Biophysical and biological properties of small linear peptides derived from crotamine, a cationic antimicrobial/antitumoral toxin with cell penetrating and cargo delivery abilities

Abstract

Crotamine is a natural polypeptide from snake venom which delivers nucleic acid molecules into cells, besides having pronounced affinity for negatively charged membranes and antifungal activity. We previously demonstrated that crotamine derived short linear peptides were not very effective as antifungal, although the non-structured recombinant crotamine was overridingly more potent compared to the native structured crotamine. Aiming to identify the features necessary for the antifungal activity of crotamine, two linear short peptides, each comprising half of the total positively charged amino acid residues of the full-length crotamine were evaluated here to show that these linear peptides keep the ability to interact with lipid membrane model systems with different phospholipid compositions, even after forming complexes with DNA. Interestingly, the presence of cysteine residues in the structure of these linear peptides highly influenced the antifungal activity, which was not associated to the lipid membrane lytic activity. In addition to the importance of the positive charges, the crucial role of cysteine residues was noticed for these linear analogs of crotamine, although the tridimensional structure and lipid membrane lytic activity observed only for native crotamine was not essential for the antifungal activity. As these peptides still keep the ability to form complexes with DNA molecules with no prejudice to their ability to bind to lipid membranes, they may be potentially advantageous as membrane translocation vector, as they do not show lipid membrane lytic activity and may harbor or not antifungal activity, by keeping or not the semi-essential amino acid cysteine in their sequence.

Effects of the Natural Peptide Crotamine from a South American Rattlesnake on Candida auris, an Emergent Multidrug Antifungal Resistant Human Pathogen

“…The action of crotamine on mitochondria was also previously demonstrated by us [13], and phenolic compounds such as flavonoids, when combined with FLC, were demonstrated to show activity against Candida tropicalis strains resistant to FLC, by promoting mitochondrial depolarization, apoptosis, and exposure of phosphatidylserine in the plasma membrane [39]. Interestingly, crotamine is also able to promote mitochondrial depolarization and apoptosis [12,13], besides having an affinity for negatively charged lipids such as phosphatidylserine [19,38]. Furthermore, although the lipid-membrane-disrupting activity of crotamine and its shorter derived peptides was previously demonstrated by us [19,38], at this point we cannot simply come to the conclusion that the negative charges in the cell membrane of FLC-resistant strains could be playing a role in the candicidal effect against the C. auris resistant strains.…”

“…Crotamine is a well-characterized polypeptide with multiple biological activities [37], including the in vitro activity against a selected panel of Candida species [23]. Crotamine is also a recognized member of the antimicrobial peptide (AMPs) class whose members are efficient in killing most microbes, and for which the development of resistance is rare, mainly due to the characteristic mechanism(s) of action based on the rapid interaction with and disruption of lipid cell membranes [15,19,38]. AMPs represent therefore a powerful drug candidate with reduced risk of resistance development and potential reduction in the duration of treatment [38].…”

“…Interestingly, crotamine is also able to promote mitochondrial depolarization and apoptosis [12,13], besides having an affinity for negatively charged lipids such as phosphatidylserine [19,38]. Furthermore, although the lipid-membrane-disrupting activity of crotamine and its shorter derived peptides was previously demonstrated by us [19,38], at this point we cannot simply come to the conclusion that the negative charges in the cell membrane of FLC-resistant strains could be playing a role in the candicidal effect against the C. auris resistant strains. In addition, the low ability of native crotamine in inhibiting the closely related C. haemulonii resistant clinical isolates also deserves special attention, and further studies are planned to clarify this selective activity of native crotamine only against the C. auris clinical strains.…”

Long term safety of targeted internalization of cell penetrating peptide crotamine into renal proximal tubular epithelial cells in vivo

“…The mechanism by which crotamine or crotamine-DNA complexes enter tumor cells involves the binding to cell surface heparan sulfate proteoglycans (HSPGs), which is followed by endocytosis 18 . The ability of crotamine to permeabilize endosomal/lysosomal vesicles confers an additional and unique advantage for this polypeptide, as gene nanocarrier 18–20,22 . Furthermore, crotamine contains a nuclear retention motif, which after liberation from endosomal/lysosomal vesicles, guides crotamine to the nucleus of transfected cells 23,24 .…”

New generation CPPs show distinct selectivity for cancer and noncancer cells

“…This peptide is similar to the nucleolar targeting peptide NrTP6, which as the name indicates, accumulates preferentially in the nucleoli of the cells. In addition to cell-penetrating and selective antifungal and antitumoral properties, 9 crotamine exhibits potent antiplasmodial activity. Hence, we term our sequence Cys-[4-Ser-crotamine(Δ10-37)] as mini-crotamine and abbreviate this in the figures as mini-crot.…”