RESUMO
BACKGROUND: Therapeutic products with coagulation factor VIII (FVIII) have a wide range of specific activities, implying presence of protein with altered structure. Previous studies showed that recombinant FVIII products (rFVIII) contain a fraction (FVIIIFT ) unable to bind von Willebrand factor (VWF) and reported to lack activity. Because of loss of function(s), FVIIIFT can be defined as a product-related impurity, whose properties and levels in rFVIII products should be investigated. OBJECTIVE: To isolate and characterize the FVIIIFT fraction in rFVIII products. METHODS: Protein fractions unable (FVIIIFT ) and able (FVIIIEL ) to bind VWF were isolated from rFVIII products using immobilized VWF affinity chromatography (IVAC) and characterized by gel electrophoresis, immunoblotting, FVIII activity test, surface plasmon resonance, mass spectrometry, and for plasma clearance in mice. RESULTS AND CONCLUSIONS: A robust IVAC methodology was developed and applied for analysis of 10 rFVIII products marketed in the United States. FVIIIFT was found at various contents (0.4%-21.5%) in all products. Compared with FVIIIEL , FVIIIFT had similar patterns of polypeptide bands by gel electrophoresis, but lower functional activity. In several representative products, FVIIIFT was found to have reduced sulfation at Tyr1680, important for VWF binding, decreased interaction with a low-density lipoprotein receptor-related protein 1 fragment, and faster plasma clearance in mice. These findings provide basic characterization of FVIIIFT and demonstrate a potential for IVAC to control this impurity in rFVIII products to improve their efficacy in therapy of hemophilia A.
Assuntos
Hemofilia A , Hemostáticos , Animais , Testes de Coagulação Sanguínea , Fator VIII , Hemofilia A/tratamento farmacológico , Camundongos , Fator de von WillebrandRESUMO
Copper is an essential cofactor of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Inherited loss-of-function mutations in several genes encoding proteins required for copper delivery to CcO result in diminished CcO activity and severe pathologic conditions in affected infants. Copper supplementation restores CcO function in patient cells with mutations in two of these genes, COA6 and SCO2, suggesting a potential therapeutic approach. However, direct copper supplementation has not been therapeutically effective in human patients, underscoring the need to identify highly efficient copper transporting pharmacological agents. By using a candidate-based approach, we identified an investigational anticancer drug, elesclomol (ES), that rescues respiratory defects of COA6-deficient yeast cells by increasing mitochondrial copper content and restoring CcO activity. ES also rescues respiratory defects in other yeast mutants of copper metabolism, suggesting a broader applicability. Low nanomolar concentrations of ES reinstate copper-containing subunits of CcO in a zebrafish model of copper deficiency and in a series of copper-deficient mammalian cells, including those derived from a patient with SCO2 mutations. These findings reveal that ES can restore intracellular copper homeostasis by mimicking the function of missing transporters and chaperones of copper, and may have potential in treating human disorders of copper metabolism.
Assuntos
Antineoplásicos/farmacologia , Cobre/deficiência , Drogas em Investigação/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Hidrazinas/farmacologia , Mitocôndrias/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Transporte Biológico/genética , Proteínas de Transporte/genética , Linhagem Celular , Coenzimas/deficiência , Cobre/uso terapêutico , Transportador de Cobre 1 , Suplementos Nutricionais , Modelos Animais de Doenças , Reposicionamento de Medicamentos , Drogas em Investigação/uso terapêutico , Fibroblastos , Humanos , Hidrazinas/uso terapêutico , Proteínas de Membrana Transportadoras/genética , Erros Inatos do Metabolismo/tratamento farmacológico , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Chaperonas Moleculares , Mutagênese Sítio-Dirigida , Mutação , Ratos , Saccharomyces cerevisiae , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Copper (Cu) is an essential cofactor for various enzymatic activities including mitochondrial electron transport, iron mobilization, and peptide hormone maturation. Consequently, Cu dysregulation is associated with fatal neonatal disease, liver and cardiac dysfunction, and anemia. While the Cu transporter ATP7A plays a major role in both intestinal Cu mobilization to the periphery and prevention of Cu over-accumulation, it is unclear how regulation of ATP7A contributes to Cu homeostasis in response to systemic Cu fluctuation. Here we show, using Cu-deficient mouse models, that steady-state levels of ATP7A are lower in peripheral tissues (including the heart, spleen, and liver) under Cu deficiency and that subcutaneous administration of Cu to these animals restore normal ATP7A levels in these tissues. Strikingly, ATP7A in the intestine is regulated in the opposite manner - low systemic Cu increases ATP7A while subcutaneous Cu administration decreases ATP7A suggesting that intestine-specific non-autonomous regulation of ATP7A abundance may serve as a key homeostatic control for Cu export into the circulation. Our results support a systemic model for how a single transporter can be inversely regulated in a tissue-specific manner to maintain organismal Cu homeostasis.
Assuntos
ATPases Transportadoras de Cobre/metabolismo , Cobre/metabolismo , Homeostase , Especificidade de Órgãos , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Células Cultivadas , Cobre/deficiência , Cobre/farmacologia , Transportador de Cobre 1 , ATPases Transportadoras de Cobre/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Fígado/citologia , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenantrolinas/farmacologia , RatosRESUMO
Copper plays key catalytic and regulatory roles in biochemical processes essential for normal growth, development, and health. Defects in copper metabolism cause Menkes and Wilson's disease, myeloneuropathy, and cardiovascular disease and are associated with other pathophysiological states. Consequently, it is critical to understand the mechanisms by which organisms control the acquisition, distribution, and utilization of copper. The intestinal enterocyte is a key regulatory point for copper absorption into the body; however, the mechanisms by which intestinal cells transport copper to maintain organismal copper homeostasis are poorly understood. Here, we identify a mechanism by which organismal copper homeostasis is maintained by intestinal copper exporter trafficking that is coordinated with extraintestinal copper levels in Caenorhabditis elegans Specifically, we show that CUA-1, the C. elegans homolog of ATP7A/B, localizes to lysosome-like organelles (gut granules) in the intestine under copper overload conditions for copper detoxification, whereas copper deficiency results in a redistribution of CUA-1 to basolateral membranes for copper efflux to peripheral tissues. Worms defective in gut granule biogenesis exhibit defects in copper sequestration and increased susceptibility to toxic copper levels. Interestingly, however, a splice isoform CUA-1.2 that lacks a portion of the N-terminal domain is targeted constitutively to the basolateral membrane irrespective of dietary copper concentration. Our studies establish that CUA-1 is a key intestinal copper exporter and that its trafficking is regulated to maintain systemic copper homeostasis. C. elegans could therefore be exploited as a whole-animal model system to study regulation of intra- and intercellular copper trafficking pathways.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Cobre/metabolismo , Homeostase , Mucosa Intestinal/metabolismo , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Intestinos/crescimento & desenvolvimento , Transporte ProteicoRESUMO
Sequential treatment with different tyrosine kinase inhibitors (TKIs) is one of the strategies for handling chronic myeloid leukemia (CML) in which dynamic change in Bcr-Abl kinase domain mutation is often an obstacle faced during TKI therapy. Here we report successful sequential therapy with different TKIs for the CML patient harboring V299L and E459K compound mutations. Molecular monitoring including quantitative analysis of BCR-ABL transcript level and mutation analysis were performed regularly for successful treatment. Additionally a drug-target complex was structurally modeled to investigate influence of amino acid substitutions on drug resistance, and to choose alternative TKI in sequential therapy, suggesting protein structural modeling can be useful approach in selecting alternative TKIs.