LyeTxI-b, a Synthetic Peptide Derived From a Spider Venom, Is Highly Active in Triple-Negative Breast Cancer Cells and Acts Synergistically With Cisplatin.

Breast cancer is the most common cancer that affects women globally and is among the leading cause of women's death. Triple-negative breast cancer is more difficult to treat because hormone therapy is not available for this subset of cancer. The well-established therapy against triple-negative breast cancer is mainly based on surgery, chemotherapy, and immunotherapy. Among the drugs used in the therapy are cisplatin and carboplatin. However, they cause severe toxicity to the kidneys and brain and cause nausea. Therefore, it is urgent to propose new chemotherapy techniques that provide new treatment options to patients affected by this disease. Nowadays, peptide drugs are emerging as a class of promising new anticancer agents due to their lytic nature and, apparently, a minor drug resistance compared to other conventional drugs (reviewed in Jafari et al., 2022). We have recently reported the cytotoxic effect of the antimicrobial peptide LyeTx I-b against glioblastoma cells (Abdel-Salam et al., 2019). In this research, we demonstrated the cytotoxic effect of the peptide LyeTx I-b, alone and combined with cisplatin, against triple-negative cell lines (MDA-MD-231). LyeTx-I-b showed a selectivity index 70-fold higher than cisplatin. The peptide:cisplatin combination (P:C) 1:1 presented a synergistic effect on the cell death and a selective index value 16 times greater than the cisplatin alone treatment. Therefore, an equi-effective reduction of cisplatin can be reached in the presence of LyeTx I-b. Cells treated with P:C combinations were arrested in the G2/M cell cycle phase and showed positive staining for acridine orange, which was inhibited by bafilomycin A1, indicating autophagic cell death (ACD) as a probable cell death mechanism. Furthermore, Western blot experiments indicated a decrease in P21 expression and AKT phosphorylation. The decrease in AKT phosphorylation is indicative of ACD. However, other studies are still necessary to better elucidate the pathways involved in the cell death mechanism induced by the peptide and the drug combinations. These findings confirmed that the peptide LyeTx I-b seems to be a good candidate for combined chemotherapy to treat breast cancer. In addition, in vivo studies are essential to validate the use of LyeTx I-b as a therapeutic drug candidate, alone and/or combined with cisplatin.

Synthetic Peptides Derived From Lycosa Erythrognatha Venom: Interaction With Phospholipid Membranes and Activity Against Resistant Bacteria.

Superbugs are a public health problem, increasing the need of new drugs and strategies to combat them. Our group has previously identified LyeTxI, an antimicrobial peptide isolated from Lycosa erythrognatha spider venom. From LyeTxI, we synthesized and characterized a derived peptide named LyeTxI-b, which has shown significant in vitro and in vivo activity. In this work, we elucidate the interaction of LyeTxI-b with artificial membranes as well as its effects on resistant strains of bacteria in planktonic conditions or biofilms. Isothermal titration calorimetry revealed that LyeTxI-b interacts more rapidly and with higher intensity with artificial vesicles, showing higher affinity to anionic vesicles, when compared to synthetic LyeTxI. In calcein experiments, LyeTxI-b caused greater levels of vesicle cleavage. Both peptides showed antibacterial activity at concentrations of µmol L-1 against 12 different clinically isolated strains, in planktonic conditions, in a concentration-dependent manner. Furthermore, both peptides elicited a dose-dependent production of reactive oxygen species in methicillin-resistant Staphylococcus aureus. In S. aureus biofilm assay, LyeTxI-b was more potent than LyeTxI. However, none of these peptides reduced Escherichia coli biofilms. Our results show LyeTxI-b as a promising drug against clinically resistant strains, being a template for developing new antibiotics.

A New Topical Eye Drop Containing LyeTxI-b, A Synthetic Peptide Designed from A Lycosa erithrognata Venom Toxin, Was Effective to Treat Resistant Bacterial Keratitis.

Bacterial keratitis is an ocular infection that can lead to severe visual disability. Staphylococcus aureus is a major pathogen of the eye. We recently demonstrated the strong antimicrobial activity of LyeTxI-b, a synthetic peptide derived from a Lycosa erithrognatha toxin. Herein, we evaluated a topical formulation (eye drops) containing LyeTxI-b to treat resistant bacterial keratitis. Keratitis was induced with intrastromal injection of 4 × 105 cells (4 µL) in New Zealand female white rabbits. Minimum inhibitory concentration (MIC) and biofilm viability were determined. LyeTxI-b ocular toxicity was evaluated through chorioallantoic membrane and Draize tests. One drop of the formulation (LyeTxI-b 28.9 µmol/L +0.5% CMC in 0.9% NaCl) was instilled into each eye four times a day, for a week. Slit-lamp biomicroscopy analysis, corneal histopathological studies and cellular infiltrate quantification through myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) detection were performed. LyeTxI-b was very effective in the treatment of keratitis, with no signs of ocular toxicity. Planktonic bacteria MIC was 3.6 µmol/L and LyeTxI-b treatment reduced biofilm viability in 90%. LyeTxI-b eliminated bacteria and reduced inflammatory cellular activity in the eyes. Healthy and treated animals showed similar NAG and MPO levels. LyeTxI-b is a potent new drug to treat resistant bacterial keratitis, showing effective antimicrobial and anti-inflammatory activity.

LyeTxI-b, a Synthetic Peptide Derived From Lycosa erythrognatha Spider Venom, Shows Potent Antibiotic Activity in Vitro and in Vivo.

The antimicrobial peptide LyeTxI isolated from the venom of the spider Lycosa erythrognatha is a potential model to develop new antibiotics against bacteria and fungi. In this work, we studied a peptide derived from LyeTxI, named LyeTxI-b, and characterized its structural profile and its in vitro and in vivo antimicrobial activities. Compared to LyeTxI, LyeTxI-b has an acetylated N-terminal and a deletion of a His residue, as structural modifications. The secondary structure of LyeTxI-b is a well-defined helical segment, from the second amino acid to the amidated C-terminal, with no clear partition between hydrophobic and hydrophilic faces. Moreover, LyeTxI-b shows a potent antimicrobial activity against Gram-positive and Gram-negative planktonic bacteria, being 10-fold more active than the native peptide against Escherichia coli. LyeTxI-b was also active in an in vivo model of septic arthritis, reducing the number of bacteria load, the migration of immune cells, the level of IL-1ß cytokine and CXCL1 chemokine, as well as preventing cartilage damage. Our results show that LyeTxI-b is a potential therapeutic model for the development of new antibiotics against Gram-positive and Gram-negative bacteria.

Efeito antimicrobiano do peptídeo Lye Tx-I associado à ß-Ciclodextrina em biofilmes de bacterias patógenas periodontais: estudo in vitro / Effect of antimicrobial Peptide Lye Tx-I associated with ß-Cyclodextrin in biofilms of bacterial periodontal patógenas: in vitro study

Biofilmes bacterianos estão associados como fator etiológico da doença periodontal, uma vez que são difíceis de controlar por meio da terapia mecânica e antibiótica convencional. As células bacterianas dos biofilmes possuem mecanismos de resistência bacteriana tornando-as difíceis de neutralizar tanto pela resposta imune do hospedeiro quanto à ação dos antibacterianos. Uma alternativa para o controle dos biofilmes bacterianos, é o uso de peptídeos antimicrobianos, uma vez que tem amplo espectro e baixa incidência de resistência bacteriana. O presente estudo investigou a atividade antibacteriana do peptídeo sintético denominado LyeTx I, originário do veneno de uma aranha (Lycosaerithognata), em sua forma livre ou associado em ßciclodextrina (ßCD) contra bactérias periodontopatógenas colonizadoras iniciais e tardias tanto em estado planctônico quanto em biofilme. A mistura entre LyeTx I e a ßCD foi caracterizada por espectroscopia de infravermelho. Para conhecer a cinética das bactérias testadas, foram feitas curvas de crescimento bacteriano tanto em estado planctônico quanto em biofilmes formados sobre pinos de polietileno no dispositivo de biofilme da Universidade de Calgary. Para confirmar a formação e estrutura dos biofilmes, os pinos foram examinados por microscopia eletrônica de varredura em diferentes tempos (2, 5, 10 dias). Em seguida, foram determinadas as concentrações inibitória mínima (CIM) e bactericida mínima (CBM) dos S. mutans, S. oralis, S. sanguinis, E. corrodens, L. acidophilus, L. casei, F. nucleatum e P. intermedia. Inicialmente, o efeito da cinética do LyeTxI nas bactérias foi determinado por meio do ensaio de curva de morte usando as CIMs. Adicionalmente, foram determinadas a concentração mínima de erradicação do biofilme (CMEB) e a porcentagem de redução da atividade metabólica tanto em biofilme jovem (2 dias) quanto para biofilme maturo (4 dias). As CIMs tanto do LyeTxI quanto do LyeTx I/ßCD foram entre 7,81- 62,5 µg/mL em estado planctônico sendo que o S. mutans, S. oralis, S. sanguinis e E. corrodens foram as mais sensíveis ao LyeTx I e ao LyeTx I/ßCD. O efeito antibacteriano do LyeTx I começou dentro dos primeiros 15 minutos e se manteve durante 10 horas de acordo com os resultados obtidos nas curvas de morte das bactérias testadas. A porcentagem de redução da atividade metabólica das células do biofilme foi determinada usando resazurina e leitura da fluorescência (λex570 nm/ λem 590 nm). Os resultados mostraram que tanto o LyeTx I quanto o LyeTx I/ßCD quando comparados à clorexidina (controle), apresentaram uma redução da atividade metabólica das células dos biofilmes de 2 dias na maior concentração (250 µg/mL), porém não houve diferença estatisticamente significativa após análise da variância e o teste Tukey de multiplex comparações entre os tratamentos testados (p>0,05). Contudo, dentre os agentes testados nenhum foi capaz de reduzir a atividade metabólica das células do biofilme de 4 dias, sendo que a clorexidina diminuiu levemente a atividade metabólica do biofilme maturo (p< 0,05). Os resultados do presente trabalho sugerem que, o peptídeo LyeTx I e o composto de inclusão LyeTx I/ßCD possuem efeito antibacteriano contra bactérias patógenas periodontais, agindo de forma bacteriostática por um período de até 10 horas. LyeTx I e LyeTx I/ßCD possuem efeito antibacteriano em biofilmes jovens (2 dias), indicando o potencial uso destes como coadjuvantes na terapia mecânica periodontal

Biofilms are involved as etiology of bacterial infections such as periodontal disease, once they difficult to control with conventional mechanic and antibiotic therapy. Biofilms cells display mechanism of resistance making them tolerant against immune response and antibiotics. Antimicrobial peptides (AMPs) are an alternative to control bacterial biofilms because their wide spectrum and low rate of bacterial resistance. Therefore, the aim of this study was to evaluate the antibacterial activity of the synthetic peptide LyeTx I (originary of the poison of a spider Lycosaerithognata), free and associated with beta-cyclodextrin (ßCD) against both, first and late colonizer bacteria in planktonic state and in biofilms. The mixture of LyeTx I and ßCDwas characterized by infrared absorption spectroscopy. Planktonic and biofilm growth curves were done to identify the kinetics of tested bacteria. Biofilms were formed on polyethylene pegs using the calgary biofilm device (CBD) of the University of Calgary. To determine whether differences in biofilm architecture and composition exist between immature biofilms to mature biofilms formed on pegs, we examined biofilms by scanning electron microscopy at 2, 5 and 10 days. Then, the minimal inhibitory concentrations (MIC) and minimal bactericidal concentration (MBC) were determined for S. mutans, S. oralis, S. sanguinis, E. corrodens, L. acidophilus, L. casei, F. nucleatum and P. intermedia. Killing kinetics assays were performed for each species at MICs to determine the kinetic of LyeTx I. Additionally, the minimal biofilm eradication concentration (MBEC) and the percent of reduction in metabolic activity were determined for young (2-day) and mature (4-day) multispecies biofilms. The band detected between 1200-1700 cm1 regions shows a decrease of functional group C-H aliphatic stretching vibrations that may be attributed to the formation of van der Waals interactions between ßCD and LyeTx I, thus confirming the presence of the association compound LyeTx I/ßCD. The MICs of the LyeTx I and the LyeTx I /ßCD were found between 62.5 - 7.81 µg /mL in planktonic state. S. mutans, S. oralis, S. sanguinis and E. corrodens were the most sensitive to LyeTx I and LyeTx I /ßCD. The antibacterial effect of LyeTx I started within the first 15 minutes and continued for 10 hours according to results of death curves on tested bacteria. The percentage reduction of metabolic activity of biofilm cells was determined using resazurin and fluorometry (λex 570 nm / λof 590 nm). LyeTx I, LyeTx I/ßCD and chlorhexidine (control) decreased the metabolic activity of 2-day biofilm cells at 250 µg/mL and there were no statistically significant difference between the treatments (p> 0.05). In contrast, any of the tested agents decreased metabolic activity of 4-day biofilm cells. Chlorhexidine was able to slightly reduce metabolic activity of mature biofilm (p<0.05). In conclusion, the LyeTx I peptide free or associated with ßCD possesses antibacterial effect against planktonic periodontal pathogenic bacteria and reduce the metabolic activity in young biofilm cells acting as a bacteriostatic agent for a period of 10 hours. Therefore, this study suggests that LyeTx I and LyeTx I/ßCD are promising antibacterial agents for the control of 2-day biofilms, which suggest their use as coadjuvant in periodontal mechanical therapy