their influence on the development of the larvae. In combination with one of the helicokinins the in vivo injection of the ACE inhibitors led to increased rates of mortality and/or reductions in pupal weight.

In vivo effects of Manduca sexta allatotropin and allatostatin on development and reproduction in the fall armyworm, Spodoptera frugiperda (Lepidoptera, Noctuidae)

Summary Two types of allato-regulating peptides, Manse-AT and Manse-AS, were injected twice a day into larvae and adults of S. frugiperda to test their effects on life table parameters in vivo. Injections of Manse-AT (1 nmol per injection) into penultimate instar larvae drastically reduced their weight gain and increased mortality. The duration of the penultimate and the last larval stage, respectively, was prolonged in those animals which survived. Injections of Manse-AT (1 or 10 nmol per injection) into last instar larvae reduced their weight gain and increased mortality in a dose-dependent manner. At the higher dose of 10 nmol no individual emerged. Injections of Manse-AS (1 nmol) into larvae of both larval stages hardly affected growth and development of the animals, whereas combined injections of Manse-AS and Manse-AT (1 nmol of each peptide) resulted in effects similar to those obtained with Manse-AT alone. Peptide injections into adult female moths shortened their life span and thereby reduced the total number of deposited eggs. The oviposition rate was reduced with Manse-AT alone and Manse-AS plus Manse-AT, whereas egg deposition in Manse-AS injected females on a per day basis was not affected during their short life span. Peptide injections into mated females did not affect juvenile hormone biosynthesis by the corpora allata ex vivo. Incubation of Manse-AT with haemolymph from last instar larvae in vitro resulted in rapid degradation of the peptide with half-lives of 1 to 5 min.

PF1022A : A novel anthelmintic cyclooctadepsipeptide. Modification and exchange of the N-methyl leucine residues

The first structure-activity relationships of the anthelmintic cyclooctadepsipeptide PF1022A have been established via a systematic exchange of the leucine residues by a series of related N-alkylated amino acids. The data presented strongly suggest that (L)-N-methyl-leucine is crucial for high in vivo activity.

Pharmacological characterisation of 5-hydroxytryptamine-induced contractile effects in the isolated gut of the lepidopteran caterpillar Spodoptera frugiperda

The indolealkylamine 5-hydroxytryptamine (5-HT, 0.1nM-1&mgr;M) caused dose-dependent increases in the number of contractions observed in guts isolated from the caterpillar Spodoptera frugiperda. Of the 5-HT analogues tested for agonist action, 2-methyl-5-HT (0.1-10&mgr;M) was a full agonist with reduced potency while alpha-methyl-5-HT (0.1-100&mgr;M), 5-carboxamidotryptamine (0.1-100&mgr;M), 5-methoxytryptamine (5-MeOT) (10nM-10&mgr;M), and tryptamine (1-100&mgr;M) were partial agonists. Incubation of isolated guts with proven mammalian 5-HT receptor antagonists showed that cyproheptadine (10nM-1&mgr;M), MDL 72222 (1-10&mgr;M), tropisetron (1-10&mgr;M) and 5-benzoyloxygramine (1-10&mgr;M) were potent non-competitive antagonists of 5-HT-induced tissue contraction. In comparison, ketanserin (0.1-1&mgr;M) was a competitive antagonist. The mammalian selective serotonin reuptake inhibitors, clomipramine (10nM-10&mgr;M) and fluoxetine (10nM-10&mgr;M) also caused non-competitive inhibition of 5-HT-induced contraction while fluvoxamine (10nM-10&mgr;M) was a weak competitive antagonist. Low doses of clomipramine (0.1&mgr;M) caused potentiation of 5-HT-induced gut contraction thereby suggesting the presence of 5-HT reuptake systems in this tissue. The contractile effects of 5-HT were inhibited by verapamil, Li(+) and H7 and potentiated by theophylline thereby indicating that L-type Ca(2+) channels, phosphatidylinositol second messengers and cAMP, respectively, are involved in 5-HT-induced tissue contraction. The 5-HT receptors mediating contractility in the gut of S. frugiperda have properties in common with mammalian 5-HT(2) and Drosophila 5-HT(dro2A/2B) receptors. In addition, these data suggest that the tissue also contains receptors that are similar to mammalian 5-ht(6) and 5-HT(7) as well as Drosophila(dro1) receptors. However, the primary amino acid sequence of these lepidopteran 5-HT receptors will have to be elucidated before full comparisons can be made.

Synthesis, conformational studies and anthelmintic activity of a constrained PF1022A analogue

A mono-proline analogue of the potent anthelmintic cyclooctadepsipeptide PF1022A has been synthesized. NMR studies in combination with molecular dynamics simulations demonstrate that the confirmation of this analogue is both rigidified and distorted compared with the asymmetric conformation of PF1022A. The backbone conformation is significant with respect to anthelmintic activity.

Structure-activity relationship of contractile effects induced by helicokinins in the isolated gut of the lepidopteran caterpillar Spodoptera frugiperda.

The diuretic helicokinins YFSPWG-amide (Hez KI), VRFSPWG-amide (Hez KII) and KVKFSAWG-amide (Hez KIII) are potent contractants of the isolated gut of the caterpillar Spodoptera frugiperda at doses ranging from 0.1 to 10nM. In comparison, the pentapeptide FSPWG-amide was a full agonist with greatly reduced potency while SPWG-amide and PWG-amide were weak partial agonists. Substitution of individual amino acids in Hez KI with alanine revealed that replacement of the [phenylalanine(2)] residue caused a large fall in potency while replacement of [tryptophan(5)] residue caused complete loss of myogenic activity. The striking fall in potency of YASPWG-amide and the lack of activity of YFSPAG-amide confirm the requirement for aromatic groups in positions 2 and 3 of the core pentapeptide as well as supporting the ideas that the active core of these peptides adopts a beta-turn when interacting with receptors, bringing together the [Phe] and [Trp] residues that are critical for activity. Neither the pentapeptide proctolin nor the potent mammalian gut contractant Substance P were able to cause contraction when applied to caterpillar gut tissue. Incubation of isolated gut tissue in the phosphodiesterase inhibitor theophylline (10-100&mgr;M) caused significant potentiation of the response to applied Hez KI. Conversely, in the presence of the L-type Ca(2+) channel blocker verapamil (10&mgr;M-1mM) or Co(2+) (1-50mM) the contractile effects of Hez KI were attentuated significantly. These data suggest that the gut of S. frugiperda contains G-protein-linked kinin receptors that utilise cyclic AMP as their second messenger system and cause contraction by promoting the entry of extracellular Ca(2+).

Syntheses of depsipeptide analogues of the insect neuropeptide proctolin

Four depsipeptide analogues of the insect neuropeptide proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) have been prepared, containing a single ester linkage between Arg(1) and Tyr(2), Tyr(2) and Leu(3), and between Pro(4) and Thr(5), respectively. A didepsipentapeptide containing an ester linkage between Tyr(2) and Leu(3) and between Pro(4) and Thr(5), has also been prepared. The depsipeptide 4 is the first example of a backbone-modified proctolin analogue which shows full myotropic activity.