D- and N-Methyl Amino Acids for Modulating the Therapeutic Properties of Antimicrobial Peptides and Lipopeptides.

Here we designed and synthesized analogs of two antimicrobial peptides, namely C10:0-A2, a lipopeptide, and TA4, a cationic α-helical amphipathic peptide, and used non-proteinogenic amino acids to improve their therapeutic properties. The physicochemical properties of these analogs were analyzed, including their retention time, hydrophobicity, and critical micelle concentration, as well as their antimicrobial activity against gram-positive and gram-negative bacteria and yeast. Our results showed that substitution with D- and N-methyl amino acids could be a useful strategy to modulate the therapeutic properties of antimicrobial peptides and lipopeptides, including enhancing stability against enzymatic degradation. The study provides insights into the design and optimization of antimicrobial peptides to achieve improved stability and therapeutic efficacy. TA4(dK), C10:0-A2(6-NMeLys), and C10:0-A2(9-NMeLys) were identified as the most promising molecules for further studies.

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.

A Comparative Study of the Antimicrobial and Structural Properties of Short Peptides and Lipopeptides Containing a Repetitive Motif KLFK.

BACKGROUND: In the last years, Antimicrobial Peptides (AMPs) and lipopeptides have received attention as promising candidates to treat infections caused by resistant microorganisms. OBJECTIVE: The main objective of this study was to investigate the effect of repetitive KLFK motifs and the attachment of aliphatic acids to the N-terminus of (KLFK)n peptides on therapeutic properties. METHODS: Minimal inhibitory concentration against Gram (+) and (-) bacteria and yeast of synthetic compounds were determined by broth microtiter dilution method, and the toxicity was evaluated by hemolysis assay. Membrane-peptide interaction studies were performed with model phospholipid membranes mimicking those of bacterial and mammalian cells by Fluorescence Spectroscopy. The secondary structure in solution and membranes was determined by Circular Dichroism. RESULTS: Our results showed that the resulting compounds have inhibitory activity against bacteria and fungi. The (KLFK)3 peptide showed the highest therapeutic index against bacterial and yeast strains, and the (KLFK)2 peptide conjugated with octanoic acid was the most active against yeasts. All the lipopeptides containing long-chain fatty acids (C14 or longer) were highly hemolytic at low concentrations. The antimicrobial activity of (KLFK)2 and (KLFK)3 lipopeptides was mainly associated with improved stability of the amphipathic secondary structure, which showed high contributions of α-helix in dipalmitoylphosphatidylglycerol (DPPG) vesicles. CONCLUSION: The repetition of the KLFK sequence and the conjugation with lipid tails allowed obtained compounds with high antimicrobial activity and low toxicity, becoming good candidates for treating infectious diseases.

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.

Biological and structural effects of the conjugation of an antimicrobial decapeptide with saturated, unsaturated, methoxylated and branched fatty acids.

The increasing bacterial resistance against conventional antibiotics has led to the search for new antimicrobial drugs with different modes of action. Cationic antimicrobial peptides (AMPs) and lipopeptides are promising candidates to treat infections because they act on bacterial membranes causing rapid destruction of sensitive bacteria. In this study, a decapeptide named A2 (IKQVKKLFKK) was conjugated at the N-terminus with saturated, unsaturated, methoxylated and methyl -branched fatty acids of different chain lengths (C8 - C20), the antimicrobial and structural properties of the lipopeptides being then investigated. The attachment of the fatty acid chain significantly improved the antimicrobial activity of A2 against bacteria, and so, endowed it with moderated antifungal activity against yeast strains belonging to genus Candida. Lipopeptides containing hydrocarbon chain lengths between C8 and C14 were the best antibacterial compounds (MIC = 0.7 to 5.8 µM), while the most active compounds against yeast were A2 conjugated with methoxylated and enoic fatty acids (11.1 to 83.3 µM). The improvement in antimicrobial activity was mainly related to the amphipathic secondary structure adopted by A2 lipopeptides in the presence of vesicles that mimic bacterial membranes. Peptide conjugation with long hydrocarbon chains (C12 or more), regardless of their structure, significantly increased toxicity towards eukaryotic cells, resulting in a loss of selectivity. These findings suggest that A2-derived lipopeptides are potential good candidates for the treatment of infectious diseases caused by bacteria and opportunistic pathogenic yeast belonging to genus Candida. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Combined analysis of cross-reacting antibodies anti-ß1AR and anti-B13 in advanced stages of Chagas heart disease.

OBJECTIVE: Autoantibodies cross-reacting with the ß1 adrenergic receptor (anti-ß1AR and anti-p2ß) and cardiac myosin antigens (anti-B13) have been related to the pathogenesis of chronic Chagas heart disease (CCHD). Studies exploring their levels in different stages are scarce. We aimed to evaluate the relationship of these autoantibodies with the clinical profile of chronic patients, especially regarding their classificatory accuracy in severe presentation with heart failure. METHODS AND RESULTS: We conducted a cross-sectional study of 155 T. cruzi-seropositive patients and 26 age- and gender-matched healthy controls. They were categorised in three stages of CCHD. Serum antibodies were measured by specific immunoassays. Symptomatic individuals showed increased levels of anti-ß1AR and anti-B13, while anti-p2ß antibodies were similar between groups. A composite logistic regression model including anti-B13, anti-ß1AR antibody levels and age was able to predict systolic heart failure yielding an area under the curve of 83% (sensitivity of 67% and specificity of 89%). CONCLUSIONS: In our study, anti-ß1AR and anti-B13 antibodies were higher in individuals with chronic Chagas heart disease stage III, mainly in those with dilated cardiomyopathy associated with systolic heart failure. Logistic regression analysis showed that both antibodies were good predictors of severe CCHD. As well as being involved in disease progression, anti-ß1AR and anti-B13 antibodies may be used as a serum marker of poor prognosis in terms of heart compromise.

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.

Contribution of the Tyr-1 in Plantaricin149a to disrupt phospholipid model membranes.

Plantaricin149a (Pln149a) is a cationic antimicrobial peptide, which was suggested to cause membrane destabilization via the carpet mechanism. The mode of action proposed to this antimicrobial peptide describes the induction of an amphipathic α-helix from Ala7 to Lys20, while the N-terminus residues remain in a coil conformation after binding. To better investigate this assumption, the purpose of this study was to determine the contributions of the Tyr1 in Pln149a in the binding to model membranes to promote its destabilization. The Tyr to Ser substitution increased the dissociation constant (KD) of the antimicrobial peptide from the liposomes (approximately three-fold higher), and decreased the enthalpy of binding to anionic vesicles from -17.2 kcal/mol to -10.2 kcal/mol. The peptide adsorption/incorporation into the negatively charged lipid vesicles was less effective with the Tyr1 substitution and peptide Pln149a perturbed the liposome integrity more than the analog, Pln149S. Taken together, the peptide-lipid interactions that govern the Pln149a antimicrobial activity are found not only in the amphipathic helix, but also in the N-terminus residues, which take part in enthalpic contributions due to the allocation at a lipid-aqueous interface.

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.

Disruption of Saccharomyces cerevisiae by Plantaricin 149 and investigation of its mechanism of action with biomembrane model systems.

The action of a synthetic antimicrobial peptide analog of Plantaricin 149 (Pln149a) against Saccharomyces cerevisiae and its interaction with biomembrane model systems were investigated. Pln149a was shown to inhibit S. cerevisiae growth by more than 80% in YPD medium, causing morphological changes in the yeast wall and remaining active and resistant to the yeast proteases even after 24 h of incubation. Different membrane model systems and carbohydrates were employed to better describe the Pln149a interaction with cellular components using circular dichroism and fluorescence spectroscopies, adsorption kinetics and surface elasticity in Langmuir monolayers. These assays showed that Pln149a does not interact with either mono/polysaccharides or zwitterionic LUVs, but is strongly adsorbed to and incorporated into negatively charged surfaces, causing a conformational change in its secondary structure from random-coil to helix upon adsorption. From the concurrent analysis of Pln149a adsorption kinetics and dilatational surface elasticity data, we determined that 2.5 muM is the critical concentration at which Pln149a will disrupt a negative DPPG monolayer. Furthermore, Pln149a exhibited a carpet-like mechanism of action, in which the peptide initially binds to the membrane, covering its surface and acquiring a helical structure that remains associated to the negatively charged phospholipids. After this electrostatic interaction, another peptide region causes a strain in the membrane, promoting its disruption.

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.

Cooperative effects in phospholipid monolayers induced by a peptide from HIV-1 capsid protein.

The study of interactions between biological molecules and model membranes is essential for the understanding of a number of physiological mechanisms involved in viral infections and dissemination. In this paper, the analysis of the interaction between a peptide from the p24 protein of Human Immunodeficiency Virus type 1 (HIV-1) and a phospholipid monolayer has pointed to a cooperative response in which very small amounts of peptide p24-1 (e.g. 0.05 mol%) can lead to measurable effects. Monolayer surface pressure and surface potential isotherms were affected for peptide concentrations as low as 0.05 mol%, with saturation at 0.5 mol%. The expansion effect from p24-1 is confirmed by changes in morphology of the monolayers using Brewster angle microscopy. Even though p24-1 is disordered in aqueous solutions, the interaction with dipalmitoyl phosphatidylcholine (DPPC) causes it to adopt an alpha-helix structure, as shown by circular dichroism (CD) data for multilamellar vesicles (MLV). The expansion of the phospholipid monolayer in a cooperative way may imply that p24-1 has potential antiviral activity, by participating in the cell rupture, with no need of specific receptors in the membrane.

Conformation of a synthetic antigenic peptide from HIV-1 p24 protein induced by ionic micelles.

We studied the interaction of the peptide AAMQMLKETINEEAAEWDRVHPVHAGPIA from the HIV-1 p24 protein in the presence of SDS (anionic) and CTABr (cationic) micelles at pH 7.0 by circular dichroism, fluorescence, and electron spin resonance (ESR). The micelles induced secondary structure as well as a blue shift in the tryptophan fluorescence emission, indicating an interaction between the peptide and the micelles. However, different contents of secondary structure elements were found when the peptide interacts with SDS or CTABr micelles. Steady-state anisotropy indicates a constraint on the rotational mobility of the tryptophan residue of the peptide upon interaction with micelles. ESR studies pointed to different locations for the peptide in either micelle. Our results suggested that at least part of the peptide might be located at the hydrophobic core of the CTABr micelles, probably at the C-terminal region, while it is more inserted into the SDS micelles.

Utilidad de la cromatografia gaseosa para la identificación de micobacterias / Utility of gas chromatography for the identification of mycobacterial species

El virus de la inmunodeficiencia humana (HIV) causa un profundo impacto sobre el problema de la tuberculosis (TBC) tanto en los países industrializados como en los en vía de desarrollo. Enfermedades graves causadas por micobacterias no tuberculosas, la mayoría correspondientes al Complejo Mycobacterium avium-intracellulare (MAC), se han vuelto muy comunes en asociación con la inmunosupresión severa. El aumento de complejidad en las enfermedades micobacterianas há estimulado el desarrollo de métodos diagnósticos más rápidos y eficientes. El presente trabajo pretende investigar la especificidad y aplicabilidad de la técnica de cromatografía gaseosa (CG) para el diagnóstico de micobacterias de importancia clínica en Argentina. Se tipificaron mediante CG 183 aislamientos clínicos de micobacterias, empleando como gold standard las pruebas bioquímicas clásicas. Del total de aislamientos analizados, 69 por ciento fueron correctamente identificados a nivel de especie y 5 por ciento incorrectamente. Si sólo se tienen en cuenta los aislamientos que pudieron ser clasificados, 93 por ciento lo fueron correctamente. Entre las especies de mayor interés clínico en Argentina, fueron correctamente identificados todos (40/50). Los aislamientos clasificables de M. tuberculosis, 15/16 aislamientos de M. bovis y 39/43 aislamientos de MAC. La CG representa una técnica rápida y de alto valor predictivo para la identificación de micobacterias en las condiciones de Argentina. Su aplicación en laboratorios de referencia es recomendable en este país u otros de Latinoamérica con una situación epidemiológica de la TBC similar y disponibilidad de esta tecnología.

Utilidad de la cromatografia gaseosa para la identificación de micobacterias / Utility of gas chromatography for the identification of mycobacterial species

El virus de la inmunodeficiencia humana (HIV) causa un profundo impacto sobre el problema de la tuberculosis (TBC) tanto en los países industrializados como en los en vía de desarrollo. Enfermedades graves causadas por micobacterias no tuberculosas, la mayoría correspondientes al Complejo Mycobacterium avium-intracellulare (MAC), se han vuelto muy comunes en asociación con la inmunosupresión severa. El aumento de complejidad en las enfermedades micobacterianas há estimulado el desarrollo de métodos diagnósticos más rápidos y eficientes. El presente trabajo pretende investigar la especificidad y aplicabilidad de la técnica de cromatografía gaseosa (CG) para el diagnóstico de micobacterias de importancia clínica en Argentina. Se tipificaron mediante CG 183 aislamientos clínicos de micobacterias, empleando como gold standard las pruebas bioquímicas clásicas. Del total de aislamientos analizados, 69 por ciento fueron correctamente identificados a nivel de especie y 5 por ciento incorrectamente. Si sólo se tienen en cuenta los aislamientos que pudieron ser clasificados, 93 por ciento lo fueron correctamente. Entre las especies de mayor interés clínico en Argentina, fueron correctamente identificados todos (40/50). Los aislamientos clasificables de M. tuberculosis, 15/16 aislamientos de M. bovis y 39/43 aislamientos de MAC. La CG representa una técnica rápida y de alto valor predictivo para la identificación de micobacterias en las condiciones de Argentina. Su aplicación en laboratorios de referencia es recomendable en este país u otros de Latinoamérica con una situación epidemiológica de la TBC similar y disponibilidad de esta tecnología. (AU)