Janusz Zabrocki

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

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 beta-turn over C-terminal pentapeptide core residues 1-4 and a transPro type I-like beta-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.

Impact of the peptide sequence on the coordination abilities of albumin-like tripeptides towards Cu2+, Ni2+ and Zn2+ ions. Potential albumin-like peptide chelators

Thermodynamic and spectroscopic studies have shown that the insertion of α-hydroxylmethylserine (HmS) residues into the N-terminal peptide motif of human serum albumin results in a very powerful chelating agent for Cu2+ and Ni2+ ions. The insertion of two HmS residues results in the HmS–HmS–His–OH/NH2 sequence, which is the most effective chelating agent based on an albumin-like sequence for both studied metal ions, especially when the C-terminal carboxylate is protected by an amide function.

Comparison of active conformations of the insectatachykinin/tachykinin and insect kinin/Tyr-W-MIF-1 neuropeptide family pairs.

Abstract: A comparison of solution conformations of active, restricted‐conformation analogues of two sequence‐similar insect/vertebrate neuropeptide family pairs shed light on the potential existence of molecular evolutionary relationships. Analogues of the locustatachykinins and the mammalian tachykinin substance P, containing a sterically hindered Aib‐NMePhe/Tyr residue block, share similar low‐energy turn conformations incorporating a cis peptide bond. Conversely, restricted conformation analogues of the insect kinins and the mammalian opiate peptide Tyr‐W‐MIF‐1, with near identical C‐terminal tetrapeptide sequences, adopt different conformations. The insect kinins adopt a cisPro 1‐4 β‐turn, in which the Phe1 is critical for bioactivity. Tyr‐W‐MIF‐1 prefers a transPro 2‐5 turn, and an additional N‐terminal Phe severely inhibits μ‐opiate receptor binding. Comparisons of the chemical/conformational requirements for receptor interaction are consistent with a distant evolutionary relationship between the insectatachykinins and tachykinins, but not between the insect kinins and Tyr‐W‐MIF‐1. Therefore, analogues of the insect kinins with pest control potential can be readily designed to avoid mammalian interactions.

Disruption of insect diapause using agonists and an antagonist of diapause hormone

The dormant state known as diapause is widely exploited by insects to circumvent winter and other adverse seasons. For an insect to survive, feed, and reproduce at the appropriate time of year requires fine coordination of the timing of entry into and exit from diapause. One of the hormones that regulates diapause in moths is the 24-aa neuropeptide, diapause hormone (DH). Among members of the Helicoverpa/Heliothis complex of agricultural pests, DH prompts the termination of pupal diapause. Based on the structure of DH, we designed several agonists that are much more active than DH in breaking diapause. One such agonist that we describe also prevents the entry into pupal diapause when administered to larvae that are environmentally programmed for diapause. In addition, we used the unique antagonist development strategy of incorporating a dihydroimidazole (“Jones”) trans-Proline mimetic motif into one of our DH agonists, thereby converting the agonist into a DH antagonist that blocks the termination of diapause. These results suggest potential for using such agents or next-generation derivatives for derailing the success of overwintering in pest species.

A C-terminal aldehyde insect kinin analog enhances inhibition of weight gain and induces significant mortality in Helicoverpa zea larvae

The first reported examples of C-terminal aldehyde analogs of an insect neuropeptide are described. They are hexapeptide insect kinin analogs Boc-VFFPWG-H and Fmoc-RFFPWG-H. Activity observed for these modified analogs in an in vitro insect diuretic assay confirms that the C-terminal aldehyde group is tolerated by an insect kinin receptor. The two analogs demonstrate greatly enhanced activity over standard C-terminal amide insect kinins in a larval weight gain inhibition assay in the corn earworm Helicoverpa zea. Treatment with Boc-VFFPWG-H led to significant increases in larval mortality at doses of 500pm (45%) and 5nm (67%). Boc-VFFPWG-H represents a lead analog in the development of novel, environmentally friendly pest insect management agents based on the insect kinin class of neuropeptides.

Diuretic and myotropic activities of N-terminal truncated analogs of Musca domestica kinin neuropeptide.

Musca kinin (Musdo-K; NTVVLGKKQRFHSWG-NH(2)) and N-terminal truncated analogs of 4-14 residues in length were assayed for diuretic and myotropic activity on housefly Malpighian tubules and hindgut, respectively. The pentapeptide was the minimum sequence required for biological activity, but it was > 5 orders of magnitude less potent than the intact peptide. The pharmacological profiles of the different analogs in the two assays were very similar, suggesting the same receptor is present on both tissues. Potency was little affected by the deletion of Asn(1), but was reduced > 10-fold after the removal of Thr(2). Deletion of the next 5 residues had relatively little effect, but after the second lysyl residue (Lys(8)) was removed potency fell by one to two orders of magnitude. There was a similar drop in potency after the removal of Arg(10), and at 100 microM the pentapeptide had only 20% of the diuretic activity of the intact peptide. The importance of Arg(10) was confirmed by comparing dose-response curves for Musdo-K [6-15] and Acheta kinin-V (AFSHWG-NH(2)) in the diuretic assay; the substitution of arginine by alanine produced a significant reduction in potency and some loss of activity.

Functional characterization of five different PRXamide receptors of the red flour beetle Tribolium castaneum with peptidomimetics and identification of agonists and antagonists

The neuropeptidergic system in insects is an excellent target for pest control strategies. One promising biorational approach is the use of peptidomimetics modified from endogenous ligands to enhance biostability and bioavailability. In this study, we functionally characterized five different G protein-coupled receptors in a phylogenetic cluster, containing receptors for PRXamide in the red flour beetle Tribolium castaneum, by evaluating a series of 70 different peptides and peptidomimetics. Three pyrokinin receptors (TcPKr-A, -B, and -C), cardioacceleratory peptide receptor (TcCAPAr) and ecdysis triggering hormone receptor (TcETHr) were included in the study. Strong agonistic or antagonistic peptidomimetics were identified, and included beta-proline (β(3)P) modification of the core amino acid residue proline and also a cyclo-peptide. It is common for a ligand to act on multiple receptors. In a number of cases, a ligand acting as an agonist on one receptor was an efficient antagonist on another receptor, suggesting complex outcomes of a peptidomimetic in a biological system. Interestingly, TcPK-A was highly promiscuous with a high number of agonists, while TcPK-C and TcCAPAr had a lower number of agonists, but a higher number of compounds acting as an antagonist. This observation suggests that a target GPCR with more promiscuity will provide better success for peptidomimetic approaches. This study is the first description of peptidomimetics on a CAPA receptor and resulted in the identification of peptidomimetic analogs that demonstrate antagonism of CAPA ligands. The PRXamide receptor assays with peptidomimetics provide useful insights into the biochemical properties of receptors.

Biostable and PEG polymer-conjugated insect pyrokinin analogs demonstrate antifeedant activity and induce high mortality in the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae)

The pyrokinins (PK) are multifunctional neuropeptides found in a variety of arthropod species, including the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae). A series of biostable pyrokinin analogs based on the shared C-terminal pentapeptide core region were fed in solutions of artificial diet to the pea aphid over a period of three days and evaluated for antifeedant and aphicidal activity. The analogs contained either modified Pro residues Oic or Hyp and or a d-amino acid in key positions to enhance resistance to tissue-bound peptidases and retain activity in a number of PK bioassays. A series of PK analogs conjugated with two lengths of polyethyleneglycol (PEG) polymers were also evaluated in the aphid feeding assay. Three of the biostable PK analogs demonstrated potent antifeedant activity, with a marked reduction in honeydew formation and very high mortality after 1 day. In contrast, a number of unmodified, natural pyrokinins and several other analogs containing some of the same structural components that promote biostability were inactive. Two of the most active analogs, Oic analog PK-Oic-1 (FT[Oic]RL-NH(2)) and PEGylated analog PK-dF-PEG(8) [(P(8))-YF[dF]PRL-NH(2)], featured aphicidal activity calculated at LC(50)s of 0.042nmol/μl [0.029μg/μl] (LT(50) of 1.0 day) and 0.126nmol/μl (LT(50) of 1.3 days), respectively, matching the potency of some commercially available aphicides. Notably, a PEGylated analog of a PK antagonist can block over 55% of the aphicidal effects of the potent PK agonist PK-Oic-1, suggesting that the aphicidal effects are mediated by a PK receptor. The mechanism of this activity has yet to be established, though the aphicidal activity of the biostable analogs may result from disruption of digestive processes by interfering with gut motility patterns, a process shown to be regulated by the PKs in other insects. The active PK analogs represent potential leads in the development of selective, environmentally friendly aphid pest control agents.

Analogs of sulfakinin-related peptides demonstrate reduction in food intake in the red flour beetle, Tribolium castaneum, while putative antagonists increase consumption.

The insect sulfakinins (SKs) constitute a family of neuropeptides that display both structural and functional similarities to the mammalian hormones gastrin and cholecystokinin (CCK). As a multifunctional neuropeptide, SKs are involved in muscle contractions as well as food intake regulation in many insects. In the red flour beetle Tribolium castaneum, the action on food intake by a series of synthetic SK analogs and one putative antagonist was investigated by injection in beetle adults. The most remarkable result was that both sulfated and non-sulfated SKs [FDDY(SO3H)GHMRFamide] inhibited food intake by about 70%. Strong activity observed for SK analogs featuring a residue that mimics the acidic nature of Tyr(SO3H) but lack the phenyl ring of Tyr, indicate that aromaticity is not a critical characteristic for this position of the peptide. SK demonstrated considerable tolerance to Ser and Ala substitution in position 8 (basic Arg), as analogs featuring these uncharged substitutions retained almost all of the food intake inhibitory activity. Also, the Phe in position 1 could be replaced by Ser without complete loss of activity. Conversely, substitution of Met by Nle in position 7 led to inactive compounds. Finally, the Caenorhabditis elegans sulfated neuropeptide-like protein-12 (NLP-12), that shares some sequence similarities with the SKs but features a Gln-Phe-amide rather than an Arg-Phe-amide at the C-terminus, elicited increased food intake in T. castaneum, which may indicate an antagonist activity. Co-injection of NLP-12 with nsSK blocked the food intake inhibitory effects of nsSK.

Aliphatic amino diacid Asu functions as an effective mimic of Tyr(SO3H) in sulfakinins for myotropic and food intake-inhibition activity in insects

The aliphatic amino diacid alpha-aminosuberic acid can function as an effective, stable mimic of the hydrolysis-susceptible Tyr(SO3H) group in sulfakinin neuropeptide analogs for both hindgut contractile activity in cockroach and food intake-inhibition activity in the desert locust. In the analog, the acidic sulfate group is replaced with an acidic carboxyl group. The degree of activity of sulfakinin analogs is correlated with the carboxyl/alpha-carbon distance in the cockroach hindgut contractile assay. The results represent an important step in the design and synthesis of biostable, sulfakinin analogs that could potentially suppress the feeding behavior of destructive insect pests of agricultural importance.