Synthesis, bioactivity and functional evaluation of linker-modified allatostatin analogs as potential insect growth regulators

Article abstract of DOI:10.1016/j.cclet.2016.02.010

Insect growth regulators play an important role in integrated pest management strategies. The FGLa-allatostatins (ASTs) are a family of neuropeptides that can inhibit juvenile hormone (JH) biosynthesis by the corpora allata (CA) of Diploptera punctata in

Full text of DOI:10.1016/j.cclet.2016.02.010

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Chinese Chemical Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Synthesis, bioactivity and functional evaluation of linker-modified
allatostatin analogs as potential insect growth regulators
Xiao-Qing Wu a, Juan Huang b, Mei-Zi Wang a, Zhe Zhang a, Xin-Lu Li a, Xin-Ling Yang a,
,
*
Stephen S. Tobe b,
*
a Department of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, China
b Department of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada M5S 3G5
A R T I C L E I N F O
A B S T R A C T
Article history:
Insect growth regulators play an important role in integrated pest management strategies. The FGLa–
allatostatins (ASTs) are a family of neuropeptides that can inhibit juvenile hormone (JH) biosynthesis by
the corpora allata (CA) of Diploptera punctata in vitro, are regarded as insect growth regulator candidates.
In the search for new potential mimics and to explore the effect of linker length on inhibiting JH
biosynthesis, a series of AST analogs were synthesized by modifying the linker of K24, which was found
to have a significant effect on JH biosynthesis in vitro in our previous study. Functional evaluation
demonstrated that all the target compounds can activate the Dippu-AstR, albeit with different potencies.
Analog L6 with the longest linker (n = 5), exhibited not only a promising effect on inhibition of JH
biosynthesis both in vitro and in vivo, but also good activity in inhibiting basal oocyte growth. Structure–
activity relationships (SAR) studies showed that longer linkers provided greater contribution to activity.
ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Received 4 January 2016
Received in revised form 23 January 2016
Accepted 2 February 2016
Available online xxx
Keywords:
Allatostatin
Insect growth regulators
Juvenile hormone
Analogs
Dippu-Ast receptor
1. Introduction
Earlier structure–activity relationship (SAR) studies demonstrat-
ed that the C-terminal pentapeptide Y/FXFGLa (X = A, N, G, and S)
Juvenile hormone (JH) production in the corpora allata (CA) of
Diploptera punctata is tightly controlled and highly predictable
during the reproductive cycle. This characteristic provides an ideal
model system to study the effect of compounds with anti-JH
actions. FGLa–allatostatins (ASTs), a family of pleiotropic neuro-
peptides, were originally identified for their ability to inhibit JH
biosynthesis by CA rapidly and reversibly [1]. Because of their
high-efficiency, selectivity and safety to non-target organisms,
FGLa–ASTs have been employed in the design of new potential
IGRs [2–4]. Although ASTs inhibit JH biosynthesis effectively in
vitro, they have some shortcomings as potential pesticides. First,
the more amino acid residues, the higher the production costs. For
ASTs, even the shortest AST peptide has six amino acids [5]. Second,
ASTs are susceptible to metabolic inactivation by peptidases in
hemolymph and midgut [3]. Third, difficulties in ASTs absorption
through cuticle could prevent their effects in vivo. Therefore, much
effort has been expended to overcome these problems and create
better AST peptidomimetics.
represents the minimum sequence capable of eliciting inhibition of
JH production in vitro [6–8]. Subsequently, based on the peptido-
mimetic approach and identification of catabolic cleavage sites of
ASTs, several AST analogs were discovered to show activity in vivo as
well as reduced susceptibility to metabolic inactivation [2–4]. As
neuropeptides, ASTs exert their effects by binding to a G protein-
coupled receptor (GPCR). In 2008, a single FGLa–AstR was isolated
and functionally characterized from D. punctata [9]. Recently, the
single receptor of D. punctata was activated with thirteen natural
FGLa–ASTs [10]. However, whether AST analogs can activate the D.
punctata receptor (Dippu-AstR) remains uncertain.
In our previous studies [11–15], four series of the pentapeptide
analogs were designed and synthesized employing the peptidomi-
metic approach to probe the SAR of the core pentapeptide region of
AST. The bioassay and hologram quantitative structure–activity
relationships (HQSAR) calculation results suggested that the potent
AST analogs should contain an aromatic group, an appropriate
length of linker, and an FGLa moiety. Subsequently, a potent AST
mimic H17 was found to have a significant inhibitory effect on JH
biosynthesis by cockroach CA both in vitro and in vivo [11].
Additionally, owing to its high activity in vitro and its molecular
flexibility, K24 was considered as another good lead. In the present
work, a series of AST analogs (Fig. 1) were synthesized by modifying
*
Corresponding authors.
E-mail addresses: yangxl@cau.edu.cn (X.-L. Yang),
stephen.tobe@utoronto.ca (S.S. Tobe).
http://dx.doi.org/10.1016/j.cclet.2016.02.010
1001-8417/ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: X.-Q. Wu, et al., Synthesis, bioactivity and functional evaluation of linker-modified allatostatin analogs
as potential insect growth regulators, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.02.010

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Table 1
Inhibitory effect on JH release (IC50) in vitro, potency in activation of Dippu-AstR
(EC50), retention time and log P calculation of AST analogs.
a
b
c
Compd.
n
IC50 (nmol/L)
EC50 (nmol/L)
tR (min)
log Pe
AST-1
L1
/
9.57d
957
1.3
2952
472.7
153.3
443.3
62.6
/
/
0
1
2
3
4
5
7.465
7.748
9.598
11.832
15.765
22.598
1.69
1.49
1.87
2.34
2.77
3.24
L2
93.5
77.0
L3
L4
69.5
37.8
27.2
L5 (K24)
L6
65.5
a
IC50 values were determined by radiochemical assay (RCA).
b
c
EC50 was determined by the Dippu-AstR activation assay in CHO-WTA11 cells.
Fig. 1. Design strategy of target analog L.
Retention time of AST analogs was determined by reversed phase high
performance liquid chromatography (HPLC).
d
IC50 value of AST 1 is as reported by Tobe et al. [5].
e
log P calculation of AST analogs was made in the Prediction System of log P
the linker of K24 and their bioactivities were evaluated for the
purpose of discovering new potential mimics and exploring the
effect of linker length on inhibition of JH biosynthesis.
Version 1.0 and done by Dr. Jianhua Yao.
20% piperidine in DMF (5 mL) for 20 min. Relative phenyl acids
were coupled to the GFGL with resin with HBTU, HOBt and DIEA in
DMF for 3 h at room temperature. Then, analogs were cleaved from
the resin with TFA containing 5% phenol, 2.5% thioanisole and 5%
water for 2 h at room temperature. The crude peptides were then
washed with anhydrous ethyl ether, and lyophilized.
2. Experimental
2.1. Synthesis of AST analogs
All the solvents and reagents were purchased from commercial
suppliers and used without further purification. The analogs
structures were confirmed with high-resolution mass spectrome-
try (HRMS) and 1H NMR. HRMS data was obtained using Agilent
Accurate-Mass-Q-TOF MS 6520 system equipped with an Electro
Spray Ionization (ESI) source. All the MS experiments were
detected in the positive ionization mode. For Q-TOF/MS conditions,
fragment and capillary voltages were kept at 130 and 3500 V,
respectively. Nitrogen was supplied as the nebulizing and drying
gas. The temperature of the drying gas was set at 300 8C. The flow
rate of the drying gas and the pressure of the nebulizer were 10 L/
min and 25 psi, respectively. Full-scan spectra were acquired over
The crude compounds were purified on a C18 reversed-phase
preparative column (250 mm  4.6 mm, 10
mm) with a flow rate
of 10 mL/min using the ratio of acetonitrile/water (v/v) 50:50
containing 0.1% TFA. UV detection was at 215 nm. Purification by
reversed phase high performance liquid chromatography (HPLC)
yielded peptides with over 95% purity. The physical and
identification data of target compounds L1–L6 are given in the
Supporting information.
2.2. Bioassays
a scan range of m/z 80–1200 at 1.03 spectra sÀ1 1H NMR spectra
.
The detailed in vitro and in vivo biological evaluations and
functional assays of Dippu-AstR in response to AST analogs are also
shown in the Supporting information.
was recorded on a Bruker AM-300 (300 MHz) spectrometer with
DMSO-d6 as the solvent and TMS as the internal standard.
Chemical shifts (d) are reported in parts per million (ppm).
General synthetic procedure: The synthesis of analogs was
illustrated in Scheme 1. GFGL with resin was synthesized from Rink
Amide-AM resin (440 mg, 0.3 mmol) using the standard Fmoc/tBu
chemistry and HBTU/HOBt protocol [15]. Incoming amino acids
were activated with HBTU (456 mg, 1.2 mmol), HOBt (163 mg,
3. Results and discussion
3.1. In vitro effect of AST analogs on the inhibition of JH biosynthesis
0.3 mmol) and DIEA (210
couplings were run for 2 h at room temperature. Removal of the N-
terminal Fmoc group from the residues was accomplished with
m
L, 0.6 mmol) in DMF (5 mL) for 5 min,
The ability of target analogs to inhibit JH biosynthesis was
evaluated in vitro using the CA of the cockroach D. punctata. As
shown in Table 1, all target analogs exhibit differing potencies as a
Scheme 1. Synthetic route of lead compound L5 (K24) and target compounds L1–L4, L6. Reagents: (a) 20% piperidine/DMF, 20 min. (b) Fmoc-
L-Leu-OH, HBTU, HOBT, DIEA, r.t.,
2 h. (c) Fmoc-Gly-OH, HBTU, HOBT, DIEA, r.t., 2 h. (d) Fmoc- -Phe-OH, HBTU, HOBT, DIEA, r.t., 2 h; (e) Fmoc-Gly-OH, HBTU, HOBT, DIEA, r.t., 2 h; (f) Relative phenyl acids,
L
HBTU, HOBT, DIEA, r.t., 3 h; (g) 20% piperidine/DMF, 20 min; then 90% TFA, 5% phenol, 2.5% H2O, 2.5% TIS, r.t., 2 h.
Please cite this article in press as: X.-Q. Wu, et al., Synthesis, bioactivity and functional evaluation of linker-modified allatostatin analogs
as potential insect growth regulators, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.02.010

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3
AST analogs induced clear dose-dependent bioluminescence
response in Dippu-AstR expressing cells, albeit with different
potencies as a consequence of linker length. None of the AST
analogs functioned as a full agonist in this cell line. CHO-WTA11
cells transfected with pcDNA3.1D (empty vector) did not show
any response to the AST analogs. The EC50 values of AST analogs in
activating Dippu-AstR ranged from 62 nmol/L to 2952 nmol/L
(Table 1). In general, the ability of AST analogs to activate Dippu-
AstR corresponded to their potencies as inhibitors of JH production
by CA. This Dippu-AstR activity-linker length relationship was
consistent with above relationship between JH inhibitory activity
and linker length. However, the AST analogs K24/L5 and L6 which
showed similar activity in inhibiting JH biosynthesis as AST 1,
exhibited much lower EC50 values in activating the Dippu-AstR
(Table 1). This suggests that L5 and L6 exhibit more resistant to
metabolic inactivation.
Fig. 2. Dose-response curves for ASTs in CHO-WTA11 cells expressing Dippu-AstR.
Data points represent the average Æ SEM of three independent measurements
performed in duplicate and are expressed as percentage of the maximal response. The
zero response level corresponds to treatment with BSA buffer only.
3.3. In vivo effect of L6 on JH biosynthesis and basal oocyte growth
L6, which displays the highest activity in inhibition of JH
biosynthesis in vitro, was used to determine its in vivo effect
following injection and topical treatment in D. punctata. As
shown in Fig. 3A, injection of L6 resulted in an apparent decrease
in JH biosynthesis of 42.6% in comparison to control values.
Moreover, fluctuation in rates of JH biosynthesis after emer-
gence from day 1 to day 8 is shown in Fig. 4A. L6 exhibited a
highly significant and sustained effect on JH biosynthesis
following topical application, although the inhibitory effect
was reduced on day 6. This indicates that appropriate
optimizations in AST structure can increase their resistance to
degradation in vivo. Simultaneously, the inhibitory effect of L6
on basal oocyte growth in adult female D. punctata was also
assessed. For injected animals, the average oocyte length was
about 0.25 mm less than that of untreated animals on day 3
(Fig. 3B). For topical assays, oocytes of treated animals
developed more slowly than in control animals (Fig. 4B). Usually
untreated females oviposit on day 7 of adult life. However, no
treated females had oviposited by day 8.
consequence of varying linker length. L1 (n = 0) shows the lowest
activity with an IC50 of 957 nmol/L, compared to other analogs. L2
to L6 are more active in inhibiting JH biosynthesis with IC50 values
in the low nanomolar range. L2 (n = 1) with IC50 of 93.5 nmol/L, is
about 10-fold activity greater than that of L1. L2, L3 (n = 2) and L4
(n = 3) exhibit slightly improved activity by a small margin when
their linker lengths increase from 1-carbon to 3-carbon. L6 (n = 5)
with an IC50 of 27.2 nmol/L displays better activity than the lead
compound K24/L5 (n = 4, IC50: 37.8 nmol/L). This result suggests
that the potency of the analogs may be related to their lipophilicity.
Generally, the lipophilicity can be characterized by the retention
time or be calculated by the prediction system. Thus, the retention
time and log P value of six AST analogs were further determined
(Table 1). The result showed that the longer linker length, the
longer retention time or the bigger log P value. This demonstrates
that longer linkers provide a positive contribution to the inhibitory
activity.
Natural ASTs exert little or no effect in vivo because of their
rapid degradation by peptidases and poor absorption through the
insect cuticle in the case of topical application [16,17]. To date, only
H17 was found to have a significant effect on JH biosynthesis by
cockroach CA, both in vitro and in vivo. In the present study, L6, has
been shown to represent another potential AST analog with
potential to act both in vitro and in vivo.
3.2. Potency of AST analogs in activation of Dippu-AstR
To study the mechanism of action of AST analogs in inhibiting
JH biosynthesis, the activity of AST analogs in activation of
Dippu-AstR was determined. AST 1, the shortest peptide of the
ASTs, was chosen as a control [5]. As shown in Fig. 2, all tested
Fig. 3. Effect of L6 on JH biosynthesis by CA (A) and oocyte growth (B) from day 3 mated female D. punctata in vivo following injection. Five microliter of L6 (final concentration
was 10 mol/L) was injected into cockroaches on day 1 and assayed on day 3. Animals injected with 5 L of H2O were used as control group. Each bar represents the
m
m
mean Æ SEM for the number of individual measurements indicated at the top of error bars. Asterisks indicate significant differences between peptide- and water-injected groups of
animals as determined by t-test: **** P < 0.0001.
Please cite this article in press as: X.-Q. Wu, et al., Synthesis, bioactivity and functional evaluation of linker-modified allatostatin analogs
as potential insect growth regulators, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.02.010

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Fig. 4. Effect f L6 on JH biosynthesis by CA (A) and basal oocyte growth (B) from day 1 to day 8 in mated female D. punctata following topical application in vivo. Animals were
topically treated with 5 L of L6 in 20% DMSO and 80% acetone on day 0. Animals were topically treated with 5 L solvent of 20% DMSO and 80% acetone without AST analogs
were used as control group. (A) Each point represents mean Æ SEM, N = 10–15. (B) Each point represents mean Æ SEM, N = 6–8.
m
m
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4. Conclusion
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In summary, a series of AST analogs were synthesized by
modifying the linker of K24 and their bioactivities were
evaluated both in vitro and in vivo. These results show that
all target analogs exhibited differing potencies in inhibiting JH
biosynthesis and activating Dippu-AstR as a consequence of
varying linker length. In particular, analog L6, with the longest
linker (n = 5), had very good effect on inhibition of JH
biosynthesis both in vitro and in vivo. Furthermore, it displayed
good activity in inhibiting basal oocyte growth. These results
suggest that L6 is a new potential IGR for cockroach control.
Structure–activity relationship suggested that longer linkers
increased their ability to inhibit JH biosynthesis.
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Acknowledgments
We are very grateful to Prof. Jozef Vanden Broeck forhis kind help
on functional assay of AstR in response of AST analogs. This study
was financially supported by the National Natural Science Founda-
tion of China (No. 21372257), the National Basic Research Program
ofChina(973Program, No. 2010CB126104)and theNatural Sciences
and Engineering Research Council of Canada. The author Xiao-qing
Wu was financially supported by China Scholarship Council (CSC) to
study in the laboratory of SST at University of Toronto, Canada.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cclet.2016.02.010.
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Please cite this article in press as: X.-Q. Wu, et al., Synthesis, bioactivity and functional evaluation of linker-modified allatostatin analogs
as potential insect growth regulators, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.02.010