Enhanced in vivo activity of peptidase-resistant analogs of the insect kinin neuropeptide family

Peptides 23 (2002) 735–745

Enhanced in vivo activity of peptidase-resistant analogs of the insect kinin neuropeptide family Ronald J. Nachmana,*, Allison Streya, Elwyn Isaacb, Nan Pryora, Juan D. Lopezc, Jin-Gen Dengd, Geoffrey M. Coaste a

Veterinary Entomology Research Laboratory, ARS, U.S. Department of Agriculture, 2881 F/B Road, College Station, TX 77845, USA b Molecular and Cellular Sciences, Faculty of Biology, University of Leeds, Leeds, LS2 9JT, UK c Areawide Pest Management Research Unit, SPARC, ARS, U.S. Department of Agriculture, 2881 F/B Road, College Station, TX 77845, USA d Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu, 610041, China e School of Biological Sciences, Birkbeck College, London WC1E 7HX, UK Received 4 September 2001; accepted 18 October 2001

Abstract The diuretic/myotropic insect kinin neuropeptides, which share the common C-terminal pentapeptide core FX1X2WG-NH2, reveal primary (X2-W) and secondary (N-terminal to F) sites of susceptibility to peptidases bound to corn earworm (H. zea) Malpighian tubule tissue. Analogs designed to enhance resistance to tissue-bound peptidases, and pure insect neprilysin and ACE, demonstrate markedly enhanced in vivo activity in a weight gain inhibition assay in H. zea, and strong in vivo diuretic activity in the housefly (M. domestica). The peptidase-resistant insect kinin analog pQK(pQ)FF[Aib]WG-NH2 demonstrates a longer internal residence time in the housefly than the native muscakinin (MK), and despite a difference of over 4 orders of magnitude in an in vitro Malpighian tubule fluid secretion assay, is equipotent with MK in an in vivo housefly diuretic assay. Aminohexanoic acid (Ahx) is shown to function as a surrogate for N-terminal Lys, while at the same time providing enhanced resistance to aminopeptidase attack. Peptidaese-resistant insect kinin analogs demonstrate enhanced inhibition of weight gain in larvae of the agriculturally destructive corn earworm moth. Potent peptidase resistant analogs of the insect kinins, coupled with an increased understanding of related regulatory factors, offer promise in the development of new, environmentally friendly pest insect control measures. © 2002 Elsevier Science Inc. All rights reserved.

1. Introduction The insect kinins share a highly conserved C-terminal pentapeptide sequence Phe-Xaa-Xbb-Trp-Gly-NH2, where Xaa can be Tyr, His, Ser or Asn, and Xbb can be Ala but is generally Ser or Pro [13,14]. The insect kinins have been isolated from a number of insects, including species of Dictyoptera, Lepidoptera, and Orthoptera. Kinin-like peptides have also been isolated from a crustacean, the shrimp Peneaus vannamei [28,32], and in a mollusk, the snail Lymnaea stagnalis [10]. The first members of this insect neuropeptide family were isolated on the basis of their ability to stimulate contractions of the isolated cockroach hindgut [12,14]. They are also potent diuretic peptides that stimulate the secretion of pimary urine by Malpighian tu* Corresponding author. Tel.: ⫹1-979-260-9315; fax: ⫹1-979-2609377. E-mail address: [email protected] (R.J. Nachman).

bules, organs involved in the regulation of fluid and ion transport. The immediate cellular response to kinin stimulation is an increase in intracellular calcium that opens a shunt conductance that allows chloride entry into the tubule lumen. A second diuretic peptide family in insects is the corticotropin-releasing factor (CRF)-related peptides that operate through cyclic-AMP as a secondary messenger. In the migratory locust (Locusta migratoria) the insect kinins and the CRF-related peptide, co-localized in locust neurosecretory cells, act synergistically to stimulate Malpighian tubule fluid secretion [6,15]. In the housefly, muscakinin has been implicated in the control of diuresis in response to hypervolemia [7] and elicits a four to five-fold increase in in vitro fluid secretion of the Malpighian tubules, more than twice the response observed with the larger CRF-related Musca-DP [6,13]. In addition, insect kinins have been identified in hemolymph [4,20] where they could act as hormones. Kinins have also been shown to inhibit protein synthesis and to mobilize lipid [11]. More recently, insect

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kinins have been reported to inhibit weight gain by larvae of the tobacco budworm (Heliothis virescens) [30]. Initial structure-activity studies with the insect kinins demonstrated that full biological activity in in vitro cockroach hindgut myotropic and cricket diuretic assays resided in the C-terminal pentapeptide, which therefore represents the active core [21]. Within this active core, a series of Ala-replacement analogs demonstrated that the Phe and Trp sidechains were critical for biological activity in both of these assays [21,26,29]. The synthesis of a rigid end-to-end cyclic analog (cyclo[AFFPWG] that was biologically active allowed detailed NMR studies of the conformation adopted by the insect kinins during receptor interaction. When data from the NMR studies were incorporated into molecular dynamics analyses, it was found that the cyclic analog could adopt two turn conformations, a predominant type VI ␤-turn over residues Phe-Phe-Pro-Trp and featuring a cisPro, and a type I-like ␤-turn encompassing residues Phe-Pro-Trp-Gly with a transPro. Further investigations with restricted conformation analogs incorporating a tetrazole moiety, a rigid mimic of a cis peptide bond, confirmed a cisPro type VI ␤-turn as the active conformation adopted by the core region when bound to receptors on cockroach hindgut and cricket Malpighian tubules [19,25,26,27,29]. An examination of the cisPro conformation indicates that the aromatic sidechains of Phe1 and Trp4, both critical for biological activity, are adjacent to one another and oriented on the same side of the mainchain backbone. In contrast, the sidechain of residue 2, a position that features a lot of variability in natural isoforms, lies on the opposite side of the mainchain backbone, extending away from the surface formed by Phe1 and Trp4 [25,26,27,29]. While the C-terminal pentapeptide retains full biological activity in in vitro cockroach hindgut myotropic and cricket Malpighian tubule fluid secretion assays, it proves to be ⬎5 orders of magnitude less potent than the intact peptide in an in vitro housefly Malpighian tubule fluid secretion assay. This difference in structural requirements for insect kinin receptor interaction is probably a function of the relative lengths and number of isoforms of the insect kinins native to the three species of insects. In the Madeira cockroach (Leucophaea maderae), all nine of the natural insect kinins are octapeptides, and in the house cricket (Acheta domesticus), the five native isoforms range from 6 – 8 residues in length. In contrast, the single insect kinin isolated from the housefly, muscakinin (MK), is 15 residues long [13]. A recent structure-activity study has demonstrated that residues Thr2, Lys8 and Arg [10], all outside the C-terminal pentapeptide core, also contribute to the potency and efficacy of MK in the housefly diuretic assay [8]. While the insect kinins have been shown to mediate critical processes such as diuresis, digestive organ contraction and larval development in insects, they are subject to rapid degradation to peptidases, such as angiotensin converting enzyme (ACE), present in the hemolymph and other insect tissues [17,18,24]. The development of peptidase-

resistant analogs of the insect kinins would provide important tools to neuroendocrinologists investigating the mechanisms by which the insect kinins function and to researchers studying novel, environmentally friendly pest insect management strategies for the future. In this study, we investigate the sequence sites susceptible to corn earworm (Heliothis zea) tissue-bound peptidase attack, and evaluate the in vivo activity of analogs designed to fortify the susceptible peptide bonds and the N-terminus. The insect kinin assays used in this study are a corn earworm larval development inhibition assay, an in vitro cricket diuretic assay, and both in vitro and in vivo housefly diuretic assays.

2. Materials and methods 2.1. Peptide synthesis Muscakinin and muscakinin fragments MK[7–15], MK[8 –15], and pQRFHSWG-NH2 [8], and the insect kinin analog [Aib] pQK(pQ)FF[Aib]WG-NH2 [24] were synthesized and purified as previously described. The helicokinin analogs VRFSSWG-NH2, pQRFS[Aib]WG-NH2, leuco/achetakinin analogs [Aib]FS[Aib]WG-NH2, [␣MeF]FS[Aib]WG-NH2, and muscakinin analogs KQRFH[Aib]WG-NH2, KQ[Aib]FH[Aib]WG-NH2 and [Ahx]KQRFH[Aib]WG-NH2 were synthesized via solid phase chemistry using Fmoc technology on an ABI 433A peptide synthesizer under previously described conditions [24]. Fmoc-Aminohexanoic acid (FmocAhx) was purchased from Neosystems Laboratoire (Strasbourg, France). The crude products were purified on a Waters C18 Sep Pak cartridge and a Delta Pak C18 reverse-phase column (8 ⫻ 100 mm, 15 ␮m particle size, 100 A pore size) on a Waters 510 HPLC controlled with a Millennium 2010 chromatography manager system (Waters, Milford, MA) with detection at 214 nm at ambient temperature. Solvent A ⫽ 0.1% aqueous trifluoroacetic acid (TFA); Solvent B ⫽ 80% aqueous acetonitrile containing 0.1% TFA. Conditions: Initial solvent consisting of 20% B was followed by the Waters linear program to 100% B over 40 min; flow rate, 2 ml/min. Delta-Pak C-18 retention times: VRFSSWG-NH2, 9 min; pQRFS[Aib]WG-NH2, 12.5 min; [Aib]FS[Aib]WG-NH2, 11 min; [␣MeF]FS[Aib]WG-NH2, 6 min; KQRFH[Aib]WGNH2, 6.25 min; KQ[Aib]FH[Aib]WG-NH2, 9.25 min; and [Ahx]KQRFH[Aib]WG-NH2, 6 min. Some of the analogs were further purified on a Waters Protein Pak I125 column (Milligen Corp., Milford, MA). Conditions: Solvent A ⫽ 95% acetonitrile made to 0.01% TFA; Solvent B ⫽ 50% aqueous acetonitrile made to 0.01% TFA; 100% A isocratic for 4 min then a linear program to 100% B over 80 min. WatPro retention times: [Aib]FS[Aib]WG-NH2, 7 min; and KQ[Aib]FH[Aib]WG-NH2, 12.25 min. These HPLC conditions have been described in detail elsewhere [24]. Amino acid analysis was carried out under previously reported

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conditions [24] and used to quantify the peptides and to confirm identity, leading to the following analyses: VRFSSWG-NH2, F[1.0], G[0.9], R[0.7], S[1.7], V[0.7]; pQRFS[Aib]WG-NH2, E[1.0], F[.0], G[0.9], R[1.2], S[1.2]; [Aib]FS[Aib]WG-NH2, F[1.0], G[1.0], S[0.9]; [␣MeF]FS[Aib]WG-NH2, F[1.0], G[1.0], S[1.0]; KQRFH[Aib]WGNH2, E[0.9], F[1.0], G[1.0], H[1.1], K[1.0], R[1.1]; KQ[Aib]FH[Aib]WG-NH2, E[1.0], F[1.0], G[1.0] H[0.8], K[1.0]; and [Ahx]KQRFH[Aib]WG-NH2, E[0.9], F[1.0], G[1.0], H[1.0], K[1.0], R[1.1]. The identity of the peptide analogs was confirmed via MALDI-MS on a Kratos Kompact Probe MALDI-MS machine (Kratos Analytical, Ltd., Manchester, UK) with the presence of the following molecular ions (MH⫹): VRFSSWG-NH2, 837.2 [calc. MH⫹: 836.4]; pQRFS[Aib]WG-NH2, 847.4 [calc. MH⫹: 847.4]; [Aib]FS[Aib]WG-NH2, 665.5 [calc. MH⫹: 665.3]; [␣MeF]FS[Aib]WG-NH2, 741.3 [calc. MH⫹: 741.4]; KQRFH[Aib]WG-NH2, 1042.0 [calc. MH⫹: 1042.5]; KQ[Aib]FH[Aib]WG-NH2, 971.5 [calc. MH⫹: 971.3]; and [Ahx]KQRFH[Aib]WG-NH2, 1155.4 [calc. MH⫹: 1155.4]. 2.2. Tissue-bound peptidase susceptibility assays Malpighian tubules were dissected from adult males of the corn earworm Heliothis zea (Heliothis premix artificial diet purchased from Stonefly Industries, Inc., Bryan, TX) and were incubated with 5 nmoles peptide in 500 ul Manduca saline [3] for 5 min up to four hours. At the end of each incubation period, the tubules were removed and 500 ul 15% trifluoroacetic acid (TFA) was added to the tube. The tubes were then vortexed and centrifuged, and the resulting supernatant was run on reversed phase HPLC (Deltabond C18 ODS 250 ⫻ 4.6 mm, 5uM, 300Å; A solvent: 0.1% TFA; B solvent: 80% acetonitrile in 0.1% TFA, 0 –100%/80 min.; 1 ml/min.). The experiment was repeated a minimum of four times. Fractions were collected, dried, and analyzed on a Kratos Kompact MALDI-MS (Kratos Analytical Ltd., Manchester, UK) to identify degradation products. The progress of the peptidase degradation was followed by monitoring the disappearance of the substrate peak.

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rate of decline of the substrate. More experimental detail is presented in a previous manuscript [18]. 2.4. Corn earworm larval development assay Newly hatched Heliothis (Helicoverpa) zea were purchased from Stonefly Industries, Inc., Bryan, TX. day 1 injections were administered to non-anesthetized 5 day old H. zea larvae weighing approximately 500 mg. These larvae were held at 28°C in individual cups of Heliothis premix artificial diet purchased from Stonefly Industries, Inc., Bryan, TX. The cups and larvae were placed on a cooling table at 4°C 10 min before injection. Each larva was injected with 0.5 microliters of aqueous test solution containing 500 pm of peptide analog using a 10 ul syringe inserted between the third and fourth segment on the dorsal surface. No bleeding was observed. A control group injected with distilled water was included in every trial. Each larva was weighed and treated at 24 h intervals until the prepupal stage was reached. This occurred at day 5 or 6 as the gut was emptied and contraction of the larva began. Pupation date was observed and recorded. The experiment was repeated five times with ten animals in each treatment group. Differences between the weight of control and treatment means were tested for statistical significance using a two-tailed, paired Student’s t test. The weight values recorded for controls and two insect kinin analogs follow. Control values are presented as 100% and treated groups are compared as a percentage of that. Standard deviation is presented in parentheses. For the control: day 1, 100 (4.7); day 2, 100 (14.9); day 3, 100 (34.7); day 4, 100 (20.3); day 5, 100 (108.4); day 6, 100 (117.3); day 7, 100 (86.2); and day 8, 100(93.2); for VRFSSWG-NH2: day 1, 96.5 (6.1); day 2, 96.6 (21.5); day 3, 94.6 (37.7); day 4,; day 4, 87.4 (25.4); day 5, 79.1 (83.5); day 6, 89.3 (136.7); day 7, 88.4 (99.5); day 8, 92.1 (93.2); and for pQRFS[Aib]WG-NH2: day 1, 102.3 (5.3); day 2, 86.6 (18.7); day 3, 74.0 (39.8); day 4, 60.3 (23.5); day 5, 52.0 (69.5); day 6, 62.5 (133.5); day 7, 75.5 (156.9); and day 8, 80.8 (152.4). Pupation times for the control (set at 100%) was 100 (1.96); for VRFSSWGNH2 it was 104.1 (1.94); and for pQRFS[Aib]WG-NH2 it was 124.0 (4.77) (P ⬍ 0.03).

2.3. Neprilysin (NEP) degradation trials 2.5. In vitro cricket fluid secretion assay Insect kinin analogs (12.5 ␮M) were incubated with pig kidney neprilysin (NEP)(a gift from Dr. A.J. Kenny, School of Biochemistry and Molecular Biology, University of Leeds) in 0.1 M Tris-HCl, pH 7.5. The greater susceptibility of VRFSSWG-NH2 to hydrolysis meant that only 3 ng of NEP was required, whereas 30 ng of NEP was used to determine the rates of hydrolysis of the other peptides. HPLC analysis of the fragments was performed using a Pharmacia Pep-S column and a linear gradient of acetonitrile (5–36%) in 0.1% TFA, at a flow rate of 0.8 ml/min. The peptides and fragments were followed using a UV monitor set at 214 nm. Rates of hydrolysis were calculated from the

This assay has been described in detail elsewhere [5]. Malpighian tubules were removed from adult female crickets 6 –12 days old and were transferred to 5 ␮l drops of bathing fluid having the following composition (in mM/ liter): NaCl, 82; KCl, 27; CaCl2, 2; MgCl2, 8.5; NaH2PO4, 4; NaOH, 11; glucose, 24; proline, 10; Hepes, 25. The pH was adjusted to 7.2 with 1 M NaOH. The bathing fluid has a higher K⫹ concentration (and lower Na⫹ concentration) than that used previously, but supports greater diuretic activity in response to kinin-stimulation, although potency is unchanged (G.M. Coast, unpublished observations). The

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dissected tubules and associated saline droplets are held under liquid paraffin. Urine escapes from the cut made close to the proximal end of the tubule and collects as a discrete droplet in the paraffin. Urine samples are collected at intervals and their volume determined from measurements of droplet diameter under a microscope. After a 40 min equilibration period, the rate of secretion was measured over two 40 min periods before and after the addition of peptide analogues. Diuretic activity is calculated as the increase in fluid secretion (⌬ nl/mm/min) and is expressed as a percentage of the response to a supramaximal dose of achetakinin-I assayed on Malpighian tubules taken from the same insect [22]. 2.6. In vitro housefly tubule secretion assay The fluid secretion assay is described in detail elsewhere [15]. Posterior-directed Malpighian tubules were dissected from the housefly, Musca domestica, and transferred to 10 ␮l drops of bathing fluid comprised of a 1:1 mixture dissection saline and Schneider’s Drosophila medium. The dissection saline had the following composition (in mM/ liter): NaCl, 135; KCl, 20; CaCl2, 2; MgCl2, 8.5; NaHCO3, 10.2; NaH2PO4, 4.3; HEPES, 15; glucose, 20. The dissected tubules and associated saline droplets are held under liquid paraffin. Urine escapes from the cut made close to the proximal end of the tubule and collects as a discrete droplet in the paraffin. Urine samples are collected at intervals and their volume determined from measurements of droplet diameter under a microscope. After a 60 min equilibration period, fluid secretion was measured over two 20 min periods before and after the addition of peptides. Diuretic activity is calculated as an increase in fluid secretion (⌬ nL/min) following peptide addition. 2.7. In vivo housefly diuretic assay (measurement of urine output) In initial studies with kinin analogues, tritiated water (THO) was used to measure water loss by houseflies in vivo as described by Coast [7]. However, with experiments lasting ⬎30 min, there is a progressive reduction in urine output and the major routes for water loss are evaporative and respiratory (G.M. Coast, unpublished observations). This makes it more difficult to obtain a reliable measure of urine output from overall THO loss. For this reason, we switched to measuring the loss of inulin since this takes place only through the excretory system. Essentially the same protocol was used for both measurements. Flies were injected intrathoracically with 0.5 ␮l saline containing either THO (⬃60,000 dpm) or 14C-inulin (⬃20,000 dpm) plus test compounds. They were then placed in 10 ml plastic scintillation vials that were immediately capped tightly. After 0.5 to 2.0 h, the vials were quenched in liquid nitrogen for 5 min. The flies were then tipped out leaving behind frozen water vapour and urine. Two millilitres of scintillant (Ecoscint

ATM National Diagnostics, Hull, UK) were added to each vial and 3H or 14C counted (Packard 2200 CA, Pangbourne, UK). Vials that contained body parts were discarded. Results for THO losses were converted to nl/min as described in Coast [7]. Inulin loss (in dpm) is expressed as a percentage of the total dpm injected.

3. Results 3.1. Heliothis zea Malpighian tubule tissue bound peptidase susceptibility trials HPLC analysis of saline solutions of helicokinin 2 analog VRFSSWG-NH2 exposed to dissected Malpighian tubules demonstrated that most of the peptide was hydrolyzed within 1 h. The principle fragments identified via MALDI-MS were VRFSS-OH and the dipeptide fragment WG-NH2, indicating that the primary hydrolysis site is between Ser and Trp. This has been previously identified as the hydrolysis site of insect kinins by angiotensin converting enzyme (ACE) from the housefly [18]. Insect kinin analogs incorporating the sterically hindered ␣,␣-disubstituted amino acid Aib as a substitute for Ser or Pro in the third position of the C-terminal pentapeptide proved resistant to housefly ACE [23,24]. Thus, a helicokinin 2 analog containing Aib and blocked at the N-terminus with a pGlu residue, pQRFS[Aib]WG-NH2 was synthesized and evaluated for susceptibility to H. zea Malpighian tubule tissuebound peptidases. This analog proved much more resistant to hydrolysis by the tissue-bound peptidases, with over 75% remaining after an exposure of 4 h. The principle fragment was identified as FS[Aib]WG-NH2, indicating that the secondary site of attack was the peptide bond between Arg and Phe. As a consequence, another insect kinin analog was synthesized which contained a second Aib residue adjacent to the secondary site of peptidase attack. The resulting analog, [Aib]FS[Aib]WG-NH2, proved to be completely resistant to the tissue-bound peptidases on the corn earworm Malpighian tubules over the 4 h period of the experiment (see Fig. 1). Such a susceptible site was not observed in previous experiments on Aib-containing insect kinin analogs subjected to housefly ACE [24]. However, the endopeptidase neprilysin (NEP) demonstrates broad specificity, preferentially hydrolyzing peptides by cleaving on the N-terminal side of hydrophobic amino acids such as the Phe (and sometimes Trp), located in the insect kinin C-terminal pentapeptide core region [9,16,33]. For this reason, the three analogs in this section were challenged with pure pig kidney NEP. 3.2. Neprilysin (NEP) degradation trials The results of NEP [9,33] degradation trials evaluating the stability of the three insect kinin analogs, VRFSSWG-

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Fig. 1. Stability of the insect kinin analog VRFSSWG-NH2 (‚), and the peptidase-resistant insect kinin analogs pQRFS[Aib]WG-NH2 (䊐) and [Aib]FS[Aib]WG-NH2 [{] to hydrolysis by peptidases bound to corn earworm (H. zea) Malpighian tubule tissue. Measurement of the amount of remaining peptide was made by HPLC at 1, 2 and 4 h. The data points represent the means of at least 4 replicates.

NH2, pQRFS[Aib]WG-NH2, and [Aib]FS[Aib]WG-NH2 essentially mirrored those obtained in the H. zea Malpighian tubule tissue-bound peptidase degradation trials in the previous section. At the 3 ng level of NEP, VRFSSWG-NH2 hydrolyzed quite rapidly at the following rate: Time 0, 100%; 10 min, 80%; 20 min, 65%; 30 min, 59%, 40 min, 45%; and 60 min, 45%. At this exposure level, the other two analogs remained stable. So, the three peptide analogs were compared at a higher NEP dose of 30 ng. The rates of hydrolysis are shown below in Table 1. The relative rates of disappearance of the three compounds estimated from the decline in parent peak over time of incubation were as follows: VRFSSWG-NH2: 132 pmol/h/ng; pQRFS[Aib]WGNH2: 1.7 pmol/h/ng; and [Aib]FS[Aib]WG-NH2: 0.6 pmol/h/ng. The stability of the two Aib-containing insect kinin analogs to pure NEP is greater than the unmodified parent compound by about 2 orders of magnitude. 3.3. Corn earworm larval development inhibition activity Recent work has demonstrated that injection of helicokinins into developing larvae of the tobacco budworm,

Table 1 Hydrolysis rates of insect kinin analogs to NEP (30 ng) Time % Parent peptide analog remaining VRFSSWG-NH2 pQRFS[Aib]WG-NH2 [Aib]FS[Aib]WG-NH2 0 1 hr 2 hr 3 hr

100% 2% 0% 0%

100% 92% 73% 71%

100% 96% 93% 89%

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Fig. 2. The effects of daily injections of 500 pmoles of the insect kinin analog VRFSSWG-NH2 (left slant) and the insect kinin peptidase-resistant analog pQRFS[Aib]WG-NH2 (right slant)on inhibition of weight gain in 5 day-old larvae of the corn earworm H. zea expressed as a percentage of saline injected control animals (solid). Each test group contained at least 10 animals and the experiment was repeated at least 5 times (***P ⬍ 0.001; *P ⬍ 0.05).

Heliothis virescens, can inhibit weight gain [30]. This in vivo insect kinin assay was chosen to test whether a peptidase-resistant analog of the insect kinins could enhance the biological response by virtue of a longer survival time within the target insect. We used 5 day-old larvae (weighing approx. 500 mg) of the related moth Heliothis (Helicoverpa) zea, the corn earworm moth, an agricultural pest of greater economic importance than H. virescens. The insect kinin analogs (0.5 nmoles) were injected daily for 5 or 6 days until pupation occurred, and weighed each day. The helicokinin II analog, VRFSSWG-NH2, demonstrated a developmental trend that reached a peak on day 5 post-treatment, with a 20% weight reduction compared with controls (see Fig. 2). But this difference was not statistically significant. In contrast, a peptidase-resistant kinin analog, pQRFS[Aib]WG-NH2, elicited a much stronger, statistically significant developmental effect, which demonstrated a 40% weight reduction on day 4 and reached a peak at day 5 of an almost 50% reduction in mean larval weight when compared with saline-injected controls (Fig. 2). The weight difference between larvae treated with the Aib kinin analog and the controls was significant (P ⬍ 0.001) for day 4 post-treatment, and for days 5–7 at a lower level of confidence (P ⬍ 0.02). From Fig. 2, the differences between the weights of treated larvae and controls would appear to become smaller. However, while pupation times for the larvae treated with the normal helicokinin analog and controls are essentially the same, the larvae treated with the Aib-containing analog show about a 25% delay (level of significance of P ⬍ 0.03) in the time of pupation. As they approach the pupal stage, the larvae reduce their feeding response and undergo contraction and loss of water. This makes the available pool of larvae in the control group appear lighter by comparison with the treated animals, which are experiencing a delay in pupation. Larvae treated

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Table 2 In vitro cricket malpighian tubule secretion activity of peptidase-resistant insect kinin analogs Insect kinin analog

pQK(pQ)Phe-Phe-Aib-Trp-GlyNH2 Aib-Phe-Ser-Aib-Trp-Gly-NH2 ␣MePhe-Phe-Ser-Aib-Trp-Gly-NH2 a

Cricket (Acheta domesticus) malpighian tubule fluid secretion EC50 (pM)a

Maximum response (%)

8.3 (5.6–12.3)

103% [24]

1.2 (0.6–2.3) 0.2 (0.2–1.7)

99% 107%

95% confidence limit (CL) values in parentheses [24].

with the double Aib insect kinin analog [Aib]FS[Aib]WGNH2 also demonstrated significant inhibition of weight gain, but this was not statistically different from the effect of pQRFS[Aib]WG-NH2. 3.4. In vitro cricket diuretic activity In Table 2, we see a comparison of the relative potency of three insect kinin analogs in a diuretic assay using cricket Malpighian tubules (the Ramsay assay). Each of these peptidase-resistant insect kinin analogs, containing the ␣,␣-disubstituted amino acids Aib and ␣-methyl-Phe (␣-MePhe), proved more potent at stimulating cricket Malpighian tubule fluid secretion than the native achetakinin peptides, which demonstrated EC50’s of between 22 and 324 pM [24]. The difference in potencies between the insect kinin analogs and the native achetakinins ranged between 1 and 2 orders of magnitude. Unfortunately, a reliable in vivo diuretic assay in the cricket could not be developed. The focus turned to an in vivo diuretic assay in the housefly, which proved quite reliable.

3.5. Housefly diuretic activity Many of the analogs reported in this paper were designed for the in vitro cricket Malpighian tubule fluid secretion assay, the reliable assay utilized at the earlier stages of the work to characterize the diuretic properties of the insect kinins. However, in the absence of a reliable cricket in vivo diuretic assay, the focus of the work shifted to a recently developed in vivo diuretic assay in the housefly [7], a pest of the poultry, dairy and swine industries [1,2,31,34]. However, while the C-terminal pentapeptide core of the insect kinins demonstrates full biological activity in the in vitro cricket Malpighian tubule secretion assay [23,25,29], it is ⬎5 orders of magnitude less potent than the native muscakinin (MK) peptide, NTVVLGKKQRFHSWG-NH2. N-terminal residues T2, K8 and R10 also contribute to receptor binding [8]. In addition to analogs designed using the in vitro cricket diuretic assay, MK analogs containing two of these three important residues were synthesized and evaluated in both in vitro and in vivo housefly diuretic assays. In Table 3, the diuretic activity of insect kinin analogs in the in vitro housefly Malpighian tubule secretion assay is presented in descending order of potency. It is evident from Table 3 that MK is considerably more potent than the fragment and peptidase-resistant analogs tested. It is some 60 fold more potent than the C-terminal fragment MK[7–15] and 150 fold more potent than the next fragment MK[8 –15]. The Aib-containing analogs [Ahx]KQRFH[Aib]WG-NH2 and KQRFH[Aib]WG-NH2 demonstrate a large additional drop in potency of between 700 and 800 fold as compared with the parent MK. The C-terminal hexapeptide fragment-analog of MK (pQRFHSWG-NH2), in which the Gln residue is cyclized to an N-terminal pGlu, and the double Aib MK analog KQ[Aib]FH[Aib]WG-NH2 are between 1700 and 1900 fold less potent than MK. Following is the cricket acheta-

Table 3 In vitro housefly malpighian tubule secretion activity of native and peptidase-resistant insect kinin analogs Insect kinin analog

NTVVLGKKQRFH[Ser]WG-NH2 (MK) KKQRFH[Ser]WG-NH2 (MK[7–15]) KQRFH[Ser]WG-NH2 (MK[8–15]) [Ahx]KQRFH[Aib]WG-NH2 KQRFH[Aib]WG-NH2 pQRFH[Ser]WG-NH2 KQ[Aib]FH[Aib]WG-NH2 AFH[Ser]WG-NH2(AK-V) [Aib]FS[Aib]WG-NH2 pQK(pQ)FF[Aib]WG-NH2 a

Housefly (Musca domestica) malpighian tubule fluid secretion EC50 (nM)a

Maximum response (⌬ nl/min)b

0.13 (0.08–0.21)

12.6 (0.6) [8]

7.6 (6.3–9.3) 19.9 (11.3–34.9) 91 (82–101) 102 (95–110) 220 (0.18–0.27) 244 (204–293) 771 (686–866) 1.5 ␮M (1.1–2.0) 8.3 ␮M (6.5–10.6)

13.3 (0.3) [8] 14.1 (0.7) [8] 12.2 (0.2) 12.4 (0.1) 14.4 (1.9) [8] 15.5 (0.4) 11.5 (0.2) [8] 13.3 (0.3) —c

95% confidence limit (CL) values in parentheses [24] Values in parentheses represent ⫾1 S.E. c Maximum response could not be determined due to low potency. At 100 ␮M, it reached 7.3(1.4) nl.min. b

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Table 4 In vivo housefly inulin clearance diuretic activity of native and peptidase-resistant insect kinin analogsa Insect kinin analog

NTVVLGKKQRFH[Ser]WG-NH2 (MK) pQK(pQ)FF[Aib]WG-NH2 [Aib]FS[Aib]WG-NH2 [Ahx]KQRFH[Aib]WG-NH2 KQ[Aib]FH[Aib]WG-NH2 pQRFH[Ser]WG-NH2 KKQRFH[Ser]WG-NH2(MK[7–15]) KQRFH[Ser]WG-NH2(MK[8–15]) AFH[Ser]WG-NH2(AK-V) KQRFH[Aib]WG-NH2 Saline control a

Housefly (Musca domestica) Inulin clearance diuretic activity % IE (1 h)b

% IE (2 h)b

1.33 ⫹ 0.10 (18)* 1.37 ⫹ 0.23 (9)* 1.29 ⫹ 0.10 (19)* 1.33 ⫹ 0.03 (6)* 0.68 ⫹ 0.16 (23) 1.04 ⫹ 0.16 (12)* 1.04 ⫹ 0.16 (9)* 0.95 ⫹ 0.13 (9)* 0.72 ⫹ 0.10 (9) 0.51 ⫹ 0.13 (11) 0.49 ⫹ 0.09 (24)

1.74 ⫹ 0.09 (18)* 1.56 ⫹ 0.13 (11)*c 1.56 ⫹ 0.08 (21)* 1.42 ⫹ 0.08 (9)* 1.31 ⫹0.09 (23)* 1.29 ⫹ 0.16 (12)* 1.23 ⫹ 0.13 (12) 1.23 ⫹ 0.15 (11) 0.78 ⫹ 0.15 (10) 0.61 ⫹ 0.13 (9) 0.92 ⫹ 0.09 (34)

All analogs were tested at a dose of 50 pmoles injected in 0.5 ␮l saline.

kinin, AFHSWG-NH2, with housefly diuretic activity 6000 fold less potent than MK. Finally, the insect kinin peptidaseresistant analogs [Aib]FS[Aib]WG-NH2 and pQK(pQ)FF[Aib]WG-NH2 demonstrate a loss of in vitro potency of over 4 orders of magnitude compared with MK. Nonetheless, the situation becomes quite different when these analogs are evaluated in an in vivo housefly diuretic assay. In Table 4, the diuretic activity of insect kinin analogs in the in vivo housefly inulin clearance (urine output) assay is presented in descending order of potency. The parent MK stimulates excretion and the percentage of injected inulin (%IE) excreted at 1 h (1.33%) and 2 h (1.74%) is about 2–3-fold greater than in saline-injected control flies. The MK fragments MK[7–15] and MK[8 –15] elicit about a 2-fold increase of urine output over control flies, with 1 h IE values of 1.04 ⫹ 0.23% and 0.90 ⫹ 0.09%, respectively. But at 2 h, the % IE is not significantly different than the saline controls. It is noteworthy, however, that the three peptidase-resistant analogs pQK(pQ)FF[Aib]WG-NH2 (Fig. 3a), [Aib]FS[Aib]WG-NH2 and [Ahx]KQRFH[Aib]WG-NH2 demonstrate strong % IE values of [1.37% (1 h), 1.56% (2 h)], [1.29% (1 h), 1.56%, (2 h)], and [1.33% (1 h), 1.42% (2 h)], respectively; each of which are not statistically different from the % IE of the native neuropeptide MK. Two of these analogs contain an Aib residue to prevent endopeptidase cleavage of the Cterminal dipeptide and a blocked N-terminus to prevent susceptibility to aminopeptidases. The other contains two Aib groups that protect two peptidase hydrolysis sites. The inulin excretion activity of the MK[8 –15] analog incorporating two Aib groups (KQ[Aib]FH[Aib]WG-NH2) is not statistically different from controls at 1 h, but is at 2 h post-injection (1.31% IE). This suggests that the peptidaseresistant analog with two sterically hindered Aib residues is remaining in the body of the insect to a greater extent than its muscakinin fragment parent (MK[8 –15]), which does not demonstrate statistically significant diuretic activity at 2 h. The hexapeptide fragment analog pQRFHSWG-NH2

features a blocked N-terminus that should protect it from aminopeptidase degradation, and demonstrates % IE values that are significantly different from the controls at both 1 and 2 h post injection [1.04% (1 h), 1.29% (2 h)]. The inulin clearance values for the achetakinin AK-V and the remaining Aib-containing analog are not significantly different from the saline controls. At 4 h post injection, pQK(pQ)FF[Aib]WG-NH2 (Fig. 3a) elicits increased inulin clearance activity over saline controls that is statistically significant (P ⬍ 0.01). The inulin clearance activity of the native MK is not statistically significant at 4 h (Fig. 3b). Further evidence that enhanced peptidase resistance can confer longer residence times within the hemolymph fly could be found in a tritiated water (THO) diuretic assay, which measures total water loss. In this experiment, 20 pmoles of muscakinin and the most active insect kinin analog in vivo, pQK(pQ)FF[Aib]WG-NH2, were injected separately into two groups of flies. For the MK treated flies, THO loss was significantly increased at 0.5 and 1 h, but not at 2 h post injection. In contrast, significant THO loss (about 2-fold more than controls) remained at 2 h post injection in the flies treated with the peptidase resistant Aib-containing analog (see Fig. 4).

4. Discussion In this study, we investigate the sites susceptible to tissue-bound peptidase hydrolysis in the diuretic/myotropic insect kinin family, and test analogs designed to enhance resistance to peptidase attack. These peptidase-resistant analogs feature the replacement of Pro or Ser in the third position of the C-terminal pentapeptide with the sterically hindered Aib residue, a modification that has been shown to be compatible with the turn conformation that is important for successful interaction with the receptor site [19]. Peptidase-resistant analogs were then evaluated in several in vivo insect kinin assays to determine whether a longer residence time within the internal environment of the target insect

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Fig. 4. Tritiated water loss at 0.5 and 2 h from flies injected with 20 pmoles of either pQK(pQ)FF[Aib]WG-NH2 or MK dissolved in 0.5 ␮l of saline. Data points are the means and vertical lines ⫾ 1 S.E. for 10 –12 determinations. At 0.5 h, water loss is significantly increased in both groups of flies compared with the saline controls, whereas at 2 h it is only increased in flies injected with pQK(pQ)FF[Aib]WG-NH2. Fig. 3. Percentage of injected inulin excreted at 1, 2 and 4 h by adult male flies injected with 50 pmoles of either pQK(pQ)FF[Aib]WG-NH2 or MK dissolved in 0.5 ␮l of saline. Data points are the means and vertical lines ⫾ 1 S.E. for a minimum of 11 replicates. Asterisks indicate points that are significantly greater than saline controls at the same time interval (***P ⬍ 0.001; **P ⬍ 0.01; *P ⬍ 0.05).

would lead to an enhanced biological response. Exposure of the helicokinin II analog VRFSSWG-NH2 to dissected Malpighian tubule tissue from the corn earworm H. zea indicates that the primary hydrolysis site from tissue-bound peptidases is the peptide bond between the active core residues Ser and Trp, resulting in the cleavage of the C-

terminal dipeptide (Fig. 5). The resulting fragments are inactive in insect diuretic, myotropic and larval development assays [21,25,29]. This has been established as the site of hydrolysis of the insect kinins to purified housefly ACE [18], and analogs incorporating the sterically hindered ␣,␣disubstituted amino acid Aib adjacent to the susceptible peptide bond have been shown to be resistant to housefly ACE [24]. Therefore, the Aib-containing helicokinin analog pQRFS[Aib]WG-NH2, which is also blocked at the Nterminus with a pGlu residue to protect against aminopeptidase attack, was exposed to the H. zea Malpighian tubule tissue. As can be seen in Fig. 1, this analog proved to be

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Fig. 5. Primary (large arrow) and secondary (small arrow) hydrolysis sites of peptidases bound to H. zea Malpighian tubule tissue for insect kinin analog VRFSSWG-NH2. Analogs designed to increase resistance to peptidases demonstrated enhanced in vivo physiological responses in a larval weight-gain inhibition and two diuretic insect bioassays.

considerably more resistant to peptidase hydrolysis than the parent peptide. While the parent helicokinin analog was degraded within 1 h, virtually all of the Aib-containing analog survived in that time frame, and over 75% remained after 2–3 h. Analysis of the fragments with MALDI-MS indicated that a secondary hydrolysis site could be found between the Arg and the Phe residues (Fig. 5). Such a susceptible site was not identified in earlier experiments with housefly ACE. Another peptidase found in insects, the endopeptidase neprilysin (NEP), does have broad specificity, preferentially hydrolyzing peptides by cleaving on the N-terminal side of hydrophobic amino acids such as the Phe, and sometimes Trp [16,31]. Both helicokinin analogs were subjected to pure NEP from the pig kidney. The parent helicokinin II analog was readily hydrolyzed with a dose of 30 ng of NEP, and was largely degraded within an hour. The Aib containing analog demonstrated much more stability under these conditions, with 92% remaining after 1 h and over 70% after 3 h (see Table 1). In order to also protect the Arg-Phe peptide bond, another Aib was incorporated adjacent to the susceptible bond via replacement of Arg, leading to the analog [Aib]-FS[Aib]WG-NH2. The double-Aib analog proved to be completely resistant to tissue-bound peptidases of the H. zea Malpighian tubule throughout the 4 h duration of the experiment (Fig. 1). In the NEP trials, the double-Aib insect kinin analog also proved much more resilient, with 89% remaining after 3 h. The three helicokinin analogs above were then evaluated in a larval development assay with the corn earworm H. zea, a major agricultural pest. Previous experiments with larvae of the related moth H. virescens have demonstrated that injection of helicokinins into developing larvae can inhibit weight gain [30]. It has been suggested that this reduction in weight gain may be due to both an increase in diuresis and also an induction of a ‘starvation signal’ that caused the animals to mobilize their own energy stores and to fail to utilize the digested diet. Experiments by Goldsworthy et al. have shown that injected doses of insect kinins can elicit increases in lipid concentrations and reductions in protein levels in the hemolymph of crickets and locusts [11]. In H. zea, we found that the injection of the helicokinin II led to an approx. 20% reduction in weight gain at day 5 of the

743

experiment as compared with control animals, but the difference was not statistically significant. Injection of the Aib-containing analog pQRFS[Aib]WG-NH2 demonstrated a much more pronounced effect on weight gain (Fig. 2). The peptidase-resistant helicokinin analog treated animals demonstrated weight gain reduction of 40% at day 4 (P ⬍ 0.001) and reached a peak at day 5 of an about a 50% (P ⬍ 0.02) reduction in mean larval weight. Statistically significant reductions in weight gain (P ⬍ 0.02) continued on both days 6 and 7, whereupon the larvae began the pupation process. While no statistically significant difference could be observed between the time of pupation of the normal insect kinin analog and the controls, a delay of about 25% (P ⬍ 0.03) was observed in the treated with the peptidase-resistant analog. A delay in pupation time is consistent with the reduced feeding activity and small size of the treated animals. Peptidase-resistance confers a clear advantage to insect kinin analogs, leading to an enhancement of the disruption of larval development in H. zea. The next focus was to evaluate peptidase resistant insect kinin analogs in an insect in vivo diuretic assay. Because a reliable in vivo diuretic assay in the initial test insect (the cricket) could not be developed, we turned to the housefly, a pest of the poultry, dairy and swine industries [1,2,31,34]. While several in vivo diuretic housefly assays were available, particularly the inulin clearance assay described in this paper, the structural requirements for in vitro housefly Malpighian tubule fluid secretion activity proved to be quite different from that of the cricket. The C-terminal pentapeptide insect kinin core retained full biological activity in the cricket Malpighian tubule fluid secretion assay, but proved to be over 5 orders of magnitude less potent in the analogous housefly diuretic assay. N-terminal residues outside of the active core region (T2, K8, and R10) also contributed to the ability of muscakinin (MK) to bind to the housefly Malpighian tubule receptor [8]. Consequently, the analogs developed around the insect kinin core demonstrated poor activity in the in vitro diuretic assay. While peptidase resistant analogs [Aib]FS[Aib]WG-NH2 and pQK(pQ)FF[Aib]WG-NH2 were shown to be 3–20 times more potent in the in vitro cricket diuretic assay than the most potent native achetakinin, they demonstrated a precipitous drop in potency of over 4 orders of magnitude in comparison with MK in the housefly in vitro diuretic assay. Even the peptidase resistant analog [Ahx]KQRFS[Aib]WG-NH2, based on the MK C-terminal nonapeptide fragment, proved about 1900 fold less potent than MK (Table 3). These analogs were nonetheless able to elicit a near maximal response in the Malpighian tubule fluid secretion assay. The in vivo housefly inulin clearance assay, a measure of urine output, provided a far different order of potency for the insect kinin analogs than was found for the in vitro Malpighian tubule fluid secretion assay in the same insect. For this assay, flies were injected with 50 pmoles of the test compounds dissolved in 0.5 ␮l saline. This will give an initial hemolymph concentration of about 22.5 ␮M, which

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is 3 times greater than the EC50 of even the least potent analog (see Table 3). The native housefly insect kinin, musckakinin (MK), demonstrated an increase of between 2–3 fold in the output of urine, as measured by inulin clearance, over the saline controls. This difference between MK and saline treated houseflies was statistically significant at 1 and 2, but not 4 h, post injection (Fig. 3b). C-terminal octapeptide (MK[8 –15]) and nonapeptide (MK[7–15]) fragments of MK also elicited an increase in urine output, but this difference was only significant at 1 h, but not 2 h, post injection. Almost all of the analogs with components that impart resistance to endopeptidases and/or aminopeptidases, based on various fragments of the MK sequence, demonstrated statistically significant increases in urine output at both 1 and 2 h postinjection. Indeed, the increases in in vivo urine output measured for the two peptidase resistant analogs at the bottom of the list in terms of in vitro diuretic potency in the housefly, pQK(pQ)FF[Aib]WG-NH2 and [Aib]FS[Aib]WG-NH2, are not statistically different from MK at 1 and 2 h post injection (Table 4). In addition, while the urine output of MK at 4 h postinjection is not statistically different from saline controls, the pQ-Aib containing analog demonstrated a statistically significant increase in urine output at the 4 h mark (P ⬍ 0.01) (Fig. 3a). The only Aib-containing analog that did not show a statistically significant increase in urine output in treated flies over controls at either 1 or 2 h after injection was KQRFH[Aib]WG-NH2. This lack of in vivo activity was probably due to the fact that the analog contains no protection from aminopeptidase attack on the integrity of the C-terminal pentapeptide core, which is required to elicit a diuretic response [8,21,26,29]. When this octapeptide analog is capped with aminohexanoic acid (Ahx), it is transformed into a highly active diuretic agent (Table 4). The difference in % IE between the two analogs is statistically significant (P ⬍ 0.001). The component Ahx lacks the N-terminal amino group that would render a peptide sequence susceptible to aminopeptidase attack. However, it does retain the amino-containing side chain of Lys, which contributes to the diuretic potency of MK at this position. This data demonstrates that Ahx can act as a surrogate of an N-terminal Lys residue while at the same time its incorporation into a peptide sequence can impart resistance to aminopeptidase degradation. Analogs such as pQK(pQ)FF[Aib]WG-NH2 and [Aib]FS[Aib]WG-NH2, which demonstrate in vivo housefly diuretic activity that is virtually equivalent to the native MK neuropeptide, despite a difference of over 4 orders of magnitude in the in vitro diuretic assay, must do so because their peptidase resistant nature enables the analogs to remain within the housefly for an extended period. Further evidence of a longer residence time was found in a tritiated water (THO) loss assay, which measures total water loss in vivo. As illustrated in Fig. 4, injection of MK into the housefly elicits an increase in THO water loss after 1 h that is statistically significant, but not after 2 h. In contrast, the most potent of the insect kinin analogs in the inulin clear-

ance assay, pQK(pQ)FF[Aib]WG-NH2, continues to elicit a significant increase in THO loss even 2 h after treatment. While evidence suggests that peptidase resistance can increase the residence time of insect kinin analogs, there nonetheless appears to be a limit to the duration of the diuretic response. The total water loss does not appear to exceed the volume of water injected following hypervolemia. Therefore, the limited duration of the diuretic response following treatment with these active peptidase resistant analogs suggests that other regulatory mechanisms, such as antidiuretic factors, are being mobilized by the housefly to prevent a severe disruption of the internal water balance. A thorough understanding of native antidiuretic factors in pest flies is clearly recommended, so that they can be overcome in order to achieve disruption of the internal water balance in concert with potent peptidase resistant insect kinin analogs. In conclusion, incorporation of components that can infer resistance to degradative peptidases in the tissues and blood of insects can enhance the in vivo biological activity of insect kinin neuropeptides. Peptidase resistant insect kinin analogs demonstrate significantly greater inhibition of weight gain, and a delay in pupation time, in larvae of the corn earworm H. zea. Evidence suggests that peptidase resistant insect kinin analogs can survive for longer periods of time within the housefly. This enhanced stability allows analogs with otherwise inferior potency in an in vitro housefly diuretic assay to demonstrate superior in vivo diuretic activity. The development of peptidase-resistant insect kinin analogs of even greater potency and an increased understanding of related regulatory mechanisms within pest insects can lead to disruption of insect kinin regulated functions and ultimately to new, environmentally friendly pest insect management strategies in the future.

Acknowledgments We wish to thank Alan Tyler (London) for technical assistance. We also acknowledge the financial assistance of a Collaborative Research Grant (No. 973325) from the North Atlantic Treaty Organization (NATO) (RJN & GMC).

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