Analgesic Peptides: From Natural Diversity to Rational Design.

Pain affects one-third of the global population and is a significant public health issue. The use of opioid drugs, which are the strongest painkillers, is associated with several side effects, such as tolerance, addiction, overdose, and even death. An increasing demand for novel, safer analgesic agents is a driving force for exploring natural sources of bioactive peptides with antinociceptive activity. Since the G protein-coupled receptors (GPCRs) play a crucial role in pain modulation, the discovery of new peptide ligands for GPCRs is a significant challenge for novel drug development. The aim of this review is to present peptides of human and animal origin with antinociceptive potential and to show the possibilities of their modification, as well as the design of novel structures. The study presents the current knowledge on structure-activity relationship in the design of peptide-based biomimetic compounds, the modification strategies directed at increasing the antinociceptive activity, and improvement of metabolic stability and pharmacodynamic profile. The procedures employed in prolonged drug delivery of emerging compounds are also discussed. The work summarizes the conditions leading to the development of potential morphine replacements.

Antheraea peptide and its analog: Their influence on the maturation of the reproductive system, embryogenesis, and early larval development in Tenebrio molitor L. beetle.

In recent years, many new immunologically active peptides from insects have been identified. Unfortunately, in most cases, their physiological functions are not fully known. One example is yamamarin, a pentapeptide isolated from the caterpillars of the Antheraea yamamai moth. This peptide has strong antiproliferative properties and is probably involved in the regulation of diapause. Additionally, antiviral activity was discovered. The results of the research presented in this paper are, to our knowledge, the first attempt to characterize the biological effects of yamamarin on the functioning of the reproductive processes and embryonic development of insects using a model species, the beetle Tenebrio molitor, a commonly known pest of grain storage. Simultaneously, we tested the possible activity of the molecule in an in vivo system. In this research, we present the multifaceted effects of yamamarin in this beetle. We show that yamamarin influences ovarian growth and development, maturation of terminal oocytes, level of vitellogenin gene transcript, the number of laid eggs, duration of embryonic development, and larval hatching. In experiments with palmitic acid-conjugated yamamarin (C16-yamamarin), we also showed that this peptide is a useful starting molecule for the synthesis of biopharmaceuticals or new peptidomimetics with gonadotropic activity and effects on embryonic development. The data obtained additionally provide new knowledge about the possible function of yamamarin in insect physiology, pointing to the important role of this pentapeptide as a regulator of reproductive processes and embryonic development in a heterologous bioassay with T. molitor.

Disruption of insect immunity using analogs of the pleiotropic insect peptide hormone Neb-colloostatin: a nanotech approach for pest control II.

This work continues our studies on the pleiotropic activity of the insect peptide Neb-colloostatin in insects. In vivo immunological bioassays demonstrated that hemocytotoxic analogs of Neb-colloostatin injected into Tenebrio molitor significantly reduced the number of hemocytes in the hemolymph and impaired phagocytosis, nodulation and phenoloxidase activities in the insects. Among the analogs tested, [Ala1]-,[Val1]-, [Hyp4]- and [Ach4]-colloostatin were particularly potent in disrupting cellular immunity in larvae, pupae and adult insects. This result suggests that the most effective analogs showed increases in the bioactivity period in the hemolymph of insects when compared to Neb-colloostatin. Recently, we demonstrated that it is possible to introduce Neb-colloostatin through the cuticle of an insect into the hemolymph when the peptide is coupled with nanodiamonds. In this study, we showed that [Ala1]-, [Val1]-, [Hyp4]- and [Ach4]-colloostatin, when complexed with nanodiamonds, may also pass through the cuticle into the hemolymph and induce long-term impairments of immunity in T. molitor at all developmental stages. Studies on the tissue selectivity and effectiveness of Neb-colloostatin analogs and efficient methods for their introduction into insects may contribute to the development of eco-friendly pest control methods based on bioactive peptidomimetics.

Argireline: Needle-Free Botox as Analytical Challenge.

Argireline-containing cosmetics attract public interest due to their confirmed reduction of facial wrinkles. Argireline is a peptide that works by inhibiting the release of neurotransmitters in the neuromuscular junction, producing a botox-like effect. Therefore, it is used as a safe needle-free alternative to botox treatment. In this work we investigated the presence of Argireline in cosmetic creams and sera by application of reversed phase liquid chromatography and tandem mass spectrometry (RP-HPLC/MS and MS/MS). The analysis revealed the presence of argireline and its oxidized form in several different cosmetics. The methionine residue in Argireline sequence was indicated as oxidation point according to neutral loss MS studies. The developed sample preparation strategy minimizes and monitors methionine oxidation, bringing to our attention the question of impact of ingredients on the stability of cosmetic product.

Short neuropeptide F signaling regulates functioning of male reproductive system in Tenebrio molitor beetle.

Neuropeptides of short neuropeptides F family (sNPF) have been identified in various arthropods. They are pleiotropic neuromolecules which so far have been mainly associated with regulation of feeding and metabolism, as well as growth and development, locomotion, circadian rhythm or learning and memory. Here, we describe the effects of Tenebrionid sNPF peptide (SGRSPSLRLRFa) on various aspects of the male reproductive physiology in the Tenebrio molitor beetle. We identified in silico the putative sNPF receptor Tenmo-sNPFR. Based on RT-PCR technique, it was shown that the receptor might be present in the male reproductive tissues of this beetle. The analysis of receptor amino acid sequence showed that it is similar to other beetle sNPFRs, as well as other insect species, and belongs rhodopsin-like G-protein-coupled receptors (GPCRs). Injections of Trica-sNPF and its shorter form Trica-sNPF(4-11) caused differentiated effects in T. molitor male reproductive tissues. After 24 h post injections, the peptides decreased the concentration of the soluble protein fraction in testes of 4- and 8-day-old beetles as well as the dry mass of these organs but only in 8-day-old individuals. The same effects were shown with regard to accessory glands. Both peptides decrease the concentration of the soluble protein fraction but do not affect the dry mass of this organ. Furthermore, injections of Trica-sNPF at the 10-7 M concentration decrease the total sperm number in the reproductive system. Surprisingly, the same concentration of the shorter form, Trica-sNPF(4-11) increased the sperm number. It was also shown that both peptides in different manner influence contractions of ejaculatory duct. The data presented in this article give new evidence that sNPFs are involved in the regulation of reproductive events in beetles, which might be the part of a larger neuropeptide network combining feeding, growth and development with the physiology of reproduction.

FMRFamide-Related Peptides Signaling Is Involved in the Regulation of Muscle Contractions in Two Tenebrionid Beetles.

Peptidergic signaling regulates various physiological processes in insects. Neuropeptides are important messenger molecules that act as neurotransmitters, neuromodulators or hormones. Neuropeptides with myotropic properties in insects are known as FMRFamide-like peptides (FaLPs). Here, we describe the myotropic effects of the endogenous FaLPs in the regulation of contractile activity of the heart, ejaculatory duct, oviduct and the hindgut in two beetle species, Tenebrio molitor and Zophobas atratus. A putative receptor was identified in silico in both species. Using RT-PCR these putative FaLPs receptors were found in the various tissues of both beetles, including visceral organs. Analysis of the amino acid sequence of the receptor indicated that it is similar to other insect FaLPs receptors and belongs to G-protein coupled receptors. A synthetic FaLP (NSNFLRFa) found as the bioanalogue of both species demonstrated concentration-dependent and organ-specific myoactive properties. The peptide had species-specific cardioactivity, in that it stimulated Z. atratus heart contractions, while slightly inhibiting that of T. molitor and had mainly myostimulatory effect on the examined visceral organs of both beetle species, with the lowest activity in the ejaculatory duct of these beetles. The peptide was the most active in the hindgut of both species, but only at high concentration of 10-5 M. The results suggest that FaLPs are potent modulators of endogenous contractile activity of the visceral muscles in beetles and may indirectly affect various physiological processes.

Copper(ii) complexes with alloferon analogues containing phenylalanine H6F and H12F stability and biological activity lower stabilization of complexes compared to analogues containing tryptophan.

Copper(ii) complex formation processes between alloferon 1 (Allo1) (H1 GVSGH6 GQH9 GVH12G) analogues where the phenylalanine residue is introduced in the place of His residue H6F and H12F have been studied by potentiometric, UV-visible, CD and EPR spectroscopic, and MS methods. For the phenylalanine analogues of alloferon 1, complex speciation has been obtained for a 1 : 1, 2 : 1 and 3 : 1 metal-to-ligand molar ratio. At physiological pH and in 1 : 1 metal-to-ligand molar ratio the phenylalanine analogues of alloferon 1 form a CuL complex similar to that of alanine analogues with the 4N{NH2,N1Im,2NIm} coordination mode. The stability of the complexes of the phenylalanine analogues is higher in comparison to those of alanine analogues, but lower in comparison to those containing tryptophan. Injection of Allo12F into insects induced prominent apoptotic changes in all hemocytes. The presence of apoptotic bodies only in the insect hemolymph testifies to the fact that Allo12F is an extremely pro-apoptotic peptide.

Sulfakinin Signalling Influences Fatty Acid Levels and Composition in Tenebrio Molitor Beetle.

BACKGROUND: Sulfakinins are arthropod neuropeptides that are structurally and functionally similar to vertebrate gastrin-cholecystokinin. Sulfakinins with sulfated tyrosine (sSK) or nonsulfated tyrosine (nSK) in the C-terminated heptapeptide XY(SO3H)GHMRFamide display different biological functions, including myotropic activity, inhibition of food intake, stimulation of digestive enzymes and regulation of carbohydrate and lipid content. OBJECTIVE: To reveal the mechanisms by which sulfakinin signalling modulates lipid homeostasis, we analysed the changes in the level and composition of fatty acids and organic compounds in the fat body and haemolymph of Tenebrio molitor larvae after nSK and sSK treatment. METHODS: Fatty acids in fat body and haemolymph of insects were analysed using Gas Chromatography - Mass Spectrometry (GC-MS). RESULTS: The direction of the changes observed for major fatty acids, 18:1 and 18:2, and the less abundant fatty acids, 16:0, 18:0, 16:1 and 14:0, was the same for unsaturated (UFAs) and saturated (SFAs) fatty acids, and elevated after nSK application in both analysed tissues. However, the action of sSK in fat body tissue evoked distinct effects and induced either significant decreases in individual fatty acids or UFAs and SFAs. Administration of nSK and sSK significantly increased the level of total organic compounds in the haemolymph, contrary to the effect of sSK in fat body, where the level of total organic compounds decreased, although changes differ between individual chemicals. CONCLUSION: Sulfakinins are engaged in the precise modulation of fatty acid levels and composition, but their action depends on the presence of sulfate group on the tyrosyl residue of the peptide what determines the different roles of these peptides in insect physiology.

Impairment of the immune response after transcuticular introduction of the insect gonadoinhibitory and hemocytotoxic peptide Neb-colloostatin: A nanotech approach for pest control.

This article shows that nanodiamonds can transmigrate through the insect cuticle easily, and the doses used were not hemocytotoxic and did not cause inhibition of cellular and humoral immune responses in larvae, pupae and adults of Tenebrio molitor. The examination of the nanodiamond biodistribution in insect cells demonstrated the presence of nanodiamond aggregates mainly in hemocytes, where nanoparticles were efficiently collected as a result of phagocytosis. To a lesser extent, nanodiamond aggregates were also detected in fat body cells, while they were not observed in Malpighian tubule cells. We functionalized nanodiamonds with Neb-colloostatin, an insect hemocytotoxic and gonadoinhibitory peptide, and we showed that this conjugate passed through the insect cuticle into the hemolymph, where the peptide complexed with the nanodiamonds induced apoptosis of hemocytes, significantly decreased the number of hemocytes circulating in the hemolymph and inhibited cellular and humoral immune responses in all developmental stages of insects. The results indicate that it is possible to introduce a peptide that interferes with the immunity and reproduction of insects to the interior of the insect body by means of a nanocarrier. In the future, the results of these studies may contribute to the development of new pest control agents.

Myotropic activity of allatostatins in tenebrionid beetles.

Neuropeptides control the functioning of the nervous system of insects, and they are the most diverse signalling molecules in terms of structure and function. Allatostatins are pleiotropic neuropeptides that are considered potent myoinhibitors of muscle contractions in insects. We investigated the effects caused by three distinct allatostatins, Dippu-AST1 (LYDFGL-NH2 from Diploptera punctata), Grybi-MIP1 (GWQDLNGGW-NH2 from Gryllus bimaculatus) and Trica-ASTC (pESRYRQCYFNPISCF-OH from Tribolium castaneum) on contractile activity of the myocardium, oviduct and hindgut of two tenebrionid beetles, Tenebrio molitor and Zophobas atratus. Studies showed that all three peptides exerted myostimulatory effects on the oviduct and hindgut of the beetles, however they did not cause any effect on myocardium. The effects of Dippu-AST1, Grybi-MIP1 and Trica-ASTC were dose-dependent and tissue and species specific. The highest stimulatory effect was caused by Trica-ASTC, showing stimulation of approximately 82% at a 10-12 M concentration and 76% at a 10-11 M concentration for T. molitor and Z. atratus, respectively. The oviduct of T. molitor was more susceptible to allatostatins than that of Z. atratus. Dippu-AST1 showed the maximum stimulating effect at 10-11 M (57%), whereas Grybi-MIP 1 at 10-10 M caused a 41% stimulation. Trica-ASTC, in both species, showed a myostimulatory effect over the whole range of tested concentrations but was most potent at a 10-12 M concentration and caused a 54% and 31.9% increase in the frequency of contractions in the oviduct of T. molitor and Z. atratus, respectively. The results suggest that allatostatins may affect the regulation of egg movement within the oviducts and movement of food in the digestive tract of beetles and do not regulate directly the activity of heart, thus being good candidate compounds in neuropeptides based pest control agents in future research.

The long-term immunological effects of alloferon and its analogues in the mealworm Tenebrio molitor.

The subject of this article is a search for the long-term immunological effects of alloferon and 3 structural analogues of alloferon, which were earlier characterized by the highest pro-apoptotic activity in Tenebrio molitor. The differences in the actions of these peptides on immune response were observed. Alloferon increased nodulation and significantly phenoloxidase activity in the hemolymph of experimentally infected T. molitor. However, [Phe(p-NH2 )1 ]- and [Phe(p-OMe)1 ]-alloferon strongly inhibited cellular and humoral defense of the mealworm against Staphylococcus aureus infection. One day after injection of these peptides, the specific biochemical and morphological hallmarks of apoptosis in bacteria-challenged hemocytes were visible; in contrast, 3 days after peptides injection in all hemocytes, caspase activation was not observed. However, these new, circulating hemocytes differed from the control and the peptide-untreated bacteria-challenged hemocytes. They had an increased adhesion that led to a separation of viable, anucleated fragments of hemocytes that retain the ability to adhere and to form long filopodia. The peptide-induced separation of hemocyte fragments may resemble the formation of platelets in mammals and perhaps play a role in sealing wounds in insects. The results of in vivo studies may suggest a long half-life of studied peptides in the hemolymph of mealworm. Moreover, we showed the importance of the N-terminal histidine residues at position one of the alloferon molecule for its immunological properties in insects. The results obtained here show that alloferon plays pleiotropic functions in insects.

High stability and biological activity of the copper(II) complexes of alloferon 1 analogues containing tryptophan.

Copper(II) complex formation processes between the alloferon 1 (Allo1) (HGVSGHGQHGVHG) analogues where the tryptophan residue is introducing in the place His residue H1W, H6W, H9W and H12W have been studied by potentiometric, UV-visible, CD and EPR spectroscopic, and MS methods. For all analogues of alloferon 1 complex speciation have been obtained for a 1:1 metal-to-ligand molar ratio and 2:1 of H1W because of precipitation at higher (2:1, 3:1 and 4:1) ratios. At physiological pH7.4 and a 1:1 metal-to-ligand molar ratio the tryptophan analogues of alloferon 1 form the CuH-1L and/or CuH-2L complexes with the 4N binding mode. The introduction of tryptophan in place of histidine residues changes the distribution diagram of the complexes formed with the change of pH and their stability constants compared to the respective substituted alanine analogues of alloferon 1. The CuH-1L, CuH-2L and CuH-3L complexes of the tryptophan analogues are more stable from 1 to 5 log units in comparison to those of the alanine analogues. This stabilization of the complexes may result from cation(Cu(II))-π and indole/imidazole ring interactions. The induction of apoptosis in vivo, in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 was studied. The biological results show that copper(II) ions in vivo did not cause any apparent apoptotic features. The most active were the H12W peptide and Cu(II)-H12W complex formed at pH7.4.

Novel analogs of alloferon: Synthesis, conformational studies, pro-apoptotic and antiviral activity.

In this study, we report the structure-activity relationships of novel derivatives of the insect peptide alloferon (H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH). The peptide structure was modified by exchanging His at position 9 or 12 for natural or non-natural amino acids. Biological properties of these peptides were determined in antiviral in vitro test against Human Herpes Virus 1 McIntrie strain (HHV-1MC) using a Vero cell line. The peptides were also evaluated for the pro-apoptotic action in vivo on hemocytes of the Tenebrio molitor beetle. Additionally, the structural properties of alloferon analogs were examined by the circular dichroism in water and methanol. It was found that most of the evaluated peptides can reduce the HHV-1 titer in Vero cells. [Ala(9)]-alloferon exhibits the strongest antiviral activity among the analyzed compounds. However, no cytotoxic activity against Vero cell line was observed for all the studied peptides. In vivo assays with hemocytes of T. molitor showed that [Lys(9)]-, [Phg(9)]-, [Lys(12)]-, and [Phe(12)]-alloferon exhibit a twofold increase in caspases activity in comparison with the native peptide. The CD conformational studies indicate that the investigated peptides seem to prefer the unordered conformation.

Copper(II) complexes of terminally free alloferon mutants containing two histidyl binding sites inside peptide chain structure and stability.

Mononuclear and polynuclear copper(II) complexes of alloferon 1 with point mutations, H1A/H12A H2N-A(1)GVSGH(6)GQH(9)GVA(12)G-COOH, H1A/H9A H2N-A(1)GVSGH(6)GQA(9)GVH(12)G-COOH, and H1A/H6A H2N-A(1)GVSGA(6)GQH(9)GVH(12)G-COOH, have been studied by potentiometric, UV-visible, CD, and EPR spectroscopy, and mass spectrometry (MS) methods. Complete complex speciation at different metal-to-ligand molar ratios ranging from 1 : 1 to 3 : 1 was obtained. Over a wide 6-8 pH range, including physiological pH 7.4, and a 1 : 1 metal-to-ligand molar ratio, the peptides studied formed a CuH-1L complex with the 4N{NH2,N(-),2NIm} coordination mode. The presence of the 4N binding site for the CuH-1L complexes prevented the deprotonation and coordination of the second amide nitrogen atom to copper(II) ions (pK-1/-2 7.83-8.07) compared to that of pentaGly (6.81). The amine nitrogen donor and two imidazole nitrogen atoms (H(6)H(9), H(6)H(12) and H(9)H(12)) can be considered to be independent metal-binding sites in the species formed. As a consequence, di- and trinuclear complexes for the metal-to-ligand 2 : 1 and 3 : 1 molar ratios dominate in the solution, respectively. For the Cu(II)-H1A/H9A and Cu(II)-H1A/H12A systems, the Cu3H-9L complexes are likely formed by the coordination of amide nitrogen atoms towards C-termini with ring sizes (7,5,5).

Distribution in Rats Internal Organs of Intraperitoneally Given 125I-Labeled Heptapeptide [2-8]-Leucopyrokinin ([2-8]-LPK), a Truncated Analog of Insect Neuropeptide Leucopyrokinin.

BACKGROUND: It was previously found that synthetic, insect-derived octapeptide leucopyrokinin (LPK) applied directly into the lateral brain ventricle induced a significant antinociceptive effect in rats. Its synthetic truncated analog heptapeptide [2-8]-leucopyrokinin displayed a stronger antinociceptive effect in comparison to native LPK. Moreover it was previously found a high accumulation of these both 125I-labeled peptides in adrenals, as well as in hypothalamus and in hippocampus of rats brain. OBJECTIVES: The aim of the present study was to assess the distribution of 125I-labeled [2-8]-leucopyrokinin in rats' internal organs an in several parts of the brain after peripheral - intraperitoneal (i.p.) administration. MATERIAL AND METHODS: The study was performed on male Wistar rats. A synthetic [2-8]-leucopyrokinin ([2-8]-LPK) was iodinated with Na125I. On the day of experiment a solution of 125I-[2-8]-LPK was i.p. injected and the next after 1 and 24 h animals were sacrificed by decapitation. Radioactivity levels in samples of parts of the brain and of internal organs were determined by counter Gamma Auto Count. RESULTS: A uniform, low accumulation 125I-[2-8]-LPK was found in evaluated samples of the brain and in internal organs. CONCLUSIONS: The results of the present study indicate a weak penetration into the brain and internal organs of intraperitoneally applied 125I-[2-8]-LPK in rats and correspond with previously determined weak biological effects of i.p. injected LPK and [2-8]-LPK.

Copper(II) complexes of terminally free alloferon peptide mutants containing two different histidyl (H(1) and H(6) or H(9) or H(12)) binding sites Structure Stability and Biological Activity.

Mono- and dinuclear copper(II) complexes of the alloferon 1 with point mutations H9A/H12A H(1)GVSGH(6)GQA(9)GVA(12)G, H6A/H12A H(1)GVSGA(6)GQH(9)GVA(12)G and H6A/H9A H(1)GVSGA(6)GQA(9)GVH(12)G have been studied by potentiometric, UV-visible, CD, EPR spectroscopic, and mass spectrometry (MS) methods. Complete complex speciation at metal-to-ligand molar ratios 1:1 and 2:1 was obtained. For all systems studied in the 5 - 6.5 pH range, the CuL complex dominates with 3N{NH2,NIm-H(1),NIm-H(6 or 9 or 12)} binding site. The stability of the CuL complexes for the ligands studied varies according to the H9A/H12A>H6A/H12A>H6A/H9A series. For the dinuclear systems the amine/imidazole nitrogen donor atoms of the histidine residue H(1) and the imidazole nitrogen atoms of H(6) or H(9) or H(12) can be considered as independent metal-binding sites in the species formed. The stability of the dinuclear complexes is higher when two coordinated copper(II) ions are closer to each other. The inductions of phenoloxidase activity and apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 have been studied. The H6A/H9A, H6A/H12A peptides displayed lower hemocytotoxic activity compared to that of alloferon 1, while the H9A/H12A analogue was not active. Among the copper(II) complexes, the most active was the Cu(II)-H9A/H12A complex formed at pH7.4 with 3N{NH2,NIm-H(1),NIm-H(6)} (CuL) and 3N{NH2,N(-),NIm-H(6)} and/or 4N{NH2,NIm-H(1),N(-),NIm-H(6)} (CuH-1L) binding sites. The Cu(II)-H6A/H9A and Cu(II)-H6A/H12A complexes were not active.

Effect of alloferon 1 on central nervous system in rats.

Alloferon 1 is an insect-derived peptide with potent antimicrobial and antitumor activity. It was isolated from blood of an experimentally infected insect, the blow fly Callifora vicina. Synthetic alloferon 1 reveals a capacity to stimulate activity of NK cells and synthesis IFN in animal and human models. Moreover, it was demonstrated antiviral and antitumor activity of alloferon 1 in mice. There are no data on influence of alloferon 1 on central nervous system. The aim of present study was to determine an effect of alloferon 1 on rats' central nervous system by some behavioral tests: open field test, hole test, score of rats irritability, and determination of memory consolidation in the water maze test. Moreover, a probable antinociceptive effect of alloferon 1 in rats was determined by a tail immersion test and hot plate test. Experiments were performed on female Wistar rats. Seven days before experiments, rats were anesthetized with ketamine and xylazine and polyethylene cannulas were implanted into the right lateral brain ventricle (i.c.v.). On the day of experiment, alloferon 1 dissolved in a volume of 5 µL of saline was injected directly i.c.v. through implanted cannulas at doses of 5-100 nmol. It was found that alloferon 1 had slight effect on locomotor and exploratory activity, induced some decrease of rat irritability and a weak impairment of rats memory (only at the low dose of 5 nmol). On the other hand, the higher dose of this peptide exerts significant antinociceptive effect. Obtained results indicate that alloferon 1 do not exert any evidently toxic effect on central nervous system in rats. Therefore, alloferon 1 may be good new drug with antitumor and antinociceptive activity.

Copper(II) complexes of alloferon 1 with point mutations (H1A) and (H9A) stability structure and biological activity.

Mono- and polynuclear copper(II) complexes of the alloferon 1 with point mutations (H1A) A(1)GVSGH(6)GQH(9)GVH(12)G (Allo1A) and (H9A) H(1)GVSGH(6)GQA(9)GVH(12)G (Allo9A) have been studied by potentiometric, UV-visible, CD, EPR spectroscopic and mass spectrometry (MS) methods. To obtain a complete complex speciation different metal-to-ligand molar ratios ranging from 1:1 to 4:1 for Allo1A and to 3:1 for Allo9A were studied. The presence of the His residue in first position of the peptide chain changes the coordination abilities of the Allo9A peptide in comparison to that of the Allo1A. Imidazole-N3 atom of N-terminal His residue of the Allo9A peptide forms stable 6-membered chelate with the terminal amino group. Furthermore, the presence of two additional histidine residues in the Allo9A peptide (H(6),H(12)) leads to the formation of the CuL complex with 4N {NH2,NIm-H(1),NIm-H(6),NIm-H(12)} binding site in wide pH range (5-8). For the Cu(II)-Allo1A system, the results demonstrated that at physiological pH7.4 the predominant complex the CuH-1L consists of the 3N {NH2,N(-),CO,NIm} coordination mode. The inductions of phenoloxidase activity and apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 were studied. The Allo1A, Allo1K peptides and their copper(II) complexes displayed the lowest hemocytotoxic activity while the most active was the Cu(II)-Allo9A complex formed at pH7.4. The results may suggest that the N-terminal-His(1) and His(6) residues may be more important for their proapoptotic properties in insects than those at positions 9 and 12 in the peptide chain.