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CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior In the continuing search for novel antibiotics, antimicrobial peptides are promising molecules, due to different mechanisms of action compared to classic antibiotics and to their selectivity for interaction with microorganism cells rather than with mammalian cells. Previously, our research group has isolated the antimicrobial peptide LyeTx I from the venom of the spider Lycosa erythrognatha. Here, we proposed to synthesize three novel shortened derivatives from LyeTx I (LyeTx I mn; LyeTx I mnΔK; LyeTx I mnΔKAc) and to evaluate their toxicity and biological activity as potential antimicrobial agents. Peptides were synthetized by Fmoc strategy and circular dichroism analysis was performed, showing that the three novel shortened derivatives may present membranolytic activity, like the original LyeTx I, once they folded as an alpha helix in 2.2.2-trifluorethanol and sodium dodecyl sulfate. In vitro assays revealed that the shortened derivative LyeTx I mnΔK presents the best score between antimicrobial (↓ MIC) and hemolytic (↑ EC50) activities among the synthetized shortened derivatives, and LUHMES cell-based NeuriTox test showed that it is less neurotoxic than the original LyeTx I (EC50 [LyeTx I mnΔK] ⋙ EC50 [LyeTx I]). In vivo data, obtained in a mouse model of septic arthritis induced by Staphylococcus aureus, showed that LyeTx I mnΔK is able to reduce infection, as demonstrated by bacterial recovery assay (∼10-fold reduction) and scintigraphic imaging (less technetium-99m labeled-Ceftizoxime uptake by infectious site). Infection reduction led to inflammatory process and pain decreases, as shown by immune cells recruitment reduction and threshold nociception increment, when compared to positive control group. Therefore, among the three shortened peptide derivatives, LyeTx I mnΔK is the best candidate as antimicrobial agent, due to its smaller amino acid sequence and toxicity, and its greater biological activity.

In the continuing search for novel antibiotics, antimicrobial peptides are promising molecules, due to different mechanisms of action compared to classic antibiotics and to their selectivity for interaction with microorganism cells rather than with mammalian cells. Previously, our research group has isolated the antimicrobial peptide LyeTx I from the venom of the spider Lycosa erythrognatha. Here, we proposed to synthesize three novel shortened derivatives from LyeTx I (LyeTx I mn; LyeTx I mnΔK; LyeTx I mnΔKAc) and to evaluate their toxicity and biological activity as potential antimicrobial agents. Peptides were synthetized by Fmoc strategy and circular dichroism analysis was performed, showing that the three novel shortened derivatives may present membranolytic activity, like the original LyeTx I, once they folded as an alpha helix in 2.2.2-trifluorethanol and sodium dodecyl sulfate. In vitro assays revealed that the shortened derivative LyeTx I mnΔK presents the best score between antimicrobial (↓ MIC) and hemolytic (↑ EC(50)) activities among the synthetized shortened derivatives, and LUHMES cell-based NeuriTox test showed that it is less neurotoxic than the original LyeTx I (EC(50) [LyeTx I mnΔK] ⋙ EC(50) [LyeTx I]). In vivo data, obtained in a mouse model of septic arthritis induced by Staphylococcus aureus, showed that LyeTx I mnΔK is able to reduce infection, as demonstrated by bacterial recovery assay (∼10-fold reduction) and scintigraphic imaging (less technetium-99m labeled-Ceftizoxime uptake by infectious site). Infection reduction led to inflammatory process and pain decreases, as shown by immune cells recruitment reduction and threshold nociception increment, when compared to positive control group. Therefore, among the three shortened peptide derivatives, LyeTx I mnΔK is the best candidate as antimicrobial agent, due to its smaller amino acid sequence and toxicity, and its greater biological activity.

Antimicrobial peptides (AMPs) have been found in all organism taxa and may play an essential role as a host defense system. AMPs are organized in various conformations, such as linear peptides, disulfide bond-linked peptides, backbone-linked peptides and circular peptides. AMPs apparently act primarily on the plasma membrane, although an increasing number of works have shown that they may also target various intracellular sites. Spider venoms are rich sources of biomolecules that show several activities, including modulation or blockage of ion channels, anti-insect, anti-cancer, antihypertensive and antimicrobial activities, among others. In spider venoms from the Lycosidae family there are many linear AMPs with a wide range of activities against several microorganisms. Due to these singular activities, some Lycosidae AMPs have been modified to improve or decrease desirable or undesirable effects, respectively. Such modifications, especially with the aim of increasing their antibiotic activity, have led to the filing of many patent applications. This review explores the abundance of Lycosidae venom AMPs and some of their derivatives, and their use as new drug models.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Peptídeos bioativos são uma grande classe de biomoléculas com diversos potenciais farmacológicos, destacando-se os peptídeos antimicrobianos que agem sobre diferentes organismos tais como bactérias, fungos, protozoários e vírus. Muitos desses peptídeos apresentaram também atividade antitumoral. Nesta tese, focamos na secreção de Pithecopus megacephalus, bem como no peptídeo LyeTx I-Des-His, uma variação sintética de LyeTx I, um peptídeo purificado da peçonha da aranha Lycosa erythrognatha, com o objetivo de encontrar peptídeos ativos principalmente contra células de câncer, especificamente do tipo triplo negativo. O fracionamento da secreção de P. megacephala, apresentou rica variedade de peptídeos e 14 destes foram sequenciados. Desse grupo três peptídeos foram sintetizados e testados contra bactérias, fungos e três linhagens de células de câncer (MDA-MB-231, MCF-7, ambas de câncer de mama e HCT-116 de câncer colorretal) e uma linhagem não câncer (HEK-293), como controle. Os resultados sugeriram aumento da atividade proliferativa de dois peptídeos. sobre células de câncer, sem aparente atividade em células HEK-293. Numa segunda parte deste trabalho, propusemos testar LyeTx I-Des-His combinado com cisplatina, em três diferentes proporções molares. A proporção peptídeo:cisplatina (P:C) 1:1, na dose do IC50, apresentou efeito sinérgico, bem como um valor de índice de seletividade maior que o da cisplatina isolada. Quando investigados os possíveis mecanismos de morte envolvidos, observamos diversos indícios de morte celular por autofagia. Este mecanismo de morte foi suportado pelos seguintes resultados: células tratadas com as combinações P:C pararam na fase G2/M do ciclo celular, células foram marcadas em vermelho com o corante pH sensível laranja de acridina e quando pré tratadas com inibidor de autofagia (Bafilomicina A1) esta correlação foi revertida. Além do mais, experimentos de western blot indicaram uma possível redução da expressão de P21 e da fosforilação de AKT. A redução da fosforilação de AKT pode ser indício de autofagia, como já descrito. Entretanto, outros estudos são necessários para melhor elucidar as vias envolvidas nas atividades desse peptídeo. Além do mais, estudos de toxicidade são essenciais na possível validação de LyeTx I-Des-His como um candidato a droga terapêutica. Esses achados demonstraram que o peptídeo LyeTx I-Des-His parece um bom candidato para ser combinado com alguns fármacos já bem estabelecidos, melhorando o combate a células de câncer, por induzir outras cascatas de morte. Bioactive peptides are a vast class of molecules with diverse biopharmaceutical potentials, including antimicrobial activity over a broad organism classes such as bacteria, fungi, protozoa and viruses. Several of these peptides have also shown antitumoral activity. In this thesis we focused on Pithecopus megacephalus frog secretion, as well as the peptide LyeTx I-Des-His, a synthetic variation of LyeTx I, a peptide purified from the venom of the spider Lycosa erythrognatha, with the objective to find bioactive peptides mainly against cancer cells, more specifically of the triple negative type. The fractionation of the P. megacephala secretion showed a rich variability on peptides and 14 of them were sequenced. From this set, three peptides were synthesized and tested against bacteria, fungi and three cancer cell lines (MDA-MB-231, MCF-7, both from breast cancer and HCT-116 from colorectal cancer) and one non-cancer cell line (HEK- 293), as a control. The results suggested an increase on proliferative activity of two peptides over cancer cells, without apparent activity on HEK-293 cells. In a second part of this work, we proposed to test LyeTx I-Des-His combined with cisplatin, in three different molar proportions. The proportion peptide:cisplatin, (P:C) 1:1, in the IC50 dose, showed synergistic effect as well as a selectivity index value, greater than cisplatin isolated treatment. When investigating the possible cell death mechanisms involved, we observed several indications of autophagic cell death process. This mechanism was supported by the following results: cells treated with P:C combinations arrested in G2/M cell cycle phase, cells were marked in red with the pH sensitive dye acridine orange and when pretreated with an autophagy inhibitor (Bafilomycin A1) this coloration was reversed. In addition, western blot experiments indicated a possible reduction of P21 expression and of AKT phosphorylation. The decreasing of AKT phosphorylation could be indicative of autophagy, as already described. However, other studies are necessary to better clarify the involved pathways on these activities of the drugs. In addition, toxicity studies are essential in a possible validation of LyeTx I-DesHis as a therapeutic drug candidate These findings demonstrated that the peptide LyeTx I-Des-His seems a good candidate to be combined with some well-established drugs, improving the combat to cancer cells, by inducing other death cascades.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Outra Agência Doenças infecciosas é uma significativa causa de morte e, infecções bacterianas aparecem entre as 10 principais causas de mortes no mundo. Os fungos também podem causar graves infecções, especialmente em pessoas imunossuprimidas. No entanto, apesar de existir um grande arsenal de antimicrobianos como os antibacterianos e antifúngicos é importante o desenvolvimento de novos fármacos, considerando-se o aparecimento de linhagens resistentes aos medicamentos existentes. Os peptídeos com atividade antimicrobiana, encontrados em diversos organismos multicelulares, são potenciais candidatos para modelos de novos antibióticos. Neste trabalho o peptídeo antimicrobiano denominado LyeTx I, obtido da peçonha da aranha Lycosa erythrognatha, foi utilizado como modelo de estudo. Sintetizou-se, pelo método de Fmoc, um peptídeo representando parte da sequência de LyeTx I, mas com diferentes modificações, obtendo-se três diferentes derivados: LyeTxImn, LyeTxImnΔK e LyeTxImnΔKAc. Estes peptídeos derivados apresentaram diferentes atividades antimicrobianas, bem como diferentes atividades hemolíticas. Esta última é um importante indicador da atividade dos peptídeos antimicrobianos em células de mamífero, isto é, peptídeos com alta atividade hemolítica em geral não são bons candidatos para antibióticos. Dos peptídeos derivados obtidos, somente LyeTxImnΔK foi mais eficaz do que o peptídeo original, LyeTx I, quando seus índices terapêuticos foram comparados.Investigou-se ainda, a estruturação desses peptídeos em meios miméticos tais como trifluoretanol, dodecil sulfato de sódio e dodecil fosfocolina, sendo encontrado padrões de estruturação condizentes com perfis de α-hélice, semelhante ao peptídeo LyeTx I original. Em conclusão, obtivemos por síntese química, um peptídeo menor, derivado da estrutura da toxina original (LyeTxI), com mais alta atividade para alguns fungos e bactérias, menos hemolítico bem como, com menor custo de produção, características fundamentais para um candidato a novo fármaco. Infectious disease is one big cause to death, where bacterium infections show among some 10 mainly death´s causes in the world. Fungi can cause severe diseases too, particularly in imunossupressed people. However, although there are a vast antimicrobial arsenal, as antibiotics antimicrobials and antimycotic against fungi is important to develop new medicines, considering the growing of resistence by microorganisms. Antimicrobial peptides found in many multicellular organisms could be good candidates to this aim. In this work, the antimicrobial peptide, LyeTx I, from the spider Lycosa erythrognatha´s venom was used as a model. Part of this molecule (LyeTxI minimized) was synthesized by the Fmoc method. Three derivatives were obtained: LyeTxImn, LyeTxImnΔK and LyeTxImnΔKAc. They showed different antimicrobial activities as well as different hemolytic activities. This last activity is an important indicator to verify if the antimicrobial peptide is also active in cells from mammals. A high hemolytic activity is not a good indicator for a peptide as a good antibiotic. But only LyeTxImnΔK was more effective than the native peptide (LyeTx I), when compared therapeutic index among them. The structuration of these peptides was also investigated in mimetic media such as trifluorethanol, sodium dodecyl sulfate and dodecyl phosphocholine, being found structuration patterns consistent with α-helix profiles, similar to the original peptide LyeTx I. Therefore, was possible the synthesis of a peptide derivative from the a spider toxin LyeTxI with higher antimicrobial activities, as well as with lower production cost, some, essential characteristics to a candidate to newtherapeutic drugs.