RESUMO
Hepatitis B virus (HBV) infection is a major global health problem with no established cure. Dedicator of cytokinesis 11 (DOCK11), known as a guanine nucleotide exchange factor (GEF) for Cdc42, is reported to be essential for the maintenance of HBV. However, potential therapeutic strategies targeting DOCK11 have not yet been explored. We have previously developed an in vitro virus method as a more efficient tool for the analysis of proteomics and evolutionary protein engineering. In this study, using the in vitro virus method, we screened and identified a novel antiasialoglycoprotein receptor (ASGR) antibody, ASGR3-10M, and a DOCK11-binding peptide, DCS8-42A, for potential use in HBV infection. We further constructed a fusion protein (10M-D42AN) consisting of ASGR3-10M, DCS8-42A, a fusogenic peptide, and a nuclear localization signal to deliver the peptide inside hepatocytes. We show using immunofluorescence staining that 10M-D42AN was endocytosed into early endosomes and released into the cytoplasm and nucleus. Since DCS8-42A shares homology with activated cdc42-associated kinase 1 (Ack1), which promotes EGFR endocytosis required for HBV infection, we also found that 10M-D42AN inhibited endocytosis of EGFR and Ack1. Furthermore, we show 10M-D42AN suppressed the function of DOCK11 in the host DNA repair system required for covalently closed circular DNA synthesis and suppressed HBV proliferation in mice. In conclusion, this study realizes a novel hepatocyte-specific drug delivery system using an anti-ASGR antibody, a fusogenic peptide, and DOCK11-binding peptide to provide a novel treatment for HBV.
Assuntos
Sistemas de Liberação de Medicamentos , Fatores de Troca do Nucleotídeo Guanina , Vírus da Hepatite B , Hepatite B , Anticorpos de Cadeia Única , Animais , DNA Circular/genética , Receptores ErbB/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hepatite B/tratamento farmacológico , Vírus da Hepatite B/patogenicidade , Vírus da Hepatite B/fisiologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Camundongos , Peptídeos/metabolismo , Anticorpos de Cadeia Única/metabolismo , Replicação Viral/genéticaRESUMO
Despite the development of new drugs for multiple myeloma (MM), the prognosis of MM patients with high-risk cytogenetic abnormalities such as t (4; 14) and del17p remains poor. We reported that a novel phenylphthalimide derivative, TC11, induced apoptosis of MM cells in vitro and in vivo, and TC11 directly bound to α-tubulin and nucleophosmin-1 (NPM1). However, TC11 showed low water solubility and poor pharmacokinetic properties. Here we synthesized a water-soluble TC11-derivative, PEG(E)-TC11, in which HOEtO-TC11 is pegylated with PEG through an ester bond, and we examined its anti-myeloma activity. We observed that PEG(E)-TC11 and its hydrolyzed product, HOEtO-TC11, induced G2/M arrest and the apoptosis of MM cells. Intraperitoneal administration of PEG(E)-TC11 to xenografted mice revealed improved pharmacokinetic properties and significantly delayed tumor growth. TC11 and its derivatives did not bind to cereblon (CRBN), which is a responsible molecule for thalidomide-induced teratogenicity. These results suggest that PEG(E)-TC11 is a good candidate drug for treating high-risk MM.
Assuntos
Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Ftalimidas/administração & dosagem , Ftalimidas/farmacocinética , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Composição de Medicamentos/métodos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Masculino , Taxa de Depuração Metabólica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Mieloma Múltiplo/patologia , Nucleofosmina , Ftalimidas/química , Polietilenoglicóis/química , Fatores de Risco , Resultado do TratamentoRESUMO
Overcoming hepatitis B virus (HBV) is a challenging problem because HBV deceives the host immune system. We have found that DENN domain-containing 2A (DENND2A) was essential for HBV maintenance, although its role remains unclear. In this study, we elucidate its function by screening a novel DENND2A-binding peptide, DENP4-3S. DENP4-3S exhibits homology to SAM and SH3 domain-containing protein 1 (SASH1), a scaffold protein involved in Toll-like receptor signaling that promotes proinflammatory cytokine production. We confirmed that DENND2A interacts with SASH1 specifically. Overexpression and knockdown experiments showed that overexpression of DENND2A suppressed the transcriptional activity of NF-κB, and the knockdown of DENND2A promoted it and the production of cytokines and interferons. Here, we constructed a fusion protein (10M-DEN3SN) consisting of an anti-asialoglycoprotein receptor antibody and DENP4-3S to deliver the peptide to hepatocytes specifically. 10M-DEN3SN inhibited the interaction between DENND2A and SASH1, and rescued SASH1 trapped by DENND2A, leading to the upregulation of NF-κB and its downstream signaling. In addition, 10M-DEN3SN suppressed HBV proliferation in PXB chimeric mice. These results with the DENND2A-binding peptide delivered into hepatocytes suggested the involvement of DENND2A, SASH, and NF-κB signaling pathway in the HBV infection and onset of hepatitis. In conclusion, this study indicates that HBV utilizes DENND2A and SASH1 to evade the immune system.IMPORTANCEHepatitis B virus (HBV) is a serious liver infection with no established cure, causing an abnormal host immune response. Here, we identified a novel peptide that interacts with DENN domain-containing 2A (DENND2A), a host factor essential for HBV maintenance. The resulting peptide showed sequence homology, revealing an interaction between DENND2A and the immune system regulator SASH1. This study suggests that DENND2A contributes to HBV infection by suppressing the cellular immune system by inhibiting SASH1. The DENND2A-binding peptide, incorporated into our hepatocyte-specific peptide delivery system, inhibited the DENND2A-SASH1 interaction and promoted the production of cytokines and interferons in cultured hepatocytes. As a consequence, the peptide suppressed HBV proliferation in humanized mice. We report new insights into the role of DENND2A and SASH1 in HBV maintenance and highlight the importance of the immune system.
Assuntos
Vírus da Hepatite B , Hepatite B , Camundongos , Animais , Vírus da Hepatite B/fisiologia , NF-kappa B/metabolismo , Transdução de Sinais , Interferons , Citocinas/metabolismo , Sistema ImunitárioRESUMO
BACKGROUND & AIMS: Hepatitis B virus (HBV) infection is difficult to cure owing to the persistence of covalently closed circular viral DNA (cccDNA). We performed single-cell transcriptome analysis of newly established HBV-positive and HBV-negative hepatocellular carcinoma cell lines and found that dedicator of cytokinesis 11 (DOCK11) was crucially involved in HBV persistence. However, the roles of DOCK11 in the HBV lifecycle have not been clarified. METHODS: The cccDNA levels were measured by Southern blotting and real-time detection polymerase chain reaction in various hepatocytes including PXB cells by using an HBV-infected model. The retrograde trafficking route of HBV capsid was investigated by super-resolution microscopy, proximity ligation assay, and time-lapse analysis. The downstream molecules of DOCK11 and underlying mechanism were examined by liquid chromatography-tandem mass spectrometry, immunoblotting, and enzyme-linked immunosorbent assay. RESULTS: The cccDNA levels were strongly increased by DOCK11 overexpression and repressed by DOCK11 suppression. Interestingly, DOCK11 functionally associated with retrograde trafficking proteins in the trans-Golgi network (TGN), Arf-GAP with GTPase domain, ankyrin repeat, and pleckstrin homology domain-containing protein 2 (AGAP2), and ADP-ribosylation factor 1 (ARF1), together with HBV capsid, to open an alternative retrograde trafficking route for HBV from early endosomes (EEs) to the TGN and then to the endoplasmic reticulum (ER), thereby avoiding lysosomal degradation. Clinically, DOCK11 levels in liver biopsies from patients with chronic hepatitis B were significantly reduced by entecavir treatment, and this reduction correlated with HBV surface antigen levels. CONCLUSIONS: HBV uses a retrograde trafficking route via EEs-TGN-ER for infection that is facilitated by DOCK11 and serves to maintain cccDNA. Therefore, DOCK11 is a potential therapeutic target to prevent persistent HBV infection.
Assuntos
Hepatite B Crônica , Hepatite B , Humanos , Vírus da Hepatite B/genética , Rede trans-Golgi/metabolismo , Hepatite B/metabolismo , Lisossomos/metabolismoRESUMO
BACKGROUND: HBV infection causes chronic liver disease and leads to the development of HCC. To identify host factors that support the HBV life cycle, we previously established the HC1 cell line that maintains HBV infection and identified host genes required for HBV persistence. METHODS: The present study focused on endothelial lipase (LIPG), which binds to heparan sulfate proteoglycans (HSPGs) in the cell membrane. RESULTS: We found HBV infection was impaired in humanized liver chimeric mouse-derived hepatocytes that were transduced with lentivirus expressing short hairpin RNA against LIPG. Long-term suppression of LIPG combined with entecavir further suppressed HBV replication. LIPG was shown to be involved in HBV attachment to the cell surface by using 2 sodium taurocholate cotransporting peptide (NTCP)-expressing cell lines, and the direct interaction of LIPG and HBV large surface protein was revealed. Heparin and heparinase almost completely suppressed the LIPG-induced increase of HBV attachment, indicating that LIPG accelerated HBV attachment to HSPGs followed by HBV entry through NTCP. Surprisingly, the attachment of a fluorescently labeled NTCP-binding preS1 probe to NTCP-expressing cells was not impaired by heparin, suggesting the HSPG-independent attachment of the preS1 probe to NTCP. Interestingly, attachment of the preS1 probe was severely impaired in LIPG knockdown or knockout cells. Inhibitors of the lipase activity of LIPG similarly impaired the attachment of the preS1 probe to NTCP-expressing cells. CONCLUSIONS: LIPG participates in HBV infection by upregulating HBV attachment to the cell membrane by means of 2 possible mechanisms: increasing HBV attachment to HSPGs or facilitating HSPG-dependent or HSPG-independent HBV attachment to NTCP by its lipase activity.
Assuntos
Hepatite B , Lipase , Animais , Camundongos , Proteoglicanas de Heparan Sulfato/genética , Heparina , Hepatite B/genética , Vírus da Hepatite B , Lipase/genéticaRESUMO
In vitro antibody-display technologies are powerful approaches for isolating monoclonal antibodies from recombinant antibody libraries. However, these display techniques require several rounds of affinity selection which is time-consuming. Here, we combined mRNA display with a microfluidic system for in vitro selection and evolution of antibodies and achieved ultrahigh enrichment efficiency of 10(6)- to 10(8)-fold per round. After only one or two rounds of selection, antibodies with high affinity and specificity were obtained from naive and randomized single-chain Fv libraries of approximately 10(12) molecules. Furthermore, we confirmed that not only protein-protein (antigen-antibody) interactions, but also protein-DNA and protein-drug interactions were selected with ultrahigh efficiencies. This method will facilitate high-throughput preparation of antibodies and identification of protein interactions in proteomic and therapeutic fields.
Assuntos
Evolução Molecular Direcionada , Região Variável de Imunoglobulina/genética , Técnicas Analíticas Microfluídicas/métodos , RNA Mensageiro/biossíntese , Animais , Linhagem Celular , Biblioteca Gênica , Humanos , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/imunologia , Camundongos , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/imunologia , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologiaRESUMO
Here we describe the application of the in vitro virus mRNA display method, which involves covalent linkage of an in vitro-synthesized antibody (phenotype) to its encoding mRNA (genotype) through puromycin, for in vitro evolution of single-chain Fv (scFv) antibody fragments. To establish the validity of this approach to directed antibody evolution, we used random mutagenesis by error-prone DNA shuffling and off-rate selection to improve the affinity of an anti-fluorescein scFv as a model system. After four rounds of selection of the library of mRNA-displayed scFv mutants, we obtained six different sequences encoding affinity-matured mutants with five consensus mutations. Kinetic analysis of the mutant scFvs revealed that the off-rates have been decreased by more than one order of magnitude and the dissociation constants were improved approximately 30-fold. The antigen-specificity was not improved by affinity maturation, but remained similar to that of the wild type. Although the five consensus mutations of the high-affinity mutants were scattered over the scFv sequence, analysis by site-directed mutagenesis demonstrated that the critical mutations for improving affinity were the two that lay within the complementarity determining regions (CDRs). Thus, mRNA display is expected to be useful for rapid artificial evolution of high-affinity diagnostic and therapeutic antibodies by optimizing their CDRs.
Assuntos
Evolução Molecular Direcionada/métodos , Região Variável de Imunoglobulina/genética , RNA Mensageiro/química , Afinidade de Anticorpos , Especificidade de Anticorpos , Ligação Competitiva , Embaralhamento de DNA , Ensaio de Imunoadsorção Enzimática , Fluoresceína/metabolismo , Biblioteca Gênica , Região Variável de Imunoglobulina/análise , Região Variável de Imunoglobulina/biossíntese , Mutagênese , Biossíntese de Proteínas , Ressonância de Plasmônio de Superfície , Transcrição GênicaRESUMO
Despite the recent advances in the treatment of multiple myeloma (MM), MM patients with high-risk cytogenetic changes such as t(4;14) translocation or deletion of chromosome 17 still have extremely poor prognoses. With the goal of helping these high-risk MM patients, we previously developed a novel phthalimide derivative, TC11. Here we report the further characterization of TC11 including anti-myeloma effects in vitro and in vivo, a pharmacokinetic study in mice, and anti-osteoclastogenic activity. Intraperitoneal injections of TC11 significantly delayed the growth of subcutaneous tumors in human myeloma-bearing SCID mice. Immunohistochemical analyses showed that TC11 induced apoptosis of MM cells in vivo. In the pharmacokinetic analyses, the Cmax was 2.1 µM at 1 h after the injection of TC11, with 1.2 h as the half-life. TC11 significantly inhibited the differentiation and function of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts in mouse osteoclast cultures using M-CSF and RANKL. We also revealed that TC11 induced the apoptosis of myeloma cells accompanied by α-tubulin fragmentation. In addition, TC11 and lenalidomide, another phthalimide derivative, directly bound to nucleophosmin 1 (NPM1), whose role in MM is unknown. Thus, through multiple molecular interactions, TC11 is a potentially effective drug for high-risk MM patients with bone lesions. The present results suggest the possibility of the further development of novel thalidomide derivatives by drug designing.
Assuntos
Mieloma Múltiplo/patologia , Ftalimidas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Xenoenxertos/efeitos dos fármacos , Humanos , Lenalidomida , Masculino , Camundongos Endogâmicos ICR , Camundongos SCID , Mieloma Múltiplo/genética , Proteínas Nucleares/química , Nucleofosmina , Osteoclastos/efeitos dos fármacos , Ftalimidas/química , Ftalimidas/farmacocinética , Talidomida/análogos & derivados , Talidomida/química , Talidomida/farmacologiaRESUMO
Fungal roselipins, discovered as inhibitors of diacylglycerol acyltransferase (DGAT), consist of three parts; highly methylated C20 fatty acid, mannose and arabinitol. Demannosyl and/or dearabinitoyl roselipins were prepared chemically or enzymatically. Demannnosyl roselipins conserved the DGAT inhibitory activity, but the others lost the activity, indicating that the arabinitoyl fatty acid core is essential for eliciting the activity.
Assuntos
Aciltransferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Ácidos Graxos/farmacologia , Gliocladium/química , Monossacarídeos/farmacologia , Antibacterianos , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Diacilglicerol O-Aciltransferase , Inibidores Enzimáticos/química , Ácidos Graxos/síntese química , Ácidos Graxos/química , Fungos/efeitos dos fármacos , Monossacarídeos/síntese química , Monossacarídeos/química , Relação Estrutura-AtividadeRESUMO
Talaromyces flavus FKI-0076, a soil isolate, was found to produce compounds which reinforce the anti-Candida albicans activity of miconazole. Four structurally related compounds, a novel one, designated actofunicone, and the knowns deoxyfunicone, vermistatin and NG-012, were isolated from the culture broth by solvent extraction, ODS column chromatography and HPLC. The structure of actofunicone was elucidated as benzoic acid, 3,5-dimethoxy-2-[[4-oxo-6-(2-acetyloxy propyl)-4H-pyran-3-yl]carbonyl]-, methyl ester by various spectroscopic analyses including NMR experiments. These compounds potentiated the anti-C. albicans activity of miconazole, decreasing the IC50 value of miconazole from 19 microM to 1.6 approximately 3.7 microM in the presence of the funicones.
Assuntos
Antifúngicos/isolamento & purificação , Candida albicans/efeitos dos fármacos , Miconazol/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Sinergismo Farmacológico , Fermentação , Testes de Sensibilidade MicrobianaRESUMO
The oncoprotein MDM2 binds to tumor suppressor protein p53 and inhibits its anticancer activity, which leads to promotion of tumor cell growth and tumor survival. Abrogation of the p53:MDM2 interaction reportedly results in reactivation of the p53 pathway and inhibition of tumor cell proliferation. We recently performed rigorous selection of MDM2-binding peptides by means of mRNA display and identified an optimal 12-mer peptide (PRFWEYWLRLME), named MDM2 Inhibitory Peptide (MIP), which shows higher affinity for MDM2 (and also its homolog, MDMX) and higher tumor cell proliferation suppression activity than known peptides. Here we determined the NMR solution structure of a MIP-MDM2 fusion protein to elucidate the structural basis of the tight binding of MIP to MDM2. A region spanning from Phe3 to Met11 of MIP forms a single α-helix, which is longer than those of the other MDM2-binding peptides. MIP shares a conserved Phe3-Trp7-Leu10 triad, whose side chains are oriented towards and fit into the hydrophobic pockets of MDM2. Additionally, hydrophobic surface patches that surround the hydrophobic pockets of MDM2 are covered by solvent-exposed MIP residues, Trp4, Tyr6, and Met11. Their hydrophobic interactions extend the interface of the two molecules and contribute to the strong binding. The potential MDM2 inhibition activity observed for MIP turned out to originate from its enlarged binding interface. The structural information obtained in the present study provides a road map for the rational design of strong inhibitors of MDM2:p53 binding.
Assuntos
Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , RNA Mensageiro/genética , Proteína Supressora de Tumor p53/antagonistas & inibidores , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação Proteica , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
Here, we used mRNA display to search for proteins that bind to FK506, a potent immunosuppressant drug, and identified spartin, a hereditary spastic paraplegia protein, from a human brain cDNA library. We demonstrated that FK506 binds to the C-terminal region of spartin and thereby inhibits the interaction of spartin with TIP47, one of the lipid droplet-associated proteins. We further confirmed that FK506 inhibits localization of spartin and its binder, an E3 ubiquitin ligase AIP4, in lipid droplets and increases the protein level of ADRP (adipose differentiation-related protein), which is a regulator of lipid homeostasis. These results strongly suggest that FK506 suppresses the proteasomal degradation of ADRP, a substrate of AIP4, by inhibiting the spartin-TIP47 interaction and thereby blocking the localization of spartin and AIP4 in lipid droplets.
Assuntos
Imunossupressores/metabolismo , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Tacrolimo/metabolismo , Proteínas de Ciclo Celular , Células HEK293 , Células HeLa , Humanos , Imunossupressores/farmacologia , Ligação Proteica , Transporte Proteico/efeitos dos fármacos , Proteínas/química , RNA Mensageiro/genética , Tacrolimo/farmacologiaRESUMO
We recently identified a novel anilinoquinazoline derivative, Q15, as a potent apoptosis inducer in a panel of human cancer cell lines and determined that Q15 targets hCAP-G2, a subunit of condensin II complex, leading to abnormal cell division. However, whether the defect in normal cell division directly results in cell death remains unclear. Here, we used an mRNA display method on a microfluidic chip to search for other Q15-binding proteins. We identified an additional Q15-binding protein, MIP-2A (MBP-1 interacting protein-2A), which has been reported to interact with MBP-1, a repressor of the c-Myc promoter. Our results indicate that Q15 inhibits the interaction between MIP-2A and MBP-1 as well as the expression of c-Myc protein, thereby inducing cell death. This study suggests that the simultaneous targeting of hCAP-G2 and MIP-2A is a promising strategy for the development of antitumor drugs as a treatment for intractable tumours.
Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Quinazolinas/farmacologia , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Compostos de Anilina/química , Compostos de Anilina/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Humanos , Técnicas Analíticas Microfluídicas , Dados de Sequência Molecular , Estrutura Molecular , Complexos Multiproteicos/metabolismo , Quinazolinas/química , Quinazolinas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Despite the introduction of newly developed drugs such as lenalidomide and bortezomib, patients with multiple myeloma are still difficult to treat and have a poor prognosis. In order to find novel drugs that are effective for multiple myeloma, we tested the antitumor activity of 29 phthalimide derivatives against several multiple myeloma cell lines. Among these derivatives, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3- dione (TC11) was found to be a potent inhibitor of tumor cell proliferation and an inducer of apoptosis via activation of caspase-3, 8 and 9. This compound also showed in vivo activity against multiple myeloma cell line KMS34 tumor xenografts in ICR/SCID mice. By means of mRNA display selection on a microfluidic chip, the target protein of TC11 was identified as nucleophosmin 1 (NPM). Binding of TC11 and NPM monomer was confirmed by surface plasmon resonance. Immunofluorescence and NPM knockdown studies in HeLa cells suggested that TC11 inhibits centrosomal clustering by inhibiting the centrosomal-regulatory function of NPM, thereby inducing multipolar mitotic cells, which undergo apoptosis. NPM may become a novel target for development of antitumor drugs active against multiple myeloma.