RESUMO
Diseases such as osteoporosis are associated with reduced bone mass. Therapies to prevent bone loss exist, but there are few that stimulate bone formation and restore bone mass. Bone morphogenetic proteins (BMPs) are members of the TGFß superfamily, which act as pleiotropic regulators of skeletal organogenesis and bone homeostasis. Ablation of the BMPR1A receptor in osteoblasts increases bone mass, suggesting that inhibition of BMPR1A signaling may have therapeutic benefit. The aim of this study was to determine the skeletal effects of systemic administration of a soluble BMPR1A fusion protein (mBMPR1A-mFc) in vivo. mBMPR1A-mFc was shown to bind BMP2/4 specifically and with high affinity and prevent downstream signaling. mBMPR1A-mFc treatment of immature and mature mice increased bone mineral density, cortical thickness, trabecular bone volume, thickness and number, and decreased trabecular separation. The increase in bone mass was due to an early increase in osteoblast number and bone formation rate, mediated by a suppression of Dickkopf-1 expression. This was followed by a decrease in osteoclast number and eroded surface, which was associated with a decrease in receptor activator of NF-κB ligand (RANKL) production, an increase in osteoprotegerin expression, and a decrease in serum tartrate-resistant acid phosphatase (TRAP5b) concentration. mBMPR1A treatment also increased bone mass and strength in mice with bone loss due to estrogen deficiency. In conclusion, mBMPR1A-mFc stimulates osteoblastic bone formation and decreases bone resorption, which leads to an increase in bone mass, and offers a promising unique alternative for the treatment of bone-related disorders.
Assuntos
Doenças Ósseas Metabólicas/prevenção & controle , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Osso e Ossos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/efeitos dos fármacos , Análise de Variância , Animais , Western Blotting , Densidade Óssea/efeitos dos fármacos , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Reabsorção Óssea/tratamento farmacológico , Osso e Ossos/anatomia & histologia , Osso e Ossos/fisiologia , Cromatografia em Gel , Clonagem Molecular , Densitometria , Eletroforese em Gel de Poliacrilamida , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Luciferases , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/efeitos dos fármacos , Osteoclastos/fisiologia , Osteoprotegerina/metabolismo , Reação em Cadeia da Polimerase , Ligante RANK/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes de Fusão/administração & dosagem , Transdução de Sinais/fisiologiaRESUMO
Activin receptor-like kinase 1 (ALK1), an endothelial cell-specific type I receptor of the TGF-ß superfamily, is an important regulator of normal blood vessel development as well as pathological tumor angiogenesis. As such, ALK1 is an important therapeutic target. Thus, several ALK1-directed agents are currently in clinical trials as anti-angiogenic cancer therapeutics. Given the biological and clinical importance of the ALK1 signaling pathway, we sought to elucidate the biophysical and structural basis underlying ALK1 signaling. The TGF-ß family ligands BMP9 and BMP10 as well as the three type II TGF-ß family receptors ActRIIA, ActRIIB, and BMPRII have been implicated in ALK1 signaling. Here, we provide a kinetic and thermodynamic analysis of BMP9 and BMP10 interactions with ALK1 and type II receptors. Our data show that BMP9 displays a significant discrimination in type II receptor binding, whereas BMP10 does not. We also report the crystal structure of a fully assembled ternary complex of BMP9 with the extracellular domains of ALK1 and ActRIIB. The structure reveals that the high specificity of ALK1 for BMP9/10 is determined by a novel orientation of ALK1 with respect to BMP9, which leads to a unique set of receptor-ligand interactions. In addition, the structure explains how BMP9 discriminates between low and high affinity type II receptors. Taken together, our findings provide structural and mechanistic insights into ALK1 signaling that could serve as a basis for novel anti-angiogenic therapies.
Assuntos
Receptores de Activinas Tipo II/química , Proteínas Morfogenéticas Ósseas/química , Fatores de Diferenciação de Crescimento/química , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Inibidores da Angiogênese/química , Inibidores da Angiogênese/uso terapêutico , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Cristalografia por Raios X , Fator 2 de Diferenciação de Crescimento , Fatores de Diferenciação de Crescimento/genética , Fatores de Diferenciação de Crescimento/metabolismo , Células HEK293 , Humanos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Endoglin (CD105), a transmembrane protein of the transforming growth factor ß superfamily, plays a crucial role in angiogenesis. Mutations in endoglin result in the vascular defect known as hereditary hemorrhagic telangiectasia (HHT1). The soluble form of endoglin was suggested to contribute to the pathogenesis of preeclampsia. To obtain further insight into its function, we cloned, expressed, purified, and characterized the extracellular domain (ECD) of mouse and human endoglin fused to an immunoglobulin Fc domain. We found that mouse and human endoglin ECD-Fc bound directly, specifically, and with high affinity to bone morphogenetic proteins 9 and 10 (BMP9 and BMP10) in surface plasmon resonance (Biacore) and cell-based assays. We performed a function mapping analysis of the different domains of endoglin by examining their contributions to the selectivity and biological activity of the protein. The BMP9/BMP10 binding site was localized to the orphan domain of human endoglin composed of the amino acid sequence 26-359. We established that endoglin and type II receptors bind to overlapping sites on BMP9. In the in vivo chick chorioallantoic membrane assay, the mouse and the truncated human endoglin ECD-Fc both significantly reduced VEGF-induced vessel formation. Finally, murine endoglin ECD-Fc acted as an anti-angiogenic factor that decreased blood vessel sprouting in VEGF/FGF-induced angiogenesis in in vivo angioreactors and reduced the tumor burden in the colon-26 mouse tumor model. Together our findings indicate an important role of soluble endoglin ECD in the regulation of angiogenesis and highlight efficacy of endoglin-Fc as a potential anti-angiogenesis therapeutic agent.
Assuntos
Inibidores da Angiogênese/farmacologia , Antígenos CD/farmacologia , Proteínas Morfogenéticas Ósseas/metabolismo , Fator 2 de Diferenciação de Crescimento/metabolismo , Fatores de Diferenciação de Crescimento/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Sítios de Ligação , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Linhagem Celular , Endoglina , Fator 2 de Diferenciação de Crescimento/genética , Fatores de Diferenciação de Crescimento/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismoRESUMO
The single transmembrane domain serine/threonine kinase activin receptor type IIB (ActRIIB) has been proposed to bind key regulators of skeletal muscle mass development, including the ligands GDF-8 (myostatin) and GDF-11 (BMP-11). Here we provide a detailed kinetic characterization of ActRIIB binding to several low and high affinity ligands using a soluble activin receptor type IIB-Fc chimera (ActRIIB.Fc). We show that both GDF-8 and GDF-11 bind the extracellular domain of ActRIIB with affinities comparable with those of activin A, a known high affinity ActRIIB ligand, whereas BMP-2 and BMP-7 affinities for ActRIIB are at least 100-fold lower. Using site-directed mutagenesis, we demonstrate that ActRIIB binds GDF-11 and activin A in different ways such as, for example, substitutions in ActRIIB Leu(79) effectively abolish ActRIIB binding to activin A yet not to GDF-11. Native ActRIIB has four isoforms that differ in the length of the C-terminal portion of their extracellular domains. We demonstrate that the C terminus of the ActRIIB extracellular domain is crucial for maintaining biological activity of the ActRIIB.Fc receptor chimera. In addition, we show that glycosylation of ActRIIB is not required for binding to activin A or GDF-11. Together, our findings reveal binding specificity and activity determinants of the ActRIIB receptor that combine to effect specificity in the activation of distinct signaling pathways.
Assuntos
Receptores de Activinas Tipo II/metabolismo , Receptores de Activinas Tipo II/química , Receptores de Activinas Tipo II/genética , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , DNA Complementar/genética , Genes Reporter , Humanos , Ligantes , Mutagênese , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/metabolismo , Miostatina/química , Miostatina/metabolismo , Plasmídeos/genética , Ativadores de Plasminogênio/química , Ativadores de Plasminogênio/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Diseases that affect the regulation of bone turnover can lead to skeletal fragility and increased fracture risk. Members of the TGF-beta superfamily have been shown to be involved in the regulation of bone mass. Activin A, a TGF-beta signaling ligand, is present at high levels in bone and may play a role in the regulation of bone metabolism. Here we demonstrate that pharmacological blockade of ligand signaling through the high affinity receptor for activin, type II activin receptor (ActRIIA), by administration of the soluble extracellular domain of ActRIIA fused to a murine IgG2a-Fc, increases bone formation, bone mass, and bone strength in normal mice and in ovariectomized mice with established bone loss. These observations support the development of this pharmacological strategy for the treatment of diseases with skeletal fragility.
Assuntos
Receptores de Activinas Tipo II/farmacologia , Osso e Ossos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Receptores de Activinas Tipo II/administração & dosagem , Receptores de Activinas Tipo II/isolamento & purificação , Animais , Fenômenos Biomecânicos , Reabsorção Óssea , Linhagem Celular , Feminino , Humanos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/farmacologia , Vértebras Lombares/efeitos dos fármacos , Camundongos , Tamanho do Órgão/efeitos dos fármacos , Ovariectomia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Solubilidade/efeitos dos fármacosRESUMO
MAP KAP kinase 2 (MK2), a Ser/Thr kinase, plays a crucial role in the inflammatory process. We have determined the crystal structures of a catalytically active C-terminal deletion form of human MK2, residues 41-364, in complex with staurosporine at 2.7 A and with ADP at 3.2 A, revealing overall structural similarity with other Ser/Thr kinases. Kinetic analysis reveals that the K(m) for ATP is very similar for MK2 41-364 and p38-activated MK2 41-400. Conversely, the catalytic rate and binding for peptide substrate are dramatically reduced in MK2 41-364. However, phosphorylation of MK2 41-364 by p38 restores the V(max) and K(m) for peptide substrate to values comparable to those seen in p38-activated MK2 41-400, suggesting a mechanism for regulation of enzyme activity.
Assuntos
Difosfato de Adenosina/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Estaurosporina/metabolismo , Sequência de Aminoácidos , Ativação Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Substâncias Macromoleculares , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Alinhamento de Sequência , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Erythropoietin (EPO) stimulates proliferation of early-stage erythrocyte precursors and is widely used for the treatment of chronic anemia. However, several types of EPO-resistant anemia are characterized by defects in late-stage erythropoiesis, which is EPO independent. Here we investigated regulation of erythropoiesis using a ligand-trapping fusion protein (ACE-536) containing the extracellular domain of human activin receptor type IIB (ActRIIB) modified to reduce activin binding. ACE-536, or its mouse version RAP-536, produced rapid and robust increases in erythrocyte numbers in multiple species under basal conditions and reduced or prevented anemia in murine models. Unlike EPO, RAP-536 promoted maturation of late-stage erythroid precursors in vivo. Cotreatment with ACE-536 and EPO produced a synergistic erythropoietic response. ACE-536 bound growth differentiation factor-11 (GDF11) and potently inhibited GDF11-mediated Smad2/3 signaling. GDF11 inhibited erythroid maturation in mice in vivo and ex vivo. Expression of GDF11 and ActRIIB in erythroid precursors decreased progressively with maturation, suggesting an inhibitory role for GDF11 in late-stage erythroid differentiation. RAP-536 treatment also reduced Smad2/3 activation, anemia, erythroid hyperplasia and ineffective erythropoiesis in a mouse model of myelodysplastic syndromes (MDS). These findings implicate transforming growth factor-ß (TGF-ß) superfamily signaling in erythroid maturation and identify ACE-536 as a new potential treatment for anemia, including that caused by ineffective erythropoiesis.
Assuntos
Receptores de Activinas Tipo II , Anemia/sangue , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Células Precursoras Eritroides/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Fatores de Diferenciação de Crescimento/efeitos dos fármacos , Hematínicos/farmacologia , Síndromes Mielodisplásicas/sangue , Proteínas Recombinantes de Fusão/farmacologia , Animais , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Modelos Animais de Doenças , Quimioterapia Combinada , Contagem de Eritrócitos , Eritropoetina/farmacologia , Fatores de Diferenciação de Crescimento/antagonistas & inibidores , Haplorrinos , Humanos , Ligantes , Camundongos , Ratos , Contagem de Reticulócitos , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/efeitos dos fármacos , Proteína Smad3/efeitos dos fármacosRESUMO
This is the first report that inhibition of negative regulators of skeletal muscle by a soluble form of activin type IIB receptor (ACE-031) increases muscle mass independent of fiber-type expression. This finding is distinct from the effects of selective pharmacological inhibition of myostatin (GDF-8), which predominantly targets type II fibers. In our study 8-wk-old C57BL/6 mice were treated with ACE-031 or vehicle control for 28 days. By the end of treatment, mean body weight of the ACE-031 group was 16% greater than that of the control group, and wet weights of soleus, plantaris, gastrocnemius, and extensor digitorum longus muscles increased by 33, 44, 46 and 26%, respectively (P<0.05). Soleus fiber-type distribution was unchanged with ACE-031 administration, and mean fiber cross-sectional area increased by 22 and 28% (P<0.05) in type I and II fibers, respectively. In the plantaris, a predominantly type II fiber muscle, mean fiber cross-sectional area increased by 57% with ACE-031 treatment. Analysis of myosin heavy chain (MHC) isoform transcripts by real-time PCR indicated no change in transcript levels in the soleus, but a decline in MHC I and IIa in the plantaris. In contrast, electrophoretic separation of total soleus and plantaris protein indicated that there was no change in the proportion of MHC isoforms in either muscle. Thus these data provide optimism that ACE-031 may be a viable therapeutic in the treatment of musculoskeletal diseases. Future studies should be undertaken to confirm that the observed effects are not age dependent or due to the relatively short study duration.
Assuntos
Receptores de Activinas Tipo II/administração & dosagem , Desenvolvimento Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Receptores de Activinas Tipo II/genética , Animais , Regulação da Expressão Gênica , Humanos , Hipertrofia , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/efeitos dos fármacos , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miostatina/antagonistas & inibidores , Tamanho do Órgão , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Fatores de Tempo , Aumento de PesoRESUMO
Activin A belongs to the TGF-beta superfamily and plays an important role in bone metabolism. It was reported that a soluble form of extracellular domain of the activin receptor type IIA (ActRIIA) fused to the Fc domain of murine IgG, an activin antagonist, has an anabolic effect on bone in intact and ovariectomized mice. The present study was designed to examine the skeletal effect of human ActRIIA-IgG1-Fc (ACE-011) in non-human primates. Young adult female Cynomolgus monkeys were given a biweekly subcutaneous injection of either 10mg/kg ACE-011 or vehicle (VEH) for 3months. Treatment effects were evaluated by histomorphometric analysis of the distal femur, femoral midshaft, femoral neck and 12th thoracic vertebrae, by muCT analysis of femoral neck and by biomarkers of bone turnover. Compared to VEH, at the distal femur ACE-011-treated monkeys had significantly increased cancellous bone volume (+93%), bone formation rate per bone surface (+166%) and osteoblast surface (+196%) indicating an anabolic action. Monkeys treated with ACE-011 also had decreased osteoclast surface and number. No differences were observed in parameters of cortical bone at the midshaft of the femur. Similar to distal femur, ACE-011-treated monkeys had significantly greater cancellous bone volume, bone formation rate and osteoblast surface at the femoral neck relative to VEH. A significant increase in bone formation rate and osteoblast surface with a decrease in osteoclast surface was observed in thoracic vertebrae. muCT analysis of femoral neck indicated more plate-like structure in ACE-011-treated monkeys. Monkeys treated with ACE-011 had no effect on serum bone-specific alkaline phosphatase and CTX at the end of the study. These observations demonstrate that ACE-011 is a dual anabolic-antiresorptive compound, improving cancellous bone volume by promoting bone formation and inhibiting bone resorption in non-human primates. Thus, soluble ActRIIA fusion protein may be useful in the prevention and/or treatment of osteoporosis and other diseases involving accelerated bone loss.
Assuntos
Ativinas/metabolismo , Densidade Óssea/fisiologia , Fêmur/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Vértebras Torácicas/metabolismo , Animais , Densidade Óssea/efeitos dos fármacos , Contagem de Células , Colágeno Tipo I/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Fêmur/efeitos dos fármacos , Macaca fascicularis , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Distribuição Aleatória , Proteínas Recombinantes de Fusão/metabolismo , Estatísticas não Paramétricas , Vértebras Torácicas/efeitos dos fármacosRESUMO
Activin receptor-like kinase-1 (ALK1) is a type I, endothelial cell-specific member of the transforming growth factor-beta superfamily of receptors known to play an essential role in modulating angiogenesis and vessel maintenance. In the present study, we sought to examine the angiogenic and tumorigenic effects mediated upon the inhibition of ALK1 signaling using a soluble chimeric protein (ALK1-Fc). Of 29 transforming growth factor-beta-related ligands screened by surface plasmon resonance, only bone morphogenetic protein (BMP9) and BMP10 displayed high-affinity binding to ALK1-Fc. In cell-based assays, ALK1-Fc inhibited BMP9-mediated Id-1 expression in human umbilical vein endothelial cells and inhibited cord formation by these cells on a Matrigel substrate. In a chick chorioallantoic membrane assay, ALK1-Fc reduced vascular endothelial growth factor-, fibroblast growth factor-, and BMP10-mediated vessel formation. The growth of B16 melanoma explants was also inhibited significantly by ALK1-Fc in this assay. Finally, ALK1-Fc treatment reduced tumor burden in mice receiving orthotopic grafts of MCF7 mammary adenocarcinoma cells. These data show the efficacy of chimeric ALK1-Fc proteins in mitigating vessel formation and support the view that ALK1-Fc is a powerful antiangiogenic agent capable of blocking vascularization.
Assuntos
Receptores de Activinas Tipo II/metabolismo , Fragmentos Fc das Imunoglobulinas/metabolismo , Neoplasias/metabolismo , Neovascularização Patológica , Proteínas Recombinantes de Fusão/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Células CHO , Cricetinae , Cricetulus , Células Endoteliais/citologia , Endotélio Vascular/citologia , Fator 2 de Diferenciação de Crescimento/metabolismo , Humanos , Camundongos , Ressonância de Plasmônio de Superfície , Telangiectasia Hemorrágica Hereditária/metabolismoRESUMO
A recent study suggests that activin inhibits bone matrix mineralization, whereas treatment of mice with a soluble form of the activin type IIA receptor markedly increases bone mass and strength. To further extend these observations, we determined the skeletal effects of inhibiting activin signaling through the ActRIIA receptor in a large animal model with a hormonal profile and bone metabolism similar to humans. Ten female cynomolgus monkeys (Macaca fascicularis) were divided into two weight-matched groups and treated biweekly, for 3 months, with either a subcutaneous injection 10 mg/kg of a soluble form of the ActRIIA receptor fused with the Fc portion of human IgG(1) (ACE-011) or vehicle (VEH). Bone mineral density (BMD), micro-architecture, compressive mechanical properties, and ash fraction were assessed at the end of the treatment period. BMD was significantly higher in ACE-011 treated individuals compared to VEH: +13% (p=0.003) in the 5th lumbar vertebral body and +15% (p=0.05) in the distal femur. In addition, trabecular volumetric bone density at the distal femur was 72% (p=0.0004) higher than the VEH-treated group. Monkeys treated with ACE-011 also had a significantly higher L5 vertebral body trabecular bone volume (p=0.002) and compressive mechanical properties. Ash fraction of L4 trabecular bone cores did not differ between groups. These results demonstrate that treatment with a soluble form of ActRIIA (ACE-011) enhances bone mass and bone strength in cynomolgus monkeys, and provide strong rationale for exploring the use of ACE-011 to prevent and/or treat skeletal fragility.
Assuntos
Receptores de Activinas Tipo II/farmacologia , Densidade Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Macaca fascicularis/metabolismo , Animais , Conservadores da Densidade Óssea/farmacologia , Feminino , HumanosRESUMO
Androgen deprivation, a consequence of hypogonadism, certain cancer treatments, or normal aging in men, leads to loss of muscle mass, increased adiposity, and osteoporosis. In the present study, using a soluble chimeric form of activin receptor type IIB (ActRIIB) we sought to offset the adverse effects of androgen deprivation on muscle, adipose tissue, and bone. Castrated (ORX) or sham-operated (SHAM) mice received either TBS [vehicle-treated (VEH)] or systemic administration of ActRIIB-mFc, a soluble fusion protein comprised of a form of the extracellular domain of ActRIIB fused to a murine IgG2aFc subunit. In vivo body composition imaging demonstrated that ActRIIB-mFc treatment results in increased lean tissue mass of 23% in SHAM mice [19.02 +/- 0.42 g (VEH) versus 23.43 +/- 0.35 g (ActRIIB-mFc), P < 0.00001] and 26% in ORX mice [15.59 +/- 0.26 g (VEH) versus 19.78 +/- 0.26 g (ActRIIB-mFc), P < 0.00001]. Treatment also caused a decrease in adiposity of 30% in SHAM mice [5.03 +/- 0.48 g (VEH) versus 3.53 +/- 0.19 g (ActRIIB-mFc), NS] and 36% in ORX mice [7.12 +/- 0.53 g (VEH) versus 4.57 +/- 0.28 g (ActRIIB-mFc), P < 0.001]. These changes were also accompanied by altered serum levels of leptin, adiponectin, and insulin, as well as by prevention of steatosis (fatty liver) in ActRIIB-mFc-treated ORX mice. Finally, ActRIIB-mFc prevented loss of bone mass in ORX mice as assessed by whole body dual x-ray absorptiometry and micro-computed tomography of proximal tibias. The data demonstrate that treatment with ActRIIB-mFc restored muscle mass, adiposity, and bone quality to normal levels in a mouse model of androgen deprivation, thereby alleviating multiple adverse consequences of such therapy.
Assuntos
Receptores de Activinas Tipo II/farmacologia , Antagonistas de Androgênios/farmacologia , Composição Corporal/efeitos dos fármacos , Densidade Óssea/efeitos dos fármacos , Receptores de Activinas Tipo II/genética , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Análise de Variância , Animais , Peso Corporal/efeitos dos fármacos , Linhagem Celular , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Leptina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Obesidade/sangue , Obesidade/prevenção & controle , Orquiectomia , Distribuição Aleatória , Proteínas Recombinantes de Fusão/farmacologia , SolubilidadeRESUMO
The effects of ACE-011 on safety, pharmacokinetics, and bone biomarkers were evaluated in healthy, postmenopausal women. Our data indicate that ACE-011 results in a sustained increase in biomarkers of bone formation and reduction in markers of bone resorption. The activin type IIA receptor (ActRIIA) is the high-affinity receptor for activin. ACE-011 is a dimeric fusion protein consisting of the extracellular domain of the human ActRIIA linked to the Fc portion of human IgG1. ACE-011 binds to activin, preventing activin from binding endogenous receptors. A randomized, double-blind, placebo-controlled study was conducted to evaluate the safety and tolerability of ACE-011. Forty-eight healthy, postmenopausal women were randomized to receive either a single dose of ACE-011 or placebo and were followed for 4 mo. Dose levels ranged from 0.01 to 3.0 mg/kg intravenously and from 0.03 to 0.1 mg/kg subcutaneously. Safety and pharmacokinetic (PK) analyses and the biological activity of ACE-011, as assessed by markers of bone turnover, and follicle stimulating hormone (FSH) levels were measured. No serious adverse events (AEs) were reported. AEs were generally mild and transient. The PK of ACE-011 was linear over the dose range studied, with a mean half-life of 24-32 days. The absorption after subcutaneous dosing was essentially complete. ACE-011 caused a rapid and sustained dose-dependent increase in serum levels of bone-specific alkaline phosphatase (BSALP) and a dose-dependent decrease in C-terminal type 1 collagen telopeptide (CTX) and TRACP-5b levels. There was also a dose-dependent decrease in serum FSH levels consistent with inhibition of activin. ACE-011 is a novel agent with biological evidence of both an increase in bone formation and a decrease in bone resorption. ACE-011 may be an effective therapy in a variety of diseases involving bone loss.
Assuntos
Pós-Menopausa/efeitos dos fármacos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacologia , Idoso , Biomarcadores/metabolismo , Reabsorção Óssea/metabolismo , Demografia , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Hormônio Foliculoestimulante/sangue , Humanos , Injeções Intravenosas , Injeções Subcutâneas , Pessoa de Meia-Idade , Osteogênese/efeitos dos fármacos , Placebos , Pós-Menopausa/sangue , Proteínas Recombinantes de Fusão/efeitos adversos , Proteínas Recombinantes de Fusão/farmacocinéticaRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurologic disease characterized by progressive weakness that results in death within a few years of onset by respiratory failure. Myostatin is a member of the TGF-beta superfamily that is predominantly expressed in muscle and acts as a negative regulator of muscle growth. Attenuating myostatin has previously been shown to produce increased muscle mass and strength in normal and disease animal models. In this study, a mouse model of ALS (SOD1(G93A) transgenic mice) was treated with a soluble activin receptor, type IIB (ActRIIB.mFc) which is a putative endogenous signaling receptor for myostatin in addition to other ligands of the TGF-beta superfamily. ActRIIB.mFc treatment produces a delay in the onset of weakness, an increase in body weight and grip strength, and an enlargement of muscle size whether initiated pre-symptomatically or after symptom onset. Treatment with ActRIIB.mFc did not increase survival or neuromuscular junction innervation in SOD1(G93A) transgenic mice. Pharmacologic treatment with ActRIIB.mFc was superior in all measurements to genetic deletion of myostatin in SOD1(G93A) transgenic mice. The improved function of SOD1(G93A) transgenic mice following treatment with ActRIIB.mFc is encouraging for the development of TGF-beta pathway inhibitors to increase muscle strength in patients with ALS.
Assuntos
Receptores de Activinas Tipo II/uso terapêutico , Esclerose Lateral Amiotrófica/tratamento farmacológico , Debilidade Muscular/tratamento farmacológico , Miostatina/antagonistas & inibidores , Receptores de Activinas Tipo II/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Peso Corporal/efeitos dos fármacos , Peso Corporal/fisiologia , Células CHO , Cricetinae , Cricetulus , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Força Muscular/efeitos dos fármacos , Força Muscular/fisiologia , Debilidade Muscular/etiologia , Debilidade Muscular/fisiopatologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Miostatina/metabolismo , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/uso terapêutico , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Resultado do TratamentoRESUMO
The nadD gene, encoding the enzyme nicotinic acid mononucleotide (NaMN) adenylyltransferase (AT), is essential for the synthesis of NAD and subsequent viability of the cell. The nadD gene in Bacillus subtilis (yqeJ) was identified by sequence homology with other bacterial nadD genes and by biochemical characterization of the gene product. NaMN AT catalyzes the reversible adenylation of both NaMN and the nicotinamide mononucleotide (NMN) but shows specificity for the nicotinate. In contrast to other known NMN ATs, biophysical characterizations reveal it to be a dimer. The NaMN AT crystal structure was determined for both the apo enzyme and product-bound form, to 2.1 and 3.2 A, respectively. The structures reveal a "functional" dimer conserved in both crystal forms and a monomer fold common to members of the nucleotidyl-transferase alpha/beta phosphodiesterase superfamily. A structural comparison with family members suggests a new conserved motif (SXXXX(R/K)) at the N terminus of an alpha-helix, which is not part of the shared fold. Interactions of the nicotinic acid with backbone atoms indicate the structural basis for specificity.