Conformational analysis of a cyclic AKH neuropeptide analog that elicits selective activity on locust versus honeybee receptor.

Neuropeptides belonging to the adipokinetic hormone (AKH) family elicit metabolic effects as their main function in insects, by mobilizing trehalose, diacylgycerol, or proline, which are released from the fat body into the hemolymph as energy sources for muscle contraction required for energy-intensive processes, such as locomotion. One of the AKHs produced in locusts is a decapeptide, Locmi-AKH-I (pELNFTPNWGT-NH2). A head-to-tail cyclic, octapeptide analog of Locmi-AKH-I, cycloAKH (cyclo[LNFTPNWG]) was synthesized to severely restrict the conformational freedom of the AKH structure. In vitro, cycloAKH selectively retains full efficacy on a pest insect (desert locust) AKH receptor, while showing little or no activation of the AKH receptor of a beneficial insect (honeybee). Molecular dynamic analysis incorporating NMR data indicate that cycloAKH preferentially adopts a type II ß-turn under micelle conditions, whereas its linear counterpart and natural AKH adopts a type VI ß-turn under similar conditions. CycloAKH, linear LNFTPNWG-NH2, and Locmi-AKH-I feature the same binding site during docking simulations with the desert locust AKH receptor (Schgr-AKHR), but differ in the details of the ligand/receptor interactions. However, cycloAKH failed to enter the binding pocket of the honeybee receptor 3D model during docking simulations. Since the locust AKH receptor has a greater tolerance than the honeybee receptor for the cyclic conformational constraint in vitro receptor assays, it could suggest a greater tolerance for a shift in the direction of the type II ß turn exhibited by cycloAKH from the type VI ß turn of the linear octapeptide and the native locust decapeptide AKH. Selectivity in biostable mimetic analogs could potentially be enhanced by incorporating conformational constraints that emphasize this shift. Biostable mimetic analogs of AKH offer the potential of selectively disrupting AKH-regulated processes, leading to novel, environmentally benign control strategies for pest insect populations.

The pro-apoptotic action of new analogs of the insect gonadoinhibiting peptide Neb-colloostatin: synthesis and structure-activity studies.

Neb-colloostatin (SIVPLGLPVPIGPIVVGPR), an insect oostatic factor found in the ovaries of the flesh fly Neobellieria bullata, strongly induces apoptosis in insect haemocytes. To explain the role of Ser(1) and Pro(4) residues of Neb-colloostatin in the pro-apoptotic activity of this peptide, the synthesis of a series of analogs was performed, such as: [Ac-Ser(1)]- (1), [d-Ser(1)]- (2), [Thr(1)]- (3), [Asp(1)]- (4), [Glu(1)]- (5), [Gln(1)]- (6), [Ala(1)]- (7), [Val(1)]- (8), [d-Pro(4)]-(9), [Hyp(4)]- (10), [Acp(4)]- (11), [Ach(4)]- (12), [Ala(4)]- (13), [Ile(4)]- (14), and [Val(4)]-colloostatin (15). All peptides were bioassayed in vivo for the pro-apoptotic action on haemocytes of Tenebrio molitor. Additionally, the structural properties of Neb-colloostatin and its analogs were examined by the circular dichroism in water and methanol. Peptides 1, 4, 5, 7, 8, 10, 12, 14, and 15 strongly induce T. molitor haemocytes to undergo apoptosis and they show about 120-230% of the Neb-colloostatin activity at a dose of 1nM. The CD conformational studies show that the investigated peptides seem to prefer the unordered conformation.

Ecdysis triggering hormone signaling in arthropods.

Ecdysis triggering hormones (ETHs) from endocrine Inka cells initiate the ecdysis sequence through action on central neurons expressing ETH receptors (ETHR) in model moth and dipteran species. We used various biochemical, molecular and BLAST search techniques to detect these signaling molecules in representatives of diverse arthropods. Using peptide isolation from tracheal extracts, cDNA cloning or homology searches, we identified ETHs in a variety of hemimetabolous and holometabolous insects. Most insects produce two related ETHs, but only a single active peptide was isolated from the cricket and one peptide is encoded by the eth gene of the honeybee, parasitic wasp and aphid. Immunohistochemical staining with antiserum to Manduca PETH revealed Inka cells on tracheal surface of diverse insects. In spite of conserved ETH sequences, comparison of natural and the ETH-induced ecdysis sequence in the honeybee and beetle revealed considerable species-specific differences in pre-ecdysis and ecdysis behaviors. DNA sequences coding for putative ETHR were deduced from available genomes of several hemimetabolous and holometabolous insects. In all insects examined, the ethr gene encodes two subtypes of the receptor (ETHR-A and ETHR-B). Phylogenetic analysis showed that these receptors fall into a family of closely related GPCRs. We report for the first time the presence of putative ETHs and ETHRs in genomes of other arthropods, including the tick (Arachnida) and water flea (Crustacea). The possible source of ETH in ticks was detected in paired cells located in all pedal segments. Our results provide further evidence of structural and functional conservation of ETH-ETHR signaling.

A novel member of the adipokinetic peptide family in a "living fossil", the ice crawler Galloisiana yuasai, is the first identified neuropeptide from the order Grylloblattodea.

This is the first report on the structural identity of a neuropeptide of the insect order Grylloblattodea. A peptide was isolated and sequenced from the retrocerebral corpora cardiaca-corpora allata complex of the ice crawler, Galloisiana yuasai. The sequence of the peptide was deduced from the multiple MS(N) electrospray mass data as that of an octapeptide: pGlu-Val-Asn-Phe-Ser-Pro-Thr-Trp amide. The retention time on reversed-phase HPLC and the CID MS(2) mass spectra of a synthetic peptide with the same primary structure were exactly the same as of the natural peptide. The sequence represents a novel peptide of the adipokinetic hormone family which contains presently 50 members. The primary structure differs in only one position to a few previously discovered AKHs. A scenario is outlined that makes it likely that the most recently discovered insect order, the Mantophasmatodea, and the Grylloblattodea are closely related.

The synthesis and biological activity of linear and cyclic analogs of the two diuretic peptides of Diploptera punctata.

We have investigated the effect of analogs of the two Dippu diuretic hormones, Dippu-DH(46) and Dippu-DH(31), on fluid secretion by Malpighian tubules of male Diploptera punctata. We synthesized analogs containing the amino acid methyl-homoserine, to replace methionine residues, to render these modified peptides less subject to oxidation. We have also synthesized C-terminal fragments and their corresponding cyclic analogs to determine their effect on fluid secretion in D. punctata. Our results indicate that the modified peptides retain significant activity in the Ramsay secretion assay. The linear fragments displayed no activity or some inhibitory activity whereas the cyclic analog fragments showed stimulatory activity, in the case of DH(46), or slight inhibitory activity, in the case of DH(31).

The cDNA for leucomyosuppressin in Blattella germanica and molecular evolution of insect myosuppressins.

Myosuppressins are a group of 10-residues FMRFamide-related peptides reported in Dictyoptera, Orthoptera, Lepidoptera and Diptera. Myosuppressins inhibit visceral muscle contractions and, in the cockroach Blattella germanica, inhibit food intake. In B. germanica, the cDNA of leucomyosuppressin (LMS) has been cloned and sequenced. The deduced precursor is 96 amino acids long and contains a single copy of LMS. Brain mRNA levels remain constant during the first reproductive cycle of adult females, whereas those in the gut show a slight decline during the time of maximal food intake. Comparison of myosuppressin precursors of different species reveals that all have the same organization. Phylogenetic analysis suggests that the precursor experienced an accelerated evolution in Lepidoptera and Diptera with respect to Dictyoptera, whereas only Lepidoptera has radical changes in the bioactive peptide.

Enantioconvergent synthesis of (-)-(S)- and (+)-(R)-2-acetyl-3,6-dihydroxycyclohex-2-enone starting from rac-6-hydroxy-3-methoxycyclohex-2-enone.

The synthesis of enantiomerically pure (-)-(S)- and (+)-(R)-2-acyl-3,6-dihydroxycyclohex-2-enone starting from diastereomerically pure N-tosyl-(S)-proline esters 3-methoxy-6-hydroxycyclohex-2-enone 1 is presented. An enantioconvergent synthesis of either (-)-(S)- and (+)-(R)-2-acyl-3,6-dihydroxycyclohex-2-enone starting with the racemic alpha-ketol 1 through a conversion of ( approximately 1:1) mixture of diastereomeric esters into one diastereomer by a repeated crystallization, followed by dimethylaminopyridine-catalyzed equilibration as key steps is described.

A third active AKH is present in the pyrgomorphid grasshoppers Phymateus morbillosus and Dictyophorus spumans.

The corpora cardiaca of the African pyrgomorphid grasshoppers Phymateus morbillosus and Dictyophorus spumans contain three adipokinetic hormones (AKHs): besides two already known AKHs, Phm-AKH-I and Scg-AKH-II (Gäde et al., 1996 [Gäde, G., Kellner, R., Rinehart, K.L., 1996. Pyrgomorphid grasshoppers of the genus Phymateus contain species-specific decapeptides of the AKH/RPCH family regulating lipid-mobilisation during flight. Physiol. Entomol. 21, 193-202]), a new AKH-III, denoted Phm-AKH-III, pGlu-Ile-Asn-Phe-Thr-Pro-Trp-Trp-NH(2), has been characterised. This is only the second AKH-III identified so far, thus, only three insect species - all of them grasshoppers - contain three active AKHs. Phm-AKH-III differs from Lom-AKH-III from the migratory locust, Locusta migratoria, only in position 2: isoleucine is present instead of leucine. The structure of the Phm-AKH-III was confirmed by synthesis, subsequent mass determination and reversed-phase high-performance liquid chromatography. The synthetic peptide also induced hyperlipaemia in D. spumans and L. migratoria.

Synthesis and biological activity of adipokinetic hormone analogues with modifications in the 4-8 region.

Several structural characteristics in the molecule of the locust adipokinetic hormone, AKH-I, have been investigated in terms of their importance in determining biologic activity. All modifications tested in this study resulted in analogues with decreased potency in comparison with the parent molecule. However, all analogues that were found to be active gave a full response, although often only at very high doses of peptide. This study has highlighted for the locust receptor(s) the vital role of the side chain of Thr(5), and the importance of positions 4 and 8. For example, when Trp(8) and Phe(4) were exchanged, the resulting analogue (Trp(4),Phe(8)-AKH-I) was one of the least active analogues tested in this study. Although Trp is tolerated quite well as a substitute for Phe(4), with only a 10-fold loss of potency, Phe is not favored as a substitute for Trp(8) (>300 times decrease in potency). On the other hand, 3-[2-napthyl] alanine (Nal) is a better substitute for Trp(8) (only a 100-fold loss in potency). We conclude that position 4 requires a phenyl ring in the side chain, and position 8 an indole ring.

Evidence for helicity in insect diuretic peptide hormones: computational analysis, spectroscopic studies, and biological assays.

The conformation of four insect diuretic hormones has been analyzed computationally using secondary structure prediction routines and comparison with structures in the Brookhaven Protein Databank. Based on this analysis, a common seven-residue peptide fragment (DVLRQRL) had a high probability of forming an alpha-helix. Circular dichroism (CD) studies found that addition of trifluoroethanol (TFE) to an aqueous solution of the seven-residue fragment induces a change from random coil to helix. Subsequent NMR studies in water-TFE (1:1) produced nOe values and 3JalphaNH coupling constants confirming a helical conformation: 3JalphaNH coupling constants for the first five residues (D1 to Q5) were all < or = 6.0 Hz and two medium-range nOe values (dalphaN (i,i+3)) were observed between V2 and Q5, and R4 and L7. The longer fragments PLDVLRQRL in water-TFE and Lom-DH 1-26 in water alone, both containing the DVLRQRL sequence of the locust (Locusta migratoria) diuretic hormone, maintained the helicity as determined by CD analysis. However, the remaining 20 residues of the locust diuretic hormone did not maintain the same amount of helicity in water and all of the truncated fragments were not biologically active.

Biological actions of synthetic locust ion transport peptide (ITP).

Locust Ion Transport Peptide (ITP) a member of the arthropod neuropeptide family which includes hyperglycemic, vitellogenesis-inhibiting, and moult-inhibiting hormones (CHH, VIH, MIH, respectively) was synthesized as proposed by Meredith et al. (1996) with terminal amidation of amino acid residue 72 and with 3 disulphide bridges. This is the first member of this family to be synthesized. Biological activities of synthetic ITP (synITP) were very similar to those previously reported for ITP purified from Schistocerca corpora cardiaca (ScgITP) and partially sequenced by Audsley et al. (1992a, b). Dose-response curves for both synITP and ScgITP on ileal transport of Cl- (measured as increased short-circuit current, delta Isc), were similar with a EC50 of 1-2 nM. The Isc time course and maximum delta Isc across ileal epithelia at different dosages of synITP and ScgITP had similar patterns as did changes in transepithelial open-circuit potential (Vt) and resistance (Rt), reflecting changes in salt transport which drives fluid absorption. Disulphide bridges were shown to be required for biological activity of synITP, which caused the same 4-fold increase in ileal fluid transport rate (Jv) as previously reported for ScgITP. Both synITP and ScgITP caused only partial stimulation of rectal Isc and had no significant effect on rectal Jv. These results indicate that the structure of ITP predicted earlier from cDNA is correct.

Synthesis, radiolabeling and biological activity of peptide oostatic hormone and its analogues.

A series of Pro peptides containing the sequence of the oostatic hormone 3d and its shorter analogues 3a-3c differing in a number of the C-terminal Pro residues was prepared for a study of its effect on oogenesis in Sarcophaga bullata Parker (Diptera). Peptides 3a-3d were synthesized in solution by the fragment condensation of Boc-Tyr-Asp(OtBu)-Pro-Ala-Pro-OH (2f) with Pro oligopeptides H-(Pro)2-5-OtBu. The amino-terminal protected pentapeptide acid 2f was prepared by a stepwise procedure from TFA.H-Ala-Pro-OMe using Boc-Pro-OH, Z-Asp(OtBu)-OSu and Boc-Tyr-OSu. The H(Z)-(Pro)2-5-OtBu oligopeptides 1a-1h were synthesized from Z-Pro-OH and H-Pro-OtBu by a combination of stepwise procedure and fragment condensation. The 125I-labeled molecules of the octapeptide 3b and decapeptide 3d were used for radiotracer distribution studies. Evidence of content of the labeled peptide material in various parts of the insect body (ovaries, head, intestine) is presented. The time distribution of the labeled material in the insect organs was correlated with results of histological analysis of ovaries treated by nonlabeled peptides. The peptides assayed affected processes of egg development in 20-60% of ovarioles. The decapeptide 3d caused changes consisting in some resorbed egg chambers and normal appearance of vitellogenic eggs, whereas the octapeptide 3b caused abnormal yolk deposition and formation of big eggs with irregular yolk granules, proliferation of follicular epithelium in some egg chambers and about the same amount of resorbed egg chambers as decapeptide. These structural differences are complementary to the different values of organ radioactivities.

Prolonged pheromonotropic activity of pseudopeptide mimics of insect pyrokinin neuropeptides after topical application or injection into a moth.

Amphiphilic pseudopeptide analogs of Phe-Thr-Pro-Arg-Leu-NH2, representing the active C-terminal core pentapeptide of the pyrokinin class of insect neuropeptides, were synthesized by replacement of phenylalanine with hydrocinnamic acid (Hca-Thr-Pro-Arg-Leu-NH2), or addition of 1-pyrenebutyric acid (Pba-Phe-Thr-Pro-Arg-Leu-NH2) or 9-fluoreneacetic acid (Fla-Phe-Thr-Pro-Arg-Leu-NH2). The pseudopeptides were found to stimulate sex pheromone biosynthesis when injected into females of the moth Heliothis virescens. Optimal pheromonotropic responses were obtained by injection of 0.25 pmol of Hca-Thr-Pro-Arg-Leu-NH2, 2.5 pmol of Pba-Thr-Pro-Arg-Leu-NH2 and 0.5 pmol of Fla-Thr-Pro-Arg-Leu-NH2. Topical application of each of the pseudopeptides in water to the cuticle of moths stimulated significant production of pheromone at a dose of 50 pmol with optimal stimulation occurring when 500 pmol were applied. The parent peptide, Phe-Thr-Pro-Arg-Leu-NH2, failed to stimulate significant production of pheromone when applied topically at a dose as high as 2000 pmol. Temporal studies indicated that Hca-Thr-Pro-Arg-Leu-NH2 stimulated significant production of pheromone for only 4 h after application where as continuous pheromone production for 18 h was observed when either Pba-Phe-Thr-Pro-Arg-Leu-NH2 or Fla-Phe-Thr-Pro-Arg-Leu-NH2 were applied to the abdomen. The results show that modification of the C-terminal active core of the insect pyrokinins, by addition of hydrophobic moieties, can result in production of pseudopeptides which effectively penetrate the insect cuticle and have prolonged physiological effects making them favorable candidates for use in development of alternative strategies for pest insect control.

Hydrophobic effects on antibacterial and channel-forming properties of cecropin A-melittin hybrids.

The design of cecropin-melittin hybrid analogues is of interest due to the similarities in the structure of the antimicrobial peptides cecropin and melittin but differences in their lytic properties. We suspected that a hydrophobic residue in position 2 of milittin (Ile8 in the hybrid) plays an important role in the activity of the 15-residue hybrid, KWKLFKKIGAVLKVL-NH2, [CA(1-7)M(2-9)NH2] and have now examined its role in the analogue toward five test bacteria. Deletion of Ile8 reduced activity, and it was not restored by lengthening to 15 residues by addition of another threonine at the C-terminus. Replacement of Ile8 by a hydrophobic leucine maintained good activity and Ala8 was equally active for four organisms, although less active against Staphylococcus aureus. Replacement by the hydrophilic Ser8 strongly reduced potency against all five organisms. Deletion of Leu15 decreased activity, but addition of Thr16 maintained good activity. The presence of hydrophobic residues appears to have a significant effect on the process of antibacterial activity. These peptide analogues showed voltage-dependent conductance changes and are capable of forming ion-pores in planar lipid bilayers. The antibacterial action of the peptides is thought to be first an ionic interaction with the anionic phosphate groups of the membrane followed by interaction with the hydrocarbon core of the membrane and subsequent reorientation into amphipathic alpha-helical peptides that form pores (ion-channels), which span the membrane. The analogue also showed an increase in alpha-helicity with an increase in hexafluoro 2-propanol concentration.

A comparative immunocytochemical study using an antiserum against a synthetic analogue of the corpora cardiaca peptide Pea-CAH-I (MI, neurohormone D) of Periplaneta americana.

An antiserum against the octapeptide Pea-CAH-I, a member of the adipokinetic hormone/red pigment-concentrating hormone family, has been produced for immunocytochemical staining in insects and various other invertebrate species. The anti-Pea-CAH-I serum stains the glandular corpora cardiaca cells of those insect species that synthesize identical or structurally similar peptides. In the corpora cardiaca of species producing peptides with a different C-terminus, these cells remain unstained. Pea-CAH-I-like immunoreactivity has also been found in neurons of the central nervous system of all invertebrate orders studied. The antiserum recognizes the C-terminal sequence Pro-Asn-Trp-NH2 of the Pea-CAH-I molecule as established by enzyme immunoassay. The widespread Pea-CAH-I-like immunoreactivity in all nervous systems of the studied animals probably does not reflect the presence of Pea-CAH-I but the occurrence of peptides carrying similar epitopes.

The use of synthetic Cecropin (D5C) in disinfecting contact lens solutions.

PURPOSE: Microbial keratitis caused by Pseudomonas aeruginosa is the most common contact lens associated corneal infection. Cecropins are microbicidal peptides isolated from the hemolymph of the Cecropia moth. Previous in vitro studies have demonstrated their efficacy against a broad spectrum of ocular pathogens. This study was designed: a) to evaluate the antimicrobial potency of three different contact lens solutions (Renu, Complete, and Opti-Free) against P. aeruginosa, and b) to evaluate the activity of the same contact lens solutions in combination with a synthetic cecropin analog, D5C, against the challenge organism in the presence of a soft contact lens. METHODS: A virulent strain of P.aeruginosa isolated from a case of ulcerative keratitis was used in the study. Three different concentrations of bacteria (10(3), 10(5) and 10(7) CFU/mL) were inoculated into the contact lens solutions and into buffered saline, which was employed as a control. The samples were incubated at 27 degrees C, and at time 0, 30, and 90 minutes, 24, 48, and 72 hours, and aliquots of the test solutions were plated and subcultured on nutrient agar. After 24 hours of incubation at 37 degrees C, colonies observed on the nutrient agar plates were counted. To study the antimicrobial efficacy of D5C (100 micrograms/mL), we used the identical test series and assay, adding a soft contact lens to the solutions and a larger inoculum of bacteria (10(9) CFU/mL). RESULTS: After 72 hours, all of the contact lens solutions tested sterilized 10(3) CFU/mL of P. aeruginosa. At 10(7) CFU/mL, they yielded greater than 2 logs of killing of the bacteria, but the solutions were not sterilized. The addition of D5C (100 micrograms/mL) to the contact lens solutions yielded greater than 3 logs of killing with a larger inoculum of bacteria in the presence of the soft contact lens. CONCLUSION: The contact lens solutions tested were effective against P. aeruginosa at 27 degrees C for up to 72 hours with an inoculum of 10(3) CFU/mL. The addition of D5C augmented their antimicrobial activity in the presence of the contact lens.