Natural Multi-Target Inhibitors of Cholinesterases and Monoamine Oxidase Enzymes with Antioxidant Potential from Skin Extracts of Hypsiboas cordobae and Pseudis minuta (Anura: Hylidae).

Alzheimer's disease (AD) is the most common cause of dementia, characterized by loss of selective neuronal and normal brain functions. Every year, ten million new cases are diagnosed worldwide. AD is a complex disease associated with all kind of different pathways, making their simultaneous modulation necessary. Nowadays anti-AD treatments are focused on enzymatic inhibitors. The study of the amphibians' skin had acquired great importance in the fields of biology and human health and represents an attractive and novel source for natural compounds with high potential in the development of new drugs. The present work exhibits the power of amphibian skins as a source of bioactive compounds. Herein we report the activity of extracts of two species from Hylidae family (H. cordobae and P. minuta) as reversible inhibitors of acetylcholinesterase and butyrylcholinesterase enzymes. Furthermore, the extracts inhibit MAO-B enzyme and showed antioxidant activities, acting on four important pathways of AD.

Leptodactylus latrans Amphibian Skin Secretions as a Novel Source for the Isolation of Antibacterial Peptides.

Amphibians´ skin produces a diverse array of antimicrobial peptides that play a crucial role as the first line of defense against microbial invasion. Despite the immense richness of wild amphibians in Argentina, current knowledge about the presence of peptides with antimicrobial properties is limited to a only few species. Here we used LC-MS-MS to identify antimicrobial peptides with masses ranging from 1000 to 4000 Da from samples of skin secretions of Leptodactylus latrans (Anura: Leptodactylidae). Three novel amino acid sequences were selected for chemical synthesis and further studies. The three synthetic peptides, named P1-Ll-1577, P2-Ll-1298, and P3-Ll-2085, inhibited the growth of two ATCC strains, namely Escherichia coli and Staphylococcus aureus. P3-Ll-2085 was the most active peptide. In the presence of trifluoroethanol (TFE) and anionic liposomes, it adopted an amphipathic α-helical structure. P2-Ll-1298 showed slightly lower activity than P3-Ll-2085. Comparison of the MIC values of these two peptides revealed that the addition of seven amino acid residues (GLLDFLK) on the N-terminal of P2-Ll-1298 significantly improved activity against both strains. P1-Ll-1577, which remarkably is an anionic peptide, showed interesting antimicrobial activity against E. coli and S. aureus strain, showing marked membrane selectivity and non-hemolysis. Due to this, P1-L1-1577 emerges as a potential candidate for the development of new antibacterial drugs.

Molecular design and synthesis of novel peptides from amphibians skin acting as inhibitors of cholinesterase enzymes.

Cholinesterases are a family of enzymes that catalyze the hydrolysis of neurotransmitter acetylcholine. There are two types of cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), which differ in their distribution in the body. Currently, cholinesterase inhibitors (ChEI) represent the treatment of choice for Alzheimer's disease (AD). In this paper, we report the synthesis and inhibitory effect on both enzymes of four new peptides structurally related to P1-Hp-1971 (amphibian skin peptide found in our previous work. Sequence: TKPTLLGLPLGAGPAAGPGKR-NH2 ). The bioassay data and cytotoxicity test show that some of the compounds possess a significant AChE and BChE inhibition and no toxic effect. The present work demonstrates that diminution of the size of the original peptide could potentially result in new compounds with significant cholinesterase inhibition activity, although it appears that there is an optimal size for the sequence. We also conducted an exhaustive molecular modeling study to better understand the mechanism of action of these compounds by combining docking techniques with molecular dynamics simulations on BChE. This is the first report about amphibian peptides and the second one of natural peptides with ChE inhibitory activity. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Antimicrobial peptides from skin secretions of Hypsiboas pulchellus (Anura: Hylidae).

The skin of many amphibians produces a large repertoire of antimicrobial peptides that are crucial in the first line of defense against microbial invasion. Despite the immense richness of wild amphibians in Argentina, knowledge about peptides with antimicrobial properties is limited to a few species. Here we used LC-MS-MS to analyze samples of Hypsiboas pulchellus skin with the aim to identify antimicrobial peptides in the mass range of 1000 to 2000 Da. Twenty-three novel sequences were identified by MS, three of which were selected for chemical synthesis and further studies. The three synthetic peptides, named P1-Hp-1971, P2-Hp-1935, and P3-Hp-1891, inhibited the growth of two ATCC strains: Escherichia coli (MIC: 16, 33, and 17 µM, respectively) and Staphylococcus aureus (MIC: 8, 66, and 17 µM, respectively). P1-Hp-1971 and P3-Hp-1891 were the most active peptides. P1-Hp-1971, which showed the highest therapeutic indices (40 for E. coli and 80 for S. aureus), is a proline-glycine-rich peptide with a highly unordered structure, while P3-Hp-1891 adopts an amphipathic α-helical structure in the presence of 2,2,2-trifluoroethanol and anionic liposomes. This is the first peptidomic study of Hypsiboas pulchellus skin secretions to allow the identification of antimicrobial peptides.

Interaction of acylated and substituted antimicrobial peptide analogs with phospholipid-polydiacetylene vesicles. Correlation with their biological properties.

A series of peptide analogs based on region 6-22 of Plantaricin 149 sequence were synthesized. The interaction between these analogs and phospholipid-polydiacetylene vesicles was investigated to evaluate the ability of the bioassay to detect differences in the interaction of the peptides with dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine vesicles, associated with amino acid substitution and N-terminal conjugation of the sequences with short fatty acids (8 and 12 carbon atoms). Fatty acid conjugation of peptides with low antimicrobial activity resulted in lipopeptides with improved activity against strains of Staphylococcus aureus and Listeria monocytogenes. The length of the fatty acid determined the bacterial specificity, and the conjugation with n-octanoic acid yielded the most active analog (C8-CT) against Staphylococcus aureus strain (MIC: 1.0 µm) while the conjugation with n-dodecanoic acid (C12-CT) was optimal for Listeria monocytogenes strain (MIC: 2.0 µm). In contrast, the substitution of Phe by Trp had an unfavorable effect on the antimicrobial activity. Hemolysis tests and membrane interaction studies with dipalmitoylphosphatidylcholine-polydiacetylene vesicles showed that lipopeptides interact to a greater extent with both biological and biomimetic membranes. Also, a good correlation was found between antimicrobial activity against Staphylococcus aureus strain and % colorimetric response values with dipalmitoylphosphatidylglycerol-polydiacetylene vesicles.

Epitope mapping of pathogenic Leptospira LipL32.

AIMS: To identify LipL32 epitopes and to evaluate their capability to recognize specific antibodies using ELISA. METHODS AND RESULTS: Epitope mapping by means of a library of overlapping peptide fragments prepared by simultaneous and parallel solid phase peptide synthesis on derivatized cellulose membranes (SPOT synthesis) was carried out. Eighty-seven overlapping decapentapeptides corresponding to the complete sequence of LipL32 were synthesized. According to spot-image intensities, the most reactive sequences were localized in regions 151-177 (sequence AAKAKPVQKLDDDDDGDDTYKEERHNK) and 181-204 (sequence LTRIKIPNPPKSFDDLKNIDTKKL). Two peptides (P1 and P2) corresponding to these sequences were synthesized, and their reactivity evaluated using ELISA test. CONCLUSIONS: Epitope identification and analysis suggested the existence of two antigenic regions within LipL32. These LipL32 reactive regions were highly conserved among antigenically variants of Leptospira spp. isolates. Peptides containing these regions (P1 and P2) showed a good capability for anti-leptospiral antibody recognition. SIGNIFICANCE AND IMPACT OF THE STUDY: This finding could have potential relevance not only for serodiagnosis but also as a starting point for the characterization of targets for vaccine design.

Systematic epitope analysis of the p26 EIAV core protein.

The major core protein of equine infectious anemia virus (EIAV), p26, is one of the primary immunogenic structural proteins during a persistent infection of horses and is highly conserved among antigenically variants of viral isolates. In order to investigate its immune profile in more detail for a better diagnostic, an epitope mapping was carried out by means of two libraries of overlapping peptide fragments prepared by simultaneous and parallel SPPS on derivatized cellulose membranes (SPOT synthesis). Polyclonal equine sera from infected horses were used for the biological assay. Particularly two promising continuous epitopes (NAMRHL and MYACRD) were localized on the C-terminal extreme of p26, region 194-222. A cyclic synthetic fragment of 29 amino acid residues containing the identified epitopes was designed and studied. A significant conformational change towards a helical structure was observed when the peptide was cyclized by a bridge between Cys198 and Cys218. This observation correlated with an improvement of its ability to be recognized by specific antibodies in an EIA (Enzyme-linked Immunosorbent assay). These results suggest that the conformationally restricted synthetic antigen adequately mimics the native structure of this region of p26 core protein.

A synthetic analog of plantaricin 149 inhibiting food-borne pathogenic bacteria:evidence for alpha-helical conformation involved in bacteria-membrane interaction.

Plantaricin-149 is a bacteriocin produced by Lactobacillus plantarum NRIC 149 (a LAB isolated from pineapple), which consists of a peptidic chain made up of 22 amino acid residues [Kato et al. J. Ferment. Bioeng. 1994; 77: 277-282]. In this work, a synthetic C-terminal amidated peptide analog denoted Pln149a was prepared by SPPS-Fmoc chemistry and the antagonistic activity against gram-positive and gram-negative bacteria was tested. The secondary structure was studied by circular dichroism (CD) and the vicinity of the tyrosine residue by fluorescence spectroscopy under different conditions. We report the results of the interaction of Pln149a with reverse micelles prepared from the amphiphilic AOT in cyclohexane. Synthetic plantaricin was active against one strain of Staphylococcus aureus and four strains of Listeria genus at pH 5.5 and 7.4 and, like its natural variant, inhibited L. plantarum ATCC 8014. The data derived from spectroscopic measurements in presence of AOT reverse micelles suggest that the secondary structure of the peptide upon interaction is an alpha-helix. In this membrane model, the hydrophobic side of the alpha-helix is inserted into the micelles, leaving the lysines exposed to the solvent and interacting with the polar moieties of AOT. The fluorescence data point out that the N-terminal tyrosine residue is close to the micellar interface.