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Prolonged pheromonotropic activity of pseudopeptide mimics of insect pyrokinin neuropeptides after topical application or injection into a moth
Regulatory Peptides 72 (1997) 161–167
Prolonged pheromonotropic activity of pseudopeptide mimics of insect pyrokinin neuropeptides after topical application or injection into a moth a, b Peter E.A. Teal *, Ronald J. Nachman a
Center for Medical, Agricultural and Veterinary Entomology, USDA, ARS, 1700 SW 23 Dr., Gainesville, FL 32604, USA b Veterinary Entomology Research Unit, FAPRL, USDA, ARS, 2881 F& B Rd., College Station, TX 77845, USA Received 16 June 1997; received in revised form 8 September 1997; accepted 18 September 1997
Abstract Amphiphilic pseudopeptide analogs of Phe-Thr-Pro-Arg-Leu-NH 2 , 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-ArgLeu-NH 2 ), or addition of 1-pyrenebutyric acid (Pba-Phe-Thr-Pro-Arg-Leu-NH 2 ) or 9-fluoreneacetic acid (Fla-Phe-Thr-Pro-Arg-LeuNH 2 ). 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-NH 2 , 2.5 pmol of Pba-Thr-Pro-Arg-Leu-NH 2 and 0.5 pmol of Fla-Thr-Pro-Arg-Leu-NH 2 . 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-NH 2 , 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-NH 2 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-ProArg-Leu-NH 2 or Fla-Phe-Thr-Pro-Arg-Leu-NH 2 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. 1997 Elsevier Science B.V. Keywords: Insect cuticle; Neuropeptides; Sex pheromone biosynthesis
1. Introduction Although the existence of specialized neurosecretory cells within the cephalic ganglia and nervous system of insects has been known for more than 60 years it was not until 1975 that the first neuropeptide, proctolin, was isolated and identified [1] followed, shortly thereafter, by identification of adipokinetic hormone from locusts by Stone et al. [2]. Identification of these neuropeptides stimulated significant research on the regulatory action of insect neuropeptides that has, over the past 10 years, resulted in isolation and identification of more than 100 *Corresponding author. Tel.: 1 1 352 374 5788; fax: 1 1 352 374 5707; e-mail: [email protected] 0167-0115 / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved. PII S0167-0115( 97 )01053-7
neuropeptides from insects representing a large number of insect orders. These neuroregulators modulate virtually all aspects of insect life including homeostasis, development, reproduction and behavior [3–5]. The broad regulatory scope of insect neuropeptides, plus the fact that the effective doses required for bioactivity are in the pico- to femtomole range, make insect neuropeptides highly attractive candidates for use in development of novel strategies for pest insect control [5–7]. Several potential methods of disrupting neuroendocrine processes for the purpose of insect pest control have been outlined by various authors ( [4–7], and references therein). These include: (1) molecular approaches in which genes for either neuropeptides or antagonists are inserted into insect-specific microorganisms and the products ex-
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pressed after infection of the insect; (2) inhibition or hyperstimulation of natural neuropeptide release by compounds that mimic neurohormone releasing factors or neurotransmitters; (3) inhibition of neuropeptide processing enzymes; (4) development of agonists or antagonists that act at the neuropeptide receptor sites. While all of these approaches have advantages, we have concentrated our efforts on designing stable agonists capable of inducing physiological responses when applied topically to the insect. Design of analogs that penetrate the integument effectively is the key to success in development of this method because the cuticular component of the epidermis is hydrophobic, which inhibits peptides from entering the body [6]. We have chosen the pyrokinin class of insect neuropeptides as model compounds for design of analogs. This class of neuropeptides regulates a wide variety of physiological functions including: hindgut and oviduct myotropic activity in cockroaches and locusts, pupariation in flies, induction of egg diapause and reddish coloration and melanization in moths and sex pheromone biosynthesis in moths and some flies (see Refs. [8,9]). Although these neuropeptides range in length from 8 to 34 amino acids, they all share the common C-terminal pentapeptide PheXxx-Pro-Arg-Leu-NH 2 (Xxx 5 Gly,Ser,Thr or Val). This common sequence forms a type 1 b-turn required for receptor recognition and is the critical portion of these peptides required for bioactivity in all physiological assays [10]. Detailed structure activity studies have indicated the importance of the variable amino acid (Xxx) in the Cterminal pentamer [11] for stimulation of sex pheromone biosynthesis in moths. For example, the pentapeptide PheGly-Pro-Arg-Leu-NH 2 failed to stimulate production of pheromone when injected into the moth Helicoverpa zea at amounts of up to 500 pmol, whereas Phe-Thr-Pro-ArgLeu-NH 2 induced significant amounts of pheromone at doses of 20 pmol and higher [11]. In fact, all Thr substituted peptides tested had greater pheromonotropic activity than did similar analogs having Val, Ser or Gly substitutions. Several were more active than the naturally occurring 33 amino acid neuropeptide, pheromone biosynthesis activating neuropeptide (PBAN), although they contained significantly fewer amino acids [11]. In our initial studies we showed that attachment of 6-phenylhexanoic acid, linked through alanine, to the amino terminus of the naturally occurring pyrokinin, Locusta myotropin II (GluGly-Asp-Phe-Thr-Pro-Arg-Leu-NH 2 ), yielded an amphiphilic pseudopeptide that penetrated the insect cuticle effectively and stimulated pheromone production in the moth Heliothis virescens [12]. Another analog of the moderately active pentapeptide, Phe-Thr-Pro-Arg-LeuNH 2 , was synthesized by replacement of the Phe with a hydrophobic carboranyl moiety lacking an N-terminal amino group [8]. This analog proved to be more potent than the native 33 amino acid pheromone biosynthesis activating neuropeptide when injected into moths and also
stimulated pheromone production when applied topically to the insect [8]. These results provided evidence that we could design amphiphilic analogs utilizing the small Cterminal pentapeptide active core with potent pheromonotropic activity. The following reports results of studies using three pseudopeptide analogs of Phe-Thr-Pro-ArgLeu-NH 2 conducted to determine if they would stimulate pheromone production when applied topically to the insect. The smallest, a pseudotetrapeptide, formed by substitution of phenylalanine by hydrocinnamic acid, was synthesized to determine if substitution of only the amine group of phenylalanine with H would impart sufficient amphiphilic character to penetrate the insect cuticle while still maintaining biological activity. The two pseudopentapeptides tested were synthesized by addition of either 1pyrenebutyric acid or 9-fluoreneacetic acid to the pentapeptide core, Phe-Thr-Pro-Arg-Leu-NH 2 . These molecules were synthesized to determine if attachment of relatively large apolar molecules to the active pentapeptide core would yield analogs that were released slowly through the cuticle and resulted in prolonged biological effects.
2. Methods and materials
2.1. Peptides and pseudopeptides Hydrocinnamic acid (Hca), 1-pyrenebutyric acid (Pba) and fluorene-9-acetic acid (Fla) were purchased from Aldrich Chemical (Milwaukee, WI). Thr-Pro-Arg (Pmc)Leu-rink amide resin complex and Phe-Thr-Pro-Arg (Pmc)-Leu-rink amide resin complex were synthesized using FMOC methodology according to previously described procedures [8]. The pseudopeptide analogs were synthesized by condensation of Hca to the Thr-Pro-Arg (Pmc)-Leu-rink amide resin complex or Pba or Fla to the Phe-Thr-Pro-Arg (Pmc)-Leu-rink amide resin complex by stirring with one equivalent of 1,3-diisopropylcarbodiimide / 1-hydroxy-7-azabenzotriazole in dimethyl sulfoxide for 4 h at room temperature. The crude pseudopeptides were cleaved from the resin and protecting groups were removed by treatment with a mixture of trifluoroacetic acid (TFA) (90%), anisole (5%), thianisole (4%) and 1,2-ethanedithiol (1%) for 1 h. The resin was removed by filtration and volatile reagents were removed by vacuum concentration with a Savant Speed Vac concentrator. Purification of the pseudopeptides was accomplished using solid phase extraction with reversed phase (C18) material and reversed phase HPLC as described elsewhere [8,12]. Peptides including PBAN and Phe-Thr-Pro-Arg-Leu-NH 2 were synthesized and purified using previously described methods [7,13]. Mass spectra of purified compounds were obtained using 1 pmol amounts with a Biosystems Perseptive MALDI MS operated at an accelerating voltage of 30 kV and quantitation was accom-
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plished by amino acid analysis after hydrolysis in 6N HCl using an Applied Biosystems 420A amino acid analyzer.
2.2. Pheromonotropic assays Pupae of the moth Heliothis virescens were obtained from a laboratory culture maintained at the Center for Medical, Agricultural and Veterinary Entomology, USDA, Gainesville, FL, and were separated by sex with the males being discarded. Females were allowed to eclose to the adult stage in 4-l containers held in environmental chambers at 25628, 65638 relative humidity with a 14 h:10 h light:dark photoperiod. Newly eclosed adults were transferred to new containers daily and provided with a 5% sucrose solution as food. All bioassays were conducted using 2-day old females during the photophase, when endogenous levels of pheromone are low or undetectable [14]. Initially, we assessed the effects of the three pseudopeptides and Phe-Thr-Pro-Arg-Leu-NH 2 in inducing sex pheromone biosynthesis by injecting them into females and comparing the amounts of pheromone produced to amounts produced when females were injected with the optimum dose of PBAN for stimulation of pheromone production (5 pmol / 20 ml, H 2 O) [14] or just H 2 O. Pseudopeptides were dissolved in H 2 O at various concentrations from 0.001 to 100.0 pmol / 20 ml and injected into the side of the abdomen. After an incubation period of 1 h the terminal abdominal segments, which contain the sex pheromone gland, were excised from the insects and extracted in 20 ml of hexane containing 1 ng ml 21 of heptadecane and nonadecane as internal standards. The amount of pheromone in individual extracts was determined by quantitating the amount of (Z)-11-hexadecenal (Z11-16:AL) using capillary gas chromatography as described elsewhere [11]. The amount of pheromone in extracts obtained from insects injected with the pseudopeptides or with only saline were converted to a percentage of the mean amount present in extracts obtained from females injected with 5 pmol of PBAN for that day. Data were analyzed using a one-way ANOVA and Tukey’s test using Statmost software (DataMost Corporation). For our initial topical application studies we conducted experiments to determine if the pentapeptide (Phe-Thr-ProArg-Leu-NH 2 ), used as a model for design of the pseudopeptides, or the pseudopeptides would penetrate the insect cuticle and stimulate pheromone production when applied to the abdomen at 1 nmol. To insure that the compounds were applied directly to the surface of the cuticle we removed the scales on the surface of the abdomen by gently rubbing the ventral surface of the abdomen on cellulose adhesive tape. Moths were held immobile, ventral side up, by clamping the wings behind the back using smooth jawed alligator clips held in modeling clay. A 1 ml drop of H 2 O containing 1 nmol of the test compound was then applied to the descaled portion of the abdomen as
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previously described [12] and the insects were incubated for 1 h. After incubation the pheromone glands were excised, extracted and the extracts were analyzed as above. Amounts of pheromone present in extracts of treated insects were compared with amounts present in control insects treated only with a 1 ml drop of H 2 O. Topical dose response studies were conducted using serial dilutions of the pseudopeptides. For these experiments 1 ml drops of H 2 O containing from 0.5 to 2000 pmol of peptide were applied to the abdomen and the preparations were allowed to incubate for 1 h prior to extraction and analysis of pheromone. Temporal activity studies were conducted by applying 1 nmol of the pseudopeptides to the abdomen in a 1 ml drop of H 2 O or just H 2 O. For incubations of up to 12 h, applications were made to different groups of moths at 2 or 1 h intervals throughout the photophase with the final group being treated 1 h prior to excision of the pheromone glands. When incubations of 18 and 24 h were conducted single applications were made 6 h after initiation of the photophase and the glands were excised 18 and 24 h later. Additionally, for these long incubation experiments, females from the same cohort were treated with either 1 nmol of the pseudopeptides or just water 17 h after the initial group of insects were treated as positive and negative controls. Data were analyzed using a one-way ANOVA and Tukey’s test. To determine if insects incubated for 24 h after application of the pseudopeptides had lost their ability to produce pheromone or if the pseudopeptides had become inactive we conducted tests in which females were injected with either water or 5 pmol of PBAN 23 h after topical application of 1 nmol of the pseudopeptides. After a 1 h incubation period, extracts of the pheromone glands were analyzed.
3. Results and discussion Studies conducted to determine if the pseudopeptides would stimulate pheromone production when injected into moths indicated that all three had pheromonotropic properties (Fig. 1 Fig. 2). The most potent of the three pseudopeptides was Hca-Thr-Pro-Arg-Leu-NH 2 . Insects injected with 0.05 pmol of this pseudopeptide produced significantly more pheromone than control insects injected with only water (t 5 8.07, 10 d.f.) and insects injected with either 0.1 pmol of Hca-Thr-Pro-Arg-Leu-NH 2 or 5 pmol of PBAN produced statistically similar amounts of pheromone (t 5 2.11, 10 d.f.). The results indicate that the pseudopeptide has superagonistic effects when compared to PBAN because 0.5 pmol of PBAN is required to stimulate production of significantly more pheromone than controls and 5.0 pmol of PBAN is required for optimal production of pheromone [12]. This pseudotetrapeptide was synthesized to determine if the amino group of Phe was critical for the activity of the C-terminal active core of
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Fig. 1. Relative amount of pheromone present in extracts obtained from females 60 min after injection of different doses of either Phe-Thr-Pro-Arg-LeuNH 2 (a), Hca-Thr-Pro-Arg-Leu-NH 2 (b), Fla-Phe-Thr-Pro-Arg-Leu-NH 2 (c) or Pba-Phe-Thr-Pro-Arg-Leu-NH 2 (d). The amount of pheromone (6SEM, n 5 6 / dose) is expressed as a percentage of the mean amount produced by females injected with 5 pmol of PBAN (100%, dashed line, n 5 6 / day) for each assay. Structures of each compound are shown above each graph for comparison.
Fig. 2. Amount of pheromone present in extracts of glands obtained 1 h after topical application of only water, or 1 nmol of either Phe-Thr-ProArg-Leu-NH 2 (FTPRL-NH2) or Hca-Thr-Pro-Arg-Leu-NH 2 (HcnTPRLNH2). Values represent means of eight replicates per treatment (6SEM). The mean amount of pheromone present in extracts from glands (n 5 8) obtained 1 h after injection of 5 pmol of PBAN is shown as the dashed line for comparison.
the pyrokinins (Phe-Thr-Pro-Arg-Leu-NH 2 ). The data show that deletion of the amino group of Phe does not reduce pheromonotropic activity because the pseudo-
peptide was, in fact, more potent than Phe-Thr-Pro-ArgLeu-NH 2 (Fig. 1). Two possible explanations for the increased activity of the pseudopeptide relative to Phe-ThrPro-Arg-Leu-NH 2 are that the pseudopeptide binds more strongly and is cleared from the receptor site less rapidly than Phe-Thr-Pro-Arg-Leu-NH 2 or that the pseudopeptide is more resistive to aminopeptidase attack within the insect. Although neither explanation is supported by experiments conducted here, temporal studies of the topically applied pseudopeptide indicate that the activity declines much more rapidly than either of the other pseudopeptides tested, suggesting that proteolytic enzymes act rapidly on this molecule (see below). Although both of the pseudopentapeptides induced production of pheromone when injected (Fig. 1) into moths, neither was as potent as Hca-Thr-Pro-Arg-LeuNH 2 . Fla-Phe-Thr-Pro-Arg-Leu-NH 2 was the more potent of the two pseudopentapeptides. This pseudopeptide stimulated production of significantly more pheromone when injected at 0.05 pmol than that present in extracts from insects injected with only water (t 5 4.84, 10 d.f.). Injection of 0.5 pmol of this pseudopeptide induced production of as much pheromone as was produced by injection of 5 pmol of PBAN (t 5 0.81, 10 d.f.). The dose response profile obtained for Pba-Phe-Thr-Pro-Arg-Leu-
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NH 2 indicated that the optimal dose for stimulation was 2.5 pmol. Injection of less than 2.5 pmol of this pseudopeptide resulted in production of only limited amounts of pheromone. This result suggested that the large hydrophobic pyrene moiety attached to the amino terminus rendered Pba-Phe-Thr-Pro-Arg-Leu-NH 2 less soluble in water. However, tests in which the pseudopeptide was dissolved in 20% acetonitrile for injection yielded a similar dose response curve (data not shown). Moths injected with PBAN dissolved in 20% acetonitrile produced as much pheromone as was produced when PBAN was dissolved in only water. This, plus the fact that HPLC analysis indicated that Pba-Phe-Thr-Pro-Arg-Leu-NH 2 was completely soluble in 20% acetonitrile, indicate that solubility of the pseudopeptide in water did not affect its activity. In our initial topical application studies we assessed the effects of application of the pentapeptide, Phe-Thr-ProArg-Leu-NH 2 , and its hydrocinnamic analog, Hca-ThrPro-Arg-Leu-NH 2 , on stimulation of pheromone production when applied at 1000 pmol in water. PBAN was not used for topical application studies because earlier work has shown that it will not stimulate pheromone production when applied topically in water [12]. As indicated in Fig. 2 the pentapeptide, Phe-Thr-Pro-Arg-Leu-NH 2 , did not stimulate production of any more pheromone during a 1 h incubation than was produced when water alone was applied. Doubling the amount of Phe-Thr-Pro-Arg-LeuNH 2 applied had no effect on pheromone production. However, the pseudotetrapeptide, Hca-Thr-Pro-Arg-LeuNH 2 , stimulated production of as much pheromone, when applied topically, as was produced when 5 pmol of PBAN was injected. In fact, dose response studies conducted using Hca-Thr-Pro-Arg-Leu-NH 2 indicated that topical application of 50 pmol induced significant production of pheromone in a 1 h period (Fig. 3). Both of the pseudopentapeptides tested also induced pheromone production when applied topically in water (Fig. 3). Dose response curves were similar for all three of the pseudopeptides, with significant stimulation of pheromone production occurring when 50 pmol were applied and optimal production being induced at a dose of 500 pmol. The fact that similar dose response profiles were obtained for all three pseudopeptides when applied topically was of interest because each had different dose response curves when injected (Fig. 1), with Hca-Thr-Pro-Arg-LeuNH 2 being considerably more potent than either of the pseudopentapeptides. One possible explanation for the differences between the topical and injection dose response curves was that Hca-Thr-Pro-Arg-Leu-NH 2 was the most polar of the three pseudopeptides and, as such, could have been the slowest to penetrate the cuticle. If this pseudotetrapeptide penetrated the cuticle more slowly than the pseudopentapeptides then it might be expected to have prolonged activity when applied topically because the effective dose necessary for stimulation of pheromone production is significantly lower than the other two
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Fig. 3. Amounts of pheromone present in extracts obtained from glands excised 1 h after topical application of different amounts of Hca-Thr-ProArg-Leu-NH 2 (a) or Fla-Phe-Thr-Pro-Arg-Leu-NH 2 (b) or Pba-Phe-ThrPro-Arg-Leu-NH 2 (c). Values represent the mean amounts determined from analysis of eight replicates / dose (6SEM).
pseudopeptides and should be maintained over long periods due to slow release into the body. To address this question we conducted studies in which we applied a dose of the pseudopeptides sufficient to readily stimulate maximum production of pheromone in a 1 h period, but allowed the preparations to incubate for periods of up to 24 h before extracting the pheromone glands. Application times for these experiments were staggered to insure that all pheromone glands were excised during the photophase when pheromone is not normally produced.
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Contrary to the above prediction, the results of these studies indicated that the activity of Hca-Thr-Pro-Arg-LeuNH 2 was maintained for the shortest length of time, being constant for the first 4 h after application and declining to resting levels by 6 h (Fig. 4). This time response profile was similar to that obtained when PBAN is injected [14] and suggests that the pseudotetrapeptide penetrates the cuticle rapidly and is subjected to rapid deactivation, presumably due to the action of peptidases. Pheromone production induced by application of 1 nmol of either Fla-Phe-Thr-Pro-Arg-Leu-NH 2 or Pba-Phe-Thr-Pro-Arg-
Fig. 4. Amounts of pheromone present in extract obtained from glands obtained at different times after topical application of 1 nmol of Hca-ThrPro-Arg-Leu-NH 2 (a) or Fla-Phe-Thr-Pro-Arg-Leu-NH 2 (b) or Pba-PheThr-Pro-Arg-Leu-NH 2 (c). Values represent the mean amounts determined from analysis of 12 replicates / dose (6SEM).
Leu-NH 2 remained high for 18 h after application. We believe that the prolonged period of activity of the two pseudopentapeptides, relative to Hca-Thr-Pro-Arg-LeuNH 2 , reflects the fact that the two pseudopentapeptides have greater resistance to peptidase attack and that the balance between the apolar and polar characteristics of Fla-Phe-Thr-Pro-Arg-Leu-NH 2 and Pba-Phe-Thr-Pro-ArgLeu-NH 2 allows for slow continuous release through the cuticle and into the insect. Thus, a constant slow release of the pseudopeptides maintains sufficient titers to induce pheromone production over a prolonged period. However, these pseudopeptides do lose their activity over time, indicating that they are eventually degraded by the insect. The prolonged period of pheromone production induced by application of Fla-Phe-Thr-Pro-Arg-Leu-NH 2 and PbaPhe-Thr-Pro-Arg-Leu-NH 2 was of interest to us because one possible use of these neuropeptide analogs for insect pest control is to inhibit the natural production of pheromone by depletion of precursors in the pheromone gland [8,12]. To determine if the ability to produce pheromone was diminished among females treated with 1 nmol of Fla-Phe-Thr-Pro-Arg-Leu-NH 2 or Pba-Phe-Thr-Pro-ArgLeu-NH 2 we incubated females for 23 h after application of the pseudopeptides and then injected either water or 5 pmol of PBAN. Pheromone glands excised and extracted 24 h after topical application of Fla-Phe-Thr-Pro-Arg-LeuNH 2 or Pba-Phe-Thr-Pro-Arg-Leu-NH 2 and 1 h after injection of PBAN contained an average of 229.1 (619.4, n 5 8) and 186.1 (624.9, n 5 8) ng of pheromone, respectively. However, extracts obtained from similarly treated females that had been injected with only water contained only 20.7 (63.4, n 5 8) and 13.9 (62.8, n 5 8) ng, respectively. The results indicate that pseudopeptides are degraded over time and that prolonged exposure to the pseudopeptides has no effect on the ability of H. virescens females to produce pheromone. However, they do not imply that depletion of pheromone precursor might occur in other species because females of H. virescens do not appear to store or use fatty acyl precursors during pheromone production [15] whereas other species do. For example, studies on pheromone biosynthesis by females of Manduca sexta have shown that continuous exposure to PBAN causes significant declines in the amounts of pheromone precursors stored as triacyl glycerols in the pheromone gland [16,17]. Therefore, these pseudopeptides may effectively deplete pheromone precursors in other species. Although our studies have centered on the abilities of the pseudopeptides to induce pheromone production, the results have other implications because the pyrokinin class of insect neuropeptides regulate a number of other physiological functions including: hindgut and oviduct myotropic activity and induction of pupariation and egg diapause [18]. Thus, the analogs tested here may have application for use in development of control strategies based on these other physiological parameters. In summary, the results of our studies have provided
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significant information on the modifications that can be incorporated into pseudopeptides to allow them to penetrate the insect cuticle while still maintaining biological activity. The results indicate that replacement of the amino group of phenylalanine with hydrogen in the pentapeptide, Phe-Thr-Pro-Arg-Leu-NH 2 , is all that is required to impart sufficient lipidic character to allow the molecule to penetrate the hydrophobic wax layer of the insect cuticle. Thus, only minor substitutions of single amino acids are required to render pyrokinins both amphiphilic and more potent than their parent peptides. The increased potency of HcaThr-Pro-Arg-Leu-NH 2 relative to the parent peptide, PheThr-Pro-Arg-Leu-NH 2 , could reflects its greater resistance to hemolymph aminopeptidase attack as this would allow more of the pseudopeptide to reach the target receptor. However, the pseudopeptide is clearly degraded rapidly as is indicated by the short period of bioactivity observed in temporal activity studies. Alternatively, the substitution of hydrocinnamic acid for phenylalanine could give the pseudopeptide greater affinity for the receptor which would result in enhanced biological activity. Although, when injected, neither Fla-Phe-Thr-Pro-Arg-Leu-NH 2 nor PbaPhe-Thr-Pro-Arg-Leu-NH 2 were as potent as Hca-Thr-ProArg-Leu-NH 2 , all three were equipotent when applied topically and the pseudopentapeptides induced pheromone production for a much longer period than did Hca-Thr-ProArg-Leu-NH 2 . This probably reflects the fact that both Fla-Phe-Thr-Pro-Arg-Leu-NH 2 and Pba-Phe-Thr-Pro-ArgLeu-NH 2 are highly resistant to aminopeptidase attack and that the apolar nature of the amino terminal fluorene or pyrene allows for slow continuous release of these molecules through the cuticle. The development of pseudopeptide analogs that have prolonged periods of bioactivity is a key element in the development of these compounds for use in insect control because it reduces the need for continuous exposure for maintenance of activity. The technology developed for the pyrokinin class of neuropeptides should be applicable to other types of insect neuropeptides. Therefore, long lasting pseudopeptide analogs of other neuropeptides, for example those inhibiting reproductive development, could be used for direct control of insect pests.
References [1] Starrat AN, Brown BE. Structure of the pentapeptide proctolin, a proposed neurotransmitter in insects. Life Sci 1975;17:1253–6. [2] Stone JV, Mordue W, Batley KE, Morris HR. Structure of locust adipokinetic hormone, a neurohormone that regulates lipid utilization during flight. Nature 1976;263:207–11.
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[3] Holman GM, Nachman RJ, Wright MS. Insect neuropeptides. Annu Rev Entomol 1990;35:201–17. [4] Kelly TJ, Masler EP, Menn JJ. Insect neuropeptides: New strategies for insect control. In: Casida JE, editor. Pesticides and Alternatives: Innovative Chemical and Biological Approaches to Pest Control. Amsterdam: Elsevier, 1990:283–97. [5] Masler EP, Kelly TJ, Menn JJ. Insect neuropeptides: Discovery and application in insect management. Arch Insect Biochem Physiol 1993;22:87–111. [6] Keeley LL, Hayes TK. Speculations on biotechnology applications for insect neuroendocrine research. Insect Biochem 1987;17:639– 51. [7] Nachman RJ, Holman GM, Haddon WF. Leads for insect neuropeptide mimetic development. Arch Insect Biochem Physiol 1993;22:181–97. [8] Nachman RJ, Teal PEA, Radel PA, Holman GM, Abernathy RL. Potent pheromonotropic / myotropic activity of a carboranyl pseudotetrapeptide analogue of the insect pyrokinin / PBAN neuropeptide family administered via injection or topical application. Peptides 1996;17:747–52. [9] Teal PEA, Abernathy RL, Nachman RJ, Fang N, Meredith JA, Tumlinson JH. Pheromone biosynthesis activating neuropeptides: Functions and chemistry. Peptides 1996;17:337–44. [10] Nachman RJ, Roberts VA, Dyson HJ, Holman GM. Active conformation of an insect neuropeptide family. Proc Natl Acad Sci USA 1991;88:4518–22. [11] Abernathy RL, Nachman RJ, Teal PEA, Yamashita O, Tumlinson JH. Pheromonotropic activity of naturally occurring pyrokinin insect neuropeptides (FXPRLamide) in Helicoverpa zea. Peptides 1995;16:215–9. [12] Abernathy RL, Teal PEA, Meredith JA, Nachman RJ. Induction of pheromone production in a moth by topical application of a pseudopeptide mimic of a pheromonotropic neuropeptide. Proc Natl Acad Sci USA 1996;93:12621–5. [13] Christensen TC, Itagaki H, Teal PEA, Jasensky R, Tumlinson JH, Hildebrand JG. Innervation and neural regulation of the sex pheromone gland in female Heliothis moths. Proc Natl Acad Sci USA 1991;88:4971–5. [14] Teal PEA, Oostendorp A, Tumlinson JH. Induction of pheromone production in females of Heliothis virescens (F.) and H. subflexa (Gn.) (Lepidoptera: Noctuidae) during the photophase. Can Entomol 1993;125:355–66. [15] Jurenka RA. Signal-transduction in the stimulation of sex pheromone biosynthesis in moths. Arch Insect Biochem Physiol 1996;33:245–58. [16] Fang N, Teal PEA, Tumlinson JH. Correlation between gylcerolipids and pheromone aldehydes in the sex pheromone gland of female tobacco hornworm moths Manduca sexta (L.). Arch Insect Biochem Physiol 1995;30:321–36. [17] Fang N, Teal PEA, Tumlinson JH. Effects of decapitation and PBAN injection on amounts of triacylglycerols in the sex pheromone gland of Manduca sexta (L.). Arch Insect Biochem Physiol 1996;32:249–60. [18] Nachman RJ, Roberts VA, Lange AB, Orchard I, Holman GM, Teal, PEA. Active confirmation and mimetic analog development for the pyrokinin-PBAN-diapause-pupariation and myosuppressin insect neuropeptide families. In: Hedin PA, Hollingworth RM, Masler EP, Miyamoto J, Tompson DG, editors. Phytochemicals for Pest Control. Am Chem Soc Symposium Series 658. Washington: American Chemical Society, 1997:277–91.
Prolonged pheromonotropic activity of pseudopeptide mimics of insect pyrokinin neuropeptides after topical application or injection into a moth a, b Peter E.A. Teal *, Ronald J. Nachman a
Center for Medical, Agricultural and Veterinary Entomology, USDA, ARS, 1700 SW 23 Dr., Gainesville, FL 32604, USA b Veterinary Entomology Research Unit, FAPRL, USDA, ARS, 2881 F& B Rd., College Station, TX 77845, USA Received 16 June 1997; received in revised form 8 September 1997; accepted 18 September 1997
Abstract Amphiphilic pseudopeptide analogs of Phe-Thr-Pro-Arg-Leu-NH 2 , 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-ArgLeu-NH 2 ), or addition of 1-pyrenebutyric acid (Pba-Phe-Thr-Pro-Arg-Leu-NH 2 ) or 9-fluoreneacetic acid (Fla-Phe-Thr-Pro-Arg-LeuNH 2 ). 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-NH 2 , 2.5 pmol of Pba-Thr-Pro-Arg-Leu-NH 2 and 0.5 pmol of Fla-Thr-Pro-Arg-Leu-NH 2 . 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-NH 2 , 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-NH 2 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-ProArg-Leu-NH 2 or Fla-Phe-Thr-Pro-Arg-Leu-NH 2 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. 1997 Elsevier Science B.V. Keywords: Insect cuticle; Neuropeptides; Sex pheromone biosynthesis
1. Introduction Although the existence of specialized neurosecretory cells within the cephalic ganglia and nervous system of insects has been known for more than 60 years it was not until 1975 that the first neuropeptide, proctolin, was isolated and identified [1] followed, shortly thereafter, by identification of adipokinetic hormone from locusts by Stone et al. [2]. Identification of these neuropeptides stimulated significant research on the regulatory action of insect neuropeptides that has, over the past 10 years, resulted in isolation and identification of more than 100 *Corresponding author. Tel.: 1 1 352 374 5788; fax: 1 1 352 374 5707; e-mail: [email protected] 0167-0115 / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved. PII S0167-0115( 97 )01053-7
neuropeptides from insects representing a large number of insect orders. These neuroregulators modulate virtually all aspects of insect life including homeostasis, development, reproduction and behavior [3–5]. The broad regulatory scope of insect neuropeptides, plus the fact that the effective doses required for bioactivity are in the pico- to femtomole range, make insect neuropeptides highly attractive candidates for use in development of novel strategies for pest insect control [5–7]. Several potential methods of disrupting neuroendocrine processes for the purpose of insect pest control have been outlined by various authors ( [4–7], and references therein). These include: (1) molecular approaches in which genes for either neuropeptides or antagonists are inserted into insect-specific microorganisms and the products ex-
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pressed after infection of the insect; (2) inhibition or hyperstimulation of natural neuropeptide release by compounds that mimic neurohormone releasing factors or neurotransmitters; (3) inhibition of neuropeptide processing enzymes; (4) development of agonists or antagonists that act at the neuropeptide receptor sites. While all of these approaches have advantages, we have concentrated our efforts on designing stable agonists capable of inducing physiological responses when applied topically to the insect. Design of analogs that penetrate the integument effectively is the key to success in development of this method because the cuticular component of the epidermis is hydrophobic, which inhibits peptides from entering the body [6]. We have chosen the pyrokinin class of insect neuropeptides as model compounds for design of analogs. This class of neuropeptides regulates a wide variety of physiological functions including: hindgut and oviduct myotropic activity in cockroaches and locusts, pupariation in flies, induction of egg diapause and reddish coloration and melanization in moths and sex pheromone biosynthesis in moths and some flies (see Refs. [8,9]). Although these neuropeptides range in length from 8 to 34 amino acids, they all share the common C-terminal pentapeptide PheXxx-Pro-Arg-Leu-NH 2 (Xxx 5 Gly,Ser,Thr or Val). This common sequence forms a type 1 b-turn required for receptor recognition and is the critical portion of these peptides required for bioactivity in all physiological assays [10]. Detailed structure activity studies have indicated the importance of the variable amino acid (Xxx) in the Cterminal pentamer [11] for stimulation of sex pheromone biosynthesis in moths. For example, the pentapeptide PheGly-Pro-Arg-Leu-NH 2 failed to stimulate production of pheromone when injected into the moth Helicoverpa zea at amounts of up to 500 pmol, whereas Phe-Thr-Pro-ArgLeu-NH 2 induced significant amounts of pheromone at doses of 20 pmol and higher [11]. In fact, all Thr substituted peptides tested had greater pheromonotropic activity than did similar analogs having Val, Ser or Gly substitutions. Several were more active than the naturally occurring 33 amino acid neuropeptide, pheromone biosynthesis activating neuropeptide (PBAN), although they contained significantly fewer amino acids [11]. In our initial studies we showed that attachment of 6-phenylhexanoic acid, linked through alanine, to the amino terminus of the naturally occurring pyrokinin, Locusta myotropin II (GluGly-Asp-Phe-Thr-Pro-Arg-Leu-NH 2 ), yielded an amphiphilic pseudopeptide that penetrated the insect cuticle effectively and stimulated pheromone production in the moth Heliothis virescens [12]. Another analog of the moderately active pentapeptide, Phe-Thr-Pro-Arg-LeuNH 2 , was synthesized by replacement of the Phe with a hydrophobic carboranyl moiety lacking an N-terminal amino group [8]. This analog proved to be more potent than the native 33 amino acid pheromone biosynthesis activating neuropeptide when injected into moths and also
stimulated pheromone production when applied topically to the insect [8]. These results provided evidence that we could design amphiphilic analogs utilizing the small Cterminal pentapeptide active core with potent pheromonotropic activity. The following reports results of studies using three pseudopeptide analogs of Phe-Thr-Pro-ArgLeu-NH 2 conducted to determine if they would stimulate pheromone production when applied topically to the insect. The smallest, a pseudotetrapeptide, formed by substitution of phenylalanine by hydrocinnamic acid, was synthesized to determine if substitution of only the amine group of phenylalanine with H would impart sufficient amphiphilic character to penetrate the insect cuticle while still maintaining biological activity. The two pseudopentapeptides tested were synthesized by addition of either 1pyrenebutyric acid or 9-fluoreneacetic acid to the pentapeptide core, Phe-Thr-Pro-Arg-Leu-NH 2 . These molecules were synthesized to determine if attachment of relatively large apolar molecules to the active pentapeptide core would yield analogs that were released slowly through the cuticle and resulted in prolonged biological effects.
2. Methods and materials
2.1. Peptides and pseudopeptides Hydrocinnamic acid (Hca), 1-pyrenebutyric acid (Pba) and fluorene-9-acetic acid (Fla) were purchased from Aldrich Chemical (Milwaukee, WI). Thr-Pro-Arg (Pmc)Leu-rink amide resin complex and Phe-Thr-Pro-Arg (Pmc)-Leu-rink amide resin complex were synthesized using FMOC methodology according to previously described procedures [8]. The pseudopeptide analogs were synthesized by condensation of Hca to the Thr-Pro-Arg (Pmc)-Leu-rink amide resin complex or Pba or Fla to the Phe-Thr-Pro-Arg (Pmc)-Leu-rink amide resin complex by stirring with one equivalent of 1,3-diisopropylcarbodiimide / 1-hydroxy-7-azabenzotriazole in dimethyl sulfoxide for 4 h at room temperature. The crude pseudopeptides were cleaved from the resin and protecting groups were removed by treatment with a mixture of trifluoroacetic acid (TFA) (90%), anisole (5%), thianisole (4%) and 1,2-ethanedithiol (1%) for 1 h. The resin was removed by filtration and volatile reagents were removed by vacuum concentration with a Savant Speed Vac concentrator. Purification of the pseudopeptides was accomplished using solid phase extraction with reversed phase (C18) material and reversed phase HPLC as described elsewhere [8,12]. Peptides including PBAN and Phe-Thr-Pro-Arg-Leu-NH 2 were synthesized and purified using previously described methods [7,13]. Mass spectra of purified compounds were obtained using 1 pmol amounts with a Biosystems Perseptive MALDI MS operated at an accelerating voltage of 30 kV and quantitation was accom-
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plished by amino acid analysis after hydrolysis in 6N HCl using an Applied Biosystems 420A amino acid analyzer.
2.2. Pheromonotropic assays Pupae of the moth Heliothis virescens were obtained from a laboratory culture maintained at the Center for Medical, Agricultural and Veterinary Entomology, USDA, Gainesville, FL, and were separated by sex with the males being discarded. Females were allowed to eclose to the adult stage in 4-l containers held in environmental chambers at 25628, 65638 relative humidity with a 14 h:10 h light:dark photoperiod. Newly eclosed adults were transferred to new containers daily and provided with a 5% sucrose solution as food. All bioassays were conducted using 2-day old females during the photophase, when endogenous levels of pheromone are low or undetectable [14]. Initially, we assessed the effects of the three pseudopeptides and Phe-Thr-Pro-Arg-Leu-NH 2 in inducing sex pheromone biosynthesis by injecting them into females and comparing the amounts of pheromone produced to amounts produced when females were injected with the optimum dose of PBAN for stimulation of pheromone production (5 pmol / 20 ml, H 2 O) [14] or just H 2 O. Pseudopeptides were dissolved in H 2 O at various concentrations from 0.001 to 100.0 pmol / 20 ml and injected into the side of the abdomen. After an incubation period of 1 h the terminal abdominal segments, which contain the sex pheromone gland, were excised from the insects and extracted in 20 ml of hexane containing 1 ng ml 21 of heptadecane and nonadecane as internal standards. The amount of pheromone in individual extracts was determined by quantitating the amount of (Z)-11-hexadecenal (Z11-16:AL) using capillary gas chromatography as described elsewhere [11]. The amount of pheromone in extracts obtained from insects injected with the pseudopeptides or with only saline were converted to a percentage of the mean amount present in extracts obtained from females injected with 5 pmol of PBAN for that day. Data were analyzed using a one-way ANOVA and Tukey’s test using Statmost software (DataMost Corporation). For our initial topical application studies we conducted experiments to determine if the pentapeptide (Phe-Thr-ProArg-Leu-NH 2 ), used as a model for design of the pseudopeptides, or the pseudopeptides would penetrate the insect cuticle and stimulate pheromone production when applied to the abdomen at 1 nmol. To insure that the compounds were applied directly to the surface of the cuticle we removed the scales on the surface of the abdomen by gently rubbing the ventral surface of the abdomen on cellulose adhesive tape. Moths were held immobile, ventral side up, by clamping the wings behind the back using smooth jawed alligator clips held in modeling clay. A 1 ml drop of H 2 O containing 1 nmol of the test compound was then applied to the descaled portion of the abdomen as
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previously described [12] and the insects were incubated for 1 h. After incubation the pheromone glands were excised, extracted and the extracts were analyzed as above. Amounts of pheromone present in extracts of treated insects were compared with amounts present in control insects treated only with a 1 ml drop of H 2 O. Topical dose response studies were conducted using serial dilutions of the pseudopeptides. For these experiments 1 ml drops of H 2 O containing from 0.5 to 2000 pmol of peptide were applied to the abdomen and the preparations were allowed to incubate for 1 h prior to extraction and analysis of pheromone. Temporal activity studies were conducted by applying 1 nmol of the pseudopeptides to the abdomen in a 1 ml drop of H 2 O or just H 2 O. For incubations of up to 12 h, applications were made to different groups of moths at 2 or 1 h intervals throughout the photophase with the final group being treated 1 h prior to excision of the pheromone glands. When incubations of 18 and 24 h were conducted single applications were made 6 h after initiation of the photophase and the glands were excised 18 and 24 h later. Additionally, for these long incubation experiments, females from the same cohort were treated with either 1 nmol of the pseudopeptides or just water 17 h after the initial group of insects were treated as positive and negative controls. Data were analyzed using a one-way ANOVA and Tukey’s test. To determine if insects incubated for 24 h after application of the pseudopeptides had lost their ability to produce pheromone or if the pseudopeptides had become inactive we conducted tests in which females were injected with either water or 5 pmol of PBAN 23 h after topical application of 1 nmol of the pseudopeptides. After a 1 h incubation period, extracts of the pheromone glands were analyzed.
3. Results and discussion Studies conducted to determine if the pseudopeptides would stimulate pheromone production when injected into moths indicated that all three had pheromonotropic properties (Fig. 1 Fig. 2). The most potent of the three pseudopeptides was Hca-Thr-Pro-Arg-Leu-NH 2 . Insects injected with 0.05 pmol of this pseudopeptide produced significantly more pheromone than control insects injected with only water (t 5 8.07, 10 d.f.) and insects injected with either 0.1 pmol of Hca-Thr-Pro-Arg-Leu-NH 2 or 5 pmol of PBAN produced statistically similar amounts of pheromone (t 5 2.11, 10 d.f.). The results indicate that the pseudopeptide has superagonistic effects when compared to PBAN because 0.5 pmol of PBAN is required to stimulate production of significantly more pheromone than controls and 5.0 pmol of PBAN is required for optimal production of pheromone [12]. This pseudotetrapeptide was synthesized to determine if the amino group of Phe was critical for the activity of the C-terminal active core of
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Fig. 1. Relative amount of pheromone present in extracts obtained from females 60 min after injection of different doses of either Phe-Thr-Pro-Arg-LeuNH 2 (a), Hca-Thr-Pro-Arg-Leu-NH 2 (b), Fla-Phe-Thr-Pro-Arg-Leu-NH 2 (c) or Pba-Phe-Thr-Pro-Arg-Leu-NH 2 (d). The amount of pheromone (6SEM, n 5 6 / dose) is expressed as a percentage of the mean amount produced by females injected with 5 pmol of PBAN (100%, dashed line, n 5 6 / day) for each assay. Structures of each compound are shown above each graph for comparison.
Fig. 2. Amount of pheromone present in extracts of glands obtained 1 h after topical application of only water, or 1 nmol of either Phe-Thr-ProArg-Leu-NH 2 (FTPRL-NH2) or Hca-Thr-Pro-Arg-Leu-NH 2 (HcnTPRLNH2). Values represent means of eight replicates per treatment (6SEM). The mean amount of pheromone present in extracts from glands (n 5 8) obtained 1 h after injection of 5 pmol of PBAN is shown as the dashed line for comparison.
the pyrokinins (Phe-Thr-Pro-Arg-Leu-NH 2 ). The data show that deletion of the amino group of Phe does not reduce pheromonotropic activity because the pseudo-
peptide was, in fact, more potent than Phe-Thr-Pro-ArgLeu-NH 2 (Fig. 1). Two possible explanations for the increased activity of the pseudopeptide relative to Phe-ThrPro-Arg-Leu-NH 2 are that the pseudopeptide binds more strongly and is cleared from the receptor site less rapidly than Phe-Thr-Pro-Arg-Leu-NH 2 or that the pseudopeptide is more resistive to aminopeptidase attack within the insect. Although neither explanation is supported by experiments conducted here, temporal studies of the topically applied pseudopeptide indicate that the activity declines much more rapidly than either of the other pseudopeptides tested, suggesting that proteolytic enzymes act rapidly on this molecule (see below). Although both of the pseudopentapeptides induced production of pheromone when injected (Fig. 1) into moths, neither was as potent as Hca-Thr-Pro-Arg-LeuNH 2 . Fla-Phe-Thr-Pro-Arg-Leu-NH 2 was the more potent of the two pseudopentapeptides. This pseudopeptide stimulated production of significantly more pheromone when injected at 0.05 pmol than that present in extracts from insects injected with only water (t 5 4.84, 10 d.f.). Injection of 0.5 pmol of this pseudopeptide induced production of as much pheromone as was produced by injection of 5 pmol of PBAN (t 5 0.81, 10 d.f.). The dose response profile obtained for Pba-Phe-Thr-Pro-Arg-Leu-
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NH 2 indicated that the optimal dose for stimulation was 2.5 pmol. Injection of less than 2.5 pmol of this pseudopeptide resulted in production of only limited amounts of pheromone. This result suggested that the large hydrophobic pyrene moiety attached to the amino terminus rendered Pba-Phe-Thr-Pro-Arg-Leu-NH 2 less soluble in water. However, tests in which the pseudopeptide was dissolved in 20% acetonitrile for injection yielded a similar dose response curve (data not shown). Moths injected with PBAN dissolved in 20% acetonitrile produced as much pheromone as was produced when PBAN was dissolved in only water. This, plus the fact that HPLC analysis indicated that Pba-Phe-Thr-Pro-Arg-Leu-NH 2 was completely soluble in 20% acetonitrile, indicate that solubility of the pseudopeptide in water did not affect its activity. In our initial topical application studies we assessed the effects of application of the pentapeptide, Phe-Thr-ProArg-Leu-NH 2 , and its hydrocinnamic analog, Hca-ThrPro-Arg-Leu-NH 2 , on stimulation of pheromone production when applied at 1000 pmol in water. PBAN was not used for topical application studies because earlier work has shown that it will not stimulate pheromone production when applied topically in water [12]. As indicated in Fig. 2 the pentapeptide, Phe-Thr-Pro-Arg-Leu-NH 2 , did not stimulate production of any more pheromone during a 1 h incubation than was produced when water alone was applied. Doubling the amount of Phe-Thr-Pro-Arg-LeuNH 2 applied had no effect on pheromone production. However, the pseudotetrapeptide, Hca-Thr-Pro-Arg-LeuNH 2 , stimulated production of as much pheromone, when applied topically, as was produced when 5 pmol of PBAN was injected. In fact, dose response studies conducted using Hca-Thr-Pro-Arg-Leu-NH 2 indicated that topical application of 50 pmol induced significant production of pheromone in a 1 h period (Fig. 3). Both of the pseudopentapeptides tested also induced pheromone production when applied topically in water (Fig. 3). Dose response curves were similar for all three of the pseudopeptides, with significant stimulation of pheromone production occurring when 50 pmol were applied and optimal production being induced at a dose of 500 pmol. The fact that similar dose response profiles were obtained for all three pseudopeptides when applied topically was of interest because each had different dose response curves when injected (Fig. 1), with Hca-Thr-Pro-Arg-LeuNH 2 being considerably more potent than either of the pseudopentapeptides. One possible explanation for the differences between the topical and injection dose response curves was that Hca-Thr-Pro-Arg-Leu-NH 2 was the most polar of the three pseudopeptides and, as such, could have been the slowest to penetrate the cuticle. If this pseudotetrapeptide penetrated the cuticle more slowly than the pseudopentapeptides then it might be expected to have prolonged activity when applied topically because the effective dose necessary for stimulation of pheromone production is significantly lower than the other two
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Fig. 3. Amounts of pheromone present in extracts obtained from glands excised 1 h after topical application of different amounts of Hca-Thr-ProArg-Leu-NH 2 (a) or Fla-Phe-Thr-Pro-Arg-Leu-NH 2 (b) or Pba-Phe-ThrPro-Arg-Leu-NH 2 (c). Values represent the mean amounts determined from analysis of eight replicates / dose (6SEM).
pseudopeptides and should be maintained over long periods due to slow release into the body. To address this question we conducted studies in which we applied a dose of the pseudopeptides sufficient to readily stimulate maximum production of pheromone in a 1 h period, but allowed the preparations to incubate for periods of up to 24 h before extracting the pheromone glands. Application times for these experiments were staggered to insure that all pheromone glands were excised during the photophase when pheromone is not normally produced.
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Contrary to the above prediction, the results of these studies indicated that the activity of Hca-Thr-Pro-Arg-LeuNH 2 was maintained for the shortest length of time, being constant for the first 4 h after application and declining to resting levels by 6 h (Fig. 4). This time response profile was similar to that obtained when PBAN is injected [14] and suggests that the pseudotetrapeptide penetrates the cuticle rapidly and is subjected to rapid deactivation, presumably due to the action of peptidases. Pheromone production induced by application of 1 nmol of either Fla-Phe-Thr-Pro-Arg-Leu-NH 2 or Pba-Phe-Thr-Pro-Arg-
Fig. 4. Amounts of pheromone present in extract obtained from glands obtained at different times after topical application of 1 nmol of Hca-ThrPro-Arg-Leu-NH 2 (a) or Fla-Phe-Thr-Pro-Arg-Leu-NH 2 (b) or Pba-PheThr-Pro-Arg-Leu-NH 2 (c). Values represent the mean amounts determined from analysis of 12 replicates / dose (6SEM).
Leu-NH 2 remained high for 18 h after application. We believe that the prolonged period of activity of the two pseudopentapeptides, relative to Hca-Thr-Pro-Arg-LeuNH 2 , reflects the fact that the two pseudopentapeptides have greater resistance to peptidase attack and that the balance between the apolar and polar characteristics of Fla-Phe-Thr-Pro-Arg-Leu-NH 2 and Pba-Phe-Thr-Pro-ArgLeu-NH 2 allows for slow continuous release through the cuticle and into the insect. Thus, a constant slow release of the pseudopeptides maintains sufficient titers to induce pheromone production over a prolonged period. However, these pseudopeptides do lose their activity over time, indicating that they are eventually degraded by the insect. The prolonged period of pheromone production induced by application of Fla-Phe-Thr-Pro-Arg-Leu-NH 2 and PbaPhe-Thr-Pro-Arg-Leu-NH 2 was of interest to us because one possible use of these neuropeptide analogs for insect pest control is to inhibit the natural production of pheromone by depletion of precursors in the pheromone gland [8,12]. To determine if the ability to produce pheromone was diminished among females treated with 1 nmol of Fla-Phe-Thr-Pro-Arg-Leu-NH 2 or Pba-Phe-Thr-Pro-ArgLeu-NH 2 we incubated females for 23 h after application of the pseudopeptides and then injected either water or 5 pmol of PBAN. Pheromone glands excised and extracted 24 h after topical application of Fla-Phe-Thr-Pro-Arg-LeuNH 2 or Pba-Phe-Thr-Pro-Arg-Leu-NH 2 and 1 h after injection of PBAN contained an average of 229.1 (619.4, n 5 8) and 186.1 (624.9, n 5 8) ng of pheromone, respectively. However, extracts obtained from similarly treated females that had been injected with only water contained only 20.7 (63.4, n 5 8) and 13.9 (62.8, n 5 8) ng, respectively. The results indicate that pseudopeptides are degraded over time and that prolonged exposure to the pseudopeptides has no effect on the ability of H. virescens females to produce pheromone. However, they do not imply that depletion of pheromone precursor might occur in other species because females of H. virescens do not appear to store or use fatty acyl precursors during pheromone production [15] whereas other species do. For example, studies on pheromone biosynthesis by females of Manduca sexta have shown that continuous exposure to PBAN causes significant declines in the amounts of pheromone precursors stored as triacyl glycerols in the pheromone gland [16,17]. Therefore, these pseudopeptides may effectively deplete pheromone precursors in other species. Although our studies have centered on the abilities of the pseudopeptides to induce pheromone production, the results have other implications because the pyrokinin class of insect neuropeptides regulate a number of other physiological functions including: hindgut and oviduct myotropic activity and induction of pupariation and egg diapause [18]. Thus, the analogs tested here may have application for use in development of control strategies based on these other physiological parameters. In summary, the results of our studies have provided
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significant information on the modifications that can be incorporated into pseudopeptides to allow them to penetrate the insect cuticle while still maintaining biological activity. The results indicate that replacement of the amino group of phenylalanine with hydrogen in the pentapeptide, Phe-Thr-Pro-Arg-Leu-NH 2 , is all that is required to impart sufficient lipidic character to allow the molecule to penetrate the hydrophobic wax layer of the insect cuticle. Thus, only minor substitutions of single amino acids are required to render pyrokinins both amphiphilic and more potent than their parent peptides. The increased potency of HcaThr-Pro-Arg-Leu-NH 2 relative to the parent peptide, PheThr-Pro-Arg-Leu-NH 2 , could reflects its greater resistance to hemolymph aminopeptidase attack as this would allow more of the pseudopeptide to reach the target receptor. However, the pseudopeptide is clearly degraded rapidly as is indicated by the short period of bioactivity observed in temporal activity studies. Alternatively, the substitution of hydrocinnamic acid for phenylalanine could give the pseudopeptide greater affinity for the receptor which would result in enhanced biological activity. Although, when injected, neither Fla-Phe-Thr-Pro-Arg-Leu-NH 2 nor PbaPhe-Thr-Pro-Arg-Leu-NH 2 were as potent as Hca-Thr-ProArg-Leu-NH 2 , all three were equipotent when applied topically and the pseudopentapeptides induced pheromone production for a much longer period than did Hca-Thr-ProArg-Leu-NH 2 . This probably reflects the fact that both Fla-Phe-Thr-Pro-Arg-Leu-NH 2 and Pba-Phe-Thr-Pro-ArgLeu-NH 2 are highly resistant to aminopeptidase attack and that the apolar nature of the amino terminal fluorene or pyrene allows for slow continuous release of these molecules through the cuticle. The development of pseudopeptide analogs that have prolonged periods of bioactivity is a key element in the development of these compounds for use in insect control because it reduces the need for continuous exposure for maintenance of activity. The technology developed for the pyrokinin class of neuropeptides should be applicable to other types of insect neuropeptides. Therefore, long lasting pseudopeptide analogs of other neuropeptides, for example those inhibiting reproductive development, could be used for direct control of insect pests.
References [1] Starrat AN, Brown BE. Structure of the pentapeptide proctolin, a proposed neurotransmitter in insects. Life Sci 1975;17:1253–6. [2] Stone JV, Mordue W, Batley KE, Morris HR. Structure of locust adipokinetic hormone, a neurohormone that regulates lipid utilization during flight. Nature 1976;263:207–11.
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[3] Holman GM, Nachman RJ, Wright MS. Insect neuropeptides. Annu Rev Entomol 1990;35:201–17. [4] Kelly TJ, Masler EP, Menn JJ. Insect neuropeptides: New strategies for insect control. In: Casida JE, editor. Pesticides and Alternatives: Innovative Chemical and Biological Approaches to Pest Control. Amsterdam: Elsevier, 1990:283–97. [5] Masler EP, Kelly TJ, Menn JJ. Insect neuropeptides: Discovery and application in insect management. Arch Insect Biochem Physiol 1993;22:87–111. [6] Keeley LL, Hayes TK. Speculations on biotechnology applications for insect neuroendocrine research. Insect Biochem 1987;17:639– 51. [7] Nachman RJ, Holman GM, Haddon WF. Leads for insect neuropeptide mimetic development. Arch Insect Biochem Physiol 1993;22:181–97. [8] Nachman RJ, Teal PEA, Radel PA, Holman GM, Abernathy RL. Potent pheromonotropic / myotropic activity of a carboranyl pseudotetrapeptide analogue of the insect pyrokinin / PBAN neuropeptide family administered via injection or topical application. Peptides 1996;17:747–52. [9] Teal PEA, Abernathy RL, Nachman RJ, Fang N, Meredith JA, Tumlinson JH. Pheromone biosynthesis activating neuropeptides: Functions and chemistry. Peptides 1996;17:337–44. [10] Nachman RJ, Roberts VA, Dyson HJ, Holman GM. Active conformation of an insect neuropeptide family. Proc Natl Acad Sci USA 1991;88:4518–22. [11] Abernathy RL, Nachman RJ, Teal PEA, Yamashita O, Tumlinson JH. Pheromonotropic activity of naturally occurring pyrokinin insect neuropeptides (FXPRLamide) in Helicoverpa zea. Peptides 1995;16:215–9. [12] Abernathy RL, Teal PEA, Meredith JA, Nachman RJ. Induction of pheromone production in a moth by topical application of a pseudopeptide mimic of a pheromonotropic neuropeptide. Proc Natl Acad Sci USA 1996;93:12621–5. [13] Christensen TC, Itagaki H, Teal PEA, Jasensky R, Tumlinson JH, Hildebrand JG. Innervation and neural regulation of the sex pheromone gland in female Heliothis moths. Proc Natl Acad Sci USA 1991;88:4971–5. [14] Teal PEA, Oostendorp A, Tumlinson JH. Induction of pheromone production in females of Heliothis virescens (F.) and H. subflexa (Gn.) (Lepidoptera: Noctuidae) during the photophase. Can Entomol 1993;125:355–66. [15] Jurenka RA. Signal-transduction in the stimulation of sex pheromone biosynthesis in moths. Arch Insect Biochem Physiol 1996;33:245–58. [16] Fang N, Teal PEA, Tumlinson JH. Correlation between gylcerolipids and pheromone aldehydes in the sex pheromone gland of female tobacco hornworm moths Manduca sexta (L.). Arch Insect Biochem Physiol 1995;30:321–36. [17] Fang N, Teal PEA, Tumlinson JH. Effects of decapitation and PBAN injection on amounts of triacylglycerols in the sex pheromone gland of Manduca sexta (L.). Arch Insect Biochem Physiol 1996;32:249–60. [18] Nachman RJ, Roberts VA, Lange AB, Orchard I, Holman GM, Teal, PEA. Active confirmation and mimetic analog development for the pyrokinin-PBAN-diapause-pupariation and myosuppressin insect neuropeptide families. In: Hedin PA, Hollingworth RM, Masler EP, Miyamoto J, Tompson DG, editors. Phytochemicals for Pest Control. Am Chem Soc Symposium Series 658. Washington: American Chemical Society, 1997:277–91.