Crotamine/siRNA Nanocomplexes for Functional Downregulation of Syndecan-1 in Renal Proximal Tubular Epithelial Cells.

Proteinuria drives progressive tubulointerstitial fibrosis in native and transplanted kidneys, mainly through the activation of proximal tubular epithelial cells (PTECs). During proteinuria, PTEC syndecan-1 functions as a docking platform for properdin-mediated alternative complement activation. Non-viral gene delivery vectors to target PTEC syndecan-1 could be useful to slow down alternative complement activation. In this work, we characterize a PTEC-specific non-viral delivery vector composed of the cell-penetrating peptide crotamine complexed with a syndecan-1 targeting siRNA. Cell biological characterization was performed in the human PTEC HK2 cell line, using confocal microscopy, qRT-PCR, and flow cytometry. PTEC targeting in vivo was carried out in healthy mice. Crotamine/siRNA nanocomplexes are positively charged, about 100 nm in size, resistant to nuclease degradation, and showed in vitro and in vivo specificity and internalization into PTECs. The efficient suppression of syndecan-1 expression in PTECs mediated by these nanocomplexes significantly reduced properdin binding (p < 0.001), as well as the subsequent complement activation by the alternative complement pathway (p < 0.001), as observed in either normal or activated tubular conditions. To conclude, crotamine/siRNA-mediated downregulation of PTEC syndecan-1 reduced the activation of the alternative complement pathway. Therefore, we suggest that the present strategy opens new venues for targeted proximal tubular gene therapy in renal diseases.

A Native CPP from Rattlesnake with Therapeutic and Theranostic Properties.

The cell-penetrating peptides (CPPs) are characterized by the ability of internalization into cells in vitro and in vivo, and the ability of these peptides can rely on a high content of positive charges, as it is the case of the native CPP crotamine. Crotamine is a polypeptide of about 42 amino acid residues with high content of basic residues as Arg and Lys. Although most of known CPPs are linear peptides, native crotamine from the venom of a South American rattlesnake has a well-defined 3D structure stabilized by three disulfide bonds which guarantee the exposure of side chains of basic amino acids. This 3D structure also protects this amphipathic polypeptide from the degradation even if administered by oral route, therefore, protecting also the biological activities of crotamine. As several different biological properties of crotamine are dependent of cell penetration, the methods mainly employed for analyzing crotamine properties as anthelminthic and antimalarial activities, antimicrobial and antitumor activities, with a unique selective cytotoxic property against actively proliferating cells, as tumor cells, were chosen based on crotamine ability of internalization mediated by its positive charge. This native cationic polypeptide is also able to efficiently carry, with no need of covalent linkage with the cargo, genetic material into cells in vitro and in vivo, suggesting its use in gene therapy. Moreover, the possibility of decorating gold nanoparticles keeping the ability of transfecting cells was demonstrated. More recently, the ability of crotamine to interfere in animal metabolism, inducing browning of adipose tissue and increasing the energy expenditure, and its application in renal therapy was demonstrated. As crotamine also accumulates specifically in tumor cells in vivo, and the potential utility of crotamine as a theranostic agent was then suggested. Therefore, diverse methodologies employed for the characterization and exploration of the therapeutic applications of this promising native CPP for remediation of several pathogenic conditions are presented here.

Regulation of monoamine levels by typical and atypical antipsychotics in Caenorhabditis elegans mutant for nuclear distribution element genes.

Mammalian nuclear distribution genes encode proteins with essential roles in neuronal migration and brain formation during embryogenesis. The implication of human nuclear distribution genes, namely nudC and NDE1 (Nuclear Distribution Element 1)/NDEL1 (Nuclear Distribution Element-Like 1), in psychiatric disorders including schizophrenia and bipolar disorder, has been recently described. The partial loss of NDEL1 expression results in neuronal migration defects, while ndel1 null knockout (KO) leads to early embryonic lethality in mice. On the other hand, loss-of-function of the orthologs of nuclear distribution element genes (nud) in Caenorhabditis elegans renders viable worms and influences behavioral endophenotypes associated with dopaminergic and serotoninergic pathways. In the present work, we evaluated the role of nud genes in monoamine levels at baseline and after the treatment with typical or atypical antipsychotics. Dopamine, serotonin and octopamine levels were significantly lower in homozygous loss-of-function mutant worms KO for nud genes compared with wild-type (WT) C. elegans at baseline. While treatment with antipsychotics determined significant differences in monoamine levels in WT, the nud KO mutant worms appear to respond differently to the treatment. According to the best of our knowledge, we are the first to report the influence of nud genes in the monoamine levels changes in response to antipsychotic drugs, ultimately placing the nuclear distribution genes family at the cornerstone of pathways involved in the modulation of monoamines in response to different classes of antipsychotic drugs.

Crotamine Cell-Penetrating Nanocarriers: Cancer-Targeting and Potential Biotechnological and/or Medical Applications.

Crotamine is a basic, 42-residue polypeptide from snake venom that has been shown to possess cell-penetrating properties. Here we describe the preparation, purification, biochemical and biophysical analysis of venom-derived, recombinant, chemically synthesized, and fluorescent-labeled crotamine. We also describe the formation and characterization of crotamine-DNA and crotamine-RNA nanoparticles; and the delivery of these nanoparticles into cells and animals. 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 living organisms such as mice, Plasmodium, and worms. As such, these nanoparticles could form the basis for a nucleic acid drug-delivery system. We also describe here the design and characterization of crotamine-functionalized gold nanoparticles, and the delivery of these nanoparticles into cells. We also evaluated the viability of using the combination of crotamine with silica nanoparticles in animal models, aiming to provide slow delivery, and to decrease the crotamine doses needed for the biological effects. In addition, the efficacy of administering crotamine orally was also demonstrated.

Long term safety of targeted internalization of cell penetrating peptide crotamine into renal proximal tubular epithelial cells in vivo.

Activated proximal tubular epithelial cells (PTECs) play a crucial role in progressive tubulo-interstitial fibrosis in native and transplanted kidneys. Targeting PTECs by non-viral delivery vectors might be useful to influence the expression of important genes and/or proteins in order to slow down renal function loss. However, no clinical therapies that specifically target PTECs are available at present. We earlier showed that a cationic cell penetrating peptide isolated from South American rattlesnake venom, named crotamine, recognizes cell surface heparan sulfate proteoglycans and accumulates in cells. In healthy mice, crotamine accumulates mainly in kidneys after intraperitoneal (ip) injection. Herein we demonstrate for the first time, the overall safety of acute or long-term treatment with daily ip administrated crotamine for kidneys functions. Accumulation of ip injected crotamine in the kidney brush border zone of PTECs, and its presence inside these cells were observed. In addition, significant lower in vitro crotamine binding, uptake and reporter gene transport and expression could be observed in syndecan-1 deficient HK-2 PTECs compared to wild-type cells, indicating that the absence of syndecan-1 impairs crotamine uptake into PTECs. Taken together, our present data show the safety of in vivo long-term treatment with crotamine, and its preferential uptake into PTECs, which are especially rich in HSPGs such as syndecan-1. In addition to the demonstrated in vitro gene delivery mediated by crotamine in HK-2 cells, the potential applicability of crotamine as prototypic non-viral (gene) delivery nanocarrier to modulate PTEC gene and/or protein expression was confirmed.

Pharmacological characterization of crotamine effects on mice hind limb paralysis employing both ex vivo and in vivo assays: Insights into the involvement of voltage-gated ion channels in the crotamine action on skeletal muscles.

The high medical importance of Crotalus snakes is unquestionable, as this genus is the second in frequency of ophidian accidents in many countries, including Brazil. With a relative less complex composition compared to other genera venoms, as those from the Bothrops genus, the Crotalus genus venom from South America is composed basically by the neurotoxin crotoxin (a phospholipase A2), the thrombin-like gyroxin (a serinoprotease), a very potent aggregating protein convulxin, and a myotoxic polypeptide named crotamine. Interestingly not all Crotalus snakes express crotamine, which was first described in early 50s due to its ability to immobilize animal hind limbs, contributing therefore to the physical immobilization of preys and representing an important advantage for the envenoming efficacy, and consequently, for the feeding and survival of these snakes in nature. Representing about 10-25% of the dry weight of the crude venom of crotamine-positive rattlesnakes, the polypeptide crotamine is also suggested to be of importance for antivenom therapy, although the contribution of this toxin to the main symptoms of envenoming process remains far unknown until now. Herein, we concomitantly performed in vitro and in vivo assays to show for the first time the dose-dependent response of crotamine-triggered hind limbs paralysis syndrome, up to now believed to be observable only at high (sub-lethal) concentrations of crotamine. In addition, ex vivo assay performed with isolated skeletal muscles allowed us to suggest here that compounds active on voltage-sensitive sodium and/or potassium ion channels could both affect the positive inotropic effect elicited by crotamine in isolated diaphragm, besides also affecting the hind limbs paralysis syndrome imposed by crotamine in vivo. By identifying the potential molecular targets of this toxin, our data may contribute to open new roads for translational studies aiming to improve the snakebite envenoming treatment in human. Interestingly, we also demonstrate that the intraplantal or intraperitoneal (ip) injections of crotamine in mice do not promote pain. Therefore, this work may also suggest the profitable utility of non-toxic analogs of crotamine as a potential tool for targeting voltage-gated ion channels in skeletal muscles, aiming its potential use in the therapy of neuromuscular dysfunctions and envenoming therapy.

Identification of snake bradykinin-potentiating peptides (BPPs)-simile sequences in rat brain--Potential BPP-like precursor protein?

Bradykinin-potentiating peptides (BPPs) from the South American pit viper snake venom were the first natural inhibitors of the human angiotensin I-converting enzyme (ACE) described. The pioneer characterization of the BPPs precursor from the snake venom glands by our group showed for the first time the presence of the C-type natriuretic peptide (CNP) in this same viper precursor protein. The confirmation of the BPP/CNP expression in snake brain regions correlated with neuroendocrine functions stimulated us to pursue the physiological correlates of these vasoactive peptides in mammals. Notably, several snake toxins were shown to have endogenous physiological correlates in mammals. In the present work, we expressed in bacteria the BPPs domain of the snake venom gland precursor protein, and this purified recombinant protein was used to raise specific polyclonal anti-BPPs antibodies. The correspondent single protein band immune-recognized in adult rat brain cytosol was isolated by 2D-SDS/PAGE and/or HPLC, before characterization by MS fingerprint analysis, which identified this protein as superoxide dismutase (SOD, EC 1.15.1.1), a classically known enzyme with antioxidant activity and important roles in the blood pressure modulation. In silico analysis showed the exposition of the BPP-like peptide sequences on the surface of the 3D structure of rat SOD. These peptides were chemically synthesized to show the BPP-like biological activities in ex vivo and in vivo pharmacological bioassays. Taken together, our data suggest that SOD protein have the potential to be a source for putative BPP-like bioactive peptides, which once released may contribute to the blood pressure control in mammals.