Biophysical studies tounravel the mechanism of action of anticancer peptides

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Synthesis, structure, and activity of the antifungal plant defensin PvD1

Publication . Skalska, Julia; Andrade, Vitor M.; Cena, Gabrielle L.; Harvey, Peta J.; Gaspar, Diana; Mello, Érica O.; Henriques, Sónia T.; Valle, Javier; Gomes, Valdirene M.; Conceição, Katia; Castanho, Miguel A. R. B.; Andreu, David

Available treatments for invasive fungal infections have limitations, including toxicity and the emergence of resistant strains. Therefore, there is an urgent need for alternative solutions. Because of their unique mode of action and high selectivity, plant defensins (PDs) are worthy therapeutic candidates. Chemical synthesis remains a preferred method for the production of many peptide-based therapeutics. Given the relatively long sequence of PDs, as well as their complicated posttranslational modifications, the synthetic route can be considered challenging. Here, we describe a total synthesis of PvD1, the defensin from the common bean Phaseolus vulgaris. Analytical, structural, and functional characterization revealed that both natural and synthetic peptides fold into a canonical CSαβ motif stabilized by conserved disulfide bonds. Moreover, synthetic PvD1 retained the biological activity against four different Candida species and showed no toxicity in vivo. Adding the high resistance of synthetic PvD1 to proteolytic degradation, we claim that conditions are now met to consider PDs druggable biologicals.

2020Artigo científico

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Biophysical and functional studies to unravel the biomedical potential of plant defensin PvD1

Publication . Skalska, Julia; Castanho, Miguel Rico Botas

Due to the unique mode of action and high selectivity, plant defensins (PDs) constitute a group of therapeutic candidates worthy of note. PDs are involved in the first-line defense system in plants having strong antimicrobial action on a wide variety of pathogens. Numerous studies highlighted the activity of several PDs against human infectious pathogens including resistant species of bacteria and viruses, and even cancer. This remarkable set of biological activities is additionally enriched by the low toxicity of healthy mammalian cells. This set of notable facets makes plant defensins interesting pharmaceutical candidates for further development. However, entry to the clinical pipeline requires a thorough characterization of the mode of action of a drug lead. Despite a reasonable understanding of the mode of action of plant defensins on microorganisms, there are very few reports elaborating on mechanisms in which PDs exert a harmful effect on cancer cells. To this end, the major pitfall concerns the production of PDs in quantities enabling detailed investigation on relevant in vitro and in vivo models. Chemical synthesis is the most frequently used method in the pharmaceutical manufactory. Nevertheless, several concerns discourage using this technique to produce more “complicated” peptides, such as plant defensins. PDs have a long sequence containing multiple cysteine residues. These residues are involved in intramolecular covalent disulfide bonds thus regulate peptide’s 3D structure. Consequently, due to the highly knotted folding, PDs have been mostly obtained through isolation from the plant extract or by heterologous expression systems. Natural peptide PvD1 is a representative of plant defensins family originating from common bean (Phaseolus vulgaris) from Brazil. This peptide shares common structural facets with other members of PDs such as the canonical CSαβ motif. PvD1 shows strong antifungal activity, yet it has been also shown to have antiprotozoal and anticancer properties, concurrently presenting a low toxicity profile on healthy human cells. Such a remarkable multifunctional action of this peptide calls for deepening the understanding of PvD1’s mode of action to further explore its medical application. Vibrant communication between cancer cells regulates their growth, development, and progression. This communication can be mediated by various signalling pathways enabling the contact between cells and extracellular matrix (ECM). One such pathway is tumour-derived exosomes (TDE). These are nano-sized vesicles that originated from the endosomal membrane and secreted by cells. As TDE can transmit genetic material and proteins they play a regulatory function in intracellular crosstalk. Moreover, they have been found to alleviate cancer survival mechanisms, such as multi-drug resistance. Hence, the development of the anticancer agent that could target TDE would envision an interesting strategy for tumour eradication. This first part of the project aimed to target the production of exosomes by breast cancer cells with natural PvD1 peptide. Here we focused on the modulatory effect of PvD1 on the expression level of CD63 and CD9 tetraspanin proteins in TDE. These proteins play an important role in controlling the formation of exosomes and are enriched in exosomal membranes. Additionally, the interaction of PvD1 with various biological membranes was followed by the combination of tailored biophysical techniques including dynamic light scattering (DLS), atomic force microscopy (AFM), and surface plasmon resonance (SPR). The innovative approach of immobilizing exosomes on the surface of the SPR sensor chip revealed that PvD1 can bind to these vesicles. Furthermore, a semi-quantitative analysis of the peptide-membrane interaction in TDE confirmed that, after binding PvD1, remains inserted and resides in the exosomal membrane. In contrast to exosomes, PvD1 does not cause perturbations of the membrane of cancer cells, presumably translocating inside the cell and targeting intracellular functions. Within this work, we contributed to broadening the understanding of the anticancer action of PvD1 and envision an innovative strategy for treating cancer. The advent of solid-phase chemical synthesis (SPPS) has enabled the easy production of pharmaceutical peptides. In addition, SPPS provides the tools for endless sequence manipulation to improve peptides’ properties or facilitate research. These comprise terminal modifications such as methylation or lipidation, introduction/deletion of amino acids, or labelling with fluorophores. Therefore, the goal of the second part of this work was to reproduce synthetically natural peptide PvD1. Analytical and structural analyses were employed in order to compare natural peptide with a synthetically obtained replica. Besides, solution-phase nuclear magnetic resonance (NMR) allowed elucidation of PvD1's structure, yet, predicted only by homology with defensin VrD2. Overlapping spectra of these two peptides and equal RP-HPLC elution time, provide strong evidence suggesting an identical structure. Moreover, to verify biological activity assays were performed on a collection of Candida species. It has been shown by in vitro and in vivo tests that both natural and synthetic PvD1 have expected antifungal activity, determined by the presence of glucosylceramide (GlcCer) in fungal membranes. Additionally, thanks to multiple disulfide bridges, tightly stabilizing the 3D structure, PvD1 preserves high resistance to proteolytic degradation. Overall, this work elaborates on the multifunctional activity of PvD1 peptide revealing a novel mechanism of anticancer action of plant defensin as well as strong antifungal properties. Adding susceptibility of this peptide to the chemical synthesis production method poses a valuable strategy for medical application either as a therapy alone or as a co-adjuvant in conventional treatments.

2021-05Tese de doutoramento

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Entidade financiadora

Fundação para a Ciência e a Tecnologia

Número da atribuição

PD/BD/114177/2016