RESUMO
Although polyethylene glycol (PEG), or "PEGylation" has become a widely applied approach for improving the efficiency of drug delivery, the immunogenicity and non-biodegradability of this synthetic polymer have prompted an evident need for alternatives. To overcome these caveats and to mimic PEG -or other natural or synthetic polymers- for the purpose of drug half-life extension, unstructured polypeptides are designed. Due to their tunable length, biodegradability, low immunogenicity and easy production, unstructured polypeptides have the potential to replace PEG as the preferred technology for therapeutic protein/peptide delivery. This review provides an overview of the evolution of unstructured polypeptides, starting from natural polypeptides to engineered polypeptides and discusses their characteristics. Then, it is described that unstructured polypeptides have been successfully applied to numerous drugs, including peptides, proteins, antibody fragments, and nanocarriers, for half-life extension. Innovative applications of unstructured peptides as releasable masks, multimolecular adaptors and intracellular delivery carriers are also discussed. Finally, challenges and future perspectives of this promising field are briefly presented. STATEMENT OF SIGNIFICANCE: Polypeptide fusion technology simulating PEGylation has become an important topic for the development of long-circulating peptide or protein drugs without reduced activity, complex processes, and kidney injury caused by PEG modification. Here we provide a detailed and in-depth review of the recent advances in unstructured polypeptides. In addition to the application of enhanced pharmacokinetic performance, emphasis is placed on polypeptides as scaffolders for the delivery of multiple drugs, and on the preparation of reasonably designed polypeptides to manipulate the performance of proteins and peptides. This review will provide insight into future application of polypeptides in peptide or protein drug development and the design of novel functional polypeptides.
Assuntos
Peptídeos , Proteínas , Peptídeos/química , Proteínas/química , Sistemas de Liberação de Medicamentos , Polímeros/química , Polietilenoglicóis/química , Tecnologia , Portadores de FármacosRESUMO
The protein ISG15 encoded by interferon-stimulated gene (ISG) 15 is the first identified member of the ubiquitin-like protein family and exists in the form of monomers and conjugated complexes. Like ubiquitin, ISG15 can mediate an ubiquitin-like modification by covalently modifying other proteins, known as ISGylation. There is growing evidence showing that both the free and conjugated ISG15 are involved in multiple key cellular processes, including autophagy, exosome secretion, DNA repair, immune regulation, and cancer occurrence and progression. In this review, we aim to further clarify the function of ISG15 and ISGylation in cancer, demonstrate the important relationship between ISG15/ISGylation and cancer, and emphasize new insights into the different roles of ISG15/ISGylation in cancer progression. This review may contribute to therapeutic intervention in cancer. However, due to the limitations of current research, the regulation of ISG15/ISGylation on cancer progression is not completely clear, thus further comprehensive and sufficient correlation studies are still needed.
Assuntos
Citocinas , Neoplasias , Humanos , Citocinas/metabolismo , Interferons , Ubiquitina/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , Neoplasias/metabolismoRESUMO
Bispecific T-cell Engager (BiTE) antibodies can redirect T-cells to tumor cells, and turn on the targeted lysis of tumor cells. However, BiTE has been challenging in solid tumors due to short plasma half-life, "off-target" effect, and immunosuppression via PD-1/PD-L1 axis. This study designed a safe, long-acting, and highly effective Protease-Activated PSTAGylated BiTE, named PAPB, which includes a shielding polypeptide domain (PSTAG), a protease-activated linker, and a BiTE core. The BiTE core consists of two scFvs targeting PD-L1 and CD3. BiTE core bound PD-L1 and CD3 in a dose-dependent manner, and PAPB can release BiTE core in response to MMP2 in the tumor microenvironment to exert antitumor activity. The plasma half-life of PAPB in mice was significantly prolonged from 2.46 h to 6.34 h of the BiTE core. In mice bearing melanoma (A375) xenografts, PAPB significantly increased infiltration of T lymphocytes in tumor tissue, and inhibited tumor proliferation without activating T-cells in the peripheral blood. Overall, the engineering protein PAPB could be a promising drug candidate for solid tumor immunotherapy.
Assuntos
Anticorpos Biespecíficos , Melanoma , Humanos , Camundongos , Animais , Complexo CD3/metabolismo , Complexo CD3/farmacologia , Linfócitos T , Melanoma/metabolismo , Imunoterapia , Peptídeo Hidrolases/metabolismo , Microambiente TumoralRESUMO
Multidrug resistance (MDR) to chemotherapeutic drugs and targeted drug delivery are recurring issues in clinical cancer treatment. Here, a multifunctional fusion protein-DNA conjugate was designed as a co-delivery vehicle for anticancer peptides and chemotherapeutic drugs to combat both drug-resistant and drug-sensitive tumor cells. The fusion protein was constructed by fusing a PsTag polypeptide, a matrix metalloproteinase 2 (MMP2)-degradable domain, and the mitochondria-targeted pro-apoptotic peptide KLAKLAKKLAKLAK. Doxorubicin was efficiently loaded into the fusion protein pre-conjugated dendrimer-like DNA nanostructure. With the incorporation of enhanced stability, tumor targeting, and controlled-release elements, the tailored nanostructure can selectively enter tumor cells and synergistically exert antitumor activity with no significant adverse effects. Thus, these protein-conjugated DNA nanocarriers could be a potential co-delivery system for protein/peptide and chemotherapeutic drugs delivery in synergistic cancer therapy.
Assuntos
Antineoplásicos , Sistemas de Liberação de Medicamentos , Neoplasias , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , DNA , Doxorrubicina , Resistencia a Medicamentos Antineoplásicos , Humanos , Metaloproteinase 2 da Matriz , Nanopartículas , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Peptídeos/químicaRESUMO
The great therapeutic potential of peptides has not yet been achieved, mainly due to their remarkably short in vivo half-life. Although conjugation to macromolecules has been an effective way of improving protein in vivo half-life, the steric hindrance of macromolecules usually reduces the in vivo efficacy of peptides. Here we report a complex delivery system made from PsTag polypeptide, polyglutamic acid chain, matrix metalloproteinase 2 (MMP2)-degradable domain and cationic cell penetrating peptide for anticancer peptide delivery. Clear evidence was shown in vitro and in vivo to demonstrate that this multifunctional protein fusing a pro-apoptotic KLAKLAKKLAKLAK (KLA), named PAK, can increase circulation time in blood, enhance accumulation at tumor sites, eliminate the PsTag domain and the polyanionic sequence when triggered by tumor overexpressing MMP2, and then expose the cell penetrating peptide to realize the potent cellular uptake of KLA. Treatment of tumor-bearing mice with PAK could markedly induce tumor cells apoptosis and inhibit tumor growth, with no significant adverse effects. These results suggest our fusion protein can be a potential delivery system for peptide delivery in cancer treatments.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacologia , Portadores de Fármacos , Neoplasias/tratamento farmacológico , Microambiente Tumoral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Proliferação de Células/efeitos dos fármacos , Peptídeos Penetradores de Células/administração & dosagem , Peptídeos Penetradores de Células/farmacocinética , Feminino , Células Hep G2 , Humanos , Células MCF-7 , Metaloproteinase 2 da Matriz/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Terapia de Alvo Molecular , Neoplasias/enzimologia , Neoplasias/patologia , Fragmentos de Peptídeos/metabolismo , Ácido Poliglutâmico/metabolismo , Domínios Proteicos , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Successful and efficient delivery of Cas9 protein and gRNA into cells is critical for genome editing and its therapeutic application. In this study, we developed an improved supercharged polypeptide (SCP) mediated delivery system based on dithiocyclopeptide linker to realize the effective genome editing in tumor cells. The fusion protein Cas9-linker-SCP (Cas9-LS) forms positively charged complexes with gRNA in vitro to provide possibilities for gRNA delivery into cells. Under the microenvironment of tumor cells, the dithiocyclopeptide linker, containing matrix metalloproteinase 2 (MMP-2) sensitive sequence and an intramolecular disulfide bond, can be completely disconnected to promote the release of Cas9 protein with the nuclear localization sequence (NLS) in the cytoplasm and transfer to the cell nucleus for highly efficient genome editing, resulting in an obvious increase of indel efficiency in comparison to fusion protein without dithiocyclopeptide linker (Cas9-SCP). Furthermore, Cas9-LS shows no significant cytotoxicity and minimal hemolytic activity. We envision that the microenvironment-responsive Cas9 protein delivery system can facilitate more efficient genome editing in tumor cells.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Endonucleases/metabolismo , Edição de Genes/métodos , Microambiente Tumoral , Humanos , Metaloproteinase 2 da Matriz/metabolismo , RNA Guia de Cinetoplastídeos/genéticaRESUMO
The efficient delivery of proteins into cells is needed to fully realize the potential of protein-based therapeutics. Current protein delivery strategies generally suffer from poor endosomal escape and low tolerance for serum. Here, the genetic fusion of a supercharged polypeptide, called SCP, to a protein provides a generic method for intracellular protein delivery. It allows efficient protein endocytosis and endosomal escape and is capable of potently delivering various proteins with a range of charges, sizes, and bioactivities into the nucleus of living cells. SCP is discovered to bind directly to the nuclear import protein importin ß1 and gains access to the nucleus. Furthermore, SCP shows minimal hemolytic activity and stability in serum and lacks toxicity and immunogenicity in vivo. Effective gene editing can be achieved by SCP-mediated delivery of Cas9 protein and guide RNA. This study may provide an efficient and useful tool for the design and development of cell-nuclear-targeted drug delivery.
Assuntos
Peptídeos Penetradores de Células/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Peptídeos Penetradores de Células/sangue , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/toxicidade , Endocitose/fisiologia , Escherichia coli/genética , Feminino , Humanos , Camundongos Endogâmicos BALB C , Estabilidade Proteica , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/toxicidade , beta Carioferinas/metabolismoRESUMO
We have previously constructed a novel polypeptide, PsTag, that should be useful in the development of biologics with properties comparable to those achievable by PEGylation, but with potentially less side effects. However, the low fermentation yields of polypeptide fusion proteins may limit the application of this technology. We suspected that when polypeptide fusion protein was expressed in E. coli, the corresponding 8 tRNAs were needed to transport a large number of repetitive 5 amino acids to the ribosomes and thus, resulting in a relative deficiency of these tRNAs. PsTag600-FGF21, a long-acting FGF21 fusion protein, was used as a model for studying the effects of these non-rare tRNAs on the efficiency of heterologous protein production in E. coli. To further enhance the expression level and facilitate purification, secretory expressions of PsTag600-FGF21 were achieved by fusion with three signal peptides. Meanwhile, a comparison of several distinctive characterizations was carried out between PsTag600-FGF21 and PEG20K-FGF21. We investigated the protective effects of PsTag600-FGF21 in a nonalcoholic steatohepatitis model induced by methionine- and choline-deficient diet. Our results showed that the provision of 8 tRNAs and secretory expression remarkably increased the expression levels of PsTag fusion protein, meanwhile there were no significant effects on E. coli growth states. PsTag600-FGF21 had a larger hydrodynamic volume, a higher affinity and a longer plasma half-life than PEG20K-FGF21, while avoiding vacuole formation in mice. In NASH mice, administration of PsTag600-FGF21 reduced hepatic steatosis, fibrosis and inflammation. Therefore, PsTag600-FGF21 with higher expression level may be further developed for potentially application in clinics.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Células 3T3-L1 , Animais , Escherichia coli/genética , Feminino , Fatores de Crescimento de Fibroblastos/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND AND PURPOSE: Non-alcoholic steatohepatitis (NASH) is the most severe form of non-alcoholic fatty liver disease and is a serious public health problem around the world. There are currently no approved treatments for NASH. FGF21 has recently emerged as a promising drug candidate for metabolic diseases. However, the disadvantages of FGF21 as a clinically useful medicine include its short plasma half-life and poor drug-like properties. Here, we have explored the effects of PsTag600-FGF21, an engineered long-acting FGF21 fusion protein, in mice with NASH and describe some of the underlying mechanisms. EXPERIMENTAL APPROACH: A long-acting FGF21 was prepared by genetic fusion with a 600 residues polypeptide (PsTag600). We used a choline-deficient high-fat diet-induced model of NASH in mice. The effects on body weight, insulin sensitivity, inflammation and levels of hormones and metabolites were studied first. We further investigated whether PsTag600-FGF21 attenuated inflammation through the Th17-IL17A axis and the associated mechanisms. KEY RESULTS: PsTag600-FGF21 dose-dependently reduced body weight, blood glucose, and insulin and lipid levels and reversed hepatic steatosis. PsTag600-FGF21 enhanced fatty acid activation and mitochondrial ß-oxidation in the liver. The profound reduction in hepatic inflammation in NASH mice following PsTag600-FGF21 was associated with inhibition of IL17A expression in Th17 cells. Furthermore, PsTag600-FGF21 depended on adiponectin to exert its suppression of Th17 cell differentiation and IL17A expression. CONCLUSIONS AND IMPLICATIONS: Our data have uncovered some of the mechanisms by which PsTag600-FGF21 suppresses hepatic inflammation and further suggest that PsTag600-FGF21 could be an effective approach in NASH treatment.
Assuntos
Adiponectina/sangue , Anti-Inflamatórios , Fatores de Crescimento de Fibroblastos , Interleucina-17/sangue , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Glicemia/análise , Peso Corporal/efeitos dos fármacos , Preparações de Ação Retardada/farmacologia , Preparações de Ação Retardada/uso terapêutico , Modelos Animais de Doenças , Fatores de Crescimento de Fibroblastos/farmacologia , Fatores de Crescimento de Fibroblastos/uso terapêutico , Glucagon/sangue , Insulina/sangue , Lipídeos/sangue , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Transdução de SinaisRESUMO
Escherichia coli extracellular expression systems have a number of advantages over other systems, such as lower pyrogen levels and a simple purification process. Various approaches, such as the generation of leaky mutants via chromosomal engineering, have been explored for this expression system. However, extracellular protein yields in leaky mutants are relatively low compared to that in intracellular expression systems and therefore need to be improved. In this work, we describe the construction, characterization, and mechanism of enhanced extracellular expression in Escherichia coli. On the basis of the localizations, functions, and transcription levels of cell envelope proteins, we systematically elucidated the effects of multiple gene deletions on cell growth and extracellular expression using modified CRISPR/Cas9-based genome editing and a FlAsH labeling assay. High extracellular yields of heterologous proteins of different sizes were obtained by screening multiple gene mutations. The enhancement of extracellular secretion was associated with the derepression of translation and translocation. This work utilized universal methods in the design of extracellular expression systems for genes not directly associated with protein synthesis that were used to generate strains with higher protein expression capability. We anticipate that extracellular expression systems may help to shed light on the poorly understood aspects of these secretion processes as well as to further assist in the construction of engineered prokaryotic cells for efficient extracellular production of heterologous proteins.
Assuntos
Sistemas CRISPR-Cas , Escherichia coli/genética , Engenharia Genética/métodos , Escherichia coli/crescimento & desenvolvimento , Matriz Extracelular/genética , Fluorescência , Deleção de Genes , Perfilação da Expressão Gênica/métodos , Interferon-alfa/genética , Interferon-alfa/metabolismo , Microrganismos Geneticamente Modificados , Peso Molecular , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND AND PURPOSE: Chemical conjugation of therapeutic proteins with polyethylene glycol (PEG) is an established strategy to extend their biological half-life (t1/2 ) to a clinically useful range. We developed a novel uncharged and unstructured recombinant polypeptide composed of five amino acids (P, S, T, A and G), named PsTag, as another approach to extend the t1/2 of human FGF21, with increased hydrodynamic radius. EXPERIMENTAL APPROACH: Human FGF21 was fused with PsTag polymers of differing lengths (200 - 600 residues). Three fusion proteins and native FGF21 were produced in Escherichia coli. The biophysical characteristics, metabolic stability, immunogenicity and pharmacokinetics in were assessed in first. In lean and diet-induced obese (DIO) mice, effects on body weight, oral glucose tolerance tests and levels of relevant hormones and metabolites were studied. KEY RESULTS: Fusion proteins were solubly expressed in E. coli and prolonged the t1/2 from 0.34h up to 12.9 h in mice. Fusion proteins were also biodegradable, thus avoiding vacuole formation, while lacking immunogenicity in mice. In DIO mice, administration of PsTag fused to FGF21 reduced body weight, blood glucose and lipids levels and reversed hepatic steatosis. CONCLUSIONS AND IMPLICATIONS: The novel recombinant polypeptide, PsTag, should be useful in the development of biological drugs with properties comparable to those achievable by PEGylation, but with potentially less side effects. In mice, fusion of FGF21 to PsTag prolonged and potentiated pharmacological effects of native FGF21, and may offer greater therapeutic effects in treatment of obesity.
Assuntos
Modelos Animais de Doenças , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/farmacocinética , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Peptídeos/química , Peptídeos/farmacocinética , Animais , Fatores de Crescimento de Fibroblastos/química , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Peptídeos/síntese químicaRESUMO
Translational efficiency in Escherichia coli is strongly influenced by mRNA secondary structure of translational initiation region (TIR). We have previously reported that the expression of heterologous protein is directly related to the minimal folding free energy (ΔG) of the local secondary structure. However, identifying biologically relevant maximum and minimum levels of expression, or exploring the optimal level between them, is a key to successful optimization of heterologous protein expression. To systematically search a large range of the ΔG of TIR, we now present a quantitative analysis of the relationship between expression level and these ΔGs. The ΔG of TIR in green fluorescent protein is found to be linearly correlated with the fluorescence intensity over a range of tenfold change. The result demonstrates that the increasing ΔG of TIR can enhance the expression level linearly with no threshold or plateau.
Assuntos
Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Iniciação Traducional da Cadeia Peptídica/genética , Dobramento de RNA , RNA Mensageiro/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Escherichia coli/genética , Expressão Gênica , RNA Mensageiro/genética , TermodinâmicaRESUMO
Though numerous strategy options are available for achieving high expression levels of genes in Escherichia coli, not every gene can be efficiently expressed in this organism. By investigating the relationship between the mRNA secondary structure of translational initiation region (TIR) and gene expression in E.coli, we establish a simple method to design sequences of appropriate TIR (from -35 to +36) that meet a specific expression level as we need. Using this method, overexpression of native human humor necrosis factor α and extracellular domain of Her2/neu protein (aa 23-146) in E. coli were achieved. Differences in expression appeared was mainly related to the efficiency of translation initiation and the stability of mRNA secondary structure, because the intracellular mRNA levels analyzed by real-time RT-PCR were quite similar. Our approach can overcome the steric hindrance of translation startup, and therefore promote translation smoothly to acquire high expression of exogenous protein.