It has been established that the more diverse a biome is, the more intricate is the chemical-ecological relationships among organisms sharing the same niche and richer is the diversity of molecules and pharmacological activities that these co-inhabiting organisms contain. Indeed, a great probability of success in the drug discovery process from nature is connected to maximal biodiversity and chemical diversity [1
Animal venoms and skin secretions are rich biological materials that contain a blend of biologically and pharmacologically active components, used by venomous and poisonous organisms for defense, predation and territorial disputes. The mechanisms via which these organisms deliver toxic components are related to how toxins have evolved and exerted their ecological functions [2
]. As a result of millions of years of evolution, the diversity of molecular structures and activities is immeasurable, with hidden functions and structures only recently disclosed by modern omics techniques associated with classical pharmacological studies [3
]. Thus, the holistic molecular genetic analyses of venomous animals utilizing, for example, transcriptome in combination with proteome studies of venom glands, have revealed not only the diversity of a set of substances produced by a given organism that inhabits a particular biome but also the uniqueness of such molecular repertoire expressed as active components of biological materials, such as venoms [4
Although marine and terrestrial biological reservoirs of active venom peptides appear crucial sources for drug discovery, examples of basic and applied research should be provided to attract the attention of the scientific community both in academia and industry. Fundamental research, development and innovation of venom components as diagnostics, therapeutics or both are not always an obvious issue for consideration for the general audience. Furthermore, molecular diversity is intrinsically related to biodiversity and pharmaceutical success in drug discovery from nature is dependent on the abundance and variety of biological resources, thus highlighting arguments for policies of environmental conservation and economic sustainability worldwide [9
A particular class of molecules from animal venom that has been under constant focus comprises biologically active (bioactive) peptides. Owing to their selectivity for corresponding cell receptors and target specificity, as well as the relative structural stability in body fluids and amenability to be genetically and synthetically engineered, natural venom peptides are promising scaffolds for conversion into biopharmaceuticals (biotherapeutics), as exemplified by cysteine-stabilized and linear helical peptides [10
]. In this review, I discussed natural peptides from animal venom and their derivatives possessing intrinsic properties such as interaction with biomembranes of target cells, access to the cytoplasm through membrane translocation and eventual accumulation in distinct organelles such as the nucleus and mitochondria. This class of special peptides, collectively called cell-penetrating peptides (CPPs), comprise full sequences, peptide segments or patches of large proteins and even encrypted subcellular localization signals, as well as rationally designed peptide chimeras, cyclic, stapled, dimeric, multivalent and self-assembled peptides and peptoid foldamers [13
In the field of toxicology, membrane translocation and cell-penetration capabilities are well-recognized phenomena mainly associated with microbial and plant toxins, such as binary bacterial toxins (e.g., diphtheria toxin, Shiga toxin and cholera toxin) and plant ribosome inhibitor proteins (e.g., ricin and abrin), which bind to their respective receptors on the eukaryotic cytoplasmic membrane and enter cells by receptor-mediated endocytosis [16
]. Less common but with a steeply increasing number of reports published over the years, animal toxins and venom peptides endowed with intrinsic properties of membrane permeation and cell internalization are currently being unraveled. These venom CPPs and derivatives, recapitulated herein, mechanistically penetrate cells by distinct pathways that involve receptor-dependent and/or receptor-independent endocytosis (non-disruptive peptides) or direct translocation through pore formation, followed by cellular uptake and intracellular compartmentalization (disruptive peptides). Hence, in this review, I collated current examples of cell-penetrating peptides derived from animal venoms and toxins, their basic research and advanced applications.
Cell-penetrating peptides (CPPs) consist of short sequences that range from few amino acids to less than 40 residues, which owing to their physicochemical and biological properties can cross lipid membranes of cells and intracellularly transport diverse types of molecular cargoes in the form of covalent conjugates or noncovalent complexes. The first CPP sequences were characterized and derived from the transactivator of transcription from HIV-1 (Tat protein), the antennapedia (Antp) homeodomain from Drosophila and
VP22 from Herpes simplex virus type I, almost three decades ago. For instance, CPPs such as Tat48–60
(residues 48–60, GRKKRRQRRRPPQ) and penetratin (Antp43–58
, RQIKIWFQNRRMKWKK), which are short-derived fragments from TAT protein and Drosophila
Antp, respectively, were able to translocate across the plasma membrane of eukaryotic cells and set the stage for the development of a new class of non-viral vectors for the intracellular delivery of compounds and, therefore, converted into archetypal CPPs [19
]. Since these initial discoveries, the field of CPPs has expanded into a prolific field of research. Databases of curated information about experimentally characterized CPPs and algorithms to predict CPPs have been created, along with the expansion of the field of CPPs [21
]. It has become evident that CPPs are mostly linear sequences but cyclic sequences have also been detected and developed. They are usually composed of positively charged amino acids with or without distributed hydrophobic residues, conferring these peptide structures with a variable but a considerable level of amphipathic and cationicity and high affinity for negatively charged lipid membranes and their components, including phospholipids and proteoglycans. In particular, in the case of linear CPPs, studies investigating the relationship between CPP secondary structures and penetrability have demonstrated that cationic helical peptides possess superior cell permeation than amphipathic helical and amphipathic random peptides, as revealed through synthetic polyarginine CPPs stabilized with different proportion of α-aminoisobutyric acid [25
]. Nevertheless, unstructured peptides in solution eventually form helices upon interaction with plasma membranes, promoting their efficient membrane insertion, translocation and cellular uptake [26
]. Hydrophobic interaction followed by membrane insertion also plays a role in the cell penetration property demonstrated by some amphipathic CPPs [27
]. Importantly, from the perspective of applications in biomedicine and pharmaceutical biotechnology, CPPs can simultaneously mediate the intracellular delivery of diverse molecular cargos, along with the membrane-crossing activity. These molecular cargoes include hydrophilic drugs, radionuclides, imaging agents (fluorescent dyes), biopolymers (nucleic acids, polypeptides), functionalized liposomes and nanoparticles (nanocrystals, light-sensitive and magnetic nanoparticles). Therefore, CPPs have attracted considerable attention not only for their application as vectors for drug delivery but also as agents for diagnostics and therapy (theranostics) in medicine [28
Regarding their mechanistic of cell penetrability, although not conclusively elucidated, it has been shown that CPPs traverse hydrophobic membranes and enter into cells through distinct routes, relying on both membrane-disruptive and non-disruptive processes, this later comprising energy-dependent, receptor-mediated and receptor-independent endocytosis, as well as energy independent, direct translocation strategies. Hence, pathways for CPP entry into cells involve: (I) non-disruptive endocytic routes, such as clathrin-mediated endocytosis, caveolae-mediated uptake, clathrin/caveolae-independent endocytosis and micropinocytosis; (II) non-disruptive membrane, direct translocation by alternative routes, like inverted micelle formation and “carpet” model penetration; (III) direct translocation by membrane-disruptive routes, such as pore-formation (toroid and barrel-stave pores) and electroporation-like permeabilization. Moreover, based on experimental evidence with numerous known CPPs, more than a single pathway of membrane permeation and cellular internalization may be involved, depending on the CPP physicochemical features, effective concentrations (i.e., the peptide-to-cell ratio) and targeted cell types [19
]. Although distinct pathways are involved in membrane translocation and internalization of several distinct CPPs investigated, the mode of membrane interaction, the efficacy of CPP-mediated cargo-dependent transport, subcellular compartmentalization and eventual adverse cytotoxic effects, depend not only on their own CPP structures but also on the position and nature of the molecular cargoes and the types of cell targets, as observed with CPPs that were conjugated with fluorescent dyes and polypeptides [32
presents examples of CPP archetypes, their sequences, origins and physicochemical and structural attributes.
Interestingly, most structural and physicochemical features of CPPs can be observed in members of another class of bioactive peptides, termed antimicrobial peptides (AMPs), which are ubiquitously found in cells, tissues and biological fluids of organisms from diverse kingdoms, including venoms of poisonous animals [37
]. Indeed, based on their structural characteristics, AMPs are membrane-active peptides that possess the ability to disrupt the plasma membrane integrity of their respective cellular targets as their principal mechanism of action, and penetrate target cells through pores [38
]. The cell penetrability of AMPs appears to reinforce their antimicrobial efficacy owing to the interaction and interference with intracellular components, including macromolecules and organelles [40
]. In this case, cellular uptake and permeability of AMPs can be associated with variable levels of cytotoxicity. However, the design and synthesis of AMPs can provide engineered peptides with tuned membrane translocation and cellular uptake capabilities, combined with lower or absent detrimental effects on membrane stability and cell viability, as exemplified by buforin II from the stomach tissue of the Asian toad Bufo bufo garagrizans
and derivates [41
] and other AMPs [42
], including some presented in this article, in following sections.
To date, native venom peptides with cell-penetrating properties have been identified in a limited number of species of insects (bees and wasps), arachnids (spider and scorpions), fishes, amphibians and snakes (elapids and pit vipers). These CPPs have dual or multiple biological activities and are derived from the venoms or skin secretions, which are toxigenic, cytotoxic or antimicrobials. Thus, in this review, the origins, basic research and advances regarding these venom peptides and related sequences, as well as their original single, dual or multiple biological activities, including cell-penetrating properties, are presented and discussed. These venom CPPs include melittin from the honeybee, anoplin and mastoparans from wasps, latarcin and lycosin from spiders, chlorotoxin, maurocalcine and imperatoxin from scorpions, cardiotoxin, crotamine, crotalicidin and elapid cathelicidin-related antimicrobial peptides (CRAMPs) from snakes, pardaxin from fish skin and bombesin from frog skin, all presented in following sections. The importance of this theme and class of bioactive peptides relies on the fact that venom peptides and toxins, as well as related sequences, have evolved for millions of years of natural history and can be considered crucial improvements in peptide design that efficiently performs their functions. Thus, recombinant products, peptide chimeras and synthetic analogs can be engineered and prepared for biomedical applications, as well as potentially converted into biopharmaceuticals.