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Projeto de investigação
BROAD-SPECTRUM FUSION INHIBITORS AGAINST ENVELOPED VIRUSES
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Broad spectrum antiviral activity for paramyxoviruses is modulated by biophysical properties of fusion inhibitory peptides
Publication . Mathieu, Cyrille; Augusto, Marcelo T.; Niewiesk, Stefan; Horvat, Branka; Palermo, Laura M.; Sanna, Giuseppina; Madeddu, Silvia; Huey, Devra; Castanho, Miguel A. R. B.; Porotto, Matteo; Santos, Nuno C.; Moscona, Anne
Human paramyxoviruses include global causes of lower respiratory disease like the parainfluenza viruses, as well as agents of lethal encephalitis like Nipah virus. Infection is initiated by viral glycoprotein-mediated fusion between viral and host cell membranes. Paramyxovirus viral fusion proteins (F) insert into the target cell membrane, and form a transient intermediate that pulls the viral and cell membranes together as two heptad-repeat regions refold to form a six-helix bundle structure that can be specifically targeted by fusion-inhibitory peptides. Antiviral potency can be improved by sequence modification and lipid conjugation, and by adding linkers between the protein and lipid components. We exploit the uniquely broad spectrum antiviral activity of a parainfluenza F-derived peptide sequence that inhibits both parainfluenza and Nipah viruses, to investigate the influence of peptide orientation and intervening linker length on the peptides' interaction with transitional states of F, solubility, membrane insertion kinetics, and protease sensitivity. We assessed the impact of these features on biodistribution and antiviral efficacy in vitro and in vivo. The engineering approach based on biophysical parameters resulted in a peptide that is a highly effective inhibitor of both paramyxoviruses and a set of criteria to be used for engineering broad spectrum antivirals for emerging paramyxoviruses.
Lipophilicity is a key factor to increase the antiviral activity of HIV neutralizing antibodies
Publication . Augusto, Marcelo T.; Hollmann, Axel; Troise, Fulvia; Veiga, Ana S.; Pessi, Antonello; Santos, Nuno C.
The HIV broadly neutralizing antibody 2F5 targets the transiently exposed epitope in the membraneproximal external region (MPER) of HIV-1 gp41, by a two-step mechanism involving the viral membraneand this viral glycoprotein. It was recently shown that 2F5 conjugation with a cholesterol moiety outsideof the antibody paratope substantially increases its antiviral activity. Additionally, the antiviral activityof D5, a human antibody that binds to the N-terminal heptad repeat (NHR) of gp41 and lacks membranebinding, was boosted by the same cholesterol conjugation. In this work, we evaluated the membrane affin-ity of both antibodies towards membranes of different compositions, using surface plasmon resonance. Acorrelation was found between membrane affinity and antiviral activity against HIV-1. We propose thatthe conjugation of cholesterol to 2F5 or D5 allows a higher degree of antibody pre-concentration at theviral membrane. This way, the antibodies become more available to bind efficiently to the gp41 epitope,blocking viral fusion faster than the unconjugated antibody. These results set up a relevant strategy toimprove the rational design of therapeutic antibodies against HIV.
Enveloped viruses : a peptide inhibition strategy
Publication . Augusto, Marcelo T.; Santos, Nuno Fernando Duarte Cordeiro Correia dos; Hollmann, Axel
The infection by enveloped viruses causes several diseases worldwide, which in some cases can develop to a chronic stage or lead to death. Overall, the lack of a cure or the emergence of strains resistant to commercial drugs is the driving force for the research and development of new alternative drugs. Peptides can be used to inhibit the fusion between enveloped viruses and host cells. The involvement of both membranes in the fusion process, aiming to deliver to the cell the viral genetic material, highlights the important role of the membrane.
Based on these findings, several membrane fusion inhibitor peptides were developed to fight HIV-1, influenza and a few paramyxoviruses, such parainfluenza and Nipah virus, were conjugated to lipid anchors. The resulting lipid anchored-peptides were found to interact much more with biomembrane model systems (liposomes) and human blood cells (erythrocytes and peripheral blood mononuclear cells) in comparison to the unconjugated peptides. Furthermore, we found a correlation between membrane binding and antiviral activity.
Polyethylenoglycol (PEG) is known for increasing the water solubility of molecules. Here, in order to improve the activity of the peptides, PEG was used as a linker between the peptide and the lipid anchor, and its effect on peptide-membrane interaction was also tested. We found that the length of the PEG influences the antiviral peptide potency, despite increasing peptide sensibility to proteases, which can be avoided with N-terminal lipid conjugation.
Influenza fusion occurs inside the endosomes after pH acidification. We developed peptides aiming to be internalized in endosomes together with the virus. Despite the use of lipid anchors to promote membrane attachment, we added a cell penetrating peptide (CPP) sequence to increase membrane crossing. We found that the CPP by itself was not enough to promote membrane permeation. However, the combination of lipid tagging and a CPP was enough to promote peptide membrane anchoring and intracellular localization, especially when tocopherol was used as anchor.
All the results obtained in the present Thesis support membranothropic behavior as a key factor in the final activity of the peptides against enveloped viruses. Furthermore, this Thesis provides important guidelines in terms of peptide design, based on the finding of a balance between peptide sequences, lipid anchor, linker length and peptide orientation.
Role of amphipathicity and hydrophobicity in the balance between hemolysis and peptide–membrane interactions of three related antimicrobial peptides
Publication . Hollmann, Axel; Martínez, Melina; Noguera, Martín E.; Augusto, Marcelo T.; Disalvo, Anibal; Santos, Nuno C.; Semorile, Liliana; Maffía, Paulo C.
Cationic antimicrobial peptides (CAMPs) represent important self defense molecules in many organisms,
including humans. These peptides have a broad spectrum of activities, killing or neutralizing many
Gram-negative and Gram-positive bacteria. The emergence of multidrug resistant microbes has stimulated
research on the development of alternative antibiotics. In the search for new antibiotics, cationic
antimicrobial peptides (CAMPs) offer a viable alternative to conventional antibiotics, as they physically
disrupt the bacterial membranes, leading to lysis of microbial membranes and eventually cell death. In
particular, the group of linear -helical cationic peptides has attracted increasing interest from clinical
as well as basic research during the last decade.
In this work, we studied the biophysical and microbiological characteristics of three new designed
CAMPs. We modified a previously studied CAMP sequence, in order to increase or diminish the hydrophobic
face, changing the position of two lysines or replacing three leucines, respectively. These mutations
modified the hydrophobic moment of the resulting peptides and allowed us to study the importance of
this parameter in the membrane interactions of the peptides. The structural properties of the peptides
were also correlated with their membrane-disruptive abilities, antimicrobial activities and hemolysis of
human red blood cells.
Antiviral lipopeptide-cell membrane Interaction Is Influenced by PEG linker length
Publication . Augusto, Marcelo T.; Hollmann, Axel; Porotto, Matteo; Moscona, Anne; Santos, Nuno C.
A set of lipopeptides was recently reported for their broad-spectrum antiviral activity against viruses belonging to the Paramyxoviridae family, including human parainfluenza virus type 3 and Nipah virus. Among them, the peptide with a 24-unit PEG linker connecting it to a cholesterol moiety (VG-PEG24-Chol) was found to be the best membrane fusion inhibitory peptide. Here, we evaluated the interaction of the same set of peptides with biomembrane model systems and isolated human peripheral blood mononuclear cells (PBMC). VG-PEG24-Chol showed the highest insertion rate and it was among the peptides that induced a larger change on the surface pressure of cholesterol rich membranes. This peptide also displayed a high affinity towards PBMC membranes. These data provide new information about the dynamics of peptide-membrane interactions of a specific group of antiviral peptides, known for their potential as multipotent paramyxovirus antivirals.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
OE
Número da atribuição
SFRH/BD/95624/2013