STUDYING AND DEVELOPING ANTIMICROBIAL PEPTIDE HYDROGELS USED IN TISSUE REGENERATION

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The antimetastatic breast cancer activity of the viral protein‐derived peptide vCPP2319 as revealed by cellular biomechanics

Publication . Oliveira, Filipa; Cavaco, Marco; Figueira, Tiago Nascimento; Valle, Javier; Neves, Vera; Andreu, David; Gaspar, Diana; Castanho, Miguel A. R. B.

The incidence of metastatic breast cancer (MBC) is increasing and the therapeutic arsenal available to fight it is insufficient. Brain metastases, in particular, represent a major challenge for chemotherapy as the impermeable nature of the blood–brain barrier (BBB) prevents most drugs from targeting cells in the brain. For their ability to transpose biological membranes and transport a broad spectrum of bioactive cargoes, cell-penetrating peptides (CPPs) have been hailed as ideal candidates to deliver drugs across biological barriers. A more ambitious approach is to have the CPP as a drug itself, capable of both killing cancer cells and interacting with the blood/brain interface, therefore blocking the onset of brain metastases. vCPP2319, a viral protein-derived CPP, has both properties as it: (a) is selective toward human breast cancer cells (MDA-MB-231) and increases cell stiffness compared to breast epithelial cells (MCF 10A) hindering the progression of metastases; and (b) adsorbs at the surface of human brain endothelial cells potentially counteracting metastatic cells from reaching the brain. Overall, the results reveal the selective anticancer activity of the peptide vCPP2319, which is also able to reside at the blood–brain interface, therefore counteracting brain penetration by metastatic cancer cells.

2021Journal article

Open access

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

Publication . Freire, João M.; Gaspar, Diana; Veiga, Ana Salomé; Castanho, Miguel A. R. B.

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.

2015Journal article

Restricted access

Structural studies of a lipid-binding peptide from tunicate hemocytes with anti-biofilm activity

Publication . Silva, Osmar N.; Alves, Eliane S. F.; Fuente-Núñez, César de la; Ribeiro, Suzana M.; Mandal, Santi M.; Gaspar, Diana; Veiga, Ana S.; Castanho, Miguel A. R. B.; Andrade, Cesar A. S.; Nascimento, Jessica M.; Fensterseifer, Isabel C. M.; Porto, William F.; Correa, Jose R.; Hancock, Robert. E. W.; Korpole, Suresh Korpole; Oliveira, Aline L.; Liao, Luciano M.; Franco, Octavio L.

Clavanins is a class of peptides (23aa) histidine-rich, free of post-translational modifications. Clavanins have been studied largely for their ability to disrupt bacterial membranes. In the present study, the interaction of clavanin A with membranes was assessed by dynamic light scattering, zeta potential and permeabilization assays. We observed through those assays that clavanin A lysis bacterial cells at concentrations corresponding to its MIC. Further, the structure and function of clavanin A was investigated. To better understand how clavanin interacted with bacteria, its NMR structure was elucidated. The solution state NMR structure of clavanin A in the presence of TFE-d3 indicated an α-helical conformation. Secondary structures, based on circular dichroism measurements in anionic sodium dodecyl sulfate (SDS) and TFE (2,2,2-trifluorethanol), in silico lipid-peptide docking and molecular simulations with lipids DPPC and DOPC revealed that clavanin A can adopt a variety of folds, possibly influencing its different functions. Microcalorimetry assays revealed that clavanin A was capable of discriminating between different lipids. Finally, clavanin A was found to eradicate bacterial biofilms representing a previously unrecognized function.

2016Journal article

Open access

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

Publication . Freire, João Miguel; Gaspar, Diana; Torre, Beatriz Garcia de la; Veiga, Ana Salomé; Andreu, David; Castanho, Miguel A. R. B.

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 atmembrane-level. Kinetic data regarding antibiotic binding and lytic actionwere 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.

2015Journal article

Restricted access