RESUMO
Ferroptosis, a non-apoptotic form of programmed cell death, is triggered by oxidative stress in cancer, heat stress in plants, and hemorrhagic stroke. A homeostatic transcriptional response to ferroptotic stimuli is unknown. We show that neurons respond to ferroptotic stimuli by induction of selenoproteins, including antioxidant glutathione peroxidase 4 (GPX4). Pharmacological selenium (Se) augments GPX4 and other genes in this transcriptional program, the selenome, via coordinated activation of the transcription factors TFAP2c and Sp1 to protect neurons. Remarkably, a single dose of Se delivered into the brain drives antioxidant GPX4 expression, protects neurons, and improves behavior in a hemorrhagic stroke model. Altogether, we show that pharmacological Se supplementation effectively inhibits GPX4-dependent ferroptotic death as well as cell death induced by excitotoxicity or ER stress, which are GPX4 independent. Systemic administration of a brain-penetrant selenopeptide activates homeostatic transcription to inhibit cell death and improves function when delivered after hemorrhagic or ischemic stroke.
Assuntos
Isquemia Encefálica , Peptídeos Penetradores de Células/farmacologia , Ferroptose/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hemorragias Intracranianas , Neurônios , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/biossíntese , Selênio/farmacologia , Acidente Vascular Cerebral , Transcrição Gênica/efeitos dos fármacos , Animais , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Hemorragias Intracranianas/tratamento farmacológico , Hemorragias Intracranianas/metabolismo , Hemorragias Intracranianas/patologia , Masculino , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Fator de Transcrição Sp1/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Fator de Transcrição AP-2/metabolismoRESUMO
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
NRF2 (nuclear factor erythroid-2-related factor 2) is a key regulator of genes involved in the cell's protective response to oxidative stress. Upon activation by disturbed redox homeostasis, NRF2 promotes the expression of metabolic enzymes to eliminate reactive oxygen species (ROS). Cell internalization of peroxisome-like artificial organelles that harbor redox-regulating enzymes was previously shown to reduce ROS-induced stress and thus cell death. However, if and to which extent ROS degradation by such nanocompartments interferes with redox signaling pathways is largely unknown. Here, we advance the design of H2O2-degrading artificial nano-organelles (AnOs) that exposed surface-attached cell penetrating peptides (CPP) for enhanced uptake and were equipped with a fluorescent moiety for rapid visualization within cells. To investigate how such AnOs integrate in cellular redox signaling, we engineered leukemic K562 cells that report on NRF2 activation by increased mCherry expression. Once internalized, ROS-metabolizing AnOs dampen intracellular NRF2 signaling upon oxidative injury by degrading H2O2. Moreover, intracellular AnOs conferred protection against ROSinduced cell death in conditions when endogenous ROS-protection mechanisms have been compromised by depletion of glutathione or knockdown of NRF2. We demonstrate CPP-facilitated AnO uptake and AnO-mediated protection against ROS insults also in the T lymphocyte population of primary peripheral blood mononuclear cells from healthy donors. Overall, our data suggest that intracellular AnOs alleviated cellular stress by the on-site reduction of ROS.
Assuntos
Peróxido de Hidrogênio , Fator 2 Relacionado a NF-E2 , Estresse Oxidativo , Espécies Reativas de Oxigênio , Transdução de Sinais , Humanos , Fator 2 Relacionado a NF-E2/metabolismo , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Células K562 , Espécies Reativas de Oxigênio/metabolismo , Oxirredução , Peptídeos Penetradores de Células/metabolismo , Peptídeos Penetradores de Células/farmacologia , Organelas/metabolismoRESUMO
Noncanonical nucleic acid structures, particularly G-quadruplexes, have garnered significant attention as potential therapeutic targets in cancer treatment. Here, the recognition of G-quadruplex DNA by peptides derived from the Rap1 protein is explored, with the aim of developing novel peptide-based G-quadruplex ligands with enhanced selectivity and anticancer activity. Biophysical techniques were employed to assess the interaction of a peptide derived from the G-quadruplex-binding domain of the protein with various biologically relevant G-quadruplex structures. Through alanine scanning mutagenesis, key amino acids crucial for G-quadruplex recognition were identified, leading to the discovery of two peptides with improved G-quadruplex-binding properties. However, despite their in vitro efficacy, these peptides showed limited cell penetration and anticancer activity. To overcome this challenge, cell-penetrating peptide (CPP)-conjugated derivatives were designed, some of which exhibited significant cytotoxic effects on cancer cells. Interestingly, selected CPP-conjugated peptides exerted potent anticancer activity across various tumour types via a G-quadruplex-dependent mechanism. These findings underscore the potential of peptide-based G-quadruplex ligands in cancer therapy and pave the way for the development of novel therapeutic strategies targeting these DNA structures.
Assuntos
Antineoplásicos , Peptídeos Penetradores de Células , Quadruplex G , Quadruplex G/efeitos dos fármacos , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Linhagem Celular Tumoral , Peptídeos/química , Peptídeos/farmacologia , Ligantes , DNA/química , DNA/metabolismo , Complexo Shelterina/metabolismo , Complexo Shelterina/química , Ligação ProteicaRESUMO
Second mitochondrial-derived activator of caspase (SMAC), also known as direct inhibitor of apoptosis-binding proteins with low pI (Diablo), is known as a pro-apoptotic mitochondrial protein released into the cytosol in response to apoptotic signals. We recently reported SMAC overexpression in cancers as essential for cell proliferation and tumor growth due to non-apoptotic functions, including phospholipid synthesis regulation. These functions may be associated with its interactions with partner proteins. Using a peptide array with 768 peptides derived from 11 selected SMAC-interacting proteins, we identified SMAC-interacting sequences. These SMAC-binding sequences were produced as cell-penetrating peptides targeted to the cytosol, mitochondria, or nucleus, inhibiting cell proliferation and inducing apoptosis in several cell lines. For in vivo study, a survivin/baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5)-derived peptide was selected, due to its overexpression in many cancers and its involvement in mitosis, apoptosis, autophagy, cell proliferation, inflammation, and immune responses, as a target for cancer therapy. Specifically, a SMAC-targeting survivin/BIRC5-derived peptide, given intratumorally or intravenously, strongly inhibited lung tumor growth, cell proliferation, angiogenesis, and inflammation, induced apoptosis, and remodeled the tumor microenvironment. The peptide promoted tumor infiltration of CD-8+ cells and increased cell-intrinsic programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) expression, resulting in cancer cell self-destruction and increased tumor cell death, preserving immune cells. Thus, targeting the interaction between the multifunctional proteins SMAC and survivin represents an innovative therapeutic cancer paradigm.
Assuntos
Proteínas Reguladoras de Apoptose , Apoptose , Proliferação de Células , Proteínas Mitocondriais , Survivina , Humanos , Survivina/metabolismo , Survivina/genética , Animais , Camundongos , Proteínas Mitocondriais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/tratamento farmacológico , Inflamação/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Ligação Proteica , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Inibidoras de Apoptose/genética , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/química , Peptídeos/farmacologia , Peptídeos/química , Terapia de ImunossupressãoRESUMO
Peptide-based cancer vaccines are widely investigated in the clinic but exhibit modest immunogenicity. One approach that has been explored to enhance peptide vaccine potency is covalent conjugation of antigens with cell-penetrating peptides (CPPs), linear cationic and amphiphilic peptide sequences designed to promote intracellular delivery of associated cargos. Antigen-CPPs have been reported to exhibit enhanced immunogenicity compared to free peptides, but their mechanisms of action in vivo are poorly understood. We tested eight previously described CPPs conjugated to antigens from multiple syngeneic murine tumor models and found that linkage to CPPs enhanced peptide vaccine potency in vivo by as much as 25-fold. Linkage of antigens to CPPs did not impact dendritic cell activation but did promote uptake of linked antigens by dendritic cells both in vitro and in vivo. However, T cell priming in vivo required Batf3-dependent dendritic cells, suggesting that antigens delivered by CPP peptides were predominantly presented via the process of cross-presentation and not through CPP-mediated cytosolic delivery of peptide to the classical MHC class I antigen processing pathway. Unexpectedly, we observed that many CPPs significantly enhanced antigen accumulation in draining lymph nodes. This effect was associated with the ability of CPPs to bind to lymph-trafficking lipoproteins and protection of CPP-antigens from proteolytic degradation in serum. These two effects resulted in prolonged presentation of CPP-peptides in draining lymph nodes, leading to robust T cell priming and expansion. Thus, CPPs can act through multiple unappreciated mechanisms to enhance T cell priming that can be exploited for cancer vaccines with enhanced potency.
Assuntos
Vacinas Anticâncer , Peptídeos Penetradores de Células , Imunogenicidade da Vacina , Linfonodos , Animais , Apresentação de Antígeno , Antígenos , Vacinas Anticâncer/imunologia , Peptídeos Penetradores de Células/farmacologia , Apresentação Cruzada , Células Dendríticas/imunologia , Linfonodos/imunologia , Camundongos , Linfócitos T/imunologia , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
BACKGROUND: The blood-brain barrier serves as a critical interface between the bloodstream and brain tissue, mainly composed of pericytes, neurons, endothelial cells, and tightly connected basal membranes. It plays a pivotal role in safeguarding brain from harmful substances, thus protecting the integrity of the nervous system and preserving overall brain homeostasis. However, this remarkable selective transmission also poses a formidable challenge in the realm of central nervous system diseases treatment, hindering the delivery of large-molecule drugs into the brain. In response to this challenge, many researchers have devoted themselves to developing drug delivery systems capable of breaching the blood-brain barrier. Among these, blood-brain barrier penetrating peptides have emerged as promising candidates. These peptides had the advantages of high biosafety, ease of synthesis, and exceptional penetration efficiency, making them an effective drug delivery solution. While previous studies have developed a few prediction models for blood-brain barrier penetrating peptides, their performance has often been hampered by issue of limited positive data. RESULTS: In this study, we present Augur, a novel prediction model using borderline-SMOTE-based data augmentation and machine learning. we extract highly interpretable physicochemical properties of blood-brain barrier penetrating peptides while solving the issues of small sample size and imbalance of positive and negative samples. Experimental results demonstrate the superior prediction performance of Augur with an AUC value of 0.932 on the training set and 0.931 on the independent test set. CONCLUSIONS: This newly developed Augur model demonstrates superior performance in predicting blood-brain barrier penetrating peptides, offering valuable insights for drug development targeting neurological disorders. This breakthrough may enhance the efficiency of peptide-based drug discovery and pave the way for innovative treatment strategies for central nervous system diseases.
Assuntos
Peptídeos Penetradores de Células , Doenças do Sistema Nervoso Central , Humanos , Barreira Hematoencefálica/química , Células Endoteliais , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/uso terapêutico , Encéfalo , Doenças do Sistema Nervoso Central/tratamento farmacológicoRESUMO
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
BACKGROUND: Treatment with tumor-targeted toxins attempts to overcome the disadvantages of conventional cancer therapies by directing a drug's cytotoxic effect specifically towards cancer cells. However, success with targeted toxins has been hampered as the constructs commonly remain bound to the outside of the cell or, after receptor-mediated endocytosis, are either transported back to the cell surface or undergo degradation in lysosomes. Hence, solutions to ensure endosomal escape are an urgent need in treatment with targeted toxins. In this work, a molecular adapter that consists of a cell penetrating peptide and two cleavable peptides was inserted into a targeted toxin between the ribosome-inactivating protein dianthin and the epidermal growth factor. Applying cell viability assays, this study examined whether the addition of the adapter further augments the endosomal escape enhancement of the glycosylated triterpenoid SO1861, which has shown up to more than 1000-fold enhancement in the past. RESULTS: Introducing the peptide adapter into the targeted toxin led to an about 12-fold enhancement in the cytotoxicity on target cells while SO1861 caused a 430-fold increase. However, the combination of adapter and glycosylated triterpenoid resulted in a more than 4300-fold enhancement and in addition to a 51-fold gain in specificity. CONCLUSIONS: Our results demonstrated that the cleavable peptide augments the endosomal escape mediated by glycosylated triterpenoids while maintaining specificity. Thus, the adapter is a promising addition to glycosylated triterpenoids to further increase the efficacy and therapeutic window of targeted toxins.
Assuntos
Endossomos , Humanos , Endossomos/metabolismo , Endossomos/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Triterpenos/farmacologia , Triterpenos/química , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologiaRESUMO
The design of a potent amyloid-ß protein (Aß) inhibitor plays a pivotal role in the prevention and treatment of Alzheimer's disease (AD). Despite endogenous transthyretin (TTR) being recognized as an Aß inhibitor, the weak inhibitory and blood brain barrier (BBB) crossing capabilities hinder it for Aß aggregation inhibition and transport. Therefore, we have herein designed a recombinant TTR by conjugating a cationic cell penetrating peptide (penetratin, Pen), which not only enabled the fusion protein, TTR-Pen (TP), to present high BBB penetration but also greatly enhanced the potency of Aß inhibition. Namely, the protein fusion made TP positively charged, leading to a potent suppression of Aß40 fibrillization at a low concentration (1.5 µM), while a TTR concentration as high as 12.5 µM was required to gain a similar function. Moreover, TP could mitigate Aß-induced neuronal death, increase cultured cell viability from 72% to 92% at 2.5 µM, and extend the lifespan of AD nematodes from 14 to 18 d. Thermodynamic studies revealed that TP, enriched in positive charges, presented extensive electrostatic interactions with Aß40. Importantly, TP showed excellent BBB penetration performance, with a 10 times higher BBB permeability than TTR, which would allow TP to enter the brain of AD patients and participate in the transport of Aß species out of the brain. Thus, it is expected that the fusion protein has great potential for drug development in AD treatment.
Assuntos
Doença de Alzheimer , Peptídeos Penetradores de Células , Humanos , Barreira Hematoencefálica/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/metabolismo , Pré-Albumina/metabolismo , Pré-Albumina/uso terapêutico , Peptídeos beta-Amiloides/metabolismo , Proteínas Recombinantes/uso terapêuticoRESUMO
Proinflammatory cytokines/chemokines are commonly regulated by RNA-binding proteins at posttranscriptional levels. Human Ag R (HuR)/embryonic lethal abnormal vision-like 1 (ELAVL1) is one of the well-characterized RNA-binding proteins that increases the stability of short-lived mRNAs, which encode proinflammatory mediators. HuR employs its nucleocytoplasmic shuttling sequence (HNS) domain, interacting with poly(ADP-ribose) polymerase 1 (PARP1), which accounts for the enhanced poly-ADP-ribosylation and cytoplasmic shuttling of HuR. Also by using its HNS domain, HuR undergoes dimerization/oligomerization, underlying the increased binding of HuR with proinflammatory cytokine/chemokine mRNAs and the disassociation of the miRNA-induced silencing complex from the targets. Therefore, competitively blocking the interactions of HuR with its partners may suppress proinflammatory mediator production. In this study, peptides derived from the sequence of the HuR-HNS domain were synthesized, and their effects on interfering HuR interacting with PARP1 and HuR itself were analyzed. Moreover, cell-penetrating TAT-HuR-HNS3 was delivered into human and mouse cells or administered into mouse lungs with or without exposure of TNF-α or LPS. mRNA levels of proinflammatory mediators as well as neutrophil infiltration were evaluated. We showed that TAT-HuR-HNS3 interrupts HuR-PARP1 interaction and therefore results in a lowered poly-ADP-ribosylation level and decreased cytoplasmic distribution of HuR. TAT-HuR-HNS3 also blocks HuR dimerization and promotes Argonaute 2-based miRNA-induced silencing complex binding to the targets. Moreover, TAT-HuR-HNS3 lowers mRNA stability of proinï¬ammatory mediators in TNF-α-treated epithelial cells and macrophages, and it decreases TNF-α-induced inflammatory responses in lungs of experimental animals. Thus, TAT-HuR-HNS3 is a promising lead peptide for the development of inhibitors to treat inï¬ammation-related diseases.
Assuntos
Peptídeos Penetradores de Células , Proteína Semelhante a ELAV 1/imunologia , MicroRNAs , Animais , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/metabolismo , Peptídeos Penetradores de Células/farmacologia , Quimiocinas/genética , Citocinas/metabolismo , Proteínas ELAV/genética , Proteínas ELAV/metabolismo , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Expressão Gênica , Camundongos , MicroRNAs/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The escalating threat of multidrug-resistant pathogens necessitates innovative approaches to combat infectious diseases. In this study, we examined peptides R23FS*, V31KS*, and R44KS*, which were engineered to include an amyloidogenic fragment sourced from the S1 protein of S. aureus, along with one or two cell-penetrating peptide (CPP) components. We assessed the antimicrobial efficacy of these peptides in a liquid medium against various strains of both Gram-positive bacteria, including S. aureus (209P and 129B strains), MRSA (SA 180 and ATCC 43300 strains), and B. cereus (strain IP 5832), and Gram-negative bacteria such as P. aeruginosa (ATCC 28753 and 2943 strains) and E. coli (MG1655 and K12 strains). Peptides R23FS*, V31KS*, and R44KS* exhibited antimicrobial activity comparable to gentamicin and meropenem against all tested bacteria at concentrations ranging from 24 to 48 µM. The peptides showed a stronger antimicrobial effect against B. cereus. Notably, peptide R44KS* displayed high efficacy compared to peptides R23FS* and V31KS*, particularly evident at lower concentrations, resulting in significant inhibition of bacterial growth. Furthermore, modified peptides V31KS* and R44KS* demonstrated enhanced inhibitory effects on bacterial growth across different strains compared to their unmodified counterparts V31KS and R44KS. These results highlight the potential of integrating cell-penetrating peptides, amyloidogenic fragments, and amino acid residue modifications to advance the innovation in the field of antimicrobial peptides, thereby increasing their effectiveness against a broad spectrum of pathogens.
Assuntos
Peptídeos Antimicrobianos , Peptídeos Penetradores de Células , Testes de Sensibilidade Microbiana , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Peptídeos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos/química , Antibacterianos/farmacologia , Antibacterianos/química , Aminoácidos/química , Desenho de Fármacos , Proteínas Amiloidogênicas/químicaRESUMO
Cell-penetrating peptides (CPPs) are invaluable tools for delivering various substances into cells by crossing biological membranes. However, the effects of cell-penetrating peptide fusion proteins on the biological activity of antibodies remain to be fully understood. Here, we engineered a recombinant protein, LP-scFv, which combines the single-chain variable region of anti-human epidermal growth factor receptor-2 with a novel and non-oxic cell-penetrating peptide as a leader peptide. The introduction of this leader peptide led to a more than twofold increase in the internalization efficiency of the single-chain antibody, as confirmed using microscopic analysis and flow cytometry. The effects of the single-chain antibodies and LP-scFv on cell viability were evaluated using the MTT assay. Both the single-chain antibodies and LP-scFv reduced the viability of BT474 and NCI-N87 cells in a dose-dependent manner while exhibiting minimal toxicity towards MCF-7 and MCF-10A cells. Further investigation into LP-scFv's mechanism revealed that the induced leader peptide does not alter the MAPK-ERK1/2 and PI3K/AKT pathways of single-chain antibodies. An enhanced antitumor activity was also confirmed in an NCI-N87 tumor xenograft model in mice with a reduction of 45.2% in tumor growth inhibition (vs. 23.1% for scFv) with a 50 mg/kg dose after orthotopic injection administration, which was equivalent to that of trastuzumab (vs. 55.7% for trastuzumab). Overall, these results indicate that LP-scFv exhibits significant permeation activity in HER2-positive cells to enhance the intracellular dose effect on antitumor activity in vitro and in vivo. This research lays the foundation for designing novel antibody-based therapies for cancer.
Assuntos
Neoplasias da Mama , Peptídeos Penetradores de Células , Anticorpos de Cadeia Única , Humanos , Animais , Camundongos , Feminino , Neoplasias da Mama/patologia , Anticorpos de Cadeia Única/farmacologia , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Linhagem Celular Tumoral , Trastuzumab/uso terapêutico , Receptor ErbB-2/metabolismo , Sinais Direcionadores de Proteínas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Upon a variety of environmental stresses, eukaryotic cells usually recruit translational stalled mRNAs and RNA-binding proteins to form cytoplasmic condensates known as stress granules (SGs), which minimize stress-induced damage and promote stress adaptation and cell survival. SGs are hijacked by cancer cells to promote cell survival and are consequently involved in the development of anticancer drug resistance. However, the design and application of chemical compounds targeting SGs to improve anticancer drug efficacy have rarely been studied. Here, we developed two types of SG inhibitory peptides (SIPs) derived from SG core proteins Caprin1 and USP10 and fused with cell-penetrating peptides to generate TAT-SIP-C1/2 and SIP-U1-Antp, respectively. We obtained 11 SG-inducing anticancer compounds from cell-based screens and explored the potential application of SIPs in overcoming resistance to the SG-inducing anticancer drug sorafenib. We found that SIPs increased the sensitivity of HeLa cells to sorafenib via the disruption of SGs. Therefore, anticancer drugs which are competent to induce SGs could be combined with SIPs to sensitize cancer cells, which might provide a novel therapeutic strategy to alleviate anticancer drug resistance.
Assuntos
Antineoplásicos , Sorafenibe , Grânulos de Estresse , Humanos , Sorafenibe/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Grânulos de Estresse/metabolismo , Células HeLa , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Peptídeos/farmacologia , Peptídeos/química , Sobrevivência Celular/efeitos dos fármacos , Ubiquitina Tiolesterase/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/químicaRESUMO
Spinal muscular atrophy (SMA) is a severe neuromuscular disorder that is caused by mutations in the survival motor neuron 1 (SMN1) gene, hindering the production of functional survival motor neuron (SMN) proteins. Antisense oligonucleotides (ASOs), a versatile DNA-like drug, are adept at binding to target RNA to prevent translation or promote alternative splicing. Nusinersen is an FDA-approved ASO for the treatment of SMA. It effectively promotes alternative splicing in pre-mRNA transcribed from the SMN2 gene, an analog of the SMN1 gene, to produce a greater amount of full-length SMN protein, to compensate for the loss of functional protein translated from SMN1. Despite its efficacy in ameliorating SMA symptoms, the cellular uptake of these ASOs is suboptimal, and their inability to penetrate the CNS necessitates invasive lumbar punctures. Cell-penetrating peptides (CPPs), which can be conjugated to ASOs, represent a promising approach to improve the efficiency of these treatments for SMA and have the potential to transverse the blood-brain barrier to circumvent the need for intrusive intrathecal injections and their associated adverse effects. This review provides a comprehensive analysis of ASO therapies, their application for the treatment of SMA, and the encouraging potential of CPPs as delivery systems to improve ASO uptake and overall efficiency.
Assuntos
Peptídeos Penetradores de Células , Atrofia Muscular Espinal , Oligonucleotídeos Antissenso , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Humanos , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/terapia , Oligonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Animais , Oligonucleotídeos/química , Oligonucleotídeos/farmacologia , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacosRESUMO
Hematopoietic cell transplantation (HCT) is often considered a last resort leukemia treatment, fraught with limited success due to microbial infections, a leading cause of mortality in leukemia patients. To address this critical issue, we explored a novel approach by synthesizing antileukemic agents containing antibacterial substances. This innovative strategy involves conjugating fluoroquinolone antibiotics, such as ciprofloxacin (CIP) or levofloxacin (LVX), with the cell-penetrating peptide transportan 10 (TP10). Here, we demonstrate that the resultant compounds display promising biologic activities in preclinical studies. These novel conjugates not only exhibit potent antimicrobial effects but are also selective against leukemia cells. The cytotoxic mechanism involves rapid disruption of cell membrane asymmetry leading to membrane damage. Importantly, these conjugates penetrated mammalian cells, accumulating within the nuclear membrane without significant effect on cellular architecture or mitochondrial function. Molecular simulations elucidated the aggregation tendencies of TP10 conjugates within lipid bilayers, resulting in membrane disruption and permeabilization. Moreover, mass spectrometry analysis confirmed efficient reduction of disulfide bonds within TP10 conjugates, facilitating release and activation of the fluoroquinolone derivatives. Intriguingly, these compounds inhibited human topoisomerases, setting them apart from traditional fluoroquinolones. Remarkably, TP10 conjugates generated lower intracellular levels of reactive oxygen species compared with CIP and LVX. The combination of antibacterial and antileukemic properties, coupled with selective cytostatic effects and minimal toxicity toward healthy cells, positions TP10 derivatives as promising candidates for innovative therapeutic approaches in the context of antileukemic HCT. This study highlights their potential in search of more effective leukemia treatments. SIGNIFICANCE STATEMENT: Fluoroquinolones are commonly used antibiotics, while transportan 10 (TP10) is a cell-penetrating peptide (CPP) with anticancer properties. In HCT, microbial infections are the primary cause of illness and death. Combining TP10 with fluoroquinolones enhanced their effects on different cell types. The dual pharmacological action of these conjugates offers a promising proof-of-concept solution for leukemic patients undergoing HCT. Strategically designed therapeutics, incorporating CPPs with antibacterial properties, have the potential to reduce microbial infections in the treatment of malignancies.
Assuntos
Antineoplásicos , Peptídeos Penetradores de Células , Leucemia , Animais , Humanos , Fluoroquinolonas/farmacologia , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Antineoplásicos/farmacologia , Antibacterianos/farmacologia , Leucemia/tratamento farmacológico , Transplante de Células , Mamíferos/metabolismoRESUMO
Parkinson's disease is a neurodegenerative movement disorder associated with the intracellular aggregation of α-synuclein (α-syn). Cytotoxicity is mainly associated with the oligomeric species (αSOs) formed at early stages in α-syn aggregation. Consequently, there is an intense focus on the discovery of novel inhibitors such as peptides to inhibit oligomer formation and toxicity. Here, using peptide arrays, we identified nine peptides with high specificity and affinity for αSOs. Of these, peptides p194, p235, and p249 diverted α-syn aggregation from fibrils to amorphous aggregates with reduced ß-structures and increased random coil content. However, they did not reduce αSO cytotoxicity and permeabilization of large anionic unilamellar vesicles. In parallel, we identified a non-self-aggregating peptide (p216), derived from the cell-penetrating peptide penetratin, which showed 12-fold higher binding affinity to αSOs than to α-syn monomers (Kdapp 2.7 and 31.2 µM, respectively). p216 reduced αSOs-induced large anionic unilamellar vesicle membrane permeability at 10-1 to 10-3 mg/ml by almost 100%, was not toxic to SH-SY5Y cells, and reduced αSOs cytotoxicity by about 20%. We conclude that p216 is a promising starting point from which to develop peptides targeting toxic αSOs in Parkinson's disease.
Assuntos
Peptídeos Penetradores de Células , Doença de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Peptídeos Penetradores de Células/isolamento & purificação , Peptídeos Penetradores de Células/farmacologia , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Linhagem Celular TumoralRESUMO
Peptides are promising therapeutic agents for COVID-19 because of their specificity, easy synthesis, and ability to be fine-tuned. We previously demonstrated that a cell-permeable peptide corresponding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike C-terminal domain (CD) inhibits the interaction between viral spike and nucleocapsid proteins that results in SARS-CoV-2 replication in vitro. Here, we used docking studies to design R-t-Spike CD(D), a more potent short cell-penetrating peptide composed of all D-form amino acids and evaluated its inhibitory effect against the replication of SARS-CoV-2 S clade and other variants. R-t-Spike CD(D) was internalized into Vero cells and Calu-3 cells and suppressed the replication of SARS-CoV-2 S clade, delta variant, and omicron variant with higher potency than the original peptide. In hemizygous K18-hACE2 mice, intratracheal administration of R-t-Spike CD(D) effectively delivered the peptide to the trachea and lungs, whereas intranasal administration delivered the peptide mostly to the upper respiratory system and stomach, and a small amount to the lungs. Administration by either route reduced viral loads in mouse lungs and turbinates. Furthermore, intranasally administered R-t-Spike CD(D) mitigated pathological change in the lungs and increased the survival of mice after infection with the SARS-CoV-2 S clade or delta variant. Our data suggest that R-t-Spike CD(D) has potential as a therapeutic agent against SARS-CoV-2 infection.
Assuntos
COVID-19 , Peptídeos Penetradores de Células , Chlorocebus aethiops , Animais , Camundongos , Peptídeos Penetradores de Células/farmacologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Células VeroRESUMO
Activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is the most aggressive form of DLBCL, with a significantly inferior prognosis due to resistance to the standard R-CHOP immunochemotherapy. Survival of ABC-DLBCL cells addicted to the constitutive activations of both canonical and noncanonical NF-κB signaling makes them attractive therapeutic targets. However, a pharmaceutical approach simultaneously targeting the canonical and noncanonical NF-κB pathway in the ABC-DLBCL cell is still lacking. Peptide-conjugated gold nanoclusters (AuNCs) have emerged unique intrinsic biomedical activities and possess a great potential in cancer theranostics. Here, we demonstrated a Au25 nanocluster conjugated by cell-penetrating peptides that can selectively repress the growth of ABC-DLBCL cells by inducing efficient apoptosis, more efficiently than glutathione (GSH)-conjugated AuNCs. The mechanism study showed that the cell-penetrating peptides enhanced the cellular internalization efficiency of AuNCs, and the selective repression in ABC-DLBCL cells is due to the inhibition of inherent constitutive canonical and noncanonical NF-κB activities by AuNCs. Several NF-κB target genes involved in chemotherapy resistance in ABC-DLBCL cells, including anti-apoptotic Bcl-2 family members and DNA damage repair proteins, were effectively down-regulated by the AuNC. The emerged novel activity of AuNCs in targeting both arms of NF-κB signaling in ABC-DLBCL cells may provide a promising candidate and a new insight into the rational design of peptide-conjugated Au nanomedicine for molecular targeting treatment of refractory lymphomas.
Assuntos
Peptídeos Penetradores de Células , Linfoma Difuso de Grandes Células B , Nanopartículas Metálicas , NF-kappa B , Humanos , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/farmacologia , Linfócitos/metabolismo , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , NF-kappa B/metabolismo , Transdução de Sinais , Nanopartículas Metálicas/químicaRESUMO
Malaria continues to impose a global health burden. Drug-resistant parasites have emerged to each introduced small-molecule therapy, highlighting the need for novel treatment approaches for the future eradication of malaria. Herein, targeted drug delivery with peptide-drug conjugates (PDCs) was investigated as an alternative antimalarial therapy, inspired by the success of emerging antibody-drug conjugates utilized in cancer treatment. A synthetic peptide derived from an innate human defense molecule was conjugated to the antimalarial drug primaquine (PQ) to produce PDCs with low micromolar potency toward Plasmodium falciparum in vitro. A suite of PDCs with different design features was developed to identify optimal conjugation site and investigate linker length, hydrophilicity, and cleavability. Conjugation within a flexible spacer region of the peptide, with a cleavable linker to liberate the PQ cargo, was important to retain activity of the peptide and drug.