 |
INTRODUCTION |
It is well established that the activation of multiple receptors
by the same peptide arises from the ability of a given peptide to exist
in different interchangeable bioactive conformations (1). To attain
receptor selectivity it is, therefore, essential to restrict the
conformational space of a peptide so it can attain one bioactive conformation.
Cyclization is an important and attractive way to restrict the
conformational space of peptidic structures (1). Conformationally restricted peptides containing medium and long range cyclizations have
been mainly prepared following the same modes of cyclization of
naturally occurring peptides. These include end-to-end,
side-chain-to-side-chain, and side-chain-to-end (2). These modes of
cyclization involve modifications of side chains and ends which in many
cases are essential for bioactivity, and therefore their modification
causes loss of activity. Also, these modes of cyclization are not
sufficient to screen effectively the conformational space available for
a linear peptide with a given sequence. To overcome these limitations, we have introduced two methods called backbone cyclization (2) and
cycloscan (3).
Backbone cyclization is a general method by which a conformational
constraint is imposed on peptides through the connection of the
N
and/or C
atoms in
the peptide backbone to each other or to side chains or to the carboxyl
and amino ends (2). Thus, the cyclization can be performed while
retaining the functionality and the activity of the side chains.
Backbone cyclization allows nine new modes of cyclization in addition
to the four modes in naturally occurring peptides. Preparation of
backbone cyclic (BBC)1
peptides involves the use of a large variety of orthogonally protected
N
and C
(
-amino-,
-carboxy-, and
-thio-alkyl) amino acids building units (4-6). Synthetic procedures have been developed to incorporate these building units into peptides using the solid phase methodology (7, 8).
The advantages of backbone cyclization over the naturally occurring
modes of cyclization are because of the immense variability of spatial
orientation of the constitute residues which result from the multiple
anchoring points within a chain or between chains. This allows us to
screen the conformational space of a given peptide in an extremely
efficient manner. Furthermore, most of the modes of backbone
cyclization do not involve chemical modifications of side chains which
are essential for biological activity. Backbone cyclization has been
shown previously to convert peptides into selective and metabolically
stable peptidomimetics with enhanced biological activity as compared
with the linear parent peptide (7-10).
A rapid method for the disclosure of a BBC peptide that closely
resembles a unique agonistic or antagonistic bioactive conformation can
be obtained by the screening of BBC libraries (cycloscan) (3). Two such
libraries were described: biased and random. In our study we used the
biased library approach. The biased library is composed of a BBC
peptide in which the primary sequence of the active linear peptide is
retained and the members of the library differ solely in their
conformation. The diversity of the biased library (also termed
"sequence-biased cycloscan") resides in: (i) the modes of backbone
cyclization (2); (ii) the position of the backbone bridge along the
peptide sequence; (iii) the size of the bridge; and (iv) the chemistry
of the bridge. Biased libraries of BBC peptide allow systematic
screening of broad ranges of the conformational space available to a
given linear peptide.
In this study we present the application of sequence-biased cycloscan
for the disclosure of small BBC agonists and antagonists of the insect
pheromone biosynthesis activating neuropeptide (PBAN) which regulates,
among other functions (for a review, see Ref. 11), sex pheromone
biosynthesis in female moths (12-14).
Sexual communication between sexes in Lepidopteran species is mediated
mainly by sex pheromones (15, 16). Sex pheromones are volatile
compounds that are used by Lepidopteran insects to attract potential
mates from a distance. In Lepidoptera, sex pheromones are synthesized
and secreted from specialized glandular cells that are located in the
inter-segmental membrane between the eighth and ninth abdominal
segments (17). Sex pheromones play an important role in the elicitation
of mating behavior in moths and are, therefore, crucial for successful
mating and maintenance of reproductive isolation. Understanding the
mechanisms that underlie sex pheromone production is of major interest
and importance.
In 1984 it was first reported (12) that sex pheromone production in
Helicoverpa zea female moths is controlled by a cerebral neuroendocrine factor which was termed PBAN. Since then, the presence of PBAN has been demonstrated in a variety of moths as well as in
non-lepidopteran species, and its mode of action has been studied in
many laboratories (for review, see Refs. 11, 13, and 14). To date, four
different PBAN molecules have been isolated from three different moth
species, and their primary structures have been determined (18-21).
Examination of the primary structure of all four peptides revealed that
they share a high degree of homology.
Detailed structure-activity relationship studies (SAR), using synthetic
PBAN and shorter peptides derived from its sequence, have revealed that
the C-terminal pentapeptide, Phe-Xxx-Pro-Arg-Leu-NH2 (Xxx = Ser), which is identical in all PBAN molecules, is the active core required for biological activity (22-31). In addition, it
has been found that this C-terminal pentapeptide (where Xxx = Ser,
Gly, Thr, Val) is homologous with the C-terminal pentapeptide sequence
of other families of insect neuropeptides: the pyrokinins (PK) and the
myotropins (MT) (myotropic peptides isolated from Leucophaea
maderae and Locusta migratoria) (32-39), and also with the C-terminal region of Pseudaletia separata
pheromonotropin (Pss-PT) (40) and Bombyx mori diapause
hormone (Bom-DH) (41).
The above mentioned group of neuropeptides has been designated recently
the pyrokinin/PBAN family. The members of this family have been found
to be responsible for a variety of physiological and behavioral
functions such as: contraction of the locust oviduct (32), myotropic
activity of the cockroach and locust hindgut (32, 42), egg diapause in
the silkworm (41), and acceleration of pupariation in the fleshfly
larvae (43), in addition to stimulation of sex pheromone biosynthesis
in female moths (12-14, 44) and melanization and reddish coloration in
moth larvae (23, 45).
Despite the intensive studies of the bio-activity of this family of
peptides with respect to sex pheromone biosynthesis and the other
functions, very little is known about the endogenous mechanism and the
structural, chemical, and cellular basis of their activity. It is still
not known which endogenous peptide(s) mediates each of these functions
in vivo, whether each function is mediated by a different
peptide and if each peptide mediates one or several functions. It is
also not clear which receptors mediate these functions, what are their
characteristics, and whether the receptors share functional homologies.
A promising approach for resolving these issues is the use of
receptor-selective agonists and/or antagonists, as was demonstrated
previously for vertebrate neuropeptides (46-49). Backbone cyclization
and cycloscan provides a good solution for this issue by introducing
conformational constraint into peptides and thus, acquiring them with
higher selectivity because of the restricted conformational space which
can be recognized only by one receptor. Backbone cyclization also
acquires peptides with higher potency, enhanced metabolic stability,
and improved bioavailability. The advantages introduced by backbone
cyclization and cycloscan as well as the availability of
bioassays for the pyrokinin/PBAN family of peptides (23, 39, 43, 44)
(which are required to obtain the essential information for the design of BBC peptides and specificity for the desired biological effects), led us to combine both approaches for the generation of
receptor-selective agonists and antagonists.
In this study we report the generation of highly potent,
conformationally constrained BBC heptapeptide agonists and antagonists which fully inhibit one of the functions mediated by PBAN: sex pheromone biosynthesis in the female moth Heliothis
peltigera.
 |
MATERIALS AND METHODS |
General--
Fmoc-protected amino acids with standard side-chain
protecting groups as well as resins and reagents for peptide synthesis were obtained from Novabiochem (Laufelingen, Switzerland). Ultrapure quality solvents were obtained from Basker. Other reagents were obtained from Aldrich. Building units for BBC peptides were prepared as
described previously (6).
Synthesis of BBC Peptides--
BBC peptide libraries were
synthesized as described before (9). Briefly, peptides were synthesized
by the Simultaneous Multiple Peptide Synthesis methodology (50) on Rink
Amide 4-methylbenzhydrylamine Resin (loading 0.55 mmol/g) using Fmoc
chemistry and N-methylpyrrolidone as solvent. The following
BBC building units were used: t-butoxycarbonyl (Boc)-NH(CH2)n-N(Fmoc)
CH2-COOH (n = 2, 3, 4, 6). Couplings were performed for 2 h by a preactivated solution of 3-fold excess of
Fmoc amino acids or building units with
bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBrOP) and
6-fold excess of diisopropylethylamine for 5 min in
N-methylpyrrolidone. Deprotection of the Boc protecting group from the N(amino alkyl) group of the building units
was carried out with Cl3SiI, prepared in situ
from SiCl4 and NaI in ACN: dichloromethane (1:1). On resin,
cyclization was achieved by 6-fold excess of
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and repeated four times. The peptides were
removed from the resin with concomitant side chain deprotection by 90%
trifluoroacetic acid (TFA) with addition of scavengers (H2O:ethanedithiol:thioanisole, 2.5:2.5:5%). The resin was
removed by filtration and the TFA evaporated to dryness by stream of
nitrogen. The residue was extracted three times with ether to remove
the scavengers. The dry crude peptide was dissolved in water and
lyophilized. The crude peptides were purified by preparative HPLC and
characterized by mass spectrometry and amino acid analysis of
hydrolisates. All peptides gave the expected mass and were pure by
analytical HPLC. Analytical data are shown in Tables I and II.
Synthesis of Hez-PBAN--
Hez-PBAN (PBAN1-33NH2)
(18) was synthesized by the Merrifield Solid-Phase Peptide Synthesis
method (51), on an ABI peptide synthesizer Model 433A, on Rink Amide
4-methylbenzhydrylamine Resin (loading 0.55 mmol/g). The peptide was
synthesized by the FastMocTM methodology and removed from
the resin with concomitant side chain deprotection by 90% TFA with
addition of scavengers (H2O:ethanedithiol:thioanisole, 2.5:2.5:5%). Peptide purity was assessed by reverse phase-HPLC (see
legend to Table I) and found to be
95%.
Pheromonotropic Bioassay--
H. peltigera moths were
reared on an artificial diet as described previously (52).
Determination of Agonistic and Antagonistic
Activity--
Agonistic and antagonistic activities of the BBC
peptides were determined as described previously (44). Agonistic
activity of BBC peptides was determined by monitoring the ability of
the injected peptides (at 1 nmol, unless otherwise indicated) to induce sex pheromone biosynthesis in females (in the absence of
PBAN1-33NH2). Females injected with 1 nmol (unless
otherwise indicated) of PBAN1-33NH2 served as a reference
for agonistic activity. Antagonistic activity was determined by
monitoring the ability of the BBC peptides (at 1 nmol) to inhibit sex
pheromone biosynthesis evoked by 0.5 pmol of exogenously injected
PBAN1-33NH2. Females injected with 0.5 pmol of
PBAN1-33NH2 served as a reference for maximal stimulation, and those injected with 0.1 M phosphate buffer served to
determine the basal pheromone biosynthesis at photophase (which did not exceed 20 ng/female). Pheromone glands were excised 2 h
post-injection, and sex pheromone was extracted and quantified by
capillary gas chromatography as described previously (22). All
experiments were performed with a minimum of ten females per treatment.
 |
RESULTS |
Design of BBC PBAN-derived Sub-libraries--
Detailed SAR
studies, using synthetic Hez-PBAN and shorter peptides derived from its
sequence, revealed that the C-terminal pentapeptide/PBAN that is common
to all members of the pyrokinin/PBAN family comprises the active core
required for biological activity (22-31). Furthermore, studies
performed in our laboratory indicated that, under certain conditions,
the hexapeptide sequence derived from Hez-PBAN
(PBAN28-33NH2: Tyr-Phe-Ser-Pro-Arg-Leu-NH2,
MINI-PBAN) is as active as the full-length PBAN (22). Based on this
sequence, a biased library of linear peptides has been designed, and a
linear lead antagonist
([Arg27-D-Phe30]PBAN27-33NH2:
Arg-Tyr-Phe-(D)Phe-Pro-Arg-Leu-NH2) has been
disclosed (53). These findings set the basis for the design and
synthesis of two sub-libraries of cyclic peptides. The structure of the first sub-library (termed the Ser sub-library, see Fig.
1A) was based on a slight
modification of MINI-PBAN
([Arg27]PBAN27-33NH2). The second
sub-library,
[Arg27-D-Phe30]PBAN27-33NH2
(termed the D-Phe sub-library, see Fig. 1B), was based on the sequence of the lead antagonist. In both sub-libraries, following the hierarchical approach for the discovery of BBC leads, the
Pro residue was replaced by the
N
(
-amino-alkyl)Gly building unit having
various lengths of the alkyl chain (Fig. 1, n = 2-4,
6). The
-amino group of the Gly building unit was connected to the
N-terminal amino group by a dicarboxylic acid spacer (Fig. 1,
m = 2-4). All the cyclic peptides in each sub-library
had the same primary sequence and the same location of the ring. The
members of each library differed from each other by the bridge size and
the position of the amide bond along the bridge. The two sub-libraries
were tested for their agonistic and antagonistic activity using a
pheromonotropic bioassay that was optimized by us for H. peltigera (44). The aim of the experiments was to discover potent
pheromonotropic BBC antagonist(s) devoid of agonistic activity and to
determine the structural requirements for the agonistic and
antagonistic activities. These SAR studies were also performed to
assist in the further design of improved non-peptidic PBAN agonists and
antagonists.
Analysis of the Antagonistic and Agonistic Activities of the BBC
Peptides--
The first part of the study involved examination of the
ability of BBC peptides from the D-Phe sub-library
(numbered 19-30, Table II) to inhibit
sex pheromone biosynthesis evoked by exogenously administered
(injected) PBAN1-33NH2 to adult female moths. The data in
Fig. 2 depict the presence of five potent
antagonistic peptides (numbers 20, 21, 22, 25, and 28) exhibiting over
50% inhibitory activity (at 1 nmol). The inhibitory potency of these peptides ranged from 55% (peptide 25) to a maximum of 96% (peptide 20).

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Fig. 2.
Inhibition of sex pheromone biosynthesis in
H. peltigera females by cyclic peptides from the
D-Phe sub-library. Peptides were injected
to H. peltigera females at a dose of 1 nmol together with
0.5 pmol of PBAN1-33NH2. Glands were excised 2-h
post-injection, and pheromone content was determined by capillary gas
chromatography as described under "Materials and Methods." The
degree of inhibition of each peptide is expressed as 100 minus the
ratio (in percentage) between the pheromone content in the gland evoked
by the injection of PBAN1-33NH2 in the presence and
absence of each of the peptides. The amount of sex pheromone evoked by
0.5 pmol of PBAN1-33NH2 ranged from 93 to 113 ng of
pheromone/female. Pheromone content was monitored with at least ten
females for each of the tested peptides.
|
|
Antagonists may be devoid of agonistic activity, or exhibit full or
partial agonistic activity. Backbone cyclization can, in principle,
convert an antagonistic linear peptide into an agonist and vice
versa. Therefore, the antagonistic peptides (numbers 20, 21, 22, 25, and 28) were tested for their agonistic activity at the same
concentration used to assess their antagonistic activity (at 1 nmol).
Agonistic activity was determined by the ability of the peptides to
evoke sex pheromone biosynthesis in H. peltigera in the
absence of PBAN1-33NH2. Three of the five antagonists
(numbers 22, 25, and 28) were devoid of agonistic activity (Fig.
3); peptide 20 had minor agonistic
activity (10%); only one peptide (number 21) exhibited high agonistic
activity (62%) at 1 nmol.

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Fig. 3.
Comparison of agonistic and antagonistic
activities of cyclic peptides from the D-Phe
sub-library. Peptides were injected at a dose of 1 nmol. Agonistic
activity was determined in H. peltigera females by the
injection of each of the peptides for 2 h; antagonistic activity
was determined as described in the legend to Fig. 2. Agonistic activity
is expressed as the ratio (in percentage) between the sex pheromone
content evoked by the injection of each of the cyclic peptides and
PBAN1-33NH2 (at 1 nmol). The amount of sex pheromone
evoked by 1 nmol of PBAN1-33NH2 ranged from 93 to 198 ng
pheromone/female.
|
|
Synthetic PBAN1-33NH2 activates pheromone biosynthesis at
doses as low as 0.3 pmol (44). Many neuropeptides are known to cause
desensitization of their receptors at concentrations above their
EC50. To exclude the possibility that the lack of agonistic activity (at 1 nmol) of the above mentioned antagonists is not because
of desensitization, their agonistic activity was measured at lower
concentrations (e.g. 1, 10, and 100 pmol). All tested peptides (numbers 20, 22, 25, and 28) exhibited very low agonistic activity at all tested concentrations (less than 10% of the activity evoked by PBAN1-33NH2, at the same concentration). All
other peptides from the D-Phe sub-library (numbers 19, 23, 24, 26, 27, 29, and 30, Table II) that exhibited low or no antagonistic
activity also failed to stimulate sex pheromone biosynthesis in
H. peltigera females (at 1 nmol). The amount of pheromone
that was found in females injected with these peptides was negligible
and ranged from 0 to a maximum of 3 ng/gland.
Further studies involved examination of the ability of the peptides
from the Ser sub-library (numbered 4-15, Table I) to evoke agonistic
activity. The data in Fig. 4 depict one
relatively potent agonist (number 14) which exhibits 75% activity, at
1 nmol, as compared with the activity of 1 nmol
PBAN1-33NH2, and the presence of two other peptides
(numbers 6 and 10) with low agonistic activity (33 and 45%, as
compared with the activity of 1 nmol PBAN1-33NH2, respectively). All other peptides in the Ser sub-library had low or no
agonistic activity compared with that of PBAN1-33NH2, when injected for 2 h. Examination of the activity of peptides 6, 10, and 14 at lower doses (1, 10, and 100 pmol) revealed very low activity
at all tested concentrations. The only peptide that exhibited any
activity was number 14 at 100 pmol. The activity was similar to that
exhibited by the linear peptides PBAN28-33NH2 and
[Arg27]PBAN27-33NH2 and lower than that of
PBAN1-33NH2 (data not shown).

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Fig. 4.
Agonistic activity of cyclic peptides from
the Ser sub-library. All experimental details are as described in
the legend to Fig. 3. Agonistic activity is expressed as the ratio (in
percentage) between the sex pheromone content evoked by the injection
of each of the cyclic peptides and PBAN1-33NH2 (at 1 nmol
for 2 h). The amount of sex pheromone evoked by 1 nmol of
PBAN1-33NH2 ranged from 86 to 191 ng of
pheromone/female.
|
|
Based on the same rationale as was mentioned above on the ability of
peptides to exhibit both agonistic and antagonistic activities, peptides of the Ser sub-library (that did not exhibit agonistic activity) were tested for their ability to act as antagonists, namely,
to inhibit sex pheromone biosynthesis evoked by exogenously administered PBAN1-33NH2. Pheromone biosynthesis was
evoked by 0.5 pmol of PBAN1-33NH2, and each peptide from
the Ser sub-library (numbers 4, 5, 7-9, and 11-13, Table I) was
tested for its antagonistic activity at a concentration of 1 nmol. None
of the tested peptides exhibited any antagonistic activity under these
experimental conditions.
The detailed SAR studies that were performed with the BBC peptides from
both libraries lead to the conclusion that peptides from the Ser
sub-library were devoid of any pheromonotropic antagonistic activity
and that antagonistic peptides were obtained only from the
D-Phe sub-library (peptides 20, 21, 22, 25, and 28) (Table III). In addition, it was found that
agonistic or partial agonistic activities were exhibited by peptides
from both sub-libraries (numbers 6, 10, and 14 from the Ser sub-library
and 21 from the D-Phe sub-library). Examination of the
correlation between the bridge size and activity revealed that six of
the eight active peptides (whether agonistic or antagonistic) had a
bridge size of m + n = 5 or 6, and peptides
with a bridge size of m + n = 4, 7, and 10 were inactive. However, not all peptides with a bridge size of
m + n = 6 exhibited activity
(e.g. peptides 8 and 23, Table III), and in general only 8 of the 24 BBC peptides described herein were found to be agonists
and/or antagonists. This is despite the fact that all of the peptides
within each library had the same parent primary sequence (either the
agonistic MINI-PBAN sequence
Ser sub-library, or the linear lead
antagonistic sequence
D-Phe sub-library). Moreover, in
some cases the biological activity was determined by the position of
the amide bond along the bridge. Peptides 21 and 25 (n = 2; m = 4 and n = 4; m = 2, respectively) were active, whereas peptide 23, which had the same
sequence, the same location of the ring in the sequence, and the same
ring size, but differed only in the position of the ring amide bond
(n = 3; m = 3), was completely inactive. Similar
results were obtained with peptides 27 and 28 (a combination of
n = 4 and m = 4 was inactive, whereas n = 6 and m = 2 was active; Table III).
We assume that the exact position of the bridge amide bond is critical
for the stabilization of the bioactive conformation by intramolecular
hydrogen bonding. The differences in agonistic and/or antagonistic
potencies exhibited by the various BBC peptides within each sub-library
illustrate the crucial importance of conformation because all the
members of each sub-library have the same parent sequence.
 |
DISCUSSION |
Selective peptide receptor antagonists are most valuable tools for
understanding the detailed pharmacology of peptide-receptor systems.
Selective antagonists of insect neuropeptides having the appropriate
properties of selectivity, metabolic stability, and bioavailability are
also good candidates for potential use as insecticides. Despite their
tremendous importance, no methodology is available for a de
novo design of selective antagonists to a certain receptor.
Moreover, there is no way to predict a priori which
"new" ligand-receptor interactions will lead to antagonists or to
agonists of greater or lesser potency. The basic problem for the design
of antagonists is to obtain a lead compound. Once an antagonistic lead
compound is obtained, its activity can be improved by conformational
constraints in combination with SAR studies.
Potent peptide antagonists have been obtained, so far, by two main
strategies: (a) antagonists derived from agonists, where the
lead antagonists are obtained by serendipity during systematic SAR
studies of the endogenous agonist; and (b) non-peptide
antagonists, where lead compounds were obtained "incidentally" or
by trial and error from natural product or chemical libraries by
multi-receptor screening (54). We have chosen to apply the first
approach. In a previous study we elucidated the active sequence of PBAN by systematic reduction of the peptide sequence by one or several amino
acid residues at a time from the N and the C termini to determine the
minimum active sequence necessary for bioactivity. This study resulted
in the disclosure of a sequence of six amino acids derived from the C
terminus of the peptide (Tyr-Phe-Ser-Pro-Arg-Leu-NH2) that
was equipotent with PBAN1-33NH2 (22). Then, we
systematically replaced L-amino acids by hydrophobic
D-amino acids (e.g. D-Phe) in the
MINI-PBAN sequence, which led to the disclosure of a linear lead
antagonist (53). The same approach led in the past to the disclosure of
antagonists of several neuropeptides such as: bombesin (55), substance
P (56), neurokinin A (57), and vasoactive intestinal peptide (58). For
review, see Refs. 59 and 60.
To obtain improved antagonists that exhibit selectivity, metabolic
stability, and bioavailability, we applied backbone cyclization and
sequence-biased cycloscan using the amino acid sequence of the linear
antagonist as a lead sequence. Two BBC sub-libraries were generated,
out of which one agonist and four potent anti-PBAN antagonists (devoid
of agonistic activity) capable of inhibiting sex pheromone biosynthesis
in H. peltigera females were disclosed. The conformational
constraint that was imposed on the BBC peptides in both sub-libraries
enabled only a few of the BBC peptides to adapt to the appropriate
bioactive conformation, even though all the peptides were based on
highly active primary sequences, further strengthening the importance
of conformational compatibility.
The BBC approach, which introduces conformational constraint on
peptides and acquires metabolic stability for them, introduces many
advantages in the design of agonists and antagonists. First, it
provides a tool to disclose selective agonists and antagonists, as was
proven in our laboratory in the case of substance P (7) and
somatostatin (8), where the application of backbone cyclization resulted in highly selective analogs for the neurokinin receptors (NK-1) and somatostatin receptors SSTR2 and SSTR5, respectively. This
approach also facilitates determination of the bioactive conformation
of PBAN and the conformational requirements for pheromonotropic agonistic and antagonistic activities that can be assessed by nuclear
magnetic resonance (NMR) studies (8, 61). This information is essential
for the further design of improved peptidomimetic and non-peptide
agonists and antagonists of PBAN with higher potency, stability, and
improved bioavailability. The metabolic stability that is acquired for
the BBC peptides is also of major importance especially for in
vivo studies, as has been proven in our laboratory using the above
anti-PBAN antagonists. Preliminary studies indicate that the BBC
peptides exhibit protracted metabolic stability
(t1/2 = 160 min compared with
t1/2 = 7 min of the linear analog
[Arg27]PBAN28-33NH2), and their injection to
H. peltigera females at scotophase resulted in a marked
decrease in pheromone biosynthesis (that lasted over 10 h) evoked
by endogenous PBAN.2 The use
of cyclization in the design of antagonists with higher selectivity and
metabolic stability has been applied successfully to other peptides
such as oxytocin, glucagon, enkephalin, and luteinizing
hormone-releasing hormone (for review, see Refs. 59 and 60, and
references therein).
The only cyclic peptide that has been generated for the pyrokinin/PBAN
family to date is an end-to-end cyclic octapeptide derived from the
C-terminal part of Lem-PK (62). The design of this peptide was based on
computational studies of the C-terminal sequence of Lem-PK and
exhibited 1 and 10% of the pheromonotropic agonistic activity of
Lem-PK and Bom-PBAN, respectively (62).
Availability of selective agonists or antagonists is of major
importance in the study of neuropeptides in general and for the study
of the pyrokinin/PBAN family of neuropeptides in particular. This
family of peptides is implicated in the regulation of critical reproductive, development, and digestive processes such as sex pheromone biosynthesis, cuticular melanization, myotropic activity, oviposition, pupariation, and diapause in moths and other insects (for
review, see Ref. 11). Studies that were performed in several laboratories including ours have shown that PBAN and members of the
pyrokinin family demonstrate considerable cross-activity (for review
see Ref. 11). Despite the intensive studies of the bioactivity of this
family of peptides, very little is known about the endogenous mechanisms and structural, chemical, and cellular basis of their activity. It is still not known which endogenous peptides mediate each
of these functions in vivo and what are the characteristics of the receptor(s) that are involved in these processes. The BBC antagonists that were generated in this study will enable us to correlate between PBAN and its physiological functions in moths and
other insects. These studies will reveal new functions mediated by the
pyrokinin/PBAN family, disclose the presence of multiple pyrokinin/PBAN
receptors and relate the exact physiological function to each peptide
and receptor. The BBC peptides of the two sub-libraries described in
this study have been tested, so far, only on their stimulatory or
inhibitory activity of sex pheromone biosynthesis in female moths.
Experiments on the ability of the BBC peptides from both sub-libraries
to inhibit or stimulate other functions mediated by the pyrokinin/PBAN
family are in progress. It is anticipated that once this study is
completed, a series of selective and nonselective agonists and
antagonists for in vivo studies will be available for the
better understanding of the cellular and physiological basis of the
pyrokinin/PBAN-mediated activities.
In summary, in this article we present a combined novel approach of
rational design and selection method for the generation of agonistic
and/or antagonistic cyclic peptides based on a linear lead peptide.
This approach led to the discovery of several antagonists and agonists
which exhibited either pheromonotropic activity or effectively
inhibited sex pheromone biosynthesis in H. peltigera female
moths. To the best of our knowledge, this is the first report on the
use of backbone cyclization for the design of insect neuropeptide
antagonists and the first report on the disclosure of PBAN
pheromonotropic antagonists. Beyond the immediate benefits that are
introduced by the cyclic peptides as selective antagonists, the
information on the bioactive conformations of the antagonists that was
gained in the course of this study may serve as a basis for the design
of improved non-peptide mimetic agonists and antagonists. Such
compounds are potential candidates for agrochemical applications, which
can serve, after formulation and preliminary field experiments, as
prototypes for the development of a novel group of highly effective, insect-specific and environmentally friendly insecticides.