From antimicrobial to anticancer peptides : a review

Antimicrobial peptides (AMPs) are part of the innate immune defense mechanism of many organisms. Although AMPs have been essentially studied and developed as potential alternatives for fighting infectious diseases, their use as anticancer peptides (ACPs) in cancer therapy either alone or in combination with other conventional drugs has been regarded as a therapeutic strategy to explore. As human cancer remains a cause of high morbidity and mortality worldwide, an urgent need of new, selective, and more efficient drugs is evident. Even though ACPs are expected to be selective toward tumor cells without impairing the normal body physiological functions, the development of a selective ACP has been a challenge. It is not yet possible to predict antitumor activity based on ACPs structures. ACPs are unique molecules when compared to the actual chemotherapeutic arsenal available for cancer treatment and display a variety of modes of action which in some types of cancer seem to co-exist. Regardless the debate surrounding the definition of structure-activity relationships for ACPs, great effort has been invested in ACP design and the challenge of improving effective killing of tumor cells remains. As detailed studies on ACPs mechanisms of action are crucial for optimizing drug development, in this review we provide an overview of the literature concerning peptides' structure, modes of action, selectivity, and efficacy and also summarize some of the many ACPs studied and/or developed for targeting different solid and hematologic malignancies with special emphasis on the first group. Strategies described for drug development and for increasing peptide selectivity toward specific cells while reducing toxicity are also discussed.The authors thank Fundação para a Ciência e a Tecnologia (FCT- MEC, Portugal) for funding—PTDC/QUI-BIO/112929/2009. Diana Gaspar also acknowledges FCT for fellowship SFRH/BPD/ 73500/2010 and A. Salome Veiga for funding within the FCT Investigator Programme (IF/00803/2012

Bacterial Biofilms in Diabetic Foot Ulcers: Potential Alternative Therapeutics

Diabetes mellitus is a major health problem that affects approximately 171 million people globally. One of its most severe complications is the development of diabetic foot ulcers (DFU). Ischemic and neurophatic lesions are of major importance for DFU onset; however, it is the infection by multidrug-resistant and biofilm-producing microorganisms, along with local microenvironmental conditions unfavorable to antibiotics action that ultimately cause infection chronicity and lower limbs amputation. Novel therapeutic protocols for DFU management are extremely urgent. Bacteriophages, probiotics and antimicrobial peptides (AMP) have recently been proposed as alternatives to currently available antibiotics. Bacteriophages are viruses that specifically infect and multiply within bacterial cells. Their ability to diffuse through polymeric matrixes makes them particularly efficient to eradicate biofilm-based bacteria. Promising results were also observed with probiotic therapy. Probiotics are well-characterized strains with the ability to compete with pathogenic microorganisms and modulate the host immune response. AMP are molecules produced by living organisms as part of their innate immune response. Unlike conventional antibiotics, AMP also act as immunomodulators and resistance to AMP was rarely observed, supporting their potential as therapeutic agents. These innovative therapeutic strategies may in the future substitute or complement antibiotherapy, ultimately contributing for the decrease in multidrug-resistant bacteria dissemination

The interplay between HIV-1 gp41, membranes and pre-fusion antiviral agents

Tese de doutoramento em Bioquímica (Biofísica Molecular), apresentada à Universidade de Lisboa através da Faculdade de Ciências, 2008The HIV-1 envelope glycoprotein complex plays an important role in viral entry. Its surface subunit gp120 is responsible for virus binding to cellular receptors, and the transmembrane subunit gp41 mediates fusion of the virus with the target cell. Different functional regions can be identified in gp41, each one with importance in the fusion process. T20 and T-1249 are two HIV-1 fusion inhibitor peptides, being T20 already approved for clinical application in AIDS treatment. The work here presented describes research on the role of biomembranes on their mode of action. A model for the role of biomembranes was proposed. Given the similarities between the HIV-1 and SARS-CoV, and the urgency to obtain licensed drugs to treat SARS, the possibility that T20 and T-1249 can work as SARS- CoV inhibitors was investigated. The results obtained show that despite the hypothesis that T20 could inhibit SARS-CoV fusion is correct, the effect may not be strong enough for practical application. A small sequence inside the HIV-1 gp41 ectodomain membrane proximal region (MPR) is commonly referred to as a cholesterol binding domain. As the MPR plays a key role in the HIV-1 fusion process and the viral envelope is rich in cholesterol, the influence of cholesterol on the interaction of the MPR derived peptides with membrane model systems was studied. 2F5 and 4E10 are two potent and broadly neutralizing antibodies against HIV-1, whose epitopes are localized in the gp41 MPR. It was proposed that these antibodies can interact with the viral membrane trough a hydrophobic surface present on their third complementarity-determining region of the heavy chain. The 2F5 and 4E10 interactions with biomembranes models, in the absence and presence of its epitopes, were investigated in order to reveal the membranes role in the antibodies mechanism of action.O vírus da imunodeficiência humana (VIH) é o agente causador do síndrome da imunodeficiência adquirida (SIDA) e o seu modo de actuação consiste, sobretudo, em infectar e destruir células T CD4 do organismo hospedeiro. Existem dois tipos do + vírus, VIH-1 e VIH-2, mas é o VIH-1 o mais virulento e o responsável pela maior parte dos casos de SIDA no mundo. O envelope do vírus consiste numa bicamada lipídica, derivada da célula hospedeira, que possui um complexo glicoproteíco que confere ao vírus a capacidade para entrar nas células alvo. Este complexo é formado pelas subunidades gp120 (subunidade superfície) e gp41 (subunidade transmembranar), que se mantêm associadas de modo não covalente e oligomerizam sob a forma de trímeros na superfície do vírus. A gp41 é uma proteína transmembranar constituída por um domínio extracelular (ectodomínio), um domínio transmembranar e um domínio intracelular (endodomínio). O ectodomínio da gp41 contém várias regiões funcionais importantes para o processo de fusão viral. O péptido de fusão, localizado no terminal N da proteína, é uma região hidrófoba e rica em resíduos de glicina. Este domínio é essencial para o processo de fusão, nomeadamente para a penetração inicial da membrana da célula alvo. No seguimento do péptido de fusão encontram-se duas regiões que têm tendência para formar superenrolamentos de hélice a. A região adjacente ao péptido de fusão é chamada NHR e a região CHR é a que precede o segmento transmembranar. Os péptidos derivados destas regiões são chamados péptidos N- e C-, respectivamente. No terminal C da proteína, entre a região CHR e o domínio transmembranar, situa-se a chamada região próxima da membrana (MPR membrane proximal region). Esta região é rica em resíduos de triptofano e parece ter também um papel chave na fusão viral. O primeiro passo no processo de infecção consiste na ligação da gp120 ao receptor (CD4) e co-receptores (CXCR4 ou CCR5) na superfície das células alvo. Esta interacção induz alterações conformacionais na gp120 e modula as interacções gp120/gp41. A gp41 sofre então uma alteração conformacional e a sua estrutura nativa transita para uma estrutura intermediária de pré- hairpin . Estas variações na gp41 incluem a exposição do péptido de fusão e a sua inserção na membrana alvo. Posteriormente dá-se uma associação entre as regiões NHR e CHR levando à formação da estrutura em hairpin (trímero em dímeros de hélices antiparalelas), activa para afusão, o que leva à aproximação das membranas celular e viral. Na estrutura em hairpina região NHR forma três hélices centrais arranjadas num superenrolamento trimérico,enquanto que a região CHR for ma três hélices exteriores que encaixam de um modoantiparalelo em cavidades hidrófobas na superfície do superenrolamento. O que ocorredesde a aproximação das membranas até à fusão completa não está clarificado masparece envolver a formação de agregados dos trímeros da proteína do envelope paraformar poros de fusão.O tratamento mais comum para os doentes com SIDA baseia-se na aplicação decombinações de drogas anti-VIH, nomeadamente inibidores do transcriptase reversa edo protease, dois enzimas virais com importância ao nível da replicação do vírus.Apesar dos benefícios do uso destas drogas, a sua utilização é limitada devido os seusefeitos tóxicos e aparecimento de estirpes resistentes. Assim, tornou-se óbvia anecessidade de desenvolvimento de drogas mais eficientes, menos tóxicas e quepermitam eliminar os efeitos adversos associados à acção das drogas no interior dascélulas.Do trabalho científico desenvolvido nos últimos anos, resultaram fármacos que têmcomo objectivo actuar ao nível da entrada do vírus nas células, nomeadamente, naligação da gp120 ao CD4, na ligação da gp120 aos co-receptores e na fusão do envelopedo vírus com a membrana alvo. Apesar da enorme quantidade de compostos emdesenvolvimento e até em fase de testes clínicos, o T20 (também chamado Enfuvirtideou pelo nome comercial da marca Fuzeon), um inibidor de fusão, é o único já aprovadopara aplicação clínica. No entanto, apesar da sua comprovada eficácia clínica, não háconsenso sobre o seu modo de actuação ao nível molecular. O mecanismo de acçãomais aceite envolve a interacção do T20 com a região NHR da gp41, na fase de pré-hairpin , impedindo a formação da estrutura em hairpin e deste modo a fusão viral coma membrana celular.As membranas biológicas são entidades complexas, constituídas principalmente porlípidos e proteínas, que limitam as células, constituindo uma barreira entre o meioexterior e interior. A base estrutural das biomembranas é a bicamada lipídica. Noentanto, a distribuição lipídica nas biomembranas pode ser assimétrica levando a que asduas camadas tenham uma composição lipídica diferente. Além disso, apesar de grandeparte dos lipídos na bicamada formarem uma mistura uniforme e homogénea nachamada fase fluida, podem ocorrer heterogeneidades laterais, ou domínios, comcomposições lipídicas distintas. Os rafts lipídicos, são domínios encontrados nasmembranas plasmáticas que são enriquecidos em colesterol e esfingolípidos. Estesdomínios existem numa fase de líquido ordenado que é mais ordenada e compacta que oresto da bicamada, em fase fluida. Tem sido proposto que os rafts podem funcionarcomo plataformas para a reunião de complexos macromoleculares associados amembranas com importância ao nível de vários processos celulares, entrada de víruscomo o VIH-1 para as células (uma vez que receptores e co-receptores se encontramprovavelmente associados a rafts ) e a formação de vírus a partir da célula infectada. Deresto, a membrana deste vírus é muito enriquecida em colesterol e esfingolípidos,relativamente aos níveis encontrados nas membranas das células alvo, sendo sugeridoque isso se deve ao facto de a formação dos novos vírus se fazer através de raftslipídicos. O colesterol possui um papel central uma vez que mantém os rafts num estadofuncional.O estudo da interacção entre péptidos e/ou proteínas e biomembranas é muitas vezesnecessário de modo a clarificar alguns processos em que ambos estejam envolvidos. Noentanto, as membranas biológicas são sistemas demasiado complexos para serem usadascomo tal neste tipo de estudo. Uma alternativa viável é o estudo destas interacções emsistemas modelo de membranas, nomeadamente vesículas lipídicas unilamelares.Podendo ser preparadas em diferentes tamanhos, foram no presente trabalho utilizadosna sua maioria vesículas unilamelares grandes com diâmetro aproximado de 100 nm.Estes sistemas miméticos de membranas biológicas permitem uma base de estudosimplificada em simultâneo com a manutenção das principais propriedades dasbiomembranas.Os estudos realizados ao longo deste trabalho foram essencialmente biofísicos,baseados em técnicas de espectroscopia de fluorescência, dado o facto de os péptidos eproteínas estudados serem intrinsecamente fluorescentes sem necessidade deacoplamento de sondas.O T20, um péptido sintético que corresponde a uma sequência linear da gp41 doVIH-1 (entre o terminal C da região CHR e o terminal N da MPR), é um potenteinibidor da fusão deste vírus. Apesar de estar aprovado para uso clínico, não existeainda consenso acerca da sua acção ao nível molecular. Investigou-se a possívelexistência de um papel para as membranas lipídicas no modo de acção do T20, sendoque este envolvimento é sugerido tanto pela estrutura secundária do péptido, como porFundação para a Ciência e a Tecnologia (bolsa SFRH/BD/14336/2003)

Shifting gear in antimicrobial and anticancer peptides biophysical studies : from vesicles to cells

© 2015 European Peptide Society and John Wiley & Sons, Ltd.Despite the intensive study on the mechanism of action of membrane-activemolecules such as antimicrobial and anticancer peptides, most of the biophysical work has been performed using artificial model systems, mainly lipid vesicles. The use of these systems allows full control of the experimental parameters, and to obtain molecular-level detail on the action of peptides, the correlation with biological action is intangible. Recently, several biophysicalmethodologies have been translated to studies using bacterial and cancer cells. Here, we review biophysical studies on the mechanism of action of antimicrobial and anticancer peptides performed directly on cells. The data in these studies allow to correlate vesicle-based and cell-based studies and fill the vesicle-cell interdisciplinary gap.Projects Ciência Sem Fronteiras PVE 171/2012 (CAPES, Brazil) and MSCA-RISE-2014-Nr644167 are acknowledged. D. Gaspar and J. Freire are recipients of fellowships SFRH/BPD/73500/2010 and SFRH/BD/70423/2010 from FCT, Portugal, respectively. A.S. Veiga acknowledges FCT, Portugal, for funding within the FCT Investigator Programme (IF/00803/2012)

Nisin influence on the antimicrobial resistance ability of canine oral enterococci

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0).Periodontal disease (PD) is one of the most common diseases in dogs. Although previous studies have shown the potential of the antimicrobial peptide nisin for PD control, there is no information regarding its influence in the development of antimicrobial resistance or horizontal gene transfer (HGT). Nisin’s mutant prevention concentration (MPC) and selection window (MSW) were determined for a collection of canine oral enterococci. Isolates recovered after the determination of the MPC values were characterized for their antimicrobial profile and its nisin minimum inhibitory and bactericidal concentrations. The potential of vanA HGT between Enterococcus faecium CCGU36804 and nine clinical canine staphylococci and enterococci was evaluated. Nisin MPC values ranged from 400 to more than 600 µg/mL. In comparison with the original enterococci collection, the isolates recovered after the determination of the nisin MPC showed increased resistance towards amoxicillin/clavulanate (5%), vancomycin (5%), enrofloxacin (10%), gentamicin (10%) and imipenem (15%). The HGT of vanA gene was not observed. This work showed that nisin selective pressure may induce changes in the bacteria’s antimicrobial resistance profile but does not influence horizontal transfer of vanA gene. To our knowledge, this is the first report of nisin’s MPC and MSW determination regarding canine enterococci.Authors would like to acknowledge the Foundation for Science and Technology (Eva Cunha PhD fellowship SFRH/BD/131384/2017) and to CIISA–Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Project UIDB/00276/2020 (Funded by FCT), for financial support of this work.info:eu-repo/semantics/publishedVersio

Characterization of Tachyplesin peptides and their cyclized analogues to improve antimicrobial and anticancer properties

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).Tachyplesin I, II and III are host defense peptides from horseshoe crab species with antimicrobial and anticancer activities. They have an amphipathic β-hairpin structure, are highly positively-charged and differ by only one or two amino acid residues. In this study, we compared the structure and activity of the three tachyplesin peptides alongside their backbone cyclized analogues. We assessed the peptide structures using nuclear magnetic resonance (NMR) spectroscopy, then compared the activity against bacteria (both in the planktonic and biofilm forms) and a panel of cancerous cells. The importance of peptide-lipid interactions was examined using surface plasmon resonance and fluorescence spectroscopy methodologies. Our studies showed that tachyplesin peptides and their cyclic analogues were most potent against Gram-negative bacteria and melanoma cell lines, and showed a preference for binding to negatively-charged lipid membranes. Backbone cyclization did not improve potency, but improved peptide stability in human serum and reduced toxicity toward human red blood cells. Peptide-lipid binding affinity, orientation within the membrane, and ability to disrupt lipid bilayers differed between the cyclized peptide and the parent counterpart. We show that tachyplesin peptides and cyclized analogues have similarly potent antimicrobial and anticancer properties, but that backbone cyclization improves their stability and therapeutic potential.This project was funded by a National Health Medical Research Council (NHMRC) project grant (APP1084965). F.V. was supported by the UQ Research Scholarship, S.T.H. is an Australian Research Council (ARC) Future Fellow (FT150100398), D.J.C. is an ARC Australian Laureate Fellow (FL150100146). Marie Skłodowska-Curie Research and Innovation Staff Exchange grant (RISE; call: H2020-MSCA-RISE-2014, grant agreement 644167) funded secondments of S.A.D. and of A.S.V. to the University of Queensland. The Translational Research Institute is supported by a grant from the Australian Government.info:eu-repo/semantics/publishedVersio

Guar gum as a new antimicrobial peptide delivery system against diabetic foot ulcers Staphylococcus aureus isolates

© 2016 The AuthorsDiabetic patients frequently develop diabetic foot ulcers (DFUs), particularly those patients vulnerable to Staphylococcus aureus opportunistic infections. It is urgent to find new treatments for bacterial infections. The antimicrobial peptide (AMP) nisin is a potential candidate, mainly due to its broad spectrum of action against pathogens. Considering that AMP can be degraded or inactivated before reaching its target at therapeutic concentrations, it is mandatory to establish effective AMP delivery systems, with the natural polysaccharide guar gum being one of the most promising. We analysed the antimicrobial potential of nisin against 23 S. aureus DFU biofilm-producing isolates. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) were determined for nisin diluted in HCl and incorporated in guar gum gel. Statistical analysis was performed using the Wilcoxon matched-pair test. Nisin was effective against all isolates, including some multidrug-resistant clinical isolates, independent of whether it is incorporated in guar gum. While differences among MIC, MBC and MBIC values were observed for HCl- and guar gum- nisin, no significant differences were found between MBEC values. Inhibitory activity of both systems seems to differ only twofold, which does not compromise guar gum gel efficiency as a delivery system. Our results highlight the potential of nisin as a substitute for or complementary therapy to current antibiotics used for treating DFU infections, which is extremely relevant considering the increase in multidrug-resistant bacteria dissemination. The guar gum gel represents an alternative, practical and safe delivery system for AMPs, allowing the development of novel topical therapies as treatments for bacterial skin infections.The authors would like to acknowledge the Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa [Project UID/CVT/00276/2013, funded by Fundação para a Ciência e Tecnologia (FCT), Portugal]. This study was also conducted with the financial support of the project PTDC/SAU-MIC/122816/2010: ‘Biofilms in diabetic foot: microbial virulence characterization and cross-talk of major isolates’, funded by the FCT, Portugal. Raquel Santos, Diogo Barros and Ana Salomé Veiga acknowledge the FCT, Portugal, respectively, for two PhD fellowships (SFRH/BD/100571/2014 and PD/BD/113457/2015) and fellowship IF/00803/2012 under the FCT Investigator Programme.info:eu-repo/semantics/publishedVersio

Effect of pH on the influenza fusion peptide properties unveiled by constant-pH molecular dynamics simulations combined with experiment

© The Author(s) 2020. Open Access. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.The influenza virus fusion process, whereby the virus fuses its envelope with the host endosome membrane to release the genetic material, takes place in the acidic late endosome environment. Acidification triggers a large conformational change in the fusion protein, hemagglutinin (HA), which enables the insertion of the N-terminal region of the HA2 subunit, known as the fusion peptide, into the membrane of the host endosome. However, the mechanism by which pH modulates the molecular properties of the fusion peptide remains unclear. To answer this question, we performed the first constant-pH molecular dynamics simulations of the influenza fusion peptide in a membrane, extending for 40 µs of aggregated time. The simulations were combined with spectroscopic data, which showed that the peptide is twofold more active in promoting lipid mixing of model membranes at pH 5 than at pH 7.4. The realistic treatment of protonation introduced by the constant-pH molecular dynamics simulations revealed that low pH stabilizes a vertical membrane-spanning conformation and leads to more frequent contacts between the fusion peptide and the lipid headgroups, which may explain the increase in activity. The study also revealed that the N-terminal region is determinant for the peptide's effect on the membrane.This work was financially supported by FCT—Fundação para a Ciência e a Tecnologia, Portugal, through projects PTDC/QUI-BIQ/114774/2009, PTDC/CCI-BIO/28200/2017 and Pest-OE/EQB/LA0004/2011. This work was also financially supported by Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI) and by national funds through FCT—Fundação para a Ciência e a Tecnologia. DL was supported by FCT post-doc fellowship SFRH/BPD/92537/2013.info:eu-repo/semantics/publishedVersio

A designed cyclic analogue of gomesin has potent activity against Staphylococcus aureus biofilms

© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected]: Infections caused by bacterial biofilms are very difficult to treat. The use of currently approved antibiotics even at high dosages often fails, making the treatment of these infections very challenging. Novel antimicrobial agents that use distinct mechanisms of action are urgently needed. Objectives: To explore the use of [G1K,K8R]cGm, a designed cyclic analogue of the antimicrobial peptide gomesin, as an alternative approach to treat biofilm infections. Methods: We studied the activity of [G1K,K8R]cGm against biofilms of Staphylococcus aureus, a pathogen associated with several biofilm-related infections. A combination of atomic force and real-time confocal laser scanning microscopies was used to study the mechanism of action of the peptide. Results: The peptide demonstrated potent activity against 24 h-preformed biofilms through a concentration-dependent ability to kill biofilm-embedded cells. Mechanistic studies showed that [G1K,K8R]cGm causes morphological changes on bacterial cells and permeabilizes their membranes across the biofilm with a half-time of 65 min. We also tested an analogue of [G1K,K8R]cGm without disulphide bonds, and a linear unfolded analogue, and found both to be inactive. Conclusions: The results suggest that the 3D structure of [G1K,K8R]cGm and its stabilization by disulphide bonds are essential for its antibacterial and antibiofilm activities. Moreover, our findings support the potential application of this stable cyclic antimicrobial peptide to fight bacterial biofilms.This project received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 828774. This work was supported by project grants funded by Fundação para a Ciência e a Tecnologia (FCT-MCTES, Portugal; UIDB/04565/2020 and PPBI-POCI-01-0145-FEDER-022122). S.A.D. acknowledges FCT for the fellowship PD/BD/114425/2016. S.T.H. is an Australian Research Council (ARC) Future Fellow (FT150100398) and is supported by the ARC Centre of Excellence for Innovations in Peptide & Protein Science (CE200100012).info:eu-repo/semantics/publishedVersio

Intracellular nucleic acid delivery by the supercharged dengue virus capsid protein

© 2013 Freire et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins.This work was supported by Fundação para a Ciência e Tecnologia – Ministério da Educação e Ciência (FCT-MEC, Portugal) [PTDC/QUI-BIQ/112929/2009], by the European Union [projects FP7-PEOPLE IRSES (MEMPEPACROSS) and FP7-HEALTH-F3-2008-223414 (LEISHDRUG)], by the Spanish Ministry of Economy and Competitiveness (SAF2011-24899), the Generalitat de Catalunya (2009 SGR 492), by the Brazilian Conselho Nacional de Desenvolvimento Científico e Tecnoloógico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and the National Institute of Science and Technology in Dengue (INCT-Dengue). JMF also acknowledges FCT-MEC for Ph.D. fellowship SFRH/BD/70423/2010