Peptide name : Crotamine

Source/Organism : South American rattlesnake

Linear/Cyclic : Linear

Chirality : Not found

Peptide length: 65

C-terminal modification: Linear

N-terminal modification : Not found

Non-natural peptide information: None

Assay type : MTT assay

Assay time : 24h

Activity : IC50 : 5 μM

Cell line : CHO-K1

Cancer type : Not specified

Other activity : Not found

Amino acid composition bar chart :

Molecular mass : 7518.9835 Dalton

Aliphatic index : 0.586

Instability index : 61.8846

Hydrophobicity (GRAVY) : -0.293

Isoelectric point : 9.5391

Charge (pH 7) : 8.6102

Aromaticity : 0.153

Molar extinction coefficient (cysteine, cystine): (13980, 14355)

Hydrophobic/hydrophilic ratio : 1.40740740

hydrophobic moment : 0.4788

Missing amino acid : T,V

Most occurring amino acid : K

Most occurring amino acid frequency : 11

Least occurring amino acid : N

Least occurring amino acid frequency : 1

Secondary structure fraction (Helix, Turn, Sheet): (0.3, 0.2, 0.2)

SMILES Notation: CC[C@H](C)[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCSC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)O)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H](Cc1c[nH]cn1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)NCC(=O)N[C@@H](Cc1c[nH]cn1)C(=O)N[C@@H](CS)C(=O)N[C@@H](Cc1ccccc1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](Cc1ccccc1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)O)[C@@H](C)CC

1 : Fadel V, et al. Automated NMR structure determination and disulfide bond identification of the myotoxin crotamine from Crotalus durissus terrificus. Toxicon. 2005; 46:759-67. doi: 10.1016/j.toxicon.2005.07.018

2 : Laure CJ. [The primary structure of crotamine (author's transl)]. Hoppe Seylers Z Physiol Chem. 1975; 356:213-5.

3 : Chang CC and Tseng KH. Effect of crotamine, a toxin of South American rattlesnake venom, on the sodium channel of murine skeletal muscle. Br J Pharmacol. 1978; 63:551-9. doi: 10.1111/j.1476-5381.1978.tb07811.x

4 : Pereira A, et al. Crotamine toxicity and efficacy in mouse models of melanoma. Expert Opin Investig Drugs. 2011; 20:1189-200. doi: 10.1517/13543784.2011.602064

5 : Peigneur S, et al. Crotamine pharmacology revisited: novel insights based on the inhibition of KV channels. Mol Pharmacol. 2012; 82:90-6. doi: 10.1124/mol.112.078188

6 : Nascimento FD, et al. Crotamine mediates gene delivery into cells through the binding to heparan sulfate proteoglycans. J Biol Chem. 2007; 282:21349-60. doi: 10.1074/jbc.M604876200

7 : Kerkis A, et al. Crotamine is a novel cell-penetrating protein from the venom of rattlesnake Crotalus durissus terrificus. FASEB J. 2004; 18:1407-9. doi: 10.1096/fj.03-1459fje

8 : Nascimento FD, et al. The natural cell-penetrating peptide crotamine targets tumor tissue in vivo and triggers a lethal calcium-dependent pathway in cultured cells. Mol Pharm. 2012; 9:211-21. doi: 10.1021/mp2000605

9 : Yount NY, et al. Selective reciprocity in antimicrobial activity versus cytotoxicity of hBD-2 and crotamine. Proc Natl Acad Sci U S A. 2009; 106:14972-7. doi: 10.1073/pnas.0904465106

10 : Rádis-Baptista G, et al. Structure and chromosomal localization of the gene for crotamine, a toxin from the South American rattlesnake, Crotalus durissus terrificus. Toxicon. 2003; 42:747-52. doi: 10.1016/j.toxicon.2003.10.019

11 : Hayashi MA, et al. Cytotoxic effects of crotamine are mediated through lysosomal membrane permeabilization. Toxicon. 2008; 52:508-17. doi: 10.1016/j.toxicon.2008.06.029

12 : Rádis-Baptista G, et al. Nucleotide sequence of crotamine isoform precursors from a single South American rattlesnake (Crotalus durissus terrificus). Toxicon. 1999; 37:973-84. doi: 10.1016/s0041-0101(98)00226-8

13 : Kerkis I, et al. Biological versatility of crotamine--a cationic peptide from the venom of a South American rattlesnake. Expert Opin Investig Drugs. 2010; 19:1515-25. doi: 10.1517/13543784.2010.534457

14 : Mancin AC, et al. The analgesic activity of crotamine, a neurotoxin from Crotalus durissus terrificus (South American rattlesnake) venom: a biochemical and pharmacological study. Toxicon. 1998; 36:1927-37. doi: 10.1016/s0041-0101(98)00117-2

15 : Yamane ES, et al. Unraveling the antifungal activity of a South American rattlesnake toxin crotamine. Biochimie. 2013; 95:231-40. doi: 10.1016/j.biochi.2012.09.019

16 : Rizzi CT, et al. Crotamine inhibits preferentially fast-twitching muscles but is inactive on sodium channels. Toxicon. 2007; 50:553-62. doi: 10.1016/j.toxicon.2007.04.026

17 : Nicastro G, et al. Solution structure of crotamine, a Na+ channel affecting toxin from Crotalus durissus terrificus venom. Eur J Biochem. 2003; 270:1969-79. doi: 10.1046/j.1432-1033.2003.03563.x

18 : Oguiura N, et al. In vitro antibacterial and hemolytic activities of crotamine, a small basic myotoxin from rattlesnake Crotalus durissus. J Antibiot (Tokyo). 2011; 64:327-31. doi: 10.1038/ja.2011.10

Paper title : Automated NMR structure determination and disulfide bond identification of the myotoxin crotamine from Crotalus durissus terrificus.

Doi : https://doi.org/10.1016/j.toxicon.2005.07.018

Abstract : Crotamine is one of four major components of the venom of the South American rattlesnake Crotalus durissus terrificus. Similar to its counterparts in the family of the myotoxins, it induces myonecrosis of skeletal muscle cells. This paper describes a new NMR structure determination of crotamine in aqueous solution at pH 5.8 and 20 degrees C, using standard homonuclear 1H NMR spectroscopy at 900MHz and the automated structure calculation software ATNOS/CANDID/DYANA. The automatic NOESY spectral analysis included the identification of a most likely combination of the six cysteines into three disulfide bonds, i.e. Cys4-Cys36, Cys11-Cys30 and Cys18-Cys37; thereby a generally applicable new computational protocol is introduced to determine unknown disulfide bond connectivities in globular proteins. A previous NMR structure determination was thus confirmed and the structure refined. Crotamine contains an alpha-helix with residues 1-7 and a two-stranded anti-parallel beta-sheet with residues 9-13 and 34-38 as the only regular secondary structures. These are connected with each other and the remainder of the polypeptide chain by the three disulfide bonds, which also form part of a central hydrophobic core. A single conformation was observed, with Pro13 and Pro21 in the trans and Pro20 in the cis-form. The global fold and the cysteine-pairing pattern of crotamine are similar to the beta-defensin fold, although the two proteins have low sequence homology, and display different biological activities.

Paper title : [The primary structure of crotamine (author's transl)].

Doi : https://doi.org/Not available

Abstract : The primary structure of crotamine, a basic toxin isolated from the venom of the South American rattle-snake Crotalus durissus terrificus has been determined. The polypeptide chain is composed of 42 residues of amino acids. Crotamine shows a molecular weight of 4900 and contains 6 half cystine, 9 lysine, 2 arginine, 2 histidine and 2 tryptophan residues.

Paper title : Effect of crotamine, a toxin of South American rattlesnake venom, on the sodium channel of murine skeletal muscle.

Doi : https://doi.org/10.1111/j.1476-5381.1978.tb07811.x

Abstract : 1 Crotamine (0.5 mug/ml) augmented the single twitch response of the rat and mouse isolated diaphragm to direct stimulation and prolonged the time course of contraction. At higher doses (10 to 50 mug/ml), contracture was observed with spontaneous fibrillation.2 The resting membrane potential of diaphragm was rapidly depolarized to about -50 mV within 5 minutes. No increase of depolarization occurred on prolongation of the incubation time or increase of crotamine concentration from 0.5 mug/ml to 50 mug/ml. The effect was not reversed by washing.3 Tetrodotoxin, low Na(+) (12 mM), Ca(2+) (10 mM) and procaine (1 mM) prevented the crotamine-depolarization. However, depolarization resumed when crotamine and the antagonists were removed.4 Low Cl(-) (8.5 mM) and pretreatment with ouabain enhanced depolarization by crotamine.5 High K(+) (25 to 50 mM) prevented the further depolarization by crotamine and the membrane potential was restored to normal on washout of crotamine with normal Tyrode solution.6 Effective membrane resistance was decreased by about 50% by crotamine.7(24)Na-influx of the rat diaphragm was increased by crotamine. (42)K-influx was slightly increased if tetrodotoxin was also present but was decreased in the absence of tetrodotoxin.8 No effect on the miniature and evoked endplate potential of the rat diaphragm was observed. Skeletal muscles from frog and chick were not affected.9 It is inferred that crotamine acts on a molecule regulating the Na(+) - permeability of the Na(+) channel of murine muscles. It is proposed that extracellular K(+) depresses the permeability of the Na(+) channel by acting on the same regulator molecule.

Paper title : Crotamine toxicity and efficacy in mouse models of melanoma.

Doi : https://doi.org/10.1517/13543784.2011.602064

Abstract : OBJECTIVES: Selective anticancer cell activity for both cell-penetrating and cationic antimicrobial peptides has previously been reported. As crotamine possesses activities similar to both of these, this study investigates crotamine's anticancer toxicity in vitro and in vivo. RESEARCH DESIGN AND METHODS: In vitro cancer cell viability was evaluated after treatment with 1 and 5 μg/ml of crotamine. In vivo crotamine cytotoxic effects in C57Bl/6J mice bearing B16-F10 primary cutaneous melanoma were tested, with two groups each containing 35 mice. The crotamine-treated group received 1 μg/day of crotamine per animal, subcutaneously which was well tolerated; the untreated group received a placebo. RESULTS: Crotamine at 5 μg/ml was lethal to B16-F10, Mia PaCa-2 and SK-Mel-28 cells and inoffensive to normal cells. In vivo crotamine treatment over 21 days significantly delayed tumor implantation, inhibited tumor growth and prolonged the lifespan of the mice. Mice in the crotamine-treated group survived at significantly higher rates (n = 30/35) than those in the untreated group (n = 7/35) (significance calculated with the Kaplan-Meier estimator). The average tumor weight in the untreated group was 4.60 g but was only about 0.27 g in the crotamine-treated mice, if detectable. CONCLUSIONS: These data warrant further exploration of crotamine as a tumor inhibition compound.

Paper title : Crotamine pharmacology revisited: novel insights based on the inhibition of KV channels.

Doi : https://doi.org/10.1124/mol.112.078188

Abstract : Crotamine, a 5-kDa peptide, possesses a unique biological versatility. Not only has its cell-penetrating activity become of clinical interest but, moreover, its potential selective antitumor activity is of great pharmacological importance. In the past, several studies have attempted to elucidate the exact molecular target responsible for the crotamine-induced skeletal muscle spasm. The aim of this study was to investigate whether crotamine affects voltage-gated potassium (K(V)) channels in an effort to explain its in vivo effects. Crotamine was studied on ion channel function using the two-electrode voltage clamp technique on 16 cloned ion channels (12 K(V) channels and 4 Na(V) channels), expressed in Xenopus laevis oocytes. Crotamine selectively inhibits K(V)1.1, K(V)1.2, and K(V)1.3 channels with an IC(50) of ∼300 nM, and the key amino acids responsible for this molecular interaction are suggested. Our results demonstrate for the first time that the symptoms, which are observed in the typical crotamine syndrome, may result from the inhibition of K(V) channels. The ability of crotamine to inhibit the potassium current through K(V) channels unravels it as the first snake peptide with the unique multifunctionality of cell-penetrating and antitumoral activity combined with K(V) channel-inhibiting properties. This new property of crotamine might explain some experimental observations and opens new perspectives on pharmacological uses.

Paper title : Crotamine mediates gene delivery into cells through the binding to heparan sulfate proteoglycans.

Doi : https://doi.org/10.1074/jbc.M604876200

Abstract : Recently we have shown that crotamine, a toxin from the South American rattlesnake Crotalus durissus terrificus venom, belongs to the family of cell-penetrating peptides. Moreover, crotamine was demonstrated to be a marker of centrioles, of cell cycle, and of actively proliferating cells. Herein we show that this toxin at non-toxic concentrations is also capable of binding electrostatically to plasmid DNA forming DNA-peptide complexes whose stabilities overcome the need for chemical conjugation for carrying nucleic acids into cells. Interestingly, crotamine demonstrates cell specificity and targeted delivery of plasmid DNA into actively proliferating cells both in vitro and in vivo, which distinguishes crotamine from other known natural cell-penetrating peptides. The mechanism of crotamine penetration and cargo delivery into cells was also investigated, showing the involvement of heparan sulfate proteoglycans in the uptake phase, which is followed by endocytosis and peptide accumulation within the acidic endosomal vesicles. Finally, the permeabilization of endosomal membranes induced by crotamine results in the leakage of the vesicles contents to the cell cytosol.

Paper title : Crotamine is a novel cell-penetrating protein from the venom of rattlesnake Crotalus durissus terrificus.

Doi : https://doi.org/10.1096/fj.03-1459fje

Abstract : Herein we report that crotamine, a small lysine- and cysteine-rich protein from the venom of the South American rattlesnake, can rapidly penetrate into different cell types and mouse blastocysts in vitro. In vivo crotamine strongly labels cells from mouse bone marrow and spleen and from peritoneal liquid, as shown by fluorescent confocal laser-scanning microscopy. Nuclear localization of crotamine was observed in both fixed and unfixed cells. In the cytoplasm, crotamine specifically associates with centrosomes and thus allows us to follow the process of centriole duplication and separation. In the nucleus, it binds to the chromosomes at S/G2 phase, when centrioles start dividing. Moreover, crotamine appears as a marker of actively proliferating cells, as shown by 5-BrdU cell-proliferation assay. Crotamine in the micromolar range proved nontoxic to any of the cell cultures tested and did not affect the pluripotency of ES cells or the development of mouse embryos.

Paper title : The natural cell-penetrating peptide crotamine targets tumor tissue in vivo and triggers a lethal calcium-dependent pathway in cultured cells.

Doi : https://doi.org/10.1021/mp2000605

Abstract : Our goal was to demonstrate the in vivo tumor specific accumulation of crotamine, a natural peptide from the venom of the South American rattlesnake Crotalus durissus terrificus, which has been characterized by our group as a cell penetrating peptide with a high specificity for actively proliferating cells and with a concentration-dependent cytotoxic effect. Crotamine cytotoxicity has been shown to be dependent on the disruption of lysosomes and subsequent activation of intracellular proteases. In this work, we show that the cytotoxic effect of crotamine also involves rapid intracellular calcium release and loss of mitochondrial membrane potential as observed in real time by confocal microscopy. The intracellular calcium overload induced by crotamine was almost completely blocked by thapsigargin. Microfluorimetry assays confirmed the importance of internal organelles, such as lysosomes and the endoplasmic reticulum, as contributors for the intracellular calcium increase, as well as the extracellular medium. Finally, we demonstrate here that crotamine injected intraperitoneally can efficiently target remote subcutaneous tumors engrafted in nude mice, as demonstrated by a noninvasive optical imaging procedure that permits in vivo real-time monitoring of crotamine uptake into tumor tissue. Taken together, our data indicate that the cytotoxic peptide crotamine can be used potentially for a dual purpose: to target and detect growing tumor tissues and to selectively trigger tumor cell death.

Paper title : Selective reciprocity in antimicrobial activity versus cytotoxicity of hBD-2 and crotamine.

Doi : https://doi.org/10.1073/pnas.0904465106

Abstract : Recent discoveries suggest cysteine-stabilized toxins and antimicrobial peptides have structure-activity parallels derived by common ancestry. Here, human antimicrobial peptide hBD-2 and rattlesnake venom-toxin crotamine were compared in phylogeny, 3D structure, target cell specificity, and mechanisms of action. Results indicate a striking degree of structural and phylogenetic congruence. Importantly, these polypeptides also exhibited functional reciprocity: (i) they exerted highly similar antimicrobial pH optima and spectra; (ii) both altered membrane potential consistent with ion channel-perturbing activities; and (iii) both peptides induced phosphatidylserine accessibility in eukaryotic cells. However, the Na(v) channel-inhibitor tetrodotoxin antagonized hBD-2 mechanisms, but not those of crotamine. As crotamine targets eukaryotic ion channels, computational docking was used to compare hBD-2 versus crotamine interactions with prototypic bacterial, fungal, or mammalian Kv channels. Models support direct interactions of each peptide with Kv channels. However, while crotamine localized to occlude Kv channels in eukaryotic but not prokaryotic cells, hBD-2 interacted with prokaryotic and eukaryotic Kv channels but did not occlude either. Together, these results support the hypothesis that antimicrobial and cytotoxic polypeptides have ancestral structure-function homology, but evolved to preferentially target respective microbial versus mammalian ion channels via residue-specific interactions. These insights may accelerate development of anti-infective or therapeutic peptides that selectively target microbial or abnormal host cells.

Paper title : Structure and chromosomal localization of the gene for crotamine, a toxin from the South American rattlesnake, Crotalus durissus terrificus.

Doi : https://doi.org/10.1016/j.toxicon.2003.10.019

Abstract : Crotamine is a 42 amino acid-long basic polypeptide, one of the major components of the South American rattlesnake, Crotalus durissus terrificus, venom. The mRNA has about 340 nucleotides and codifies a pre-crotamine, including the signal peptide, the mature crotamine, and a final lysine. In this report, we describe the crotamine gene with 1.8 kb organized into three exons separated by a long phase-1 (900 bp) and a short phase-2 (140 bp) introns. Exon 1 includes the 5'-untranslated region and codifies the first 19 amino acids of the signal peptide. Exon 2 codifies 42 amino acids, three belonging to the signal peptide and 39 to the mature crotamine. Exon 3 codifies the last three amino acids of the mature toxin and the terminal lysine. The crotamine gene was mapped by in situ hybridization to the end of the long arm of chromosome 2, the intensity of signals differing between the two homologues. This may reflect a difference in gene copy numbers between chromosomes, a possible explanation for the variable amounts of crotamine found in the venom.

Paper title : Cytotoxic effects of crotamine are mediated through lysosomal membrane permeabilization.

Doi : https://doi.org/10.1016/j.toxicon.2008.06.029

Abstract : Crotamine, one of the main toxic components of Crotalus durissus terrificus venom, is a small non-enzymatic basic polypeptide, which causes hind limb paralysis and necrosis of muscle cells. It is well-known that several toxins penetrate into the cytosol through endocytosis, although in many cases the mechanism by which this occurs has not been fully investigated. Recently, using low concentrations of crotamine, we demonstrated the uptake of this toxin into actively proliferative cells via endocytosis, an event that ensues crotamine binding to cell membrane heparan sulfate proteoglycans. Thus, crotamine can be regarded as a cell-penetrating peptide that, additionally, has been shown to be able of delivering some biologically active molecules into various cells. Herein, we investigate one of the mechanisms by which crotamine exerts its cytotoxic effects by following its uptake into highly proliferative cells, as CHO-K1 cells. Crotamine accumulation in the acidic endosomal/lysosomal vesicles was observed within 5 in after treatment of these cells with a cytotoxic concentration of this toxin, a value determined here by classical MTT assay. This accumulation caused disruption of lysosomal vesicles accompanied by the leakage of these vesicles contents into the cytosol. This lysosomal lysis also promoted the release of cysteine cathepsin and an increase of caspase activity in the cytoplasm. This chain of events seems to trigger a cell death process. Overall, our data suggest that lysosomes are the primary targets for crotamine cytotoxicity, a proposal corroborated by the correlation between both the kinetics and concentration-dependence of crotamine accumulation in lysosome compartments and the cytotoxic effects of this protein in CHO-K1 cells. Although crotamine is usually regarded as a myotoxin, we observed that intraperitoneal injection of fluorescently labeled crotamine in living mice led to significant and rapid accumulation of this toxin in the cell cytoplasm of several tissues, suggesting that crotamine cytotoxicity might not be restricted to muscle cells.

Paper title : Nucleotide sequence of crotamine isoform precursors from a single South American rattlesnake (Crotalus durissus terrificus).

Doi : https://doi.org/10.1016/s0041-0101(98)00226-8

Abstract : A cDNA phage library was constructed from venom glands of a single adult specimen of crotamine-plus Crotalus durissus terrificus (South American rattlesnake) captured in a known region. Fifteen crotamine positive clones were isolated using a PCR-based screening protocol and sequenced. These complete cDNAs clones were grouped for maximal alignment into six distinct nucleotide sequences. The crotamine cDNAs, with 340-360 bases, encompass open reading frame of 198 nucleotides with 5' and 3' untranslated regions of variable size, signal peptide sequence, one crotamine isoform message, and putative poly(A+) signal. Of these six different crotamine cDNA precursors, two predict the identical amino acid sequence previously described by Laure (1975), and the other four a crotamine isoform precursor where the Leucine residue at position 19 is replaced by isoleucine by a single base change. On the other hand, nucleotide variation was observed in the 5' and 3' untranslated regions, with one interesting variant containing an 18 base pair deletion at the 5' untranslated region which results in the usual ATG initiator being replaced by the rarely used GUG start codon. Comparison by Northern blot analysis of poly(A+) RNA from venom glands of a crotamine-plus specimen to total and poly(A+) RNA from a crotamine-minus snake indicated that crotamine transcripts were not expressed in the crotamine-minus specimen.

Paper title : Biological versatility of crotamine--a cationic peptide from the venom of a South American rattlesnake.

Doi : https://doi.org/10.1517/13543784.2010.534457

Abstract : IMPORTANCE OF THE FIELD: Molecules isolated from animals, insects, plants or microorganisms can provide prototypes for design of biopharmaceutical products. Some venom toxins and their derivatives are used in medicine, while others provide templates for development of new drugs. AREAS COVERED IN THIS REVIEW: The mild toxin, crotamine, a small basic low-molecular-weight polypeptide purified from the venom of a South American rattlesnake, Crotalus durissus terrificus. Crotamine was discovered more than 50 years ago and only in the past six years has its exceptional biological versatility been demonstrated. Particularly, its cell-penetrating ability, which allows crotamine to cross cell membranes and to accumulate in the nucleus; its use for intracellular vesicle tracking and as a cell cycle marker and its capability for delivering DNA into replicating mammalian cells. Both antimicrobial action and potential selective antitumor activity of crotamine have also been found. WHAT THE READER WILL GAIN: Multidisciplinary approaches and pathways of discovery placed crotamine in a rare category of versatile biomolecules, in which concentration, molecular target preference, structural ancestry and specificity toward biological membranes play an integral role. TAKE HOME MESSAGE: Crotamine is a druggable peptide with high potential for use as an imaging agent for detecting dividing cells, for intracellular delivery of hydrophilic biomolecules, and as an alternative chemotherapeutic compound against aggressive types of cancer.

Paper title : The analgesic activity of crotamine, a neurotoxin from Crotalus durissus terrificus (South American rattlesnake) venom: a biochemical and pharmacological study.

Doi : https://doi.org/10.1016/s0041-0101(98)00117-2

Abstract : Crotamine, a 4.88 kDa neurotoxic protein, has been purified to apparent homogeneity from Crotalus durissus venom by gel filtration on Sephadex G-75. When injected (i.p. or s.c.) in adult male Swiss mice (20-25 g), it induced a time-dose dependent analgesic effect which was inhibited by naloxone, thus suggesting an opioid action mechanism. When compared with morphine (4 mg/kg), crotamine, even in extremely low doses (133.4 microg/kg, i.p., about 0.4% of a LD50 is approximately 30-fold more potent than morphine (w/w) as an analgesic. On a molar basis it is more than 500-fold more potent than morphine. It is also much more potent than the lower molecular weight crude fractions of the same venom. The antinociceptive effects of crotamine and morphine were assayed by the hot plate test and by the acetic acid-induced writhing method. Therefore, both central and peripheral mechanisms should be involved. Histopathological analysis of the brain, liver, skeletal muscles, stomach, lungs, spleen, heart, kidneys and small intestine of the crotamine injected mice did not show any visible lesion in any of these organs by light microscopy. Since crotamine accounted for 22% (w/w) of the desiccated venom, it was identified as its major antinociceptive low molecular weight peptide component.

Paper title : Unraveling the antifungal activity of a South American rattlesnake toxin crotamine.

Doi : https://doi.org/10.1016/j.biochi.2012.09.019

Abstract : Crotamine is a highly basic peptide from the venom of Crotalus durissus terrificus rattlesnake. Its common gene ancestry and structural similarity with the β-defensins, mainly due to an identical disulfide bond pattern, stimulated us to assess the antimicrobial properties of native, recombinant, and chemically synthesized crotamine. Antimicrobial activities against standard strains and clinical isolates were analyzed by the colorimetric microdilution method showing a weak antibacterial activity against both Gram-positive and Gram-negative bacteria [MIC (Minimum Inhibitory Concentration) of 50->200 μg/mL], with the exception of Micrococcus luteus [MIC ranging from 1 to 2 μg/mL]. No detectable activity was observed for the filamentous fungus Aspergillus fumigatus and Trichophyton rubrum at concentrations up to 125 μg/mL. However, a pronounced antifungal activity against Candida spp., Trichosporon spp., and Cryptococcus neoformans [12.5-50.0 μg/mL] was observed. Chemically produced synthetic crotamine in general displayed MIC values similar to those observed for native crotamine, whereas recombinant crotamine was overridingly more potent in most assays. On the other hand, derived short linear peptides were not very effective apart from a few exceptions. Pronounced ultrastructure alteration in Candida albicans elicited by crotamine was observed by electron microscopy analyses. The peculiar specificity for highly proliferating cells was confirmed here showing potential low cytotoxic effect of crotamine against nontumoral mammal cell lines (HEK293, PC12, and primary culture astrocyte cells) compared to tumoral B16F10 cells, and no hemolytic activity was observed. Taken together these results suggest that, at low concentration, crotamine is a potentially valuable anti-yeast or candicidal agent, with low harmful effects on normal mammal cells, justifying further studies on its mechanisms of action aiming medical and industrial applications.

Paper title : Crotamine inhibits preferentially fast-twitching muscles but is inactive on sodium channels.

Doi : https://doi.org/10.1016/j.toxicon.2007.04.026

Abstract : Crotamine is a peptide toxin from the venom of the rattlesnake Crotalus durissus terrificus that induces a typical hind-limb paralysis of unknown nature. Hind limbs have a predominance of fast-twitching muscles that bear a higher density of sodium channels believed until now to be the primary target of crotamine. Hypothetically, this makes these muscles more sensitive to crotamine and would explain such hind-limb paralysis. To challenge this hypothesis, we performed concentration vs. response curves on fast (extensor digitorum longus (EDL)) and slow (soleus) muscles of adult male rats. Crotamine was tested on various human Na+ channel isoforms (Na(v)1.1-Na(v)1.6 alpha-subunits) expressed in HEK293 cells in patch-clamp experiments, as well as in acutely dissociated dorsal root ganglion (DRG) neurons. Also, the behavioral effects of crotamine intoxication were compared with those of a muscle-selective sodium channel antagonist mu-CgTx-GIIIA, and other sodium-acting toxins such as tetrodotoxin alpha- and beta-pompilidotoxins, sea anemone toxin BcIII, spider toxin Tx2-6. Results pointed out that EDL was more susceptible to crotamine than soleus under direct electrical stimulation. Surprisingly, electrophysiological experiments in human Na(v)1.1 to Na(v)1.6 Na+ channels failed to show any significant change in channel characteristics, in a clear contrast with former studies. DRG neurons did not respond to crotamine. The behavioral effects of the toxins were described in detail and showed remarkable differences. We conclude that, although differences in the physiology of fast and slow muscles may cause the typical crotamine syndrome, sodium channels are not the primary target of crotamine and therefore, the real mechanism of action of this toxin is still unknown.

Paper title : Solution structure of crotamine, a Na+ channel affecting toxin from Crotalus durissus terrificus venom.

Doi : https://doi.org/10.1046/j.1432-1033.2003.03563.x

Abstract : Crotamine is a component of the venom of the snake Crotalus durissus terrificus and it belongs to the myotoxin protein family. It is a 42 amino acid toxin cross-linked by three disulfide bridges and characterized by a mild toxicity (LD50 = 820 micro g per 25 g body weight, i.p. injection) when compared to other members of the same family. Nonetheless, it possesses a wide spectrum of biological functions. In fact, besides being able to specifically modify voltage-sensitive Na+ channel, it has been suggested to exhibit analgesic activity and to be myonecrotic. Here we report its solution structure determined by proton NMR spectroscopy. The secondary structure comprises a short N-terminal alpha-helix and a small antiparallel triple-stranded beta-sheet arranged in an alphabeta1beta2beta3 topology never found among toxins active on ion channels. Interestingly, some scorpion toxins characterized by a biological activity on Na+ channels similar to the one reported for crotamine, exhibit an alpha/beta fold, though with a beta1alphabeta2beta3 topology. In addition, as the antibacterial beta-defensins, crotamine interacts with lipid membranes. A comparison of crotamine with human beta-defensins shows a similar fold and a comparable net positive potential surface. To the best of our knowledge, this is the first report on the structure of a toxin from snake venom active on Na+ channel.

Paper title : In vitro antibacterial and hemolytic activities of crotamine, a small basic myotoxin from rattlesnake Crotalus durissus.

Doi : https://doi.org/10.1038/ja.2011.10

Abstract : Crotamine, a myotoxin from the venom of South American rattlesnake, is structurally related to β-defensins, antimicrobial peptides (AMPs) found in vertebrate animals. Here, we tested the antibacterial properties of crotamine and found that it killed several strains of Escherichia coli, with the MICs ranging from 25 to 100 μg ml⁻¹. Time-kill and bacterial membrane permeabilization assays revealed that killing of bacteria by crotamine occurred within 1 h and reached the maximum by 2 h. Additionally, the anti-E. coli activity of crotamine was completely abolished with 12.5 mM NaCl. Furthermore, the three intramolecular disulfide bonds of crotamine appeared dispensable for its antibacterial activity. The reduced form of crotamine was active against E. coli as well. However, crotamine showed no or weak activity up to 200 μg ml⁻¹ against other species of Gram-negative and Gram-positive bacteria. Crotamine showed no appreciable hemolytic activity to erythrocytes. Our studies revealed that crotamine is also an AMP that kills bacteria through membrane permeabilization. However, crotamine appears to have a narrow antibacterial spectrum, distinct from many classical β-defensins, reinforcing the notion that crotamine originated from the β-defensin gene lineage, but has undergone significant functional diversification.