Active diuretic peptidomimetic insect kinin analogs that contain β-turn mimetic motif 4-aminopyroglutamate and lack native peptide bonds

Peptides 34 (2012) 262–265

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Active diuretic peptidomimetic insect kinin analogs that contain ␤-turn mimetic motif 4-aminopyroglutamate and lack native peptide bonds Ronald J. Nachman a,∗ , Krzysztof Kaczmarek a,b , Janusz Zabrocki a,b , Geoffrey M. Coast a,c,∗∗ a

Areawide Pest Management Research Unit, Southern Plains Agricultural Research Center, ARS, U.S. Department of Agriculture, 2881 F/B Rd., College Station, TX 77845, USA Institute of Organic Chemistry, Technical University of Lodz, 90-924 Lodz, Poland c Department of Biological Sciences, Birkbeck, University of London, London WC1E 7HX, UK b

a r t i c l e

i n f o

Article history: Received 10 August 2011 Received in revised form 28 September 2011 Accepted 28 September 2011 Available online 5 October 2011 Keywords: Insect management Reduced peptide bond Cricket Malpighian tubule Diuresis

a b s t r a c t The multifunctional ‘insect kinins’ of arthropods share the evolutionarily conserved C-terminal pentapeptide core sequence Phe-X1 -X2 -Trp-Gly-NH2 , where X1 = His, Asn, Ser, or Tyr and X2 = Ser, Pro, or Ala. Insect kinins regulate diuresis in many species of insects, including the house cricket, Acheta domesticus. Insect kinins, however, are susceptible to fast enzymatic degradation by endogenous peptidases that severely limit their potential use as tools for pest control or for endocrinological studies. To enhance resistance to peptidases, the core insect kinin sequence was structurally modified in this study to replace native peptide bonds susceptible to proteolytic degradation. These modifications include incorporation of two stereochemical variants of the ␤-turn mimetic motif 4-aminogutamate in place of the X1 -X2 residues, insertion of a reduced peptide bond between residues Trp-Gly, and replacement of the Phe residue with a hydrocinnamyl group. The resulting biostable, peptidomimetic analogs contain no native peptide bonds and yet retain significant diuretic activity in an in vitro cricket Malpighian tubule fluid secretion assay, matching the efficacy of a native A. domesticus kinin (Achdo-KI). These novel analogs represent ideal new tools for endocrinologists studying arthropod kinin regulated processes in vivo, and provide leads in the development of novel, environmentally friendly pest insect management agents capable of disruption of the critical processes that kinins regulate. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Due to their specificity and their high activity at extremely low concentrations, neuropeptides have been studied as potential leads for the development of new environmentally friendly pest control agents. However, the natural compounds cannot be directly used, as they are susceptible to degradation by endogenous peptidases present in the insect digestive system, outer surfaces of many tissues and circulating in the hemolymph (blood) [3,4,14]. With an understanding of both chemical and conformational requirements responsible for neuropeptide biological activity, the design of analogs containing unnatural moieties that overcome these limitations is attainable [22]. In diverse species, insect kinins stimulate hindgut contractions, diuresis, digestive enzyme release, and probably regulate larval weight gain [1,2,5,8,16,21,27,28]. The endogenous insect

∗ Corresponding author. Tel.: +1 979 260 9315; fax: +1 979 260 9377. ∗∗ Corresponding author at: Department of Biological Sciences, Birkbeck, University of London, London WC1E 7HX, UK. Tel.: +44 208 245 2046; fax: +44 7759056495. E-mail addresses: [email protected] (R.J. Nachman), [email protected] (G.M. Coast). 0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.09.021

kinins are 6–23 amino acids long neuropeptides characterized by the evolutionarily conserved C-terminal pentapeptide Phe-X1 X2 -Trp-Gly-NH2 , where X1 = His, Asn, Ser, or Tyr and X2 = Ser, Pro, or Ala [6,7,26,33]. This C-terminal pentapeptide kinin core is the minimum sequence required for full cockroach myotropic and cricket diuretic activity in in vitro bioassays [16,19] and for bioluminescence response in CHO-K1 cells expressing kinin receptors [9,24,32]. In fact, the active core sequence Phe1 -Tyr2 Pro3 -Trp4 -Gly5 -NH2 is equipotent with the parent nonapeptide (SGADFYPWGa) in the cockroach myotropic and cricket diuretic assays. Within the active core sequence, the aromatic residues Phe1 and Trp4 are crucial for activity in both bioassay systems, whereas position 2 tolerates wide variations in side chain character [2,16]. Although the kinin active core is highly conserved, residues outside of the C-terminal pentapeptide sequence may be needed for high affinity receptor binding. In the housefly (Musca domestica), for example, the full length peptide (Musdo-K) is >5 orders of magnitude more potent in a diuretic assay than the C-terminal pentapeptide. This suggests it may be possible to design analogs with high specificity for target species. Conformational studies suggest that the active conformation adopted by the C-terminal pentapeptide active core of the insect kinins at the cricket Malpighian tubule receptor site is the cis-Pro

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type VI ␤-turn over residues 1–4 [23,25]. Analogs incorporating the tetrazole and 4-aminopyroglutamate (APy) motifs, mimics of a cis-peptide bond and a type VI ␤-turn, elicited significant diuretic activity and provided evidence for this preferred active conformation [18,23,25]. An evaluation of all four stereochemical variants of the APy motif in the insect kinin core sequence in the cricket Malpighian tubule secretion assay revealed that the analog (2R,4S)APy (EC50 = 7 × 10−9 M) was an order of magnitude more potent than the other three analogs, including the parent analog (2S,4S)APy (EC50 = 1.4 × 10−7 M) [12]. The parentheses preceding the APy denote the stereochemistry at the two chiral centers at positions 2 and 4 within the 4-aminopyroglutamate ring. Unfortunately, insect kinin peptides are unsuitable as pest control agents and/or research tools for insect neuroendocrinologists due to susceptibility to degradation by both exo- and endopeptidases on tissue surfaces and in the hemolymph (blood) and gut, as well as an inability to efficiently penetrate the outer cuticular layer of the insect. Two susceptible hydrolysis sites in insect kinins [18] have been reported. The primary site is between the Pro3 and Trp4 residues and the secondary site is N-terminal to the Phe1 residue in natural, extended insect kinin sequences. In this study, we synthesized simplified mimetic analogs of the insect kinin Cterminal pentapeptide core that incorporate the (2R,4S)-APy and (2S,4S)-APy motifs, a ‘reduced peptide bond’ linkage ([r]) [17,30] between residues Trp4 -Gly5 , and feature a hydrocinnamyl (Hca) group as a replacement and mimic of the Phe1 residue. The two analogs (listed below) were evaluated on the cricket Malpighian tubule fluid secretion assay. The biostable, mimetic analogs contain no native peptide bonds that would be susceptible to degradation by exo- and endopeptidases. They represent a significant step toward the development of completely non-peptide mimetic agonists. In addition, they provide leads in the development of novel, environmentally friendly pest insect management agents capable of disruption of the critical processes that the insect kinins regulate. Hca-APy(2S,4S)-Trp[r]Gly-NH2 (1796) Hca-APy(2R,4S)-Trp[r]Gly-NH2 (1797) 2. Materials and methods 2.1. Peptidomimetic analog synthesis Synthesis was performed via a combination of solution and solid phase chemistry [10–12,18]. Fmoc-Gly-OH (Applied Biosystem, Foster City, CA) was attached to Rink Amide resin (0.25 mM, 0.70 mm/g, 100–200 mesh, NOVABIOCHEM, San Diego, CA) followed by removal of the Fmoc group. Both steps were performed on an ABI433A peptide synthesizer (Applied Biosystem, Foster City, CA). Fmoc-Tryptophanol (AnaSpec Inc., Freemont, CA) was oxidized to an Fmoc-Tryptophanal with Dess-Martin periodinane method (0.3 M solution in DCM; Aldrich, St. Louis, Mo) according to a previously described procedure [15,29]. This aldehyde was used for reductive alkylation of the N-terminal amino group of the glycine residue by treatment of the intermediate aldimine with NaBH3 CN in trimethyl orthoformate. This step was adapted from a published procedure [30], which was elaborated by us for the reductive alkylation of the N-terminal amino group in a 4-aminopyroglutamate residue by means of 4-(4-benzyloxy)benzaldehyde and/or benzaldehyde using Solid Phase Peptide Synthesis (SPPS). Standard removal of the Fmoc protection from the tryptophanyl residue was accomplished by means of 4-methylpiperidine, followed by standard washings (N-Methylpyrrolidone [NMP], methanol [MeOH], NMP). The secondary amino group arising from reductive alkylation was left unprotected during the rest of the synthesis, expecting that this amino group would not be acylated either

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in the next step by activated Fmoc-4-aminopyroglutamate or in the last step by an activated dihydrocinnamyl residue due to steric hindrance. The Fmoc-APy(2S,4S)-OH and Fmoc-APy(2R,4S)OH required in the next step were obtained using a previously described method [10–12]. The next step of the synthesis was Fmoc-APy(2S,4S)-OH or Fmoc-APy(2R,4S)-OH acylation with the free primary alpha-amino group of a tryptophyl residue, which was accomplished by double coupling using 1.5 equiv. Fmoc- AA/O - (7 - Azabenzotriazol - 1 - yl)-N,N,N ,N -tetramethyluronium hexafluorophosphate (HATU)/1-Hydroxy-7aza-benzotriazole (HOAt)/2,4,6collidine/NMP. After the Fmoc deprotection step already described, the next step of the synthesis was acylation with hydrocinnamic acid activated by HATU in the presence of 2,4,6-collidine in NMP. The products from the resin were cleaved by means of trifluoracetic acid containing 2.5% Triisopropylsilane (TIS) and 2.5% water, and then precipitated by pouring filtered TFA cleavage solution slowly into cold diethyl ether. The peptidomimetic analogs were purified on a Waters C18 Sep Pak cartridge and a Delta-Pak C18 reverse-phase column (8 mm × 100 mm, 15 ␮m particle size, 100 A pore size) on a Watres 510 HPLC controlled with a Millennium 2010 chromatography manager system (Waters, Milford, MA) with detection at 214 nm at ambient temperature [21]. 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 (tR in min) – 1796 (Hca-APy(2S,4S)-Trp[r]Gly-NH2 ): 13.4; 1797 (Hca-APy(2R,4S)Trp[r]Gly-NH2 ): 12.0. The peptidomimetic analogs were further purified on a Waters Protein Pak I125 column (7.8 mm × 300 mm) (Milligen Corp., Milford, MA). Conditions: Flow rate: 2.0 ml/min; 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 (tR in min) – 1796 (Hca-APy(2S,4S)-Trp[r]Gly-NH2 ): 6.0; 1797 (Hca-APy(2R,4S)-Trp[r]Gly-NH2 ): 6.25. Amino acid analysis was carried out under previously reported conditions [21], with the exception of a prolonged hydrolysis period of 6 h, and used to quantify the peptidomimetic analogs and to confirm identity, leading to the following analyses – 1796 (Hca-APy(2S,4S)-Trp[r]Gly-NH2 ): G[1.0]; 1797 (Hca-APy(2R,4S)Trp[r]Gly-NH2 ): G[1.0]. Identities of the peptidomimetic analogs were confirmed via MALDI-TOF-MS on a Kratos Kompact Probe MALDI-TOFMS machine (Kratos Analytical, Ltd., Manchester, UK) with the presence of the following molecular ions [MH+ ] – 1796 (Hca-APy(2S,4S)-Trp[r]Gly-NH2 ): 504.8 (calc: 504); 1797 (HcaAPy(2R,4S)-Trp[r]Gly-NH2 ): 504.9 (calc: 504). 2.2. Cricket Malpighian tubule secretion bioassay Crickets were reared as described [1,2] and fed a diet of turkey starter crumbs. Water was provided ad libitum. Malpighian tubules were removed from 6 to 12 day-old adult virgin females. Single tubules were isolated in vitro as described [2]. After 40 min equilibration period, the bathing fluid was changed and the rate of secretion, in picoliters per millimeter length of tubule per minute (pL/mm/min), was determined over 40 min (control rate). Thereafter, the bathing fluid was exchanged for one containing the peptide analog and the rate of secretion was determined over a second 40 min period (experimental rate). Diuretic activity was calculated as the percentage increase in the rate of secretion between the first and second measurements. Each assay included a group of tubules that were challenged with a supramaximal concentration (10 nM) of Achdo-KI during the experimental period. This allowed the diuretic activity of test analogs to be compared with that of

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O H2N

NH

N

N H

O

H N

N H

O

NH2

O

O

O N H

NH O

O

NH N H

H N

O NH2

Fig. 1. A structural comparison of the insect kinin C-terminal pentapeptide core sequence [top] and the two stereoisomeric peptidomimetic analogs Hca-APy(2S,4S)-Trp␺[CH2 NH]Gly-NH2 (Hca-APy(2S,4S)-Trp[r]Gly-NH2 ) (1796), Hca-APy(2R,4S)-Trp␺[CH2 NH]Gly-NH2 (Hca-APy(2R,4S)-Trp[r]Gly-NH2 ) (1797) [bottom]. The two analogs differ only in the stereochemistry at the chiral center designated by a wavy bond between the ring and the carbonyl group within the middle box, and can therefore be represented by a single structure (bottom). The three boxes highlight the regions of the parent pentapeptide that were modified in the peptidomimetic analogs. In the box on left, it can be seen that the analogs lack the N-terminal amino group; as the Phe residue is replaced with hydrocinnamic acid rather than another amino acid. Highlighted by the central box, the amino acids PhePro are replaced with the 4-aminopyroglutamic acid motif that locks in a cis-amide bond and mimics a type VI ␤-turn. In the box on the right, it can be seen that the carboxyl group of the peptide bond is replaced with a methylene group, yielding a reduced peptide bond between the Trp and Gly residues. The two analogs lack the native peptide bonds present in the parent insect kinin pentapeptide that render it susceptible to peptidase degradation.

Achdo-KI. All experiments were performed at room temperature (21–24 ◦ C) [2]. 3. Results The two stereochemical variant insect kinin mimetic analogs 1796 and 1797 containing (2S,4S)-APy (APy) (1796) and (2R,4S)APy (Apy) (1797) (see Fig. 1), respectively, were evaluated on isolated Malpighian tubules of the house cricket Acheta domesticus. The rate of secretion by unstimulated tubules was 285 pL/mm/min and results are expressed as the percentage increase in secretion following the addition of test compounds. The two biostable analogs retained significant diuretic activity on the cricket Malpighian tubule fluid secretion assay (Fig. 2). Both retained activity at a threshold concentration between 1 and 10 ␮M; and while the activity of 1797 appeared to be somewhat greater than that of 1796, this difference was not statistically significant under these experimental conditions. For comparison, native Achdo-KI at 10 nM is also included in Fig. 2. The maximal response (efficacy) of the two analogs and the natural Achdo-KI are statistically equivalent, although the latter is 3 orders of magnitude more potent. 4. Discussion Evaluations of the two stereochemical variant insect kinin mimetic analogs 1796 and 1797 containing (2S,4S)-APy (APy) (1796) and (2R,4S)-APy (Apy) (1797) (Fig. 1) on isolated Malpighian tubules of the house cricket A. domesticus were undertaken to

Fig. 2. In vitro Acheta domesticus Malpighian tubule secretion assay of the peptidomimetic insect kinin analogs 1796 (Hca-APy(2S,4S)-Trp[r]Gly-NH2 ) and 1797 (Hca-APy(2R,4S)-Trp[r]Gly-NH2 ) tested at 1 and 10 ␮M concentrations, and the native Achdo-KI (AK-I) (10 nM). Results are expressed as the percentage increase in the rate of secretion following the addition of test compounds. Bars indicate the mean and vertical lines +1 s.e.m. for the number of replicates shown in parentheses.

determine if they could retain the fluid secretion stimulatory response observed for the natural insect kinins and parent APy analogs. Despite the major structural modifications present in these mimetic analogs in comparison with the natural insect kinins and even the parent APy analogs, the two biostable analogs retained significant diuretic activity on the cricket Malpighian tubule fluid secretion assay (Fig. 2). While the potency of the modified analogs is less than the parent compound, their maximal response (efficacy) and that of natural Achdo-KI are statistically equivalent. The two peptidomimetic APy analogs of the insect kinin C-terminal pentapeptide active core feature major structural modifications in comparison with the natural sequence, including substitution of the Phe1 residue with Hca, replacement of the peptide linkage between the C-terminal Trp4 -Gly5 residue block with an isosteric reduced bond [17,31] and a cyclic APy linkage as a replacement for the Xaa2 -Pro3 dipeptide block (Fig. 1). As a consequence, the analogs contain no native peptide linkages. The Hca moiety blocks the amino terminus and prevents aminopeptidase attack, whereas reduced bonds contain no hydrolyzable amide linkage [17,31]. The unnatural APy moiety represents a severe departure from the amino acid block that it replaces, making it less susceptible to peptidase attack [10], and in addition this moiety is not flanked by an ␣-amino acid. A previous paper has reported the synthesis of ‘simplified’ analogs of the insect kinins based on an amino piperidinone carboxylate scaffold that also lack native peptide bonds, though these peptidomimetic analogs showed extremely weak stimulatory activity at a concentration of 10 ␮M in the in vitro cricket Malpighian tubule fluid secretion assay that fell far short of the maximal response of Achdo-KI [13]. Whereas 1796 and 1797 match the maximal response of Achdo-KI and almost double the rate of tubule secretion, three of the ‘simplified’ analogs detailed in the previous report manage to elicit only between 10 and 26% of the efficacy of the native peptide [13]. The two peptidomimetic analogs lack the native peptide bonds that would be expected to render them susceptible to hydrolysis by either exo- or endo-peptidases in the hemolymph (blood) or tissues of pest insects. A diuretic agonist that the target insect is unable to inactivate via normal proteolytic pathways offers the potential to disrupt the water balance critical for insect survival. Previous reports indicate that while other insect kinin analogs with enhanced biostability show potency levels that are even several orders of magnitude less than native peptides in in vitro Malpighian tubule secretion assays they nonetheless match the potency of the

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native peptides in an in vivo housefly diuretic assay [20]. Interestingly, biostable insect kinin analogs containing Aib residues have recently been reported to demonstrate potent oral aphicidal effects that match the potency of at least some commercial aphicides [28]. In summary, the active biostable peptidomimetic insect kinin analogs described in this study match the efficacy of the native kinin neuropeptide, Achdo-KI, in an in vitro diuretic assay, although they are less potent. They represent a significant step toward the development of completely non-peptide agonists of this critical regulatory peptide family. Secondly, these novel analogs and/or 2nd generation agents, either in isolation or in combination with biostable analogs of other neuropeptide classes that also regulate aspects of diuretic, antidiuretic, digestive, reproductive and/or developmental processes, represent potential leads in the development of selective, environmentally friendly pest insect control agents capable of disrupting those critical processes. Acknowledgements We wish to thank Allison Strey and Nan Pryor (USDA, College Station, TX) for able technical assistance. We also acknowledge financial assistance from the North Atlantic Treaty Organization (NATO) (RJN, GMC, KK, and JZ) Collaborative Research Grant (#LST.CLG.979226) and the USDA/DOD DWFP Research Initiative (#00500-32000-001-01R) (RJN). References [1] Coast GM. The regulation of primary urine production in insects. In: Coast GM, Webster SG, editors. Recent advances in arthropod endocrinology. Cambridge, UK: Cambridge University Press; 1998. p. 189–209. [2] Coast GM, Holman GM, Nachman RJ. The diuretic activity of a series of cephalomyotropic neuropeptides, the achetakinins, on isolated Malpighian tubules of the house cricket, Acheta domesticus. J Insect Physiol 1990;36:481–8. [3] Cornell MJ, Williams TA, Lamango NS, Coates D, Corvol P, Soubier F, Hoheisel J, Lehrach H, Isaac RE. Cloning and expression of an evolutionary conserved single-domain angiotensin converting enzyme from Drosophila melanogaster. J Biol Chem 1995;270:13613–9. [4] Gäde G, Goldsworthy GJ. Insect peptide hormones: a selective review of their physiology and potential application for pest control. Pest Manag Sci 2003;59:1063–75. [5] Harshini S, Manchu V, Sunitha VB, Sreekumar S, Nachman RJ. In vitro release of amylase by culekinins in two insects: Opsinia arenosella (Lepidoptera) and Rhynchophorus ferrugineus (Coleoptera). Trends Life Sci 2003;17:61–4. [6] Holman GM, Cook BJ, Nachman RJ. Isolation, primary structure, and synthesis of leucokinins VII and VIII: the final members of this new family of chephalomyotropic peptides isolated from head extracts of Leucophaea maderae. Comp Biochem Physiol 1987;C88:31–4. [7] Holman GM, Nachman RJ, Coast GM. Isolation, characterization and biological activity of a diuretic myokinin neuropeptide from the housefly Musca domestica. Peptides 1999;20:1–10. [8] Holman GM, Nachman RJ, Wright MS. Insect neuropeptides. Annu Rev Entomol 1990;35:201–17. [9] Holmes SP, Barhoumi R, Nachman RJ, Pietrantonio PV. Functional analysis of a G protein-coupled receptor from the southern cattle tick Boophilus microplus (Acari: Ixodidae) identifies it as the first arthropod myokinin receptor. Insect Mol Biol 2003;12:27–38. [10] Kaczmarek K, Chung NN, Schiller PW, Zabrocki J. A novel cis-peptide bond motif inducing type VI ␤-turn. Synthesis and biological evaluation of conformationally restricted enkephalin and morphiceptin analogues. In: Peptides 2002 (Proceedings of the 27th European peptide symposium). 2003. p. 158–9. [11] Kaczmarek K, Kaleta M, Chung NN, Schiller PW, Zabrocki J. A novel cis-peptide bond motif inducing ␤-turn type VI. The synthesis of enkephalen analogues modified with 4-aminopyroglutamic acid. Acta Biochim Pol 2001;48:1159–63.

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