A Potential Insect Growth Regulator: Synthesis and Bioactivity of an Allatostatin Mimic

Peptides 30 (2009) 1249–1253

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Peptides

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A potential insect growth regulator: Synthesis and bioactivity of an allatostatin mimic

Zhen-peng Kai a,b, Juan Huang a, Stephen S. Tobe b,*, Xin-ling Yang a a Department of Applied Chemistry, College of Science, China Agricultural University, Beijing, 100193, PR China b Department of Cell and Systems Biology, University of Toronto, 25 Harbord St., Toronto, ON, Canada M5S 3G5

ARTICLE INFO ABSTRACT

Article history: Insect growth regulators play an important role in Integrated Pest Management systems. Cockroach- Received 18 February 2009 type allatostatins (FGLamides) (ASTs), which are a family of basic peptides first isolated from brains of Received in revised form 15 March 2009 Diploptera punctata were originally discovered on the basis of their ability to inhibit the production of Accepted 17 March 2009 juvenile hormone by the corpora allata. For this reason, the ASTs can be regarded as possible IGR Available online 26 March 2009 candidates for pest control although the absence of effect in vivo, rapid degradation and high production costs of the natural peptides preclude their use in pest management. However, we have synthesized a Keywords: new AST mimic, H17, from the pentapeptide C-terminal active core of the AST. This mimic is able to Allatostatin significantly inhibit the biosynthesis of JH by cockroach CA in vitro (IC value: 12 nM) and in vivo IGR 50 Juvenile hormone following injection (IC50 value: 33 nM). H17 also shows a highly significant inhibition of JH production in Biosynthesis topical cuticular assays in vivo. Our results suggest that H17 has potential as an IGR for cockroach Cockroach control. Peptidomimetic ß 2009 Elsevier Inc. All rights reserved.

1. Introduction allatostatin receptors and elicits inhibition of JH production [4,9]. Garside et al. [3] studied the effects of injection of Dippu- Insect growth regulators (IGRs) are compounds that can AST 5 or Dippu-AST 6 analogs on rates of JH biosynthesis in vitro regulate the growth of insect pests and play an important role and on basal oocyte growth of mated female cockroaches, D. in Integrated Pest Management systems. As commercial IGRs have punctata. These authors found several peptidomimetics that some shortcomings such as the slowness of insecticidal activity, inhibited JH biosynthesis and oocyte growth significantly although pest resistance and limitations in their application, it has become the effects of injection were monitored only at one specific age. necessary to develop new IGRs that have new structures and new Piulachs et al. [8] also synthesized Dippu-AST 5 analogs that can targets to address these problems. inhibit JH biosynthesis and vitellogenin production by the fat body Cockroach-type (FGLa) allatostatins (ASTs) are a family of basic of the cockroach Blattella germanica. All the analogs were designed peptides first isolated from brains of Diploptera punctata that were with Dippu-AST 5 and Dippu-AST 6 as the lead compounds and originally found to inhibit the production of juvenile hormone (JH) both are octapeptides. In the present study, for purposes of by the corpora allata (CA) [13]. ASTs are potent inhibitors of JH potential application, we have focused on pentapeptide active core biosynthesis in cockroaches [5] and they therefore represent mimics, to reduce the production cost. potential leads for the discovery of new IGRs. However, ASTs also Our approach to the design of pseudopeptide analogs differs have some shortcomings such as lack of effect in vivo, rapid from those of Piulachs et al. [8] and Garside et al. [3], by focusing on degradation in vivo, and high production cost [3,7]. These issues the active core region and by making simple substitutions in this preclude the use of ASTs themselves in pest management. region to reduce costs of synthesis. Piulachs et al. [8], for example, Previous structure–activity studies demonstrated that the C- altered the nature of the peptide bond, thereby reducing the terminal pentapeptide Y/FXFGL-NH2 is the ‘active core’ region of susceptibility of the bond to hydrolysis. However, these com- the cockroach-type ASTs; this region probably binds to the pounds involved modifications outside the core region targeted in the present work and are considerably more difficult to synthesize. Other substitutions were made outside of the core region, which

* Corresponding author. Tel.: +1 416 978 3517; fax: +1 416 978 3522. differs from our focus. Nachman et al. [7] and Garside et al. [3] E-mail addresses: [email protected] (S.S. Tobe), [email protected] replaced Phe-Gly residues in the pentapeptide with unnatural (X.-l. Yang). amino acids to reduce degradation by endoproteases, whereas in

Fig. 1. Synthesis of H17. Reagents and conditions: (a) (E)-3-(4-nitrophenyl)acrylic acid, HBTU, HOBt, DIEA, DMF, room temperature, 3 h; (b) TFA, 5% phenol, 2.5% thioanisole, 5% water, room temp, 2 h. The crude peptide was purified on a C18 reversed-phase column with a flow rate of 1 mL/min using acetonitrile/water (80:20) containing 0.06% TFA as an ion-pairing reagent. UV detection was at 215 nm. The structure of H17 was confirmed by the presence of the following molecular ions using an 1100 series LC/MSD Trap (VL) (Agilent Technologies, USA). H17: 589.3 (calc. MNa+: 589.6). our research, the Tyr/Phe in the pentapeptide was replaced by (E)- 2.3. Bioassays 3-(4-nitrophenyl)acrylic acid to prevent degradation by amino- peptidases. This new AST mimic (H17) has potent effects in vitro 2.3.1. In vitro assays and in vivo on the inhibition of JH biosynthesis in the cockroach. All radiochemical assays for JH biosynthesis were performed This disruption in JH biosynthesis in the present study appears to using individual pairs of CA from day 7 mated females. Compounds be reflected in attenuated basal oocyte growth. were dissolved in medium 199 for assay as described previously [14,15]. Compounds were used in the bioassay on the same day 2. Methods that the samples were prepared. The solutions were discarded at the end of each day. Rates of JH release were determined using 2.1. Synthesis of peptidomimetic H17 the modified in vitro radiochemical assay [14,15]. This assay measures the incorporation of the radiolabeled S-methyl moiety of GFGL with resin was synthesized from Rink Amide-AM resin radiolabeled methionine into JH III in the final step of biosynthesis using the standard Fmoc/tBu chemistry and HBTU/HOBt (O- by CA maintained in vitro. CA were incubated for 3 h in 100 mLof Benzotriazole-N,N,N0,N0-tetramethyl-uronium-hexafluoro-phos- medium 199 (GIBCO, 1.3 mM Ca2+, 2% Ficoll, methionine-free) phate/1-hydroxybenzotriazole anhydrate) protocol [1].Incoming containing L[14C-S-methyl] methionine (40 mM, specific radio- amino acids were activated with HOBt, HBTU and DIEA in DMF for activity 1.48–2.03 GBq/mmol (Amersham)). Samples were 5 min, and couplings were run for 2 h. Removal of the N-terminal extracted and JH release determined. Each data point on the Fmoc group from the residues was accomplished with 20% dose–response figure represents replicate incubations of 10–27 piperidine in DMF for 20 min. experimental CA compared to control CA (i.e. no analog added). H17:(E)-3-(4-nitrophenyl)acrylic acid was coupled to the GFGLwithresinwithHOBt,HBTUandDIEAinDMFfor3hat 2.3.2. In vivo assays room temperature (Fig. 1). The peptide was cleaved from the resin with TFA containing 5% phenol, 2.5% thioanisole and 5% 2.3.2.1. Injection. H17 was dissolved in DMSO and then diluted to a water for 2 h. The crude peptide was purified on a C18 reversed- final DMSO concentration of 0.3% in H2O (stock final concentration phase column with a flow rate of 1 mL/min using acetonitrile/ of H17 was 10 mM). Injections were administered using a 26 gauge water (80:20) containing 0.06% TFA as an ion-pairing reagent. 10 mL Hamilton syringe. The needle was inserted into the UV detection was at 215 nm. The structure of H17 was membranous joint between the coxa and femur on the metathor- confirmed by the presence of the following molecular ions acic leg. Control insects were similarly injected, but with 5 mLof using an 1100 series LC/MSD Trap (VL) (Agilent Technologies, double distilled water. Five mLofH17 (at concentrations ranging USA). H17: 589.3 (calc. MNa+: 589.6). between 10 mM and 1 nM, serially diluted in H2O from the stock) (E)-3-(4-Nitrophenyl)acrylic acid was prepared using the were injected into D. punctata females at day 1, and animals were method of Sun et al. [12]. Rink Amide-AM resin (0.52 mmol/g assayed for JH biosynthesis at day 3 as described (see Section substitution), HOBt, HBTU, DIEA (N,N0-diisopropyl ethylamine), 2.3.1). All injections were administered to non-anesthetized mated TFA (trifluoroacetic acid) and Fmoc-protected amino acids were female D. punctata. Assuming a hemolymph volume of 50 mL for purchased from GL Biochem (Shanghai) Ltd. HPLC grade DMF (N,N- day 1 adult female D. punctata [6], the final concentrations of the dimethyl-formamide), DCM (dichloromethane) and acetonitrile were purchased from DIMA Technology Inc. (USA). Thioanisole, Table 1 99% was purchased from ACROS, USA. Age at oviposition following treatment with H17a,b.

Age (days) Number of animals ovipositing 2.2. Animals Controlsc Injection Topical applicationd

Newly emerged mated female D. punctata (day 0) were 7 24 (100%) 24 (100%) 0 isolated from stock cultures. Mating was conﬁrmed by the 8 4 (27%) presence of a spermatophore. Stocks and isolated females, fed 9 5 (33%) 10 3 (20%) Lab Chow and water ad libitum were kept at 27 1 8C, relative 11 2 (13%) humidity 50 5% with a 12 h light/12 h dark cycle. Unless a otherwise noted (Table 1 and Fig. 5), basal oocyte lengths were Female D. punctata were treated on day 0 for tropical application and on day 1 for injection. measured at the time of assay. Measurement of oocyte lengths in b Percent animals ovipositing are shown in parentheses. control animals conﬁrmed that oocyte growth in these animals was c Only values from injected control animals are shown. normal. d One animal (7%) died during the course of the experiment. Z.-p. Kai et al. / Peptides 30 (2009) 1249–1253 1251 injected analog in the hemolymph were approximately 0.1– 1000 nM. Each group of H17-injected animals was compared with a group of water-injected animals treated concurrently.

2.3.2.2. Topical application. H17 solution (5 mL) was applied to the dorsal abdomen of D. punctata females at day 0. The H17 concentration (20% DMSO and 80% acetone) used in the bioassays was 20 mM. Each animal received 0.1 nmol H17 in the topical cuticular assays. These animals were assayed for JH biosynthesis at day 3 using the method described in Section 2.3.1.

2.3.3. Oocyte growth measurements To determine if the inhibition of JH production by H17 treatment was reflected in a physiological process dependent upon titer of JH, the lengths of basal oocytes of D. punctata females were measured on each day of the first gonadotrophic cycle following treatment of the Fig. 4. The inhibitory effect in vivo of H17 on JH biosynthesis by CA following topical animals with mimic or control substance using either injection or application of H17. 0.1 nmol H17 was applied to the dorsal abdominal segments in 5 mL of 20% DMSO and 80% acetone of female D. punctata at day 0 and were assayed topical treatment as described above. The lengths of basal oocytes of on day 3. Each bar represents the mean SEM for the number of individual all experimental and control animals were measured using a measurements indicated at the top of error bars. Asterisks indicate significant dissecting microscope and ocular micrometer. differences between peptide- and water-injected groups of animals as determined by Dunnett’s multiple comparison test following one-way ANOVA: ***p < 0.0001. 3. Results 3.2. Effect of H17 on JH biosynthesis in vivo 3.1. Effect of H17 on JH biosynthesis in vitro In addition to its effect in vitro, H17 also showed a significant Treatment of adult female D. punctata with the AST peptido- effect on JH production following treatment in vivo. In the injection mimetic H17 showed a significant effect on JH production in all bioassay, the IC50 value of H17 was 33 nM (Fig. 3). In the topical treated animals. The dose response curve of for H17 in vitro cuticular assay, H17 also demonstrated a highly significant effect treatment is shown in Fig. 2 and the IC50 (inhibitory concentra- on JH biosynthesis (Fig. 4), albeit only one time point was tion50 defined as the concentration of inhibitor that provokes a measured (day 3). This age was chosen because rates of JH response halfway between the baseline and maximum response) biosynthesis increase rapidly at this time, shortly after the onset of for H17 treatment in vitro was 12 nM. This result demonstrated vitellogenesis. Accordingly, the knockdown of JH production that H17 has a significant effect in vitro, when compared with the would be particularly apparent at this time if the analog were pentapeptide (the IC50 value of YDFGLa is 130 nM—unpublished). This IC50 value for H17 is similar to those for the native Dippu-AST 1, Dippu-AST 3 and Dippu-AST 13 with values of 9.6 nM, 12.5 nM and 12.8 nM, respectively [18].

Fig. 2. Effect of H17 on JH biosynthesis by CA from day 7 mated female cockroaches in vitro as a function of dose. Each point represents mean SEM. N = 14–16.

Fig. 5. Basal oocyte lengths of female D. punctata following treatment with H17 using either (A) injection or (B) topical application. (A) Animals were injected with Fig. 3. Effect of H17 on JH biosynthesis by CA from day 3 mated female cockroaches 5 mLof1mM H17 on day 1. (B) Animals were topically treated with 0.1 nmol H17 in in vivo as a function of dose. Cockroaches were injected with the doses shown on 20% DMSO and 80% acetone on day 0. Each point represents mean SEM. N = 3–6 day 0, and assayed on day 3. Each point represents mean SEM. N = 20–33. individuals. In all cases, the error bars were smaller than the symbols showing means. 1252 Z.-p. Kai et al. / Peptides 30 (2009) 1249–1253 exerting an inhibitory effect. Each animal in the topical cuticular degradation by the aminopeptidase, and the C-terminal amino assay was treated with 0.1 nmol H17. group blocks degradation by carboxylpeptidases simulta- neously. The low molecular weight and hydrophobic nature of 3.3. Oocyte growth following treatment in vivo H17 appear to improve its absorption through cockroach cuticle and its transport in hemolymph. These results demonstrate that Untreated and control animals usually oviposit on day 7 of H17 has considerable potential as an IGR for cockroach control. adult life [11,16]. The effect of H17 on basal oocyte growth in We are continuing to study the use of H17 as a cockroach control adult females was also assessed following injection or topical agent. treatment. The difference in lengths of oocytes of D. punctata females which were treated topically was readily apparent in 5. Conclusion comparison to controls (Fig. 5B). However, the differences observed following injection were less obvious (Fig. 5A). The We report an AST mimic H17, which has a significant effect on results are also shown in Table 1. For the injected animals, all the JH biosynthesis by cockroach CA both in vitro and in vivo. Good females (both control and H17-injected) oviposited at day 7. For bioactivities of H17 in both injection and topical bioassays suggest topical assays, no animals oviposited at day 7, 27% oviposited at that H17 has considerable potential as an IGR for cockroach day 8, 33% oviposited at day 9, 20% oviposited at day 10 and 13% control. Our results also demonstrate that insect neuropeptide oviposited at day 11, whereas7% of animals died during the mimics can be potential IGRs. course of the experiment. Acknowledgements 4. Discussion We thank Dr. Tie-niu Kang for providing the (E)-3-(4- The use of peptidomimetics to disrupt physiological pro- nitrophenyl)acrylic acid. Financial support was provided by the cesses in pest insects represents a promising avenue for the National Natural Science Foundation of China (20672138 and development of new pest control agents. Surprisingly, the 20272080), the National Basic Research Program of China peptidomimetic H17 is more effective than the pentapeptide (2003CB114400), the National High Technology Research and core region of the ASTs in inhibiting the production of JH in vitro Development Program of China (2006AA10A201), the Natural (IC for H17 = 12 nM and 130 nM for YDFGLa). In fact, its 50 Sciences and Engineering Research Council of Canada and bioactivity is similar to some of the natural but less potent ASTs the China Postdoctoral Science Foundation funded project [18] (see also Section 3). This demonstrates that appropriate (20070420049). changes in AST structure can improve the binding ability of the ASTtothereceptorsand/orincreaseresistancetodegradation References in vivo. It is striking that the inhibition of JH production by H17 [1] Chan WG, White PD. Fmoc solid phase peptide synthesis. 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