Chapter 15
Pseudopeptide Mimetic Analogs of Insect Neuropeptides 1
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Downloaded by UCSF LIB CKM RSCS MGMT on November 24, 2014 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0551.ch015
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Ronald J. Nachman , Jefferson W. Tilley , Timothy K. Hayes , G. Mark Holman , and Ross C. Beier 1
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Food Animal Protection Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, College Station, TX 77845 Roche Research Center, Hoffmann-LaRoche, Inc., Nutley, NJ 07110 Department of Entomology, Texas A&M University, College Station, TX 77843
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Pseudopeptide analogs that feature acyl group, reduced-bond, and/or stable chemical-construct replacements for amino acids or peptide linkages mimic the biological activity of the insect kinin, sulfakinin, and myosuppressin peptide families. The non-peptide mimetic CP96,345-1 demonstrated both agonism and antagonism of the cockroach hindgut myostimulatory activity of substance P, which shows sequence homology with the insectlocustatachykinins.Biological evaluation of a rigid cyclic analog reveals the conformational preference of the pyrokinin/PBAN active-core during interaction with hindgut/oviduct contractile and pheromone production receptors. The importance of information on the conformational requirements for neuropeptide activity for the development of peptidomimetics is also discussed. Strategies for the development of future pest insect management agents from insect neuropeptides have been previously advocated and outlined because these chemical messengers regulate critical processes in insects (1-3). However, insect neuropeptides in and of themselves hold little promise as traditional insect control agents because of their susceptibility to enzymatic degradation in the target insect, lability under environmental conditions, and inability to pass through the hydrophobic insect cuticle. The removal of the peptide nature (i.e., the constituent peptide bonds) of insect neuropeptides leading to pseudopeptide and nonpeptide analogs represents a strategy that could overcome these limitations. In this paper, we discuss pseudopeptide mimetic analogs that have been developed for selected insect neuropeptide families, including the insect kinins, sulfakinins, myosuppressins, and insect tachykinins. First members of these families were isolated from the cockroach or locust on the basis of their ability to either stimulate or inhibit contractions of the isolated cockroach/hindgut. Subsequently, these peptide families have been found in a range of insect species and associated with a variety of different physiological responses. The paper closes 0097-6156/94/0551-0210$06.00/0 © 1994 American Chemical Society In Natural and Engineered Pest Management Agents; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
15. NACHMAN ET AL. Pseudopeptide Mimetic Analogs of Insect Neuropeptides with a discussion on the conformational preference of the pyrokinin active-core for interaction with hindgut/oviduct myostimulatory and pheromonotropic receptor sites, and on the utility of this information for the development of potent pseudopeptide and nonpeptide mimetic agonists and antagonists.
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Insect Kinins Members of the insect kinin peptide family have been isolated from such diverse sources as the cockroach (Leucophaea maderae). locust (Locusta migratoria). cricket (Acheta domesticus). and mosquito (Culex salinarius) (1,4) and share the conserved C-terminal pentapeptide sequence Phe-X^X^Trp-Gly-NHj [X = Asn, His, Ser, or Tyr, X = Pro or Ser]. All of the insect kinins demonstrate myostimulatory properties on the isolated cockroach hindgut preparation (5) at potent-thresholds of between 10 and 10~ M. Achetakinins, leucokinins, and culekinin depolarizing peptide stimulate fluid secretion in Malpighian tubules of the cricket and mosquito (5-7). Therefore, the kinins may regulate water and ion balance in addition to hindgut motility in a variety of insects. The C-terminal pentapeptide sequence common to the insect kinins is all that is required to elicit a physiological response in myotropic and diuretic assays. In particular, the active core sequence Phe-Tyr-Pro-Trp-Gly-NH is equipotent with the parent nonapeptide in hindgut myotropic (8) and cricket Malpighian tubule secretion (6) assays. Within the active core pentapeptide, the aromatic residues Phe and Tip are of paramount importance for myotropic and diuretic activity (68), whereas position 2 tolerates wide variations in side chain character ranging from acidic to basic or hydrophobic to hydrophilic. Aromatic residues, such as Tyr or Phe, in the variable position 2 promote the highest potencies in myotropic and Malpighian tubulefluidsecretion assays (6-8). Superagonist activity has been observed for the unnatural hexapeptides Ala-PhePhe-Pro-Trp-Gly-NH (1) and Ar^-Phe-Phe-Pro-Trp-Gly-NH (Nachman et al., unpublished data) in cockroach hindgut myotropic and cricket Malpighian tubule fluid secretion assays, with the latter analog demonstrating at least 550-fold greater myotropic potency than the pentapeptide Phe-Phe-Pro-Trp-Gly-NH and/or natural leucokinins of longer chainlength. The leucokinin analog Ala-Tyr-Ser-Trp-GlyNH , which replaces one of the two critical aromatic residues with an Ala, appears to antagonize the activity of the leucokinins/achetakinins in the cricket Malpighian fluid secretion assay at concentrations of between 0.1 and 1 μΜ or higher (Coast and Nachman, unpublished data). This result underscores the importance of the Trp residue to the receptor-binding affinity and biological activity of the insect kinins. 1
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Pseudopeptide Analogs. Nonpeptide modification of the insect kinin pentapeptide core, leading to analogs with retention of biological activity, was first achieved by replacing the amide bond (-C(O)NH-) between Phe and Phe of the sequence PhePhe-Ser-Trp-Gly-NH with a reduced-bond linkage (-CH NH-). The resulting pseudopeptides Phe ^[CH -NH] Phe-Ser-Trp-Gly-NH (Figure 1) (9) and Phe i^[CH -NH] Phe-Pro-Trp-Gly-NH demonstrated threshold values of about 3 nM, which represents a significant 1 % of the activity of the parent pentapeptides. These pseudotripeptides were followed by the synthesis of a series of simple aryl, acyl 1
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In Natural and Engineered Pest Management Agents; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
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NATURAL AND ENGINEERED PEST M A N A G E M E N T AGENTS
pseudopeptides of the core pentapeptide Phe-Tyr-Pro-Trp-Gly-NH (Figure 2). The amino acid or amino acid blocks Phe, Phe-Tyr, and Phe-Tyr-Pro were replaced with the aryl, acyl groups hydrocinnamic acid (Hca), 6-phenylhexanoic acid (6Pha), and 9-phenylnonanoic acid (9Pna), respectively. In each of the resulting pseudopeptide analogs Hça-Tyr-Pro-Trp-Gly-NH , 6Pha-Pro-Trp-Gly-NH , and 9Pna-Trp-GlvNH , the distance between the critical N-terminal phenyl ring and the Trp residue was kept approximately constant via the introduction of methylene groups. An evaluation of the myotropic potency of the pseudopeptide 6Pha-Trp-Gly-NH was also undertaken. Remarkably, the pseudotetrapeptide Hca-Tyr-Pro-Trp-Gly-NH demonstrated a threshold concentration of 58 pM and an EDso of 10 nM on the isolated cockroach hindgut preparation, retaining 70% of the potency of the parent pentapeptide. In addition, this pseudotetrapeptide analog retained 86% of the maximal contractile response of the core pentapeptide. This result demonstrated that the N-terminal amino group is of little consequence to the interaction between the insect kinin core peptide and the receptor site. With a threshold concentration of 8 nM and an ED of 2 μΜ, the pseudotripeptide proved to be about 2 orders of magnitude less potent than the pseudotetrapeptide, but retained 98% of the maximal myostimulatory activity of the parent pentapeptide. The two pseudodipeptide analogs 9Pna-Trp-Glv-NH and 6Pha-Trp-Gly-NH demonstrated a further drop in potency of about 2 orders of magnitude when compared with the pseudotripeptide (10). Nevertheless, despite major modifications, i.e. the replacement of three Nterminal core-amino acids with a simple aryl acyl group, these pseudodipeptides did not completely lose myostimulatory activity. As 9Pna-Trp-Gly-NH contains 3 more methylene groups than does 6Pha-Trp-Gly-NH , the similar potencies of the two analogs suggests that the receptor exhibits tolerance towards modification of the distance between the phenyl ring of the acyl group and the Trp residue. Taking the modification process one step further, the remaining peptide bond between Trp and Gly of 6Pha-Trp-Glv-NH was replaced with a reduced-amide linkage (-CH NH-). The resulting non-peptide mimic 6Pha-Trp ^[CH -NH]GlyNH (Figure 3) was observed to elicit low but discernable myostimulatory activity on the isolated cockroach hindgut assay and is expected to be resistant to exopeptidase degradation (Nachman, unpublished data). Using a similar strategy, a methyl group was placed on the amide bond between Trp and Gly of 6Pha-TrpGly-NH to increase steric hindrance at this linkage. The resulting non-peptide mimic 6Pha-Trp-Sar-NH (Figure 3) (Sar = sarcosine or N-methyl glycine) retained myostimulatory activity at a threshold concentration of 10 μΜ. The additional steric hindrance at the amide linkage of this pseudopeptide analog will increase resistance to peptidase degradation (Nachman, unpublished data). Pseudopeptide analogs of the insect kinin family involving modifications of the C-terminus have also been reported. The C-terminal acid forms of the insect kinin sequences are inactive, perhaps as a result of unfavorable interaction between the negatively-charged carboxyl moiety and the myotropic receptor. However, the methyl ester and N-methyl amide analogs of the Ala-Phe-Phe-Pro-Trp-Gly kinin sequence proved active, although at a reduced level. These two analogs demonstrated reductions in myostimulatory potency over the parent peptide of about 2 orders of magnitude, with threshold concentrations in the 0.75 - 1.0 nM range. The Ν,Ν-dimethylated amide analog proved completely inactive in the cockroach 2
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Downloaded by UCSF LIB CKM RSCS MGMT on November 24, 2014 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0551.ch015
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In Natural and Engineered Pest Management Agents; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
15.
Pseudopeptide Mimetic Analogs of Insect Neuropeptides
NACHMAN ET AL.
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Downloaded by UCSF LIB CKM RSCS MGMT on November 24, 2014 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0551.ch015
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