RESUMO
Leucine residues are commonly found in the hydrophobic face of antimicrobial peptides (AMPs) and are crucial for membrane permeabilization, leading to the cell death of invading pathogens. Melittin, which contains four leucine residues, demonstrates broad-spectrum antimicrobial properties but also significant cytotoxicity against mammalian cells. To enhance the cell selectivity of melittin, this study synthesized five analogs by replacing leucine with its structural isomer, 6-aminohexanoic acid. Among these analogs, Mel-LX3 exhibited potent antibacterial activity against both Gram-positive and Gram-negative bacteria. Importantly, Mel-LX3 displayed significantly reduced hemolytic and cytotoxic effects compared to melittin. Mechanistic studies, including membrane depolarization, SYTOX green uptake, FACScan analysis, and inner/outer membrane permeation assays, demonstrated that Mel-LX3 effectively permeabilized bacterial membranes similar to melittin. Notably, Mel-LX3 showed robust antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Furthermore, Mel-LX3 effectively inhibited biofilm formation and eradicated existing biofilms of MDRPA. With its improved selective antimicrobial and antibiofilm activities, Mel-LX3 emerges as a promising candidate for the development of novel antimicrobial agents. We propose that the substitution of leucine with 6-aminohexanoic acid in AMPs represents a significant strategy for combating resistant bacteria.
Assuntos
Antibacterianos , Biofilmes , Meliteno , Staphylococcus aureus Resistente à Meticilina , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa , Meliteno/farmacologia , Meliteno/química , Biofilmes/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Humanos , Hemólise/efeitos dos fármacos , Ácido Aminocaproico/química , Ácido Aminocaproico/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , AnimaisRESUMO
Hirudo nipponia, a blood-sucking leech native to East Asia, possesses a rich repertoire of active ingredients in its saliva, showcasing significant medical potential due to its anticoagulant, anti-inflammatory, and antibacterial effects against human diseases. Despite previous studies on the transcriptomic and proteomic characteristics of leech saliva, which have identified medicinal compounds, our knowledge of tissue-specific transcriptomes and their spatial expression patterns remains incomplete. In this study, we conducted an extensive transcriptomic profiling of the salivary gland tissue in H. nipponia based on de novo assemblies of tissue-specific transcriptomes from the salivary gland, teeth, and general head region. Through gene ontology (GO) analysis and hierarchical clustering, we discovered a novel set of anti-coagulant factors-i.e., Hni-Antistasin, Hni-Ghilanten, Hni-Bdellin, Hni-Hirudin-as well as a previously unrecognized immune-related gene, Hni-GLIPR1 and uncharacterized salivary gland specific transcripts. By employing in situ hybridization, we provided the first visualization of gene expression sites within the salivary gland of H. nipponia. Our findings expand on our understanding of transcripts specifically expressed in the salivary gland of blood-sucking leeches, offering valuable resources for the exploration of previously unidentified substances with medicinal applications.
Assuntos
Hirudo medicinalis , Sanguessugas , Animais , Perfilação da Expressão Gênica , Hirudo medicinalis/genética , Hirudo medicinalis/metabolismo , Sanguessugas/genética , Sanguessugas/metabolismo , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Proteômica , Glândulas Salivares/metabolismoRESUMO
Polo-like kinase 1 (PLK1), which is crucial in cell cycle regulation, is considered a promising anticancer drug target. Herein, we present the N-degron pathway-based proteolysis targeting chimera (PROTAC) for PLK1 degradation, targeting the Polo-box domain (PBD). We identified DD-2 as the most potent PROTAC that selectively induces PLK1 degradation in cancer cells, including HeLa and nonsmall cell lung cancer (NSCLC), through the N-degron pathway. DD-2 exhibited significant in vitro anticancer effects, inducing G2/M arrest and apoptosis in HeLa and NSCLC cell lines. DD-2 showed significant tumor growth inhibition in a xenograft mouse model using HeLa and NSCLC cell lines, highlighting its potential in cancer treatment. Furthermore, the combination of DD-2 with tyrosine kinase inhibitor (TKI), osimertinib, effectively suppressed tumor growth in double-mutated H1975 cell lines, emphasizing DD-2's potential in combination cancer therapies. Collectively, this study demonstrates the potential of the N-degron pathway, especially using DD-2, for targeted cancer therapies.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Animais , Camundongos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Ciclo Celular , Quimera de Direcionamento de Proteólise , Proteínas Serina-Treonina Quinases , Quinase 1 Polo-Like , Apoptose , Degrons , Linhagem Celular Tumoral , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Pontos de Checagem da Fase G2 do Ciclo Celular , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Abstract Introduction Methicillin-resistant staphylococcus aureus is an emerging problem for the treatment of chronic suppurative otitis media, and also for pediatric tympanostomy tube otorrhea. To date, there are no effective topical antibiotic drugs to treat methicillin-resistant staphylococcus aureus otorrhea. Objective In this study, we evaluated the ototoxicity of topical KR-12-a2 solution on the cochlea when it is applied topically in the middle ear of guinea pigs. Methods The antimicrobial activity of KR-12-a2 against methicillin-resistant staphylococcus aureus strains was examined by using the inhibition zone test. Topical application of KR-12-a2 solution, gentamicin and phosphate buffered saline were applied in the middle ear of the guinea pigs after inserting ventilation tubes. Ototoxicity was assessed by auditory brainstem evoked response and scanning electron microscope examination. Results KR-12-a2 produced an inhibition zone against methicillin-resistant staphylococcus aureus from 6.25 µg. Hearing threshold in the KR-12-a2 and PBS groups were similar to that before ventilation tube insertion. However, the gentamicin group showed elevation of the hearing threshold and there were statistically significant differences compared to the phosphate buffered saline or the KR-12-a2 group. In the scanning electron microscope findings, the KR-12-a2 group showed intact outer hair cells. However, the gentamicin group showed total loss of outer hair cells. In our experiment, topically applied KR-12-a2 solution did not cause hearing loss or cochlear damage in guinea pigs. Conclusion In our experiment, topically applied KR-12-a2 solution did not cause hearing loss or cochlear damage in guinea pigs. The KR-12-a2 solution can be used as ototopical drops for treating methicillin-resistant staphylococcus aureus otorrhea; however, further evaluations, such as the definition of optimal concentration and combination, are necessary.
Resumo Introdução O staphylococcus aureus resistente à meticilina é um problema emergente não só para a otite média supurativa crônica, mas também para casos de otorreia crônica em crianças com tubo de ventilação. Até o momento, não há antibióticos tópicos efetivos para a otorreia causada por staphylococcus aureus resistente à meticilina. Objetivo Nesse estudo, avaliamos a ototoxicidade da solução tópica de KR-12-a2 na cóclea quando aplicada topicamente na orelha média de cobaias. Método A atividade antimicrobiana de KR-12-a2 contra cepas de staphylococcus aureus resistente à meticilina foi avaliada utilizando-se o teste de zona de inibição de crescimento. Foram aplicados na orelhas médias de 3 grupos de cobaias, ou solução tópica de KR-12-a2, ou gentamicina ou solução salina tamponada com fosfato após timpanostomia. A ototoxicidade foi avaliada através do exame auditivo de potencial evocado auditivo de tronco encefálico e por microscopia eletrônica de varredura. Resultados O KR-12-a2 produziu uma zona de inibição contra o staphylococcus aureus resistente à meticilina a partir de 6,25 µg. Alterações do limiar de audição no grupo KR-12-a2 e no grupo com solução salina foram semelhantes aos observados antes da inserção do tubo de ventilação. No entanto, o grupo gentamicina apresentou um limiar auditivo mais elevado, estatisticamente significativo em comparação ao grupo solução salina ou ao grupo KR-12-a2. Nos achados da microscopia eletrônica, o grupo KR-12-a2 apresentou células ciliadas externas intactas. No entanto, o grupo gentamicina apresentou perda total das células ciliadas externas. Em nosso experimento, a solução de KR-12-a2 aplicada topicamente não causou perda auditiva ou dano coclear em cobaias. Conclusão Em nosso experimento, a solução de KR-12-a2 aplicada topicamente não causou perda auditiva ou dano coclear em cobaias. A solução de KR-12-a2 pode ser utilizada como gotas otológicas para o tratamento da otorreia causada por staphylococcus aureus resistente à meticilina; no entanto, são necessárias outras avaliações, para a definição da concentração e das associações ideais.