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The potential biotechnological and biomedical applications of the animal venom components are widely recognized. Indeed, many components have been used either as drugs or as templates/prototypes for the development of innovative pharmaceutical drugs, among which many are still used for the treatment of human diseases. A specific South American rattlesnake, named Crotalus durissus terrificus, shows a venom composition relatively simpler compared to any viper or other snake species belonging to the Crotalus genus, although presenting a set of toxins with high potential for the treatment of several still unmet human therapeutic needs, as reviewed in this work. In addition to the main toxin named crotoxin, which is under clinical trials studies for antitumoral therapy and which has also anti-inflammatory and immunosuppressive activities, other toxins from the C. d. terrificus venom are also being studied, aiming for a wide variety of therapeutic applications, including as antinociceptive, anti-inflammatory, antimicrobial, antifungal, antitumoral or antiparasitic agent, or as modulator of animal metabolism, fibrin sealant (fibrin glue), gene carrier or theranostic agent. Among these rattlesnake toxins, the most relevant, considering the potential clinical applications, are crotamine, crotalphine and gyroxin. In this narrative revision, we propose to organize and present briefly the updates in the accumulated knowledge on potential therapeutic applications of toxins collectively found exclusively in the venom of this specific South American rattlesnake, with the objective of contributing to increase the chances of success in the discovery of drugs based on toxins., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Gyroxin is a serine protease displaying a thrombin-like activity found in the venom of the South American rattlesnake Crotalus durissus terrificus. Typically, intravenous injection of purified gyroxin induces a barrel rotation syndrome in mice. The serine protease thrombin activates platelets aggregation by cleaving and releasing a tethered N-terminus peptide from the G-protein-coupled receptors, known as protease-activated receptors (PARs). Gyroxin also presents pro-coagulant activity suggested to be dependent of PARs activation. In the present work, the effects of these serine proteases, namely gyroxin and thrombin, on PARs were comparatively studied by characterizing the hydrolytic specificity and kinetics using PARs-mimetic FRET peptides. We show for the first time that the short (sh) and long (lg) peptides mimetizing the PAR-1, -2, -3, and -4 activation sites are all hydrolyzed by gyroxin exclusively after the Arg residues. Thrombin also hydrolyzes PAR-1 and -4 after the Arg residue, but hydrolyzes sh and lg PAR-3 after the Lys residue. The kcat/KM values determined for gyroxin using sh and lg PAR-4 mimetic peptides were at least 2150 and 400 times smaller than those determined for thrombin, respectively. For the sh and lg PAR-2 mimetic peptides the kcat/KM values determined for gyroxin were at least 6500 and 2919 times smaller than those determined for trypsin, respectively. The kcat/KM values for gyroxin using the PAR-1 and -3 mimetic peptides could not be determined due to the extreme low hydrolysis velocity. Moreover, the functional studies of the effects of gyroxin on PARs were conducted in living cells using cultured astrocytes, which express all PARs. Despite the ability to cleavage the PAR-1, -2, -3, and -4 peptides, gyroxin was unable to activate the PARs expressed in astrocytes as determined by evaluating the cytosolic calcium mobilization. On the other hand, we also showed that gyroxin is able to interfere with the activation of PAR-1 by thrombin or by synthetic PAR-1 agonist in cultured astrocytes. Taken together, the data presented here allow us showing that gyroxin cleaves PARs-mimetic peptides slowly and it does not induce activation of PARs in astrocytes. Although gyroxin does not mobilize calcium it was shown to interfere with PARs activation by thrombin and PAR-1 agonist. The determination of gyroxin enzymatic specificity and kinetics on PAR-1, -2, -3, and -4 will potentially help to fill the gap in the knowledge in this field, as the PARs are still believed to have a key role for the gyroxin biological effects., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Gyroxin is one of main serine proteases of Crotalus durissus terrificus venom, representing about 2% of the protein content in the crude venom. It is a 33 kDa glycoprotein with 3.8% by weight of sugar moiety. This toxin induces hemotoxicity in mice and a neurological condition called barrel rotation syndrome. In the present work, we report the molecular cloning of five new nucleotide sequences from a cDNA library of the venom glands of a single specimen of C. d. terrificus. These sequences have been analyzed in silico with respect to their cDNA organization and similarity with other snake venom serine proteases (SVSPs). We also describe a rapid and efficient method for screening vectors for mammalian cell expression, based on the fact that SVSPs are difficult-to-express toxins due to the presence of several disulfide bonds and glycosylation in their structures. Thus, one of the Gyroxin cDNAs was subcloned into pSectag2 HygroA and pED vectors and used to transfect COS-7 cells. Expression of the functional recombinant Gyroxin isoform was achieved with this cell line with esterase activity in the conditioned culture medium, as revealed by immunoblot of secreted protein and standard anti-crotalic serum from Butantan Institute.

The primary goal of this study was to determine whether Tx2-5, a sodium channel selective toxin obtained from the venom of the spider Phoneutria nigriventer, produced penile erection by means of nitric oxide mechanism. Toxin identity was analyzed by MALDI-TOF, ES-MS and N-terminal amino acid sequencing. Pretreating mice with the non-selective nitric oxide synthase (NOS) inhibitor N(omega)-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and the selective neuronal-NOS inhibitor 7-Nitroindazole (7-NI) prior to Tx2-5 i.p. (10 microg/25 g mouse) injection challenged the hypothesis above. Controls were injected with the D-isomer or DMSO or saline. Results demonstrated that L-NAME inhibited penile erections in about half the animals treated, while 7-NI completely abolished this effect. Interestingly 7-NI also abolished all the other symptoms of intoxication induced by Tx2-5, including salivation, respiratory distress and death. Tx2-5 killed all the animals of the control group and no one in the 7-NI-treated group. We conclude that (1) intraperitoneal injections of Tx2-5 induce a toxic syndrome that include penile erection, hypersalivation and death by respiratory distress or pulmonary edema; (2) pretreatment with the non-selective NOS inhibitor L-NAME reduces the penile erection and partially protects from the lethal effects of Tx2-5; (3) pretreatment with the nNOS-selective inhibitor 7-NI completely abolishes all the toxic effects of Tx2-5, including penile erection and death suggesting that nNOS is the major player in this intoxication; (4) toxins from other animals that affect sodium channels in the same way as Tx2-5 and induce similar toxic syndromes may have as a major common target, the activation of nitric oxide synthases.