RESUMO
Among carbohydrate-processing enzymes, Jack bean α-mannosidase (JBα-man) is the glycosidase with the best responsiveness to the multivalent presentation of iminosugar inhitopes. We report, in this work, the preparation of water dispersible gold nanoparticles simultaneously coated with the iminosugar deoxynojirimycin (DNJ) inhitope and simple monosaccharides (ß-d-gluco- or α-d-mannosides). The display of DNJ at the gold surface has been modulated (i) by using an amphiphilic linker longer than the aliphatic chain used for the monosaccharides and (ii) by presenting the inhitope, not only in monomeric form, but also in a trimeric fashion through combination of a dendron approach with glyconanotechnology. The latter strategy resulted in a strong enhancement of the inhibitory activity towards JBα-man, with a Ki in the nanomolar range (Ki = 84 nM), i.e., more than three orders of magnitude higher than the monovalent reference compound.
Assuntos
1-Desoxinojirimicina/administração & dosagem , Canavalia/enzimologia , Inibidores Enzimáticos/administração & dosagem , Ouro/química , Nanopartículas Metálicas/administração & dosagem , alfa-Manosidase/antagonistas & inibidores , 1-Desoxinojirimicina/química , Inibidores Enzimáticos/química , Nanopartículas Metálicas/químicaRESUMO
Multivalent mannosides with inherent macrophage recognition abilities, built on ß-cyclodextrin, RAFT cyclopeptide or peptide dendrimer cores, trigger selective inhibition of lysosomal ß-glucocerebrosidase or α-mannosidase depending on valency and topology, offering new opportunities in multitargeted drug design.
Assuntos
Desenho de Fármacos , Manosídeos/química , Glucosilceramidase/antagonistas & inibidores , Lectinas/química , Macrófagos/metabolismo , Manosídeos/metabolismo , Peptídeos Cíclicos/química , alfa-Manosidase/antagonistas & inibidores , beta-Ciclodextrinas/químicaRESUMO
The best multivalent effects observed in glycosidase inhibition have been achieved so far with jack bean α-mannosidase (JBα-man) using iminosugar clusters based on weakly binding mismatching active-site-directed inhibiting epitopes (inhitopes) in the d-gluco series. Here, we synthesize and evaluate as JBα-man inhibitors a series of mono- to 14-valent glycoimidazoles with inhitopes displaying inhibition values up to the range of hundreds of nMs to study the impact of inhitope affinity on the multivalent effect. The most potent inhibitor of the series, a 14-valent mannoimidazole derivative, inhibits JBα-man with a nanomolar Ki value (2 ± 0.5 nM) and binding enhancements observed are, at best, relatively small (up to 25-fold on a valency-corrected basis). The results of this study support the fact that JBα-man-inhitope affinity and the strength of the inhibitory multivalent effect evolve in the opposite direction. The major impact of the glycoimidazole-based inhitope is found on the binding scenario; most of the synthesized mannoimidazole clusters as well as a 14-valent glucoimidazole derivative prove to be tight binding inhibitors of JBα-man.
Assuntos
Fabaceae/enzimologia , Imidazóis/química , Imidazóis/farmacologia , alfa-Manosidase/antagonistas & inibidores , Domínio Catalítico , Estrutura Molecular , Ligação ProteicaRESUMO
Cervical cancer, caused by high oncogenic risk Human Papillomavirus (HPV) infection, continues to be a public health problem, mainly in developing countries. Using peptide phage display as a tool to identify potential molecular targets in HPV associated tumors, we identified α-mannosidase, among other enriched sequences. This enzyme is expressed in both tumor and inflammatory compartment of the tumor microenvironment. Several studies in experimental models have shown that its inhibition by swainsonine (SW) led to inhibition of tumor growth and metastasis directly and indirectly, through activation of macrophages and NK cells, promoting anti-tumor activity. Therefore, the aim of this work was to test if swainsonine treatment could modulate anti-tumor immune responses and therefore interfere in HPV associated tumor growth. Validation of our biopanning results showed that cervical tumors, both tumor cells and leukocytes, expressed α-mannosidase. Ex vivo experiments with tumor associated macrophages showed that SW could partially modulate macrophage phenotype, decreasing CCL2 secretion and impairing IL-10 and IL-6 upregulation, which prompted us to proceed to in vivo tests. However, in vivo, SW treatment increased tumor growth. Investigation of the mechanisms leading to this result showed that SW treatment significantly induced the accumulation of myeloid derived suppressor cells in the spleen of tumor bearing mice, which inhibited T cell activation. Our results suggested that SW contributes to cervical cancer progression by favoring proliferation and accumulation of myeloid cells in the spleen, thus exacerbating these tumors systemic effects on the immune system, therefore facilitating tumor growth.
Assuntos
Proliferação de Células/efeitos dos fármacos , Swainsonina/farmacologia , Neoplasias do Colo do Útero/patologia , alfa-Manosidase/metabolismo , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Quimiocina CCL2/metabolismo , Progressão da Doença , Feminino , Humanos , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Leucócitos/citologia , Leucócitos/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Supressoras Mieloides/citologia , Células Supressoras Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/metabolismo , Swainsonina/uso terapêutico , Microambiente Tumoral/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/virologia , alfa-Manosidase/antagonistas & inibidoresRESUMO
Inhibition of the biosynthesis of complex N-glycans in the Golgi apparatus is one of alternative ways to suppress growth of tumor tissue. Eight N-benzyl substituted 1,4-imino-l-lyxitols with basic functional groups (amine, amidine, guanidine), hydroxyl and fluoro groups were prepared, optimized their syntheses and tested for their ability to inhibit several α-mannosides from the GH family 38 (GMIIb, LManII and JBMan) as models for human Golgi and lysosomal α-mannoside II. All compounds were found to be selective inhibitors of GMIIb. The most potent structure bearing guanidine group, inhibited GMIIb at the micromolar level (Kiâ¯=â¯19⯱â¯2⯵M) while no significant inhibition (>2â¯mM) of LManII and JBMan was observed. Based on molecular docking and pKa calculations this structure may form two salt bridges with aspartate dyad of the target enzyme improving its inhibitory potency compared with other N-benzyl substituted derivatives published in this and previous studies.
Assuntos
Inibidores Enzimáticos/química , Imino Açúcares/química , Álcoois Açúcares/química , alfa-Manosidase/antagonistas & inibidores , Animais , Domínio Catalítico , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Ensaios Enzimáticos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Imino Açúcares/síntese química , Imino Açúcares/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Álcoois Açúcares/síntese química , Álcoois Açúcares/metabolismo , alfa-Manosidase/química , alfa-Manosidase/metabolismoRESUMO
A novel supramolecular multivalent glycosidase inhibitor was constructed based on the amphiphilic deoxynojirimycin derivative FA-DNJ. FA-DNJ self-assembled into spherical assemblies with the diameters between 103 nm and 137 nm under different pH values (pH 2-7) and showed a potent glycosidase effect against α-mannosidase with a Ki value of 0.11 µM, an effect that increased approximately 330-fold compared with that of miglitol. In addition, FA-DNJ exhibited a hypoglycaemic effect in mice.
Assuntos
Inibidores Enzimáticos/química , Ácidos Graxos/química , Glucosamina/análogos & derivados , Hipoglicemiantes/química , alfa-Manosidase/antagonistas & inibidores , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/química , 1-Desoxinojirimicina/metabolismo , Animais , Glicemia/análise , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Glucosamina/química , Teste de Tolerância a Glucose , Concentração de Íons de Hidrogênio , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacologia , Maltose/administração & dosagem , Camundongos , alfa-Manosidase/metabolismoRESUMO
Understanding the enzyme reaction mechanism can lead to the design of enzyme inhibitors. A Claisen rearrangement was used to allow conversion of an α-1,4-disaccharide into an α-1,3-linked glycosyl carbasugar to target the endo-α-mannosidase from the GH99 glycosidase family, which, unusually, is believed to act through a 1,2-anhydrosugar "epoxide" intermediate. Using NMR and X-ray crystallography, it is shown that glucosyl carbasugar α-aziridines can act as reasonably potent endo-α-mannosidase inhibitors, likely by virtue of their shape mimicry and the interactions of the aziridine nitrogen with the conserved catalytic acid/base of the enzyme active site.
Assuntos
Carbaçúcares/farmacologia , Dissacarídeos/farmacologia , Inibidores de Glicosídeo Hidrolases/farmacologia , alfa-Manosidase/antagonistas & inibidores , Carbaçúcares/síntese química , Carbaçúcares/química , Dissacarídeos/química , Relação Dose-Resposta a Droga , Inibidores de Glicosídeo Hidrolases/síntese química , Inibidores de Glicosídeo Hidrolases/química , Estrutura Molecular , Relação Estrutura-Atividade , alfa-Manosidase/metabolismoRESUMO
Multivalent design of glycosidase inhibitors is a promising strategy for the treatment of diseases involving enzymatic hydrolysis of glycosidic bonds in carbohydrates. An essential prerequisite for successful applications is the atomic-level understanding of how outstanding binding enhancement occurs with multivalent inhibitors. Herein we report the first high-resolution crystal structures of the Jack bean α-mannosidase (JBα-man) in apo and inhibited states. The three-dimensional structure of JBα-man in complex with the multimeric cyclopeptoid-based inhibitor displaying the largest binding enhancements reported so far provides decisive insight into the molecular mechanisms underlying multivalent effects in glycosidase inhibition.
Assuntos
alfa-Manosidase/metabolismo , Sítios de Ligação , Canavalia/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Imino Açúcares/química , Imino Açúcares/metabolismo , Estrutura Terciária de Proteína , Zinco/química , Zinco/metabolismo , alfa-Manosidase/antagonistas & inibidoresRESUMO
Three Golgi mannosidases (GMs), namely Golgi α-mannosidases IA, IB, and IC, remove mannose residues from N-glycans and regulate the quality control and transportation of nascent proteins. GM inhibitors regulate several biological events such as cell-cell communication, differentiation, and apoptosis in cancer cells. As a result, GM inhibitor-based therapies have gained significant attention for cancer treatment. However, to date, no GM inhibitor has been approved and none is in clinical development for anti-cancer treatment. Meanwhile, drug repositioning plays an important role in identifying potential inhibitors that vary in molecular structure and properties to bypass much of the early cost and time. We performed a drug repositioning screen of a compound library that included approved drugs. The estrogen receptor antagonists tamoxifen and raloxifene inhibited human GMs at the cellular level. Sulindac, a nonsteroidal anti-inflammatory drug, also inhibited GMs. Our results demonstrated the efficacy of this screening strategy and revealed lead compounds for anti-cancer drug development.
Assuntos
Reposicionamento de Medicamentos , Inibidores Enzimáticos/farmacologia , Complexo de Golgi/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , alfa-Manosidase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Fluorescência , Células HeLa , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , alfa-Manosidase/metabolismoRESUMO
Kifunensine, a potent and selective inhibitor of class I α-mannosidases, prevents α-mannosidases I from trimming mannose residues on glycoproteins, thus resulting in oligomannose-type glycans. We report for the first time that through one-time vacuum infiltration of kifunensine in plant tissue, N-linked glycosylation of a recombinant protein transiently produced in whole-plants shifted completely from complex-type to oligomannose-type. Fc-fused capillary morphogenesis protein 2 (CMG2-Fc) containing one N-glycosylation site on the Fc domain, produced in Nicotiana benthamiana whole plants, served as a model protein. The CMG2-Fc fusion protein was produced transiently through vacuum agroinfiltration, with and without kifunensine at a concentration of 5.4 µM in the agroinfiltration suspension. The CMG2-Fc N-glycan profile was determined using LC-MS/MS with a targeted dynamic multiple reaction monitoring (MRM) method. The CMG2-Fc expression level in the infiltrated plant tissue and the percentage of oligomannose-type N-glycans for kifunensine treated plants was 874 mg/kg leaf fresh weight (FW) and 98.2%, respectively, compared to 717 mg/kg leaf FW and 2.3% for untreated plants. Oligomannose glycans are amenable to in vitro enzymatic modification to produce more human-like N-glycan structures that are preferred for the production of HIV-1 viral vaccine and certain monoclonal antibodies. This method allows glycan modifications using a bioprocessing approach without compromising protein yield or modification of the primary sequence, and could be expanded to other small molecule inhibitors of glycan-processing enzymes. For recombinant protein targeted for secretion, kifunensine treatment allows collection of glycoform-modified target protein from apoplast wash fluid (AWF) with minimal plant-specific complex N-glycan at higher starting purity and concentration than in whole-leaf extract, thus simplifying the downstream processing.
Assuntos
Alcaloides/farmacologia , Inibidores Enzimáticos/farmacologia , Glicoproteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Vacinas contra a AIDS/biossíntese , Agrobacterium/genética , Agrobacterium/metabolismo , Anticorpos Monoclonais/biossíntese , Glicosilação/efeitos dos fármacos , HIV-1/imunologia , Humanos , Manose/metabolismo , Receptores de Peptídeos/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/microbiologia , Vácuo , alfa-Manosidase/antagonistas & inibidoresRESUMO
An efficient and practical strategy for the synthesis of unknown azetidine iminosugars (2S,3R,4S)-2-((R)-1,2-dihydroxyethyl)-3-hydroxy-4-(hydroxymethyl)azetidine 2, (2S,3r,4R)-3-hydroxy-2,4-bis(hydroxymethyl)azetidine 3 and (2S,3R,4S)-3-hydroxy-4-(hydroxymethyl)-N-methylazetidine-2-carboxylic acid 4, starting from the d-glucose has been reported. The methodology involves preparation of the 3-amino-N-benzyloxycarbonyl-3-deoxy-6-O-tert-butyldimethylsillyl-1,2-O-isopropylidene-α-d-glucofuranose 9, which was converted to the C-5-OMs derivative 11. Intramolecular nucleophilic displacement of the C-5-OMs group with in situ generated 3-amino functionality provided the required key azetidine ring skeletons 10 with additional hydroxymethyl group. Removal of 1,2-acetonide protection, followed by reduction and hydrogenolysis afforded azetidine iminosugar 2. Alternatively, removal of 1,2-acetonide group and chopping of C1-anomeric carbon gave C2-aldehyde that on reduction or oxidation followed by hydrogenolysis gave 2,4-bis(hydroxymethyl) azetidine iminosugars 3 and N-methylazetidine-2-carboxylic acid 4 respectively. The glycosidase inhibitory activity of 2-4 iminosugars was screened against various glycosidase enzymes and compared with a standard miglitol. Amongst synthesized targets, the compound 2 was found to be more potent amyloglucosidase inhibitor than miglitol. These results were supported by molecular docking studies.
Assuntos
Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Imino Açúcares/farmacologia , Simulação de Acoplamento Molecular , Aspergillus niger/enzimologia , Canavalia/enzimologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Escherichia coli/enzimologia , Glicosídeo Hidrolases/metabolismo , Hidroxilação , Imino Açúcares/síntese química , Imino Açúcares/química , Estrutura Molecular , Relação Estrutura-Atividade , alfa-Galactosidase/antagonistas & inibidores , alfa-Galactosidase/metabolismo , alfa-Manosidase/antagonistas & inibidores , alfa-Manosidase/metabolismoRESUMO
Novel pyrrolidine-based multivalent iminosugars, synthesized by a CuAAC approach, have shown remarkable multivalent effects towards jack bean α-mannosidase and a Golgi α-mannosidase from Drosophila melanogaster, as well as a good selectivity with respect to a lysosomal α-mannosidase, which is important for anticancer applications. STD NMR and molecular modeling studies supported a multivalent mechanism with specific interactions of the bioactive iminosugars with Jack bean α-mannosidase. TEM studies suggested a binding mode that involves the formation of aggregates, which result from the intermolecular cross-linked network of interactions between the multivalent inhibitors and two or more dimers of JBMan heterodimeric subunits.
Assuntos
Pirrolidinas/metabolismo , alfa-Manosidase/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Drosophila melanogaster/enzimologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Imino Açúcares/síntese química , Imino Açúcares/química , Imino Açúcares/metabolismo , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Transmissão , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Pirrolidinas/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , alfa-Manosidase/antagonistas & inibidores , alfa-Manosidase/genéticaRESUMO
KEY POINTS: Ionotropic glutamate receptor (iGluR) subunits are N-glycosylated at 4-12 sites, and Golgi processing produces mature receptors that contain high-mannose, hybrid and complex oligosaccharides. N-glycosylation is crucial for receptor biogenesis, influences receptor trafficking and provides a binding site for carbohydrate binding proteins. Glycan moieties are large, polar and occasionally charged, and they are attached at sites along iGluRs that position them for involvement in the structural changes underlying gating. Altering glycan content on kainate receptors (KARs), a subfamily of iGluRs, changes functional properties of the receptor, such as desensitization, recovery from desensitization and deactivation. We report the first observation that the charged trisaccharide HNK-1 is conjugated to native KARs, and we find that it substantially alters recombinant KAR functional properties. Our results show that the molecular composition of N-glycans can influence KAR biophysical properties, revealing a potential mechanism for fine-tuning the function of these receptors. ABSTRACT: Ionotropic glutamate receptors (iGluRs) are tetrameric proteins with between four and 12 consensus sites for N-glycosylation on each subunit, which potentially allows for a high degree of structural diversity conferred by this post-translational modification. N-glycosylation is required for proper folding of iGluRs in mammalian cells, although the impact of oligosaccharides on the function of successfully folded receptors is less clear. Glycan moieties are large, polar, occasionally charged and mediate many protein-protein interactions throughout the nervous system. Additionally, they are attached at sites along iGluR subunits that position them for involvement in the structural changes underlying gating. In the present study, we show that altering glycan content on kainate receptors (KARs) changes the functional properties of the receptors in a manner dependent on the identity of both the modified sugars and the subunit composition of the receptor to which they are attached. We also report that native KARs carry the complex capping oligosaccharide human natural killer-1. Glycosylation patterns probably differ between cell types, across development or with pathologies, and thus our findings reveal a potential mechanism for context-specific fine-tuning of KAR function through diversity in glycan structure.
Assuntos
Polissacarídeos/química , Receptores de Ácido Caínico/química , Receptores de Ácido Caínico/fisiologia , Alcaloides/farmacologia , Animais , Feminino , Glicosilação , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Ácido Caínico/genética , Swainsonina/farmacologia , alfa-Manosidase/antagonistas & inibidoresRESUMO
The varied yet family-specific conformational pathways used by individual glycoside hydrolases (GHs) offer a tantalising prospect for the design of tightly binding and specific enzyme inhibitors. A cardinal example of a GH-family-specific inhibitor, and one that finds widespread practical use, is the natural product kifunensine, which is a low-nanomolar inhibitor that is selective for GH family 47 inverting α-mannosidases. Here we show, through quantum-mechanical approaches, that kifunensine is restrained to a "ring-flipped" 1 C4 conformation with another accessible, but higher-energy, region around the 1,4 B conformation. The conformations of kifunensine in complex with a range of GH47 enzymes-including an atomic-level resolution (1â Å) structure of kifunensine with Caulobacter sp. CkGH47 reported herein and with GH family 38 and 92 α-mannosidases-were mapped onto the kifunensine free-energy landscape. These studies revealed that kifunensine has the ability to mimic the product state of GH47 enzymes but cannot mimic any conformational states relevant to the reaction coordinate of mannosidases from other families.
Assuntos
Alcaloides/química , Caulobacter/enzimologia , alfa-Manosidase/química , Calorimetria , Conformação Molecular , Teoria Quântica , alfa-Manosidase/antagonistas & inibidoresRESUMO
BACKGROUND & AIMS: Human tumors and liver cancer cell lines express the product of a fusion between the first 13 exons in the mannosidase α class 2A member 1 gene (MAN2A1) and the last 6 exons in the FER tyrosine kinase gene (FER), called MAN2A1-FER. We investigated whether MAN2A1-FER is expressed by human liver tumors and its role in liver carcinogenesis. METHODS: We performed reverse transcription polymerase chain reaction analyses of 102 non-small cell lung tumors, 61 ovarian tumors, 70 liver tumors, 156 glioblastoma multiform samples, 27 esophageal adenocarcinomas, and 269 prostate cancer samples, as well as 10 nontumor liver tissues and 20 nontumor prostate tissues, collected at the University of Pittsburgh. We also measured expression by 15 human cancer cell lines. We expressed a tagged form of MAN2A1-FER in NIH3T3 and HEP3B (liver cancer) cells; Golgi were isolated for analysis. MAN2A1-FER was also overexpressed in PC3 or DU145 (prostate cancer), NIH3T3 (fibroblast), H23 (lung cancer), and A-172 (glioblastoma multiforme) cell lines and knocked out in HUH7 (liver cancer) cells. Cells were analyzed for proliferation and in invasion assays, and/or injected into flanks of severe combined immunodeficient mice; xenograft tumor growth and metastasis were assessed. Mice with hepatic deletion of PTEN were given tail-vein injections of MAN2A1-FER. RESULTS: We detected MAN2A1-FER messenger RNA and fusion protein (114 kD) in the hepatocellular carcinoma cell line HUH7, as well as in liver tumors, esophageal adenocarcinoma, glioblastoma multiforme, prostate tumors, non-small cell lung tumors, and ovarian tumors, but not nontumor prostate or liver tissues. MAN2A1-FER protein retained the signal peptide for Golgi localization from MAN2A1 and translocated from the cytoplasm to Golgi in cancer cell lines. MAN2A1-FER had tyrosine kinase activity almost 4-fold higher than that of wild-type FER, and phosphorylated the epidermal growth factor receptor at tyrosine 88 in its N-terminus. Expression of MAN2A1-FER in 4 cell lines led to epidermal growth factor receptor activation of BRAF, MEK, and AKT; HUH7 cells with MAN2A1-FER knockout had significant decreases in phosphorylation of these proteins. Cell lines that expressed MAN2A1-FER had increased proliferation, colony formation, and invasiveness and formed larger (>2-fold) xenograft tumors in mice, with more metastases, than cells not expressing the fusion protein. HUH7 cells with MAN2A1-FER knockout formed smaller xenograft tumors, with fewer metastases, than control HUH7 cells. HUH7, A-172, and PC3 cells that expressed MAN2A1-FER were about 2-fold more sensitive to the FER kinase inhibitor crizotinib and the epidermal growth factor receptor kinase inhibitor canertinib; these drugs slowed growth of xenograft tumors from MAN2A1-FER cells and prevented their metastasis in mice. Hydrodynamic tail-vein injection of MAN2A1-FER resulted in rapid development of liver cancer in mice with hepatic disruption of Pten. CONCLUSIONS: Many human tumor types and cancer cell lines express the MAN2A1-FER fusion, which increases proliferation and invasiveness of cancer cell lines and has liver oncogenic activity in mice.
Assuntos
Antineoplásicos/farmacologia , Transformação Celular Neoplásica/genética , Fusão Gênica , Neoplasias Hepáticas/genética , Proteínas de Fusão Oncogênica/genética , Oncogenes , Proteínas Tirosina Quinases/genética , alfa-Manosidase/genética , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Crizotinibe , Relação Dose-Resposta a Droga , Ativação Enzimática , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Complexo de Golgi/enzimologia , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , Camundongos SCID , Morfolinas/farmacologia , Células NIH 3T3 , Invasividade Neoplásica , Transplante de Neoplasias , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/metabolismo , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pirazóis/farmacologia , Piridinas/farmacologia , Interferência de RNA , Fatores de Tempo , Transfecção , Carga Tumoral , alfa-Manosidase/antagonistas & inibidores , alfa-Manosidase/metabolismoRESUMO
Locoism threatens the sustainable development of animal husbandry in areas around the world with intensified desertification, especially in the western United States, western China, Canada, and Mexico, among other countries. This study was conducted to discover potential serum biomarkers in locoweed-poisoned rabbits and lay a foundation for early diagnosis of locoism. We performed iTRAQ labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS), comparing locoweed-poisoned rabbits and healthy controls. A total of 78 differentially-expressed proteins (fold change > 1.5 or < 0.67) were identified in the locoweed-poisoned rabbits compared to healthy controls. We found that 57.70% of differentially-expressed proteins were functionally related, and through bioinformatics analysis, we were able to construct a network mainly in complement and coagulation cascades. Significant differences in thrombospondin 4 (THBS4), kininogen 1 (KNG1), hemoglobin (HBB), and complement factor I (CFI) between locoweed poisoned animals and controls were found (P < 0.05) and validated by western blotting. These results suggested that locoweed could damage neurocytes, lower immunity, and form thrombi in rabbits. Our study proposes potential biomarkers for locoism diagnosis and also provides a new experimental basis to understand the pathogenesis of locoism.
Assuntos
Proteínas Sanguíneas/análise , Oxytropis/intoxicação , Proteoma/análise , Animais , Biomarcadores/sangue , Feminino , Masculino , Proteômica , Coelhos , Swainsonina/farmacologia , alfa-Manosidase/antagonistas & inibidoresRESUMO
Human Golgi α-mannosidase II (GMII), a zinc ion co-factor dependent glycoside hydrolase (E.C.3.2.1.114), is a pharmaceutical target for the design of inhibitors with anti-cancer activity. The discovery of an effective inhibitor is complicated by the fact that all known potent inhibitors of GMII are involved in unwanted co-inhibition with lysosomal α-mannosidase (LMan, E.C.3.2.1.24), a relative to GMII. Routine empirical QSAR models for both GMII and LMan did not work with a required accuracy. Therefore, we have developed a fast computational protocol to build predictive models combining interaction energy descriptors from an empirical docking scoring function (Glide-Schrödinger), Linear Interaction Energy (LIE) method, and quantum mechanical density functional theory (QM-DFT) calculations. The QSAR models were built and validated with a library of structurally diverse GMII and LMan inhibitors and non-active compounds. A critical role of QM-DFT descriptors for the more accurate prediction abilities of the models is demonstrated. The predictive ability of the models was significantly improved when going from the empirical docking scoring function to mixed empirical-QM-DFT QSAR models (Q(2)=0.78-0.86 when cross-validation procedures were carried out; and R(2)=0.81-0.83 for a testing set). The average error for the predicted ΔGbind decreased to 0.8-1.1kcalmol(-1). Also, 76-80% of non-active compounds were successfully filtered out from GMII and LMan inhibitors. The QSAR models with the fragmented QM-DFT descriptors may find a useful application in structure-based drug design where pure empirical and force field methods reached their limits and where quantum mechanics effects are critical for ligand-receptor interactions. The optimized models will apply in lead optimization processes for GMII drug developments.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Neoplasias/tratamento farmacológico , Relação Quantitativa Estrutura-Atividade , alfa-Manosidase/química , Sítios de Ligação/efeitos dos fármacos , Desenho de Fármacos , Inibidores Enzimáticos/química , Complexo de Golgi/enzimologia , Humanos , Ligantes , Modelos Moleculares , Neoplasias/enzimologia , Teoria Quântica , alfa-Manosidase/antagonistas & inibidoresRESUMO
Betulinic acid (3ß-hydroxy-20(29)-lupen-28-oic acid), oleanolic acid (3ß-hydroxy-olean-12-en-28-oic acid), and ursolic acid (3ß-hydroxy-urs-12-en-28-oic acid) are close structural isomers of natural pentacyclic triterpenoid carboxylic acids. We recently identified a unique biological effect of ursolic acid, its inhibition of the intracellular trafficking of glycoproteins. In the present study, we demonstrated that betulinic acid and oleanolic acid did not inhibit the interleukin-1α-induced expression of cell-surface intercellular adhesion molecule-1 (ICAM-1) in human lung carcinoma A549 cells. Nevertheless, betulinic acid and, to a lesser extent, oleanolic acid interfered with N-linked glycan modifications to ICAM-1 in a similar manner to castanospermine (an inhibitor of endoplasmic reticulum α-glucosidases I and II), but not swainsonine (an inhibitor of Golgi α-mannosidase II). Consistent with these results, betulinic acid and oleanolic acid inhibited yeast α-glucosidase activity, but not Jack bean α-mannosidase activity. Thus, to the best of our knowledge, this is the first study to show that betulinic acid and oleanolic acid interfere with N-linked glycan modifications to ICAM-1, but not its intracellular transport to the cell surface.
Assuntos
Inibidores de Glicosídeo Hidrolases/farmacologia , Molécula 1 de Adesão Intercelular/metabolismo , Ácido Oleanólico/farmacologia , Triterpenos/farmacologia , Linhagem Celular Tumoral , Humanos , Indolizinas/farmacologia , Molécula 1 de Adesão Intercelular/biossíntese , Interleucina-1alfa/farmacologia , Triterpenos Pentacíclicos , Transporte Proteico/efeitos dos fármacos , Swainsonina/farmacologia , alfa-Manosidase/antagonistas & inibidores , Ácido BetulínicoRESUMO
Ligand-based and structure-based methods were applied in combination to exploit the physicochemical properties of 2,3-dideoxy hex-2-enopyranosid-4-uloses against Mycobacterium tuberculosis H37Rv. Statistically valid 3D-QSAR models with good correlation and predictive power were obtained with CoMFA steric and electrostatic fields (r(2) = 0.797, q(2) = 0.589) and CoMSIA with combined steric, electrostatic, hydrophobic and hydrogen bond acceptor fields (r(2) = 0.867, q(2) = 0.570) based on training set of 33 molecules with predictive r(2) of 0.808 and 0.890 for CoMFA and CoMSIA respectively. The results illustrate the requirement of optimal alkyl chain length at C-1 position and acceptor groups along hydroxy methyl substituent of C-6 to enhance the anti-tubercular activity of the 2,3-dideoxy hex-2-enopyranosid-4-uloses while any substitution at C-3 position exert diminishing effect on anti-tubercular activity of these enulosides. Further, homology modeling of M. tuberculosis alpha-mannosidase followed by molecular docking and molecular dynamics simulations on co-complexed models were performed to gain insight into the rationale for binding affinity of selected inhibitors with the target of interest. The comprehensive information obtained from this study will help to better understand the structural basis of biological activity of this class of molecules and guide further design of more potent analogues as anti-tubercular agents.
Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Desoxiaçúcares/química , Desoxiaçúcares/farmacologia , Mycobacterium tuberculosis/enzimologia , alfa-Manosidase/antagonistas & inibidores , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Relação Quantitativa Estrutura-Atividade , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , alfa-Manosidase/química , alfa-Manosidase/metabolismoRESUMO
Swainsonine (SW) is the principal toxic ingredient of locoweeds, which can cause intensive vacuolar degeneration because of α-mannosidase inhibition after animal ingestion. While SW can lead to obvious liver damage in vivo, the mechanism of hepatotoxic damage caused by SW is not clear. Therefore, BRL-3A cells were treated for 24, 48, and 72 h with SW at various concentrations (0, 700, 900, 1100 µg/mL). The α-mannosidase (AMAN) activity was determined in BRL-3A cells using an enzyme substrate technique. The expression of mRNA and proteins of GM II (MAN2A1) and LAM (MAN2B1) in BRL-3A cells was detected by qPCR and Western-blot. The results showed that SW could significantly reduce the activity of AMAN in a time-dose effect relationship. Compared with the control group, the activity of AMAN significantly decreased only in the group treated with 1100 µg/mL SW for 24 h (P < 0.01), but the activity decreased significantly (P < 0.05 or P < 0.01) in all experimental groups treated for 48 or 72 h. SW also significantly reduced the expression of MAN2A1 and MAN2B1 mRNA and proteins in a time-dose effect relationship (P < 0.05 or P < 0.01), while the inhibition of SW was stronger for MAN2B1 than for MAN2A1. These results suggest that SW can significantly reduce the activity and expression of α-mannosidase thus causing SW-induced hepatotoxic damage.