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1.
Bioorg Med Chem Lett ; 110: 129886, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38996938

RESUMO

(+)-Plakevulin A (1), an oxylipin isolated from an Okinawan sponge Plakortis sp. inhibits enzymatic inhibition of DNA polymerases (pols) α and δ and exhibits cytotoxicity against murine leukemia (L1210) and human cervix carcinoma (KB) cell lines. However, the half-maximal inhibitory concentration (IC50) value for cytotoxicity significantly differed from those observed for the enzymatic inhibition of pols α and ß, indicating the presence of target protein(s) other than pols. This study demonstrated cytotoxicity against human promyelocytic leukemia (HL60), human cervix epithelioid carcinoma (HeLa), mouse calvaria-derived pre-osteoblast (MC3T3-E1), and human normal lung fibroblast (MRC-5) cell lines. This compound had selectivity to cancer cells over normal ones. Among these cell lines, HL60 exhibited the highest sensitivity to (+)-plakevulin A. (+)-Plakevulin A induced DNA fragmentation and caspase-3 activation in HL60 cells, indicating its role in apoptosis induction. Additionally, hydroxysteroid 17-ß dehydrogenase 4 (HSD17B4) was isolated from the HL60 lysate as one of its binding proteins through pull-down experiments using its biotinylated derivative and neutravidin-coated beads. Moreover, (+)-plakevulin A suppressed the activation of interleukin 6 (IL-6)-induced signal transducer and activator of transcription 3 (STAT3). Because the knockdown or inhibition of STAT3 induces apoptosis and HSD17B4 regulates STAT3 activation, (+)-plakevulin A may induce apoptosis in HL60 cell lines by suppressing STAT3 activation, potentially by binding to HSD17B4. The present findings provide valuable information for the mechanism of its action.

2.
Photodiagnosis Photodyn Ther ; 45: 103898, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38008301

RESUMO

We synthesized a new silyl porphyrin derivative conjugated with 6-deoxy-6-sulfo-α-d-glucopyranose (SGlc). Conjugation with SGlc improved A549 cellular uptake without significant changes in the photophysical and photochemical properties and subcellular localization. This improved cellular uptake led to enhanced photodynamic activity. Furthermore, conjugation with SGlc suppressed dark toxicity. These advantages were not observed for a conjugate with a glucose molecule. These results indicated that the conjugation with SGlc is a promising strategy for enhancing photodynamic efficacy.


Assuntos
Fotoquimioterapia , Porfirinas , Humanos , Fármacos Fotossensibilizantes/farmacologia , Fotoquimioterapia/métodos , Células A549 , Glucose , Porfirinas/farmacologia
3.
J Antibiot (Tokyo) ; 76(2): 75-82, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36513753

RESUMO

Vanitaracin A, an anti-hepatitis B virus polyketide, has been previously isolated from Talaromyces sp. In the present study, we searched for novel compounds in the culture broth obtained from a vanitaracin A-producing fungus under various conditions. Three novel compounds (vanitaracin C, vanitaraphilone A, and 2-hydroxy-4-(hydroxymethyl)-6-methylbenzaldehyde) were isolated, and their structures were determined using spectroscopic methods (1D/2D NMR and MS). In addition, the antiviral spectrum of vanitaracin A was examined by measuring its antiviral activities against rabies virus, Borna disease virus 1, and bovine leukemia virus. This compound exhibited antiviral activity against bovine leukemia virus, which is the causative agent of enzootic bovine leukosis. The anti-bovine leukemia virus effects of other compounds isolated from the vanitaracin A-producing fungus, namely, vanitaracins B and C, vanitaraphilone A, and 2-hydroxy-4-(hydroxymethyl)-6-methylbenzaldehyde, were also evaluated. Vanitaracin B, vanitaraphilone A and 2-hydroxy-4-(hydroxymethyl)-6-methylbenzaldehyde were also found to exhibit activity against bovine leukemia virus. These findings reveal the broad-spectrum antiviral activity of the vanitaracin scaffold and suggest several candidates for the development of anti-bovine leukemia virus drugs.


Assuntos
Leucemia , Policetídeos , Talaromyces , Animais , Bovinos , Humanos , Antivirais/química , Estrutura Molecular , Policetídeos/farmacologia , Talaromyces/química
4.
J Antibiot (Tokyo) ; 75(2): 92-97, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35034105

RESUMO

Vanitaracin A is an anti-hepatitis B virus (anti-HBV) compound isolated from the culture broth of the fungus Talaromyces sp. Vanitaracin A inhibits the entry of HBV into target cells with sub-micromolar IC50 values. While a structure-activity relationship study is highly desirable, a synthetic approach still needs to be developed, which is difficult because the absolute configurations of the six stereogenic centers in the structure of vanitaracin A have not yet been determined. In the present study, we used the crystalline sponge method to clarify the configuration of the compound after determining the absolute configuration of the secondary alcohol using a modified Mosher ester method. Combining these analyses with the NOESY spectrum of vanitaracin A and NMR analyses of the crude side-chain carboxylic acid obtained by the alkaline hydrolysis of vanitaracin A revealed the absolute configurations of all stereogenic centers in this important compound.


Assuntos
Antivirais/química , Vírus da Hepatite B/efeitos dos fármacos , Policetídeos/química , Antivirais/farmacologia , Hidrólise , Espectroscopia de Ressonância Magnética , Conformação Molecular , Estrutura Molecular , Policetídeos/farmacologia , Relação Estrutura-Atividade , Talaromyces/química
5.
J Radiat Res ; 63(1): 19-29, 2022 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-34738103

RESUMO

Malignant pleural mesothelioma (MPM) is a highly malignant disease that develops after asbestos exposure. Although the number of MPM cases is predicted to increase, no effective standard therapies have been established. The novel radiosensitizer α-sulfoquinovosyl-acylpropanediol (SQAP) enhances the effects of γ-radiation in human lung and prostate cancer cell lines and in animal models. In this study, we explored the radiosensitizing effect of SQAP and its mechanisms in MPM. The human MPM cell lines MSTO-211H and MESO-4 were implanted subcutaneously into the backs and thoracic cavities of immunodeficient KSN/Slc mice, then 2 mg/kg SQAP was intravenously administered with or without irradiation with a total body dose of 8 Gy. In both the orthotopic and ectopic xenograft murine models, the combination of irradiation plus SQAP delayed the implanted human MSTO-211H tumor growth. The analysis of the changes in the relative tumor volume of the MSTO-211H indicated a statistically significant difference after 8 Gy total body combined with 2 mg/kg SQAP, compared to both the untreated control (P = 0.0127) and the radiation treatment alone (P = 0.0171). After the treatment in each case, immunostaining of the harvested tumors revealed decreased cell proliferation, increased apoptosis and normalization of tumor blood vessels in the SQAP- and irradiation-treated group. Furthermore, hypoxia-inducible factor (HIF) 1 mRNA and protein expression were decreased, indicating reoxygenation in this group. In conclusion, SQAP improved hypoxic conditions in tumor tissue and may elicit a radiosensitizing effect in malignant mesothelioma models.


Assuntos
Antineoplásicos , Mesotelioma Maligno , Mesotelioma , Neoplasias Pleurais , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Humanos , Masculino , Mesotelioma/tratamento farmacológico , Mesotelioma/metabolismo , Mesotelioma/radioterapia , Camundongos , Camundongos Nus , Neoplasias Pleurais/tratamento farmacológico , Neoplasias Pleurais/metabolismo , Neoplasias Pleurais/radioterapia , Tolerância a Radiação
6.
Transl Oncol ; 15(1): 101285, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34839108

RESUMO

α-Sulfoquinovosylacyl-1,3-propanediol (SQAP) is a semi-synthetic derivative of natural sulfoglycolipid that sensitizes tumors to external-beam radiotherapy. How SQAP affects internal radiotherapy, however, is not known. Here, we investigated the effects of SQAP for radioimmunotherapy (RIT) targeting tissue factor (TF) in a stroma-rich refractory pancreatic cancer mouse model, BxPC-3. A low dose of SQAP (2 mg/kg) increased tumor uptake of the 111In-labeled anti-TF antibody 1849, indicating increased tumor perfusion. The addition of SQAP enhanced the growth-inhibitory effect of 90Y-labeled 1849 without leading to severe body weight changes, allowing for the dose of 90Y-labeled 1849 to be reduced to half that when used alone. Histologic analysis revealed few necrotic and apoptotic cells, but Ki-67-positive proliferating cells and increased vascular formation were detected. These results suggest that the addition of a low dose of SQAP may improve the therapeutic efficacy of TF-targeted RIT by increasing tumor perfusion, even for stroma-rich refractory pancreatic cancer.

7.
Bioorg Med Chem Lett ; 52: 128391, 2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34601028

RESUMO

Sulfoquynovosylacyl propanediol (SQAP; 1) has been developed as a radiosensitizer (anti-cancer agent) for solid tumors, but it was easily cleaved in vivo and had a problem of short residence time. We synthesized a novel compound of a SQAP derivative (3-octadecanoxypropyl 6-deoxy-6-sulfo-α-d-glucopyranoside: ODSG; 2) to solve these problems not easily cleaved by lipase. ODSG (2) cytotoxicity was investigated in vitro, resulting in low toxicity like SQAP (1).


Assuntos
Lipase/metabolismo , Radiossensibilizantes/farmacologia , Células A549 , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Radiossensibilizantes/química , Radiossensibilizantes/metabolismo , Relação Estrutura-Atividade
8.
Bioorg Med Chem ; 41: 116203, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34015702

RESUMO

Epo-C12 is a synthetic derivative of epolactaene, isolated from Penicillium sp. BM 1689-P. Epo-C12 induces apoptosis in human acute lymphoblastoid leukemia BALL-1 cells. In our previous studies, seven proteins that bind to Epo-C12 were identified by a combination of pull-down experiments using biotinylated Epo-C12 (Bio-Epo-C12) and mass spectrometry. In the present study, the effect of Epo-C12 on peroxiredoxin 1 (Prx 1), one of the proteins that binds to Epo-C12, was investigated. Epo-C12 inhibited Prx 1 peroxidase activity. However, it did not suppress its chaperone activity. Binding experiments between Bio-Epo-C12 and point-mutated Prx 1s suggest that Epo-C12 binds to Cys52 and Cys83 in Prx 1. The present study revealed that Prx 1 is one of the target proteins through which Epo-C12 exerts an apoptotic effect in BALL-1 cells.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Peroxirredoxinas/antagonistas & inibidores , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Inibidores Enzimáticos , Compostos de Epóxi/química , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Estrutura Molecular , Mutação , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Polienos/química
9.
Biosci Biotechnol Biochem ; 85(1): 85-91, 2021 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-33577659

RESUMO

Sulfoglycolipid, SQAP, is a radiosensitizing agent that makes tumor cells more sensitive to radiation therapy. A previous study revealed that SQAP induced the degradation of hypoxia-inducible factor-1α (HIF-1α) and inhibited angiogenesis in a hepatoma model mouse. Herein, we examined the biological activities of SQAP against hepatocarcinoma cells under low oxygen conditions. Cell growth inhibition of SQAP under hypoxic conditions was significantly higher than that under normoxic conditions. In addition, SQAP was found to impair the expression of histone deacetylase (HDAC) under low oxygen conditions. Our present data suggested that SQAP induced the degradation of HIF-1α and then decreased the expression of HDAC1. Unlike known HDAC inhibitors, SQAP increased the acetylation level of histone in cells without inhibition of enzymatic activity of HDACs. Our data demonstrated hypoxia-specific unique properties of SQAP.


Assuntos
Morte Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicolipídeos/química , Glicolipídeos/farmacologia , Histona Desacetilase 1/metabolismo , Hipóxia Tumoral/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Histonas/metabolismo , Humanos
10.
Expert Opin Drug Discov ; 15(10): 1199-1211, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32660284

RESUMO

INTRODUCTION: Our understanding of the mechanism of action of bioactive small molecules contributes to the research and development of new medical drugs, as well as elucidating the pathological mechanisms underlying various diseases. Researchers in this field are committed to a very ambitious goal: the discovery of novel therapeutic compounds along with their molecular targets. To achieve this goal, new methodological developments are indispensable. AREAS COVERED: This review gives an update on the advancements of phage display (PD) technology in the past decade (2011-2020) for determining the targets of the small molecule therapeutics. In particular, other than providing a brief overview of this field of research, we focus on reporting the research trends and the results solely obtained using this strategy. EXPERT OPINION: Despite the development of bioinformatics tools and artificial intelligence (AI)-mediated methods, affinity-guided information obtained experimentally are still indispensable to identify drug-protein interactions. By taking advantage of small-molecule-oriented PD methods and their improvements, the extension of the druggable proteome will be further expanded, providing new opportunities to generate small-molecule therapeutics.


Assuntos
Técnicas de Visualização da Superfície Celular , Desenvolvimento de Medicamentos/métodos , Terapia de Alvo Molecular , Inteligência Artificial , Biologia Computacional , Descoberta de Drogas/métodos , Humanos , Bibliotecas de Moléculas Pequenas
11.
Biosci Biotechnol Biochem ; 84(2): 217-227, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31589093

RESUMO

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) and 4'-ethynyl-2'-deoxyadenosine (EdA) are nucleoside analogues which inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. EdAP, a cyclosaligenyl (cycloSal) phosphate derivative of EdA, inhibits the replication of the influenza A virus. The common structural feature of these compounds is the ethynyl group at the 4'-position. In this study, these nucleoside analogues were prepared by a common synthetic strategy starting from the known 1,2-di-O-acetyl-D-ribofuranose. Biological evaluation of EdAP revealed that this compound reduced hepatitis B virus (HBV) replication dose-dependently without cytotoxicity against host cells tested in this study.


Assuntos
Antivirais/síntese química , Nucleotídeos de Desoxiadenina/síntese química , Desoxiadenosinas/síntese química , Vírus da Hepatite B/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Linhagem Celular , Nucleotídeos de Desoxiadenina/farmacologia , Desoxiadenosinas/farmacologia , Vírus da Hepatite B/fisiologia , Humanos
12.
Bioorg Med Chem ; 27(23): 115149, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31679979

RESUMO

Pyrenocine A, a phytotoxin, was found to exhibit cytotoxicity against cancer cells with an IC50 value of 2.6-12.9 µM. Live cell imaging analysis revealed that pyrenocine A arrested HeLa cells at the M phase with characteristic ring-shaped chromosomes. Furthermore, as a result of immunofluorescence staining analysis, we found that pyrenocine A resulted in the formation of monopolar spindles in HeLa cells. Monopolar spindles are known to be induced by inhibitors of the kinesin motor protein Eg5 such as monastrol and STLC. Monastrol and STLC induce monopolar spindle formation and M phase arrest via inhibition of the ATPase activity of Eg5. Interestingly, our data revealed that pyrenocine A had no effect on the ATPase activity of Eg5 in vitro, which suggested the compound induces a monopolar spindle by an unknown mechanism. Structure-activity relationship analysis indicates that the enone structure of pyrenocine A is likely to be important for its cytotoxicity. An alkyne-tagged analog of pyrenocine A was synthesized and suppressed proliferation of HeLa cells with an IC50 value of 2.3 µM. We concluded that pyrenocine A induced monopolar spindle formation by a novel mechanism other than direct inhibition of Eg5 motor activity, and the activity of pyrenocine A may suggest a new anticancer mechanism.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Fuso Acromático/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Células HeLa , Humanos , Neoplasias/tratamento farmacológico , Pirimidinas/farmacologia , Pironas/farmacologia , Tionas/farmacologia
13.
Molecules ; 24(14)2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31319565

RESUMO

Influenza A viruses leading to infectious respiratory diseases cause seasonal epidemics and sometimes periodic global pandemics. Viral polymerase is an attractive target in inhibiting viral replication, and 4'-ethynyladenosine, which has been reported as a highly potent anti-human immunodeficiency virus (HIV) nucleoside derivative, can work as an anti-influenza agent. Herein, we designed and synthesized a 4'-ethynyl-2'-deoxyadenosine 5'-monophosphate analog called EdAP (5). EdAP exhibited potent inhibition against influenza virus multiplication in Madin-Darby canine kidney (MDCK) cells transfected with human α2-6-sialyltransferase (SIAT1) cDNA and did not show any toxicity toward the cells. Surprisingly, this DNA-type nucleic acid analog (5) inhibited the multiplication of influenza A virus, although influenza virus is an RNA virus that does not generate DNA.


Assuntos
Antivirais/farmacologia , Nucleotídeos de Desoxiadenina/farmacologia , Desoxiadenosinas/síntese química , Influenza Humana/tratamento farmacológico , Animais , Antivirais/síntese química , Antivirais/química , Nucleotídeos de Desoxiadenina/síntese química , Nucleotídeos de Desoxiadenina/química , Desoxiadenosinas/química , Desoxiadenosinas/farmacologia , Cães , Células HEK293 , Humanos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/patogenicidade , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Replicação Viral/efeitos dos fármacos
14.
Sci Rep ; 9(1): 9283, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31243315

RESUMO

Chloramphenicol (Cm) is a broad-spectrum classic antibiotic active against prokaryotic organisms. However, Cm has severe side effects in eukaryotes of which the cause remains unknown. The plant pathogenic fungus Magnaporthe oryzae, which causes rice blast, forms an appressorium to infect the host cell via single-cell differentiation. Chloramphenicol specifically inhibits appressorium formation, which indicates that Cm has a novel molecular target (or targets) in the rice blast fungus. Application of the T7 phage display method inferred that MoDullard, a Ser/Thr-protein phosphatase, may be a target of Cm. In animals Dullard functions in cell differentiation and protein synthesis, but in fungi its role is poorly understood. In vivo and in vitro analyses showed that MoDullard is required for appressorium formation, and that Cm can bind to and inhibit MoDullard function. Given that human phosphatase CTDSP1 complemented the MoDullard function during appressorium formation by M. oryzae, CTDSP1 may be a novel molecular target of Cm in eukaryotes.


Assuntos
Cloranfenicol/farmacologia , Magnaporthe/efeitos dos fármacos , Oryza/microbiologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Antifúngicos/farmacologia , Bacteriófago T7 , Diferenciação Celular , DNA Fúngico , Deleção de Genes , Teste de Complementação Genética , Humanos , Magnaporthe/enzimologia , Mutação , Biblioteca de Peptídeos , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Doenças das Plantas/microbiologia , Plasmídeos/genética , RNA Fúngico
15.
J Biol Chem ; 294(19): 7942-7965, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30926603

RESUMO

endo-ß-1,2-Glucanase (SGL) is an enzyme that hydrolyzes ß-1,2-glucans, which play important physiological roles in some bacteria as a cyclic form. To date, no eukaryotic SGL has been identified. We purified an SGL from Talaromyces funiculosus (TfSGL), a soil fungus, to homogeneity and then cloned the complementary DNA encoding the enzyme. TfSGL shows no significant sequence similarity to any known glycoside hydrolase (GH) families, but shows significant similarity to certain eukaryotic proteins with unknown functions. The recombinant TfSGL (TfSGLr) specifically hydrolyzed linear and cyclic ß-1,2-glucans to sophorose (Glc-ß-1,2-Glc) as a main product. TfSGLr hydrolyzed reducing-end-modified ß-1,2-gluco-oligosaccharides to release a sophoroside with the modified moiety. These results indicate that TfSGL is an endo-type enzyme that preferably releases sophorose from the reducing end of substrates. Stereochemical analysis demonstrated that TfSGL is an inverting enzyme. The overall structure of TfSGLr includes an (α/α)6 toroid fold. The substrate-binding mode was revealed by the structure of a Michaelis complex of an inactive TfSGLr mutant with a ß-1,2-glucoheptasaccharide. Mutational analysis and action pattern analysis of ß-1,2-gluco-oligosaccharide derivatives revealed an unprecedented catalytic mechanism for substrate hydrolysis. Glu-262 (general acid) indirectly protonates the anomeric oxygen at subsite -1 via the 3-hydroxy group of the Glc moiety at subsite +2, and Asp-446 (general base) activates the nucleophilic water via another water. TfSGLr is apparently different from a GH144 SGL in the reaction and substrate recognition mechanism based on structural comparison. Overall, we propose that TfSGL and closely-related enzymes can be classified into a new family, GH162.


Assuntos
Proteínas Fúngicas/química , Glicosídeo Hidrolases/química , Microbiologia do Solo , Talaromyces/enzimologia , Relação Estrutura-Atividade , Especificidade por Substrato
16.
Xenobiotica ; 49(3): 346-362, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29543539

RESUMO

Sulfoquinovosylacylpropanediol (SQAP) is a novel potent radiosensitizer that inhibits angiogenesis in vivo and results in increased oxigenation and reduced tumor volume. We investigated the distribution, metabolism, and excretion of SQAP in male KSN-nude mice transplanted with a human pulmonary carcinoma, Lu65. For the metabolism analysis, a 2 mg (2.98 MBq)/kg of [glucose-U-14C]-SQAP (CP-3839) was intravenously injected. The injected SQAP was decomposed into a stearic acid and a sulfoquinovosylpropanediol (SQP) in the body. The degradation was relatively slow in the carcinoma tissue.1,3-propanediol[1-14C]-SQAP (CP-3635) was administered through intravenous injection of a 1 mg (3.48 MBq)/kg dose followed by whole body autoradiography of the mice. The autoradiography analysis demonstrated that SQAP rapidly distributed throughout the whole body and then quickly decreased within 4 hours except the tumor and excretion organs such as liver, kidney. Retention of SQAP was longer in tumor parts than in other tissues, as indicated by higher levels of radioactivity at 4 hours. The radioactivity around the tumor had also completely disappeared within 72 hours.


Assuntos
Glicolipídeos/farmacocinética , Radiossensibilizantes/farmacocinética , Administração Intravenosa , Animais , Autorradiografia , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Glicolipídeos/administração & dosagem , Glicolipídeos/uso terapêutico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos Nus , Radiossensibilizantes/administração & dosagem , Radiossensibilizantes/uso terapêutico , Espectrometria de Massas em Tandem
17.
J Biol Chem ; 293(51): 19559-19571, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30381393

RESUMO

Viruses hijack and modify host cell functions to maximize viral proliferation. Hepatitis C virus (HCV) reorganizes host cell metabolism to produce specialized membrane structures and to modify organelles such as double-membrane vesicles and enlarged lipid droplets (LDs), thereby enabling virus replication and assembly. However, the molecular bases of these host-HCV interactions are largely unknown. Here, using a chemical screen, we demonstrate that the benzamide derivative flutamide reduces the host capacity to produce infectious HCV. Flutamide disrupted the formation of enlarged LDs in HCV-infected cells, thereby abolishing HCV assembly. We also report that aryl hydrocarbon receptor (AhR), a known flutamide target, plays a key role in mediating LD accumulation and HCV production. This AhR function in lipid production was also observed in HCV-uninfected Huh-7 cells and primary human hepatocytes, suggesting that AhR signaling regulates lipid accumulation independently of HCV infection. We further observed that a downstream activity, that of cytochrome P450 1A1 (CYP1A1), was the primary regulator of AhR-mediated lipid production. Specifically, blockade of AhR-induced CYP1A1 up-regulation counteracted LD overproduction, and overproduction of CYP1A1, but not of CYP1B1, in AhR-inactivated cells restored lipid accumulation. Of note, HCV infection up-regulated the AhR-CYP1A1 pathway, resulting in the accumulation of enlarged LDs. In conclusion, we demonstrate that the AhR-CYP1A1 pathway has a significant role in lipid accumulation, a hallmark of HCV infection that maximizes progeny virus production. Our chemical-genetic analysis reveals a new strategy and lead compounds to control hepatic lipid accumulation as well as HCV infection.


Assuntos
Citocromo P-450 CYP1A1/metabolismo , Hepacivirus/fisiologia , Metabolismo dos Lipídeos , Receptores de Hidrocarboneto Arílico/metabolismo , Montagem de Vírus , Linhagem Celular , Flutamida/farmacologia , Hepacivirus/efeitos dos fármacos , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Ligação Proteica , Montagem de Vírus/efeitos dos fármacos
18.
Cancer Res ; 78(24): 6828-6837, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30301838

RESUMO

: Hypoxic zones in solid tumors contribute to radioresistance, and pharmacologic agents that increase tumor oxygenation prior to radiation, including antiangiogenic drugs, can enhance treatment response to radiotherapy. Although such strategies have been applied, imaging assessments of tumor oxygenation to identify an optimum time window for radiotherapy have not been fully explored. In this study, we investigated the effects of α-sulfoquinovosylacyl-1,3-propanediol (SQAP or CG-0321; a synthetic derivative of an antiangiogenic agent) on the tumor microenvironment in terms of oxygen partial pressure (pO2), oxyhemoglobin saturation (sO2), blood perfusion, and microvessel density using electron paramagnetic resonance imaging, photoacoustic imaging, dynamic contrast-enhanced MRI with Gd-DTPA injection, and T2*-weighted imaging with ultrasmall superparamagnetic iron oxide (USPIO) contrast. SCCVII and A549 tumors were grown by injecting tumor cells into the hind legs of mice. Five days of daily radiation (2 Gy) combined with intravenous injection of SQAP (2 mg/kg) 30 minutes prior to irradiation significantly delayed growth of tumor xenografts. Three days of daily treatment improved tumor oxygenation and decreased tumor microvascular density on T2*-weighted images with USPIO, suggesting vascular normalization. Acute effects of SQAP on tumor oxygenation were examined by pO2, sO2, and Gd-DTPA contrast-enhanced imaging. SQAP treatment improved perfusion and tumor pO2 (ΔpO2: 3.1 ± 1.0 mmHg) and was accompanied by decreased sO2 (20%-30% decrease) in SCCVII implants 20-30 minutes after SQAP administration. These results provide evidence that SQAP transiently enhanced tumor oxygenation by facilitating oxygen dissociation from oxyhemoglobin and improving tumor perfusion. Therefore, SQAP-mediated sensitization to radiation in vivo can be attributed to increased tumor oxygenation. SIGNIFICANCE: A multimodal molecular imaging study evaluates pharmacological alteration of the tumor microenvironment to improve radiation response.


Assuntos
Imagem Molecular/métodos , Imagem Multimodal/métodos , Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Microambiente Tumoral , Células A549 , Acústica , Inibidores da Angiogênese/farmacologia , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Gadolínio/química , Gadolínio DTPA/química , Glicolipídeos/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Hipóxia , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C3H , Microcirculação , Transplante de Neoplasias , Neoplasias/metabolismo , Oxigênio/química , Oxigênio/metabolismo , Fotoquímica , Tolerância a Radiação , Radioterapia
19.
Methods Mol Biol ; 1795: 159-172, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29846927

RESUMO

Identification of target proteins that directly bind to bioactive small molecule is of great interest in terms of clarifying the mode of action of the small molecule as well as elucidating the biological phenomena at the molecular level. Of the experimental technologies available, T7 phage display allows comprehensive screening of small molecule-recognizing amino acid sequence from the peptide libraries displayed on the T7 phage capsid. Here, we describe the T7 phage display strategy that is combined with quartz-crystal microbalance (QCM) biosensor for affinity selection platform and bioinformatics analysis for small molecule-recognizing short peptides. This method dramatically enhances efficacy and throughput of the screening for small molecule-recognizing amino acid sequences without repeated rounds of selection. Subsequent execution of bioinformatics programs allows combinatorial and comprehensive target protein discovery of small molecules with its binding site, regardless of protein sample insolubility, instability, or inaccessibility of the fixed small molecules to internally located binding site on larger target proteins when conventional proteomics approaches are used.


Assuntos
Bacteriófago T7/genética , Técnicas Biossensoriais , Técnicas de Visualização da Superfície Celular , Biologia Computacional , Descoberta de Drogas , Bibliotecas de Moléculas Pequenas , Sequência de Aminoácidos , Biologia Computacional/métodos , Descoberta de Drogas/métodos , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Análise de Sequência de DNA , Software
20.
Biosci Biotechnol Biochem ; 82(3): 442-448, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29447077

RESUMO

Neoechinulin A is an indole alkaloid with several biological activities. We previously reported that this compound protects neuronal PC12 cells from cytotoxicity induced by the peroxynitrite generator 3-morpholinosydnonimine (SIN-1), but the target proteins and precise mechanism of action of neoechinulin A were unclear. Here, we employed a phage display screen to identify proteins that bind directly with neoechinulin A. Our findings identified two proteins, chromogranin B and glutaredoxin 3, as candidate target binding partners for the alkaloid. QCM analyses revealed that neoechinulin A displays high affinity for both chromogranin B and glutaredoxin 3. RNA interference-mediated depletion of chromogranin B decreased the sensitivity of PC12 cells against SIN-1. Our results suggested chromogranin B is a plausible target of neoechinulin A.


Assuntos
Cromogranina B/metabolismo , Glutarredoxinas/metabolismo , Alcaloides Indólicos/metabolismo , Fármacos Neuroprotetores/metabolismo , Biblioteca de Peptídeos , Piperazinas/metabolismo , Animais , Cromogranina B/deficiência , Cromogranina B/genética , Inativação Gênica , Glutarredoxinas/deficiência , Glutarredoxinas/genética , Alcaloides Indólicos/farmacologia , Fármacos Neuroprotetores/farmacologia , Células PC12 , Piperazinas/farmacologia , Ligação Proteica , Ratos
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