Lenka Borovičková

Toxicity study of antimicrobial peptides from wild bee venom and their analogs toward mammalian normal and cancer cells.

Recently, we have isolated and characterized remarkable antimicrobial peptides (AMPs) from the venom reservoirs of wild bees. These peptides (melectin, lasioglossins, halictines and macropin) and their analogs display high antimicrobial activity against Gram-positive and -negative bacteria, antifungal activity and low or moderate hemolytic activity. Here we describe cytotoxicity of the above-mentioned AMPs and some of their analogs toward two normal cell lines (human umbilical vein endothelial cells, HUVEC, and rat intestinal epithelial cells, IEC) and three cancer cell lines (HeLa S3, CRC SW 480 and CCRF-CEM T). HeLa S3 cells were the most sensitive ones (concentration causing 50% cell death in the case of the most toxic analogs was 2.5-10 μM) followed by CEM cells. For the other cell lines to be killed, the concentrations had to be four to twenty times higher. These results bring promising outlooks of finding medically applicable drugs on the basis of AMPs. Experiments using fluorescently labeled lasioglossin III (Fl-VNWKKILGKIIKVVK-NH(2)) as a tracer confirmed that the peptides entered the mammalian cells in higher quantities only after they reached the toxic concentration. After entering the cells, their concentration was the highest in the vicinity of the nucleus, in the nucleolus and in granules which were situated at very similar places as mitochondria. Experiments performed using cells with tetramethylrhodamine labeled mitochondria showed that mitochondria were fragmented and lost their membrane potential in parallel with the entrance of the peptides into the cell and the disturbance of the cell membrane.

New potent antimicrobial peptides from the venom of Polistinae wasps and their analogs

Four new peptides of the mastoparan family, characterized recently in the venom of three neotropical social wasps collected in the Dominican Republic, Polistes major major, Polistes dorsalis dorsalis and Mischocyttarus phthisicus were synthesized and tested for antimicrobial potency against Bacillus subtilis, Staphylococcus aureus, Escherichia coli (E.c.) and Pseudomonas aeruginosa, and for hemolytic and mast cells degranulation activities. As these peptides possess strong antimicrobial activity (minimal inhibitory concentration (MIC) values against Bacillus subtillis and E.c. in the range of 5-40 microM), we prepared 40 of their analogs to correlate biological activities, especially antimicrobial, with the net positive charge, hydrophobicity, amphipathicity, peptide length, amino acid substitutions at different positions of the peptide chain, N-terminal acylation and C-terminal deamidation. Circular dichroism spectra of the peptides measured in the presence of trifluoroethanol or SDS showed that the peptides might adopt alpha-helical conformation in such anisotropic environments.

Novel antimicrobial peptides from the venom of the eusocial bee Halictus sexcinctus (Hymenoptera: Halictidae) and their analogs

Two novel antimicrobial peptides, named halictines, were isolated from the venom of the eusocial bee Halictus sexcinctus. Their primary sequences were established by ESI-QTOF mass spectrometry, Edman degradation and enzymatic digestion as Gly-Met-Trp-Ser-Lys-Ile-Leu-Gly-His-Leu-Ile-Arg-NH2 (HAL-1), and Gly-Lys-Trp-Met-Ser-Leu-Leu-Lys–His-Ile-Leu-Lys-NH2 (HAL-2). Both peptides exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria but also noticeable hemolytic activity. The CD spectra of HAL-1 and HAL-2 measured in the presence of trifluoroethanol or SDS showed ability to form an amphipathic α-helical secondary structure in an anisotropic environment such as bacterial cell membrane. NMR spectra of HAL-1 and HAL-2 measured in trifluoroethanol/water confirmed formation of helical conformation in both peptides with a slightly higher helical propensity in HAL-1. Altogether, we prepared 51 of HAL-1 and HAL-2 analogs to study the effect of such structural parameters as cationicity, hydrophobicity, α-helicity, amphipathicity, and truncation on antimicrobial and hemolytic activities. The potentially most promising analogs in both series are those with increased net positive charge, in which the suitable amino acid residues were replaced by Lys. This improvement basically relates to the increase of antimicrobial activity against pathogenic Pseudomonas aeruginosa and to the mitigation of hemolytic activity.

Melectin: a novel antimicrobial peptide from the venom of the cleptoparasitic bee Melecta albifrons.

A novel antimicrobial peptide designated melectin was isolated from the venom of the cleptoparasitic bee Melecta albifrons. Its primary sequence was established as H‐Gly‐Phe‐Leu‐Ser‐Ile‐Leu‐Lys‐Lys‐Val‐Leu‐Pro‐Lys‐Val‐Met‐Ala‐His‐Met‐Lys‐NH2 by Edman degradation and ESI‐QTOF mass spectrometry. Synthetic melectin exhibited antimicrobial activity against both Gram‐positive and ‐negative bacteria and it degranulated rat peritoneal mast cells, but its hemolytic activity was low. The CD spectra of melectin measured in the presence of trifluoroethanol and sodium dodecyl sulfate showed a high content α‐helices, which indicates that melectin can adopt an amphipathic α‐helical secondary structure in an anisotropic environment such as the bacterial cell membrane. To envisage the role of the proline residue located in the middle of the peptide chain on biological activity and secondary structure, we prepared several melectin analogues in which the Pro11 residue was either replaced by other amino acid residues or was omitted. The results of biological testing suggest that a Pro kink in the α‐helical structure of melectin plays an important role in selectivity for bacterial cells. In addition, a series of N‐ and C‐terminal‐shortened analogues was synthesized to examine which region of the peptide is related to antimicrobial activity.

Lasioglossins: Three Novel Antimicrobial Peptides from the Venom of the Eusocial Bee Lasioglossum laticeps (Hymenoptera: Halictidae)

Three novel structurally related pentadecapeptides, named lasioglossins, were isolated from the venom of the eusocial bee Lasioglossum laticeps. Their primary sequences were established as H‐Val‐Asn‐Trp‐Lys‐Lys‐Val‐Leu‐Gly‐Lys‐Ile‐Ile‐Lys‐Val‐Ala‐Lys‐NH2 (LL‐I), H‐Val‐Asn‐Trp‐Lys‐Lys‐Ile‐Leu‐Gly‐Lys‐Ile‐Ile‐Lys‐Val‐Ala‐Lys‐NH2 (LL‐II) and H‐Val‐Asn‐Trp‐Lys‐Lys‐Ile‐Leu‐Gly‐Lys‐Ile‐Ile‐Lys‐Val‐Val‐Lys‐NH2 (LL‐III). These lasioglossins exhibited potent antimicrobial activity against both Gram‐positive and Gram‐negative bacteria, low haemolytic and mast cell degranulation activity, and a potency to kill various cancer cells in vitro. The lasioglossin CD spectra were measured in the presence of trifluoroethanol and sodium dodecyl sulfate solution and indicated a high degree of α‐helical conformation. NMR spectroscopy, which was carried out in trifluoroethanol/water confirmed a curved α‐helical conformation with a concave hydrophobic and convex hydrophilic side. To understand the role of this bend on biological activity, we studied lasioglossin analogues in which the Gly in the centre of the molecule was replaced by other amino acid residues (Ala, Lys, Pro). The importance of the N‐terminal part of the molecule to the antimicrobial activity was revealed through truncation of five residues from both the N and C termini of the LL‐III peptide. C‐terminal deamidation of LL‐III resulted in a drop in antimicrobial activity, but esterification of the C terminus had no effect. Molecular modelling of LL‐III and the observed NOE contacts indicated the possible formation of a bifurcated H‐bond between hydrogen from the Lys15 CONH peptide bond and one H of the C‐terminal CONH2 to the Ile11 oxygen atom. Such interactions cannot form with C‐terminal esterification.

Melectin MAPs: the influence of dendrimerization on antimicrobial and hemolytic activity

The recently described antimicrobial peptide melectin (MEP, GFLSILKKVLPKVMAHMK-NH2) exhibits high antimicrobial activity against Gram-positive and Gram-negative bacteria. Here we describe the synthesis and biological activities of 23 new analogues of MEP. We studied the influence of dimerization and tetramerization (MAP-constructs of MEP) on the antimicrobial and hemolytic activities, as well as the role of Met in positions 14 and 17 of the peptide chain. Oxidation of the Met to Met(O) and Met(O2) decreases antimicrobial activity of all tested bacteria if the peptide is in the monomeric form, however, only to Staphylococcus aureus if in the form of dimer or tetramer. Dimerization and tetramerization increase the undesirable hemolytic activity of the peptides. Interestingly, substitution of Leu for Val in position 6 leads to the decrease of hemolytic activity. Introduction of the isosteric amino acid Nle into positions 14 or 17 or both leads to slight increase of hemolytic activity under preservation of high antimicrobial activities. Unfortunately, dimerization again leads to an increase of hemolytic activity.

The antifungal effect of peptides from hymenoptera venom and their analogs

As the occurrence of Candida species infections increases, so does resistance against commonly-used antifungal agents. It is therefore necessary to look for new antifungal drugs. This study investigated the antifungal activity of recently isolated, synthesized and characterized antimicrobial α-helical amphipathic peptides (12–18 amino acids long) from the venom of hymenoptera (melectin, lasioglossins I, II, and III, halictines I and II) as well as a whole series of synthetic analogs. The minimal inhibitory concentrations (MICs) against different Candida species (C. albicans, C. krusei, C. glabrata, C. tropicalis and C. parapsilosis) of the natural peptides amounted to 4–20 µM (7–40 mg/l). The most active were the synthetic analog all-D-lasioglossin III and lasioglossin III analog KNWKK-Aib-LGK-Aib-IK-Aib-VK-NH2. As shown using a) colony forming unit determination on agar plates, b) the efflux of the dye from rhodamine 6B-loaded cells, c) propidium iodide and DAPI staining, and d) fluorescently labeled antimicrobial peptide (5(6)-carboxyfluorescein lasioglossin-III), the killing of fungi by the peptides studied occurs within minutes and might be accompanied by a disturbance of all membrane barriers. The peptides represent a promising lead for the development of new, effective antifungal drugs.

Novel analogues of arginine vasopressin containing α-2-indanylglycine enantiomers in position 2

Continuing our efforts to obtain potent and selective analogues of AVP we synthesized and pharmacologically evaluated ten new compounds modified at position 2 with α‐2‐indanylglycine or its D‐enantiomer (Igl or D‐Igl, respectively). All the peptides were tested for pressor, antidiuretic, and in vitro uterotonic activities. We also determined the binding affinity of these compounds to human OT receptor. The Igl2 substitution resulted in a significant change of the pharmacological profile of the peptides. The new analogues were moderate or potent OT antagonists (pA2 values ranging from 7.19 to 7.98) and practically did not interact with V1a and V2 receptors. It is worth emphasizing that these new peptides were exceptionally selective. On the other hand, the D‐Igl2 substituted counterparts turned out to be weak antagonists of the pressor response to AVP and displayed no antidiuretic activity. Some of the results were unexpected, e.g. dual activity in the rat uterotonic test in vitro: the D‐Igl peptides showed a strong antioxytocic potency (pA2 values ranging from 7.70 to 8.20) at low concentrations and full agonism at high concentrations. The results provided useful information about the SAR of AVP analogues. Copyright

Design, Synthesis and Structure–Activity Relationship of New Arginine Vasopressin Analogues Containing Proline Derivatives in Position 2

In this study, we present the synthesis and pharmacological properties of new analogues of arginine vasopressin modified in the N‐terminal part of the molecule with proline derivatives: indoline‐2‐carboxylic acid (Ica) and (2S,4R)‐4‐(naphthalene‐2‐ylmethyl)pyrrolidine‐2‐carboxylic acid. All the peptides were tested for pressor, antidiuretic and in vitro uterotonic activities. We also determined their binding affinity to the human oxytocin receptor. The Ica2 substitution resulted in two moderately potent and selective antioxytocic agents: [Mpa1, Ica2, D‐Arg8]VP and [Mpa1,Ica2,Val4,D‐Arg8]VP (pA2 = 7.09 and 7.50, respectively). On the other hand, peptides modified with (2S,4R)‐4‐(naphthalene‐2‐ylmethyl)pyrrolidine‐2‐carboxylic acid, apart from their moderate antioxytocic activity, turned out to be weak antagonists of the pressor response to arginine vasopressin. The results of this study provide useful information about the structure–activity relationship of arginine vasopressin analogues and can help to design compounds with desired biological properties.

In vitro binding and in vivo biodistribution studies of the neuroprotective peptide humanin using [125I]humanin derivatives

Humanin (HN) and HN-derivatives are a family of peptides first reported in the last decade with potent in vitro and in vivo neuroprotective activity, which is mediated through a not completely elucidated mechanism. Recently, our group has evaluated the effect of various HN-derivatives on the 3-quinuclidinyl benzilate (QNB)-induced impairment of spatial orientation and memory in rats, by employing the T-maze test. In the present work four new, tyrosine containing HN-derivatives were synthesized (Y-PAGASRLLLTGEIDLP, peptide I; Y-PAGASRLLLLTGEIDLP, peptide II; Y-SALLRSIPAPAGASRLLLTGEIDLP, peptide III; Y-SALLRSIPAPAGASRLLLLTGEIDLP, peptide IV). The neuroprotective action of these peptides was evaluated in the T-maze test and the most active among them (peptides I and III) was radiolabeled with (125)I. The pure monoradioiodinated peptides were used in: (i) in vitro binding studies with various neuronal cell lines and with brain and stomach membranes from rats and mice and (ii) in vivo biodistribution studies in rats and mice. Moreover, the metabolic stability of the above radiolabeled peptides was studied. Under the experimental conditions used, our data do not confirm the existence of specific binding sites for HN on the neuronal tissue. Nevertheless, they are setting the basis for further relevant studies aiming at the clarification of the mode of the neuroprotective action of HN-peptides.