RESUMEN
Mutant activin receptor-like kinase-2 (ALK2) was reported to be closely associated with the pathogenesis of fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG), and therefore presents an attractive target for therapeutic intervention. Through in silico virtual screenings and structure-activity relationship studies assisted by X-ray crystallographic analyses, a novel series of bis-heteroaryl pyrazole was identified as potent inhibitors of ALK2 (R206H). Derived from in silico hit compound RK-59638 (6a), compound 18p was identified as a potent inhibitor of ALK2 (R206H) with good aqueous solubility, liver microsomal stability, and oral bioavailability.
Asunto(s)
Receptores de Activinas Tipo I/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Administración Oral , Animales , Área Bajo la Curva , Disponibilidad Biológica , Simulación por Computador , Cristalografía por Rayos X , Semivida , Humanos , Microsomas Hepáticos/efectos de los fármacos , Estructura Molecular , Miositis Osificante/enzimología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Espectroscopía de Protones por Resonancia Magnética , Pirazoles/administración & dosificación , Pirazoles/química , Pirazoles/farmacocinética , Solubilidad , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-ActividadRESUMEN
Fibrodysplasia ossificans progressiva (FOP) is a rare and intractable disorder characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor gain-of-function mutations in ACVR1 (FOP-ACVR1), a type I receptor for bone morphogenetic proteins. Despite numerous studies, no drugs have been approved for FOP. Here, we developed a high-throughput screening (HTS) system focused on the constitutive activation of FOP-ACVR1 by utilizing a chondrogenic ATDC5 cell line that stably expresses FOP-ACVR1. After HTS of 5,000 small-molecule compounds, we identified two hit compounds that are effective at suppressing the enhanced chondrogenesis of FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) and suppressed the heterotopic ossification (HO) of multiple model mice, including FOP-ACVR1 transgenic mice and HO model mice utilizing FOP-iPSCs. Furthermore, we revealed that one of the hit compounds is an mTOR signaling modulator that indirectly inhibits mTOR signaling. Our results demonstrate that these hit compounds could contribute to future drug repositioning and the mechanistic analysis of mTOR signaling.
Asunto(s)
Miositis Osificante/enzimología , Miositis Osificante/patología , Osificación Heterotópica/enzimología , Osificación Heterotópica/patología , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Receptores de Activinas Tipo I/metabolismo , Animales , Benzodioxoles/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Ratones SCID , Ratones Transgénicos , Oxazoles/farmacología , Pirimidinas/farmacología , Quinazolinas/farmacología , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Triazoles/farmacología , Urea/análogos & derivados , Urea/farmacologíaRESUMEN
Severe variants of fibrodysplasia ossificans progressiva (FOP) affect <2% of all FOP patients worldwide, but provide an unprecedented opportunity to probe the phenotype-genotype relationships that propel the pathology of this disabling disease. We evaluated two unrelated children who had severe reduction deficits of the hands and feet with absence of nails, progressive heterotopic ossification, hypoplasia of the brain stem, motor and cognitive developmental delays, facial dysmorphology, small malformed teeth, and abnormal hair development. One child had sensorineural hearing loss, microcytic anemia, and a tethered spinal cord and the other had a patent ductus arteriosus and gonadal dysgenesis with sex reversal (karyotype 46, XY female). Both children had an identical mutation in ACVR1 c.772A>G; p.Arg258Gly (R258G), not previously described in FOP. Although many, if not most, FOP mutations directly perturb the structure of the GS regulatory subdomain and presumably the adjacent αC helix, substitution with glycine at R258 may directly alter the position of the helix in the kinase domain, eliminating a key aspect of the autoinhibitory mechanism intrinsic to the wild-type ACVR1 kinase. The high fidelity phenotype-genotype relationship in these unrelated children with the most severe FOP phenotype reported to date suggests that the shared features are due to the dysregulated activity of the mutant kinase during development and postnatally, and provides vital insight into the structural biology and function of ACVR1 as well as the design of small molecule inhibitors.
Asunto(s)
Anomalías Múltiples/patología , Receptores de Activinas Tipo I/genética , Mutación , Miositis Osificante/patología , Anomalías Múltiples/diagnóstico , Anomalías Múltiples/enzimología , Anomalías Múltiples/genética , Receptores de Activinas Tipo I/metabolismo , Sustitución de Aminoácidos , Femenino , Expresión Génica , Estudios de Asociación Genética , Genotipo , Humanos , Lactante , Cariotipo , Modelos Moleculares , Miositis Osificante/diagnóstico , Miositis Osificante/enzimología , Miositis Osificante/genética , Fenotipo , Estructura Terciaria de Proteína , Índice de Severidad de la EnfermedadRESUMEN
Bone morphogenetic protein (BMP) receptor kinases are tightly regulated to control development and tissue homeostasis. Mutant receptor kinase domains escape regulation leading to severely degenerative diseases and represent an important therapeutic target. Fibrodysplasia ossificans progressiva (FOP) is a rare but devastating disorder of extraskeletal bone formation. FOP-associated mutations in the BMP receptor ALK2 reduce binding of the inhibitor FKBP12 and promote leaky signaling in the absence of ligand. To establish structural mechanisms of receptor regulation and to address the effects of FOP mutation, we determined the crystal structure of the cytoplasmic domain of ALK2 in complex with the inhibitors FKBP12 and dorsomorphin. FOP mutations break critical interactions that stabilize the inactive state of the kinase, thereby facilitating structural rearrangements that diminish FKBP12 binding and promote the correct positioning of the glycine-serine-rich loop and αC helix for kinase activation. The balance of these effects accounts for the comparable activity of R206H and L196P. Kinase activation in the clinically benign mutant L196P is far weaker than R206H but yields equivalent signals due to the stronger interaction of FKBP12 with R206H. The presented ALK2 structure offers a valuable template for the further design of specific inhibitors of BMP signaling.
Asunto(s)
Receptores de Activinas Tipo I/química , Miositis Osificante/enzimología , Receptores de Activinas Tipo I/antagonistas & inhibidores , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo I/metabolismo , Secuencias de Aminoácidos , Animales , Proteína Morfogenética Ósea 4/fisiología , Dominio Catalítico , Cristalografía por Rayos X , Activación Enzimática , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Ratones , Modelos Moleculares , Mutación Missense , Miositis Osificante/genética , Unión Proteica , Pirazoles/química , Pirimidinas/química , Transducción de Señal , Tacrolimus/farmacología , Proteína 1A de Unión a Tacrolimus/antagonistas & inhibidores , Proteína 1A de Unión a Tacrolimus/química , Proteína 1A de Unión a Tacrolimus/metabolismoRESUMEN
A single recurrent mutation in the regulatory subdomain of a bone morphogenetic protein type I receptor kinase has been linked to heterotopic ossification in classic fibrodysplasia ossificans progressiva (FOP). As a result of a substitution at 1 residue by only 1 other side chain (Arg206His) in just 1 of the 4 type I BMP receptors (ALK2/ACVR1), soft connective tissues progressively metamorphose through an endochondral process into cartilage that is replaced by bone. The substitution of arginine for histidine, also a basic residue yet with the singular property of ionization/protonation over the physiological pH range, led to the hypothesis of an aberrant, pH-sensitive switch mechanism for the ligand-independent activation of BMP signaling through the mutant receptor kinase in patients presenting with classic FOP. To test a potential aspect of the putative pH-dependent mechanism, i.e. loss of autoinhibition of the kinase mediated by the inhibitory protein FKBP12, in vitrointeraction analyses with purified wild-type and R206H ALK2 kinase and FKBP12 proteins were performed. Interactions between the kinases and inhibitory proteins were analyzed qualitatively and quantitatively by native gel electrophoresis and HPLC size exclusion chromatography and with an optical biosensor (Octet; ForteBio). Binding of inhibitory protein by the R206H mutant was diminished 3-fold relative to the wild type kinase at a physiological pH, yet below this value (<~7.5) pronounced nonspecific interactions, particularly with the mutant, prevented comparative evaluations. In conclusion, substitution with histidine leads to partial loss of inhibition of the mutant type I receptor through diminished binding of FKBP12, which may act as a gradient reader in morphogenetic contexts.
Asunto(s)
Receptores de Activinas Tipo I/metabolismo , Sustitución de Aminoácidos/genética , Miositis Osificante/complicaciones , Miositis Osificante/enzimología , Osificación Heterotópica/complicaciones , Osificación Heterotópica/enzimología , Proteína 1A de Unión a Tacrolimus/metabolismo , Técnicas Biosensibles , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Humanos , Interferometría , Unión ProteicaRESUMEN
Fibrodysplasia ossificans progressiva (FOP) is a rare disabling disease characterized by heterotopic ossification for which there is currently no treatment available. FOP has been linked recently to a heterozygous R206H mutation in the bone morphogenetic protein (BMP) type I receptor activin receptor-like kinase 2 (ALK2). Expression of the mutant ALK2-R206H receptor (FOP-ALK2) results in increased phosphorylation of the downstream Smad1 effector proteins and elevated basal BMP-dependent transcriptional reporter activity, indicating that FOP-ALK2 is constitutively active. FOP-ALK2-induced transcriptional activity could be blocked by overexpressing either of the inhibitory Smads, Smad6 or -7, or by treatment with the pharmacological BMP type I receptor inhibitor dorsomorphin. However, in contrast to wild-type ALK2, FOP-ALK2 is not inhibited by the negative regulator FKBP12. Mesenchymal cells expressing the FOP-ALK2 receptor are more sensitive to undergoing BMP-induced osteoblast differentiation and mineralization. In vivo bone formation was assessed by loading human mesenchymal stem cells (hMSCs) expressing the ALK2-R206H receptor onto calcium phosphate scaffolds and implantation in nude mice. Compared with control cells FOP-ALK2-expressing cells induced increased bone formation. Taken together, the R206H mutation in ALK2 confers constitutive activity to the mutant receptor, sensitizes mesenchymal cells to BMP-induced osteoblast differentiation, and stimulates new bone formation. We have generated an animal model that can be used as a stepping stone for preclinical studies aimed at inhibiting the heterotopic ossification characteristic of FOP.
Asunto(s)
Receptores de Activinas Tipo I/genética , Proteínas Morfogenéticas Óseas/farmacología , Células Madre Mesenquimatosas/citología , Mutación/genética , Miositis Osificante/genética , Osteoblastos/citología , Osteogénesis/efectos de los fármacos , Receptores de Activinas Tipo I/metabolismo , Sustitución de Aminoácidos/genética , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Fosfatos de Calcio/farmacología , Bovinos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Coristoma/metabolismo , Coristoma/patología , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/enzimología , Ratones , Proteínas Mutantes/metabolismo , Miositis Osificante/enzimología , Miositis Osificante/patología , Osteoblastos/efectos de los fármacos , Andamios del TejidoRESUMEN
Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant congenital disorder characterized by progressive heterotopic bone formation in muscle tissues. A common mutation among FOP patients has been identified in ALK2, ALK2(R206H), which encodes a constitutively active bone morphogenetic protein (BMP) receptor. Recently, a unique mutation of ALK2, ALK2(G356D), was identified to be a novel mutation in a Japanese FOP patient who had unique clinical features. Over-expression of ALK2(G356D) induced phosphorylation of Smad1/5/8 and activated Id1-luc and alkaline phosphatase activity in myoblasts. However, the over-expression failed to activate phosphorylation of p38, ERK1/2, and CAGA-luc activity. These ALK2(G356D) activities were weaker than those of ALK2(R206H), and they were suppressed by a specific inhibitor of the BMP-regulated Smad pathway. These findings suggest that ALK2(G356D) induces heterotopic bone formation via activation of a BMP-regulated Smad pathway. The quantitative difference between ALK2(G356D) and ALK2(R206H) activities may have caused the phenotypic differences in these patients.
Asunto(s)
Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo I/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Mutación , Miositis Osificante/enzimología , Miositis Osificante/genética , Receptores de Activinas Tipo I/antagonistas & inhibidores , Sustitución de Aminoácidos , Animales , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/agonistas , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/antagonistas & inhibidores , Diferenciación Celular , Glicina/genética , Glicina/metabolismo , Humanos , Ligandos , Ratones , Desarrollo de Músculos/efectos de los fármacos , Desarrollo de Músculos/genética , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Osteoblastos/efectos de los fármacos , Pirazoles/farmacología , Pirimidinas/farmacología , Transducción de Señal , Proteínas Smad/metabolismoRESUMEN
UNLABELLED: FOP is a disorder in which skeletal muscle is progressively replaced with bone. FOP lymphocytes, a model system for exploring the BMP pathway in these patients, exhibit a defect in BMPRIA internalization and increased activation of downstream signaling, suggesting that altered BMP receptor trafficking underlies ectopic bone formation in this disease. INTRODUCTION: Fibrodysplasia ossificans progressiva (FOP) is a severely disabling disorder characterized by progressive heterotopic ossification of connective tissues. Whereas the genetic defect and pathophysiology of this condition remain enigmatic, BMP4 mRNA and protein are overexpressed, and mRNAs for a subset of secreted BMP antagonists are not synthesized at appropriate levels in cultured lymphocytes from FOP patients. These data suggest involvement of altered BMP signaling in the disease. In this study, we investigate whether the abnormality is associated with defective BMP receptor function in lymphocytes. MATERIALS AND METHODS: Cell surface proteins were quantified by fluorescence-activated cell sorting (FACS). Protein phosphorylation was assayed by immunoprecipitation and immunoblotting. Protein synthesis and degradation were examined by [35S]methionine labeling and pulse-chase assays. mRNA was detected by RT-PCR. RESULTS: FOP lymphocytes expressed 6-fold higher levels of BMP receptor type IA (BMPRIA) on the cell surface compared with control cells and displayed a marked reduction in ligand-stimulated internalization and degradation of BMPRIA. Moreover, in control cells, BMP4 treatment increased BMPRIA phosphorylation, whereas BMPRIA showed ligand-insensitive constitutive phosphorylation in FOP cells. Our data additionally support that the p38 mitogen-activated protein kinase (MAPK) signaling pathway is a major BMP signaling pathway in these cell lines and that expression of inhibitor of DNA binding and differentiation 1 (ID-1), a transcriptional target of BMP signaling, is enhanced in FOP cells. CONCLUSIONS: These data extend our previous observations of misregulated BMP4 signaling in FOP lymphocytes and show that cell surface overabundance and constitutive phosphorylation of BMPRIA are associated with a defect in receptor internalization. Altered BMP receptor trafficking may play a significant role in FOP pathogenesis.
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Proteínas Morfogenéticas Óseas/metabolismo , Membrana Celular/enzimología , Miositis Osificante/enzimología , Osteogénesis , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Proteína Morfogenética Ósea 4 , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1 , Membrana Celular/inmunología , Células Cultivadas , Humanos , Miositis Osificante/fisiopatología , Fosforilación , Proteínas Serina-Treonina Quinasas/análisis , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento/análisis , Receptores de Factores de Crecimiento/genética , Transducción de SeñalRESUMEN
To assess the role and status of telomerase activity in benign bone tumors and tumor-like lesions, we performed telomerase assays in four giant cell tumors of bone, four fibrous dysplasias, three osteochondromas, three aneurysmal bone cysts, two osteoblastomas, one juvenile bone cyst and one myositis ossificans. A very sensitive non-radioactive TRAP assay was applied. Low level activity was detected in 7 of 18 tumor samples (38.9%), and high level activity was not detected in any of the cases. Telomerase activity was observed in all patients with osteochondromas, in two of the three aneurysmal bone cysts, in one of the four giant cell tumors of bone and in one of the four fibrous dysplasias, but not in osteoblastomas, juvenile bone cyst and myositis ossificans. Although the origin of this enzyme is still unclear, it might play a role in precancerous immortalization of benign bone tumors. Other possible reasons explaining the occurrence of telomerase activity, such as migrating lymphocytes or contamination of immortalized non-tumor cells, should not be ruled out. Telomerase activity, however, does exist in those samples having no malignant phenotype, for which reason telomerase assays are not always useful for the clinical and diagnostic approach in benign bone tumors. Determination of the telomerase status in benign lesions may contribute to a better understanding of the regulation mechanism of telomerase activity during progression of bone tumors.
Asunto(s)
Neoplasias Óseas/enzimología , Neoplasias de Tejido Óseo/enzimología , Telomerasa/metabolismo , Adolescente , Adulto , Quistes Óseos Aneurismáticos/enzimología , Quistes Óseos Aneurismáticos/genética , Quistes Óseos Aneurismáticos/patología , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Línea Celular , Niño , Femenino , Displasia Fibrosa Ósea/enzimología , Displasia Fibrosa Ósea/genética , Displasia Fibrosa Ósea/patología , Tumor Óseo de Células Gigantes/enzimología , Tumor Óseo de Células Gigantes/genética , Tumor Óseo de Células Gigantes/patología , Humanos , Masculino , Persona de Mediana Edad , Miositis Osificante/enzimología , Miositis Osificante/genética , Miositis Osificante/patología , Neoplasias de Tejido Óseo/genética , Neoplasias de Tejido Óseo/patología , Osteocondroma/enzimología , Osteocondroma/genética , Osteocondroma/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosAsunto(s)
Fosfatasa Alcalina/metabolismo , Miositis Osificante/enzimología , Piel/enzimología , Adolescente , Adulto , Anciano , Envejecimiento , Células Cultivadas , Niño , Preescolar , Femenino , Feto , Fibroblastos/enzimología , Humanos , Lactante , Persona de Mediana Edad , Embarazo , Valores de ReferenciaRESUMEN
The cytoplasm of tumor cells from a subdermal nodule in a patient with fibrodysplasia ossificans progressiva (FOP) exhibited intense enzymatic activity in cryostat sections processed for demonstration of alkaline phosphatase. Nuclear heterochromatin and nucleoli, particularly in the area of the dense component, also showed strong reactivity. Finely minced blocks from the lesion of the patient with FOP revealed lighter reactivity which, in the tumor cells, avoided membrane limited spaces and appeared to be confined to hyaloplasm. Extracellular spaces disclosed very little or no reactivity and specimens from the patient's uninvolved skin lacked staining. The tumor cells from the subdermal nodule did not exhibit increased acid phosphatase activity. Cells (L-FOP) derived from a subdermal nodule and grown by tissue culture techniques also synthesized large amounts of prostaglandin E-like material and alkaline phosphatase. The amounts of prostaglandin E-like material synthesized by these L-FOP cells was reduced by more than 31 per cent by the antiinflammatory drugs indomethacin and sodium meclofenmate. Also, the production of alkaline phosphatase by these L-FOP cells was reduced by more than 40 per cent by ethane-1-hydroxyl-1,1-diphosphonate. Addition of prostaglandin E to nonlesion cells did not result in increased alkaline phosphatase activity.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Miositis Osificante/enzimología , Fosfatasa Alcalina/análisis , Nucléolo Celular/enzimología , Células Cultivadas , Citoplasma/enzimología , Ácido Etidrónico/farmacología , Femenino , Aparato de Golgi/enzimología , Humanos , Indometacina/farmacología , Lactante , Macrófagos/enzimología , Ácido Meclofenámico/farmacología , Prostaglandinas E/biosíntesisRESUMEN
By light microscopy the subdermal nodule of a patient with fibrodysplasia ossificans progressiva (FOP) had a fibromatoid histologic appearance. The cytoplasm of the cells stained strongly for mannose-rich glycoprotein with the concanavalin A-horseradish peroxidase (con A-HRP) method. The tumors also exhibited abundant hyaluronidase-digestible mucopolysaccharide in the interstitium with various basic staining reagents. This material appeared to consist principally of hyaluronic acid or chondroitin sulfate with few or mainly masked sulfate esters. At the ultrastructural level, cells interpreted as the tumor cells in the subdermal nodule from the patient displayed extremely hyperplastic granular reticulum and well-developed Golgi elements and appeared very active in synthesis and secretion of protein. The material in the dilated cisternae of the granular reticulum stained for glycoprotein with the con-A-HRP method. Macrophages which comprised the other main cell type in the nodules commonly contacted the tumor cells and occasionally evidenced engulfment of these cells. The intercellular matrix of the nonossified subdermal nodule exhibited greatly increased mucosubstance and, by electron microscopy, showed an unusual network of dialyzed iron-reactive acid muco-substance in the interstitium.
Asunto(s)
Miositis Osificante/patología , Fosfatasa Alcalina/metabolismo , División Celular , Colágeno/metabolismo , Femenino , Fibroblastos/ultraestructura , Glicosaminoglicanos/metabolismo , Aparato de Golgi/ultraestructura , Humanos , Lactante , Uniones Intercelulares/ultraestructura , Macrófagos/ultraestructura , Miositis Osificante/enzimología , Miositis Osificante/metabolismo , Prostaglandinas E/metabolismo , Piel/ultraestructuraRESUMEN
Alkaline phosphatase activity in four strains of cultured skin fibroblasts obtained from a patient with fibrodysplasia ossificans progressiva was at the low normal range. The enzyme activity in normal fibroblasts significantly increased at late confluency. It appears that the high levels of alkaline phosphatase activity reported in biopsies of lesions are not genetically determined but are secondary events of local tissue reaction.