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PBAN neuropeptide family administered via injection or topical application
Peptides, Vol. 17,No. 5, pp. 747-752, 1996 Published by Elsevier Science Inc. Printed in the USA. All rights reserved 0196.9781/96 $15.00 + .OO
PI1 SO196-9781(96)00111-8
ELSEVIER
Potent Phleromonotropic /Myotropic Activity of a Carboranyl Pseudotetrapeptide Analogue of the Insect Pyrokinin/PBAN Neuropeptide Family Administered via Injection or Topical Application RONALD
J. NACHMAN,*”
PETER E. A. TEAL,t PEGGY A. RADEL,$ RELLA L. ABERNATHYZ
G. MARK
HOLMAN*
AND
*Veterinary Entomology Research Unit, FAPRL, USDA-ARS, 2881 F&B Road, College Station, TX 77845, USA flnsect Attractants, Behavior and Basic Biology Research Laboratory, USDA-ARS, Gainesville, FL 32611, USA $Department of Pharmaceutical Chemistry, U.C. San Francisco, CA 94143, USA Received
6 February
1996
NACHMAN, R. J., P. E. A. TEAL, P. A. RADEL, G. M. HOLMAN AND R. L. ABERNATHY. Potent pheromonotropic/ myotropic activity qf a carboranyl pseudotetrapeptide analogue of the insect pyrokinin/PBAN neuropeptide family administered via injection or topical application. PEPTIDES 17(5) 747-752, 1996.-A pseudotetrapeptide analogue of the pyrokinin/PBAN or FXPRLamide family (Cbe-Thr-Pro-Arg-Leu-NH*; Cbe = 2-o-carboranylethanoyl-), in which the phenyl ring of the Phe side chain is replaced with the hydrophobic cage-like o-carborane moiety, was synthesized and found to be IO-fold more potent than cockroach leucopyrokinin on an isolated cockroach hindgut bioassay system. In contrast with the naturally occurring peptide, the myostimulatory activity could not be immediately reversed following a saline rinse, providing evidence that the pseudopeptide analogue binds very strongly to the receptor. Once the analogue reaches the receptor, strong receptor binding characteristics may allow it to avoid inactivation by hemolymph peptidases. Although it has an eightfold smaller sequence than the endogenous 33membered pheromone biosynthesis activating neuropeptide (PBAN), the carboranyl analogue is IO-fold more potent in an in vivo pheromonotropic bioassay of the female tobacco budworm moth Heliothis virescens, demonstrating that the small, C-terminal pentapeptide pyrokinin core analogue contains all the structural information necessary to fully activate pyrokinin receptors. In contrast with PBAb’, the amphiphylic carboranyl analogue elicits pheromone production following topical application in aqueous solution to the lateral abdominal surface of H. virescens, providing a noninvasive means of inducing pheromone production in moths. The analogue can potentially serve as a useful tool to insect researchers studying, and/or attempting to disrupt, physiological processes regulated by pyrokinin-like neuropeptides in insects. A possible role for this and related pyrokinin analogues in future pest insect management strategies is briefly discussed. Tobacco
budworm
Cockroach
Peptidomimetic
Irreversible
THE pyrokinin family of insect neuropeptides is widespread in several insect orders and has been associated with a variety of different physiological functions (29). Although the first pyrokinin, leucopyrokinin (LPI<), was originally identified as a hindgut and oviduct myotropin of the cockroach Leucophaea maderae (7), other members have since been isolated from other insect species on the basis of their ability to stimulate contractions of the oviduct of the locust (26-28,30,3 1), sex pheromone biosynthesis in the corn earworm (25) and silkworm ( IO, 11)) reddish coloration and melanization in silkworm larvae ( 15)) and diapause induction in silkworm moth eggs (9). Although the members of this class of insect neuropeptides vary from 8 to 34 amino acids in length (29), they share the common C-terminal
binding
Hindgut
pentapeptide Phe-X-Pro-Arg-Leu-NH2 (X = Gly, Ser, Thr, Val) (9). The common C-terminal pentapeptide accounts for the considerable cross-activity observed for members of this family of peptides from diverse sources in the different physiological systems (2,6,13,14,17,19). Studies on an active, rigid cyclic pyrokinin analogue indicate that successful receptor interaction involves a p-turn over residues X-Pro-Arg-Leu-NH, of the C-terminal pentapeptide core (21,22). Although this pentapeptide represents the active core (minimum residue fragment) for biological activity in myotropic ( 18), pheromonotropic (8), melanization ( 14), and diapause induction assays ( 19), the observed potency is generally only a fraction of that of the respective parent pyrokinin peptide.
’ Requests for reprints should be addressed to Ronald J. Nachman.
747
748
NACHMAN
Although members of the pyrokinin class of peptides can influence a number of physiological processes critical to insects, they are unsuitable as pest insect control agents and/or tools for insect neuroendocrinologists due to their large sequence, susceptibility to peptidases in the hemolymph and gut, and poor adaptation for passage through the exterior cuticle or gut wall (20). In this article, we report on the synthesis and biological evaluation of a pseudotetrapeptide analogue of the pyrokinin core region that incorporates the hydrophobic carborane moiety as a replacement for the phenyl ring of the Phe residue. The resulting tetrapeptide analogue, protected from aminopeptidase attack, is evaluated in an isolated cockroach hindgut assay and via injection in an in vivo pheromonotropic assay in the tobacco budworm moth Heliothis virescens in comparison with the endogenous 8membered LPK and 33-membered pheromone biosynthesis activating neuropeptide (PBAN) , respectively. The amphiphylic pseudopeptide analogue is also evaluated in pheromonotropic trals in which an aqueous solution is topically applied to the lateral abdominal surface of H. virescens. METHOD
Pseudopeptide
Synthesis
Cbe or 2-o-carboranylethanoic acid was synthesized according to a previously described procedure (23,24). The pyrokinin pseudotetrapeptide analogue was synthesized by condensation of Cbe to the Thr-Pro-Arg(Pmc)-Leu-rink amide resin complex with one equivalent of a 1,3-diisopropylcarbodiimide/ l-hydroxy-7-azabenzotriazole mixture in dimethyl sulfoxide for 4 h according to previously described procedures. The Thr-ProArg( Pmc)-Leu-rink amide resin complex was synthesized via FMOC methodology as previously described ( 16). Protected amino acid starting materials were purchased from Advanced Chemtech (Louisville, KY). The crude product was cleaved from the resin by stirring the resin complex with a mixture of trifluoroacetic acid (95%), anisole (5%). thioanisole (4%), and 1,2-ethanedithiol (1%) for 1 h at RT. The resin was filtered and volatile reagents were removed in vacua on a Savant Speed Vat concentrator at 40°C. The product was purified on a Waters C 18 Sep Pak cartridge and a Delta Pak Cl8 reverse-phase column on a Waters Model 510 HPLC controlled with a Millenium 2010 chromatography manager system (Waters, Milford, MA) with detection at 214 nm at ambient temperature. Solvent A = 0.1% aqueous trifluoroacetic acid (TFA); solvent B = 80% aqueous acetonitrile containing 0.1% TFA. Conditions: initial solvent consisting of 20% B was followed by Waters linear program 6100% B over 40 min; flow rate 2 ml/min. Retention time of carboranyl pseudotetrapaptide Cbe-Thr-Pro-Arg-Leu-NH, : 2 1.5 min. The pure pseudotetrapeptide was analyzed and quantitated via amino acid analysis under previously described procedures (16), revealing the following analyses: L( l.O), P( l.O), R(0.9), and T ( 1.O) . Fast atom bombardment (FAB ) mass spectra were obtained by adding 10 pg of pseudopeptide to glycerol ( 1.5 ~1) on a copper probe, followed by bombardment with 8KV Xe atoms on a Kratos MS-50 mass spectrometer (Kratos, Manchester, UK). The structural identity was confirmed by the presence of the following molecular ions (MH+ ) : Cbe-Thr-Pro-Arg-LeuNH2, 685.7 (talc. MH+: 685.52). The molecular ion revealed an ion distribution consistent with the presence of 10 boron atoms. Cockroach
Myotropic
Leucophaea
onies maintained
Bioassay
maderae
cockroaches were taken from stock colat 27°C and fed dry dog food ad lib. Cockroach
ET AL.
hindguts, free of central nervous system (CNS) tissue, were dissected, immersed in saline, and prepared for recording of myogenie activity as previously described (8). Threshold concentrations were determined for each analogue by adding a known quantity of peptide [dissolved in 0.5 ml bioassay saline) to the bioassay chamber containing the hindgut and observing the response on a Gould 2200s oscillograph recorder. The quantity of the peptide analogues was calculated from the values obtained for Phe in the amino acid analysis. The threshold concentration was defined as the minimum concentration of peptide in the chamber required to evoke an observable change in the frequency (50%) or amplitude of contractions (5%) within 1 min and sustained for 3 min. Threshold concentrations were obtained from five cockroach hindguts on 5 consecutive days for each peptide. Quantitative data for dose-response plots were obtained as previously described (8). Heliothis
Virescens
Pupae of H. virescens were obtained from a laboratory colony maintained at the Insect Attractants, Behavior and Basic Biology Research Laboratory, USDA-ARS, in Gainesville, FL. The pupae were separated by sex and the males were discarded. After eclosion, the females were held in 4-l cages and provided with cotton wicks soaked with 5% sucrose solution. The insects were held at 25 t 2°C 65 + 3% relative humidity with a 12: 12 (L:D) photoperiod. Virgin females were used for bioassays 2 and 3 days after eclosion during the 7-8th hour of the photophase when endogenous levels of pheromone are very low ( 1) . Pheromonotropic
Assay (Injection)
biosynthesis activating neuropeptide from H. zea and the carboranyl pseudopeptide Cbe-Thr-ProArg-Leu-NH? were dissolved in physiological saline (3). Female H. virescens insects were injected through the side of the third abdominal segment using a 26-ga needle with 20 ~1 of the above solutions. Each female received a dose of 0.01, 0.03,0.1, 0.3, 1, 3, 10, 30, 100, or 300 pmol. After 1 h of incubation, the terminal abdominal segments, which contain the pheromone gland, were excised and placed in 10 11 of hexane containing 10 ng each of internal standards (octadecane and nonadecane) used for quantitation and calculation of relative retention indices. The pheromone gland extracts were analyzed for the amount of (2) 1 1-hexadecenal (Zl 1- 16:AL), the major sex pheromone component of H. virescens ( 12) by capillary gas chromatography. Hewlett-Packard 5890 gas chromatographs were equipped with splitless injectors, flame ionization detectors, and 30 m X 0.25 mm (i.d.) fused silica SPB 10 (polar) or SPB 1 (apolar) columns (Supelco). Conditions of chromatography were as follows: initial temperature = 60°C; injector operated in the splitless mode for 0.5 min; oven temperature increased by 30”C/min after 1 min; final temperature = 190°C (SPB 10) or 200°C (SPB 1) ; helium carrier gas at a linear flow velocity of 18 cm/s. A Nelson Analytical 3000” acquisition system (Cupertino, CA) was used to collect and analyze the data (34). After the dose response of Hez-PBAN was completed, the 5 pmol dose was chosen to use for comparison with the carboranyl pseudopeptide assayed here. Insects were also injected with saline alone as a control. The pseudopeptide was prepared by dilution with saline as described above. Based on the dose-response for the activity of Hez-PBAN, 10 doses were chosen to assess the pheromonotropic activity of the carboranyl pseudopeptide. Females were injected with 20 ~1 containing 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, or 300 pmol. Each bioassay consisted of one dose of several different peptides along with saline and Pheromone
( Hez-PBAN)
INSECT
ANALOGUE
IN HINDGUT
749
ASSAY
amount of pheromone determined for the injected insects.
by the method described
above
RESULTS
The carboranyl pseudotetrapeptide analogue Cbe-Thr-ProArg-Leu-NH, ( Cbe= 2-o-carboranylethanoyl or Cb-CH2-CH2C(O)- [Cb = o-carborane] ) (Fig. 1) of the pyrokinin/PBAN family demonstrates potent activity on an isolated Leucophaea maderae cockroach hindgut bioassay at a threshold concentration of 70 pM (Table 1) , over 30 and 10 times more potent than the parent pentapeptide core Phe-Thr-Pro-Arg-Leu-NH2 and the Smer LPK, respectively ( 18). Whereas LPK and its fragments demonstrate reversible stimulation of contractions of the isolated cockroach hindgut, the potent myostimulatory activity of the carboranyl analogue could not be readily reversed even following repeated saline rinses (Fig. 2). The myostimulatory activity arising from LPK immediately vanished following a saline rinse, whereas that from the pseudopeptide remained stable and only gradually subsided over a 20-min period to reach a resting state. This slow reduction of myostimulatory activity could result from either downregulation of the receptor or degradation following very slow dissociation from the binding site. This same pattern is observed with LPK that is not rinsed from the isolated hindgut preparation. The pseudopeptide analogue also elicited pheromone production following injection into the female tobacco budworm moth Heliothis virescens. Dose-response data (Fig. 3 ) reveal that the small carboranyl analogue demonstrated an ED5,, of 0.1 pmol/ female and elicits a 100% response at 2.5 pmol/female, more potent than the 33-membered PBAN molecule endogenous to Helicoverpa . By contrast, the pyrokinin pentapeptide core fragment Phe-Thr-Pro-Arg-Leu-NH2 elicits a truncated maximal response in the related moth H. zea of only 30% of the pheromone titer of PBAN at a larger dose of 70 pmol/female (2). In another comparison, an 1800-fold greater dose of the pyrokinin pentapeptide core is required to achieve a 30% pheromonotropic titre in H. zea than for Hez-PBAN in H. zea or for either the carborany1 analogue or Hez-PBAN in H. virescens. No data exist for pheromone production of the pentapeptide core fragment in H. virescens, but a comparison of existing dose-response data for
FIG. 1.In the carbonane pseuclotetrapeptide analogue (above), the phenyl ring of the Phe side chain of the insect pyrokinin C-terminal pentapeptide core (below) is replaced with the cage-like or ball-shaped, hydrophobic carborane moiety.
Hez-PBAN controls. The amount of pheromone present in extracts of the pheromone glands of females from each group was compared with that produc:ed by females injected with 5 pmol of Hez-PBAN during that particular bioassay. After all the bioassays were completed, the amount of pheromone produced by each insect was calculated as a percentage of the amount of pheromone produced by femal’es treated with Hez-PBAN. Data were analyzed using one-way ANOVA and t-test with Statview@. Variability was reported as SEM. Pheromonotropic Assay (Topical) The amphiphilic carboranyl analogue Cbe-Thr-Pro-Arg-LeuNH, was dissolved in water and solutions at concentrations of 5, 10, 25, 60, and 200 pmol/yl were prepared. The cuticular scales along the lateral side of the abdomen were removed with a l-ml plastic pipet tip attached to house vacuum with a rubber hose. A low vacuum was applied lto the apparatus and the pipet tip was gently passed over the cuticle until the scales were drawn by suction. After the scales were removed, 1 ~1 of peptide solution was applied to the surface of the cuticle and smoothed evenly over the prepared surface (1). The insects were allowed to incubate 1 h and then the pheromone gland was excised and the
TABLE COMPARATIVE
ACTIVITY
1
OF THE CARBORANYL PYROKININ ANALOG Cbe-&-Pro-Arg-La-NH2 AND NATURAL HINDGUT MYOTROPIC AND Heliothis MOTH PHEROMONOTROPIC BIOASSAYS
PYROKININS
IN COCKROACH
Pheromone Production in Female Tobacco Budworm Moth (Heliofhis virescens)
Peptide Analogue
Cbe-Thr-Pro-Arg-Leu-NH*? Phe-Thr-Pro-Arg-Leu-NH2 pQTSPhe-Thr-Pro-Arg-Leu-NH* (leucopyrokinin[LPK]) HezPBANs
Via Injection
Myostimulation of the Isolated Cockroach (Leucophaea maderae) Hindgut Threshold Concentration (PM)
ED,o
Maximal Response*
ED1o
Maximal Response*
(pm&
(pm&
(pm00
@mot)
70 2,200 (18)
60
5Of 0 (2)
650 (18) 2,600 (20)
0.1
5$ 1.0
Via Topical Application
2.5
(2) (33)
70$9
(2)
25 -
70$ 5.0
(2) (33)
Inactive’
* Maximal pheromone production relative to 5 pmol PBAN via injection. 7 Cbe = 2-o-carboranylethanoyl-. $ Data from the related corn earworm moth H. zea. $ Maximal response is a truncated 30% of that elicited by HezPBAN. ( HezPBAN = LSDDMPATPADQEMYRQDPEQIDSRTKYPhe-Ser-Pro-Arg-Leu-NH~. # Topical application of aqueous solutions of HezPBAN at a 100 pmol dose does not lead to production production over a control grcup at 15 mitt, 1 h, 2 h, or even 3 h following treatment.
-
of statistically
different
Inactive’
levels of pheromone
750
NACHMAN
*
Time (min.)
FIG. 2. The stimulation of spontaneous contractions of the isolated cockroach hindgut elicited by the endogenous peptide LPK is immediately reversed by a saline rinse (top). In contrast, the myostimulatory response elicited by the carborane pseudotetrapeptide cannot be reversed by one or more saline rinses (bottom). The myostimulatory activity elicited by the pseudopeptide analogue gradually subsides over about 20 min, presumably as a result of downregulation of the receptor. The up arrow indicates the point of application of the test peptide solution and the down arrow signifies the saline rinse. See the Method section.
the two moths suggests that the response profile is similar ( 1,2,33). Dose-response data for pheromone production following topical application of the amphiphilic carboranyl analogue in a l-p1 volume of aqueous solution to the lateral abdominal surface of H. virescens is illustrated in Fig. 4. The pseudopeptide analogue elicits pheromone production at an EDS0 of 25 pmol/ female and approaches a 100% maximal response at 60 pmol/ female 15 mitt following topical application to the surface of the moth. Topical application of aqueous solutions of PBAN at a 100 pmol dose does not lead to production of statistically different levels of pheromone production in H. virescens over a control group at 15 min, or 1, 2, and even 3 h following treatment ( 1).
ET AL.
The pseudopeptide demonstrates potent activity on the isolated Leucophaea maderae hindgut bioassay with a threshold response at 70 pM, 30 and 10 times more active than the Cterminal pentapeptide core and the endogenous &membered LPK, respectively. When this small tetrapeptide analogue is injected into the female tobacco budwotm moth Helicoverpa virescens, it can elicit pheromone production with an ED50 of 0.1 pmol/female, lo-fold more potent than the much larger 33-membered Hez-PBAN peptide. The potent characteristics of the pseudopeptide analogue in the in vivo pheromonotropic assay are likely due in part to its blocked N-terminus, which would protect it from aminopeptidase attack. Hez-PBAN is not blocked at the N-terminus. However, the isolated (in vitro) hindgut is suspended in a saline solution and the effect of dissolved peptide samples occurs virtually instantaneously following introduction into the chamber. Relative potency of sample peptide analogues in this assay, therefore, is largely due to receptor interaction characteristics rather than peptidase enzyme stability. Yet the carboranyl analogue is 10 times more potent than the N-terminally blocked cockroach pyrokinin LPK. Thus, the potency of this analogue is probably also due to its strong receptor binding and/or interaction characteristics. The potency and efficacy observed for this small fragment analogue demonstrate that the C-terminal pentapeptide core region of the natural pyrokinins, regardless of sequence length, contains all the structual information necessary to fully activate the receptor sites. Naturally occurring pyrokinin peptides form a dynamic equilibrium between a bound and a solution state in the hemolymph, where they can be inactivated by both soluble and tissue-bound peptidases. The carboranyl analogue is conceivably afforded greater protection from encounters with peptidases by virtue of its propensity to remain in a receptor-bound state. Carboranyl analogues of insect neuropeptides represent an alternate class of analogues that can avoid peptidase attack. Although traditional analogue strategies involve the incorporation of peptidase resistant components into natural peptide sequences, carboranyl analogues can potentially
Dose response of carborane pseudopeptide in Hefiofhis virescens as percent of amount produced with 5 pmol PBAN z
no-
DISCUSSION
The carboranyl pseudotetrapeptide analogue Cbe-Thr-ProArg-Leu-NH2 features a replacement of the phenyl ring of the Phe residue of the C-terminal pentapeptide core region Phe-ThrPro-Arg-Leu-NHn of the pyrokinin/PBAN insect neuropeptide family. The dodecahedral volume created by rotation of the planar phenyl ring over 360” is mimicked by the ball-shaped or cagelike carborane structure (Fig. 1) . It is capable of mimicking the aromatic character of the phenyl ring, but is a considerably more hydrophobic moiety (35). The greater surface area and hydrophobic character provided by the carboranyl cage moiety in comparison with the planar phenyl group may explain the nearly irreversible nature of the myostimulatory response on the isolated cockroach hindgut (Fig. 2) elicited by this pseudopeptide analogue. Although the hindgut myostimulation induced by the cockroach pyrokinin LPK subsides immediately following a saline rinse, the myostimulatory effects of the carboranyl analogue cannot be reversed even following repeated saline rinses. Probably as a consequence of downregulation of the receptor, the myostimulatory effect only slowly subsides over a period of more than 20 min following the rinse. The strong receptor binding characteristics observed for this pseudopeptide make it unique among analogues of the prokinin/PBAN peptide family.
m
I
0
0.01
0.03
0.1
0.3
1
3
10
30
100
300
Injected dose per female (pmoi) 3. Effect of dose of carborane pseudopeptide on stimulation of pheromone production when injected into virgin females of H. virescens. Pheromone glands were excised and extracted 60 min after injection of the pseudopeptide. The amount of pheromone produced in response to each dose is expressed as a percentage of the amount of pheromone produced by females injected with 5 pmol of synthetic Hez-PBAN (53.4 2 7.1 ng pheromone), the optimal dose for stimulation of pheromone production. Dashed lines represent the response (100%) (horizontal) from the 5 pmol dose (vertical) of Hez-PBAN used for that particular bioassay. Values are means of eight replicates per dose (? SEM). See the Method section. FIG.
INSECT
ANALOGUE
IN HINDGUT
751
ASSAY
Dose Response of TopicallyApplied Carborane Analog
”
0
so
1W
150
2ca
260
Amcunt of An&g Applied (pmol) FIG. 4. Effect of dose of carborane
pseudopeptide applied topically in 1 ~1 of Hz0 on production of pheromone by virgin females of H. virescens. Pheromone glands were excised and extracted 60 min after application of the pseudopeptide. Values are means of 10 replicates per dose (t SEM), except for 6 pmol (n = 5). See the Method section.
avoid inactivation by peptidases because of strong receptor binding characteristics. The presence of the hydophobic carborane moiety coupled with the hydrophyllic, positively charged Arg residue confers amphiphylic properties to the pseudopeptide analogue. Aqueous solutions of the carboranyl peptide readily “wet” the lateral abdominal surface of female H. virescens. These topically applied analogue solutions also elicit pheromone production after about 15 min with an E& of 22; pmol/female and a maximal response
at about 60 pmol/female. In contrast, 100 pmol doses of HezPBAN in aqueous solution at 15 min, or 1, 2, and 3 h following topical application led to no statistical increase in pheromone production ( 1). The ability of the analogue to transmigrate through the cuticular surface of the moth means that female moths can be induced to produce pheromone without using invasive procedures such as injection. Therefore, the carboranyl analogue can potentially serve as a useful tool to insect researchers studying aspects of reproduction, endocrinology, and behavior of not only H. virescens and H. zea but a wide range of moth species. For example, PBAN was used as a tool in the identification of the pheromone components of Plodia interpunctella (32). Injection of PBAN has also been used to study the biosynthetic pathways for pheromones (4). The carboranyl analogue could replace PBAN in situations such as these and be delivered in a noninvasive manner. Continuous stimulation of pheromone production by PBAN has been shown to deplete the triacylglyceride precursors of pheromone components in Manduca sexta (5). In the future, continuous stimulation of pheromone production may be induced via timed release penetration of the cuticle by topical agents such as the carboranyl pseudopeptide. Should continuous stimulation of pheromone by topically applied agents lead to low levels of pheromone release in moths, the result could be the disruption of species propagation by impairing the ability of males to locate females. Alternatively, should an agent bind strongly and desensitize the PBAN receptor, the result could also lead to low levels of pheromone production and disruption of reproduction. Future experiments to determine the extent to which the analogue can alter insect physiology or behavior can aid in the assessment of its potential for integration into future pest insect management strategies. ACKNOWLEDGEMENTS
We wish to acknowledge the able technical assistance Tara Peterson, Gabriel Rothrock, and David Dent.
of Hugo Hein,
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rapid and sensitive bioassay tool for the isolation of insect myotropins of other insect species. Insect Biochem. 2 I ( 1) : 107- 112; 199 1. Imai, K.; Konno, T.; Nakagawa, Y.; et al. Isolation and structure of diapause hormone of the silkworm, Bombyx mori. Proc. Jpn. Acad. [B] 67:98-101; 1991. Kitamura, A.: Nagasawa, H.; Kataoka, H.; Ando, T.; Suzuki, A. Amino acid sequence of pheromone biosynthesis activating neuropeptide(PBAN-II) of the silkmoth, Bombyx mori. Agric. Biol. Chem. 54~2495-2497; 1990. Kitamura, A.; Nagasawa, H.; Kataoka, H.; et al. Amino acid sequence of pheromone-biosynthesis-activating neuropeptide (PBAN) __ of the silkworm, Bombyx mori. Biochem. giophys. Res. Commun. 163:520-526; 1989. Klun, J. A.; Bierl-Leonhardt, B. A.; Plimmer, J. R.; et al. Sex pheromone chemistry of the female tobacco budworm moth, H. virestens. J. Chem. Ecol. 6:177-183; 1980. Kuniyoshi, H.; Nagasawa, R. A.; Suzuki, A.; Nachman, R. J.; Holman, G. M. Cross-activity between pheromone biosynthesis activating neuropeptide (PBAN) and myotropic pyrokinin insect peptides. Biosci. Biotech. Biochem. 56: 167- 168; 1992. Matsumoto, S.; Fonagy, A.; Schoofs, L.; et al. Induction of cuticular melanization in the armyworm larvae, Pseudaletia separuru , by insect myotropic peptides possessing FXPRLamide at the C-terminus. J. Pestic. Sci. 18:127-129; 1993. Matsumoto, S.; Kitamura, A.; Nagasawa, H.; et al. Functional diversity of a neurohormone produced by the suboesophageal ganglion: Molecular identity of melanization and reddish coloration hormone and pheromone biosynthesis activating neuropeptide. J. Insect Physiol. 36:427-432; 1990.
752
16. Nachman, R. J.; Coast, G. M.; Holman, G. J.; Beier, R. C. Diuretic activity of C-terminal group analogs of the insect kinins in A&eta domesticus. Peptides 16:809-813; 1995. 17. Nachman, R. J.; Holman, G. M. Myotropic insect neuropeptide families from the cockroach Leucophaea maderue: Structure-activity relationships. In: Menn. J. J.; Kelly, T. J.; Masler, E. P., eds. Insect neuropeptides: Chemistry, biology and action. Washington, DC: American Chemical Society: 1991: 194-214. 18. Nachman, R. J.: Holman, G. M.; Cook, B. J. Active fragments and analogs of the insect neuropeptide leucopyrokinin: Structure-function studies. Biochem. Biophys. Res. Commun. 137:936-942; 1986. 19. Nachman, R. J.; Holman, G. M.; Schoofs, L.; Yamashita, 0. Silkworm diapause induction activity of myotropic pyrokinin (FXPRLamide) insect neuropeptides. Peptides 14:1043- 1048; 1993. 20. Nachman, R. J.; Holman, G. M.; Haddon, W. F. Leads for insect neuropeptide mimetic development. Arch. Insect Biochem. Physiol. 22:181-197; 1993. 2 I. Nachman, R. .I., Kuniyoshi, H.; Roberts. V. A.; Holman, G. M.; Suzuki, A. Active conformation of the pyrokinin/PBAN neuropeptide family for pheromone biosynthesis in the silkworm. Biochem. Biophys. Res. Commun. 193:661-666; 1993. 22. Nachman, R. J.; Roberts, V. A.; Dyson, H. J.; Holman, G. M.: Tainer, J. A. Active conformation of an insect neuropeptide family. Proc. Natl. Acad. Sci. USA 88:45 18-4522; 1991. 23. Radel, P. A.; Kahl. S. B. Enantioselective synthesis of an unnatural amino acid, L-carboranylalanine. In: Barth, R., ed. Proceedings Fifth International Symposium Neutron capture therapy. New York: Plenum Press; 1993~277. 24. Radel. P.: Kahl, S. B. Enantioselective synthesis of L- and D-carboranylalanine. J. Org. Chem. (submitted). 25. Raina, A. K.; Jaffe. H.; Kempe, T. G.; et al. Identification of a neuropeptide hormone that regulates sex pheromone production in female moths. Science 244:796-798; 1989. 26. Schoofs, L.; Holman, G. M..; Hayes, T. K.: et al. Isolation. identification. and synthesis of locustamyotropin (Lom-MT), a novel biologically active insect neuropeptide. Peptides 11:427-433: 1990.
NACHMAN
ET AL.
27. Schoofs, L.; Holman. G. M.; Hayes. T. K.: Nachman, R. J.: DeLoof, A. Isolation and identification of locustapyrokinin, a myotropic peptide from Locusta migratoria. Gen. Comp. Endocrinol. 81:97-104: 1991. 28. Schoofs, L.; Holman, G. M.; Hayes, T. K.: Nachman, R. J.; DeLoof, A. Isolation, identification, and synthesis of locustamyotropin II, an additional neump-eptide of Locusta migraforia: Members of the cephalomyotropic peptide family. Insect Biochem. 20:479-484; 1990. 29. Schoofs, L.; Holman. G. M.; Nachman, R. J.; Hayes, T. K.; DeLoof, A. Structure, function and distribution of insect myotropic peptides. In: Davey. K., G.; Peter, R. E.; Tobe. S. S.. eds. Perspectives in comparative endocrinology. Ottawa: National Research Council of Canada; 1994:155- 165. 30. Schoofs, L.: Holman, G. M.; Hayes, T. K.; Nachman, R. J.; Kochansky. J. P.; DeLoof, A. Isolation, identification and synthesis of locustamyotropin III and IV, two additional neuropeptides of Locusta migratoria: Members of the locustamyotropin peptide family. Insect Biochem. Mol. Biol. 22447-452; 1992. 3 I, Schoofs, L.; Holman, G. M.; Nachman, R. J.: Proost. P.; VonDamme, J.; DeLoof. A. Isolation, identification and synthesis of locustapyrokinin II and Locusta migrutoria, another member of the FXPRLamide peptide family. Comp. Biochem. Physiol. [C] 106:103-109; 1993. 32. Teal, P. E. A.; Heath, R. R.; Dueben, B. D.; Coffelt, J. A.; Vick, K. W. Production and release of [E,Z]-9,12_tetradecadienal by sex pheromone glands of females of Plodia interpunctella [Lepidoptera Pyralidae]. J. Chem. Ecol. 21:787-799; 1995. 33. Teal, P. E. A.; Oostendorp, A.; Tumlinson, J. H. Induction of pheromone production in females of Heliothis virescens (F.) and H. subflexa (Gn.) (Lepidoptera: Noctuidae) during the photophase. Can. Entomol. 125:355-366: 1993. 34. Teal, P. E. A.: Tumlinson, J. H.; Oberlander, H. Neural regulation of sex pheromone biosynthesis in Heliothis moths. Proc. Natl. Acad. Sci. USA 86:2488-2492; 1989. 35. Varadajaran, A.; Hawthorne, F. Novel carboranyl amino acids and peptides: Reagents for antibody modification and subsequent neutron-capture studies. Bioconjugate Chem. 2:242-253; 199 I.
PI1 SO196-9781(96)00111-8
ELSEVIER
Potent Phleromonotropic /Myotropic Activity of a Carboranyl Pseudotetrapeptide Analogue of the Insect Pyrokinin/PBAN Neuropeptide Family Administered via Injection or Topical Application RONALD
J. NACHMAN,*”
PETER E. A. TEAL,t PEGGY A. RADEL,$ RELLA L. ABERNATHYZ
G. MARK
HOLMAN*
AND
*Veterinary Entomology Research Unit, FAPRL, USDA-ARS, 2881 F&B Road, College Station, TX 77845, USA flnsect Attractants, Behavior and Basic Biology Research Laboratory, USDA-ARS, Gainesville, FL 32611, USA $Department of Pharmaceutical Chemistry, U.C. San Francisco, CA 94143, USA Received
6 February
1996
NACHMAN, R. J., P. E. A. TEAL, P. A. RADEL, G. M. HOLMAN AND R. L. ABERNATHY. Potent pheromonotropic/ myotropic activity qf a carboranyl pseudotetrapeptide analogue of the insect pyrokinin/PBAN neuropeptide family administered via injection or topical application. PEPTIDES 17(5) 747-752, 1996.-A pseudotetrapeptide analogue of the pyrokinin/PBAN or FXPRLamide family (Cbe-Thr-Pro-Arg-Leu-NH*; Cbe = 2-o-carboranylethanoyl-), in which the phenyl ring of the Phe side chain is replaced with the hydrophobic cage-like o-carborane moiety, was synthesized and found to be IO-fold more potent than cockroach leucopyrokinin on an isolated cockroach hindgut bioassay system. In contrast with the naturally occurring peptide, the myostimulatory activity could not be immediately reversed following a saline rinse, providing evidence that the pseudopeptide analogue binds very strongly to the receptor. Once the analogue reaches the receptor, strong receptor binding characteristics may allow it to avoid inactivation by hemolymph peptidases. Although it has an eightfold smaller sequence than the endogenous 33membered pheromone biosynthesis activating neuropeptide (PBAN), the carboranyl analogue is IO-fold more potent in an in vivo pheromonotropic bioassay of the female tobacco budworm moth Heliothis virescens, demonstrating that the small, C-terminal pentapeptide pyrokinin core analogue contains all the structural information necessary to fully activate pyrokinin receptors. In contrast with PBAb’, the amphiphylic carboranyl analogue elicits pheromone production following topical application in aqueous solution to the lateral abdominal surface of H. virescens, providing a noninvasive means of inducing pheromone production in moths. The analogue can potentially serve as a useful tool to insect researchers studying, and/or attempting to disrupt, physiological processes regulated by pyrokinin-like neuropeptides in insects. A possible role for this and related pyrokinin analogues in future pest insect management strategies is briefly discussed. Tobacco
budworm
Cockroach
Peptidomimetic
Irreversible
THE pyrokinin family of insect neuropeptides is widespread in several insect orders and has been associated with a variety of different physiological functions (29). Although the first pyrokinin, leucopyrokinin (LPI<), was originally identified as a hindgut and oviduct myotropin of the cockroach Leucophaea maderae (7), other members have since been isolated from other insect species on the basis of their ability to stimulate contractions of the oviduct of the locust (26-28,30,3 1), sex pheromone biosynthesis in the corn earworm (25) and silkworm ( IO, 11)) reddish coloration and melanization in silkworm larvae ( 15)) and diapause induction in silkworm moth eggs (9). Although the members of this class of insect neuropeptides vary from 8 to 34 amino acids in length (29), they share the common C-terminal
binding
Hindgut
pentapeptide Phe-X-Pro-Arg-Leu-NH2 (X = Gly, Ser, Thr, Val) (9). The common C-terminal pentapeptide accounts for the considerable cross-activity observed for members of this family of peptides from diverse sources in the different physiological systems (2,6,13,14,17,19). Studies on an active, rigid cyclic pyrokinin analogue indicate that successful receptor interaction involves a p-turn over residues X-Pro-Arg-Leu-NH, of the C-terminal pentapeptide core (21,22). Although this pentapeptide represents the active core (minimum residue fragment) for biological activity in myotropic ( 18), pheromonotropic (8), melanization ( 14), and diapause induction assays ( 19), the observed potency is generally only a fraction of that of the respective parent pyrokinin peptide.
’ Requests for reprints should be addressed to Ronald J. Nachman.
747
748
NACHMAN
Although members of the pyrokinin class of peptides can influence a number of physiological processes critical to insects, they are unsuitable as pest insect control agents and/or tools for insect neuroendocrinologists due to their large sequence, susceptibility to peptidases in the hemolymph and gut, and poor adaptation for passage through the exterior cuticle or gut wall (20). In this article, we report on the synthesis and biological evaluation of a pseudotetrapeptide analogue of the pyrokinin core region that incorporates the hydrophobic carborane moiety as a replacement for the phenyl ring of the Phe residue. The resulting tetrapeptide analogue, protected from aminopeptidase attack, is evaluated in an isolated cockroach hindgut assay and via injection in an in vivo pheromonotropic assay in the tobacco budworm moth Heliothis virescens in comparison with the endogenous 8membered LPK and 33-membered pheromone biosynthesis activating neuropeptide (PBAN) , respectively. The amphiphylic pseudopeptide analogue is also evaluated in pheromonotropic trals in which an aqueous solution is topically applied to the lateral abdominal surface of H. virescens. METHOD
Pseudopeptide
Synthesis
Cbe or 2-o-carboranylethanoic acid was synthesized according to a previously described procedure (23,24). The pyrokinin pseudotetrapeptide analogue was synthesized by condensation of Cbe to the Thr-Pro-Arg(Pmc)-Leu-rink amide resin complex with one equivalent of a 1,3-diisopropylcarbodiimide/ l-hydroxy-7-azabenzotriazole mixture in dimethyl sulfoxide for 4 h according to previously described procedures. The Thr-ProArg( Pmc)-Leu-rink amide resin complex was synthesized via FMOC methodology as previously described ( 16). Protected amino acid starting materials were purchased from Advanced Chemtech (Louisville, KY). The crude product was cleaved from the resin by stirring the resin complex with a mixture of trifluoroacetic acid (95%), anisole (5%). thioanisole (4%), and 1,2-ethanedithiol (1%) for 1 h at RT. The resin was filtered and volatile reagents were removed in vacua on a Savant Speed Vat concentrator at 40°C. The product was purified on a Waters C 18 Sep Pak cartridge and a Delta Pak Cl8 reverse-phase column on a Waters Model 510 HPLC controlled with a Millenium 2010 chromatography manager system (Waters, Milford, MA) with detection at 214 nm at ambient temperature. Solvent A = 0.1% aqueous trifluoroacetic acid (TFA); solvent B = 80% aqueous acetonitrile containing 0.1% TFA. Conditions: initial solvent consisting of 20% B was followed by Waters linear program 6100% B over 40 min; flow rate 2 ml/min. Retention time of carboranyl pseudotetrapaptide Cbe-Thr-Pro-Arg-Leu-NH, : 2 1.5 min. The pure pseudotetrapeptide was analyzed and quantitated via amino acid analysis under previously described procedures (16), revealing the following analyses: L( l.O), P( l.O), R(0.9), and T ( 1.O) . Fast atom bombardment (FAB ) mass spectra were obtained by adding 10 pg of pseudopeptide to glycerol ( 1.5 ~1) on a copper probe, followed by bombardment with 8KV Xe atoms on a Kratos MS-50 mass spectrometer (Kratos, Manchester, UK). The structural identity was confirmed by the presence of the following molecular ions (MH+ ) : Cbe-Thr-Pro-Arg-LeuNH2, 685.7 (talc. MH+: 685.52). The molecular ion revealed an ion distribution consistent with the presence of 10 boron atoms. Cockroach
Myotropic
Leucophaea
onies maintained
Bioassay
maderae
cockroaches were taken from stock colat 27°C and fed dry dog food ad lib. Cockroach
ET AL.
hindguts, free of central nervous system (CNS) tissue, were dissected, immersed in saline, and prepared for recording of myogenie activity as previously described (8). Threshold concentrations were determined for each analogue by adding a known quantity of peptide [dissolved in 0.5 ml bioassay saline) to the bioassay chamber containing the hindgut and observing the response on a Gould 2200s oscillograph recorder. The quantity of the peptide analogues was calculated from the values obtained for Phe in the amino acid analysis. The threshold concentration was defined as the minimum concentration of peptide in the chamber required to evoke an observable change in the frequency (50%) or amplitude of contractions (5%) within 1 min and sustained for 3 min. Threshold concentrations were obtained from five cockroach hindguts on 5 consecutive days for each peptide. Quantitative data for dose-response plots were obtained as previously described (8). Heliothis
Virescens
Pupae of H. virescens were obtained from a laboratory colony maintained at the Insect Attractants, Behavior and Basic Biology Research Laboratory, USDA-ARS, in Gainesville, FL. The pupae were separated by sex and the males were discarded. After eclosion, the females were held in 4-l cages and provided with cotton wicks soaked with 5% sucrose solution. The insects were held at 25 t 2°C 65 + 3% relative humidity with a 12: 12 (L:D) photoperiod. Virgin females were used for bioassays 2 and 3 days after eclosion during the 7-8th hour of the photophase when endogenous levels of pheromone are very low ( 1) . Pheromonotropic
Assay (Injection)
biosynthesis activating neuropeptide from H. zea and the carboranyl pseudopeptide Cbe-Thr-ProArg-Leu-NH? were dissolved in physiological saline (3). Female H. virescens insects were injected through the side of the third abdominal segment using a 26-ga needle with 20 ~1 of the above solutions. Each female received a dose of 0.01, 0.03,0.1, 0.3, 1, 3, 10, 30, 100, or 300 pmol. After 1 h of incubation, the terminal abdominal segments, which contain the pheromone gland, were excised and placed in 10 11 of hexane containing 10 ng each of internal standards (octadecane and nonadecane) used for quantitation and calculation of relative retention indices. The pheromone gland extracts were analyzed for the amount of (2) 1 1-hexadecenal (Zl 1- 16:AL), the major sex pheromone component of H. virescens ( 12) by capillary gas chromatography. Hewlett-Packard 5890 gas chromatographs were equipped with splitless injectors, flame ionization detectors, and 30 m X 0.25 mm (i.d.) fused silica SPB 10 (polar) or SPB 1 (apolar) columns (Supelco). Conditions of chromatography were as follows: initial temperature = 60°C; injector operated in the splitless mode for 0.5 min; oven temperature increased by 30”C/min after 1 min; final temperature = 190°C (SPB 10) or 200°C (SPB 1) ; helium carrier gas at a linear flow velocity of 18 cm/s. A Nelson Analytical 3000” acquisition system (Cupertino, CA) was used to collect and analyze the data (34). After the dose response of Hez-PBAN was completed, the 5 pmol dose was chosen to use for comparison with the carboranyl pseudopeptide assayed here. Insects were also injected with saline alone as a control. The pseudopeptide was prepared by dilution with saline as described above. Based on the dose-response for the activity of Hez-PBAN, 10 doses were chosen to assess the pheromonotropic activity of the carboranyl pseudopeptide. Females were injected with 20 ~1 containing 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, or 300 pmol. Each bioassay consisted of one dose of several different peptides along with saline and Pheromone
( Hez-PBAN)
INSECT
ANALOGUE
IN HINDGUT
749
ASSAY
amount of pheromone determined for the injected insects.
by the method described
above
RESULTS
The carboranyl pseudotetrapeptide analogue Cbe-Thr-ProArg-Leu-NH, ( Cbe= 2-o-carboranylethanoyl or Cb-CH2-CH2C(O)- [Cb = o-carborane] ) (Fig. 1) of the pyrokinin/PBAN family demonstrates potent activity on an isolated Leucophaea maderae cockroach hindgut bioassay at a threshold concentration of 70 pM (Table 1) , over 30 and 10 times more potent than the parent pentapeptide core Phe-Thr-Pro-Arg-Leu-NH2 and the Smer LPK, respectively ( 18). Whereas LPK and its fragments demonstrate reversible stimulation of contractions of the isolated cockroach hindgut, the potent myostimulatory activity of the carboranyl analogue could not be readily reversed even following repeated saline rinses (Fig. 2). The myostimulatory activity arising from LPK immediately vanished following a saline rinse, whereas that from the pseudopeptide remained stable and only gradually subsided over a 20-min period to reach a resting state. This slow reduction of myostimulatory activity could result from either downregulation of the receptor or degradation following very slow dissociation from the binding site. This same pattern is observed with LPK that is not rinsed from the isolated hindgut preparation. The pseudopeptide analogue also elicited pheromone production following injection into the female tobacco budworm moth Heliothis virescens. Dose-response data (Fig. 3 ) reveal that the small carboranyl analogue demonstrated an ED5,, of 0.1 pmol/ female and elicits a 100% response at 2.5 pmol/female, more potent than the 33-membered PBAN molecule endogenous to Helicoverpa . By contrast, the pyrokinin pentapeptide core fragment Phe-Thr-Pro-Arg-Leu-NH2 elicits a truncated maximal response in the related moth H. zea of only 30% of the pheromone titer of PBAN at a larger dose of 70 pmol/female (2). In another comparison, an 1800-fold greater dose of the pyrokinin pentapeptide core is required to achieve a 30% pheromonotropic titre in H. zea than for Hez-PBAN in H. zea or for either the carborany1 analogue or Hez-PBAN in H. virescens. No data exist for pheromone production of the pentapeptide core fragment in H. virescens, but a comparison of existing dose-response data for
FIG. 1.In the carbonane pseuclotetrapeptide analogue (above), the phenyl ring of the Phe side chain of the insect pyrokinin C-terminal pentapeptide core (below) is replaced with the cage-like or ball-shaped, hydrophobic carborane moiety.
Hez-PBAN controls. The amount of pheromone present in extracts of the pheromone glands of females from each group was compared with that produc:ed by females injected with 5 pmol of Hez-PBAN during that particular bioassay. After all the bioassays were completed, the amount of pheromone produced by each insect was calculated as a percentage of the amount of pheromone produced by femal’es treated with Hez-PBAN. Data were analyzed using one-way ANOVA and t-test with Statview@. Variability was reported as SEM. Pheromonotropic Assay (Topical) The amphiphilic carboranyl analogue Cbe-Thr-Pro-Arg-LeuNH, was dissolved in water and solutions at concentrations of 5, 10, 25, 60, and 200 pmol/yl were prepared. The cuticular scales along the lateral side of the abdomen were removed with a l-ml plastic pipet tip attached to house vacuum with a rubber hose. A low vacuum was applied lto the apparatus and the pipet tip was gently passed over the cuticle until the scales were drawn by suction. After the scales were removed, 1 ~1 of peptide solution was applied to the surface of the cuticle and smoothed evenly over the prepared surface (1). The insects were allowed to incubate 1 h and then the pheromone gland was excised and the
TABLE COMPARATIVE
ACTIVITY
1
OF THE CARBORANYL PYROKININ ANALOG Cbe-&-Pro-Arg-La-NH2 AND NATURAL HINDGUT MYOTROPIC AND Heliothis MOTH PHEROMONOTROPIC BIOASSAYS
PYROKININS
IN COCKROACH
Pheromone Production in Female Tobacco Budworm Moth (Heliofhis virescens)
Peptide Analogue
Cbe-Thr-Pro-Arg-Leu-NH*? Phe-Thr-Pro-Arg-Leu-NH2 pQTSPhe-Thr-Pro-Arg-Leu-NH* (leucopyrokinin[LPK]) HezPBANs
Via Injection
Myostimulation of the Isolated Cockroach (Leucophaea maderae) Hindgut Threshold Concentration (PM)
ED,o
Maximal Response*
ED1o
Maximal Response*
(pm&
(pm&
(pm00
@mot)
70 2,200 (18)
60
5Of 0 (2)
650 (18) 2,600 (20)
0.1
5$ 1.0
Via Topical Application
2.5
(2) (33)
70$9
(2)
25 -
70$ 5.0
(2) (33)
Inactive’
* Maximal pheromone production relative to 5 pmol PBAN via injection. 7 Cbe = 2-o-carboranylethanoyl-. $ Data from the related corn earworm moth H. zea. $ Maximal response is a truncated 30% of that elicited by HezPBAN. ( HezPBAN = LSDDMPATPADQEMYRQDPEQIDSRTKYPhe-Ser-Pro-Arg-Leu-NH~. # Topical application of aqueous solutions of HezPBAN at a 100 pmol dose does not lead to production production over a control grcup at 15 mitt, 1 h, 2 h, or even 3 h following treatment.
-
of statistically
different
Inactive’
levels of pheromone
750
NACHMAN
*
Time (min.)
FIG. 2. The stimulation of spontaneous contractions of the isolated cockroach hindgut elicited by the endogenous peptide LPK is immediately reversed by a saline rinse (top). In contrast, the myostimulatory response elicited by the carborane pseudotetrapeptide cannot be reversed by one or more saline rinses (bottom). The myostimulatory activity elicited by the pseudopeptide analogue gradually subsides over about 20 min, presumably as a result of downregulation of the receptor. The up arrow indicates the point of application of the test peptide solution and the down arrow signifies the saline rinse. See the Method section.
the two moths suggests that the response profile is similar ( 1,2,33). Dose-response data for pheromone production following topical application of the amphiphilic carboranyl analogue in a l-p1 volume of aqueous solution to the lateral abdominal surface of H. virescens is illustrated in Fig. 4. The pseudopeptide analogue elicits pheromone production at an EDS0 of 25 pmol/ female and approaches a 100% maximal response at 60 pmol/ female 15 mitt following topical application to the surface of the moth. Topical application of aqueous solutions of PBAN at a 100 pmol dose does not lead to production of statistically different levels of pheromone production in H. virescens over a control group at 15 min, or 1, 2, and even 3 h following treatment ( 1).
ET AL.
The pseudopeptide demonstrates potent activity on the isolated Leucophaea maderae hindgut bioassay with a threshold response at 70 pM, 30 and 10 times more active than the Cterminal pentapeptide core and the endogenous &membered LPK, respectively. When this small tetrapeptide analogue is injected into the female tobacco budwotm moth Helicoverpa virescens, it can elicit pheromone production with an ED50 of 0.1 pmol/female, lo-fold more potent than the much larger 33-membered Hez-PBAN peptide. The potent characteristics of the pseudopeptide analogue in the in vivo pheromonotropic assay are likely due in part to its blocked N-terminus, which would protect it from aminopeptidase attack. Hez-PBAN is not blocked at the N-terminus. However, the isolated (in vitro) hindgut is suspended in a saline solution and the effect of dissolved peptide samples occurs virtually instantaneously following introduction into the chamber. Relative potency of sample peptide analogues in this assay, therefore, is largely due to receptor interaction characteristics rather than peptidase enzyme stability. Yet the carboranyl analogue is 10 times more potent than the N-terminally blocked cockroach pyrokinin LPK. Thus, the potency of this analogue is probably also due to its strong receptor binding and/or interaction characteristics. The potency and efficacy observed for this small fragment analogue demonstrate that the C-terminal pentapeptide core region of the natural pyrokinins, regardless of sequence length, contains all the structual information necessary to fully activate the receptor sites. Naturally occurring pyrokinin peptides form a dynamic equilibrium between a bound and a solution state in the hemolymph, where they can be inactivated by both soluble and tissue-bound peptidases. The carboranyl analogue is conceivably afforded greater protection from encounters with peptidases by virtue of its propensity to remain in a receptor-bound state. Carboranyl analogues of insect neuropeptides represent an alternate class of analogues that can avoid peptidase attack. Although traditional analogue strategies involve the incorporation of peptidase resistant components into natural peptide sequences, carboranyl analogues can potentially
Dose response of carborane pseudopeptide in Hefiofhis virescens as percent of amount produced with 5 pmol PBAN z
no-
DISCUSSION
The carboranyl pseudotetrapeptide analogue Cbe-Thr-ProArg-Leu-NH2 features a replacement of the phenyl ring of the Phe residue of the C-terminal pentapeptide core region Phe-ThrPro-Arg-Leu-NHn of the pyrokinin/PBAN insect neuropeptide family. The dodecahedral volume created by rotation of the planar phenyl ring over 360” is mimicked by the ball-shaped or cagelike carborane structure (Fig. 1) . It is capable of mimicking the aromatic character of the phenyl ring, but is a considerably more hydrophobic moiety (35). The greater surface area and hydrophobic character provided by the carboranyl cage moiety in comparison with the planar phenyl group may explain the nearly irreversible nature of the myostimulatory response on the isolated cockroach hindgut (Fig. 2) elicited by this pseudopeptide analogue. Although the hindgut myostimulation induced by the cockroach pyrokinin LPK subsides immediately following a saline rinse, the myostimulatory effects of the carboranyl analogue cannot be reversed even following repeated saline rinses. Probably as a consequence of downregulation of the receptor, the myostimulatory effect only slowly subsides over a period of more than 20 min following the rinse. The strong receptor binding characteristics observed for this pseudopeptide make it unique among analogues of the prokinin/PBAN peptide family.
m
I
0
0.01
0.03
0.1
0.3
1
3
10
30
100
300
Injected dose per female (pmoi) 3. Effect of dose of carborane pseudopeptide on stimulation of pheromone production when injected into virgin females of H. virescens. Pheromone glands were excised and extracted 60 min after injection of the pseudopeptide. The amount of pheromone produced in response to each dose is expressed as a percentage of the amount of pheromone produced by females injected with 5 pmol of synthetic Hez-PBAN (53.4 2 7.1 ng pheromone), the optimal dose for stimulation of pheromone production. Dashed lines represent the response (100%) (horizontal) from the 5 pmol dose (vertical) of Hez-PBAN used for that particular bioassay. Values are means of eight replicates per dose (? SEM). See the Method section. FIG.
INSECT
ANALOGUE
IN HINDGUT
751
ASSAY
Dose Response of TopicallyApplied Carborane Analog
”
0
so
1W
150
2ca
260
Amcunt of An&g Applied (pmol) FIG. 4. Effect of dose of carborane
pseudopeptide applied topically in 1 ~1 of Hz0 on production of pheromone by virgin females of H. virescens. Pheromone glands were excised and extracted 60 min after application of the pseudopeptide. Values are means of 10 replicates per dose (t SEM), except for 6 pmol (n = 5). See the Method section.
avoid inactivation by peptidases because of strong receptor binding characteristics. The presence of the hydophobic carborane moiety coupled with the hydrophyllic, positively charged Arg residue confers amphiphylic properties to the pseudopeptide analogue. Aqueous solutions of the carboranyl peptide readily “wet” the lateral abdominal surface of female H. virescens. These topically applied analogue solutions also elicit pheromone production after about 15 min with an E& of 22; pmol/female and a maximal response
at about 60 pmol/female. In contrast, 100 pmol doses of HezPBAN in aqueous solution at 15 min, or 1, 2, and 3 h following topical application led to no statistical increase in pheromone production ( 1). The ability of the analogue to transmigrate through the cuticular surface of the moth means that female moths can be induced to produce pheromone without using invasive procedures such as injection. Therefore, the carboranyl analogue can potentially serve as a useful tool to insect researchers studying aspects of reproduction, endocrinology, and behavior of not only H. virescens and H. zea but a wide range of moth species. For example, PBAN was used as a tool in the identification of the pheromone components of Plodia interpunctella (32). Injection of PBAN has also been used to study the biosynthetic pathways for pheromones (4). The carboranyl analogue could replace PBAN in situations such as these and be delivered in a noninvasive manner. Continuous stimulation of pheromone production by PBAN has been shown to deplete the triacylglyceride precursors of pheromone components in Manduca sexta (5). In the future, continuous stimulation of pheromone production may be induced via timed release penetration of the cuticle by topical agents such as the carboranyl pseudopeptide. Should continuous stimulation of pheromone by topically applied agents lead to low levels of pheromone release in moths, the result could be the disruption of species propagation by impairing the ability of males to locate females. Alternatively, should an agent bind strongly and desensitize the PBAN receptor, the result could also lead to low levels of pheromone production and disruption of reproduction. Future experiments to determine the extent to which the analogue can alter insect physiology or behavior can aid in the assessment of its potential for integration into future pest insect management strategies. ACKNOWLEDGEMENTS
We wish to acknowledge the able technical assistance Tara Peterson, Gabriel Rothrock, and David Dent.
of Hugo Hein,
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