RESUMO
The methylotrophic yeast Pichia pastoris is known as an efficient host for the production of heterologous proteins. While N-linked protein glycosylation is well characterized in P. pastoris there is less knowledge of the patterns of O-glycosylation. O-glycans produced by P. pastoris consist of short linear mannose chains, which in the case of recombinant biopharmaceuticals can trigger an immune response in humans. This study aims to reveal the influence of different cultivation strategies on O-mannosylation profiles in P. pastoris. Sixteen different model proteins, produced by different P. pastoris strains, are analyzed for their O-glycosylation profile. Based on the obtained data, human serum albumin (HSA) is chosen to be produced in fast and slow growth fed batch fermentations by using common promoters, PGAP and PAOX1 . After purification and protein digestion, glycopeptides are analyzed by LC/ESI-MS. In the samples expressed with PGAP it is found that the degree of glycosylation is slightly higher when a slow growth rate is used, regardless of the efficiency of the producing strain. The highest glycosylation intensity is observed in HSA produced with PAOX1 . The results indicate that the O-glycosylation level is markedly higher when the protein is produced in a methanol-based expression system.
Assuntos
Pichia , Fermentação , Glicosilação , Humanos , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SaccharomycetalesRESUMO
Pichia pastoris has emerged in the past years as a promising host for recombinant protein and biopharmaceutical production. In the establishment of high cell density fed-batch biomanufacturing, screening phase and early bioprocess development (based on microplates and shake flasks) still represent a bottleneck due to high-cost and time-consuming procedures as well as low experiment complexity. In the present work, a screening protocol developed for P. pastoris clone selection is implemented in a multiplexed microfluidic device with 15 µL cultivation chambers able to operate in perfusion mode and monitor dissolved oxygen content in the culture in a non-invasive way. The setup allowed us to establish carbon-limited conditions and evaluate strain responses to different input variables. Results from micro-scale perfusion cultures are then compared with 1L fed-batch fermentation. The best producer in terms of titer and productivity is rapidly identified after 12 h from inoculation and the results confirmed by lab-scale fermentation. Moreover, the physiological analyses of the strains under different conditions suggested how more complex experimental conditions are achievable despite the relatively easy, straight-forward, and cost-effective experimental setup. Implementation and standardization of these micro-scale protocols could reduce the demand for lab-scale bioreactor cultivations thus accelerating the development of protein production processes.
Assuntos
Reatores Biológicos , Pichia , Células Clonais/metabolismo , Fermentação , Perfusão , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SaccharomycetalesRESUMO
Dopamine reuptake inhibitors have been shown to improve cognitive parameters in various tasks and animal models. We recently reported a series of modafinil analogues, of which the most promising, 5-((benzhydrylsulfinyl)methyl) thiazole (CE-123), was selected for further development. The present study aims to characterize pharmacological properties of CE-123 and to investigate the potential to enhance memory performance in a rat model. In vitro transporter assays were performed in cells expressing human transporters. CE-123 blocked uptake of [3H] dopamine (IC50â¯=â¯4.606⯵M) while effects on serotonin (SERT) and the norepinephrine transporter (NET) were negligible. Blood-brain barrier and pharmacokinetic studies showed that the compound reached the brain and lower elimination than R-modafinil. The Pro-cognitive effect was evaluated in a spatial hole-board task in male Sprague-Dawley rats and CE-123 enhances memory acquisition and memory retrieval, represented by significantly increased reference memory indices and shortened latency. Since DAT blockers can be considered as indirect dopamine receptor agonists, western blotting was used to quantify protein levels of dopamine receptors D1R, D2R and D5R and DAT in the synaptosomal fraction of hippocampal subregions CA1, CA3 and dentate gyrus (DG). CE-123 administration in rats increased total DAT levels and D1R protein levels were significantly increased in CA1 and CA3 in treated/trained groups. The increase of D5R was observed in DG only. Dopamine receptors, particularly D1R, seem to play a role in mediating CE-123-induced memory enhancement. Dopamine reuptake inhibition by CE-123 may represent a novel and improved stimulant therapeutic for impairments of cognitive functions.
Assuntos
Compostos Benzidrílicos/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Aprendizagem/efeitos dos fármacos , Rememoração Mental/efeitos dos fármacos , Nootrópicos/farmacologia , Memória Espacial/efeitos dos fármacos , Animais , Compostos Benzidrílicos/química , Compostos Benzidrílicos/farmacocinética , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular , Inibidores da Captação de Dopamina/química , Inibidores da Captação de Dopamina/farmacocinética , Avaliação Pré-Clínica de Medicamentos , Humanos , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Modafinila , Simulação de Acoplamento Molecular , Estrutura Molecular , Atividade Motora/efeitos dos fármacos , Nootrópicos/química , Nootrópicos/farmacocinética , Ratos Sprague-Dawley , Receptores Dopaminérgicos/metabolismoRESUMO
A series of compounds have been shown to enhance cognitive function via the dopaminergic system and indeed the search for more active and less toxic compounds is continuing. It was therefore the aim of the study to synthetise and test a novel heterocyclic compound for cognitive enhancement in a paradigm for working memory. Specific and effective dopamine re-uptake inhibition DAT (IC50=4,1±0,8µM) made us test this compound in a radial arm maze (RAM) in the rat. CE-125 (4-((benzhydrylsulfinyl)methyl)-2-cyclopropylthiazole), was tested for dopamine (DAT), serotonin and norepinephrine re-uptake inhibition by a well-established system. The working memory index (WMI) was evaluated in male Sprague Dawley rats that were intraperitoneally injected with CE-125 (1 or 10mg/kg body weight). In order to evaluate basic neurotoxicity, the open field, elevated plus maze, rota rod studies and the forced swim test were carried out. Frontal cortex was taken at the last day of the RAM test and dopamine receptors D1R and D2R, DAT and phosphorylated DAT protein levels were determined. On the 10th day both doses were increasing the WMI as compared to the vehicle-treated group. In both, trained and treated groups, D1R levels were significantly reduced while D2R levels were unchanged. DAT levels were comparable between all groups while phosphorylated DAT levels were increased in the trained group treated with 1mg/kg body weight. CE-125 as a probably non-neurotoxic compound and specific reuptake inhibitor was shown to increase performance (WMI) and modulation of the dopaminergic system is proposed as a possible mechanism of action.
Assuntos
Inibidores da Captação de Dopamina/farmacologia , Lobo Frontal/efeitos dos fármacos , Memória de Curto Prazo/efeitos dos fármacos , Nootrópicos/farmacologia , Receptores de Dopamina D1/metabolismo , Sulfóxidos/farmacologia , Tiazóis/farmacologia , Animais , Cromatografia Líquida de Alta Pressão , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores da Captação de Dopamina/síntese química , Inibidores da Captação de Dopamina/química , Lobo Frontal/metabolismo , Células HEK293 , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Memória de Curto Prazo/fisiologia , Estrutura Molecular , Atividade Motora/efeitos dos fármacos , Nootrópicos/síntese química , Nootrópicos/química , Fosforilação , Ratos Sprague-Dawley , Receptores de Dopamina D2/metabolismo , Sulfóxidos/síntese química , Sulfóxidos/química , Tiazóis/síntese química , Tiazóis/químicaRESUMO
Modafinil is a wake promoting compound with high potential for cognitive enhancement. It is targeting the dopamine transporter (DAT) with moderate selectivity, thereby leading to reuptake inhibition and increased dopamine levels in the synaptic cleft. A series of modafinil analogues have been reported so far, but more target-specific analogues remain to be discovered. It was the aim of this study to synthesize and characterize such analogues and, indeed, a series of compounds were showing higher activities on the DAT and a higher selectivity toward DAT versus serotonin and norepinephrine transporters than modafinil. This was achieved by substituting the amide moiety by five- and six-membered aromatic heterocycles. In vitro studies indicated binding to the cocaine pocket on DAT, although molecular dynamics revealed binding different from that of cocaine. Moreover, no release of dopamine was observed, ruling out amphetamine-like effects. The absence of neurotoxicity of a representative analogue may encourage further preclinical studies of the above-mentioned compounds.