RESUMO
Telomere length plays a crucial role in cellular aging and the risk of diseases. Unlike normal cells, cancer cells can extend their own survival by maintaining telomere stability through telomere maintenance mechanism. Therefore, regulating the lengths of telomeres have emerged as a promising approach for anti-cancer treatment. In this study, we introduce a nanoscale octopus-like structure designed to induce physical entangling of telomere, thereby efficiently triggering telomere dysfunction. The nanoscale octopus, composed of eight-armed PEG (8-arm-PEG), are functionalized with cell penetrating peptide (TAT) to facilitate nuclear entry and are covalently bound to N-Methyl Mesoporphyrin IX (NMM) to target G-quadruplexes (G4s) present in telomeres. The multi-armed configuration of the nanoscale octopus enables targeted binding to multiple G4s, physically disrupting and entangling numerous telomeres, thereby triggering telomere dysfunction. Both in vitro and in vivo experiments indicate that the nanoscale octopus significantly inhibits cancer cell proliferation, induces apoptosis through telomere entanglement, and ultimately suppresses tumor growth. This research offers a novel perspective for the development of innovative anti-cancer interventions and provides potential therapeutic options for targeting telomeres.
Assuntos
Apoptose , Telômero , Telômero/metabolismo , Apoptose/efeitos dos fármacos , Humanos , Animais , Linhagem Celular Tumoral , Camundongos , Quadruplex G/efeitos dos fármacos , Camundongos Nus , Polietilenoglicóis/química , Proliferação de Células/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Neoplasias/patologia , Neoplasias/tratamento farmacológico , Feminino , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Nanoestruturas/químicaRESUMO
BACKGROUND: Cell-penetrating peptides (CPPs) are effective for delivering therapeutic molecules with minimal toxicity. This study focuses on the use of penetratin, a well-characterized CPP, to deliver a DNA vector encoding short hairpin RNA (shRNA) targeting the respiratory syncytial virus (RSV) F gene into infected cells. RSV is known to cause severe lower respiratory infections in infants and poses significant risks to immunocompromised individuals and the elderly. We evaluated the antiviral efficacy of the penetratin-shRNA complex by comparing its ability to inhibit RSV replication and induce apoptosis with ribavirin treatment. METHODS: Penetratin-shRNA complexes were prepared at different ratios and analyzed using gel retardation assays, dynamic light scattering, and zeta potential measurements. The complexes were tested in HEp-2 and A549 cells for transfection efficiency, cytotoxicity, viral load, and apoptosis using plaque assays, real-time reverse transcription-polymerase chain reaction (RT-PCR), DNA fragmentation, propidium iodide staining, and caspase 3/7 activation assays. RESULTS: The gel shift assay determined that a 20:1 CPP-to-shRNA ratio was optimal for effective complexation, resulting in particles with a size of 164 nm and a zeta potential of 8.7 mV. Transfection efficiency in HEp-2 cells was highest at this ratio, reaching up to 93%. The penetratin-shRNA complex effectively silenced the RSV F gene, reduced viral titers, and decreased DNA fragmentation and apoptosis in infected cells. CONCLUSION: Penetratin effectively delivers shRNA targeting the RSV F gene, significantly reducing viral load and preventing apoptosis without toxicity. This approach surpasses Lipofectamine and shows potential for future therapeutic interventions, especially when combined with ribavirin, against RSV infection.
Assuntos
Antivirais , Apoptose , Peptídeos Penetradores de Células , RNA Interferente Pequeno , Replicação Viral , Humanos , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/química , Apoptose/efeitos dos fármacos , Antivirais/farmacologia , Replicação Viral/efeitos dos fármacos , RNA Interferente Pequeno/genética , Linhagem Celular , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/fisiologiaRESUMO
Monoclonal antibody therapies for cancer have demonstrated extraordinary clinical success in recent years. However, these strategies are thus far mostly limited to specific cell surface antigens, even though many disease targets are found intracellularly. Here we report studies on the humanization of a full-length, nucleic acid binding, monoclonal lupus-derived autoantibody, 3E10, which exhibits a novel mechanism of cell penetration and tumor specific targeting. Comparing humanized variants of 3E10, we demonstrate that cell uptake depends on the nucleoside transporter ENT2, and that faster cell uptake and superior in vivo tumor targeting are associated with higher affinity nucleic acid binding. We show that one human variant retains the ability of the parental 3E10 to bind RAD51, serving as a synthetically lethal inhibitor of homology-directed repair in vitro. These results provide the basis for the rational design of a novel antibody platform for therapeutic tumor targeting with high specificity following systemic administration.
Assuntos
Rad51 Recombinase , Humanos , Animais , Rad51 Recombinase/antagonistas & inibidores , Rad51 Recombinase/metabolismo , Rad51 Recombinase/imunologia , Camundongos , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/químicaRESUMO
The current selection of ligands for both proteins of interest (POI) and E3 ubiquitin ligase significantly restricts the scope of targeted protein degradation (TPD) technologies. This study introduces cell-penetrating peptide-induced chimera conjugates (cp-PCCs) targeting the DHHC3 enzyme involved in PD-L1 palmitoylation. This approach disrupts PD-L1's immunosuppressive function, enhancing anti-tumor immunity. We developed cp-PCCs to degrade DHHC3, directly linking DHHC3-mediated PD-L1 palmitoylation to PD-L1 stability on tumor cells. Our research utilized both in vitro assays and in vivo experiments in immune checkpoint blockade-resistant mouse models. We focused on a CRBN-based cp-PCC named PCC16, which demonstrated a DC50 of 102 nmol for DHHC3 degradation and significantly reduced PD-L1 levels. In resistant models, PCC16 not only robustly downregulated PD-L1 but also exhibited substantial anti-tumor activity in vivo without significant toxicity. This outperformed traditional inhibitors, showcasing the potential of cp-PCC technology to bypass current PROTAC limitations. Our findings suggest that cp-PCCs offer a promising method for targeting PD-L1 through DHHC3 inhibition and support their continued exploration as a versatile tool in cancer immunotherapy, especially for tumors resistant to standard treatments.
Assuntos
Antígeno B7-H1 , Peptídeos Penetradores de Células , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/química , Animais , Humanos , Antígeno B7-H1/metabolismo , Camundongos , Linhagem Celular Tumoral , Proteólise/efeitos dos fármacos , Aciltransferases/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores de Checkpoint Imunológico/farmacologia , Feminino , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
The intrinsically disordered protein MeCP2 is a global transcriptional regulator encoded by the MECP2 gene. Although the structured domains of MeCP2 have been the subject of multiple studies, its unstructured regions have not been that extensively characterized. In this work, we show that MeCP2 possesses properties akin to those of supercharged proteins. By utilizing its unstructured portions, MeCP2 can successfully transduce across cell membranes and localize to heterochromatic foci in the nuclei, displaying uptake levels a third lower than a MeCP2 construct fused to the cell-penetrating peptide TAT. MeCP2 uptake can further be enhanced by the addition of compounds that promote endosomal escape following cellular trafficking by means of macropinocytosis. Using a combination of in silico prediction algorithms and live-cell imaging experiments, we mapped the sequence in MeCP2 responsible for its cellular incorporation, which bears a striking resemblance to TAT itself. Transduced MeCP2 was shown to interact with HDAC3. These findings provide valuable insight into the properties of MeCP2 and may be beneficial for devising future protein-based treatment strategies.
Assuntos
Membrana Celular , Histona Desacetilases , Proteína 2 de Ligação a Metil-CpG , Proteína 2 de Ligação a Metil-CpG/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/química , Humanos , Membrana Celular/metabolismo , Membrana Celular/química , Histona Desacetilases/metabolismo , Histona Desacetilases/química , Histona Desacetilases/genética , Células HEK293 , Transporte Proteico , Peptídeos Penetradores de Células/metabolismo , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/genéticaRESUMO
This review presents current knowledge related to the voltage-dependent anion channel-1 (VDAC1) as a multi-functional mitochondrial protein that acts in regulating both cell life and death. The location of VDAC1 at the outer mitochondrial membrane (OMM) allows control of metabolic cross-talk between the mitochondria and the rest of the cell, and also enables its interaction with proteins that are involved in metabolic, cell death, and survival pathways. VDAC1's interactions with over 150 proteins can mediate and regulate the integration of mitochondrial functions with cellular activities. To target these protein-protein interactions, VDAC1-derived peptides have been developed. This review focuses specifically on cell-penetrating VDAC1-based peptides that were developed and used as a "decoy" to compete with VDAC1 for its VDAC1-interacting proteins. These peptides interfere with VDAC1 interactions, for example, with metabolism-associated proteins such as hexokinase (HK), or with anti-apoptotic proteins such as Bcl-2 and Bcl-xL. These and other VDAC1-interacting proteins are highly expressed in many cancers. The VDAC1-based peptides in cells in culture selectively affect cancerous, but not non-cancerous cells, inducing cell death in a variety of cancers, regardless of the cancer origin or genetics. They inhibit cell energy production, eliminate cancer stem cells, and act very rapidly and at low micro-molar concentrations. The activity of these peptides has been validated in several mouse cancer models of glioblastoma, lung, and breast cancers. Their anti-cancer activity involves a multi-pronged attack targeting the hallmarks of cancer. They were also found to be effective in treating non-alcoholic fatty liver disease and diabetes mellitus. Thus, VDAC1-based peptides, by targeting VDAC1-interacting proteins, offer an affordable and innovative new conceptual therapeutic paradigm that can potentially overcome heterogeneity, chemoresistance, and invasive metastatic formation.
Assuntos
Diabetes Mellitus , Neoplasias , Canal de Ânion 1 Dependente de Voltagem , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Animais , Diabetes Mellitus/metabolismo , Diabetes Mellitus/tratamento farmacológico , Peptídeos/farmacologia , Peptídeos/química , Peptídeos/uso terapêutico , Peptídeos/metabolismo , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/metabolismo , Peptídeos Penetradores de Células/química , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Ligação ProteicaRESUMO
One of the bottlenecks to bringing new therapies to the clinic has been a lack of vectors for delivering novel therapeutics in a targeted manner. Cell penetrating peptides (CPPs) have received a lot of attention and have been the subject of numerous developments since their identification nearly three decades ago. Known for their transduction abilities, they have generally been considered inert vectors. In this review, we present a schema for their classification, highlight what is known about their mechanism of transduction, and outline the existing literature as well as our own experience, vis a vis the intrinsic anti-inflammatory properties that certain CPPs exhibit. Given the inflammatory responses associated with viral vectors, CPPs represent a viable alternative to such vectors; furthermore, the anti-inflammatory properties of CPPs, mostly through inhibition of the NF-κB pathway, are encouraging. Much more work in relevant animal models, toxicity studies in large animal models, and ultimately human trials are needed before their potential is fully realized.
Assuntos
Anti-Inflamatórios , Peptídeos Penetradores de Células , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Humanos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Animais , Inflamação/metabolismo , Inflamação/tratamento farmacológico , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Cell-penetrating peptides (CPPs) enter the cell by two different mechanisms-endocytosis followed by endosomal escape and direct translocation at the plasma membrane. The mechanism of direct translocation remains unresolved. In this work, the direct translocation of nonaarginine (R9) and two cyclic CPPs (CPP12 and CPP17) into Jurkat cells was monitored by time-lapse confocal microscopy. Our results provide direct evidence that all three CPPs translocate across the plasma membrane by a recently discovered vesicle budding-and-collapse (VBC) mechanism. Membrane translocation is preceded by the formation of nucleation zones. Up to four different types of nucleation zones and three variations of the VBC mechanism were observed. The VBC mechanism reconciles the enigmatic and conflicting observations in the literature.
Assuntos
Membrana Celular , Peptídeos Penetradores de Células , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Humanos , Membrana Celular/metabolismo , Células Jurkat , Transporte ProteicoRESUMO
Purpose: Effective mucosal delivery of drugs continues to pose a significant challenge owing to the formidable barrier presented by the respiratory tract mucus, which efficiently traps and clears foreign particulates. The surface characteristics of micelles dictate their ability to penetrate the respiratory tract mucus. In this study, polymeric micelles loaded with insulin (INS) were modified using mucus-penetrative polymers. Methods: We prepared and compared polyethylene glycol (PEG)-coated micelles with micelles where cell-penetrating peptide (CPP) is conjugated to PEG. Systematic investigations of the physicochemical and aerosolization properties, performance, in vitro release, mucus and cell penetration, lung function, and pharmacokinetics/pharmacodynamics (PK/PD) of polymeric micelles were performed to evaluate their interaction with the respiratory tract. Results: The nano-micelles, with a particle size of <100 nm, exhibited a sustained-release profile. Interestingly, PEG-coated micelles exhibited higher diffusion and deeper penetration across the mucus layer. In addition, CPP-modified micelles showed enhanced in vitro cell penetration. Finally, in the PK/PD studies, the micellar solution demonstrated higher maximum concentration (Cmax) and AUC0-8h values than subcutaneously administered INS solution, along with a sustained blood glucose-lowering effect that lasted for more than 8 h. Conclusion: This study proposes the use of mucus-penetrating micelle formulations as prospective inhalation nano-carriers capable of efficiently transporting peptides to the respiratory tract.
Assuntos
Peptídeos Penetradores de Células , Insulina , Micelas , Polietilenoglicóis , Insulina/administração & dosagem , Insulina/farmacocinética , Insulina/química , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Animais , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacocinética , Humanos , Tamanho da Partícula , Administração por Inalação , Masculino , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Ratos Sprague-Dawley , Muco/química , Muco/metabolismo , Muco/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Glicemia/efeitos dos fármacos , Glicemia/análiseRESUMO
Intracellular delivery of large molecule cargo via cell penetrating peptides (CPPs) is an inefficient process and despite intense efforts in past decades, improvements in efficiency have been marginal. Utilizing a standardized and comparative analysis of the delivery efficiency of previously described cationic, anionic, and amphiphilic CPPs, we demonstrate that the delivery ceiling is accompanied by irreparable plasma membrane damage that is part of the uptake mechanism. As a consequence, intracellular delivery correlates with cell toxicity and is more efficient for smaller peptides than for large molecule cargo. The delivery of pharmaceutically relevant cargo quantities with acceptable toxicity thus seems hard to achieve with the CPPs tested in our study. Our results suggest that any engineered intracellular delivery system based on conventional cationic or amphiphilic CPPs, or the design principles underlying them, needs to accept low delivery yields due to toxicity limiting efficient cytoplasmic uptake. Novel peptide designs based on detailed study of uptake mechanisms are required to overcome these limitations.
Assuntos
Membrana Celular , Peptídeos Penetradores de Células , Citoplasma , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Membrana Celular/metabolismo , Humanos , Citoplasma/metabolismo , Sistemas de Liberação de Medicamentos , Células HeLa , AnimaisRESUMO
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.
Assuntos
Mucosa Nasal , Polifenóis , Taninos , Taninos/química , Polifenóis/química , Polifenóis/administração & dosagem , Mucosa Nasal/metabolismo , Mucosa Nasal/imunologia , Animais , Nanopartículas/química , Humanos , Peptídeos Penetradores de Células/química , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/química , Adesivos/química , Depuração Mucociliar/efeitos dos fármacos , Imunoglobulina A , CamundongosRESUMO
HxTx-Hv1h, a neurotoxic peptide derived from spider venom, has been developed for use in commercial biopesticide formulations. Cell Penetrating Peptides (CPPs) are short peptides that facilitate the translocation of various biomolecules across cellular membranes. Here, we evaluated the aphidicidal efficacy of a conjugated peptide, HxTx-Hv1h/CPP-1838, created by fusing HxTx-Hv1h with CPP-1838. Additionally, we aimed to establish a robust recombinant expression system for HxTx-Hv1h/CPP-1838. We successfully achieved the secretory production of HxTx-Hv1h, its fusion with Galanthus nivalis agglutinin (GNA) forming HxTx-Hv1h/GNA and HxTx-Hv1h/CPP-1838 in yeast. Purified HxTx-Hv1h exhibited contact toxicity against Megoura crassicauda, with a 48 h median lethal concentration (LC50) of 860.5 µg/mL. Fusion with GNA or CPP-1838 significantly enhanced its aphidicidal potency, reducing the LC50 to 683.5 µg/mL and 465.2 µg/mL, respectively. The aphidicidal efficacy was further improved with the addition of surfactant, decreasing the LC50 of HxTx-Hv1h/CPP-1838 to 66.7 µg/mL-over four times lower compared to HxTx-Hv1h alone. Furthermore, we engineered HxTx-Hv1h/CPP-1838 multi-copy expression vectors utilizing the BglBrick assembly method and achieved high-level recombinant production in laboratory-scale fermentation. This study is the first to document a CPP fusion strategy that enhances the transdermal aphidicidal activity of a natural toxin like HxTx-Hv1h and opens up the possibility of exploring the recombinant production of HxTx-Hv1h/CPP-1838 for potential applications.
Assuntos
Peptídeos Penetradores de Células , Neurotoxinas , Venenos de Aranha , Venenos de Aranha/química , Venenos de Aranha/genética , Venenos de Aranha/toxicidade , Animais , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/química , Neurotoxinas/toxicidade , Neurotoxinas/farmacologia , Neurotoxinas/genética , Lectinas de Plantas/farmacologia , Lectinas de Plantas/genética , Lectinas de Plantas/química , Inseticidas/farmacologia , Inseticidas/toxicidadeRESUMO
Targeted protein degradation through the lysosomal pathway has attracted increasing attention and expanded the scope of degradable proteins. However, the endogenous lysosomal degradation strategies are mainly based on antibodies or nanobodies. Effective small molecule lysosomal degraders are still rather rare. Herein, a new lysosomal degradation approach, termed peptide-mediated small molecule lysosome-targeting chimeras (PSMLTACs), was developed by the incorporation of small molecule ligands with a lysosome-sorting NPGY motif containing the cell-penetrating peptide. PSMLTACs were successfully applied to degrade both membrane and intracellular targets. In particular, the PSMLTAC strategy demonstrated higher degradation efficiency on membrane target PD-L1 and intracellular target PDEδ than corresponding PROTAC degraders. Taken together, this proof-of-concept provides a convenient and effective strategy for targeted protein degradation.
Assuntos
Lisossomos , Proteólise , Lisossomos/metabolismo , Humanos , Proteólise/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Peptídeos Penetradores de Células/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/metabolismo , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inibidores , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/metabolismoRESUMO
Radiolabelled autologous leukocytes have been used for the clinical diagnosis of inflammation and infection. To develop a stable and efficient radiopharmaceutical for labelling leukocytes, we prepared a novel radioiodinated cell-penetrating peptide, 125I-TAT, using a bi-functional linker. 125I-TAT was stable for two days under three different temperature conditions of -20 °C, 4 °C, and 40 °C, with its radiochemical purity remaining over 99%. Iodinated TAT was non-toxic to leukocytes with an IC50 value of over 100 µM. The labelling efficiency of 125I-TAT using 1ï½107 cells ranged from 27% to 53% when the three leukocyte cell lines were pre-treated with DMSO. This is comparable to the labelling efficiency recommended by the guideline for conventional labelling agents using 2ï½108 cells. Radioiodinated cell-penetrating peptide may be an improved radiopharmaceutical for white blood cell scans by further optimization.
Assuntos
Radioisótopos do Iodo , Leucócitos , Compostos Radiofarmacêuticos , Humanos , Compostos Radiofarmacêuticos/farmacocinética , Peptídeos Penetradores de Células/química , Marcação por Isótopo/métodosRESUMO
We previously reported that a linear cationic 12-amino acid cell-penetrating peptide (CPP) was bactericidal for Neisseria gonorrhoeae. In this study, our objectives were to determine the effect of cyclization of the linear CPP on its antibacterial activity for N. gonorrhoeae and cytotoxicity for human cells. We compared the bactericidal effect of 4-hour treatment with the linear CPP to that of CPPs cyclized by a thioether or a disulfide bond on human challenge and multi-drug resistant (MDR) strains of N. gonorrhoeae grown in cell culture media with 10% fetal bovine serum (FBS). The effect of lipooligosaccharide (LOS) sialylation on bactericidal activity was analyzed. We determined the ability of the CPPs to treat human cells infected in vitro with N. gonorrhoeae, to reduce the inflammatory response of human monocytic cells to gonococci, to kill strains of three commensal Neisseria species, and to inhibit gonococcal biofilms. The cyclized CPPs killed 100% of gonococci from all strains at 100 µM and >90% at 20 µM and were more potent than the linear form. The thioether-linked but not the disulfide-linked CPP was less cytotoxic for human cervical cells compared to the linear CPP. LOS sialylation had minimal effect on bactericidal activity. In treating infected human cells, the thioether-linked CPP at 20 µM killed >60% of extra- and intracellular bacteria and reduced TNF-α expression by THP-1 cells. The potency of the CPPs for the pathogenic and the commensal Neisseria was similar. The thioether-linked CPP partially eradicated gonococcal biofilms. Future studies will focus on determining efficacy in the female mouse model of gonorrhea.IMPORTANCENeisseria gonorrhoeae remains a major cause of sexually transmitted infections with 82 million cases worldwide in 2020, and 710,151 confirmed cases in the US in 2021, up 25% from 2017. N. gonorrhoeae can infect multiple tissues including the urethra, cervix, rectum, pharynx, and conjunctiva. The most serious sequelae are suffered by infected women as gonococci ascend to the upper reproductive tract and cause pelvic inflammatory disease, chronic pelvic pain, and infertility in 10%-20% of women. Control of gonococcal infection is widely recognized as increasingly challenging due to the lack of any vaccine. N. gonorrhoeae has quickly developed resistance to all but one class of antibiotics and the emergence of multidrug-resistant strains could result in untreatable infections. As such, gonorrhea is classified by the Center for Disease Control (CDC) as an urgent public health threat. The research presented herein on new therapeutics for gonorrhea has identified a cyclic cell-penetrating peptide (CPP) as a potent molecule targeting N. gonorrhoeae.
Assuntos
Antibacterianos , Peptídeos Penetradores de Células , Gonorreia , Neisseria gonorrhoeae , Neisseria gonorrhoeae/efeitos dos fármacos , Humanos , Gonorreia/tratamento farmacológico , Gonorreia/microbiologia , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/química , Antibacterianos/farmacologia , Antibacterianos/química , Animais , Camundongos , Feminino , Biofilmes/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Ciclização , Lipopolissacarídeos/metabolismo , Arginina/farmacologia , Arginina/químicaRESUMO
Despite ongoing research on antimicrobial peptides (AMPs) and cell-penetrating peptides (CPPs), their precise translocation mechanism remains elusive. This includes Buforin 2 (BF2), a well-known AMP, for which spontaneous translocation across the membrane has been proposed but a high barrier has been calculated. Here, we used computer simulations to investigate the effect of a nonequilibrium situation where the peptides are adsorbed on one side of the lipid bilayer, mimicking experimental conditions. We demonstrated that the asymmetric membrane adsorption of BF2 enhances its translocation across the lipid bilayer by lowering the energy barrier by tens of kJ mol-1. We showed that asymmetric membrane adsorption also reduced the free energy barrier of lipid flip-flop but remained unlikely even at BF2 surface saturation. These results provide insight into the driving forces behind membrane translocation of cell-penetrating peptides in nonequilibrium conditions, mimicking experiments.
Assuntos
Bicamadas Lipídicas , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Adsorção , Terpenos/química , Terpenos/farmacologia , Simulação de Dinâmica Molecular , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Termodinâmica , Membrana Celular/metabolismo , Membrana Celular/química , ProteínasRESUMO
Proteolysis targeting chimeras (PROTAC) are bifunctional chimeric molecules capable of directly degrading binding proteins through the ubiquitin-proteasome pathway. PROTACs have demonstrated significant potential in overcoming drug resistance and targeting previously untreatable targets. However, several limitations still need to be addressed, including their high molecular weight resulting in poor membrane permeability and bioavailability. In this study, we proposed that cancer-targeted penetrating peptides could enhance the cell permeability of PROTACs. We developed 26 novel targeted penetrating peptides for leukemia and lymphoma cells, among which C9C-f(3Bta) and Cyclo-C9C-R exhibited superior membrane permeability, targetability, and stability. By combining C9C-f(3Bta) and Cyclo-C9C-R with IMA-PROTAC, we effectively enhanced the anti-proliferative activity of IMA-PROTAC, facilitated degradation of Bcr-Abl protein in K562 cells, and reduced downstream STAT5 phosphorylation. Furthermore, the combined application promoted cell apoptosis while blocking G1 phase progression. HPLC-MRM-MS revealed that the combination of C9C-f(3Bta) or Cyclo-C9C-R with IMA-PROTAC significantly enhanced intracellular IMA-PROTAC content. In summary, our proof-of-concept study validated the hypothesis that combining PROTACs with targeted penetrating peptides can improve protein degradation efficiency as well as anti-proliferative capabilities.
Assuntos
Antineoplásicos , Apoptose , Proliferação de Células , Leucemia , Linfoma , Proteólise , Humanos , Leucemia/tratamento farmacológico , Leucemia/patologia , Leucemia/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Proteólise/efeitos dos fármacos , Linfoma/tratamento farmacológico , Linfoma/patologia , Linfoma/metabolismo , Proliferação de Células/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Relação Estrutura-Atividade , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/síntese química , Estrutura Molecular , Células K562 , Relação Dose-Resposta a Droga , Sistemas de Liberação de MedicamentosRESUMO
Cell-penetrating peptides (CPPs) are crucial for delivering macromolecules such as nucleic acids into cells. This study investigates the effectiveness of dual-modified penetratin peptides, focusing on the impact of stapling structures and an endosomal escape domain (EED) on enhancing intracellular uptake. Some CPPs were synthesized with an EED at either the N- or C-terminus and stapling structures, and then complexed with plasmid DNA (pDNA) to evaluate their cellular uptake. Results revealed that the combination of stapling and an EED significantly improved delivery efficiency, primarily via macropinocytosis and clathrin-mediated endocytosis. These findings underscore the importance of optimizing CPP sequences for effective nucleic acid delivery systems.
Assuntos
Peptídeos Penetradores de Células , Endossomos , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/síntese química , Peptídeos Penetradores de Células/farmacologia , Humanos , Endossomos/metabolismo , DNA/química , Plasmídeos , Células HeLaRESUMO
Glioblastoma is a common brain tumor that poses considerable challenges in drug delivery. In this study, we investigated the potential of cell-based nanoparticles for targeted drug delivery to the glioblastoma sites. The anticancer drug of temozolomide (TMZ)-loaded T7-cholesterol nanoparticle micelles efficiently delivered nanoparticles to neutrophils and, subsequently, to the tumors. T7 is a cell-penetrating peptide that enhances the delivery of T7/TMZ to the target cells. T7 also serves as a transferrin target peptide, enabling targeted delivery to tumors. T7-conjugated cholesterol can self-assemble into micelles in aqueous solution and attach to the membrane of neutrophils. We confirmed that T7/TMZ nanoparticle micelles were efficiently located inside the neutrophils. Thereafter, T7/TMZ-conveyed neutrophils were administered to a glioblastoma mouse model, enabling neutrophils to penetrate the blood-brain barrier and deliver drugs directly to the tumor site. We evaluated the drug delivery efficiency and therapeutic effects of intravenous injection of T7/TMZ-conveyed neutrophils to a glioblastoma mouse model. These results demonstrate the promising role of neutrophil-based nanoparticle delivery systems in the targeted therapy of glioblastoma.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Nanopartículas , Neutrófilos , Temozolomida , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/metabolismo , Animais , Camundongos , Temozolomida/química , Temozolomida/farmacologia , Neutrófilos/metabolismo , Neutrófilos/efeitos dos fármacos , Humanos , Nanopartículas/química , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Micelas , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Colesterol/química , Portadores de Fármacos/química , Peptídeos Penetradores de Células/química , Fragmentos de Peptídeos , Colágeno Tipo IVRESUMO
Peptide-based drug development is a promising direction due to its excellent biological activity, minimal immunogenicity, high in vivo stability, and efficient tissue penetrability. GV1001, an amphiphilic peptide, has proven effective as an anti-cancer vaccine, but its effect on osteoblast differentiation is unknown. To identify proteins interacting with GV1001, biotin-conjugated GV1001 was constructed and confirmed by mass spectrometry. Proteomic analyses were performed to determine GV1001's interaction with osteogenic proteins. GV1001 was highly associated with peptidyl-prolyl isomerase A and co-immunoprecipitation assays revealed that GV1001 bound to peptidyl-prolyl cis-trans isomerase 1 (Pin1). GV1001 significantly increased alkaline phosphatase (ALP) activity, bone nodule formation, and the expression of osteogenic gene markers. GV1001-induced osteogenic activity was enhanced by Pin1 overexpression and abolished by Pin1 knockdown. GV1001 increased the protein stability and transcriptional activity of Runx2 and Osterix. Importantly, GV1001 administration enhanced bone mass density in the OVX mouse model, as verified by µCT analysis. GV1001 demonstrated protective effects against bone loss in OVX mice by upregulating osteogenic differentiation via the Pin1-mediated protein stabilization of Runx2 and Osterix. GV1001 could be a potential candidate with anabolic effects for the prevention and treatment of osteoporosis.