cis-peptide bond mimetic tetrazole analogs of the insect kinins identify the active conformation

Peptides 23 (2002) 709 –716

cis-peptide bond mimetic tetrazole analogs of the insect kinins identify the active conformation Ronald J. Nachmana,*, Janusz Zabrockia,b, Jacek Olczakb, Howard J. Williamsc, Guillermo Moynac,d, A. Ian Scottc, Geoffrey M. Coaste a

Veterinary Entomology Research Laboratory, ARS, U.S. Department of Agriculture, 2881 F/B Road, College Station, TX 77845, USA b Technical University of Lodz, 90 –924 Lodz, Poland c Department of Chemistry, Texas A&M University, College Station, TX 77840, USA d Department of Chemistry & Biochemistry, University of the Sciences in Philadelphia, Philadelphia, PA 19104, USA e School of Biological Sciences, Birkbeck College, London WC1E 7HX, UK Received 4 September 2001; accepted 21 September 2001

Abstract The insect kinin neuropeptides have been implicated in the regulation of water balance, digestive organ contraction, and energy mobilization in a number of insect species. A previous solution conformation study of an active, restricted-conformation cyclic analog, identified two possible turn conformations as the likely active conformation adopted by the insect kinins at the receptor site. These were a cisPro type VI ␤-turn over C-terminal pentapeptide core residues 1– 4 and a transPro type I-like ␤-turn over core residues 2–5, present in a ratio of 60:40. Synthesis and evaluation of the diuretic activity of insect kinin analogs incorporating a tetrazole moiety, which mimics a cis peptide bond, identifies the active conformation as the former. The discovery of a receptor interaction model can lead to the development of potent agonist and antagonist analogs of the insect kinins. Indeed, in this study a tetrazole analog with D stereochemistry has been shown to demonstrate partial antagonism of the diuretic activity of natural insect kinins, providing a lead for more potent and effective antagonists of this critical neuropeptide family. The future development of mimetic agonists and antagonists of insect kinin neuropeptides will provide important tools to neuroendocrinologists studying the mechanisms by which they operate and to researchers developing new, environmentally friendly pest insect control strategies. © 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 [8]. They 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 Penaeus vannamei [19,22], and in a mollusk, the snail Lymnaea stagnalis [5]. The first members of this insect neuropeptide family were isolated on the basis of their ability to stimulate contractions of the isolated cockroach hindgut [7,9]. However they are also potent diuretic peptides that stimulate the secretion of primary urine by Malpighian tubules, organs involved in the * Corresponding author. Tel.: ⫹1-979-260-9315; fax: ⫹1-979-2609377. E-mail address: [email protected] (R.J. Nachman).

regulation of salt and water balance. 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 [3,8]. In the housefly, muscakinin has been implicated in the control of diuresis in response to hypovolemia [4] 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 [3,8]. In addition, insect kinins have been identified in hemolymph [1,12] where they could act as hormones. Kinins have also been shown to inhibit protein synthesis and to mobilize lipid [6]. More recently, insect kinins have been reported to inhibit weight

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Fig. 1. Low energy conformation of cyclo[Ala-Phe-Phe-Pro-Trp-Gly] found by computational search on Convex and Cray XMP supercomputers with programs Discover and Insight [17,18,20] in the predominant cisPro type VI ␤-turn over active core residues 1– 4 (Phe-Phe-Pro-Trp). This turn conformation was more energetically favored and more consistent with structure-activity data than the other possible conformation, a transPro type I-like turn over core residues 2–5 [17,18,20] [Reproduced from Fig. 5 in reference 17 with permission from the New York Academy of Sciences, New York, NY].

gain by larvae of the tobacco budworm (Heliothis virescens) [21]. Structurally, the insect kinins require an intact C-terminal pentapeptide sequence for full cockroach myotropic and cricket diuretic activity, which therefore represents the active core [14]. An Ala-replacement analog series of the insect kinin active core region confirms the importance of the Phe and Trp sidechains, because these are the only two replacements which lead to complete loss of myotropic and diuretic activity [15,18,20]. Due to decreased conformational freedom, active cyclic analogs are more useful for defining the receptor-bound conformation than are linear analogs. Analysis of the conformations adopted by the endto-end, cyclic insect kinin analog cyclo(Ala-Phe-Phe-ProTrp-Gly), in which distance and angle constraints obtained from aqueous NMR spectra were incorporated into molecular dynamics calculations, indicated the presence of two turn types over two distinct sets of residues within the active core pentapeptide. The more rigid of the two conformations featured a cisPro in the third position of a type-VI ␤-turn over core residues 1– 4, or Phe-Phe-Pro-Trp (Fig. 1). ROESY spectra supported a well-defined C␤-exo/C␥-endo pucker for the cisPro ring that was observed in unrestrained molecular dynamics for this cyclic analog. The other less rigid turn system involved a transPro and encompassed residues 2–5, or Phe-Pro-Trp-Gly. From unrestrained molecular dynamics calculations, the most favorable cisPro structure had an intramolecular energy about 7 kcal/mole lower than the most favorable transPro structure, consistent with NMR data that indicated that the cisPro structure was the predominant conformation in solution by a 60:40 ratio [17,18,20]. This is in agreement with systematic studies on linear peptides with Pro3 in which the flanking aromatic residues promote the formation of type VI ␤-turns in aque-

ous solution. Such turns are further enhanced when small, hydrophyllic residues (i.e. Asp, Ser, Thr, Gly or Asn) follow the aromatic-Pro-aromatic motif [23], as occurs in the cyclic insect kinin analog. The molecular modeling studies further indicate that interactions between the aromatic sidechains in positions 1 and 4 help to stabilize the turn over residues 1– 4 containing the cisPro configuration, which might otherwise be expected to be less energetically favorable than transPro. The cyclic analog is about 20-fold more potent than the putative degradation product Phe-Phe-Pro-Trp-Gly-Ala-OH in both myotropic and diuretic insect assays, demonstrating that the observed activity arises from the cyclic motif rather than linear by-products of in situ peptidase degradation during the course of the assays. In addition, the diuretic activity of the cyclic analog is equivalent to the linear appended analog Phe-Phe-Pro-Trp-Gly-Ala-NH2 [17,18, 20]. Therefore, the conformational studies on this cyclic analog provide strong evidence that the kinins adopt a turn conformation about the Pro residue in the active core during receptor interaction. Further evidence consistent with the ␤-turn over residues 1– 4 as the likely active conformation was obtained in a study of the solution conformation of the linear, conformationally constrained insect kinin pentapeptide analog PhePhe-Aib-Trp-Gly-NH2 [16] The sterically hindered ␣,␣disubstituted residue Aib replaces the Pro (and/or Ser) at the third position of the pentapeptide core region. Structure sets consistent with ROESY NMR distance constraints obtained by restrained simulated annealing in vacuo indicate a predominant population of a ␤-turn involving the Phe1-Trp4 region [11]; evidence of a turn in the Phe2-Gly5 region was also observed, as was the case with the cyclic kinin analog [17,18,20]. Although the available evidence (including structureactivity studies) was most supportive of the 1– 4 ␤-turn as the active receptor bound conformation [17,18,20], the 2–5 ␤-turn could not be dismissed as a candidate. in order to obtain more definitive evidence for the active conformation, active synthetic analogs that could preferentially form one of the two available conformations over the other were needed. In this paper, we report on the synthesis, insect diuretic activity and solution conformation of a C-terminal pentapeptide active-core insect kinin analog incorporating a tetrazole moiety, known to mimic a cis peptide bond and preferentially induce the formation of a type VI ␤-turn [10,24,25].

2. Materials and methods 2.1. Insect kinin tetrazole analog synthesis 2.1.1. General methods Melting points were determined on a Thomas-Hoover capillary melting point apparatus and are uncorrected. FAB mass spectra were obtained on a Finnigan MAT95 spec-

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trometer equipped with a capillaritron gas gun from Phrasor Scientific (Duarte, CA). HPLC was performed on a SpectraPhysics instrument with an SP8800 ternary pump, using a Vydac C18 column, 0.46 ⫻ 25 cm, particle size 5 ␮m, at a flow rate of 1 ml/min and solvents (A) 0.05% trifluoroacetic acid in H2O and (B) 0.038% trifluoroacetic acid in methanol/H2O (90:10). The final purification of the two tetrazole analog products was completed 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 C ⫽ 0.1% aqueous trifluoroacetic acid (TFA); Solvent D ⫽ 80% aqueous acetonitrile containing 0.1% TFA. Conditions: Initial solvent consisting of 20% D was followed by the Waters linear program to 100% D over 40 min; flow rate, 2 ml/min. This was followed by purification on a Waters Protein Pak I125 column (Milligen Corp., Milford, MA). Conditions: Solvent E ⫽ 95% acetonitrile made to 0.01% TFA; Solvent F ⫽ 50% aqueous acetonitrile made to 0.01% TFA; 100% E isocratic for 4 min then a linear program to 100% F over 80 min. Optical rotations were measured in a 1-dm cell (1 ml) on a Horiba high speed automatic polarimeter at 589 nm (Na D line). The tetrazole analogs of the insect kinin C-terminal pentapeptide active core were synthesized via solution phase chemistry in a stepwise manner as indicated below. 1. Z-Phe-⌿(CN4)-Ala-OBzl (1), C27H27N5O4, MW. 485.50 This compound (1.90 g, 3.91 mmol) was synthesized from the corresponding dipeptide in a way described previously [25] in a yield 39.7%. 2. HBr.H-Phe-⌿(CN4)-Ala-OBzl (2), C20H22N5O2Br, MW. 444.32 Compound 1 (0.907 g, 1.87 mmol) was deprotected by HBr in AcOH as described previously [25]. The crude hydrobromide (883 mg) was used for subsequent coupling without further purification. 3. Boc-Phe-Phe-⌿(CN4)-Ala-OBzl (3), C33H38N6O5, MW. 598.67 Compound 2 was coupled with Boc-Phe-OH with the use of mixed anhydride method as follows: Boc-Phe-OH (265 mg, 1 mmol) was dissolved in 3 ml of CH2Cl2. NMM (0.112 ml, 1 mmol) was added and the solution was cooled to ⫺15°C. Isobutyl chloroformate (0.135 ml, 1 mmol) was added, and the mixture was allowed to stir at that temperature for 20 min. Hydrobromide 2 (435 mg) was added and, after 5 min of stirring, the second equivalent of NMM (0.112 ml, 1 mmol) was added dropwise over 15 min. The mixture was stirred at ⫺15°C for additional 30 min and then it was allowed to warm to ambient temperature. After two hours the reaction mixture was diluted with 50 ml of ethyl

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acetate, and washed with 1M KHSO4 (2⫻, 10 ml), 5% NaHCO3 (2⫻, 10 ml) and brine (2⫻, 10 ml) and dried overnight over MgSO4. 457 mg (0.763 mmol, yield 76.3%) of product as an amorphous solid was secured after purification on silica gel in AcOEt/hexanes (3:5) solvent system. HPLC purity: 93.2%, tR ⫽ 21.9 min (50 –70%B in 25 min) [␣]D ⫽ ⫺52.8 (c 0.54, MeOH); mp. 138 –140°C 4. Boc-Phe-Phe-⌿(CN4)-Ala-OH MW. 508.55

(4),

C26H32N6O5,

Compound 3 (427 mg, 0.713 mmol) was dissolved in MeOH and hydrogenated (with hydrogen contained in a balloon) over palladium on charcoal for 4 h. The reaction progress can be conveniently followed by TLC (AcOEt/ hexanes). After the spot of starting material on TLC had disappeared, the catalyst was filtered off and the solvent removed in vacuo. The resulting white solid was triturated with hexanes and filtered. Yield 233 mg (0.458 mmol, 64.3%) HPLC purity: 98.6%, tR ⫽ 8.4 min (50 –70%B in 25 min) 5. Z-Phe-⌿(CN4)-D-Ala-OBzl (5), C27H27N5O4, MW. 485.50 This compound was synthesized analogously to its diastereoisomeric counterpart (1) as it has been described previously [1] starting from 8 mmol of Z-Phe-D-Ala-OBzl. Chromatographical purification (AcOEt/hexanes, 1:2) afforded 1.73 g (3.56 mmol, 44.5%) of product as a colorless oil. HPLC purity: 95.0%, tR ⫽ 23.2 min (50 –70%B in 25 min) [␣]D ⫽ ⫹40.3 (c 0.76, MeOH). 6. HBr.H-Phe-⌿(CN4)-D-Ala-OBzl (6), C20H22N5O2Br, MW. 444.32 Compound 5 (1.65 g, 3.40 mmol) was deprotected in the same manner as the L-Ala containing analog and the resulting hydrobromide (1.56 g) was used in the next step without any purification. 7. Boc-Phe-Phe-⌿(CN4)-D-Ala-OBzl (7), C33H38N6O5, MW. 598.67 The peptide bond forming reaction was performed in an essentially identical way as in the case of compound 3 at 1 mmol scale. Yield 351 mg (0.586 mmol, 58.6%). HPLC purity: 99.5%, tR ⫽ 16.7 min (50 –90%B in 25 min) [␣]D ⫽ ⫹32.7 (c 0.80, MeOH); mp. 75–77°C 8. Boc-Phe-Phe-⌿(CN4)-D-Ala-OH (8), C26H32N6O5, MW. 508.55

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Hydrogenation of benzyl ester 7 (320 mg, 0.534 mmol) provided compound 8 in a 87.3% (237 mg, 0.467 mmol) yield. HPLC purity: 95.9%, tR ⫽ 7.8 min (50 –70%B in 25 min) [␣]D ⫽ ⫹12.6 (c 0.79, MeOH); mp. 88 –90°C The N-protected C-terminal dipeptide (Boc-Trp-GlyMBHA), common for both designed analogs, was synthesized on MBHA resin at 0.4 mmol scale according to wellknown procedures. After the last coupling step, the peptide bearing resin was carefully washed with DMF (3⫻), CH2Cl2 (3⫻), MeOH (3⫻), Et2O (3⫻) and dried overnight in a dessicator. It was then split into two equal portions (the theoretical peptide loading was 0.2 mmol for each). Both portions were Boc-deprotected separately by means of 50% TFA in CH2Cl2 and subsequently washed with CH2Cl2 (3⫻), MeOH (3⫻), CH2Cl2 (3⫻) and DMF (3⫻). 9. H-Phe-Phe-⌿(CN4)-Ala-Trp-Gly-NH2 (9), C34H38N10O4, MW. 650.70 To the first portion of peptidyloresin H-Trp-Gly-MBHA compound 4 (102 mg, 0.2 mmol), BOP (89 mg, 0.2 mmol) and DIPEA (0.102 ml, 0.6 mmol) were added and the reactants were shaken together for 18 h. After that time the peptidyloresin was washed with DMF (3x) and the coupling reaction was repeated with the half amount of each of the reagents. After 18 h the peptidyloresin was washed with DMF (3⫻), CH2Cl2 (3⫻), MeOH (3⫻), and CH2Cl2 (3⫻) and deprotected with 50% TFA in CH2Cl2 (25⬘ and 5⬘). After being carefully washed and dried overnight in a dessicator over P2O5, the peptidyloresin was treated for 1 h with liquid HF at 0°C in the presence of anisole. The crude peptide was purified by preparative HPLC (Vydac C-18 column, gradient 30 – 60% B in 25 min; detection at 220 nm) to yield 15 mg of pure tetrazole analog. HPLC: tR ⫽ 11.4 min (30 – 60%B, 25 min); for DeltaPak C18 column: tR ⫽ 13 min; and Watpro I125 column: tR ⫽ 6.5 min. FAB-MS: 651.5 (M ⫹ H⫹), 15.3% 10. H-Phe-Phe-⌿(CN4)-D-Ala-Trp-Gly-NH2 (10), C34H38N10O4, MW. 650.70 Using the second portion of H-Trp-Gly-MBHA resin and compound 8, the 3⫹2 coupling was accomplished by means of BOP reagent as it was done in the case of L-Ala containing peptide. The remaining steps of deprotection, cleavage and purification of the peptide were identical to those performed during the synthesis of compound 9. Yield 8 mg. HPLC: for Vydac C-18 column: tR ⫽ 8.8 min (30 – 60%B, 25 min); for Delta-Pak C-18 column: tR ⫽ 12.5 min; and Watpro I125 column: tR ⫽ 10.5 min. FAB-MS: 651.5 (M ⫹ H⫹), 7.2%

2.2. Fluid secretion assay This assay has been described in detail elsewhere [2,3]. 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 dissected tubules and associated saline droplets are held under liquid paraffin. Urine escapes from a 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 (10 nM) of achetakinin-I assayed on Malpighian tubules taken from the same insect [13]. To investigate possible antagonist activity, tubules were challenged with 0.1 nM achetakinin-I together with different concentrations of the test compound. Diuretic activity is here expressed as a percentage of the response to 0.1 nM achetakinin-I alone. 2.3. NMR spectroscopy/molecular dynamics calculations NMR spectra were acquired on a Bruker ARX500 500 MHz instrument. Samples were ⬃1 mmol in 10% D2O, water suppression was by presaturation, and a 5 mm HCN probe was used. Coupling constants were measured from 1-D spectra. Most peak assignments were made using TOCSY spectra, but ambiguous peaks from amino acids containing aromatic moieties required intra-residue ROESY peaks for final determination. Molecular modeling was performed using Sybyl Tripos software with modifications developed in our laboratories, running on SGI Indigo and O2 computers. Distance constraints were determined from intensities of ROESY peaks measured by number of contours, with strong set to 1.8 –2.7 A, medium 1.8 –3.3A and weak 1.8 –5 A. Simulated annealing consisted of a suitable number of cycles of heating to 1000 deg. for 10.00 fs, then exponential cooling to 200 deg over 1000 fs. after energy minimization, 5 or 10 lowest energy forms were superimposed and rms differences were calculated. Distance geometry allowed production of greatly differing starting conformers for the above calculations, preventing detection of only local minima. Tripos or Wiener et al. force field was used for energy calculations.

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Fig. 3. Superposition of the four lowest energy conformations of the tetrazole-containing insect kinin analog Phe-Phe-␺[CN4]Ala-Trp-Gly-NH2 from extended molecular dynamics incorporating distance constraints obtained from two-dimensional NMR spectroscopy. Molecular modeling was performed using Sybyl Tripos softeware running on SGI Indigo and O2 computers. The figure shows the insect kinin analog incorporating a tetrazole group, a mimic of a cis peptide bond, in an open turn over the core residues 1– 4.

antagonist, reducing the diuretic response of a 0.1 nM solution of the natural cricket achetakinin-I by 50% at an IC50 of 4.4. ⫻ 10⫺7 M (Fig. 2b). 3.2. NMR spectroscopic/molecular dynamics conformational analysis Fig. 2 Dose-response curves for the insect kinin tetrazole-containing analogs Phe-Phe-␺[CN4]Ala-Trp-Gly-NH2 (A) and Phe-Phe-␺[CN4]D-AlaTrp-Gly-NH2 (B) in a cricket Malpighian tubule fluid secretion assay. Agonist activity is expressed as a percentage of the diuretic (⌬ nl/min) of 10 nM achetakinin, whereas antagonist activity is a percentage of the response obtained with 0;1 nM achetakinin I alone. Sigmoidal dose-response curves with variable slopes were fitted by non-linear regression using GraphPad Prism. Data points are the means of 6 – 8 determinations.

3. Results 3.1. Diuretic activity The tetrazole-containing insect kinin analogs, Phe-Phe␺[CN4]Ala-Trp-Gly-NH2 were synthesized in both L and D forms with respect to the ␣-carbon of the Ala residue. Evaluation of the diuretic activity of these analogs in a cricket Malpighian tubule fluid secretion assay demonstrated that the L version of the insect kinin tetrazole analog retains maximum activity with an EC50 of 3.4 ⫻ 10⫺7 M (Fig. 2a). In marked contrast, the D version was a partial agonist stimulating secretion by 50% of the maximum with an EC50 of 1.9 ⫻ 10⫺7 M. The D version was also a partial

Two dimensional ROESY and TOCSY NMR spectra were taken on the insect kinin tetrazole analog Phe-Phe␺[CN4]Ala-Trp-Gly-NH2, featuring L stereochemistry at the Ala. Unusual chemical shifts include Trp-4 N-H and Phe-2 N-H peaks which occur at 5.20 and 5.41 ␦ respectively, presumably due to orientation on the plane of the tetrazole moiety. With the exception of the Phe-1, all ␤ hydrogens are well separated, indicating diverse environments and a lack of free rotation. Notable nOe interactions were strong couplings between the Trp N-H and Ala H␣ and CH3 and the Gly N-H to Trp H␣. A temperature gradient study indicated that no permanent hydrogen bonds were present. Extended molecular dynamics on the L-Ala tetrazole analog indicated that the pseudopeptide demonstrated some flexibility in movement, but as expected could readily adopt an open 1– 4 turn (see Fig. 3). This alternates with a larger turn over the entire mainchain, not seen in the other biologically active, restricted-conformation analogs of the insect kinins. Of the two ␤-turns observed in the cyclic insect kinin analog [17,18,20], the tetrazole analog can easily form the 1– 4 turn (cisPro type VI ␤-turn in the cyclic analog), but not the 2–5 turn (transPro type I ␤-turn in the

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cyclic analog). The biologically active conformation must at least resemble the 1– 4 turn shown in Fig. 3, as great deviations from this form, particularly formation of a 2–5 ␤-turn, are not energetically favored.

4. Discussion Previous studies on the solution conformation of a conformationally rigid, cyclic insect kinin analog, active in myotropic and diuretic assays, demonstrated that two different turn conformations within the C-terminal pentapeptide core were present. These consisted of a cisPro type VI ␤-turn over core residues 1– 4 (Phe-Phe-Pro-Trp) and a transPro type I-like turn over core residues 2–5 (Phe-ProTrp-Gly) [11,17,18,20]. Although the predominant turn in aqueous solution was the cisPro type VI turn by a 60:40 ratio, and was most consistent with extensive structureactivity relationship studies, the transPro turn could not be excluded as a candidate for the active conformation adopted by the insect kinins during successful receptor interaction. To provide definitive evidence of the active conformation of the insect kinins, conformationally rigid analogs that could discriminate between the two candidate conformations had to be synthesized and evaluated in the diuretic bioassay. In a tetrazole moiety the peptide bond [-C(⫽O)N(H)-] is mimicked by a [-C(⫽N)N(N-)-] moiety and tied into a cis orientation by an additional nitrogen in the cyclic ring (Fig. 4). Furthermore, the tetrazole has been shown to mimic the type VI ␤-turn, which contains a cis oriented peptide bond preceding the Pro in the third position of the turn [10,24,25]. Formation of a 2–5 transPro type I turn would be strongly discouraged in a tetrazole analog of the insect kinin pentapeptide core. Insect kinin analogs incorporating the tetrazole moiety, Phe-Phe␺[CN4]Ala-Trp-Gly-NH2, were synthesized in two versions; with either L or D stereochemistry about the Ala ␣-carbon. These were then both evaluated for in vitro diuretic activity in a cricket Malpighian tubule fluid secretion bioassay. The agonist activity of the L isomer indicates that it interacted successfully with the Malpighian tubule receptor site despite the inherent rigidity of the analog. In addition, the D version appears to be able to bind to the receptor, although its ability to activate the receptor is considerably diminished. The aggregate results provide strong evidence that the active conformation adopted by the insect kinins at the cricket Malpighian tubule receptor site is the cisPro type VI ␤-turn over residues 1– 4, rather than the transPro turn over residues 2–5. This highly significant evidence for the active insect kinin turn allows for the further characterization of the interaction of the insect kinins with the Malpighian tubule receptor site which regulates fluid and ion transport in a number of insect species. The insect kinin C-terminal core in a type VI turn places the two sidechains of the Phe1 and Trp4 on the same side of the structure where they can interact as a continuous aromatic surface with the receptor site. Conversely, the sidechain of

Fig. 4. An illustration of the tetrazole group (bottom), which can serve as a mimic of a cis oriented peptide bond (top) [10,24,25].

residue 2 lies on the opposite face pointing away from the receptor surface, which explains why this position is tolerant of considerable change [17,18,20]. This insect kinin receptor interaction model is illustrated in Fig. 5. The study has already demonstrated that the incorporation of a tetrazole moiety with D stereochemistry on the Ala ␣-carbon into the active core can provide a lead candidate for an antagonist of the insect kinins at the cricket Malpighian tubule receptor site. The identification of the active core conformation opens further possibilities for the design of

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[4] [5]

[6]

[7]

Fig. 5. A receptor interaction model of an insect kinin C-terminal pentapeptide core region in a 1– 4 turn, the active conformation. It is shown binding to the cricket Malpighian tubule receptor. The aromatic sidechains of Phe1 and Trp4, both critical for biological activity, are adjacent to one another, oriented on the same side of the backbone, and can present a collective aromatic surface to the receptor. In contrast sidechain 2 (in this case a Tyr) lies on the opposite side of the mainchain backbone, and perhaps from the receptor surface as well. Position 2 shows natural variation in the native insect kinin isoforms, and structure-activity studies indicate that this position tolerates a wide range of unnatural substitutions, from basic to acidic, and from hydrophobic to hydrophilic, without complete loss of biological activity.

[8]

[9]

[10]

[11]

potent agonists and antagonists of the insect kinins, which could serve not only as important tools for neuroendocrinologists investigating the mechanisms by which these neuropeptides regulate critical physiological processes, but also provide the basis for selective, environmentally friendly pest arthropod control strategies based on insect kinin neuropeptides.

[12]

[13]

[14]

Acknowledgments We wish to thank Allison Strey (College Station), Nan Pryor (College Station) and 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, JZ, JO & GMC).

[15]

[16]

[17]

References [18] [1] Chung JS, Goldsworthy GJ, Coast GM. Haemolymph and tissue titres of achetakinins in the house cricket Acheta domesticus: effect of starvation and dehydration. J Exp Biol 1994;193:307–19. [2] Coast GM. Fluid secretion by single isolated Malpighian tubules of the house cricket, Acheta domesticus, and their response to diuretic hormone. Physiol Entomol 1988;13:381–91. [3] Coast GM. The regulation of primary urine production in insects. In: Coast GM, Webster SG, editors. Recent advances in arthropod en-

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