The insect myokinin (leucokinin-like) neuropeptide family includes peptides that have different physiological effects such as the induction of hindgut myotropic activity and stimulation of urine ...production. The C-terminal pentamer of myokinins Phe-X-(Ser/Pro/Ala)-Trp-Gly-amide X=Phe, His, Asn, Ser or Tyr, had been previously determined as the minimum fragment able to elicit a functional response. The receptor(s) for these insect neuropeptides has not yet been identified. In order to characterize the Malpighian tubule leucokinin-like peptide receptor(s) from the yellow fever mosquito (
Aedes aegypti), a leucokinin photoaffinity analogue (LPA) of sequence dAla-dTyr-Bpa-dLys-Phe-Phe-Ser-Trp-Gly-amide was designed based on structure/activity relationships for leucokinins. LPA caused depolarization of the transepithelial voltage (TEV) in female Malpighian tubule, confirming the activity of the peptide. The effective concentration to give half the maximum depolarization (EC
50) was 17 nM. The
125I-LPA was then used to characterize leucokinin binding proteins in female Malpighian tubule membranes. It specifically labeled and saturated a protein(s) of about 54 kDa as shown by SDS-PAGE/autoradiography and by competition experiments with excess unlabeled leucokinin analogues.
125I-LPA bound to the 54 kDa protein(s) with a K
d value of 13±3 nM in agreement with the EC
50 for the TEV bioassay. Altogether these data suggest that the 54 kDa protein is an
Aedes-leucokinin receptor. This is the first characterization of an insect leucokinin receptor and reveals that LPA is a powerful tool to label insect myokinin receptors.
Abstract 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. 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 α,α-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.
The production of juvenile hormone III (JH III) by the corpora allata of the cockroach Diploptera punctata is regulated in part by peptides originating from the brain. One group of these peptides, ...termed allatostatins, reversibly inhibits the biosynthesis of JH in vitro. Allatostatin 4 (AST4: Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-amide) is the smallest member of the AST family yet defined and was used as the benchmark peptide for these initial structure-activity studies. Two initial analog series of AST4 were examined for the ability of each analog to inhibit JH biosynthesis by corpora allata in vitro. Each analog series consisted of analogs that contained a single amino acid change from the native AST4 sequence. The first series contained Ala replacement analogs and the second contained analogs with D-amino acid replacements. The first analog series used Ala replacements to help indicate which amino acid side chains were most important for inhibition of JH biosynthesis. The most important side chain appeared to be Leu8 followed by Phe6 and Tyr4. Additionally, the D-amino acid series suggested that a secondary structural element(s) at the C-terminus of AST4 could be important to the biological activity.
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. ...Eight different analogs of the insect kinin C-terminal pentapeptide active core in which the critical residues Phe
1, Pro
3 and Trp
4 are replaced with β
3-amino acid and/or their β
2-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 β
2Trp
4 and double-replacement analog β
3Phe
2, β
3Pro
3 of the insect kinins proved to be selective agonists for the tick receptor, whereas single-replacement analog β
3Pro
3 and double-replacement analog β
3Phe, β
3Pro
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.
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 β-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-βA-1 and PK-βA-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-βA-4 represents a biostable, non-selective agonist in all four bioassays, essentially matching the potency of a natural PK in pupariation assay. Analog PK-βA-2 is a potent agonist in the melanotropic assay, demonstrating full efficacy at 1
pmol. In some cases, the structural changes imparted to the analogs modify the physiological responses. Analog PK-βA-3 is a non-selective agonist in all four bioassays. The analog PK-βA-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.
The pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family plays a multifunctional role in an array of important physiological processes in a variety of insects. An active core ...analog containing an (
E)-alkene,
trans-Pro isosteric component was evaluated in four disparate PK/PBAN bioassays in four different insect species. These bioassays include pheromone biosynthesis in the moth
Heliothis peltigera, melanization in the larval
Spodoptera littoralis, pupariation acceleration in the larval fly
Neobellieria bullata, and hindgut contraction in the cockroach
Leucophaea maderae. The conformationally constrained analog demonstrated activity equivalent to parent PK/PBAN peptides of equal length in all four PK/PBAN bioassays, and matched and/or approached the activity of peptides of natural length in three of them. In the melanization bioassay, the constrained analog exceeded the efficacy (maximal response) of the natural PBAN1-33 by a factor of 2 (at 1
nmol). The results provide strong evidence for the orientation of Pro and the core conformation adopted by PK/PBAN neuropeptides during interaction with receptors associated with a range of disparate PK/PBAN bioassays. The work further identifies a scaffold with which to design mimetic PK/PBAN analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting PK/PBAN-regulated systems.
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) Holmes et al., Insect Mol Biol 9:457-465 (2000); Holmes et al., Insect Mol Biol 12:27-38 (2003), and the dengue vector, the mosquito Aedes aegypti (L.) Pietrantonio et al., Insect Mol Biol 14:55-67 (2005), 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 Phe¹ and Trp⁴ 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, FFAibWGa, 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 FFAibWGa 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.
The antagonistic properties of a few linear and backbone cyclic (BBC) conformationally constraint peptide libraries and their analogs, were tested for the ability to inhibit pyrokinin/pheromone ...biosynthesis activating neuropeptide (PK/PBAN) mediated functions: sex pheromone biosynthesis in
Heliothis peltigera female moths, cuticular melanization in
Spodoptera littoralis larvae, pupariation in the fleshfly
Neobellieria bullata and hindgut contraction in
Leucophaea maderae, elicited by exogenously injected PBAN, pheromonotropin (PT), leucopyrokinin (LPK), myotropin (MT) or by the endogenous peptides. The data revealed differential inhibitory patterns within the same assay with different elicitors (in both the pheromonotropic and melanotropic assays) and among the different functions and disclosed selective antagonists, hinting at the possibility that the receptors that mediate those functions may differ from one another structurally.
The diuretic/myotropic insect kinin neuropeptides, which share the common C-terminal pentapeptide core FX
1X
2WG-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)FFAibWG-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.
Five native pyrokinin-like peptides (Neb-PK-1, Neb-PK-2, Neb-PVK-1, L
9Neb-PVK-2, I
9Neb-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 I
9Neb-PVK-2 proved inactive, whereas L
9Neb-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
≅
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.
