RESUMO
Tunicamycins (TUN) are well-defined, Streptomyces-derived natural products that inhibit protein N-glycosylation in eukaryotes, and by a conserved mechanism also block bacterial cell wall biosynthesis. TUN inhibits the polyprenylphosphate-N-acetyl-hexosamine-1-phospho-transferases (PNPT), an essential family of enzymes found in both bacteria and eukaryotes. We have previously published the development of chemically modified TUN, called TunR1 and TunR2, that have considerably reduced activity on eukaryotes but that retain the potent antibacterial properties. A mechanism for this reduced toxicity has also been reported. TunR1 and TunR2 have been tested against mammalian cell lines in culture and against live insect cells but, until now, no in vivo evaluation has been undertaken for vertebrates. In the current work, TUN, TunR1, and TunR2 are investigated for their relative toxicity and antimycobacterial activity in zebrafish using a well-established Mycobacterium marinum (M. marinum) infection system, a model for studying human Mycobacterium tuberculosis infections. We also report the relative ability to activate the unfolded protein response (UPR), the known mechanism for the eukaryotic toxicity observed with TUN treatment. Importantly, TunR1 and TunR2 retained their antimicrobial properties, as evidenced by a reduction in M. marinum bacterial burden, compared to DMSO-treated zebrafish. In summary, findings from this study highlight the characteristics of recently developed TUN derivatives, mainly TunR2, and its potential for use as a novel anti-bacterial agent for veterinary and potential medical purposes.
Assuntos
Infecções por Mycobacterium não Tuberculosas , Mycobacterium marinum , Tunicamicina , Animais , Humanos , Antibacterianos/farmacologia , Mamíferos , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium marinum/fisiologia , Tunicamicina/química , Tunicamicina/análogos & derivados , Peixe-Zebra/microbiologia , Fosfotransferases/químicaRESUMO
N-glycosylation, a common post-translational modification, is widely acknowledged to have a significant effect on protein stability and folding. N-glycosylation is a complex process that occurs in the endoplasmic reticulum (ER) and requires the participation of multiple enzymes. GlcNAc-1-P-transferase (GPT) is essential for initiating N-glycosylation in the ER. Tunicamycin is a natural product that inhibits N-glycosylation and produces ER stress, and thus it is utilized in research. The molecular mechanism by which GPT triggers N-glycosylation is discussed in this review based on the GPT structure. Based on the structure of the GPT-tunicamycin complex, we also discuss how tunicamycin reduces GPT activity, which prevents N-glycosylation. This review will be highly useful for understanding the role of GPT in the N-glycosylation of proteins, as well as presents a potential for considering tunicamycin as an antibiotic treatment.
Assuntos
Estresse do Retículo Endoplasmático , Processamento de Proteína Pós-Traducional , Glicosilação , Tunicamicina/farmacologia , Tunicamicina/química , Retículo EndoplasmáticoRESUMO
A short total synthesis of tunicamycinâ V (1), a non-selective phosphotransferase inhibitor, is achieved via a Büchner-Curtius-Schlotterbeck type reaction. Tunicamycinâ V can be synthesized in 15 chemical steps from D-galactal with 21 % overall yield. The established synthetic scheme is operationally very simple and flexible to introduce building blocks of interest. The inhibitory activity of one of the designed analogues 28 against human dolichyl-phosphate N-acetylglucosaminephosphotransferase 1 (DPAGT1) is 12.5â times greater than 1. While tunicamycins are cytotoxic molecules with a low selectivity, the novel analogue 28 displays selective cytostatic activity against breast cancer cell lines including a triple-negative breast cancer.
Assuntos
Antineoplásicos , Citostáticos , Antineoplásicos/farmacologia , Humanos , Tunicamicina/química , Tunicamicina/farmacologiaRESUMO
Although natural products are rich sources for drug discovery, only a small percentage of natural products themselves have been approved for clinical use, thus it is necessary to modulate various properties, such as efficacy, toxicity, and metabolic stability. A question in natural product drug discovery is how to logically design natural product derivatives with desired biological properties. This review describes our recent studies regarding the medicinal chemistry of tunicamycin. Tunicamycin inhibits bacterial phospho-N-acetylmuramic acid (MurNAc)-pentapeptide translocase (MraY), which is an essential enzyme in bacteria and a good target for antibacterial drug discovery. The usefulness of tunicamycin as antibacterial agents is limited by off-target inhibition of human UDP-N-acetylglucosamine (GlcNAc): polyprenol phosphate translocase (GPT). We positioned the total synthesis of tunicamycin as a starting point for the research and have accomplished the synthesis of tunicamycin V by using the Achmatowicz reaction, [3,3] sigmatropic rearrangement of allyl cyanate, and stereoselective glycosylation as key reactions. Next, the minimum structural requirements for tunicamycin V for MraY inhibition were established by systematic structure-activity relationship studies with truncated analogs of tunicamycin V. Our collaborative study elucidated a crystal structure of human GPT in complex with tunicamycin. This structural information was then exploited to rationally design an MraY-specific inhibitor of tunicamycin V in which the GlcNAc moiety was modified to a MurNAc amide. The analog was identified as a highly selective MraYAA inhibitor.
Assuntos
Produtos Biológicos , Transferases , Proteínas de Bactérias/química , Produtos Biológicos/química , Humanos , Transferases/química , Transferases/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Tunicamicina/química , Tunicamicina/metabolismo , Tunicamicina/farmacologiaRESUMO
BACKGROUND: Heterologous expression of secondary metabolite gene clusters is used to achieve increased production of desired compounds, activate cryptic gene clusters, manipulate clusters from genetically unamenable strains, obtain natural products from uncultivable species, create new unnatural pathways, etc. Several Streptomyces species are genetically engineered for use as hosts for heterologous expression of gene clusters. S. lividans TK24 is one of the most studied and genetically tractable actinobacteria, which remain untapped. It was therefore important to generate S. lividans chassis strains with clean metabolic backgrounds. RESULTS: In this study, we generated a set of S. lividans chassis strains by deleting endogenous gene clusters and introducing additional φC31 attB loci for site-specific integration of foreign DNA. In addition to the simplified metabolic background, the engineered S. lividans strains had better growth characteristics than the parental strain in liquid production medium. The utility of the developed strains was validated by expressing four secondary metabolite gene clusters responsible for the production of different classes of natural products. Engineered strains were found to be superior to the parental strain in production of heterologous natural products. Furthermore, S. lividans-based strains were better producers of amino acid-based natural products than other tested common hosts. Expression of a Streptomyces albus subsp. chlorinus NRRL B-24108 genomic library in the modified S. lividans ΔYA9 and S. albus Del14 strains resulted in the production of 7 potentially new compounds, only one of which was produced in both strains. CONCLUSION: The constructed S. lividans-based strains are a great complement to the panel of heterologous hosts for actinobacterial secondary metabolite gene expression. The expansion of the number of such engineered strains will contribute to an increased success rate in isolation of new natural products originating from the expression of genomic and metagenomic libraries, thus raising the chance to obtain novel biologically active compounds.
Assuntos
Antibacterianos/biossíntese , Produtos Biológicos , Metabolismo Secundário/genética , Streptomyces lividans/genética , Actinobacteria/genética , Actinobacteria/metabolismo , Antibacterianos/química , Bacteriocinas/biossíntese , Bacteriocinas/química , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Clonagem Molecular , Engenharia Genética/métodos , Família Multigênica , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/química , Streptomyces lividans/metabolismo , Tunicamicina/biossíntese , Tunicamicina/químicaRESUMO
Tunicamycin E (1), featuring a methyl substitution at C-10', was isolated from marine-derived Streptomyces xinghaiensis SCSIO S15077 originated from the South China Sea sediment together with six known compounds, tunicamycin B (2), tunicamycin X (3), tunicamycin A (4), streptovirudin D2 (5), tunicamycin C (6), and tunicamycin C3 (7). The structure of compound 1 was elucidated by detailed spectroscopic data analyses. All the compounds exhibited strong to moderate antibacterial activity against Gram-positive bacteria Bacillus thuringiensis BT01 and B. thuringiensis W102 with MIC values ranging from 0.008 to 2 µg/mL. Moreover, compounds 1-7 exhibited moderate antifungal activity against Candida albicans ATCC 96901 and C. albicans CMCC (F) 98001 with MIC values ranging from 2 to 32 µg/mL. This is the first report that tunicamycins exhibit antimicrobial activities against B. thuringiensis, C. albicans CMCC (F) 98001 and a fluconazole resistant strain C. albicans ATCC 96901.
Assuntos
Anti-Infecciosos/isolamento & purificação , Streptomyces/química , Tunicamicina/isolamento & purificação , Anti-Infecciosos/química , Bacillus thuringiensis/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , China , Testes de Sensibilidade Microbiana , Estrutura Molecular , Tunicamicina/química , Tunicamicina/farmacologiaRESUMO
Liposidomycin is a uridyl liponucleoside antibiotic isolated from Streptomyces griseosporeus RK-1061. It was discovered by Isono in 1985, who had previously isolated and developed a related peptidyl nucleoside antibiotic, polyoxin, a specific inhibitor of chitin synthases, as a pesticide. He subsequently isolated liposidomycin, a specific inhibitor of bacterial peptidoglycan biosynthesis from actinomycetes, using a similar approach to the discovery of polyoxin. Liposidomycin has no cytotoxicity against BALB/3T3 cells but has antimicrobial activity against Mycobacterium spp. through inhibition of MraY (MurX) [phospho-N-acetylmuramoyl-pentapeptide transferase (translocase I, EC 2.7.8.13)]. Since the discovery of liposidomycin, several liposidomycin-type antibiotics, including caprazamycin, A-90289, and muraminomycin, have been reported, and their total synthesis and/or biosynthetic cluster genes have been studied. Most advanced, a semisynthetic compound derived from caprazamycin, CPZEN-45, is being developed as an antituberculosis agent. Translocase I is an interesting and tractable molecular target for new antituberculosis and antibiotic drug discovery against multidrug-resistant bacteria. This review is dedicated to Dr Isono on the occasion of his 88th birthday to recognize his role in the study of nucleoside antibiotics.
Assuntos
Aminoglicosídeos/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Peptidoglicano/metabolismo , Aminoglicosídeos/química , Animais , Antibacterianos/síntese química , Azepinas/química , Azepinas/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Quitina Sintase/antagonistas & inibidores , Inibidores Enzimáticos/química , Camundongos , Nucleosídeos de Pirimidina/química , Nucleosídeos de Pirimidina/farmacologia , Transferases/antagonistas & inibidores , Transferases (Outros Grupos de Fosfato Substituídos) , Tunicamicina/química , Tunicamicina/farmacologia , Uracila/análogos & derivados , Uracila/química , Uracila/farmacologia , Uridina/análogos & derivados , Uridina/química , Uridina/farmacologiaRESUMO
The ß-lactams are the most widely used group of antibiotics in human health and agriculture, but this is under threat due to the persistent rise of pathogenic resistance. Several compounds, including tunicamycin (TUN), can enhance the antibacterial activity of the ß-lactams to the extent of overcoming resistance, but the mammalian toxicity of TUN has precluded its use in this role. Selective hydrogenation of TUN produces modified compounds (TunR1 and TunR2), which retain the enhancement of ß-lactams while having much lower mammalian toxicity. Here we show that TunR1 and TunR2 enhance the antibacterial activity of multiple ß-lactam family members, including penems, cephems, and third-generation penicillins, to a similar extent as does the native TUN. Eleven of the ß-lactams tested were enhanced from 2 to >256-fold against Bacillus subtilis, with comparable results against a penicillin G-resistant strain. The most significant enhancements were obtained with third-generation aminothiazolidyl cephems, including cefotaxime, ceftazidime, and cefquinome. These results support the potential of low toxicity tunicamycin analogs (TunR1 and TunR2) as clinically valid, synergistic enhancers for a broad group of ß-lactam antibiotics.
Assuntos
Cefalosporinas/farmacologia , Tunicamicina/análogos & derivados , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Bioensaio , Linhagem Celular , Cefalosporinas/administração & dosagem , Cricetinae , Sinergismo Farmacológico , Humanos , Larva/efeitos dos fármacos , Estrutura Molecular , Spodoptera/efeitos dos fármacos , Tunicamicina/administração & dosagem , Tunicamicina/química , Tunicamicina/farmacologiaRESUMO
A liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantification of tunicamycin in rat plasma as per regulatory guideline. Chromatography of tunicamycin and the IS in the processed plasma samples was achieved on an X-Terra phenyl column using a binary gradient (mobile phase A, acetonitrile and mobile phase B, 5 mm ammonium formate) elution at a flow rate of 0.6 ml/min. LC-MS/MS was operated under the multiple reaction monitoring mode using the electrospray ionization technique in positive ion mode and the transitions of m/z 817.18 â 596.10, 831.43 â 610.10, 845.29 â 624.10, 859.23 â 638.10 and 309.24 â 163.20 were used to quantitate homologs A-D and the IS, respectively. The total chromatographic run time was 4.5 min. The correlation coefficient (r2 ) was >0.99 for all homologs with accuracy 90.7-107.4% and precision 0.74-15.1%. The recovery of homologs was 78.6-90.2%. No carryover was observed and the matrix effect was minimal. Tunicamycin four homologs were found to be stable on the bench-top for 6 h, for up to three freeze-thaw cycles, in the injector for 24 h and for 1 month at -80°C. The applicability of the validated method has been demonstrated in a rat pharmacokinetic study.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Tunicamicina/sangue , Tunicamicina/farmacocinética , Animais , Estabilidade de Medicamentos , Limite de Detecção , Modelos Lineares , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Tunicamicina/químicaRESUMO
Endocannabinoid (eCB)-binding receptors can be modulated by several ligands and membrane environment, yet the effect of glycosylation remains to be assessed. In this study, we used human neuroblastoma SH-SY5Y cells to interrogate whether expression, cellular localization, and activity of eCB-binding receptors may depend on N-linked glycosylation. Following treatment with tunicamycin (a specific inhibitor of N-linked glycosylation) at the non-cytotoxic dose of 1 µg/mL, mRNA, protein levels and localization of eCB-binding receptors, as well as N-acetylglucosamine (GlcNAc) residues, were evaluated in SH-SY5Y cells by means of quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), fluorescence-activated cell sorting (FACS), and confocal microscopy, respectively. In addition, the activity of type-1 and type-2 cannabinoid receptors (CB1 and CB2) was assessed by means of rapid binding assays. Significant changes in gene and protein expression were found upon tunicamycin treatment for CB1 and CB2, as well as for GPR55 receptors, but not for transient receptor potential vanilloid 1 (TRPV1). Deglycosylation experiments with N-glycosidase-F and immunoblot of cell membranes derived from SH-SY5Y cells confirmed the presence of one glycosylated form in CB1 (70 kDa), that was reduced by tunicamycin. Morphological studies demonstrated the co-localization of CB1 with GlcNAc residues, and showed that tunicamycin reduced CB1 membrane expression with a marked nuclear localization, as confirmed by immunoblotting. Cleavage of the carbohydrate side chain did not modify CB receptor binding affinity. Overall, these results support N-linked glycosylation as an unprecedented post-translational modification that may modulate eCB-binding receptors' expression and localization, in particular for CB1.
Assuntos
Endocanabinoides/genética , Neuroblastoma/tratamento farmacológico , Receptores de Canabinoides/química , Tunicamicina/farmacologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Endocanabinoides/química , Endocanabinoides/farmacologia , Citometria de Fluxo , Glicosilação/efeitos dos fármacos , Humanos , Ligantes , Microscopia Confocal , Neuroblastoma/genética , Neuroblastoma/patologia , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Receptores de Canabinoides/genética , Canais de Cátion TRPV/genética , Tunicamicina/químicaRESUMO
Elucidating a structure-activity relationship study by evaluating a series of truncated analogues is a simple but important and effective tactic in medicinal chemistry based on natural products with a large and complex chemical structure. In this study, a series of truncated analogues of tunicamycin V were designed and synthesized and their MraY inhibitory activity was investigated in order to gain insight into the effect of these moieties on MraY inhibition.
Assuntos
Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Transferases/antagonistas & inibidores , Tunicamicina/química , Antibacterianos/síntese química , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Desenho de Fármacos , Concentração Inibidora 50 , Staphylococcus aureus/enzimologia , Transferases/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos) , Tunicamicina/metabolismoRESUMO
Collapse of the protein homeostasis (proteostasis) can lead to accumulation and aggregation of unfolded proteins, which has been found to associate with a number of disease conditions including neurodegenerative diseases, diabetes and inflammation. Here we report a maleimide-functionalized tetraphenylethene (TPE)-derivatized fluorescent dye, TPE-NMI, which shows fluorescence turn-on property upon reacting with unfolded proteins in vitro and in live cells under proteostatic stress conditions. The level of unfolded proteins can be measured by flow cytometry and visualized with confocal microscopy.
Assuntos
Corantes Fluorescentes/química , Maleimidas/química , Proteínas/química , Estilbenos/química , Animais , Linhagem Celular Tumoral , Corantes Fluorescentes/síntese química , Camundongos , Microscopia Confocal , Agregados Proteicos , Desdobramento de Proteína , Espectrometria de Fluorescência , Tunicamicina/químicaRESUMO
Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic "lipid-altered" tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.
Assuntos
Antibióticos Antituberculose/farmacologia , Defeitos Congênitos da Glicosilação/metabolismo , Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/química , Animais , Antibióticos Antituberculose/química , Sítios de Ligação , Defeitos Congênitos da Glicosilação/genética , Inibidores Enzimáticos/química , Feminino , Células HEK293 , Células Hep G2 , Humanos , Metabolismo dos Lipídeos , Camundongos , Simulação de Acoplamento Molecular , Mutação , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Ligação Proteica , Células Sf9 , Spodoptera , Tunicamicina/química , Tunicamicina/farmacologia , Uridina Difosfato Ácido Glucurônico/química , Uridina Difosfato Ácido Glucurônico/metabolismoRESUMO
The rapid growth of antibiotic-resistant bacterial infections is of major concern for human health. Therefore, it is of great importance to characterize novel targets for the development of antibacterial drugs. One promising protein target is MraY (UDP-N-acetylmuramyl-pentapeptide: undecaprenyl phosphate N-acetylmuramyl-pentapeptide-1-phosphate transferase or MurNAc-1-P-transferase), which is essential for bacterial cell wall synthesis. Here, we summarize recent breakthroughs in structural studies of bacterial MraYs and the closely related human GPT (UDP-N-acetylglucosamine: dolichyl phosphate N-acetylglucosamine-1-phosphate transferase or GlcNAc-1-P-transferase). We present a detailed comparison of interaction modes with the natural product inhibitors tunicamycin and muraymycin D2. Finally, we speculate on possible routes to design an antibacterial agent in the form of a potent and selective inhibitor against MraY.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Proteínas de Bactérias/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Peptidoglicano/biossíntese , Transferases/antagonistas & inibidores , Animais , Antibacterianos/síntese química , Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Inibidores Enzimáticos/síntese química , Humanos , Modelos Moleculares , Nucleosídeos/química , Nucleosídeos/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Conformação Proteica , Relação Estrutura-Atividade , Transferases/química , Transferases/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Tunicamicina/química , Tunicamicina/farmacologiaRESUMO
N-linked glycosylation is a predominant post-translational modification of protein in eukaryotes, and its dysregulation is the etiology of several human disorders. The enzyme UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosaminephosphotransferase (GlcNAc-1-P-transferase or GPT) catalyzes the first and committed step of N-linked glycosylation in the endoplasmic reticulum membrane, and it is the target of the natural product tunicamycin. Tunicamycin has potent antibacterial activity, inhibiting the bacterial cell wall synthesis enzyme MraY, but its usefulness as an antibiotic is limited by off-target inhibition of human GPT. Our understanding of how tunicamycin inhibits N-linked glycosylation and efforts to selectively target MraY are hampered by a lack of structural information. Here we present crystal structures of human GPT in complex with tunicamycin. Structural and functional analyses reveal the difference between GPT and MraY in their mechanisms of inhibition by tunicamycin. We demonstrate that this difference could be exploited to design MraY-specific inhibitors as potential antibiotics.
Assuntos
Antibacterianos/química , Inibidores Enzimáticos/química , Transferases (Outros Grupos de Fosfato Substituídos)/química , Tunicamicina/química , Antibacterianos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Inibidores Enzimáticos/metabolismo , Glicosilação , Humanos , Magnésio/química , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Especificidade por Substrato , Transferases/química , Transferases/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Tunicamicina/metabolismoRESUMO
Akt, also known as protein kinase B (PKB), is a serine/threonine kinase that promotes survival and growth in response to extracellular signals. Akt1 has been demonstrated to play vital roles in cardiovascular diseases, but the role of Akt2 in cardiomyocytes is not fully understood. This study investigated the effect of Akt2 knockdown on tunicamycin (TM)-induced cytotoxicity in cardiomyocytes and the underlying mechanisms with a focus on the JNK-Wnt pathway. TM treatment significantly increased the expression of Akt2 at both mRNA and protein levels, which was shown to be mediated by the induction of reactive oxygen species (ROS). Knockdown of Akt2 expression via siRNA transfection markedly increased cell viability, decreased lactate dehydrogenase (LDH) release and reduced cell apoptosis after TM exposure. The results of western blot showed that downregulation of Akt2 also attenuated the TM-induced activation of the unfolded protein response (UPR) factors and ER stress associated pro-apoptotic proteins. In addition, Si-Akt2 transfection partially prevented the TM-induced decrease in nuclear localization of ß-catenin. By using the selective inhibitor SP-600,125 to inhibit JNK phosphorylation, we found that knockdown of Akt2-induced protection and inhibition of ER stress was mediated by reversing TM-induced decrease of Wnt through the JNK pathway. In summary, these data suggested that Akt2 play a pivotal role in regulating cardiomyocyte survival during ER stress by modulating the JNK-Wnt pathway.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Tunicamicina/farmacologia , Apoptose/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Modelos Moleculares , Estrutura Molecular , Miócitos Cardíacos/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Relação Estrutura-Atividade , Tunicamicina/química , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Tunicamycin is a Streptomyces-derived inhibitor of eukaryotic protein N-glycosylation and bacterial cell wall biosynthesis, and is a potent and general toxin by these biological mechanisms. The antibacterial activity is dependent in part upon a π-π stacking interaction between the tunicamycin uridyl group and a specific Phe residue within MraY, a tunicamycin-binding protein in bacteria. We have previously shown that reducing the tunicamycin uridyl group to 5,6-dihydrouridyl (DHU) significantly lowers its eukaryotic toxicity, potentially by disrupting the π-stacking with the active site Phe. The present report compares the catalytic hydrogenation of tunicamycin and uridine with various precious metal catalysts, and describe optimum conditions for the selective production of N-acyl reduced tunicamycin or for tunicamycins reduced in both the N-acyl and uridyl double bonds. At room temperature, Pd-based catalysts are selective for the N-acyl reduction, whereas Rh-based catalysts favor the double reduction to provide access to fully reduced tunicamycin. The reduced DHU is highly base-sensitive, leading to amide ring opening under mild alkaline conditions.
Assuntos
Antibacterianos/química , Glicosilação/efeitos dos fármacos , Hidrogenação/efeitos dos fármacos , Tunicamicina/química , Antibacterianos/farmacologia , Catálise , Parede Celular/metabolismo , Oxirredução , Streptomyces/metabolismo , Tunicamicina/farmacologiaRESUMO
Tunicamycins (TUN) are inhibitors of the UDP-HexNAc: polyprenol-P HexNAc-1-P transferase family of enzymes, which initiate the biosynthesis of bacterial peptidoglycan and catalyze the first step in eukaryotic protein N-glycosylation. The TUN are therefore general and potent toxins to both eukaryotes and prokaryotes. Screening a library of synthetic TUN against Bacillus and yeast identified TUN that are antibacterial, but have significantly reduced eukaryotic toxicity. One of these (Tun-15:0) differs from the native TUN control only by the lack of the conjugated double bond in the tunicaminyl N-acyl group. Tun-15:0 also showed reduced inhibition for protein N-glycosylation in a Pichia-based bioassay. Natural TUN was subsequently modified by chemically reducing the N-acyl double bond (TunR1) or both the N-acyl and uridyl double bonds (TunR2). TunR1 and TunR2 retain their antibacterial activity, but with considerably reduced eukaryotic toxicity. In protein N-glycosylation bioassays, TunR1 is a less potent inhibitor than native TUN and TunR2 is entirely inactive. Importantly, the less toxic TunR1 and TunR2 both enhance the antibacterial activity of ß-lactams: oxacillin by 32- to 64-fold, comparable with native TUN, and with similar enhancements for methicillin and penicillin G. Hence, the modified TUNs, TunR1 and TunR2, are potentially important as less-toxic synergistic enhancers of the ß-lactams.
Assuntos
Antibacterianos/farmacologia , Tunicamicina/farmacologia , beta-Lactamas/farmacologia , Antibacterianos/química , Antibacterianos/toxicidade , Sinergismo Farmacológico , Eucariotos/efeitos dos fármacos , Glicosilação/efeitos dos fármacos , Meticilina/administração & dosagem , Meticilina/farmacologia , Oxacilina/administração & dosagem , Oxacilina/farmacologia , Penicilina G/administração & dosagem , Penicilina G/farmacologia , Tunicamicina/química , Tunicamicina/toxicidade , beta-Lactamas/administração & dosagemRESUMO
Nonalcoholic fatty liver disease (NAFLD) covers a spectrum from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. Free fatty acids (FFA) induce steatosis and lipo-toxicity and correlate with severity of NAFLD. In this study we aimed to investigate the role of exogenous and endogenous ALR (augmenter of liver regeneration) for FFA induced ER (endoplasmatic reticulum) -stress and lipoapoptosis. Primary human hepatocytes or hepatoma cells either treated with recombinant human ALR (rhALR, 15kDa) or expressing short form ALR (sfALR, 15kDa) were incubated with palmitic acid (PA) and analyzed for lipo-toxicity, -apoptosis, activation of ER-stress response pathways, triacylglycerides (TAG), mRNA and protein expression of lipid metabolizing genes. Both, exogenous rhALR and cytosolic sfALR reduced PA induced caspase 3 activity and Bax protein expression and therefore lipotoxicity. Endogenous sfALR but not rhALR treatment lowered TAG levels, diminished activation of ER-stress mediators C-Jun N-terminal kinase (JNK), X-box binding protein-1 (XBP1) and proapoptotic transcription factor C/EBP-homologous protein (CHOP), and reduced death receptor 5 protein expression. Cellular ALR exerts its lipid lowering and anti-apoptotic actions by enhancing FABP1, which binds toxic FFA, increasing mitochondrial ß-oxidation by elevating the mitochondrial FFA transporter CPT1α, and decreasing ELOVL6, which delivers toxic FFA metabolites. We found reduced hepatic mRNA levels of ALR in a high fat diet mouse model, and of ALR and FOXA2, a transcription factor inducing ALR expression, in human steatotic as well as NASH liver samples, which may explain increased lipid deposition and reduced ß-oxidation in NASH patients. Present study shows that exogenous and endogenous ALR reduce PA induced lipoapoptosis. Furthermore, cytosolic sfALR changes mRNA and protein expression of genes regulating lipid metabolism, reduces ER-stress finally impeding progression of NASH.