Desenvolvimento de complexos de Eu e Tb com aplicação imagiológica e terapêutica no cancro

Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2013Os lantanídeos (Ln) têm sido muito estudados devido às suas propriedades fluorescentes únicas, tais como um grande desvio de Stokes e tempos de decaimentos longos. A síntese de complexos de Európio (Eu) e Térbio (Tb) tem um grande número de aplicações possíveis na área da medicina. Com a descoberta recente de compostos orgânicos (8-hidroxiquinolina) que actuam especificamente em células cancerígenas, através do recrutamento de cobre, reduzindo a proliferação e provocando a apoptose, a síntese de complexos de Ln que actuam em células cancerígenas tem ganho interesse. O objectivo do presente trabalho foi sintetizar complexos de Eu/Tb (8 com e 8 sem cobre) que possam recrutar cobre (Cu) especificamente em células cancerígenas, tendo uma acção terapêutica e funcionando como biosondas. Os complexos, para além do Ln, possuem uma molécula que promove o efeito antena (2,2’-bipiridina), e têm uma ligando adicional que recruta Cu nas células tumorais (testados quatro ligandos diferentes). Os complexos foram caracterizados através da espectroscopia vibracional, confirmando a estrutura dos mesmos. Espectroscopia de UV/Vis e de fluorescência foram também realizadas, para estudar as propriedades fotoluminescentes dos complexos. Os mesmos demonstraram que possuem excelentes propriedades fluorescentes, especialmente C2 para os complexos de Eu e C9 para os de Tb, podendo assim funcionar como biosondas. O recrutamento de Cu por parte dos complexos foi confirmado por espectroscopia de UV/Vis, o que deixou em aberto a possibilidade do recrutamento de Cu poder acontecer de forma específica para células cancerígenas. Os efeitos na viabilidade celular por parte dos complexos foram estudados em células HeLa e A549, utilizando um método colorimétrico (MTT). Os complexos sem Cu não interferiram na viabilidade celular, contrariamente aos complexos bimetálicos, que reduziram significativamente a viabilidade. Os estudos de fluorescência em meio celular permitiram confirmar os resultados obtidos em solução. Concluindo, os complexos de Eu/Tb sintetizados podem funcionar como biosondas e como moléculas anticancerígenas.Lanthanides (Ln) have been studied extensively because of their unique fluorescent properties, such as a long Stokes shift, exceptionally decay times and long excitable wavelength. The complexation of these metals, especially europium (Eu) and terbium (Tb), has a large number of applications in medicine. With a discovery of an organic compound (8-hydroxyquinoline) that target cancer cells specifically, by recruiting copper, reducing their proliferation and leading to apoptosis, the synthesis of the lanthanides complexes that target cancer cells has been gaining a major interest. The objective of the current work was synthetize Eu/Tb complexes (8 with and 8 without copper) that can recruit copper specifically from cancer cells, having a therapeutic action and working like imaging probe. The complexes, besides the metal, have a 2,2’-bypyridine molecule that promotes the antenna effect for the Ln, and have one additional ligand that targets and recruits copper in tumours cells (four different ligands were tested). The complexes were characterized with vibrational spectroscopy, confirming the structure of them. Fluorescent and UV/Vis spectroscopy were also done, to study the photoluminescent properties of the complexes. The complexes have shown that have great fluorescent properties, especially C2 for Eu complexes, and C9 for Tb complexes, proving that can function as bioprobe. The recruiting of Cu by the complexes was confirmed by UV/Vis spectroscopy, which leaves open the possible function of recruiting Cu specifically in cancer cells. Proliferation and cytotoxic effects of the complexes were studied in HeLa and A549 cells, using a colorimetric method (MTT). Complexes without copper don’t interfere with the viability of the cells, contrary to the complexes with copper, which reduce the viability. Fluorescent studies were also done in cellular medium, confirming the results obtained in solution. Concluding, the complexes of Eu/Tb synthetize can function as bioprobes and as anticancer molecules

EcDBS1R4, an antimicrobial peptide effective against Escherichia coli with in vitro fusogenic ability

©2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open accessarticle distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (http://creativecommons.org/licenses/by/4.0/)Discovering antibiotic molecules able to hold the growing spread of antimicrobial resistance is one of the most urgent endeavors that public health must tackle. The case of Gram-negative bacterial pathogens is of special concern, as they are intrinsically resistant to many antibiotics, due to an outer membrane that constitutes an effective permeability barrier. Antimicrobial peptides (AMPs) have been pointed out as potential alternatives to conventional antibiotics, as their main mechanism of action is membrane disruption, arguably less prone to elicit resistance in pathogens. Here, we investigate the in vitro activity and selectivity of EcDBS1R4, a bioinspired AMP. To this purpose, we have used bacterial cells and model membrane systems mimicking both the inner and the outer membranes of Escherichia coli, and a variety of optical spectroscopic methodologies. EcDBS1R4 is effective against the Gram-negative E. coli, ineffective against the Gram-positive Staphylococcus aureus and noncytotoxic for human cells. EcDBS1R4 does not form stable pores in E. coli, as the peptide does not dissipate its membrane potential, suggesting an unusual mechanism of action. Interestingly, EcDBS1R4 promotes a hemi-fusion of vesicles mimicking the inner membrane of E. coli. This fusogenic ability of EcDBS1R4 requires the presence of phospholipids with a negative curvature and a negative charge. This finding suggests that EcDBS1R4 promotes a large lipid spatial reorganization able to reshape membrane curvature, with interesting biological implications herein discussed.This research was funded by Fundação para a Ciência e a Tecnologia—Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal), Marie Skłodowska-Curie Research and Innovation Staff Exchange (MSCA-RISE, European Union) project INPACT (call H2020-MSCA-RISE-2014, grant agreement 644167), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil), Fundação de Amparo a Pesquisa do Distrito Federal (FAPDF, Brazil) and Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT, Brazil). M.M. and M.R.F. also acknowledge FCT-MCTES fellowships SPRH/BD/128290/2017 and SPRH/BD/100517/2014, respectively.info:eu-repo/semantics/publishedVersio

Design and mechanistic characterization of novel antimicrobial and anticancer peptides

The medical field related to bacterial infections and cancer are currently facing currently one of the biggest challenges, mostly due to conventional treatments inefficiency after years of overuse and misuse in clinics. Cases of multi-resistant bacterial infections are increasing every year, according to World Health Organization (WHO), explained by resistant microorganisms’ predominance after antibiotic usage and limited pharmaceutical development of new drugs. As for cancer therapeutics, unspecific treatments that promote severe side effects had another reported consequence, increased cancer resistance, prolonging patients’treatment. As a result, new alternatives are necessary to fight these challenges, such as antimicrobial peptides (AMPs) and anticancer peptides (ACPs). These peptides physical-chemical properties, such as small amino acid sequence, amphipathicity and positive net charge, allow them to act selectively at specific cell membranes, mostly due to electrostatic interactions (cationic vs. anionic membranes). Besides, they can be used against different targets, with reported activity against bacteria, viruses, fungi and cancer cells. In the last case, they are dependent of cancer cell membrane phosphatidylserine (PS) translocation from internal to external membrane leaflet, which increases the negative cell surface charge. Throughout the work here presented, we focused on new AMPs designed according to two different strategies: (i) Pa-MAP 2 and Pa-MAP 1.9, synthetic AMPs redesign from a natural protein from the polar fish Pleuronectes americanus (winter flounder), and (ii) EcAMP1R4, PaDBS1R1 and PaDBS1R6, synthetic peptides designed through a bioinformatics algorithm that considers chemical properties and activity efficiency. In both cases, a multidisciplinary approach was performed, using biophysics and cell biology techniques to study their activity in vitro, using membrane models, bacterial and cancer cell lines, and in vivo infection models. Considering the Pa-MAP peptide family (Pa-MAP 2 and Pa-MAP 1.9), with a minimal inhibitory concentration (MIC) of 3.2 and 6.0 μM against Escherichia coli, respectively, they were shown to be efficient against a multi-resistant strain from a clinical isolates, inclusively with promising results demonstrated with an in vivo infection mice model. Nevertheless, only Pa-MAP 1.9 showed to have dual activity (AMP and ACP), being tested in two different cell lines, HeLa and HCT-166. Despite its efficiency in promoting cancer cell death, Pa-MAP 1.9 showed a different mechanistic behaviour for the cell lines tested, promoting total cell death after 6 h of incubation (IC50 of 51.8 ± 1.23 μM) and membrane homeostasis destabilization. As for the synthetic peptides (EcAMP1R4, PaDBS1R1 and PaDBS1R6), their antimicrobial activity was confirmed in vitro, according to bioinformatics studies, with MIC values against E. coli of 11.7, 1.5 and 8.0 μM, respectively. In vivo studies were also performed for the last two peptides, confirming their potential as future antimicrobial drug molecules. Their dynamics after membrane interaction was likewise studied, either using bacteria cells or lipid vesicles, showing that different biomembrane properties are destabilized, which could determinate AMP efficiency. Structure conversion to a-helix after membrane interaction showed to be the first step for peptide activity. Concluding, the work discussed in this thesis resulted in new peptide molecules that are effective AMPs and, one of them (Pa-MAP 1.9), also as ACP. Their activity was characterized in vitro and in vivo through new approaches, with the objective of identifying new insights that may help in future peptide design. Even with the promising results achieved so far, their potential use in therapeutics need to be further tested, considering their efficiency, but also their applicability, focusing on patients and in the pharmaceutical industry needs

Neuropeptide receptors as potential pharmacological targets for obesity

© 2018 Elsevier Inc. All rights reserved.Obesity is a chronic multifactorial disease, characterized by an excessive accumulation of adipose tissue. It is usually the result of excessive food intake and/or low energy expenditure. Obesity can be triggered by lifestyle, nutritional, genetic, environmental, hormonal and psychological factors. Several strategies are used to treat obesity, including dietary reeducation, with balanced food intake, increased physical exercise, in order to promote energy expenditure and to overcome the insufficiency in weight reduction by other strategies, and administration of drugs. However, these medications are associated to undesirable side effects, resulting in a high withdrawal rate. Several studies have been focused on the development of compounds that act in the hypothalamic region where the center of the regulation of hunger and satiety is located. Some of them target the activity of endogenous peptides, such as ghrelin pancreatic polypeptide, peptide YY and neuropeptide Y, as well as their receptors. This review addresses the importance of understanding the neuropeptide/peptide hormones and their receptors for the development of novel anti-obesity compounds that may aid in weight reduction as a promising alternative for the treatment of obesity.This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil), Conselho Nacional de Pesquisa e Desenvolvimento (CNPq, Brazil), Fundação de Amparo à Pesquisa do Distrito Federal (FAPDF, Brazil), Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT, Brazil), Fundação para a Ciência e a Tecnologia –Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal), including the fellowships SFRH/BD/100517/2014 to M.R.F., and Marie Skłodowska-Curie Research and Innovation Staff Exchange (MSCA-RISE, European Union) project INPACT (call H2020-MSCA-RISE-2014, grant agreement 644167) funding to M.R.F. and N.C.S.info:eu-repo/semantics/publishedVersio

New frontiers for anti-biofilm drug development

© 2016 Elsevier Inc. All rights reservedPathogenic microbial biofilm, a consortium of microbial cells protected by a self-produced polymer matrix, is considered a worldwide challenge due to the inherent antibiotic resistance conferred by its lifestyle. Living, as it does, in a community of microbial organisms in a clinical situation, makes it responsible for severe and dangerous cases of infection. Combating this organisation of cells usually requires high antibiotic doses for a prolonged time, and these approaches often fail, contributing to infection persistence. In addition to therapeutic limitations, biofilms can be a source of infections when they grow inmedical devices. The challenge imposed by biofilms has mobilised researchers in the entire world to prospect or develop alternatives to control biofilms. In this context, this review summarises the new frontiers that could be used in clinical circumstances in order to prevent or eliminate pathogenic biofilms.This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil), Conselho Nacional de Pesquisa e Desenvolvimento (CNPq, Brazil), Fundação de Amparo à Pesquisa do Distrito Federal (FAPDF, Brazil), Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT, Brazil), Fundação para a Ciência e a Tecnologia –Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal), including the fellowcship SPRH/BD/ 100517/2014 to M.R.F., and Marie Skłodowska-Curie Research and Innovation Staff Exchange (MSCA-RISE, European Union) project INPACT (call H2020-MSCA-RISE-2014, grant agreement 644167).info:eu-repo/semantics/publishedVersio

Psd2 pea defensin shows a preference for mimetic membrane rafts enriched with glucosylceramide and ergosterol

© 2019 Elsevier B.V. All rights reservedPsd2 is a pea defensin with 47 amino acid residues that inhibits the growth of fungal species by an uncharacterized mechanism. In this work, Psd2 interactions with model membranes mimicking the lipid compositions of different organisms were evaluated. Protein-lipid overlay assays indicated that Psd2 recognizes Fusarium solani glucosylceramide (GlcCerF.solani) and ergosterol (Erg) in addition to phosphatidylcholine (POPC) and some phosphatidylinositol species, such as PtdIns (3)P, (5)P and (3,5)P2, suggesting that these lipids may play important roles as Psd2 targets. Assays using lipid vesicles were also performed to study the behaviour and dynamics that occur after peptide-membrane interactions. Surface plasmon resonance analysis showed that Psd2 has a higher affinity for pure POPC and POPC-based vesicles containing GlcCer and Erg at a 70:30 proportion than for vesicles containing cholesterol (Chol). Partition experiments by fluorescence spectroscopy showed a decrease in Trp42 quantum yield of Psd2 in the presence of GlcCerF.solani and Erg, individually or in simultaneously enriched membranes. The partition coefficient (Kp) obtained indicated a Psd2 partition preference for this vesicles, confirmed by quenching assays using acrylamide and 5/16-doxyl-stearic acid. Furthermore, we showed that the presence of C8C9 double bonds and a methyl group at position C9 of the sphingoid base backbone of GlcCer was relevant to Psd2 activity against Aspergillus nidulans. These results are consistent with the selectivity of Psd2 against fungi and its lack of toxicity in human erythrocytes. Psd2 represents a promising natural compound for the treatment of fungal infections.The authors acknowledge Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasil), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, Brasil) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasil) for funding, including fellowship to V.S.G.A. Authors also acknowledge Fundação para a Ciência e a Tecnologia – Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal) for funding, including fellowship SFRH/BD/100517/2014 to M.R.F., Marie Sklodowska-Curie Research and Innovation Staff Exchange (MSCARISE, European Union) project INPACT (call H2020-MSCA-RISE-2014, grant agreement 644167), and FP7-PEOPLE IRSES (International Research Staff Exchange Scheme, European Union).info:eu-repo/semantics/publishedVersio

Structural and functional evaluation of the palindromic alanine-rich antimicrobial peptide Pa-MAP2

© 2016 Published by Elsevier B.V.Recently, several peptides have been studied regarding the defence process against pathogenic microorganisms, which are able to act against different targets, with the purpose of developing novel bioactive compounds. The present work focuses on the structural and functional evaluation of the palindromic antimicrobial peptide Pa-MAP2, designed based on the peptide Pa-MAP from Pleuronectes americanus. For a better structural understanding, molecular modelling analyses were carried out, together with molecular dynamics and circular dichroism, in different media. Antibacterial activity against Gram-negative and positive bacteria was evaluated, as well as cytotoxicity against human erythrocytes, RAW 264.7, Vero and L6 cells. In silico docking experiments, lipid vesicle studies, and atomic force microscopy (AFM) imaging were carried out to explore the activity of the peptide. In vivo studies on infected mice were also done. The palindromic primary sequence favoured an α-helix structure that was pH dependent, only present on alkaline environment, with dynamic N- and C-terminals that are stabilized in anionic media. Pa-MAP2 only showed activity against Gram-negative bacteria, with a MIC of 3.2 μM, and without any cytotoxic effect. In silico, lipid vesicles and AFM studies confirm the preference for anionic lipids (POPG, POPS, DPPE, DPPG and LPS), with the positively charged lysine residues being essential for the initial electrostatic interaction. In vivo studies showed that Pa-MAP2 increases to 100% the survival rate of mice infected with Escherichia coli. Data here reported indicated that palindromic Pa-MAP2 could be an alternative candidate for use in therapeutics against Gram-negative bacterial infections.This work was supported by the Brazilian funding agencies CNPq, CAPES, FADPDF, FINEP and FUNDECT, by Fundação para a Ciência e a Tecnologia — Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal), and by Marie Skłodowska-Curie Research and Innovation Staff Exchange (MSCA-RISE, European Union) project INPACT (call H2020-MSCA-RISE-2014, grant agreement 644167). MRF also acknowledges FCT-MCTES fellowship SPRH/BD/100517/2014.info:eu-repo/semantics/publishedVersio

Selective antibacterial activity of the cationic peptide PaDBS1R6 against Gram-negative bacteria

© 2019 Elsevier B.V. All rights reserved.Infections caused by Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa foremost among them, constitute a major worldwide health problem. Bioinformatics methodologies are being used to rationally design new antimicrobial peptides, a potential alternative for treating these infections. One of the algorithms used to develop antimicrobial peptides is the Joker, which was used to design the peptide PaDBS1R6. This study evaluates the antibacterial activities of PaDBS1R6 in vitro and in vivo, characterizes the peptide interaction to target membranes, and investigates the PaDBS1R6 structure in contact with mimetic vesicles. Moreover, we demonstrate that PaDBS1R6 exhibits selective antimicrobial activity against Gram-negative bacteria. In the presence of negatively charged and zwitterionic lipids the structural arrangement of PaDBS1R6 transits from random coil to α-helix, as characterized by circular dichroism. The tertiary structure of PaDBS1R6 was determined by NMR in zwitterionic dodecylphosphocholine (DPC) micelles. In conclusion, PaDBS1R6 is a candidate for the treatment of nosocomial infections caused by Gram-negative bacteria, as template for producing other antimicrobial agents.This work was supported by Fundação para a Ciência e a Tecnologia – Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal) project PTDC/BBB-BQB/3494/2014, Marie Skłodowska-Curie Research and Innovation Staff Exchange (MSCA-RISE, European Union) project INPACT (call H2020-MSCA-RISE-2014, grant agreement 644167), Ramon Areces Foundation (to CFN), DTRA (HDTRA1-15-1-0050 to TKL), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP #2016/24413-0 to MDTT), Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG # 201710267000062 to ESFA) and Fundação de Apoio à Pesquisa do Distrito Federal (FAPDF). MRF acknowledges FCT-MCTES fellowship SFRH/BD/100517/2014. ONS holds a postdoctoral scholarship from National Council of Technological and Scientific Development (CNPq) and Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT) – Brazil [300583/2016-8]. MHC acknowledges fellowship 141518/2015-4 (CNPq) and 88881.134423/2016-01 (CAPES).info:eu-repo/semantics/publishedVersio