RESUMO
The automation of organic compound synthesis is pivotal for expediting the development of such compounds. In addition, enhancing development efficiency can be achieved by incorporating autonomous functions alongside automation. To achieve this, we developed an autonomous synthesis robot that harnesses the power of artificial intelligence (AI) and robotic technology to establish optimal synthetic recipes. Given a target molecule, our AI initially plans synthetic pathways and defines reaction conditions. It then iteratively refines these plans using feedback from the experimental robot, gradually optimizing the recipe. The system performance was validated by successfully determining synthetic recipes for three organic compounds, yielding that conversion rates that outperform existing references. Notably, this autonomous system is designed around batch reactors, making it accessible and valuable to chemists in standard laboratory settings, thereby streamlining research endeavors.
RESUMO
Genetic variants drive the evolution of traits and diseases. We previously modeled these variants as small displacements in fitness landscapes and estimated their functional impact by differentiating the evolutionary relationship between genotype and phenotype. Conversely, here we integrate these derivatives to identify genes steering specific traits. Over cancer cohorts, integration identified 460 likely tumor-driving genes. Many have literature and experimental support but had eluded prior genomic searches for positive selection in tumors. Beyond providing cancer insights, these results introduce a general calculus of evolution to quantify the genotype-phenotype relationship and discover genes associated with complex traits and diseases.
Assuntos
Cálculos , Neoplasias , Evolução Biológica , Aptidão Genética , Genótipo , Humanos , Modelos Genéticos , Neoplasias/genética , Fenótipo , Seleção GenéticaRESUMO
The phosphoinositide-3-kinase like kinases (PIKK) such as ATM and ATR play a key role in initiating the cellular DNA damage response (DDR). One key ATM target is the cyclin-dependent kinase inhibitor p27Kip1 that promotes G1 arrest. ATM activates p27Kip1-induced arrest in part through phosphorylation of p27Kip1 at Serine 140. Here we show that this site is dephosphorylated by the type 2C serine/threonine phosphatase, WIP1 (Wildtype p53-Induced Phosphatase-1), encoded by the PPM1D gene. WIP1 has been shown to dephosphorylate numerous ATM target sites in DDR proteins, and its overexpression and/or mutation has often been associated with oncogenesis. We demonstrate that wildtype, but not phosphatase-dead WIP1, efficiently dephosphorylates p27Kip1 Ser140 both in vitro and in cells and that this dephosphorylation is sensitive to the WIP1-specific inhibitor GSK 2830371. Increased expression of wildtype WIP1 reduces stability of p27Kip1 while increased expression of similar amounts of phosphatase-dead WIP1 has no effect on p27Kip1 protein stability. Overexpression of wildtype p27Kip1 reduces cell proliferation and colony forming capability relative to the S140A (constitutively non-phosphorylated) form of p27. Thus, WIP1 plays a significant role in homeostatic modulation of p27Kip1 activity following activation by ATM.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteína Fosfatase 2C/metabolismo , Serina/metabolismo , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/genética , Células HEK293 , Humanos , Células MCF-7 , Mutação/genética , Fosfopeptídeos/metabolismo , Fosforilação , Estabilidade Proteica , Reprodutibilidade dos Testes , Ensaio Tumoral de Célula-TroncoRESUMO
Scientific progress depends on formulating testable hypotheses informed by the literature. In many domains, however, this model is strained because the number of research papers exceeds human readability. Here, we developed computational assistance to analyze the biomedical literature by reading PubMed abstracts to suggest new hypotheses. The approach was tested experimentally on the tumor suppressor p53 by ranking its most likely kinases, based on all available abstracts. Many of the best-ranked kinases were found to bind and phosphorylate p53 (P value = 0.005), suggesting six likely p53 kinases so far. One of these, NEK2, was studied in detail. A known mitosis promoter, NEK2 was shown to phosphorylate p53 at Ser315 in vitro and in vivo and to functionally inhibit p53. These bona fide validations of text-based predictions of p53 phosphorylation, and the discovery of an inhibitory p53 kinase of pharmaceutical interest, suggest that automated reasoning using a large body of literature can generate valuable molecular hypotheses and has the potential to accelerate scientific discovery.
Assuntos
Indexação e Redação de Resumos , Quinases Relacionadas a NIMA/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Células HCT116 , Células HEK293 , Humanos , Quinases Relacionadas a NIMA/genética , Processamento de Linguagem Natural , Fosforilação , PubMed , Proteína Supressora de Tumor p53/genéticaRESUMO
Here we report that the lncRNA LINC00052 expression correlates positively with HER3/ErbB3 levels in breast cancer cells. Gene silencing of LINC00052 diminished both LINC00052 and HER3 expression and reduced cancer cell growth in vitro and in vivo. LINC00052 overexpression promoted cancer cell growth in vitro and in vivo and increased HER3-mediated downstream signaling. Importantly, neutralization of HER3 signaling with HER3 targeting monoclonal antibodies blocked LINC00052 mediated cancer cell proliferation in vitro and tumor growth in vivo, suggesting LINC00052 promoting cancer growth through HER3 signaling. Taken together, our results indicate that high LINC00052 levels predict activation of HER3-mediated signaling, and LINC00052 expression level may serve as a potential biomarker for HER3 targeted antibody cancer therapies.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Proliferação de Células , RNA Longo não Codificante/metabolismo , Receptor ErbB-3/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Antineoplásicos Imunológicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Células HEK293 , Humanos , Células MCF-7 , Camundongos Nus , Fosforilação , RNA Longo não Codificante/genética , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Studies have demonstrated that cancer-associated matrix metalloproteinases (MMP) can generate single peptide bond cleavages in the hinge region of immunoglobulin G1 (IgG1). This study investigated the cleavage of endogenous IgGs by MMPs in the tumor microenvironment and the consequences of the IgG hinge cleavage for humoral immunity. EXPERIMENTAL DESIGN: We investigated the occurrence of single peptide bond cleaved IgGs (scIgG) in tumor tissues and plasma samples collected from a cohort of breast cancer patients (n = 60). Samples from healthy people (n = 20) were used as the control. Antibody hinge cleavage was detected by multiple assays, including IHC, ELISA, and flow cytometry. A correlation analysis was conducted between scIgG levels and patient clinical parameters. RESULTS: Levels of scIgGs in tumors were significantly higher than in normal tissues. In addition, scIgG levels in tumors were enriched compared with that in the plasma of the same patients. The appearance of scIgGs in tumor tissues was associated with altered host IgG content and decreased IgG1. Increased tumor scIgGs were found to be positively correlated with adverse clinical factors, such as elevated tumor-associated macrophages, increased expression of MMP9 and other MMPs, and local metastasis to axillary lymph nodes. CONCLUSIONS: The study contributes to mounting evidence for the presence of hinge-cleaved antibodies with reduced Fc immune effector function in the tumor microenvironment. The results highlight a link between tumor scIgGs and poor patient outcomes, and reveal a component of compromised humoral immunity within tumors that could point to new immunotherapeutic strategies to rescue host immunity.
Assuntos
Anticorpos/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Microambiente Tumoral/imunologia , Animais , Anticorpos/metabolismo , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Xenoenxertos , Humanos , Imunidade Humoral , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Metástase Linfática , Macrófagos/imunologia , Macrófagos/patologia , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinases da Matriz/metabolismo , Camundongos , Neoplasias/metabolismo , Proteólise , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
ERBB3/HER3 is emerging as a molecular target for various cancers. HER3 is overexpressed and activated in a number of cancer types under the conditions of acquired resistance to other HER family therapeutic interventions such as tyrosine kinase inhibitors and antibody therapies. Regulation of the HER3 expression and signaling involves numerous HER3 interacting proteins. These proteins include PI3K, Shc, and E3 ubiquitin ligases NEDD4 and Nrdp1. Furthermore, recent identification of a number of HER3 oncogenic mutations in colon and gastric cancers elucidate the role of HER3 in cancer development. Despite the strong evidence regarding the role of HER3 in cancer, the current understanding of the regulation of HER3 expression and activation requires additional research. Moreover, the lack of biomarkers for HER3-driven cancer poses a big challenge for the clinical development of HER3 targeting antibodies. Therefore, a better understanding of HER3 regulation should improve the strategies to therapeutically target HER3 for cancer therapy.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Receptor ErbB-3/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Animais , Carcinogênese/genética , Neoplasias do Colo/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Medicina Baseada em Evidências , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Terapia de Alvo Molecular , Mutação/genética , Receptor ErbB-3/genética , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/genéticaRESUMO
Human serum albumin (HSA) is a cysteine rich molecule that is most abundant in human blood plasma. To remain viable in the market due to lower marketing costs for HSA, it is important to produce a large quantity in an economical manner by recombinant technology. The objective of this study was to maximize recombinant HSA (rHSA) production using a Mut(s) Pichia pastoris strain by fermentation process optimization. We evaluated the impact of process parameters on the production of rHSA, including induction cell density (wet cell weight, g/L) and the control of specific growth rate at induction. In this study, we demonstrated that induction cell density is a critical factor for high level production of rHSA under controlled specific growth rate. We observed higher specific productivities at higher induction cell densities (285 g/L) and at lower specific growth rates (0.0022-0.0024/h) during methanol induction phase, and achieved the broth titer of rHSA up to 10 g/L. The temperature shift from 24 to 28(o) C was effective to control the specific growth rate at low level (≤0.0024/h) during methanol induction phase while maintaining high specific productivity [0.0908 mgrHSA /(gwcw h)].
Assuntos
Biotecnologia/métodos , Pichia/genética , Proteínas Recombinantes/metabolismo , Albumina Sérica/metabolismo , Técnicas de Inativação de Genes , Humanos , Fenótipo , Pichia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Albumina Sérica/química , Albumina Sérica/genéticaRESUMO
The methylotrophic yeast, Pichiapastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, either N-linked or O-linked, can elicit an immune response or enable the expressed protein to bind to mannose receptors, thus reducing their efficacy. Previously we have reported the elimination of ß-linked glycans in this organism. In the current report we have focused on reducing the O-linked mannose content of proteins produced in P. pastoris, thereby reducing the potential to bind to mannose receptors. The initial step in the synthesis of O-linked glycans in P. pastoris is the transfer of mannose from dolichol-phosphomannose to a target protein in the yeast secretory pathway by members of the protein-O-mannosyltransferase (PMT) family. In this report we identify and characterize the members of the P. pastoris PMT family. Like Candida albicans, P. pastoris has five PMT genes. Based on sequence homology, these PMTs can be grouped into three sub-families, with both PMT1 and PMT2 sub-families possessing two members each (PMT1 and PMT5, and PMT2 and PMT6, respectively). The remaining sub-family, PMT4, has only one member (PMT4). Through gene knockouts we show that PMT1 and PMT2 each play a significant role in O-glycosylation. Both, by gene knockouts and the use of Pmt inhibitors we were able to significantly reduce not only the degree of O-mannosylation, but also the chain-length of these glycans. Taken together, this reduction of O-glycosylation represents an important step forward in developing the P. pastoris platform as a suitable system for the production of therapeutic glycoproteins.
Assuntos
Manosiltransferases/genética , Pichia/enzimologia , Pichia/genética , Candida albicans/enzimologia , Candida albicans/genética , Técnicas de Inativação de Genes , Genes Fúngicos , Glicosilação , Manosiltransferases/metabolismo , Filogenia , Pichia/metabolismo , Polissacarídeos/metabolismoRESUMO
Production of recombinant proteins is affected by process conditions, where transcriptional regulation of Pichia pastoris alcohol oxidase 1 (PpAOX1) promoter has been a key factor to influence expression levels of proteins of interest. Here, we demonstrate that the AOX1 promoter and peroxisome biogenesis are regulated based on different process conditions. Two types of GFP-fusion proteins, Ub-R-GFP (short-lived GFP in the cytosol) and GFP-SKL (peroxisomal targeting GFP), were successfully used to characterize the time-course of the AOX1 promoter and peroxisome biogenesis, respectively. The activity of the AOX1 promoter and peroxisome biogenesis was highly subjected to different fermentation process conditions - methanol-limited condition at normoxy (ML), switched feeding of carbon sources (e.g., glucose and methanol) under carbon-limited condition at normoxy (SML), and oxygen-limited (OL) condition. The AOX1 promoter was most active under the ML, but less active under the OL. Peroxisome biogenesis showed a high dependency on methanol consumption. In addition, the proliferation of peroxisomes was inhibited in a medium containing glucose and stimulated in the methanol phase under a carbon-limited fed-batch culture condition. The specific productivity of a monoclonal antibody (qp) under the AOX1 promoter was higher at 86h of induction in the ML than in the OL (0.026 vs 0.020mgg(-1)h(-1)). However, the oxygen-limited condition was a robust process suitable for longer induction (180h) due to high cell fitness. Our study suggests that the maximal production of a recombinant protein is highly dependent on methanol consumption rate that is affected by the availability of methanol and oxygen molecules.
Assuntos
Aldeído Oxidase/genética , Fermentação , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Peroxissomos/metabolismo , Pichia/genética , Proteínas Recombinantes de Fusão/biossíntese , Técnicas de Cultura Celular por Lotes/métodos , Reatores Biológicos , Células Cultivadas , Glucose/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Metanol/metabolismo , Oxigênio/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genéticaRESUMO
Dimerization among the EGFR family of tyrosine kinase receptors leads to allosteric activation of the kinase domains of the partners. Unlike other members in the family, the kinase domain of HER3 lacks key amino acid residues for catalytic activity. As a result, HER3 is suggested to serve as an allosteric activator of other EGFR family members which include EGFR, HER2 and HER4. To study the role of intracellular domains in HER3 dimerization and activation of downstream signaling pathways, we constructed HER3/HER2 chimeric receptors by replacing the HER3 kinase domain (HER3-2-3) or both the kinase domain and the C-terminal tail (HER3-2-2) with the HER2 counterparts and expressed the chimeric receptors in Chinese hamster ovary (CHO) cells. While over expression of the intact human HER3 transformed CHO cells with oncogenic properties such as AKT/ERK activation and increased proliferation and migration, CHO cells expressing the HER3-2-3 chimeric receptor showed significantly reduced HER3/HER2 dimerization and decreased phosphorylation of both AKT and ERK1/2 in the presence of neuregulin-1 (NRG-1). In contrast, CHO cells expressing the HER3-2-2 chimeric receptor resulted in a total loss of downstream AKT activation in response to NRG-1, but maintained partial activation of ERK1/2. The results demonstrate that the intracellular domains play a crucial role in HER3's function as an allosteric activator and its role in downstream signaling.
Assuntos
Espaço Intracelular/enzimologia , Multimerização Proteica , Receptor ErbB-2/química , Receptor ErbB-3/química , Receptor ErbB-3/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Células CHO , Movimento Celular , Proliferação de Células , Cricetinae , Cricetulus , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Modelos Moleculares , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-3/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
BACKGROUND: Yeast mating provides an efficient means for strain and library construction. However, biotechnological applications of mating in the methylotrophic yeast Pichia pastoris have been hampered because of concerns about strain stability of P. pastoris diploids. The aim of the study reported here is to investigate heterologous protein expression in diploid P. pastoris strains and to evaluate diploid strain stability using high cell density fermentation processes. RESULTS: By using a monoclonal antibody as a target protein, we demonstrate that recombinant protein production in both wild-type and glycoengineered P. pastoris diploids is stable and efficient during a nutrient rich shake flask cultivation. When diploid strains were cultivated under bioreactor conditions, sporulation was observed. Nevertheless, both wild-type and glycoengineered P. pastoris diploids showed robust productivity and secreted recombinant antibody of high quality. Specifically, the yeast culture maintained a diploid state for 240 h post-induction phase while protein titer and N-linked glycosylation profiles were comparable to that of a haploid strain expressing the same antibody. As an application of mating, we also constructed an antibody display library and used mating to generate novel full-length antibody sequences. CONCLUSIONS: To the best of our knowledge, this study reports for the first time a comprehensive characterization of recombinant protein expression and fermentation using diploid P. pastoris strains. Data presented here support the use of mating for various applications including strain consolidation, variable-region glycosylation antibody display library, and process optimization.
Assuntos
Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Biomassa , Diploide , Fermentação , Haploidia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/metabolismo , Pichia/crescimento & desenvolvimento , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Yeast is capable of performing posttranslational modifications, such as N- or O-glycosylation. It has been demonstrated that N-glycans play critical biological roles in therapeutic glycoproteins by modulating pharmacokinetics and pharmacodynamics. However, N-glycan sites on recombinant glycoproteins produced in yeast can be underglycosylated, and hence, not completely occupied. Genomic homology analysis indicates that the Pichia pastoris oligosaccharyltransferase (OST) complex consists of multiple subunits, including OST1, OST2, OST3, OST4, OST5, OST6, STT3, SWP1, and WBP1. Monoclonal antibodies produced in P. pastoris show that N-glycan site occupancy ranges from 75-85 % and is affected mainly by the OST function, and in part, by process conditions. In this study, we demonstrate that N-glycan site occupancy of antibodies can be improved to greater than 99 %, comparable to that of antibodies produced in mammalian cells (CHO), by overexpressing Leishmania major STT3D (LmSTT3D) under the control of an inducible alcohol oxidase 1 (AOX1) promoter. N-glycan site occupancy of non-antibody glycoproteins such as recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) was also significantly improved, suggesting that LmSTT3D has broad substrate specificity. These results suggest that the glycosylation status of recombinant proteins can be improved by heterologous STT3 expression, which will allow for the customization of therapeutic protein profiles.
Assuntos
Glicoproteínas/metabolismo , Glicosilação , Pichia/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetinae , Expressão Gênica , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Humanos , Leishmania major/enzimologia , Leishmania major/genética , Engenharia Metabólica , Proteínas Recombinantes/metabolismoRESUMO
Aberrant expression and activation of EGFR and ERBB2 (HER2) have been successfully targeted for cancer therapeutics. Recent evidence from both basic and clinical studies suggests that ERBB3 (HER3) serves as a key activator of downstream signaling through dimerization with other ERBB proteins and plays a critical role in the widespread clinical resistance to EGFR and HER2 targeting cancer therapies. As a result, HER3 is actively pursued as an antibody therapeutic target for cancer. Ligand binding is thought to be a prerequisite for dimerization of HER3 with other ERBB proteins, which results in phosphorylation of its c-terminal tyrosine residues and activation of downstream AKT and MAPK signaling pathways. In this study, we report that an anti-HER2 monoclonal antibody (HER2Mab), which blocks HER2 dimerization with HER3, induces HER3 dimerization with EGFR in both low and high HER2 expressing cancer cells. Treatment of the low HER2 expressing MCF7 cancer cells with HER2Mab promoted cell proliferation and migration in the absence of HER3 ligand stimulation. Follow-up studies revealed that HER2Mab-induced HER3 signaling via EGFR/HER3 dimerization and activation of downstream AKT signaling pathways. These results suggest that equilibrium of dimerization among the ERBB proteins can be perturbed by HER2Mab and HER3 plays a key role in sensing the perturbation.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Neoplasias/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Transdução de Sinais , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Receptores ErbB/metabolismo , Humanos , Ligantes , Células MCF-7 , Neuregulina-1/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/química , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/químicaRESUMO
The NO and cGMP signaling pathways are of broad physiological and pathological significance. We compared the NO/soluble guanylyl cyclase (sGC)/cGMP pathway in human glioma tissues and cell lines with that of healthy control samples and demonstrated that sGC expression is significantly lower in glioma preparations. Our analysis of GEO databases (National Cancer Institute) further revealed a statistically significant reduction of sGC transcript levels in human glioma specimens. On the other hand, the expression levels of particulate (membrane) guanylyl cyclases (pGC) and cGMP-specific phosphodiesterase (PDE) were intact in the glioma cells that we have tested. Pharmacologically manipulating endogenous cGMP generation in glioma cells through either stimulating pGC by ANP/BNP, or blocking PDE by 3-isobutyl-1-methylxanthine/zaprinast caused significant inhibition of proliferation and colony formation of glioma cells. Genetically restoring sGC expression also correlated inversely with glioma cells growth. Orthotopic implantation of glioma cells transfected with an active mutant form of sGC (sGCα1ß1(Cys105)) in athymic mice increased the survival time by 4-fold over the control. Histological analysis of xenografts overexpressing α1ß1(Cys105) sGC revealed changes in cellular architecture that resemble the morphology of normal cells. In addition, a decrease in angiogenesis contributed to glioma inhibition by sGC/cGMP therapy. Our study proposes the new concept that suppressed expression of sGC, a key enzyme in the NO/cGMP pathway, may be associated with an aggressive course of glioma. The sGC/cGMP signaling-targeted therapy may be a favorable alternative to chemotherapy and radiotherapy for glioma and perhaps other tumors.
Assuntos
Antineoplásicos/metabolismo , Regulação Enzimológica da Expressão Gênica , Glioma/enzimologia , Glioma/prevenção & controle , Guanilato Ciclase/biossíntese , Receptores Citoplasmáticos e Nucleares/biossíntese , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Glioma/patologia , Guanilato Ciclase/fisiologia , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica/patologia , Invasividade Neoplásica/prevenção & controle , Receptores Citoplasmáticos e Nucleares/fisiologia , Guanilil Ciclase Solúvel , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Mammalian cell culture systems are used predominantly for the production of therapeutic monoclonal antibody (mAb) products. A number of alternative platforms, such as Pichia engineered with a humanized N-linked glycosylation pathway, have recently been developed for the production of mAbs. The glycosylation profiles of mAbs produced in glycoengineered Pichia are similar to those of mAbs produced in mammalian systems. This report presents for the first time the comprehensive characterization of an anti-human epidermal growth factor receptor 2 (HER2) mAb produced in a glycoengineered Pichia, and a study comparing the anti-HER2 from Pichia, which had an amino acid sequence identical to trastuzumab, with trastuzumab. The comparative study covered a full spectrum of preclinical evaluation, including bioanalytical characterization, in vitro biological functions, in vivo anti-tumor efficacy and pharmacokinetics in both mice and non-human primates. Cell signaling and proliferation assays showed that anti-HER2 from Pichia had antagonist activities comparable to trastuzumab. However, Pichia-produced material showed a 5-fold increase in binding affinity to FcγIIIA and significantly enhanced antibody dependant cell-mediated cytotoxicity (ADCC) activity, presumably due to the lack of fucose on N-glycans. In a breast cancer xenograft mouse model, anti-HER2 was comparable to trastuzumab in tumor growth inhibition. Furthermore, comparable pharmacokinetic profiles were observed for anti-HER2 and trastuzumab in both mice and cynomolgus monkeys. We conclude that glycoengineered Pichia provides an alternative production platform for therapeutic mAbs and may be of particular interest for production of antibodies for which ADCC is part of the clinical mechanism of action.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais/imunologia , Pichia/genética , Receptor ErbB-2/imunologia , Proteínas Recombinantes/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Área Sob a Curva , Ligação Competitiva/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Fucose/metabolismo , Engenharia Genética , Humanos , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Pichia/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica/imunologia , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacologia , Trastuzumab , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To humanize the glycosylation pathway in the yeast Pichia pastoris, we developed several combinatorial genetic libraries and used them to properly localize active eukaryotic mannosidases and sugar transferases. Here we report the details of the fusion of up to 66 N-terminal targeting sequences of fungal type II membrane proteins to 33 catalytic domains of heterologous glycosylation enzymes. We show that while it is difficult to predict which leader/catalytic domain will result in the desired activity, analysis of the fusion protein libraries allows for the selection of the leader/catalytic domain combinations that function properly. This combinatorial approach, together with a high-throughput screening protocol, has allowed us to humanize the yeast glycosylation pathway to secrete human glycoprotein with complex N-glycosylation.
Assuntos
Retículo Endoplasmático/enzimologia , Glucosiltransferases/metabolismo , Complexo de Golgi/enzimologia , Manosidases/metabolismo , Pichia/enzimologia , Engenharia de Proteínas , Glucosiltransferases/genética , Manosidases/genética , Pichia/genética , Sinais Direcionadores de Proteínas/genética , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
BACKGROUND: Administration of L-nil, a selective inhibitor of inducible nitric oxide synthase (iNOS), improves ileus in an animal model of resuscitation induced intestinal edema. The purpose of this study was to elucidate the iNOS/nitric oxide (NO) signal transduction pathway in intestinal edema. MATERIALS AND METHODS: Male Sprague Dawley rats were divided into two groups; CONTROL and RESUS+VH (edema, 80 cc/kg normal saline (resuscitation) with mesenteric venous hypertension). iNOS mRNA and protein, iNOS activity, NO tissue levels, soluble guanylyl cyclase (sGC) expression, and cyclic guanosine monophosphate (cGMP) levels were measured. As a functional endpoint, we evaluated intestinal contractile strength and frequency in L-nil treated animals. RESULTS: Edema was associated with increased iNOS mRNA and protein expression without subsequent increases in iNOS activity or tissue NO levels. There was no significant change in sGC expression or increase in cGMP induced by edema. Administration of L-nil did not decrease edema development or preserve contractile strength, but increased contractile frequency. CONCLUSION: Hydrostatic intestinal edema is not associated with increased iNOS activity or tissue NO levels. Administration of L-nil in edema increases intestinal contractile frequency. This may represent a potential mechanism for the amelioration of ileus seen with the administration of L-nil.
Assuntos
GMP Cíclico/metabolismo , Edema/metabolismo , Motilidade Gastrointestinal , Enteropatias/enzimologia , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Animais , Guanilato Ciclase/metabolismo , Pressão Hidrostática , Imuno-Histoquímica , Lisina/análogos & derivados , Masculino , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Our previous studies demonstrate a differential expression of nitric oxide (NO) signaling components in ES cells and our recent study demonstrated an enhanced differentiation of ES cells into myocardial cells with NO donors and soluble guanylyl cyclase (sGC) activators. Since NO-cGMP pathway exhibits a diverse role in cancer, we were interested in evaluating the role of the NO-receptor sGC and other components of the pathway in regulation of the tumor cell proliferation. Our results demonstrate a differential expression of the sGC subunits, NOS-1 and PKG mRNA and protein levels in various human cancer models. In contrast to sGC alpha(1), robust levels of sGC beta(1) were observed in OVCAR-3 (ovarian) and MDA-MB-468 (breast) cancer cells which correlated well with the sGC activity and a marked increase in cGMP levels upon exposure to the combination of a NO donor and a sGC activator. NOC-18 (DETA NONOate; NO donor), BAY41-2272 (3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine); sGC activator), NOC-18+BAY41-2272, IBMX (3-isobutyl-1-methylxanthine; phosphodiesterase inhibitor) and 8-bromo-cGMP (cGMP analog) caused growth inhibition and apoptosis in various cancer cell lines. To elucidate the molecular mechanisms involved in growth inhibition, we evaluated the effect of activators/inhibitors on ERK phosphorylation. Our studies indicate that BAY41-2272 or the combination NOC-18+BAY41-2272 caused inhibition of the basal ERK1/2 phosphorylation in OVCAR-3 (high sGC activity), SK-OV-3 and SK-Br-3 (low sGC activity) cell lines and in some cases the inhibition was rescued by the sGC inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). These studies suggest that the effects of activators/inhibitors of NO-sGC-cGMP in tumor cell proliferation is mediated by both cGMP-dependent and independent mechanisms.