RESUMEN
The controlled expression of two or more proteins at a defined and stable ratio remains a substantial challenge, particularly in the bi- and multispecific antibody field. Achieving an optimal ratio of protein subunits can facilitate the assembly of multimeric proteins with high efficiency and minimize the production of by-products. In this study, we propose a solution based on alternative splicing, enabling the expression of a tunable and predefined ratio of two distinct polypeptide chains from the same pre-mRNA under the control of a single promoter. The pre-mRNA used in this study contains two open reading frames situated on separate exons. The first exon is flanked by two copies of the chicken troponin intron 4 (cTNT-I4) and is susceptible to excision from the pre-mRNA by means of alternative splicing. This specific design enables the modulation of the splice ratio by adjusting the strength of the splice acceptor. To illustrate this approach, we developed constructs expressing varying ratios of GFP and dsRED and extended their application to multimeric proteins such as monoclonal antibodies, achieving industrially relevant expression levels (>1 g/L) in a 14-day fed-batch process. The stability of the splice ratio was confirmed by droplet digital PCR in a stable pool cultivated over a 28-day period, while product quality was assessed via intact mass analysis, demonstrating absence of product-related impurities resulting from undesired splice events. Furthermore, we showcased the versatility of the construct by expressing two subunits of a bispecific antibody of the BEAT® type, which contains three distinct subunits in total.
Asunto(s)
Empalme Alternativo , Animales , Subunidades de Proteína/genética , Humanos , Pollos , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/biosíntesis , Células CHO , Exones/genética , Cricetulus , Proteínas Fluorescentes Verdes/genética , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/biosíntesis , Precursores del ARN/genéticaRESUMEN
The inhibition of 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1), which catalyzes the conversion of inactive 11-ketoglucocorticoids to active 11beta-hydroxyglucocorticoids, emerged as promising strategy to treat symptoms of the metabolic syndrome, including obesity and type 2 diabetes. In this study the leaves of the anti-diabetic medicinal plant loquat (Eriobotrya japonica) were phytochemically investigated following hints from a pharmacophore-based virtual screening and a bioactivity-guided approach. Determination of the 11beta-HSD1 and 11beta-HSD2 inhibitory activities in cell lysates revealed triterpenes from the ursane type as selective, low micro-molar inhibitors of 11beta-HSD1, that is, corosolic acid (1), 3-epicorosolic acid methyl ester (4), 2-alpha hydroxy-3-oxo urs-12-en-28-oic acid (6), tormentic acid methyl ester (8), and ursolic acid (9). Importantly, a mixture of loquat constituents with moderate activities displayed a pronounced additive effect. By means of molecular modeling studies and the identification of the 11beta-HSD1-inhibiting 11-keto-ursolic acid (17) and 3-acetyl-11-keto-ursolic acid (18) a structure-activity relationship was deduced for this group of pentacyclic triterpenes. The mechanism of action elucidated in the present work together with the previously determined pharmacological activities provides these natural products with an astonishing multi-targeted anti-diabetic profile.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Eriobotrya/química , Bioensayo , Línea Celular , Sistemas de Administración de Bases de Datos , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-ActividadRESUMEN
We describe a mammalian expression construct (SPLICELECT™) that allows the redirection of a proportion of a secreted protein onto the cell surface using alternative splicing: whereas the majority of the RNA is spliced into a transcript encoding a secreted protein, a weak splice donor site yields a secondary transcript encoding, in addition, a C-terminal transmembrane domain. The different sequence elements can be modified in order to modulate the level of cell surface display and of secretion in an independent manner. In this work, we demonstrated that the cell surface display of stable cell lines is correlated with the level of the secreted protein of interest, but also with the level of heterodimerization in the case of a bispecific antibody. It was also shown that this construct may be useful for rapid screening of multiple antibody candidates in binding assays following transient transfection. Thus, the correlation of product quantity and quality of the secreted and of membrane-displayed product in combination with the flexibility of the construct with regards to cell surface display/secretion levels make SPLICELECT™ a valuable tool with many potential applications, not limited to industrial cell line development or antibody engineering.
Asunto(s)
Empalme Alternativo , Separación Celular/métodos , Técnicas de Visualización de Superficie Celular/métodos , Análisis de la Célula Individual/métodos , Animales , Células Cultivadas , HumanosRESUMEN
Cell populations that are exclusively composed by descendants from a defined single parental progenitor are referred to as "monoclonal", "clonal" or more correctly as "clonally derived cell population". Clonal derivation of cell lines used in the manufacturing of recombinant biologics is a regulatory requirement that aims to ensure a robust process and consistent quality throughout the life cycle of a product. Clonal derivation of cell lines is usually ensured by the process (e.g. two subsequent rounds of limiting dilution). Here we present an approach to analytically assess the probability of clonal derivation of existing cell populations. Using target locus amplification (TLA) followed by next generation sequencing (NGS), unique genetic features can be identified, for example the integration site of the plasmid in the host cell genome or plasmid-plasmid junctions of the plasmids used for expression of recombinant biologics. Whereas a direct assessment of clonal derivation using TLA/NGS data is challenging due to limitations in specificity, confirmed clonally derived populations generated from the cell line population can be analyzed by qPCR for the presence of the unique genetic features identified by TLA/NGS. In the present study, a statistical analysis allowed the demonstration that two independently generated CHO cell lines were clonally derived with an upper 95% confidence interval limit of a potentially present contaminating population of 1.3%.
Asunto(s)
Células Clonales/citología , Análisis de Secuencia de ADN/métodos , Transgenes , Animales , Células CHO , Cricetulus , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Plásmidos/genética , Reacción en Cadena de la PolimerasaRESUMEN
Glucocorticoids play an essential role in the regulation of key physiological processes, including immunomodulation, brain function, energy metabolism, electrolyte balance and blood pressure. Exposure to naturally occurring compounds or industrial chemicals that impair glucocorticoid action may contribute to the increasing incidence of cognitive deficits, immune disorders and metabolic diseases. Potentially, "glucocorticoid disruptors" can interfere with various steps of hormone action, e.g. hormone synthesis, binding to plasma proteins, delivery to target cells, pre-receptor regulation of the ratio of active versus inactive hormones, glucocorticoid receptor (GR) function, or export and degradation of glucocorticoids. Several recent studies indicate that such chemicals exist and that some of them can cause multiple toxic effects by interfering with different steps of hormone action. For example, increasing evidence suggests that organotins disturb glucocorticoid action by altering the function of factors that regulate the expression of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) pre-receptor enzymes, by direct inhibition of 11beta-HSD2-dependent inactivation of glucocorticoids, and by blocking GR activation. These observations emphasize on the complexity of the toxic effects caused by such compounds and on the need of suitable test systems to assess their effects on each relevant step.
Asunto(s)
Contaminantes Ambientales/efectos adversos , Glucocorticoides/farmacología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/antagonistas & inhibidores , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , Animales , Humanos , Compuestos Orgánicos de Estaño/efectos adversos , Receptores de Glucocorticoides/metabolismoRESUMEN
17ß-Hydroxysteroid dehydrogenase type 3 and 5 (17ß-HSD3 and 17ß-HSD5) catalyze testosterone biosynthesis and thereby constitute therapeutic targets for androgen-related diseases or endocrine-disrupting chemicals. As a fast and efficient tool to identify potential ligands for 17ßHSD3/5, ligand- and structure-based pharmacophore models for both enzymes were developed. The models were evaluated first by in silico screening of commercial compound databases and further experimentally validated by enzymatic efficacy tests of selected virtual hits. Among the 35 tested compounds, 11 novel inhibitors with distinct chemical scaffolds, e.g. sulfonamides and triazoles, and with different selectivity properties were discovered. Thereby, we provide several potential starting points for further 17ß-HSD3 and 17ß-HSD5 inhibitor development. Article from the Special issue on Targeted Inhibitors.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , 3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Descubrimiento de Drogas , Inhibidores Enzimáticos/metabolismo , Hidroxiprostaglandina Deshidrogenasas/antagonistas & inhibidores , 17-Hidroxiesteroide Deshidrogenasas/química , 17-Hidroxiesteroide Deshidrogenasas/genética , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/genética , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Cristalografía por Rayos X , Bases de Datos Factuales , Inhibidores Enzimáticos/química , Células HEK293 , Humanos , Hidroxiprostaglandina Deshidrogenasas/química , Hidroxiprostaglandina Deshidrogenasas/genética , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Ligandos , Estructura MolecularRESUMEN
Elevated glucocorticoids are a key risk factor for metabolic diseases, and the glucocorticoid-activating enzyme 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) represents a promising therapeutic target. We measured the potential of six traditional antidiabetic medicinal plants extracts to inhibit 11beta-HSD1 activity and glucocorticoid receptor (GR) activation in transfected HEK-293 cells. Leave extracts of Eriobotrya japonica preferentially inhibited 11beta-HSD1 over 11beta-HSD2. Extracts of roasted but not native coffee beans preferentially inhibited 11beta-HSD1 over 11beta-HSD2, emphasizing the importance of sample preparation. Thus, natural compounds inhibiting 11beta-HSD1 may contribute to the antidiabetic effect of the investigated plant extracts.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , Coffea , Eriobotrya , Hipoglucemiantes/farmacología , Extractos Vegetales/farmacología , Receptores de Glucocorticoides/antagonistas & inhibidores , Línea Celular , Humanos , Medicina Tradicional , Hojas de la Planta , SemillasRESUMEN
Treatment of so-called civilization diseases, including some forms of cancer, immune-related diseases and metabolic disorders, represent a major challenge in the industrialized world. In addition to genetic predisposition, behavior and exposure to xenobiotics contribute to these diseases. Here, we review existing evidence for an association of environmental chemicals with disturbed glucocorticoid- and mineralocorticoid-regulated physiological processes. Impaired activity of glucocorticoids and mineralocorticoids can contribute to several diseases, including neurological diseases, immune disorders and metabolic syndrome. Recent studies provide evidence for the existence of environmental chemicals that are able to disrupt the function of these hormones at different levels of their action. Therefore, potential interferences with these hormones should be considered for safety assessment of chemicals. Compared with the extensive knowledge on chemicals interfering with estrogen or androgen responses, the study of glucocorticoid and mineralocorticoid disruptors is an emerging field of research, and the identification of relevant xenobiotics and their underlying mechanisms of toxicity remains a major challenge.
Asunto(s)
Contaminantes Ambientales/efectos adversos , Contaminantes Ambientales/metabolismo , Glucocorticoides/metabolismo , Mineralocorticoides/metabolismo , Animales , Contaminantes Ambientales/química , Glucocorticoides/antagonistas & inhibidores , Glucocorticoides/química , Humanos , Enfermedades del Sistema Inmune/etiología , Enfermedades del Sistema Inmune/metabolismo , Enfermedades del Sistema Inmune/prevención & control , Síndrome Metabólico/etiología , Síndrome Metabólico/metabolismo , Síndrome Metabólico/prevención & control , Mineralocorticoides/antagonistas & inhibidores , Mineralocorticoides/química , Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/metabolismo , Enfermedades del Sistema Nervioso/prevención & controlRESUMEN
BACKGROUND: Organotins are highly toxic and widely distributed environmental chemicals. Dibutyltin (DBT) is used as stabilizer in the production of polyvinyl chloride plastics, and it is also the major metabolite formed from tributyltin (TBT) in vivo. DBT is immunotoxic, however, the responsible targets remain to be defined. Due to the importance of glucocorticoids in immune-modulation, we investigated whether DBT could interfere with glucocorticoid receptor (GR) function. METHODOLOGY: We used HEK-293 cells transiently transfected with human GR as well as rat H4IIE hepatoma cells and native human macrophages and human THP-1 macrophages expressing endogenous receptor to study organotin effects on GR function. Docking of organotins was used to investigate the binding mechanism. PRINCIPAL FINDINGS: We found that nanomolar concentrations of DBT, but not other organotins tested, inhibit ligand binding to GR and its transcriptional activity. Docking analysis indicated that DBT inhibits GR activation allosterically by inserting into a site close to the steroid-binding pocket, which disrupts a key interaction between the A-ring of the glucocorticoid and the GR. DBT inhibited glucocorticoid-induced expression of phosphoenolpyruvate carboxykinase (PEPCK) and tyrosine-aminotransferase (TAT) and abolished the glucocorticoid-mediated transrepression of TNF-alpha-induced NF-kappaB activity. Moreover, DBT abrogated the glucocorticoid-mediated suppression of interleukin-6 (IL-6) and TNF-alpha production in lipopolysaccharide (LPS)-stimulated native human macrophages and human THP-1 macrophages. CONCLUSIONS: DBT inhibits ligand binding to GR and subsequent activation of the receptor. By blocking GR activation, DBT may disturb metabolic functions and modulation of the immune system, providing an explanation for some of the toxic effects of this organotin.