Peptidomimetics in the discovery of new insect growth regulators: Studies on the structure-activity relationships of the core pentapeptide region of allatostatins

Article abstract of DOI:10.1021/jf200085d

Cockroach-type allatostatins (ASTs) were discovered in cockroaches through their capacity to inhibit the production of juvenile hormone by the corpora allata (CA). ASTs were considered as potential insect growth regulator (IGR) candidates, but several disadvantages, including the absence of the effect in vivo and rapid degradation in vivo, precluded their application in pest management. The CA were selected as the target, and the core pentapeptide region (YDFGL) was chosen as the lead sequence in the search for new IGRs based on the allatostatins. We designed and synthesized 24 analogues, which mimicked each amino acid of the core region, to determine structure-activity relationships and the possibility of shortening the ASTs in the core region while retaining activity. The results suggest that the sequence FGLa is more important than Y/FX because Y/FX mimics show strong effects in vitro and in vivo. In particular, compound I3 was synthesized by substitution of Y/FX with 6-phenylhexnoic acid and exhibits higher activity in vitro than the complete core region. Furthermore, compound I3 has a clear effect in vivo on juvenile hormone (JH) biosynthesis of Diploptera punctata females, providing a possible application for cockroach management. On the basis of the structure-activity relationship of pentapeptide analogues, a general structure of potential potent AST analogues is proposed here. A new approach using peptidomimetics in the discovery of IGRs is demonstrated in our study.

Full text of DOI:10.1021/jf200085d

ARTICLE
pubs.acs.org/JAFC
Peptidomimetics in the Discovery of New Insect Growth Regulators:
Studies on the Structure-Activity Relationships of the Core
Pentapeptide Region of Allatostatins
Zhen-peng Kai,†,‡,§ Yong Xie,†,‡ Juan Huang,‡,§ Stephen S. Tobe,*,§ Jin-rui Zhang,§ Yun Ling,‡ Li Zhang,‡
Yi-chen Zhao,‡ and Xin-ling Yang*,‡
‡Department of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, People’s Republic of China
§Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, Ontario M5S 3G5, Canada
ABSTRACT: Cockroach-type allatostatins (ASTs) were discovered in cockroaches through their capacity to inhibit the production
of juvenile hormone by the corpora allata (CA). ASTs were considered as potential insect growth regulator (IGR) candidates, but
several disadvantages, including the absence of the effect in vivo and rapid degradation in vivo, precluded their application in pest
management. The CA were selected as the target, and the core pentapeptide region (YDFGL) was chosen as the lead sequence in the
search for new IGRs based on the allatostatins. We designed and synthesized 24 analogues, which mimicked each amino acid of the
core region, to determine structure-activity relationships and the possibility of shortening the ASTs in the core region while
retaining activity. The results suggest that the sequence FGLa is more important than Y/FX because Y/FX mimics show strong
effects in vitro and in vivo. In particular, compound I3 was synthesized by substitution of Y/FX with 6-phenylhexnoic acid and
exhibits higher activity in vitro than the complete core region. Furthermore, compound I3 has a clear effect in vivo on juvenile
hormone (JH) biosynthesis of Diploptera punctata females, providing a possible application for cockroach management. On the
basis of the structure-activity relationship of pentapeptide analogues, a general structure of potential potent AST analogues is
proposed here. A new approach using peptidomimetics in the discovery of IGRs is demonstrated in our study.
KEYWORDS: Insect growth regulators, peptidomimetics, allatostatins, pentapeptide, analogues, juvenile hormone, structure-
activity relationships
species, raise the possibility that ASTs could be potential leads for
the discovery of new IGRs. In cockroaches, cockroach-type ASTs
inhibit JH production at very low doses in vitro but have little
promise as insect control agents. The major limitations of the ASTs,
which have precluded their use for insect control, include their rapid
degradation by peptidases and poor penetration through insect
cuticle.12 To find analogues that retain activity but overcome these
limitations as promising leads in the development of IGRs, studies
on structural modifications and structure-activity relationships
(SARs) have been carried out in recent years.
Previous structure-activity studies demonstrated that the
C-terminal pentapeptide, Tyr/Phe-Xaa-Phe-Gly-Leu-NH2 (Y/
FXFGLa), which was shared in all identified neuropeptides, was
regarded as the minimum sequence to inhibit JH production and
the “active core” region of the cockroach-type ASTs.13 This region
probably binds to the allatostatin receptors and elicits inhibition of JH
production.13,14 Using the Ala scanning technique, Hayes et al.13
indicated that Leu8, Phe6, and Tyr4 in Dippu-AST 4 (DRLYSFGLa)
were the most important side chains for inhibition of JH biosynthesis.
By altering the nature of the peptide bond with the aim of reducing
the susceptibility of the bond to hydrolysis, Piulachs et al.8 designed
ketomethylene and methyleneamino pseudo-peptide analogues of
allatostatin Dippu-AST 5 (DRLYSFGLa). Bioassay showed that
1. INTRODUCTION
Insect growth regulators (IGRs) are compounds that can
regulate the growth of insects.1,2 Juvenile hormone analogues
(JHAs), molting hormone analogues (MHAs), and chitin synth-
esis inhibitors are three principal groups of IGRs.3,4 Owing to the
virtue of high potency, good selectivity, and environmental safety
for nontarget organisms, IGRs are regarded as “the ideal pesti-
cides in the 21st century” and are playing an important role in
integrated pest management systems.3 The discovery of new
IGRs and potential IGR targets has therefore become increas-
ingly important to meet the requirements of pest management in
agriculture and public health.
In many insect species, the reduction in juvenile hormone
(JH) production profoundly influences insect growth, metamor-
phosis, and reproduction. JH plays a significant role in insect physio-
logical processes,5 and therefore, any compound that influences the
JH production can be regarded as a potential lead for insect control
agents. Allatostatins (ASTs), members of a family of insect neuro-
peptides originally isolated from the cockroach Diploptera punctata,
can inhibit the in vitro biosynthesis of JH by the corpora allata (CA)
and thereby control insect maturation and egg production.6,7 These
AST molecules, 6-18 amino acid peptides, also exhibit other
biological properties, such as modulation of myotropic activity in
both gut8 and dorsal vessel,9 inhibition of vitellogenin production in
the fat body,10 and stimulation of carbohydrate enzyme activity in
the midgut.11 These abilities to influence a number of physiological
processes, especially inhibition of JH biosynthesis in some insect
Received: January 7, 2011
Accepted: January 12, 2011
Published: February 18, 2011
r
2011 American Chemical Society
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Figure 1. Principle of the design of AST analogues. The core pentapeptide region was divided into three parts, Tyr/Phe-Xaa (series I), Phe-Gly (series
II), and Leu-NH2 (series III), which were replaced by organic groups, restricting conformation components and different amino acids to determine the
possibility of shortening the ASTs and the SARs of the core region, respectively.
both analogues were similarly active to the model peptides with
respect to the inhibition of JH in vitro biosynthesis from CA of virgin
Blattella germanica. To find analogues that have resistance to cata-
bolism and retain inhibition of JH biosynthesis, Nachman et al.12,15,16
designed a series of allatostatin analogues by replacing the first, third,
or fourth amino acid residues of the C-terminal pentapeptide with
sterically hindered amino acids or aromatic acids. These analogues
showed significant in vitro inhibition of JH biosynthesis. The ana-
logue AST(b)Φ2 (Hca-Asn-Phe-Cpa-Leu-NH2, with Hca = hydro-
cinnamyl- and Cpa = cyclopropyl-Ala-) exhibited high resistance to
degradation by enzymes in hemolymph and crude membrane
preparation of brain and midgut and retained significant biological
activity in vitro and in vivo.12,15 The above efforts reveal the possibility
for the discovery of IGRs that are neuropeptide-like but not readily
degradable by peptidases.
However, the question of which amino acids of the core
pentapeptide are most important to activity remains unresolved, and
therefore, it is unclear if amino acids of the core region could be
substituted further. The present work attempts to answer the above
issues. In the present study, 24 analogues that mimicked amino acids
of the core region pentapeptide were designed with the peptidomi-
metic approach (PA), one method widely used in the discovery of
peptide-based pharmaceuticals,17-20 by replacing portions of the
peptides with unnatural or natural structures to overcome the shor-
tage of natural peptides while retaining activity. The design strategy is
shown in Figure 1. The three types of analogues were named as
mimics of the Tyr/Phe-Xaa region (series I), the Phe-Gly region
(series II), and the Leu-NH2 region (series III). The in vitro effects
on JH biosynthesis of all 24 compounds were tested, and the in vivo
effects of some of the analogues were also evaluated. The SARs of the
analogues were subsequently analyzed.
2. EXPERIMENTAL SECTION
2.1. Synthesis. 2.1.1. Materials. Rink Amide-AM resin (0.52 mmol/g
substitution), O-benzotriazole-N,N,N0,N0-tetramethyluronium hexafluoro-
phosphate (HOBt), 1-hydroxybenzotriazole anhydrate (HBTU), N,N0-
diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), Fmoc-protected
amino acids, Fmoc-1-amnocyclohexanecarboxylic acid, and Fmoc-L-Tic-OH
were purchased from GL Biochem, Ltd. (Shanghai, China). High-perfor-
mance liquid chromatography (HPLC)-grade N,N-dimethylformamide
(DMF), dichloromethane (DCM), and acetonitrile were purchased from
Dima Technology, Inc. (Richmond Hill, Ontario, Canada). Thioanisole,
phenol, benzaldehyde, phenylmethanamine, hydrocinnamic acid, 6-phenyl-
hexanoic acid, butanedioic anhydride, butyraldehyde, 3-methylbutanal, and
NaBH4 were purchased from Acros (Geel, Belgium).
2.1.2. Synthesis of Peptides. The Dippu-AST 1 and pentapeptide
were synthesized from Rink Amide-AM resin (198 mg, 0.1 mmol) using the
standard Fmoc/tBu chemistry and HBTU/HOBt protocol.25 Incoming
amino acids were activated with HOBt (41 mg, 0.3 mmol), HBTU (114 mg,
0.3 mmol), and DIEA (105 μL, 0.6 mmol) in DMF (5 mL) for 5 min, and
couplings were run for 2 h. Removal of the N-terminal Fmoc group from the
residues was accomplished with 20% piperidine in DMF (5 mL) for 20 min.
The peptides were cleaved from the resin with TFA (4 mL) containing 5%
phenol, 2.5% thioanisole, and 5% water for 2 h.
2.1.3. Synthesis of Series I Analogues
DFGL with Resin and FGL with Resin. The two key intermediates
were synthesized from Rink Amide-AM resin using the standard Fmoc/
tBu chemistry and HBTU/HOBt protocol as the above method in
section 2.1.2.
I1. Hydrocinnamic acid (45 mg, 0.3 mmol) was coupled to the
DFGL with resin (0.1 mmol) with HOBt, HBTU, and DIEA in DMF for
3 h at room temperature. I1 was cleaved from the resin with TFA
containing 5% phenol, 2.5% thioanisole, and 5% water for 2 h.
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Scheme 1. Synthesis of I4 and I5a
Scheme 2. Synthesis of III2a
a Conditions: (a) HBTU, HOBt, DIEA, DMF, room temperature, and 3
h and (b) TFA, 5% phenol, 2.5% thioanisole, 5% water, room tempera-
ture, and 2 h.
a Conditions: (a) butanedioic anhydride, DMAP, DMF, room tempera-
ture, and 3 h; (b) PhCH2NH2, HBTU, HOBt, DIEA, DMF, room
temperature, and 24 h; and (c) TFA, 5% phenol, 2.5% thioanisole, 5%
water, room temperature, and 2 h.
Scheme 3. Synthesis of III3a
I2. Fmoc-Asp-OH (107 mg, 0.3 mmol) was coupled to the FGL with
resin (0.1 mmol) with HOBt, HBTU, and DIEA in DMF for 3 h at room
temperature. I2 was cleaved from the resin with TFA containing 5%
phenol, 2.5% thioanisole, and 5% water for 2 h.
I3. 6-Phenylhexanoic acid (58 mg, 0.3 mmol) was coupled to the
FGL with resin (0.1 mmol) with HOBt, HBTU, and DIEA in DMF for 3
h at room temperature. I3 was cleaved from the resin with TFA
containing 5% phenol, 2.5% thioanisole, and 5% water for 2 h.
I4. Butanedioic anhydride (30 mg, 0.3 mmol) was coupled to the
FGL with resin (0.1 mmol) with 4-dimethylaminopyridine (DMAP) (37
mg, 0.3 mmol) dissolved in DMF (5 mL) for 2 h at room temperature. I4
was cleaved from the resin with TFA containing 5% phenol, 2.5%
thioanisole, and 5% water for 2 h (Scheme 1).
I5. Phenylmethanamine (54 mg, 0.5 mmol) was coupled to the
intermediate I4 with resin (0.1 mmol) with HOBt, HBTU, and DIEA
in DMF for 24 h at room temperature. I5 was cleaved from the resin with
TFA containing 5% phenol, 2.5% thioanisole, and 5% water for 2 h
(Scheme 1).
a Conditions: (a) HBTU, HOBt, DIEA, DMF, room temperature, and 3
h and (b) TFA, 5% phenol, 2.5% thioanisole, 5% water, room tempera-
ture, and 2 h.
Scheme 4. Synthesis of III4a
2.1.4. Synthesis of Series II Analogues
II1-II11. They were obtained with the relevant Fmoc-protected
amino acids using the same method as in section 2.1.2.
2.1.5. Synthesis of Series III Analogues
III1. III1 was synthesized from Wang resin (67 mg, 0.1 mmol) using
the standard Fmoc/tBu chemistry and HBTU/HOBt protocol. Incom-
ing amino acids were activated with HOBt, HBTU, and DIEA in DMF
for 5 min, and couplings were run for 3 h. Removal of the N-terminal
Fmoc group from the residues was accomplished with 20% piperidine in
DMF for 20 min. III1 were cleaved from the resin with TFA containing
5% phenol, 2.5% thioanisole, and 5% water for 2 h.
III2. Butyraldehyde (43 mg, 0.5 mmol) was coupled to Rink Amide-
AM resin (198 mg, 0.1 mmol) with acetic acid (4 μL) in DMF (5 mL) for
4 h at room temperature. The resin was reduced by NaBH4 (38 mg, 1
mmol) with methanol (0.4 mL) in DMF (5 mL) for 2 h at room
temperature. Incoming amino acids were activated with HOBt, HBTU,
and DIEA in DMF for 5 min, and couplings were run for 3 h. Removal of
the N-terminal Fmoc group from the residues was accomplished with
20% piperidine in DMF for 20 min. III2 were cleaved from the resin with
TFA containing 5% phenol, 2.5% thioanisole, and 5% water for 2 h
(Scheme 2).
III3. Benzaldehyde (53 mg, 0.5 mmol) was coupled to Rink Amide-
AM resin (198 mg, 0.1 mmol) with acetic acid (4 μL) in DMF (5 mL) for
4 h at room temperature. The resin was reduced by NaBH4 (38 mg, 1
mmol) with methanol (0.4 mL) in DMF (5 mL) for 2 h at room
temperature. Incoming amino acids were activated with HOBt, HBTU,
and DIEA in DMF for 5 min, and couplings were run for 3 h. Removal of
the N-terminal Fmoc group from the residues was accomplished with
20% piperidine in DMF for 20 min. III3 was cleaved from the resin with
TFA containing 5% phenol, 2.5% thioanisole, and 5% water for 2 h
(Scheme 3).
a Conditions: (a) HBTU, HOBt, DIEA, DMF, room temperature, and 3
h and (b) TFA, 5% phenol, 2.5% thioanisole, 5% water, room tempera-
ture, and 2 h.
III4. 3-Methylbutanal (43 mg, 0.5 mmol) was coupled to Rink Amide-
AM resin (198 mg, 0.1 mmol) with acetic acid (4 μL) in DMF (5 mL) for 4
h at room temperature. The resin was reduced by NaBH4 (38 mg, 1 mmol)
with methanol (0.4 mL) in DMF (5 mL) for 2 h at room temperature.
Incoming amino acids were activated with HOBt, HBTU, and DIEA in
DMF for 5 min, and couplings were run for 3 h. Removal of the N-terminal
Fmoc group from the residues was accomplished with 20% piperidine in
DMF for 20 min. III4 were cleaved from the resin with TFA containing 5%
phenol, 2.5% thioanisole, and 5% water for 2 h (Scheme 4).
III5-III8. They were obtained with the relevant Fmoc-protected
amino acids using the same method as in section 2.1.2.
All of the crude compounds were purified on a C18 reversed-phase
preparative column with a flow rate of 10 mL/min using acetonitrile/water
(50:50) containing 0.06% TFA as an ion-pairing reagent. UV detection was
at 215 nm.21,25 The purity of each purified compound was greater than 95%.
The structures of the analogues were confirmed by the presence of the
following molecular ions using a 1100 series LC/MSD trap (VL) (Agilent
Technologies, Santa Clara, CA). The structures, purity, and mass spectro-
metry (MS) data of all target compounds are shown in Table 1.
2.2. Bioassays. 2.2.1. Animals. Newly emerged mated females of
D. punctata (day 0) were isolated from stock cultures. Mating was
confirmed by the presence of a spermatophore. Stocks and isolated
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Table 1. Structures, Purity, MS Results, and IC50 Values of Analogues
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Table 1. Continued
females, fed Lab Chow and water ad libitum, were kept at 27 ( 1 °C and
relative humidity of 50 ( 5% with a 12 h light/12 h dark cycle.
2.2.2. Bioassays in Vitro. All radiochemical assays for JH biosynthesis
were performed using individual pairs of CA from day 7 mated females.
Compounds were dissolved in medium 199 for assay as described
previously.22,23 All test compounds were assayed on the same day that
the samples were prepared. The solutions were discarded at the end of
each day. Rates of JH release were determined using the modified in vitro
radiochemical assay.22,23 This assay measures the incorporation of the
radiolabeled S-methyl moiety of radiolabeled methionine into JH III in
the final step of biosynthesis by CA maintained in vitro. CA were
incubated for 3 h in 100 μL of medium 199 (GIBCO, 1.3 mM Ca2þ, 2%
Ficoll, methionine-free) containing L-[14C-S-methyl]methionine (40
μM, specific radioactivity of 1.48-2.03 GBq/mmol, Amersham). Sam-
ples were extracted, and the JH release was determined. Each data point
on the dose-response figures represent replicate incubations of 10-27
experimental CA compared to control CA (i.e., no analogue added).
2.2.3. Bioassays in Vivo. The pentapeptides, I1, I3, and I4 were
dissolved in H2O (stock final concentration of 10 μM). A total of 5 μL
was injected into D. punctata females on day 1, and animals were assayed
for JH biosynthesis at day 3.15 The assay for JH biosynthesis is identical
to the in vitro assay described above. All injections were administered to
non-anaesthetized mated female D. punctata. Assuming a hemolymph
volume of 50 μL for day 1 adult female D. punctata,24 the concentrations
of the injected analogue in the hemolymph were approximately 1 μM.
Injections were administered using a 26-gauge 10 μL Hamilton syringe.
The needle was inserted into the membranous joint between the coxa
and femur on the metathoracic leg. For control samples, insects were
injected with 5 μL of double-distilled water. Each group of compound-
injected animals was compared to a group of water-injected animals
treated concurrently.
core” pentapeptide, retained significant in vitro activity. This
demonstrates that the C-terminal pentapeptide could be the lead.
In this study, a pentapeptide, YDFGLa, was synthesized and
investigated as the lead compound. The native Dippu-AST 1,
LYDFGLa, was chosen as the control. Treatment of adult female
D. punctata with the pentapeptide showed a significant effect on
JH production in vitro, with an IC50 value of 0.13 μM. The IC50
value for native Dippu-AST 1 was 0.008 μM. Inhibition of JH
biosynthesis by the pentapeptide is 16-fold less than that of
Dippu-AST 1, but the structure of the pentapeptide is simpler
than that of the natural AST. A good lead compound in pesticide
discovery should be bioactive and easily modified. Therefore,
taking the pentapeptide as the lead compound and Dippu-AST 1
as the control, three series of analogues were designed and
synthesized successfully in the present work.
3.2. Design and Effects of Series I. In series I, aromatic acids
and dicarboxylic fatty acids were used as mimics of the Tyr/Phe-
Xaa region of pentapeptide. In previous work, AST(b)Φ2, in
which Tyr was replaced by 3-phenylpropanoic acid, showed
resistance to catabolism and high activity.12,15 In our work, we
chose aromatic acids to mimic the Tyr/Phe-Xaa region to design
I1, I2, I3, and I5. In comparison to the lead (pentapeptide in
Table 1), these analogues showed significant inhibition of JH
biosynthesis. Compound I1, in which 3-phenylpropanoic acid
mimicked the Tyr of the core region, had the same IC50 value as
that of the pentapeptide. The bioactivity of I2 was almost 10-fold
less than that of the pentapeptide, probably because the hydro-
phobic group of (9H-fluoren-9-yl) methyl hydrogen carbonate is
larger than either Tyr or Phe. In the structures of I3 and I5,
6-phenylhexanoic acid and 3-(benzylcarbamoyl)propanoic acid
were used to mimic the Y/FX sequence, respectively. The
bioactivity of I3 was higher than that of the pentapeptide, and
the IC50 value of I5 was similar to that of the pentapeptide.
Compound I4, in which succinic acid was used to replace the Xaa
sequence of XFGLa, was also able to inhibit JH production. A
comparison of the activities of I4 and I5 indicates that the
aromatic group (benzene ring) contributes to elevate biological
activity to the analogues. The significant activity of series I
analogues, which only contained the “FGLa” region, suggests
that the Y/FX region could be substituted in analogue design,
3. RESULTS AND DISCUSSION
3.1. Choice of Lead Peptide. The length of the lead peptide
directly affects the difficulty in designing peptidomimetics; i.e.,
the longer the lead peptide, the more difficult the design. The
cockroach-type ASTs in D. punctata contain 6-18 amino acid
residues. To choose an appropriate lead peptide is the first step in
the AST peptidomimetic approach. Previous studies12,14-16
showed that AST analogues, which modify or protect the “active
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Table 2. Log P Prediction Results of Series I
Figure 2. Inhibitory effects in vivo of the pentapeptide and AST
analogues on JH biosynthesis by CA. The concentrations were 1 μM
in the hemolymph of female cockroach D. punctata at day 1 and 1.1 μM
at day 3. Each column represents the mean ( scanning electron
microscopy (SEM) for the number of individual measurements indi-
cated at the top of error bars. Asterisks indicate significant differences
between peptide- and water-injected groups of animals as determined by
Dunnett’s multiple comparison test following one-way analysis of
variation (ANOVA): (/) 0.01 < p < 0.05 and (//) 0.001 < p < 0.01.
Significance determined by a Student’s t test was as follows: pentapep-
tide, not significant (NS); I1 and I4, <0.05; and I3, <0.01.
a The PSLogP was developed by Shanghai Institute of Organic Chem-
istry, Chinese Academy of Sciences. All of the predicted values were
performed by Dr. Jian-hua Yao.
represent a plausible active conformation, in agreement with
the study using protein secondary structural prediction methods
and energy minimizations.13 In addition, Nachman et al.16
synthesized allatostatin analogues incorporating β-turn-promot-
ing moieties, some of which exhibited activity similar to the
model peptide. Thus, series II analogues incorporating turn-
promoting moieties were designed with the replacement of the
Phe-Gly region by aminoindane carboxylic acid (Aic), L-1,2,3,4-
tetrahydroisoquino line-3-carboxylic acid (Tic), L-proline (Pro),
Ala, Met, Val, and 1-aminocyclohexanecarboxylic acid to validate
the hypothesis of the turn conformation in the core region.
After incorporation of turn-promoting moieties, II1, II2, II3,
and II4 were designed as peptidomimetics, in which the FG
moiety was mimicked by Aic and L-Tic (Table 1). The phenyl is
coupled by methylene in Aic and Tic. The IC50 value of II1 is 10-
fold less than that of the pentapeptide, whereas the bioactivity of II2
is almost 18-fold less than that of Dippu-AST 1. Inhibition of JH
biosynthesis by II4 is 160-fold less than that of Dippu-AST 1. A
dose-response curve for II3 with respect to the inhibition of JH
biosynthesis in vitro could not be generated because of the low levels
of inhibition. Similarly, in analogue II5, the Phe was replaced with
Pro to hold the turn conformation. The IC50 values of II5 could not
be calculated because they showed no effect.
and the presence of an aromatic group could help to retain the
significant activity.
Although the natural ASTs exert a strong in vitro inhibitory
effect on JH biosynthesis by cockroach CA, they have little or no
effect in vivo because of the major limitations, including their rapid
degradation by peptidases and poor penetration through the insect
cuticle.12 Accordingly, the effect in vivo on JH biosynthesis of the
pentapeptide and series I analogues I1, I3, and I4 was determined.
The concentrations of the compounds were 1 μM in the hemo-
lymph of D. punctata females at day 1 and 1.1 μM at day 3. Figure 2
shows that the pentapeptide has no effect in vivo but the three
analogues do exert some effect in vivo. Previous work showed that
the analogue AST(b)Φ2 containing only four true amino acids
(XFGLa) retain in vitro and in vivo activity.12 It is striking that I4,
which contains only three true amino acids (FGLa), also had some
effect in vivo. Compound I3, whose in vitro bioactivity was
significantly better than that of pentapeptide, showed an effect in
vivo. This compound offers considerable potential for the use of
allatostatin analogues in cockroach management.
Hemolymph enzymes primarily cleave the peptide in the
N-terminal region outside of the peptide core region, and cleavage
by membrane preparation of brain, gut, and CA occurs between the
residues Tyr-Xaa.17,25 In series I, both cleavages are prevented
because Tyr is replaced by a nonpeptide structure. The log P values
of series I were calculated by the prediction system of log P
(PSLogP) and are shown in Table 2. The log P value of all of the
series I analogues is higher than that of lead pentapeptide and
Dippu-AST 1. This indicates that it is easier for these compounds to
penetrate the insect cuticle than the pentapeptide, and conse-
quently, they exhibit an effect in vivo.
In earlier studies, Ala13 and Cpa16 were used to replace Gly.
These analogues showed lower activity than their leads. In ana-
logues II6-II11, the Gly was replaced with Pro, Ala, Met, Val, and
1-aminocyclohexanecarboxylic acid, respectively, in which residues
are larger organic groups than Gly. These analogues have no effect
on JH biosynthesis. The IC50 values of series II analogues suggest
that the phenyl group in the FG region is critical to bioactivity and its
position must be considered carefully in the design of analogues.
The free space of the Gly residue should also remain small. If some
bigger organic groups, such as Ala, Cpa, Met, Val, Pro, or 1-amino-
cyclohexanecarboxylic acid are used to replace Gly, the analogue
should be inactive or less active.
3.3. Design and Effects of Series II. D-Amino acid replace-
ments suggested that a secondary structural element(s) at the C
terminus of Dippu-AST 5 could be important to the biological
activity. A turn in the active core in residues XFGL could
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’ REFERENCES
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Figure 3. General approach for the design of potential AST analogues.
Aromatic groups mimic the phenol and phenyl groups of the Y/F residue
in the core region. The linker is the bridge between the aromatic group
and the FGLa moiety.
3.4. Design and Effects of Series III. In series III, we
synthesized III1, which shares the same sequence with Dippu-
AST 1 but without the C-terminal amide to validate the function
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the inhibition of JH biosynthesis, suggesting that the hydrogen
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region can be summarized as follows: (1) The Y/FX region can
be substituted in the new potent AST analogues, and the
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Phe region. (2) The Phe-Gly region could also be modified,
although this did not result in significant activity. (3) The
C-terminal Leu-NH2 region must appear in all new bioactive
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formula for potential potent AST analogues in Figure 3. Sub-
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phenyl groups of the Y/F residue in the core region. The linker in
Figure 3 should be the bridge between the aromatic group and
the FGLa moiety. Asp, Gly, and some organic segments can be
the linker, but the length and most suitable structures of the
linker require further study. These AST mimics should be helpful
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’ AUTHOR INFORMATION
Corresponding Author
*E-mail: yangxl@cau.edu.cn (X.-L.Y); Phone: 86-10-62732223.
Fax: 86-10-62732223; or E-mail: stephen.tobe@utoronto.ca (S.S.T).
Phone: 416-978-3517. Fax: 416-978-3522.
Author Contributions
†Both of these authors contributed equally to this paper.
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Design of peptides, proteins, and peptidomimetics in chi space. Biopo-
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of nonpeptides from peptide ligands for peptide receptors. Methods
Enzymol. 2002, 343, 91–123.
Funding Sources
Financial support was provided by the National Basic Research
Program of China (2010CB126104 and 2003CB114400), the
National Natural Science Foundation of China (20972185), the
National Scientific and Technology Supporting Program of
China (2011BAE06B05-5), and the Natural Sciences and En-
gineering Research Council of Canada.
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design. Tetrahedron 1993, 49, 3547–3558.
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