Vipericidinas e derivados: caracterização de alvo molecular em Trypanosoma cruzi, avaliação antiparasitária e antitumoral, visando o uso biotecnológico dos peptídeos terapêuticos.

Abstract

Antimicrobial peptides related cathelicidins-vipericidins are involved in the innate immune response in the defense system of many organisms, and a mode of action rapid, and thus can operate in conjunction with existing and / or regimens constitute an alternative to conventional antibiotics drugs. The present study of thesis has as goals two aspects: first to characterize the target gene of T.cruzi, which interacts in silico with the vipericidin: Cyclophilin A (TcCyP19) in sensitive and resistant strains of Trypanosoma cruzi to benznidazole; second to evaluate in vitro antiparasitic and antitumor activity, as well as evaluate the cytotoxicity of vipericidinas and its derivatives, aiming at the use biotechnological of these peptides. In silico study allowed identifying a cyclophilin that interacts with the antimicrobial peptide GA650- (lutzicidina) from the venom of pit vipers. This finding is of particular interest given that our research group had conducted one study proteomic identifying the cyclophilin-A as an overexpressed protein (10 x) in resistant populations of T. cruzi compared with their pair-sensitive benznidazole. Therefore Cyclophilin A (TcCyP19), a peptidyl - prolyl isomerase - key molecule with diverse biological functions, including molecular folding, stress response, immune modulation and signal transduction - comprises a potential drug target, and thus was selected for study. Phylogenetic analysis revealed a clear divergence between the sequences of Cyclophilin-A trypanosomatids and mammals - which makes it a differential target. A combination of the Southern blot analysis and real-time RT-PCR (qPCR) showed that TcCyP19 is a single copy gene located in the chromosomal bands ranging in size from 0.68 to 2.2 Mb, depending on the strain T. cruzi. Northern blot and qPCR indicated that mRNA levels TcCyP19 were twice higher in populations of T. cruzi BZ resistant than its sensitive counterpart pair. Similarly, the expression of CyP19 protein, as determined by two-dimensional electrophoresis / western blot gel was increased in the order of resistant populations T.cruzi to BZ. Altogether, these data indicate that Cyclophilin A (TcCyP19) expression is up-regulated at transcriptional and translational levels in T. cruzi populations which were in vitro-induced and in-vivo selected for resistance to BZ. These strains were used to evaluate the antiparasitic activity of vipericidinas. Besides the antiparasitic activity, our group determined the inhibitory action of vipericidinas against bacteria and fungi. Activity assay in vitro anti-Trypanosoma cruzi epimastigotes, trypomastigote and intracellular amastigotes, it was observed that Vipericidin (crotalicidin – GA645 peptide) had dose-dependent cytotoxic effect against epimastigotes of Trypanosoma cruzi (24h IC50 = 4.72 uM; IC50 48h = 4:37 mM). To trypomastigotes and intracellular amastigotes only had activity of crotalicidin vipericidin following cell cytotoxicity and the remaining samples were inactive. For trypomastigote and amastigote forms only vipericidin GA645-crotalicidin have activity followed by cell cytotoxicity and the other samples were inactive. For to evaluate anticancer action in this study, we identify a nine-residue cryptic oligopeptide, KRFKKFFKK (EVP50) that is repeatedly encoded in tandem within vipericidin sequences. EVP50 addition exhibited a potent lethal toxicity to zebrafish (Danio rerio) (DL = 10 mM), only when the N-terminal peptide was chemically conjugated to rhodamine B (RhoB). In vitro, RhoB-conjugated EVP50 exhibits a concentration-dependent antitumor effect toward MCF-7 breast cancer cells. In MCF-7 cells, the RhoB-conjugated EVP50 nonapeptide accumulated in the cytoplasm and the nucleus within minutes. In the cytoplasm, the EVP50 RhoB-induced influx of extracellular calcium, intracellular calcium release and membrane lesions after incubation with micromolar concentrations of EVP50 conjugated with the fluorescent compound. The naïve unconjugated peptide was devoid of toxic and cytotoxic activities in our models in vivo and in vitro. Thus, the conjugate’s interference with calcium homeostasis, its nuclear function and its induced membrane dysfunction (lesion and vacuolization) seem to act in concert to break the cell circuitry. This synergistic mechanism of toxicity was restricted to structurally modified vipericidin nonapeptide. As a whole, this study contributes to the understanding of the molecular target (cyclophilin-A) that interacts with vipericidin peptide and a potential of this class of peptides in medical biotechnology against T. cruzi and anticancer activity when the peptide is conjugated.