RESUMO
Lactylation is a lactate-induced post-translational modification best known for its roles in epigenetic regulation. Herein, we demonstrate that MRE11, a crucial homologous recombination (HR) protein, is lactylated at K673 by the CBP acetyltransferase in response to DNA damage and dependent on ATM phosphorylation of the latter. MRE11 lactylation promotes its binding to DNA, facilitating DNA end resection and HR. Inhibition of CBP or LDH downregulated MRE11 lactylation, impaired HR, and enhanced chemosensitivity of tumor cells in patient-derived xenograft and organoid models. A cell-penetrating peptide that specifically blocks MRE11 lactylation inhibited HR and sensitized cancer cells to cisplatin and PARPi. These findings unveil lactylation as a key regulator of HR, providing fresh insights into the ways in which cellular metabolism is linked to DSB repair. They also imply that the Warburg effect can confer chemoresistance through enhancing HR and suggest a potential therapeutic strategy of targeting MRE11 lactylation to mitigate the effects.
Assuntos
Proteínas de Ligação a DNA , Proteína Homóloga a MRE11 , Reparo de DNA por Recombinação , Humanos , DNA , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Recombinação Homóloga , Proteína Homóloga a MRE11/metabolismo , Ácido Láctico/metabolismoRESUMO
Cell-penetrating peptides (CPPs) are short protein segments that can transport cargos into cells. Although CPPs are widely studied as potential drug delivery tools, their role in normal cell physiology is poorly understood. Early during infection, the L2 capsid protein of human papillomaviruses binds retromer, a cytoplasmic trafficking factor required for delivery of the incoming non-enveloped virus into the retrograde transport pathway. Here, we show that the C terminus of HPV L2 proteins contains a conserved cationic CPP that drives passage of a segment of the L2 protein through the endosomal membrane into the cytoplasm, where it binds retromer, thereby sorting the virus into the retrograde pathway for transport to the trans-Golgi network. These experiments define the cell-autonomous biological role of a CPP in its natural context and reveal how a luminal viral protein engages an essential cytoplasmic entry factor.
Assuntos
Proteínas do Capsídeo/metabolismo , Peptídeos Penetradores de Células/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/genética , Endossomos/metabolismo , Complexo de Golgi/virologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/fisiologia , Humanos , Mutagênese , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/genética , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Ligação Viral , Internalização do VírusRESUMO
The accumulation of irreparable cellular damage restricts healthspan after acute stress or natural aging. Senescent cells are thought to impair tissue function, and their genetic clearance can delay features of aging. Identifying how senescent cells avoid apoptosis allows for the prospective design of anti-senescence compounds to address whether homeostasis can also be restored. Here, we identify FOXO4 as a pivot in senescent cell viability. We designed a FOXO4 peptide that perturbs the FOXO4 interaction with p53. In senescent cells, this selectively causes p53 nuclear exclusion and cell-intrinsic apoptosis. Under conditions where it was well tolerated in vivo, this FOXO4 peptide neutralized doxorubicin-induced chemotoxicity. Moreover, it restored fitness, fur density, and renal function in both fast aging XpdTTD/TTD and naturally aged mice. Thus, therapeutic targeting of senescent cells is feasible under conditions where loss of health has already occurred, and in doing so tissue homeostasis can effectively be restored.
Assuntos
Envelhecimento/patologia , Antibióticos Antineoplásicos/efeitos adversos , Peptídeos Penetradores de Células/farmacologia , Doxorrubicina/efeitos adversos , Envelhecimento/efeitos dos fármacos , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/farmacologia , Apoptose , Proteínas de Ciclo Celular , Linhagem Celular , Sobrevivência Celular , Senescência Celular/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacologia , Feminino , Fibroblastos/citologia , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/metabolismo , Humanos , Corpos de Inclusão/efeitos dos fármacos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Rim/efeitos dos fármacos , Rim/fisiologia , Fígado/efeitos dos fármacos , Fígado/fisiologia , Masculino , Camundongos , Síndromes de Tricotiodistrofia/tratamento farmacológico , Proteína Supressora de Tumor p53/metabolismoRESUMO
The novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), responsible for coronavirus disease 2019 (COVID-19), can trigger dysregulated immune responses known as the cytokine release syndrome (CRS), leading to severe organ dysfunction and respiratory distress. Our study focuses on developing an improved cell-permeable nuclear import inhibitor (iCP-NI), capable of blocking the nuclear transport of inflammation-associated transcription factors, specifically nuclear factor kappa B (NF-κB). By fusing advanced macromolecule transduction domains and nuclear localization sequences from human NF-κB, iCP-NI selectively interacts with importin α5, effectively reducing the expression of proinflammatory cytokines. In mouse models mimic SARS-CoV-2-induced pneumonitis, iCP-NI treatment demonstrated a significant decrease in mortality rates by suppressing proinflammatory cytokine production and immune cell infiltration in the lungs. Similarly, in hamsters infected with SARS-CoV-2, iCP-NI effectively protected the lung from inflammatory damage by reducing tumor necrosis factor-α, interleukin-6 (IL-6), and IL-17 levels. These promising results highlight the potential of iCP-NI as a therapeutic approach for COVID-19-related lung complications and other inflammatory lung diseases.
Assuntos
COVID-19 , Camundongos , Animais , Humanos , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , SARS-CoV-2 , NF-kappa B/metabolismo , Inflamação , Citocinas/metabolismo , Peptídeos/metabolismoRESUMO
Insulin resistance (IR) is the key pathophysiological cause of type 2 diabetes, and inflammation has been implicated in it. The death domain (DD) of the adaptor protein, MyD88 plays a crucial role in the transduction of TLR4-associated inflammatory signal. Herein, we have identified a 10-residue peptide (M10), from the DD of MyD88 which seems to be involved in Myddosome formation. We hypothesized that M10 could inhibit MyD88-dependent TLR4-signaling and might have effects on inflammation-associated IR. Intriguingly, 10-mer M10 showed oligomeric nature and reversible self-assembly property indicating the peptide's ability to recognize its own amino acid sequence. M10 inhibited LPS-induced nuclear translocation of NF-κB in L6 myotubes and also reduced LPS-induced IL-6 and TNF-α production in peritoneal macrophages of BALB/c mice. Remarkably, M10 inhibited IL-6 and TNF-α secretion in diabetic, db/db mice. Notably, M10 abrogated IR in insulin-resistant L6 myotubes, which was associated with an increase in glucose uptake and a decrease in Ser307-phosphorylation of IRS1, TNF-α-induced JNK activation and nuclear translocation of NF-κB in these cells. Alternate day dosing with M10 (10 and 20â mg/kg) for 30 days in db/db mice significantly lowered blood glucose and improved glucose intolerance after loading, 3.0 g/kg glucose orally. Furthermore, M10 increased insulin and adiponectin secretion in db/db mice. M10-induced glucose uptake in L6 myotubes involved the activation of PI3K/AKT/GLUT4 pathways. A scrambled M10-analog was mostly inactive. Overall, the results show the identification of a 10-mer peptide from the DD of MyD88 with anti-inflammatory and anti-diabetic properties, suggesting that targeting of TLR4-inflammatory pathway, could lead to the discovery of molecules against IR and diabetes.
Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Animais , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Glicemia , Domínio de Morte , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Inflamação/tratamento farmacológico , Insulina/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos/toxicidade , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Peptídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The advancement of effective nasal mucoadhesive delivery faces challenges due to rapid mucociliary clearance (MCC). Conventional studies have employed mucoadhesive materials, mainly forming spherical nanoparticles, but these offer limited adhesion to the nasal mucosa. This study hypothesizes that a 2D nanoscale structure utilizing adhesive polyphenols can provide a superior strategy for countering MCC, aligning with the planar mucosal layers. We explore the use of tannic acid (TA), a polyphenolic molecule known for its adhesive properties and ability to form complexes with biomolecules. Our study introduces an unprecedented 2D nanopatch, assembled through the interaction of TA with green fluorescent protein (GFP), and cell-penetrating peptide (CPP). This 2D nanopatch demonstrates robust adhesion to nasal mucosa and significantly enhances immunoglobulin A secretions, suggesting its potential for enhancing nasal vaccine delivery. The promise of a polyphenol-enabled adhesive 2D nanopatch signifies a pivotal shift from conventional spherical nanoparticles, opening new pathways for delivery strategies through respiratory mucoadhesion.
RESUMO
Given the pathological role of Tau aggregation in Alzheimer's disease (AD), our laboratory previously developed the novel Tau aggregation inhibitor peptide, RI-AG03. As Tau aggregates accumulate intracellularly, it is essential that the peptide can traverse the cell membrane. Here we examine the cellular uptake and intracellular trafficking of RI-AG03, in both a free and liposome-conjugated form. We also characterize the impact of adding the cell-penetrating peptide (CPP) sequences, polyarginine (polyR) or transactivator of transcription (TAT), to RI-AG03. Our data show that liposome conjugation of CPP containing RI-AG03 peptides, with either the polyR or TAT sequence, increased cellular liposome association three-fold. Inhibition of macropinocytosis modestly reduced the uptake of unconjugated and RI-AG03-polyR-linked liposomes, while having no effect on RI-AG03-TAT-conjugated liposome uptake. Further supporting macropinocytosis-mediated internalization, a 'fair' co-localisation of the free and liposome-conjugated RI-AG03-polyR peptide with macropinosomes and lysosomes was observed. Interestingly, we also demonstrate that RI-AG03-polyR detaches from liposomes following cellular uptake, thereby largely evading organellar entrapment. Collectively, our data indicate that direct membrane penetration and macropinocytosis are key routes for the internalization of liposomes conjugated with CPP containing RI-AG03. Our study also demonstrates that peptide-liposomes are suitable nanocarriers for the cellular delivery of RI-AG03, furthering their potential use in targeting Tau pathology in AD.
Assuntos
Peptídeos Penetradores de Células , Lipossomos , Nanopartículas , Pinocitose , Proteínas tau , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Lipossomos/química , Humanos , Proteínas tau/metabolismo , Proteínas tau/química , Nanopartículas/química , Pinocitose/efeitos dos fármacos , Peptídeos/química , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Lisossomos/metabolismo , Sistemas de Liberação de Medicamentos/métodosRESUMO
Corneal neovascularization (CoNV) is a major cause of visual impairment worldwide. Currently, available treatment options have limited efficacy and are associated with adverse effects due to biological barriers and clearance mechanisms. To address this challenge, a novel topical delivery system is developed-Gel 2_1&Eylea-an aflibercept-loaded eye-drop hydrogel mediated with cell-penetrating peptide 1. Gel 2_1&Eylea demonstrates superior membrane permeability, increased stability, and prolonged drug retention time on the ocular surface, and thus may improve drug efficacy. In a rabbit CoNV model, Gel 2_1&Eylea significantly reduces the density of neovascularization with no adverse effects on normal corneoscleral limbal vessels, demonstrating high efficacy and biocompatibility. This work identifies a promising treatment for CoNV which has the potential to benefit other ocular neovascular diseases.
Assuntos
Peptídeos Penetradores de Células , Neovascularização da Córnea , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão , Animais , Coelhos , Neovascularização da Córnea/tratamento farmacológico , Hidrogéis , Soluções Oftálmicas/uso terapêuticoRESUMO
Cell-penetrating peptides are known to penetrate cells through endocytosis and translocation. The two pathways are hardly distinguished in current cell assays. We developed a reliable, simple and robust method to distinguish and quantify independently the two routes. The assay requires (DABCYL) 4-(dimethylaminoazo)benzene-4-carboxylic acid- and (CF) carboxyfluorescein-labeled peptides. When the labeled peptide is intact, the fluorescence signal is weak thanks to the dark quenching property of DABCYL. A 10-fold higher fluorescence signal is measured when the labeled peptide is degraded. By referring to a standard fluorescent curve according to the concentration of the hydrolyzed peptide, we have access to the internalized peptide quantity. Therefore, cell lysis after internalization permits to determine the total quantity of intracellular peptide. The molecular state of the internalized peptide (intact or degraded), depends on its location in cells (cytosol vs endo-lysosomes), and can be blocked by boiling cells. This boiling step results indeed in denaturation and inhibition of the cellular enzymes. The advantage of this method is the possibility to quantify translocation at 37 °C and to compare it to the 4 °C condition, where all endocytosis processes are inhibited. We found that ranking of the translocation efficacy is DABCYL-R6-(ϵCF)Kâ«DABCYL-R4-(ϵCF)K≥CF-R9.
Assuntos
Peptídeos Penetradores de Células , Citosol , Endossomos , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Endossomos/metabolismo , Humanos , Citosol/metabolismo , Fluoresceínas/química , Endocitose , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Células HeLaRESUMO
Protein kinase B (AKT1) is a serine/threonine kinase that regulates fundamental cellular processes, including cell survival, proliferation, and metabolism. AKT1 activity is controlled by two regulatory phosphorylation sites (Thr308, Ser473) that stimulate a downstream signaling cascade through phosphorylation of many target proteins. At either or both regulatory sites, hyperphosphorylation is associated with poor survival outcomes in many human cancers. Our previous biochemical and chemoproteomic studies showed that the phosphorylated forms of AKT1 have differential selectivity toward peptide substrates. Here, we investigated AKT1-dependent activity in human cells, using a cell-penetrating peptide (transactivator of transcription, TAT) to deliver inactive AKT1 or active phospho-variants to cells. We used enzyme engineering and genetic code expansion relying on a phosphoseryl-transfer RNA (tRNA) synthetase (SepRS) and tRNASep pair to produce TAT-tagged AKT1 with programmed phosphorylation at one or both key regulatory sites. We found that all TAT-tagged AKT1 variants were efficiently delivered into human embryonic kidney (HEK 293T) cells and that only the phosphorylated AKT1 (pAKT1) variants stimulated downstream signaling. All TAT-pAKT1 variants induced glycogen synthase kinase (GSK)-3α phosphorylation, as well as phosphorylation of ribosomal protein S6 at Ser240/244, demonstrating stimulation of downstream AKT1 signaling. Fascinatingly, only the AKT1 variants phosphorylated at S473 (TAT-pAKT1S473 or TAT-pAKT1T308,S473) were able to increase phospho-GSK-3ß levels. Although each TAT-pAKT1 variant significantly stimulated cell proliferation, cells transduced with TAT-pAKT1T308 grew significantly faster than with the other pAKT1 variants. The data demonstrate differential activity of the AKT1 phospho-forms in modulating downstream signaling and proliferation in human cells.
RESUMO
BACKGROUND: Heat shock proteins (HSPs) as an adjuvant induce antigen-specific immunity through facilitating antigen presentation and stimulating T cells. In this study, the immunostimulatory properties of two major fragments of Hsp70 (N-Hsp70(aa 1-387) with ATPase property and C-Hsp70 (aa 508-641) with peptide-binding capacity) and the full length of Hsp27 as vaccine adjuvants were evaluated to boost HIV-1 Nef antigen-specific immunity in both in vitro and in vivo experiments. METHODS: At first, the nanoparticles harbouring DNA fusion constructs (i.e. N-Hsp70-Nef, C-Hsp70-Nef and Hsp27-Nef) complexed with HIV Rev (34-50) cell-penetrating peptide were generated to deliver DNA into the cells. Then, the recombinant Nef, Hsp27-Nef, N-Hsp70-Nef and C-Hsp70-Nef proteins were generated in E.coli expression system. Next, the immunostimulatory properties of these fusion constructs were evaluated in both in vitro and in vivo studies. Finally, the secretion of main cytokines from single-cycle replicable (SCR) HIV-1 virion-exposed splenocytes was investigated. RESULTS: Our data showed that the stable and non-toxic DNA/Rev nanoparticles could successfully deliver the genes of interest into the cells. Moreover, higher secretion of antibodies and cytokines was detected in mice receiving the Hsp-Nef constructs than in mice receiving Nef antigen. The C-Hsp70 was also superior for inducing Nef-specific Th1 and CTL immunity compared with N-Hsp70 and Hsp27. The T-cell activity was maintained in the SCR-exposed splenocytes, especially the splenocytes of mice receiving the C-Hsp70-Nef regimen. CONCLUSION: Altogether, these findings demonstrate the significance of Hsps as enhancers of antigen-specific immunity. Notably, the C-Hsp70 region showed better adjuvant properties for inducing cellular immunity in the improvement of HIV-1 therapeutic vaccines.
Assuntos
Infecções por HIV , HIV-1 , Vacinas , Camundongos , Animais , Humanos , HIV-1/genética , Infecções por HIV/prevenção & controle , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico , Adjuvantes Imunológicos/farmacologia , Citocinas , DNARESUMO
Messenger ribonucleic acid (mRNA)-based gene therapy has great potential for cancer gene therapy. However, the effectiveness of mRNA in cancer therapy needs to be further improved, and the delivery efficiency and instability of mRNA limit the application of mRNA-based products. Both the delivery efficiency can be elevated by cell-penetrating peptide modification, and the immune response can be enhanced by tumor cell lysate stimulation, representing an advantageous strategy to expand the effectiveness of mRNA gene therapy. Therefore, it is vital to exploit a vector that can deliver high-efficiency mRNA with codelivery of tumor cell lysate to induce specific immune responses. We previously reported that DMP cationic nanoparticles, formed by the self-assembly of DOTAP and mPEG-PCL, can deliver different types of nucleic acids. DMP has been successfully applied in gene therapy research for various tumor types. Here, we encapsulated tumor cell lysates with DMP nanoparticles and then modified them with a fused cell-penetrating peptide (TAT-iRGD) to form an MLSV system. The MLSV system was loaded with encoded Bim mRNA, forming the MLSV/Bim complex. The average size of the synthesized MLSV was 191.4 nm, with a potential of 47.8 mV. The MLSV/mRNA complex promotes mRNA absorption through caveolin-mediated endocytosis, with a transfection rate of up to 68.6% in B16 cells. The MLSV system could also induce the maturation and activation of dendritic cells, obviously promoting the expression of CD80, CD86, and MHC-II both in vitro and in vivo. By loading the encoding Bim mRNA, the MLSV/Bim complex can inhibit cell proliferation and tumor growth, with inhibition rates of up to 87.3% in vitro. Similarly, the MLSV/Bim complex can inhibit tumor growth in vivo, with inhibition rates of up to 78.7% in the B16 subcutaneous tumor model and 63.3% in the B16 pulmonary metastatic tumor model. Our results suggest that the MLSV system is an advanced candidate for mRNA-based immunogene therapy.
Assuntos
Peptídeos Penetradores de Células , Melanoma , Nanopartículas Multifuncionais , Nanopartículas , Humanos , Melanoma/genética , Melanoma/terapia , Transfecção , Terapia Genética , Linhagem Celular TumoralRESUMO
The purpose of our research is to develop functional additives that enhance mucosal absorption of biologics, such as peptide/protein and antibody drugs, to provide their non-to-poor invasive dosage forms self-managed by patients. Our previous in vivo and in vitro studies demonstrated that the intranasal absorption of biologics in mice was significantly improved when coadministered with oligoarginines anchored chemically to hyaluronic acid via a glycine spacer, presumably through syndecan-4-mediated macropinocytosis under activation by oligoarginines. The present mouse experiments first revealed that diglycine-L-tetraarginine-linked hyaluronic acid significantly enhanced the intranasal absorption of sulpiride, which is a poor-absorptive organic compound with a low molecular weight. However, similar enhancement was not observed for levofloxacin, which has a similarly low molecular weight but is a well-absorptive organic compound, probably because its absorption was mostly dominated by passive diffusion. The subsequent monkey experiments revealed that there was no species difference in the absorption-enhancing ability of diglycine-L-tetraarginine-linked hyaluronic acid for not only organic compounds but also biologics. This was presumably because the expression levels of endocytosis-associated membrane proteins on the nasal mucosa in monkeys were almost equivalent to those in mice, and poorly membrane-permeable/membrane-impermeable drugs were mainly absorbed via syndecan-4-mediated macropinocytosis, regardless of animal species. Drug concentrations in the brain assessed in mice and monkeys and those in the cerebral spinal fluids (CSFs) assessed in monkeys indicated that drugs would be delivered from the systemic circulation to the central nervous system by crossing the blood-brain and the blood-CSF barriers under coadministration with the hyaluronic acid derivative. In line with our original hypothesis, this new set of data supported that our oligoarginine-linked hyaluronic acid would locally perform on the mucosal surface and enhance the membrane permeation of drugs under its colocalization.
Assuntos
Ácido Hialurônico , Animais , Ácido Hialurônico/química , Camundongos , Masculino , Administração Intranasal , Mucosa Nasal/metabolismo , Mucosa Nasal/efeitos dos fármacos , Macaca fascicularis , Absorção Nasal/efeitos dos fármacos , Arginina/químicaRESUMO
This study investigated crotamine (CTA), a peptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, known for its exceptional cell penetration potential. The objective was to explore the antibacterial and antifungal activity of CTA, its ability to inhibit efflux pumps and evaluate the effectiveness of its pharmacological combination with antibiotics and antifungals. In microbiological assays, CTA in combination with antibiotics was tested against strains of S. aureus and the inhibition of NorA, Tet(K) and MepA efflux pumps was also evaluated. CTA alone did not present clinically relevant direct antibacterial action, presenting MIC > 209.7 µM against strains S. aureus 1199B, IS-58, K2068. The standard efflux pump inhibitor CCCP showed significant effects in all negative relationships to assay reproducibility. Against the S. aureus 1199B strain, CTA (20.5 µM) associated with norfloxacin diluted 10 × (320.67 µM) showed a potentiating effect, in relation to the control. Against the S. aureus IS-58 strain, the CTA associated with tetracycline did not show a significant combinatorial effect, either with 2304 or 230.4 µM tetracycline. CTA at a concentration of 2.05 µM associated with ciprofloxacin at a concentration of 309.4 µM showed a significant potentiating effect. In association with EtBr, CTA at concentrations of 2.05 and 20.5 µM potentiated the effect in all strains tested, reducing the prevention of NorA, Tet(K) and MepA efflux pumps. In the C. albicans strain, a potentiating effect of fluconazole (334.3 µM) was observed when combined with CTA (2.05 µM). Against the C. tropicalis strain, a significant effect was also observed in the association of fluconazole 334.3 µM, where CTA 2.05 µM considerably reduced fungal growth and decreased the potentiation of fluconazole. Against the C. krusei strain, no significant potentiating effect of fluconazole was obtained by CTA. Our results indicate that CTA in pharmacological combination potentiates the effects of antibiotics and antifungal. This represents a new and promising antimicrobial strategy for treating a wide variety of infections.
Assuntos
Antibacterianos , Antifúngicos , Venenos de Crotalídeos , Crotalus , Testes de Sensibilidade Microbiana , Antifúngicos/farmacologia , Antifúngicos/química , Antibacterianos/farmacologia , Venenos de Crotalídeos/farmacologia , Animais , Staphylococcus aureus/efeitos dos fármacos , Sinergismo Farmacológico , Candida albicans/efeitos dos fármacos , Serpentes PeçonhentasRESUMO
Many reports have shown that stabilization of secondary structure by stapling functional peptides enhances the intracellular bioactivity. However, no report has discussed the correlation between stabilization and biological activity based on the configuration of amino acid residues used as anchors for stapling. To clarify this, we investigated the helix content and apoptotic efficiency of an apoptosis-inducing peptide, Bim, and four stapled Bim peptides containing stapling-related Cys residues introduced with different configurations within the sequence. The results demonstrated that the configuration of Cys residues in stapled Bim peptides affected the secondary structure and intracellular activity of the peptides, and furthermore, there was a correlation between these latter two variables.
RESUMO
Cell-penetrating peptides (CPPs) with better biomolecule delivery properties will expand their clinical applications. Using the MLCPP2.0 machine algorithm, we screened multiple candidate sequences with potential cellular uptake ability from the nuclear localization signal/nuclear export signal database and verified them through cell-penetrating fluorescent tracing experiments. A peptide (NCR) derived from the Rev protein of the caprine arthritis-encephalitis virus exhibited efficient cell-penetrating activity, delivering over four times more EGFP than the classical CPP TAT, allowing it to accumulate in lysosomes. Structural and property analysis revealed that a high hydrophobic moment and an appropriate hydrophobic region contribute to the high delivery activity of NCR. Trastuzumab emtansine (T-DM1), a HER2-targeted antibody-drug conjugate, could improve its anti-tumor activity by enhancing targeted delivery efficiency and increasing lysosomal drug delivery. This study designed a new NCR vector to non-covalently bind T-DM1 by fusing domain Z, which can specifically bind to the Fc region of immunoglobulin G and effectively deliver T-DM1 to lysosomes. MTT results showed that the domain Z-NCR vector significantly enhanced the cytotoxicity of T-DM1 against HER2-positive tumor cells while maintaining drug specificity. Our results make a useful attempt to explore the potential application of CPP as a lysosome-targeted delivery tool.
RESUMO
The recently developed mRNA-based coronavirus SARS-CoV-2 vaccines highlighted the great therapeutic potential of the mRNA technology. Although the lipid nanoparticles used for the delivery of the mRNA are very efficient, they showed, in some cases, the induction of side effects as well as the production of antibodies directed against particle components. Thus, the development of alternative delivery systems is of great interest in the pursuit of more effective mRNA treatments. In the present work, we evaluated the mRNA transfection capacities of a series of cationic histidine-rich amphipathic peptides derived from LAH4. We found that while the LAH4-A1 peptide was an efficient carrier for mRNA, its activity was highly serum sensitive. Interestingly, modification of this cell penetrating peptide at the N-terminus with two tyrosines or with salicylic acid allowed to confer serum resistance to the carrier.
Assuntos
RNA Mensageiro , RNA Mensageiro/genética , RNA Mensageiro/química , Humanos , SARS-CoV-2/química , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Soro/química , Soro/metabolismo , Transfecção/métodos , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Nanopartículas/química , Peptídeos/química , Animais , COVID-19RESUMO
Eye drops, including solutions and suspensions, are essential dosage forms to treat ophthalmic diseases, with poorly water-soluble drugs typically formulated as ophthalmic suspensions. In addition to low bioavailability, suspensions exhibit limited efficacy, safety, and usability due to the presence of drug particles. Improving bioavailability can reduce the drug concentrations and the risk of problems associated with suspended drug particles. However, practical penetration enhancers capable of improving bioavailability remain elusive. Herein, we focused on penetratin (PNT), a cell-penetrating peptide (CPP) that promotes active cellular transport related to macromolecule uptake, such as micropinocytosis. According to the in vitro corneal uptake study using a reconstructed human corneal epithelial tissue model, LabCyte CORNEA-MODEL24, PNT enhanced the uptake of Fluoresbrite® YG carboxylate polystyrene microspheres without covalent binding. In an ex vivo porcine eye model, the addition of 10 µM PNT to rebamipide ophthalmic suspension markedly improved the corneal uptake of rebamipide; however, the addition of 100 µM PNT was ineffective due to potentially increased particle size by aggregation. This article provides basic information on the application of PNT as a penetration enhancer in ophthalmic suspensions, including the in vitro and ex vivo studies mentioned above, as well as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay and storage stability at different pH values.
Assuntos
Peptídeos Penetradores de Células , Córnea , Soluções Oftálmicas , Suspensões , Animais , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/administração & dosagem , Soluções Oftálmicas/administração & dosagem , Humanos , Córnea/metabolismo , Córnea/efeitos dos fármacos , Suínos , Quinolonas/administração & dosagem , Quinolonas/farmacocinética , Quinolonas/química , Administração Oftálmica , Disponibilidade Biológica , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/metabolismo , Tamanho da Partícula , Alanina/análogos & derivadosRESUMO
BACKGROUND: The exogenous delivery of miRNA to mimic and restore miRNA-34a activity in various cancer models holds significant promise in cancer treatment. Nevertheless, its effectiveness is often impeded by challenges, including a short half-life, propensity for off-target accumulation, susceptibility to inactivation by blood-based enzymes, concerns regarding patient safety, and the substantial cost associated with scaling up. As a means of overcoming these barriers, we propose the development of miRNA-loaded Tat-A86 nanoparticles by virtue of Tat-A86's ability to shield the loaded agent from external environmental factors, reducing degradation and inactivation, while enhancing circulation time and targeted accumulation. RESULTS: Genetically engineered Tat-A86, featuring 16 copies of the interleukin-4 receptor (IL-4R)-binding peptide (AP1), Tat for tumor penetration, and an elastin-like polypeptide (ELP) for presenting target ligands and ensuring stability, served as the basis for this delivery system. Comparative groups, including Tat-E60 and A86, were employed to discern differences in binding and penetration. The designed ELP-based nanoparticle Tat-A86 effectively condensed miRNA, forming stable nanocomplexes under physiological conditions. The miRNA/Tat-A86 formulation bound specifically to tumor cells and facilitated stable miRNA delivery into them, effectively inhibiting tumor growth. The efficacy of miRNA/Tat-A86 was further evaluated using three-dimensional spheroids of lewis lung carcinoma (LLC) as in vitro model and LLC tumor-bearing mice as an in vivo model. It was found that miRNA/Tat-A86 facilitates effective cell killing by markedly improving miRNA penetration, leading to a substantial reduction in the size of LLC spheroids. Compared to other controls, Tat-A86 demonstrated superior efficacy in suppressing the growth of 3D cellular aggregates. Moreover, at equivalent doses, miRNA-34a delivered by Tat-A86 inhibited the growth of LLC cells in allograft mice. CONCLUSIONS: Overall, these studies demonstrate that Tat-A86 nanoparticles can deliver miRNA systemically, overcoming the basic hurdles impeding miRNA delivery by facilitating both miRNA uptake and stability, ultimately leading to improved therapeutic effects.
Assuntos
Elastina , MicroRNAs , Nanopartículas , Peptídeos , Animais , MicroRNAs/genética , Elastina/química , Camundongos , Peptídeos/química , Humanos , Nanopartículas/química , Linhagem Celular Tumoral , Neoplasias/terapia , Neoplasias/tratamento farmacológico , Portadores de Fármacos/química , Feminino , Polipeptídeos Semelhantes à ElastinaRESUMO
Cell-penetrating peptides (CPPs) serve as potent vehicles for delivering membrane-impermeable compounds, including nucleic acids, into cells. In a previous study, we reported the successful intracellular delivery of small interfering RNAs (siRNAs) with negligible cytotoxicity using a peptide containing an unnatural amino acid (dipropylglycine). In the present study, we employed the same seven peptides as the previous study to evaluate their efficacy in delivering plasmid DNA (pDNA) intracellularly. Although pDNA and siRNA are nucleic acids, they differ in size and biological function, which may influence the optimal peptide sequences for their delivery. Herein, three peptides demonstrated effective pDNA transfection abilities. Notably, only one of the three peptides previously exhibited efficient gene-silencing effect with siRNA. These findings validate our hypothesis and offer insights for the personalized design of CPPs for the delivery of pDNA and siRNA.