Síntese, estudos conformacionais e atividade biológica de novos análogos do peptídeo antimicrobiano gomesina

• Ano de defesa: 2016
• Autor(a) principal: Mendes, Roberta Brandao [UNIFESP]
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Gomesin; Peptide synthesis; Antimicrobial activity; Hemolytic activity; Antitumor activity; GUVs ite; Gomesina; Síntese de peptídeos; atividade antimicrobiana atividade hemolítica atividade antitumoral GUVs ite
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=3686566; http://repositorio.unifesp.br/handle/11600/46895

Gomesin (ZCRRLCYKQRCVTYCRGR-NH2) is an antimicrobial peptide isolated from hemocytes of the Brazilian spider Acanfhoscurria gomesiana. The molecule has four cysteines that form two intramolecular disulfide bridges at positions 2.15 and 6.11. Gm has a broad spectrum of action and a high toxicity against human erythrocytes. This study aimed to the synthesis of new analogues of gomesin to evaluate their structure-activity and their mechanisms of action in comparison to Gm. Thus, linear and cyclic analogs were synthesized, Ac-Gm2-15, Ac-[D-Lys8]-Gm2-15, Ac-Ifrp", D-Lys8]-Gm2-15, Ac- [Thr2,6,11,15]-Gm2_1A5c, -[ Thr2,6,11,15,D-Lys8]-Gm2_1a5nd Ac-[Thr2,6,11,15,D-Pro9]-Gm2_1b5y using the solid phase peptides synthesis in a t-Boc strategy. Then peptides were cleaved, purified and characterized. Antimicrobial activity were evaluated against C. albicans, B. megaferium, P. aeruginosa, P. expansum, C. neoformans, A. niger, Cladosporium sp and S. cerevisiae; hemolytic activity was determined against human erythrocytes; Peptides degradation resistance in human plasma and antitumoral activity against K562 cells were also evaluated. Structural analysis by Circular Dichroism spectroscopy and peptide/vesicle interaction using giant unilamellar vesicles and optical microscopy were also performed. Thermodynamical studies of the interaction peptide I lipid using large unilamellar vesicles (LUVs) was done by isothermal titration calorimetry (ITC). Our results showed that analogues, Ac-Gm2-15, Ac-[D-Lys8]-Gm2-15 and Ac-IIrp", D-Lys8]-Gm2-15, presented the best antimicrobial activity, particularly against microorganisms A. nigerand S. cerevisiae, their performance were even better than Gm. Ali showed low hemolytic activity even at 100 IJM concentration. They were quite resistant to degradation. Fro the Circular Dichroism studies, we observed that the cyclic compounds showed conformations similar than to gomesin, they also assume a p-hairpin conformation. On the other hand, linear analogues showed a prevalence of random conformation. The Iytic mechanism of action of Gm and Ac-Gm2-15 showed to be very similar, both of them caused a disruption of the membrane. Ac-[Thr2,6,11,15]-Gm2_1p5resented a pore formation mechanism. From our results, we had confirmed the crucial importance of the disulfide bridges in the Iytic mechanism of action; in fact its removal causes a decrease in the antimicrobial, hemolytic and antitumor activities. The disulfide bridges also have a great contribution to the stability and strength the action against plasma protease. Gomesin fragments should have less potency, maybe due to the lower amount of charge and also because of the removal of amino acids in the N- and C-terminal portions