RESUMO
Diamond-Blackfan anaemia (DBA) is a rare congenital disease causing severe anaemia and progressive bone marrow failure. The majority of patients carry mutations in ribosomal proteins, which leads to depletion of erythroid progenitors in the bone marrow. As many as 40% of all DBA patients receive glucocorticoids to alleviate their anaemia. However, despite their use in DBA treatment for more than half a century, the therapeutic mechanisms of glucocorticoids remain largely unknown. Therefore we sought to study disease specific effects of glucocorticoid treatment using a ribosomal protein s19 (Rps19) deficient mouse model of DBA. This study determines for the first time that a mouse model of DBA can respond to glucocorticoid treatment, similar to DBA patients. Our results demonstrate that glucocorticoid treatment reduces apoptosis, rescues erythroid progenitor depletion and premature differentiation of erythroid cells. Furthermore, glucocorticoids prevent Trp53 activation in Rps19-deficient cells- in a disease-specific manner. Dissecting the therapeutic mechanisms behind glucocorticoid treatment of DBA provides indispensible insight into DBA pathogenesis. Identifying mechanisms important for DBA treatment also enables development of more disease-specific treatments of DBA.
Assuntos
Anemia de Diamond-Blackfan/tratamento farmacológico , Eritropoese/efeitos dos fármacos , Prednisolona/uso terapêutico , Proteínas Ribossômicas/deficiência , Proteína Supressora de Tumor p53/fisiologia , Adolescente , Anemia de Diamond-Blackfan/sangue , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/genética , Dexametasona/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Células Precursoras Eritroides/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Prednisolona/farmacologia , Quimera por Radiação , Proteínas Ribossômicas/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Regulação para Cima/efeitos dos fármacos , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/genéticaRESUMO
Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplastic anemia, characterized by macrocytic anemia, reticulocytopenia, and severely reduced numbers of erythroid precursors in the bone marrow. For more than fifty years, glucocorticoids have remained the main option for pharmacological treatment of DBA. While continuous glucocorticoid administration increases hemoglobin levels in a majority of DBA patients, it also causes severe side effects. There is therefore a great need for more specific and effective treatments to boost or replace the use of glucocorticoids. Over the years, many alternative therapies have been tried out, but most of them have shown to be ineffective. Here we review previous and current attempts to develop such alternative therapies for DBA. We further discuss how emerging knowledge regarding the pathological mechanism in DBA and the therapeutic mechanism of glucocorticoids treatment may reveal novel drug targets for DBA treatment.
Assuntos
Anemia de Diamond-Blackfan/terapia , Anemia de Diamond-Blackfan/genética , Ensaios Clínicos como Assunto , Terapia Genética , Glucocorticoides/administração & dosagem , Glucocorticoides/efeitos adversos , Humanos , MutaçãoRESUMO
Diamond-Blackfan anemia (DBA) is a rare genetic disorder in which patients present a scarcity of erythroid precursors in an otherwise normocellular bone marrow. Most, but not all, patients carry mutations in ribosomal proteins such as RPS19, suggesting that compromised mRNA translation and ribosomal stress are pathogenic mechanisms causing depletion of erythroid precursors. To gain further insight to disease mechanisms in DBA, we performed a custom short hairpin RNA (shRNA) based screen against 750 genes hypothesized to affect DBA pathophysiology. Among the hits were two shRNAs against the erythroid specific heme-regulated eIF2α kinase (HRI), which is a negative regulator of mRNA translation. This study shows that shRNA-mediated HRI silencing or loss of one HRI allele improves expansion of Rps19-deficient erythroid precursors, as well as improves the anemic phenotype in Rps19-deficient animals. We found that Rps19-deficient erythroblasts have elevated levels of unbound intracellular heme, which is normalized by HRI heterozygosity. Additionally, targeting elevated heme levels by treating cells with the heme scavenger alpha-1-microglobulin (A1M), increased proliferation of Rps19-deficient erythroid precursors and decreased heme levels in a disease-specific manner. HRI heterozygosity, but not A1M treatment, also decreased the elevated p53 activity observed in Rps19-deficient cells, indicating that p53 activation is caused by ribosomal stress and aberrant mRNA translation and not heme overload in Rps19-deficiency. Together, these findings suggest that targeting elevated heme levels is a promising new treatment strategy for DBA.
Assuntos
alfa-Globulinas/uso terapêutico , Anemia de Diamond-Blackfan/terapia , Heme/análise , Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Deleção de Genes , Inativação Gênica , Terapia Genética , Heme/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/uso terapêutico , Proteínas Ribossômicas/genéticaRESUMO
To understand the molecular basis of sequential N-dealkylation by cytochrome P450 2B enzymes, we studied the binding of amidopyrine (AP) as well as the metabolites of this reaction, desmethylamidopyrine (DMAP) and aminoantipyrine (AAP), using the X-ray crystal structure of rabbit P450 2B4 and two nuclear magnetic resonance (NMR) techniques: saturation transfer difference (STD) spectroscopy and longitudinal (T(1)) relaxation NMR. Results of STD NMR of AP and its metabolites bound to P450 2B4 were similar, suggesting that they occupy similar niches within the enzyme's active site. The model-dependent relaxation rates (R(M)) determined from T(1) relaxation NMR of AP and DMAP suggest that the N-linked methyl is closest to the heme. To determine the orientation(s) of AP and its metabolites within the P450 2B4 active site, we used distances calculated from the relaxation rates to constrain the metabolites to the X-ray crystal structure of P450 2B4. Simulated annealing of the complex revealed that the metabolites do indeed occupy similar hydrophobic pockets within the active site, while the N-linked methyls are free to rotate between two binding modes. From these bound structures, a model of N-demethylation in which the N-linked methyl functional groups rotate between catalytic and noncatalytic positions was developed. This study is the first to provide a structural model of a drug and its metabolites complexed to a cytochrome P450 based on NMR and to provide a structural mechanism for how a drug can undergo sequential oxidations without unbinding. The rotation of the amide functional group might represent a common structural mechanism for N-dealkylation reactions for other drugs such as the local anesthetic lidocaine.