RESUMO
2K4L is a rationally designed analog of the short α-helical peptide temporin-1CEc, a natural peptide isolated and purified from the skin secretions of the Chinese brown frog Rana chensinensis by substituting amino acid residues. 2K4L displayed improved and broad-spectrum antibacterial activity than temporin-1CEc in vitro. Here, the antibacterial and anti-inflammatory activities of 2K4L in macrophages, C. elegans and mice were investigated. The results demonstrated that 2K4L could enter THP-1 cells to kill a multidrug-resistant Acinetobacter baumannii strain (MRAB 0227) and a sensitive A. baumannii strain (AB 22933), as well as reduce proinflammatory responses induced by MRAB 0227 by inhibiting NF-κB signaling pathway. Similarly, 2K4L exhibited strong bactericidal activity against A. baumannii uptake into C. elegans, extending the lifespan and healthspan of the nematodes. Meanwhile, 2K4L alleviated the oxidative stress response by inhibiting the expression of core genes in the p38 MAPK/PMK-1 signaling pathway and downregulating the phosphorylation level of p38, thereby protecting the nematodes from damage by A. baumannii. Finally, in an LPS-induced septic model, 2K4L enhanced the survival of septic mice and decreased the production of proinflammatory cytokines by inhibiting the signaling protein expression of the MAPK and NF-κB signaling pathways and protecting LPS-induced septic mice from a lethal inflammatory response. In conclusion, 2K4L ameliorated LPS-induced inflammation both in vitro and in vivo.
Assuntos
Acinetobacter baumannii , Caenorhabditis elegans , Lipopolissacarídeos , Macrófagos , Choque Séptico , Animais , Caenorhabditis elegans/efeitos dos fármacos , Camundongos , Acinetobacter baumannii/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Choque Séptico/tratamento farmacológico , Choque Séptico/induzido quimicamente , Choque Séptico/metabolismo , NF-kappa B/metabolismo , Peptídeos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos/química , Humanos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Antibacterianos/farmacologia , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Estresse Oxidativo/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno , Proteínas de Caenorhabditis elegansRESUMO
Breast cancer is a leading cause of mortality and the most prevalent form of malignant tumor among women worldwide. Breast cancer cells exhibit an elevated glycolysis and altered glucose metabolism. Moreover, these cells display abnormal glycosylation patterns, influencing invasion, proliferation, metastasis, and drug resistance. Consequently, targeting glycolysis and mitigating abnormal glycosylation represent key therapeutic strategies for breast cancer. This review underscores the importance of protein glycosylation and glucose metabolism alterations in breast cancer. The current research efforts in developing effective interventions targeting glycolysis and glycosylation are further discussed.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/metabolismo , Glicosilação , Glicólise , Glucose/metabolismo , Linhagem Celular Tumoral , Proliferação de CélulasRESUMO
The emergence of multidrug- or extremely drug-resistant M. tuberculosis strains has made very few drugs available for current tuberculosis treatment. Antimicrobial peptides can be employed as a promising alternative strategy for TB treatment. Here, we designed and synthesized a series of peptide sequences based on the structure-activity relationships of natural sequences of antimicrobial peptides. The peptide W3R6 and its analogs were screened and found to have potent antimycobacterial activity against M. smegmatis, and no hemolytic activity against human erythrocytes. The evidence from the mechanism of action study indicated that W3R6 and its analogs can interact with the mycobacterial membrane in a lytic manner and form pores on the outer membrane of M. smegmatis. Significant colocalization of D-W3R6 with mycobacterial DNA was observed by confocal laser scanning microscopy and DNA retardation assays, which suggested that the antimycobacterial mechanism of action of the peptide was associated with the unprotected genomic DNA of M. smegmatis. In general, W3R6 and its analogs act on not only the mycobacterial membrane but also the genomic DNA in the cytoplasm, which makes it difficult for mycobacteria to generate resistance due to the peptides having two targets. In addition, the peptides can effectively eliminate M. smegmatis cells from infected macrophages. Our findings indicated that the antimicrobial peptide W3R6 could be a novel lead compound to overcome the threat from drug-resistant M. tuberculosis strains in the development of potent AMPs for TB therapeutic applications.
Assuntos
Mycobacterium , Tuberculose , Humanos , Antituberculosos/farmacologia , Peptídeos Antimicrobianos , Peptídeos/farmacologia , Peptídeos/químicaRESUMO
Acute lung injury (ALI) is a life-threatening disease that is generally attributable to an uncontrolled inflammatory response in the lung, but there is a lack of effective treatments. At present, regulating the inflammatory response has become an important strategy for treating ALI. In the present study, LK2(6)A(L), a peptide derived from the natural antimicrobial peptide temporin-1CEa, inhibited lipopolysaccharide (LPS)-induced expression of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and NO in RAW264.7 cells. Herein, the anti-inflammatory mechanism of LK2(6)A(L) was investigated. The RNA-sequencing (RNA-seq) results showed that LK2(6)A(L) significantly inhibited the TLR4-mediated NF-κB and MAPK signaling pathways in LPS-induced RAW264.7 cells. The results of co-immunoprecipitation (Co-IP), pull-down experiment, confocal laser scanning microscopy, and surface plasmon resonance (SPR) suggested that MD2 was the direct target of LK2(6)A(L). Chemical inhibition of MD2 and its knockdown abolished the anti-inflammatory effect of LK2(6)A(L). Molecular dynamic simulation indicated that LK2(6)A(L) could bind to the active domain of the MD2 hydrophobic pocket via six hydrogen bonds. The truncated peptides were designed based on analysis of the molecular docking of LK2(6)A(L) to MD2. The truncated peptide IS-7 showed strong affinity to MD2 and a remarkable inhibitory effect on pro-inflammatory factors that was comparable to the effect of LK2(6)A(L). Finally, LK2(6)A(L) and IS-7 relieved inflammatory symptoms and lung tissue destruction in the ALI mouse model. Overall, our study suggested that LK2(6)A(L) showed promising anti-inflammatory activity by targeting MD2, and the amino acid domain 7-13 was an important area that binds with MD2 and also an anti-inflammatory active region. LK2(6)A(L) and IS-7 may be potential new treatments for ALI and other acute inflammatory diseases.
Assuntos
Lesão Pulmonar Aguda , Lipopolissacarídeos , Camundongos , Animais , Lipopolissacarídeos/efeitos adversos , Simulação de Acoplamento Molecular , Peptídeos Antimicrobianos , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Anti-Inflamatórios/efeitos adversos , Diferenciação CelularRESUMO
Chensinin-1b and its lipoanalogs demonstrate different anticancer activities against selected cancer cells, and the anticancer activity of PA-C1b is improved up to 3-fold compared with that of the parent peptide chensinin-1b. However, detailing the mechanism of action of these peptides is required to better understand the structure-function relationship. In this study, chensinin-1b and PA-C1b were selected as the representative peptides to investigate the mode of action in cancer cells. The results indicated that the boundary of the cell membrane was broken when the cells were treated with chensinin-1b, while that of cells treated with PA-C1b remained intact based on morphological changes. Apoptosis assays indicated that PA-C1b induced MCF-7 cancer cell apoptosis, while chensinin-1b mainly damaged the cell membrane. MCF-7 cancer cells treated with the peptides induced the loss of mitochondrial membrane potential, and cytochrome c was released from mitochondria, but PA-C1b enhanced ROS generation. Additionally, PA-C1b uptake occurred via an energy-dependent pathway and was inhibited by selected endocytosis inhibitors. Furthermore, treatment of MCF-7 cells with PA-C1b suppressed Bcl-2 mRNA levels and increased Bax mRNA levels, upregulated the expression of the proapoptotic protein Bax and downregulated the expression of the antiapoptotic protein Bcl-2. These results indicate that the anticancer mechanism of AMPs may be considerably affected by only a slight difference in the hydrophobicity of the two peptides; and such a study may facilitate the design of novel peptide-based anticancer agents.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Antimicrobianos/metabolismo , Testes de Sensibilidade Microbiana/métodos , Espécies Reativas de Oxigênio/metabolismo , Feminino , Humanos , Interações Hidrofóbicas e HidrofílicasRESUMO
Mobilization of hippocampal neurogenesis has been considered as a potential strategy for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD). In present study, we evaluated both the neuroprotective effects and the effects on the proliferation and differentiation of APP-overexpressing neural stem cells (APP-NSCs) by Jujuboside A (JuA) in vitro. Our results demonstrated that JuA (50 µM) decreased apoptosis and suppressed oxidative stress damage of APP-NSCs. JuA (50 µM) upregulated the secretion of brain-derived neurotrophic factor and promoted the proliferation and neuronal differentiation of APP-NSCs. Moreover, JuA (50 µM) upregulated Wnt-3a and ß-catenin protein expression, and enhanced the expression of downstream genes Ccnd1, Neurod1 and Prox1. However, XAV-939, an inhibitor of the Wnt/ß-catenin signaling pathway, inhibited these positive effects of JuA. Taken together, these findings suggest that JuA promote proliferation and neuronal differentiation of APP-NSCs partly by activating the Wnt/ß-catenin signaling pathway. We hope that this study will provide a viable strategy for the treatment of AD.
Assuntos
Proliferação de Células , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese , Saponinas/farmacologia , Via de Sinalização Wnt , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Compostos Heterocíclicos com 3 Anéis/farmacologia , Hipocampo/citologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , beta Catenina/metabolismoRESUMO
Antimicrobial peptides/DNA complexes were designed based on AMPs chensinin-1b and its corresponding lipo-chensinin-1b conjugated with an aliphatic acid with different chain lengths and therapeutic genes. The main goal of such a complex includes two aspects: first, antimicrobial peptides deliver therapeutic genes to cancer cells and genes expressed in targeted tissue for cancer gene therapy, and, second, the antimicrobial peptide kills cancer cells when used alone as an anticancer agent. This study presents a model composed of chensinin-1b and its lipo-chensinin-1b and eGFP plasmids, which were used as reporter genes, and the final peptide/eGFP plasmid complexes were analyzed by TEM and DLS. The gene transfection efficiency of the complex was evaluated by a microplate reader, FACS and CLSM. Compared with Lipo2000, the antimicrobial peptide showed specific selectivity for transfection against cancer cells and mammalian cells. The peptides chensinin-1b and lipo-chensinin-1b binding with the eGFP plasmid displayed optimal transfection efficiencies at a mass ratio of 8. In addition, PA-C1b can deliver p53-eGFP plasmids into MCF-7 cancer cells, and the proliferation of cells was inhibited and even caused cell death. Overall, PA-C1b was screened and found to have the highest transfection efficiency for gene delivery and good cellular uptake capability. The in vivo transfection ability of PA-C1b was investigated using a tumor-bearing mouse model, and the transfection efficiency reflected by the fluorescence of expressed GFP was determined by in vivo imaging. Conclusively, the antimicrobial peptide PA-C1b could be used as the nonviral vector with high efficiency for cancer gene therapy.
Assuntos
Técnicas de Transferência de Genes , Neoplasias , Acilação , Animais , DNA/genética , Camundongos , Neoplasias/genética , Neoplasias/terapia , Plasmídeos/genética , Proteínas Citotóxicas Formadoras de PorosRESUMO
Temporin-1CEa, which is isolated from the skin secretions of the Chinese brown frog Rana chensinensis, exhibits broad-spectrum antimicrobial activity against gram-positive and gram-negative bacteria and antitumor activity. LK2(6) and LK2(6)A(L) are the analogs of temporin-1CEa obtained by replacing amino acids and displayed an improved anticancer activity. In the present study, the anti-inflammatory activity and mechanism of action of temporin-1CEa and its analogs LK2(6) and LK2(6)A(L) in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages were investigated. The results showed that temporin-1CEa and its analogs decreased the production of the cytokines tumor necrosis factor-α and interleukin-6 by inhibiting the protein expression of nuclear factor-κB and mitogen-activated protein kinase and the MyD88-dependent signaling pathway. Isothermal titration calorimetry studies revealed that temporin-1CEa, LK2(6) and LK2(6)A(L) exhibited binding affinities to LPS, an important inflammatory inducer, with Kd values of 0.1, 0.03 and 0.06 µM, respectively. Circular dichroism and zeta potential experiments showed that temporin-1CEa and its analogs interacted with LPS by electrostatic binding between the positively charged peptides and negatively charged LPS, resulting in the neutralization of LPS toxicity.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Macrófagos/efeitos dos fármacos , Fator 88 de Diferenciação Mieloide/metabolismo , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Ranidae/metabolismo , Transdução de Sinais/efeitos dos fármacos , Pele/química , Animais , Anti-Inflamatórios/farmacologia , Linhagem Celular , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Células RAW 264.7RESUMO
Antimicrobial peptide PA-C1b (chensinin-1b conjugated with palmitic acid) showed potent anticancer activity with no obvious hemolytic activity, which made it a potential agent for treating cancers. However, after in vivo administration, peptides can be degraded by proteases because there is no effective protection. In this study, a tumor-targeting photoresponsive antimicrobial peptide delivery system was developed, and the peptide PA-C1b labeled with the dye sulfo-cyanine7 (Cy7) was loaded into mesoporous silica nanoparticles (MSNs). The final MSN@Cy7-PA-C1b nanoparticles were wrapped by graphene oxide (GO), and then folic acid was conjugated to the surface of the MSNs for targeting purposes. The final MSN@Cy7-PA-C1b@FA-GO nanoparticles were constructed to allow light-mediated peptide release and folate receptor-targeted cancer therapy. The Cy7 dye serves as a real-time indicator, and GO acts as a gatekeeper to prevent leakage of the loaded peptides in the absence of near-infrared light irradiation. Upon light irradiation, the GO wrapping detaches, and the photoresponsive peptide delivery system works well both in in vitro cell experiments and during in vivo administration in mouse tumor experiments. The construction of the MSN@Cy7-PA-C1b@FA-GO platform provides a novel approach to deliver antimicrobial peptides in vivo for the treatment of infections by pathogenic microorganisms and cancers.
Assuntos
Nanopartículas , Neoplasias , Animais , Sistemas de Liberação de Medicamentos , Camundongos , Fototerapia , Proteínas Citotóxicas Formadoras de Poros , Porosidade , Dióxido de SilícioRESUMO
Multidrug-resistant bacteria present an important threat to human health. In this study, due to the weak antimicrobial activity of chensinin-1b against multidrug-resistant (MDR) bacteria, three lipo-chensinin-1b peptides, including OA-C1b, LA-C1b and PA-C1b, were designed and their activities against MDR bacteria were examined. Both the OA-C1b and LA-C1b peptides exhibited potent antimicrobial activity against selected multidrug-resistant bacterial strains. In addition to the direct disruption of bacterial membranes by antimicrobial peptides, it has also been proposed that DNA is a superior intracellular target for antimicrobial peptides. ctDNA was used as a model to investigate the binding properties of DNA and lipo-chensinin-1b peptides using a variety of biophysical methods. The kinetics results of both UV-Vis and CD spectroscopy suggested that the interaction between lipo-chensinin-1b peptides and ctDNA was concentration-dependent and resulted in an increase in polynucleotide helicity. Viscosity measurements, Trp fluorescence and iodide quenching experiments indicated that nonclassical groove binding and electrostatic binding interaction modes were utilized when the peptides interacted with the ctDNA. In addition, the formation of peptide-ctDNA complexes was monitored using dynamic light scattering experiments, during which the peptide exhibited the ability to neutralize the negative charges on the surface of the ctDNA. These results promote the possibility of designing peptide-based antibiotics targeted to DNA.
Assuntos
Antibacterianos/química , Bactérias/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos , Sítios de Ligação/efeitos dos fármacos , DNA Tumoral Circulante , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Testes de Sensibilidade Microbiana , Espectrometria de Fluorescência , ViscosidadeRESUMO
Antimicrobial peptides have broad-spectrum killing activities against bacteria, enveloped viruses, fungi and several parasites via cell membrane permeation and exhibit primarily immunomodulatory and anti-infective functions in their interactions with host cells. However, the mechanism underlying their anti-inflammatory activity remains to be elucidated. L-K6, an analog of temporin-1CEb isolated from the skin secretion of Rana chensinensis, has demonstrated a wide range of antimicrobial activities against gram-negative and gram-positive bacteria. In this study, the potent anti-inflammatory mechanism of L-K6 and its analogs in lipopolysaccharide (LPS)-stimulated human macrophage U937 cells were evaluated. We found that L-K6 suppressed the expression of inflammatory factors by two downstream signaling components in the MyD88-dependent pathway, including the mitogen-activated protein kinases (MAPKs) and the NF (nuclear factor)-κB signaling pathway, but its analog L-K5, which had the same amino acid sequence as L-K6 but no Lys residue at the -COOH terminal, only inhibited the phosphorylation of I-κB and NF-κB. Importantly, L-K6 and L-K5 were actively taken up by U937 cells through an independent cell membrane disruption mechanism and were eventually localized to the perinuclear region. The L-K6 uptake process was mediated by endocytosis, but L-K5 was specifically taken up by U937 cells via TLR4 endocytosis. Our results demonstrated that L-K6 can neutralize LPS and diassociate LPS micelles to inhibit LPS from triggering the proinflammatory signaling pathway, and by partially inhibiting inflammatory responses by the intracellular target. However, L-K5 may mainly inhibit proinflammatory responses by intracellular reporters to modulate the NF-κB signaling pathway.
Assuntos
Anti-Infecciosos/farmacologia , Inflamação/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Citocinas/genética , Citocinas/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Leucina/química , Lipopolissacarídeos , Lisina/química , Macrófagos/metabolismo , Camundongos , Peptídeos/síntese química , Peptídeos/química , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Células U937RESUMO
The threshold hydrophobicity and amphipathic structure of the peptidic chain are important for the biological function of antimicrobial peptides. Chensinin-1b exhibits broad-spectrum bactericidal activity with no hemolytic activity but has almost no anticancer ability against the selected cancer cell lines. In this study, the conjugation of aliphatic acid was designed with different lengths of N-terminal of chensinin-1b, the antimicrobial activity of the resulting lipo-chensinin-1b was examined, in which OA-C1b showed much stronger activity than those of cheninin-1b and the other two lipopeptides. The membrane interaction between the lipo-chensinin-1b and real/mimetic bacterial cell membrane was investigated. Electrostatic interactions between the lipo-chensinin-1b and lipopolysaccharides were detected by isothermal titration calorimetry and the binding affinities were 10.83⯵M, 8.77⯵M and 7.35⯵M for OA-C1b, LA-C1b and PA-C1b, respectively. The antimicrobial activity and membrane interaction ability of the lipo-chensinin-1b followed this order: OA-C1bâ¯>â¯chensinin-1bâ¯>â¯LA-C1bâ¯>â¯PA-C1b. In addition, the lipo-chensinin-1b also exhibited lytic activity against various cancer cells and demonstrated the ability to inhibit LPS-stimulated cytokine release from human U937â¯cells. The CD spectra indicated that the helical or ß-strands contents were existed as the main components in TFE or LPS solution, respectively. The self-assembly behavior was trigged by the solution pH and affected by the length of carbon chain, in which chensinin-1b, OA-C1b, LA-C1b and PA-C1b formed micelles at neutral pH and the micelle size increased for chensinin-1b, OA-C1b and LA-C1b. PA-C1b formed nanofibers in an acidic environment indicated by TEM experiments, and the peptides formed aggregates in an acidic environment and re-dissociated when the pH was adjusted to neutral.
Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Lipopeptídeos/farmacologia , Peptídeos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipopeptídeos/síntese química , Lipopeptídeos/química , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/química , Relação Estrutura-AtividadeRESUMO
Cell surface binding and internalization are critical for the specific targeting and biofunctions of some cationic antimicrobial peptides (CAPs) with anticancer activities. However, the detailed cellular process for CAPs interacting with cancer cells and the exact molecular basis for their anticancer effects are still far from being fully understood. In the present study, we examined the cell surface binding, uptaking and anti-cancer activity of L-K6, a lysine/leucine-rich CAP, in human MCF-7 breast cancer cells. We found that L-K6 preferentially interact with MCF-7 cells. This tumor-targeting property of L-K6 might be partially due to its interactions with the surface exposed and negatively charged phosphatidylserine. Subsequently, L-K6 could internalize into MCF-7 cells mainly through a clathrin-independent macropinocytosis, without significant cell surface disruption. Finally, the internalized L-K6 induced a dramatic nuclear damage and MCF-7 cell death, without significant cytoskeleton disruption and mitochondrial impairment. This cytotoxicity of L-K6 against MCF-7 cancer cells could be further confirmed by using a mouse xenograft model. In summary, all these findings outlined the cellular process and cytotoxicity of L-K6 in MCF-7 cancer cells, and might help understand the complicated interactions between CAPs and cancer cells.
Assuntos
Antineoplásicos/farmacologia , Membrana Celular/metabolismo , Leucina , Lisina , Peptídeos/metabolismo , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Endocitose , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Leucina/química , Lisina/química , Células MCF-7 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Peptídeos/química , Permeabilidade , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Nosocomial infections caused by Pseudomonas aeruginosa are difficult to treat due to the low permeability of its outer membrane as well as to its remarkable ability to acquire further resistance to antibiotics. Chensinin-1b exhibited antibacterial activity against the tested multiple-drug-resistant bacteria with a MIC ranging between 1.56 and 50µM, except E. cloacae strain 0320 (MREC0320), P. fluorescens strain 0322 (MRPF0322) and E. aerogenes strain 0320 (MREA0320). However, the MIC (25µM) of chensinin-1b to multiple-drug-resistant P. aeruginosa strain (MRPA 0108) was 16-fold higher than that observed to P. aeruginosa susceptible strain CGMCC 1.860 (PA1860). Chensinin-1b was able to disturb the integration of the cytoplasmic membrane of PA1860 and MRPA0108 cells similarly, but the outer membrane permeability of MRPA0108 cells was significantly lower. This low permeability was associated with increased expression of lipopolysaccharide (LPS) in the outer membrane and a decrease in negatively charged phospholipids in the outer membrane leaflet. In addition, the biofilm of MRPA0108 was responsible for the reduced susceptibility to chensinin-1b. A higher concentration of chensinin-1b (12.5µM) was required to maximally inhibit the formation of MRPA0108 biofilm. Notably, chensinin-1b inhibited the formation of MRPA0108 biofilm at concentrations below its MIC value by down-regulating the level of PelA, algD, and PslA gene transcription. Importantly, chensinin-1b had a significant antibacterial effect against MRPA0108 in vivo. Administration of chensinin-1b to mice infected with MRPA 0108 significantly increased survival by 50-70%. Moreover, chensinin-1b reduced the production of pro-inflammatory mediators and correspondingly reduced lung and liver tissue damage in the mouse model of septic shock induced by MRPA 0108. Collectively, these results suggest that chensinin-1b could be an effective antibiotic against multiple-drug-resistant bacterial strains.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Peptídeos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Animais , Antibacterianos/química , Antibacterianos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos , Biofilmes/crescimento & desenvolvimento , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Potenciais da Membrana/efeitos dos fármacos , Camundongos Endogâmicos , Testes de Sensibilidade Microbiana , Microscopia Confocal , Peptídeos/química , Peptídeos/uso terapêutico , Conformação Proteica , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/fisiologia , Pseudomonas aeruginosa/ultraestrutura , Sepse/tratamento farmacológico , Sepse/microbiologiaRESUMO
The natural peptide chensinin-1 doesnot exhibit its desired biological properties. In this study, the mutant MC1-1 was designed by replacing Gly in the chensinin-1 sequence with Trp. Mutants MC1-2 and MC1-3 were designed based on the MC1-1 sequence to investigate the specific role of His residues. The mutated peptides presented α-helicity in a membrane-mimetic environment and exhibited broad-spectrum antimicrobial activities; in contrast to Trp residues, His residues were dispensable for interacting with the cell membrane. The interactions between the mutant peptides and lipopolysaccharide (LPS) facilitated the ingestion of peptides by Gram-negative bacteria. The binding affinities of the peptides were similar, at approximately 10 µM, but ΔH for MC1-2 was -7.3 kcal.mol-1, which was 6-9 folds higher than those of MC1-1 and MC1-3, probably due to the conformational changes. All mutant peptides demonstrated the ability to inhibit LPS-induced tumour-necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release from murine RAW264.7 cells. In addition, the representative peptide MC1-1showed better inhibition of serum TNF-α and IL-6 levels compared to polymyxin B (PMB), a potent binder and neutralizer of LPS as positive control in LPS-challenged mice model. These data suggest that the mutant peptides could be promising molecules for development as chensinin-based therapeutic agents against sepsis.
Assuntos
Anti-Infecciosos/farmacologia , Anti-Inflamatórios/farmacologia , Proteínas Mutantes/farmacologia , Mutação , Peptídeos/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos , Bactérias Gram-Negativas/efeitos dos fármacos , Interleucina-6/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Peptídeos/química , Peptídeos/genética , Conformação Proteica em alfa-Hélice , Células RAW 264.7 , Fator de Necrose Tumoral alfa/metabolismoRESUMO
UNLABELLED: Chensinin-1b shows a potent and broad-spectrum bactericidal activity and no hemolytic activity and thus is a potential therapeutic agent against bacterial infection. The NMR structure of chensinin-1b consists of a partially α-helical region (residues 8-14) in a membrane-mimic environment that is distinct from other common antimicrobial peptides. However, further analysis of the structural features of chensinin-1b is required to better understand its bactericidal activity. In this study, a series of N- and C-terminally truncated or amino acid-substituted chensinin-1b analogues were synthesized. Next, the bactericidal activity and bacterial membrane effects of the analogues were investigated. The results indicated that the N-terminal residues play a more significant role than the C-terminal residues in the antimicrobial activity of chensinin-1b. The removal of five amino acids from the C-terminus of chensinin-1b did not affect its biological properties, but helix disruption significantly decreased bactericidal activity. The substitution of positively charged residues increased the helicity and antimicrobial activity of the peptide. We also identified a novel analogue [R(4),R(10)]C1b(3-13) that exhibited similar bactericidal properties with its parent peptide chensinin-1b. Electrostatic interactions between the selected analogues and lipopolysaccharides or cells were detected using isothermal titration calorimetry or zeta potential. The thermodynamic parameters ΔH and ΔS for [R(4),R(10)]C1b(3-13) were -20.48kcalmol(-1) and -0.0408kcalmol(-1)deg(-1), respectively. Chensinin-1b yielded similar results of -26.36kcalmol(-1) and -0.0559kcalmol(-1)deg(-1) for ΔH and ΔS, respectively. These results are consistence with their antimicrobial activities. Lastly, membrane depolarization studies showed that selected analogues exerted bactericidal activity by damaging the cytoplasmic membrane. STATEMENT OF SIGNIFICANCE: Antimicrobial peptide chensinin-1b is a candidate for the development of new drugs and a template for the design of synthetic analogues. It mainly exhibits a random coil conformation in membrane environment, and in this manuscript, we characterized the structure of chensinin-1b using NMR spectroscopy, its structure is different than the structures of magainin 2, which has an α-helical conformation and indolicidin, which has a random coil structure. The structural features of chensinin-1b that are required for its potent bactericidal activity were also elucidated. Based on these data, we can fully understand the structure-activity relationship of such peptide and identified a novel analogue with properties that make it an attractive topic for future therapeutic research.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/crescimento & desenvolvimento , Peptídeos/química , Peptídeos/farmacologia , Peptídeos Catiônicos Antimicrobianos , Humanos , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
A series of synthesized Trp-containing antimicrobial peptides showed significantly different antimicrobial activity against Gram-negative bacteria despite having similar components and amino acid sequences and the same net positive charge and hydrophobicity. Lipopolysaccharide (LPS) in the outer membrane is a permeability barrier to prevent antimicrobial peptides from crossing into Gram-negative bacteria. We investigated the interaction of five Trp-containing peptides, I1W, I4W, L5W, L11W and L12W, with LPS using circular dichroism (CD), IR spectroscopy, isothermal titration calorimetry (ITC), dynamic light scattering (DLS), zeta-potential measurements and confocal laser scanning microscopy, to address whether bacterial LPS is responsible for the different susceptibilities of Gram-negative bacteria to Trp-containing peptides. Our data indicate that I1W and I4W penetrated the LPS layer and killed Gram-negative bacteria by a "self-promoted uptake" pathway in which the peptides first approach LPS by electrostatic forces and then dissociate LPS micelle. This process results in disorganization of the LPS leaflet and promotes the ability of the peptide to cross the outer membrane into the inner membrane and disrupt the cytoplasmic membrane. Although L5W, L11W and L12W strongly bind to LPS bilayers and depolarize bacterial cytoplasmic membranes, similar to I1W and I4W, they are unable to destabilize LPS aggregates and traverse through the tightly packed LPS molecules. This study increases our understanding of the mechanism of action of these peptides in the LPS outer membrane and will help in the development of a potent broad-spectrum antibiotic for future therapeutic purposes. STATEMENT OF SIGNIFICANCE: Tryptophan (Trp) residues show a strong preference for the interfacial region of biological membranes, and this property endows Trp-containing peptides with the unique ability to interact with the surface of bacterial cell membranes. In this manuscript, we report the membrane interaction of Trp-containing peptide to address whether bacterial LPS is responsible for the different susceptibilities of Gram-negative bacteria to Trp-containing peptides. Based on the data collected, we propose a molecular mechanism for the peptide-LPS interactions that allows the peptides to traverse or prevents them from transversing the LPS layer and the target inner membrane. The data should help in the development of a potent broad-spectrum antibiotic for future therapeutic purposes.
Assuntos
Antibacterianos/farmacologia , Endotoxinas/metabolismo , Escherichia coli/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Testes de Neutralização , Peptídeos/farmacologia , Triptofano/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/química , Morte Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Dicroísmo Circular , Camundongos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Peptídeos/química , Células RAW 264.7 , Esferoplastos/efeitos dos fármacos , TermodinâmicaRESUMO
We have previously reported that temporin-1CEa, a cationic antimicrobial peptide, exerts preferential cytotoxicity toward cancer cells. However, the exact molecular mechanism for this cancer-selectivity is still largely unknown. Here, we found that the negatively charged phosphatidylserine (PS) expressed on cancer cell surface serves as a target for temporin-1CEa. Our results indicate that human A375 melanoma cells express 50-fold more PS than non-cancerous HaCaT cells. The expression of cell surface PS in various cancer cell lines closely correlated with their ability to be recognized, bound and killed by temporin-1CEa. Additionally, the cytotoxicity of temporin-1CEa against A375 cells can be ameliorated by annexin V, which binds to cell surface PS with high affinity. Moreover, the data of isothermal titration calorimetry assay further confirmed a direct binding of temporin-1CEa to PS, at a ratio of 1:5 (temporin-1CEa:PS). Interestingly, the circular dichroism spectra analysis using artificial biomembrane revealed that PS not only provides electrostatic attractive sites for temporin-1CEa but also confers the membrane-bound temporin-1CEa to form α-helical structure, therefore, enhances the affinity and membrane disrupting ability of temporin-1CEa. In summary, these findings suggested that the melanoma cells expressed PS may serve as a promising target for temporin-1CEa or other cationic anticancer peptides.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , Melanoma/tratamento farmacológico , Fosfatidilserinas/metabolismo , Animais , Anexina A5/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Dicroísmo Circular , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/metabolismo , Camundongos , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Eletricidade EstáticaRESUMO
Lipopolysaccharide (LPS) plays a critical role in the pathogenesis of sepsis caused by gram-negative bacterial infections. Therefore, LPS-neutralizing molecules would have important clinical applications. Chensinin-1, a novel antimicrobial peptide with atypical structural features, was found in the skin secretions of the Chinese brown frog Rana chensinensis. To understand the role of LPS in the bacterial susceptibility to chensinin-1 and to investigate its anti-endotoxin effects, the interactions of chensinin-1 with LPS were investigated in this study using circular dichroism, in situ IR, isothermal titration calorimetry, and zeta potential. This study is the first to use in situ IR spectroscopy to evaluate the secondary structural changes of this peptide. The capacity of chensinin-1 to block the LPS-dependent cytokine secretion of macrophages was also investigated. Our results show that chensinin-1 can form α-helical structures in LPS suspensions. LPS can affect the antimicrobial activity of chensinin-1, and chensinin-1 was able to mitigate the effects of LPS. These data may facilitate the development of antimicrobial peptides with potent antimicrobial and anti-endotoxin activities.
Assuntos
Anti-Infecciosos/química , Lipopolissacarídeos/química , Peptídeos/química , Animais , Peptídeos Catiônicos Antimicrobianos , Células Cultivadas , Dicroísmo Circular , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Peptídeos/efeitos dos fármacos , Células RAW 264.7 , RanidaeRESUMO
Antimicrobial peptides (AMPs) with non-specific membrane disrupting activities are thought to exert their antimicrobial activity as a result of their cationicity, hydrophobicity and α-helical or ß-sheet structures. Chensinin-1, a native peptide from skin secretions of Rana chensinensis, fails to manifest its desired biological properties because its low hydrophobic nature and an adopted random coil structure in a membrane-mimetic environment. In this study, chensinin-1b was designed by rearranging the amino acid sequence of its hydrophilic/polar residues on one face and its hydrophobic/nonpolar residues on the opposite face according to its helical diagram, and by replacing three Gly residues with three Trp residues. Introduction of Trp residues significantly promoted the binding of the peptide to the bacterial outer membrane and exerted bactericidal activity through cytoplasmic membrane damage. Chensinin-1b demonstrates higher antimicrobial activity and greater cell selectivity than its parent peptide, chensinin-1. The electrostatic interactions between chensinin-1b and lipopolysaccharide (LPS) may have facilitated the uptake of the peptide into Gram-negative cells and be also helpful to disrupt the bacterial cytoplasmic membrane, as evidenced by depolarisation of the membrane potential and leakage of calceins from the liposomes of Escherichia coli and Staphylococcus aureus. Chensinin-1b was also found to penetrate mouse skin and was also effective in vivo, as measured by hydroxyproline levels in a wound infection mouse model, and could therefore act as an anti-infective agent for wound healing.