Ana Salomé Veiga

Anticancer β-Hairpin Peptides: Membrane-Induced Folding Triggers Activity

Several cationic antimicrobial peptides (AMPs) have recently been shown to display anticancer activity via a mechanism that usually entails the disruption of cancer cell membranes. In this work, we designed an 18-residue anticancer peptide, SVS-1, whose mechanism of action is designed to take advantage of the aberrant lipid composition presented on the outer leaflet of cancer cell membranes, which makes the surface of these cells electronegative relative to the surface of noncancerous cells. SVS-1 is designed to remain unfolded and inactive in aqueous solution but to preferentially fold at the surface of cancer cells, adopting an amphiphilic β-hairpin structure capable of membrane disruption. Membrane-induced folding is driven by electrostatic interaction between the peptide and the negatively charged membrane surface of cancer cells. SVS-1 is active against a variety of cancer cell lines such as A549 (lung carcinoma), KB (epidermal carcinoma), MCF-7 (breast carcinoma), and MDA-MB-436 (breast carcinoma). However, the cytotoxicity toward noncancerous cells having typical membrane compositions, such as HUVEC and erythrocytes, is low. CD spectroscopy, appropriately designed peptide controls, cell-based studies, liposome leakage assays, and electron microscopy support the intended mechanism of action, which leads to preferential killing of cancerous cells.

Cell‐penetrating peptides: A tool for effective delivery in gene‐targeted therapies

The current landscapes of novel therapeutic approaches rely mostly on gene‐targeted technologies, enabling to fight rare genomic diseases, from infections to cancer and hereditary diseases. Although, reaching the action‐site for this novel treatments requires to deliver nucleic acids, or other macromolecules into cells, which may pose difficult tasks to pharmaceutical companies. To overcome this technological limitation, a wide variety of vectors have been developed in the past decades and have proven to be successful in delivering various therapeutics. Cell‐penetrating peptides (CPP) have been one of the technologies widely studied and have been increasingly used to transport small RNA/DNA, plasmids, antibodies, and nanoparticles into cells. Despite the already proved huge potential that these peptide‐based approaches may suggest, few advances have been put to pharmacological or clinical use. This review will describe the origin, development, and usage of CPP to deliver therapeutic agents into cells, with special emphasis on their current application to gene‐therapies. Specifically, we will describe the current trials being conducted to treat cancer, gene disorders, and autoimmune diseases using CPP‐based therapies.

Interactions of HIV-1 antibodies 2F5 and 4E10 with a gp41 epitope prebound to host and viral membrane model systems

Point of recognition: Surface plasmon resonance was used to study the role of the viral membrane in HIV immunogen recognition by the monoclonal antibodies 2F5 and 4E10. The different behaviour of the antibodies towards membranes and immunogens embedded within membranes, provide insight into the emerging membrane‐based or membrane‐conformation strategies of immunogen design.

Anti-HIV-1 antibodies 2F5 and 4E10 interact differently with lipids to bind their epitopes

Objectives:2F5 and 4E10 are two broadly neutralizing monoclonal antibodies (mAbs) targeting the membrane proximal external region (MPER) of HIV-1 gp41 envelope protein. This region, which contacts the viral membrane, is highly conserved and has been regarded as a promising target for vaccine development. We aimed to clarify the basis of 2F5 and 4E10 molecular interactions with epitope cores in MPER and lipid bilayers. Design:Microscopy-based approaches were used to infer and quantify the effects of both mAbs on membranes, in the presence and absence of the epitope cores. Supported lipid bilayers (SLBs), with and without phase separation, were used as membrane models. Fluorescent-labeled and nonlabeled MPER-derived peptides containing both 2F5 and 4E10 epitopes were used. Methods:mAbs 2F5 and 4E10 membrane interactions, in the presence or absence of MPER-derived peptides, were evaluated by combined atomic force and confocal microscopies. Results:Both mAbs form lipid-segregated aggregates on SLBs and do not induce other significant membrane perturbations. Furthermore, the affinity of MPER toward membranes is differently affected by both mAbs and correlates with the mAbs–epitope core lipid interactions. 2F5 is able to dock the MPER peptide on the membrane, whereas 4E10 extracts the MPER from the lipid bilayer. Conclusion:The results reveal the molecular details underneath 2F5/4E10 membrane-epitope binding and a model is proposed to explain the differential mAbs neutralization efficacies, which relates to the exposure of the epitopes in the lipid bilayers and the role of the lipids in mAb–epitope binding.

Monitoring antibacterial permeabilization in real time using time-resolved flow cytometry

Despite the intensive study of antibiotic-induced bacterial permeabilization, its kinetics and molecular mechanism remain largely elusive. A new methodology that extends the concept of the live-dead assay in flow cytometry to real time-resolved detection was used to overcome these limitations. The antimicrobial activity of pepR was monitored in time-resolved flow cytometry for three bacterial strains: Escherichia coli (ATCC 25922), E. coli K-12 (CGSC Strain 4401) and E. coli JW3596-1 (CGSC Strain 11805). The latter strain has truncated lipopolysaccharides (LPS) in the outer membrane. This new methodology provided information on the efficacy of the antibiotics and sheds light on their mode of action at membrane-level. Kinetic data regarding antibiotic binding and lytic action were retrieved. Membrane interaction and permeabilization events differ significantly among strains. The truncation of LPS moieties does not hamper AMP binding but compromises membrane disruption and bacterial killing. We demonstrated the usefulness of time-resolved flow cytometry to study antimicrobial-induced permeabilization by collecting kinetic data that contribute to characterize the action of antibiotics directly on bacteria.

Nucleic acid delivery by cell penetrating peptides derived from dengue virus capsid protein : design and mechanism of action

Cell penetrating peptides (CPPs) can be used as drug delivery systems for different therapeutic molecules. In this work two novel CPPs, pepR and pepM, designed from two domains of the dengue virus (DENV) capsid protein, were studied for their ability to deliver nucleic acids into cells as non‐covalently bound cargo. Translocation studies were performed by confocal microscopy in HepG2, BHK and HEK cell lineages, astrocytes and peripheral blood mononuclear cells. Combined studies in HepG2 cells, astrocytes and BHK cells, at 4 and 37 °C or using specific endocytosis inhibitors, revealed that pepR and pepM use distinct internalization routes: pepM translocates lipid membranes directly, while pepR uses an endocytic pathway. To confirm these results, a methodology was developed to monitor the translocation kinetics of both peptides by real‐time flow cytometry. Kinetic constants were determined, and the amount of nucleic acids delivered was estimated. Additional studies were performed in order to understand the molecular bases of the peptide‐mediated translocation. Peptide–nucleic acid and peptide–lipid membrane interactions were studied quantitatively based on the intrinsic fluorescence of the peptides. pepR and pepM bound ssDNA to the same extent. Partition studies revealed that both peptides bind preferentially to anionic lipid membranes, adopting an α‐helical conformation. However, fluorescence quenching studies suggest that pepM is deeply inserted into the lipid bilayer, in contrast with pepR. Moreover, only pepM is able to promote the fusion and aggregation of vesicles composed of zwitterionic lipids. Altogether, the results show that DENV capsid protein derived peptides serve as good templates for novel CPP‐based nucleic acid delivery strategies, defining different routes for cell entry.

Intracellular Nucleic Acid Delivery by the Supercharged Dengue Virus Capsid Protein

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.

Rethinking the capsid proteins of enveloped viruses: multifunctionality from genome packaging to genome transfection

Regardless of the debate on whether there is a place for viruses in the tree of life, it is consensual that they co‐evolve with their hosts under the pressure of genome minimization. The abundance of multifunctional viral structural proteins is a consequence of this pressure. The molecular key to multifunctionality is the existence of intrinsically disordered domains together with ordered domains in the same protein. Capsid proteins, the hallmark of viruses, are not exceptions because they have coexisting ordered and disordered domains that are crucial for multifunctionality. It is also frequent to find supercharged proteins (i.e. proteins for which the net charge per unit molecular mass is > +0.75/kDa) among viral capsid proteins. All flaviviruses having annotated proteins in the ExPASy Viralzone database have supercharged capsid proteins. Moreover, cell‐penetrating sequences/domains are frequent in viral proteins, even when they are not supercharged. Altogether, the findings strongly suggest that the ability to translocate membranes was acquired, conserved and optimized throughout the evolution of some viral proteins as part of their multifunctionality. The fitness of capsid proteins to translocate membranes carrying genomes was experimentally demonstrated with dengue virus capsid protein. This protein is potentially able to help the fusion process and translocate the RNA genome across the hemifused membrane formed by the viral envelope and the endosomal membrane. In addition, one of the cell‐penetrating domains of the capsid protein also has antibacterial activity. This may be reminiscent of parasitic bacteria–bacteria competition for the same host and shed light on the origins of enveloped viruses.

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

Despite the intensive study on the mechanism of action of membrane‐active molecules 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 biophysical methodologies 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. Copyright

An Insight on the Leading HIV Entry Inhibitors

The main strategies nowadays to fight AIDS rely on chemical therapy to inhibit the reverse transcriptase or protease of HIV. However, a synthetic 36 amino-acids peptide that blocks the entry of the virus in the target cells (enfuvirtide) has recently reached approval for clinical application. This molecule may probably be just the leader of a new generation of drugs that is about to emerge to interrupt the first step in the HIV life cycle, i.e. preventing the virus from actually entering cells. This paper reviews the enfuvirtide path from clinical trials to the attempts to detail its molecular-level mode of action. It is commonly accepted that this peptide would block the fusion between viral and cell plasma membrane through binding to the N-terminal heptad repeat (NHR) region of the viral protein gp41. However, there has been growing evidence that this model of action may be unrealistic, the action of enfuvirtide being more complex and diverse than initially thought. Membrane-assisted local concentration increase and interference with gp120/co-receptor docking may also contribute for the inhibitory action of the peptide. Selected HIV-entry inhibitors on clinical trials are presented to characterize the future drugs in the market in this class.