Crotamine

RNA synthesis in the venom glands of Crotalus durissus terrificus was stimulated by the manual extraction of the venom (milking). RNA was extracted from venom glands activated by milking and fractionated by centrifugation through sucrose density gradients. Template activity for protein synthesis and base composition of the RNA fractions were studied. RNA fractions that sediment between 18S and 4S had the highest template activity. The base composition analysis indicated that the 28S and 18S rRNA have a C+G content of 65.4 and 58% respectively. The ;melting’ temperature (T(m)) of DNA in 0.15m-NaCl-0.015m-trisodium citrate, pH7.0, was 85 degrees C, corresponding to a C+G content of 38%. The base ratio of the RNA fractions that showed a high template activity was intermediate between that of rRNA and homologous DNA. The possible role of these fractions in the synthesis of the two main toxins (crotoxin and crotamine) of the South American rattlesnake’s venom is discussed.

De Lucca FL., et al. (1974) Characterization of ribonucleic acids from the venom glands of Crotalus durissus terrifucus (Ophidia, Reptilia) after manual extraction of the venom. Studies on template activity and base composition. Biochem J. PMID: 4463939

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. Sincecrotamine accounted for 22% (w/w) of the desiccated venom, it was identified as its major antinociceptive low molecular weight peptide component.

 

Mancin AC., et al. (1998)  The analgesic activity of crotamine, a neurotoxin from Crotalus durissus terrificus (South American rattlesnake) venom: a biochemical and pharmacological study. Toxicon. PMID: 9839677

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.

Kerkis A., et al. (2004) Crotamine is a novel cell-penetrating protein from the venom of rattlesnake Crotalus durissus terrificus. FASEB J. PMID: 15231729

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.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. 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. 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.

Kerkis I., et al. (2010) Biological versatility of crotamine a cationic peptide from the venom of a South American rattlesnake. Expert Opin Investig Drugs. PMID: 21062230

Animal venoms comprise a naturally selected cocktail of bioactive peptides/proteins and other molecules, each of which playing a defined role thanks to the highly specific interactions with diverse molecular targets found in the prey. Research focused on isolation, structural, and functional characterizations of novel natural biologics (bioactive peptides/proteins from natural sources) has a long way to go through from the basic science to clinical applications. Herein, we overview the structural and functional characteristics of the myoneurotoxin crotamine, firstly isolated from the South American rattlesnake venom. Crotamine is the first venom peptide classified as a natural cell penetrating and antimicrobial peptide (CPP and AMP) with a more pronounced antifungal activity. In contrast to other known natural CPPs and AMPs, crotamine demonstrates a wide spectrum of biological activities with potential biotechnological and therapeutic values. More recent studies have demonstrated the selective in vitro anticancer activity of crotamine. In vivo, using a murine melanoma model, it was shown that crotamine delays tumor implantation, inhibits tumor cells proliferation, and also increases the survival of mice engrafted with subcutaneous melanoma. The structural and functional properties and also the possible biotechnological applications of minimized molecules derived from crotamine are also discussed.

Kerkis I., et al. (2014) State of the art in the studies on crotamine, a cell penetrating peptide from South American rattlesnake. Biomed Res Int. PMID: 24551848

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.

Nascimento FD (2012) The natural cell-penetrating peptide crotamine targets tumor tissue in vivo and triggers a lethal calcium-dependent pathway in cultured cells. Mol Pharm. PMID: 22142367

Crotamine is a basic, 42-residue polypeptide derived from snake venom that has been shown to possess cell-penetrating properties. Crotamine forms nanoparticles with a variety of DNA and RNA molecules, and crotamine-plasmid DNA nanoparticles are selectively delivered into actively proliferating cells in culture or in mice. As such, these nanoparticles could form the basis for a nucleic acid drug-delivery system. Here we describe the preparation, purification, and biochemical and biophysical analysis of venom-derived, recombinant, chemically synthesized, and fluorescent-labeled crotamine; the formation and characterization of crotamine-DNA and -RNA nanoparticles; and the delivery of these nanoparticles into cells and animals.

Hayashi MA., et al. (2012) Crotamine: a novel cell-penetrating polypeptide nanocarrier with potential anti-cancer and biotechnological applications. Methods Mol Biol. PMID: 22791447