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 [4,5,6,7,8].

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 [10,11,12]. 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 [13,14,15].

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 [16,17,18]. 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 Tat^48–60 (residues 48–60, GRKKRRQRRRPPQ) and penetratin (Antp^43–58, 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 [19,20]. 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 [21,22,23,24]. 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 [28,29,30,31].

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 [19,20,32]. 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 [32–36].

2. Cell-Penetrating Peptides from Animal Venoms and Toxins

To date, native venom peptides with cell-penetrating properties have been identified in a limited number of species of insects (bees and wasps), arachnids (spiders and scorpions), fish, 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. 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 (Table 1).

Table 1. Poisonous and venomous organisms that produce toxins with cell penetrability that originate cell-penetrating peptides

Scientific name	Common name	Toxin or toxin template	Obs.
Insects			Table 2, below
Apis mellifera	European honey bee	Melittin
Anoplius samariensis	Japanese solitary wasp	Anoplin
Vespula lewisii	Korean leather-jacket	Mastoparan
Arachnids			Table 3
Lachesana tarabevi	Central Asian spider	Latarcin-1
Lycosa singorensis	Wolf spider	Lycosin-1
Leiurus quinquestriatus	Israeli yellow scorpion	Chlorotoxin
Scorpio maurus palmatus	Tunisian scorpion	Maurocalcine
Pandinus imperator	African scorpion	Imperatoxin-A
Hadrurus gertschi	Mexican scorpion	Hadrucalcin
Urodacus manicatus	Australian Black Rock scorpion	Wasabi receptor toxin
Fish
Pardachirus sp	Mole sole fishes	Pardaxin	Table 4
Amphibian			Table 4
Bombina bombina	European fire-bellied toad	Bombesin
Snakes			Table 4
Crotalus durissus terrificus	South American rattlesnake	crotamine
		crotalicidin
Naja sp	Cobras	Cardiotoxin

 

The importance of this 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. Importantly, native animal venom peptides and toxins with cell-penetrating activity have been characterized and CPPs derived from them conceived through a variety of structural modifications of their native sequences. These structural modifications of cell-penetrating venom peptides include peptide downsizing, amino acid residue replacement, structural stabilization and production of chimeras, which mostly reduced peptide cytotoxicity, maintained the target selectivity and enhanced the cellular penetrability and the capacity of intracellular cargo transport and delivery.

Tables 2 to 4 summarize examples of known venom peptides, toxins and derivatives with cell-penetrating properties, their overall structures, mode of cell entry, and conveyed compounds.

Table 2. Examples of peptides from insect (honey bee and wasp) venom and derivatives with cell-penetrating property.

Peptide	Sequence a	Size	Major Structural Features b	Mechanism(s) of Cell Penetration c	Cargo Delivery d	Ref.
Honey bee
Mellitin (MEL)	GIGAVLKVLTTGLPALISWIKRKRQQ-NH2	26	Amphipathic α-helix	DT, PF	FD	[49]
Tat/CM18 hybrid			Chimera	DT, MD, ND	CI	[52]
MEL-d(KLAKLAK)2	GIGAVLKVLTTGLPALISWIKRKRQQGGGGS-d[KLAKLAKKLAKLAK]	45	Chimera	DT (?)	AP	[53]
p5RHH	VLTTGLPALISWIRRRHRRHC	21	C-terminal fragment	Endocytosis (?)	DNA polyplexes	[54,55]
p5RWR	VLTTGLPALISWIKRKRQQRWRRRR	25	C-terminal fragment	Endocytosis (?)	DNA polyplexes	[54,55]
MT20	GIGAVLKVLTTGLPALISWI	20	Hydrophobic helix	DT, MD	DNA polyplexes	[56]
FL20	GIGAILKVLATGLPTLISWI	20	Hydrophobic helix	DT, MD	DNA polyplexes	[56]
Melittin [1–14]	GIGAVLKVLTTGLP	14	N-terminal fragment	DT	NC, LP	[57]
Wasp
Anoplin	GLLKRIKTLL-NH2	10	Amphipathic α-helix	DT, PF	FD	[60]
Anoplin dimer	(GLLKRIKTLL-NH2)2	20	C-N terminal dimer	DT, PF	-	[61]
Mastoparan	INLKALAALAKKIL-NH2	14	Amphipathic α-helix	DT, PF	-	[64]
Mastoparan-X	INWKGIAAMAKKLL-NH2	14	Amphipathic α-helix	DT, PF	-	[65]
Transportan	GWTLNSAGYLLGKINLKALAALAKKIL-NH2	27	Chimera	multiple	CI	[66]
TP10	AGYLLGKINLKALAALAKKIL-NH2	21	Shorter transportan	multiple	CI	[70,77]
TP10-5	AGYLLGKINLKKLAKL(Aib)KKIL-NH2	21	Helical stabilized	DT	FD	[71]
Transportan 9dR	GWTLNSAGYLLGKINLKALAALAKKIL(dR)9	36	Chimera	Endocytosis (?)	siRNA	[73]
Mitoparan	INLKKLAKL(Aib)KKIL-NH2	14	Mastoparan analog	DT	FD, AP	[78–80]

Notes: ^a-NH_2, amidated peptide; anoplin C-N terminal dimer, as prepared by intermolecular triazole bridge; Aib, α-aminoisobutyric acid; ^bParticular structural characteristics of native, designed or analogous peptides; C-N terminal dimer, intermolecular dimerization involving the carboxyl-terminus of one peptide and the amino-terminus of the other; ^cDT, direct translocation; PF, pore-forming; MD, membrane-disruptive cell uptake; NR, non-disruptive penetration; RM, receptor-mediated endocytosis; HI, hydrophobic insertion; multiple, more than a single mechanism of cell entry, that can involve direct translocation and endocytosis; ^dFD, fluorescent dyes; CI, diverse types of cell impermeant cargoes; AP, apoptotic peptides; NC, nanocrystals; LP, large proteins; RD, radionuclides; EZ, enzymes; IR, infrared; “-,” not applicable.

 

Table 3. Examples of peptides and derivatives from arachnid venoms with cell-penetrating and cargo delivery capacity.

Peptide	Sequence	Size	Major structural features	Mechanism(s) of cell Penetration	Cargo delivery	Ref.
Spider
Latarcin-1 (Ltc1)	SMWSGMWRRKLKKLRNALKKKLKGE	25	cationic α-helix	DT, PF	FD	[81]
Ltc1-decapeptide (LDP)	KWRRKLKKLR	10	Designed	DT	FD	[82]
LDP-NLS	KWRRKLKKLRPKKKRKV	17	Chimera	ED	FD, EZ	[83]
Lycosin-I	RKGWFKAMKSIAKFIAKEKLKEHL	24	Amphipathic α-helix	DT, ND	FD, NP	[87,88]
R-lycosin-I	Ac-RGWFRAMRSIARFIARERLREHL-amide	24	Lys-->Arg analog	ED	FD	[89]
Scorpion
Chlorotoxin (CTX)	MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR	36	ICKfold/knottin	ED	FD, RD	[91–93]
[K15R/K23R]CTX	MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR	36	Mutant analog	ED	FD	[95]
[K15R/K23R/Y29W]CTX	MCMPCFTTDHQMARRCDDCCGGRGRGKCWGPQCLCR	36	Mutant analog	ED	FD	[95]
Maurocalcine (MCa)	GDCLPHLKLCKENKDCCSKKCKRRGTNIEKRCR	33	ICK fold/knottin	DT, ND	CI, NP	[99–101]
MCaUF1–9-C	GDAbuLPHLKLC	10	Truncated, unfold	DT	FD	[105]
Imperatoxin A (IpTxa)	GDCLPHLKRCKADNDCCGKKCKRRGTNAEKRCR	33	ICK fold/knottin	n	FD	[109]
Hadrucalcin (HdCa)	SEKDCIKHLQRCRENKDCCSKKCSRRGTNPEKRCR	35	ICK fold/knottin	DT, ND	-	[110]
HadUF1−11 (H11)	SEKDAbuIKHLQR-C	12	N-terminal fragment	DT, ND, ED (?)	NP	[111]
WaTx	ASPQQAKYCYEQCNVNKVPFDQCYQMCSPLERS	33	CS-helical hairpin	DT, ND	FD	[112]

Notes: Ac, acetylated; -NH_2, amidated; Abu, Lys®Arg replacement; C-S, cysteine stabilized α-helix; DT, direct translocation; PF, pore-forming; ED, endocytosis; ND, non-disruptive penetration; RM, receptor-mediated endocytosis; HI, hydrophobic insertion; multiple, more than a single mechanism of cell entry, that can involve direct translocation and endocytosis; “n“, not informed; FD, fluorescent dyes; EZ, enzymes; NP, nanoparticles; RD, radionuclides; CI, diverse types of cell impermeant cargoes; “-“, not applicable.

 

Table 4. Examples of peptides and derivatives from fish and amphibian secretion and snake venom, with cell-penetrating and cargo delivery capacity.

Peptide	Sequence a	Size	Major Structural Features b	Mechanism(s) of Cell Penetration c	Cargo Delivery d	Ref.
Fish
Ac-Pardaxin P5	Ac-GFFALIPKIISSPLFKTLLSAVGSALSSSGDQE	33	Hydrophobic helix	DT, PF	FD	[117]
Amphibian
bombesin	EQKLGNQWAVGHLM-NH2	14	helical hairpin	RM	-	[118–120]
Tat-K3-bombesin1-14	RKKRRQRRRGGCGEQKLGNQWAVGHLM-NH2	27	Chimera	RM	RD	[124]
Snake
Crotamine	YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGSG	42	β-defensin fold	multiple	FD, CI	[138]
NrTP1	YKQCHKKGGKKGSG	14	N«C splice variant	multiple	FD	[146,147]
NrTP6	YKQSHKKGGKKGSG	14	NrTP1 CysDSer	multiple	FD, EZ	[148]
DY676-NrTP6	C-YKQSHKKGGKKGSG	15	Extra Cys		IR	[152]
CyLop-1	CRWRWKCCKK	10	Encrypted sequence	DT, ED	FD	[145]
Crotalicidin	KRFKKFFKKVKKSVKKRLKKIFKKPMVIGVTIPF	34	Amphipathic α-helix	DT, PF	FD	[176]
Ctn[15–34]	KKRLKKIFKKPMVIGVTIPF-NH2	20	C-terminal fragment	DT, PF	FD	[170,176]
cathelicidin- BF30	KFFRKLKKSVKKRAKEFFKKPRVIGVSIPF	30	Shorter analog	DT, PF	-
Cardiotoxin	LKCNKLIPLAYKTCPAGKNLCYKMFMVSNKTVPVKRGCID ACPKNSLLVKYVCCNTDRCN	60	Three-finger fold	DT, HI	FD	[179]
PLA2 C-terminal	KKYKAYFKFKCKK-NH2	13	C-terminal fragment	DT, MD	-	[185]
PLA2 fragment K®R	RRYRAYFRFRCRR-NH2	13	Mutant K-->R	DT, MD	-	[185]
BPP-13a	<EGGWPRPGEIPP	12	Native	DT (?)	-	[187]
desPyr-BPP13a	GGWPRPGPEIPP	12	Mutant analog	DT (?)	FD	[187]

Notes: ^aAc, acetylated; -NH_2, amidated; <E, pyroglutamic; DT, direct translocation; ^bN«C, a spliced derivative of crotamine, comprising the N-terminal residues 1 to 9 and C-terminal 38 to 42 ([1–9] ~ [38–42]); NrTP6, an NrTP1 with a Cys-Ser replacement; DY676, near-infrared dye; ^cDT, direct translocation; PF, pore-forming; RM, receptor-mediated endocytosis; multiple, more than a single mechanism of cell entry, that can involve direct translocation and endocytosis; ED, endocytosis; HI, hydrophobic insertion; MD, membrane-disruptive cell uptake; ^dFD, fluorescent dyes; RD, radionuclides; EZ, enzymes; IR, infrared dye; “-“ not applicable;

These designed CPPs from venom peptides have found applications in biomedicine and biopharmaceutical biotechnology, in diagnostics to detect populations of diseased cells or tissues, in therapy to induce selective cell death through molecular delivery of cell-impermeant drugs, organic compounds, radionuclides and crystal, and metallic nanoparticles. The discoveries of cell-penetrating venom peptides and animal toxins will continue to contribute to the basic and applied research in this expanding field of CPPs derived from components of poisonous and venomous animals. The cell-penetrating venom peptides and toxin derivatives have expanded the library of animal toxins that act intracellularly and have presently served and will be useful in the future, for a variety of biomedical and biotechnological applications.