RESUMO
Secreted frizzled related protein-1 (sFRP-1) inhibitors have the potential to be used for the treatment of osteoporosis or other bone related disorders, since the level of sFRP-1 affects osteoblast apoptosis and proliferation. From high throughput screening, we have identified a class of iminooxothiazolidines as sFRP-1 inhibitors. Structure-activity relationships were established for various regions of the scaffold along with the biochemical characterization of this class to probe selectivity, binding and ex vivo activity.
Assuntos
Osteogênese/fisiologia , Proteínas/isolamento & purificação , Calcificação Fisiológica , Diferenciação Celular/fisiologia , Células Cultivadas , Receptores Frizzled/antagonistas & inibidores , Receptores Frizzled/isolamento & purificação , Peptídeos e Proteínas de Sinalização Intracelular , Estrutura Molecular , Proteínas/antagonistas & inibidores , Ligante RANKRESUMO
Canonical Wnt signaling has been demonstrated to increase bone formation, and Wnt pathway components are being pursued as potential drug targets for osteoporosis and other metabolic bone diseases. Deletion of the Wnt antagonist secreted frizzled-related protein (sFRP)-1 in mice activates canonical signaling in bone and increases trabecular bone formation in aged animals. We have developed small molecules that bind to and inhibit sFRP-1 in vitro and demonstrate robust anabolic activity in an ex vivo organ culture assay. A library of over 440,000 drug-like compounds was screened for inhibitors of human sFRP-1 using a cell-based functional assay that measured activation of canonical Wnt signaling with an optimized T-cell factor (TCF)-luciferase reporter gene assay. One of the hits in this screen, a diarylsulfone sulfonamide, bound to sFRP-1 with a K(D) of 0.35 microM in a tryptophan fluorescence quenching assay. This compound also selectively inhibited sFRP-1 with an EC(50) of 3.9 microM in the cell-based functional assay. Optimization of this high throughput screening hit for binding and functional potency as well as metabolic stability and other pharmaceutical properties led to improved lead compounds. One of these leads (WAY-316606) bound to sFRP-1 with a K(D) of 0.08 microM and inhibited it with an EC(50) of 0.65 microM. Moreover, this compound increased total bone area in a murine calvarial organ culture assay at concentrations as low as 0.0001 microM. This work demonstrates the feasibility of developing small molecules that inhibit sFRP-1 and stimulate canonical Wnt signaling to increase bone formation.
Assuntos
Osteogênese/efeitos dos fármacos , Proteínas/antagonistas & inibidores , Sulfonamidas/farmacologia , Proteínas Wnt/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Técnicas de Cultura de Órgãos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteócitos/citologia , Osteócitos/efeitos dos fármacos , Proteínas/genética , Proteínas/metabolismo , Crânio/citologia , Crânio/efeitos dos fármacos , Espectrometria de Fluorescência , Sulfonamidas/químicaRESUMO
Ror2 is a receptor tyrosine kinase, the expression of which increases during differentiation of pluripotent stem cells to osteoblasts and then declines as cells progress to osteocytes. To test whether Ror2 plays a role in osteoblastogenesis, we investigated the effects of Ror2 overexpression and down-regulation on osteoblastic lineage commitment and differentiation. Expression of Ror2 in pluripotent human mesenchymal stem cells (hMSCs) by adenoviral infection caused formation of mineralized extracellular matrix, which is the ultimate phenotype of an osteogenic tissue. Concomitantly, Ror2 over-expression inhibited adipogenic differentiation of hMSCs as monitored by lipid formation. Ror2 shifted hMSC fate toward osteoblastogenesis by inducing osteogenic transcription factor osterix and suppressing adipogenic transcription factors CCAAT/enhancer-binding protein alpha and peroxisome proliferator activated receptor gamma. Infection with Ror2 virus also strongly promoted matrix mineralization in committed osteoblast-like MC3T3-E1 cells. Expression of Ror2 in a human preosteocytic cell line by stable transfection also promoted further differentiation, as judged by inhibited alkaline phosphatase activity, potentiated osteocalcin secretion, and increased cellular apoptosis. In contrast, down-regulation of Ror2 expression by short hairpin RNA essentially abrogated dexamethasone-induced mineralization of hMSCs. Furthermore, down-regulation of Ror2 expression in fully differentiated SaOS-2 osteosarcoma cells inhibited alkaline phosphatase activity. We conclude that Ror2 initiates commitment of MSCs to osteoblastic lineage and promotes differentiation at early and late stages of osteoblastogenesis. Finally, using a mouse calvariae ex vivo organ culture model, we demonstrate that these effects of Ror2 result in increased bone formation, suggesting that it may also activate mature osteoblasts.
Assuntos
Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Osteogênese , Células-Tronco Pluripotentes/citologia , Receptores de Superfície Celular/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Calcificação Fisiológica/fisiologia , Diferenciação Celular , Linhagem Celular , Dexametasona/farmacologia , Regulação para Baixo , Glucocorticoides/farmacologia , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Técnicas de Cultura de Órgãos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , PPAR gama/metabolismo , Células-Tronco Pluripotentes/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase , Crânio/citologia , Fator de Transcrição Sp7 , Fatores de Transcrição/metabolismoRESUMO
Ror2 is an orphan receptor tyrosine kinase that plays crucial roles in developmental morphogenesis, particularly of the skeleton. We have identified human Ror2 as a novel regulator of canonical Wnt signaling in osteoblastic (bone-forming) cells with selective activities, enhancing Wnt1 but antagonizing Wnt3. Immunoprecipitation studies demonstrated physical interactions between human Ror2 and mammalian Wnt1 and Wnt3. Functionally, Ror2 antagonized Wnt1- and Wnt3-mediated stabilization of cytosolic beta-catenin in osteoblastic cells. However, Ror2 had opposing effects on a more distal step of canonical Wnt signaling: it potentiated Wnt1 activity but inhibited Wnt3 function as assessed by changes in Wnt-responsive reporter gene activity. Despite binding to Ror2, neither Wnt1 nor Wnt3 altered receptor activity as assessed by levels of Ror2 autophosphorylation. The ability of Ror2 to regulate canonical Wnt signaling in osteoblastic cells should have physiological consequences in bone, because Wnt signaling is known to modulate osteoblast survival and differentiation. Expression of Ror2 mRNA was highly regulated in a biphasic manner during human osteoblast differentiation, being virtually undetectable in pluripotent stem cells, increasing 300-fold in committed preosteoblasts, and disappearing again in osteocytes. Furthermore, Ror2 expression in osteoblasts was suppressed by the Wnt antagonist, secreted frizzled-related protein 1. The regulated expression of Ror2 during osteoblast differentiation, its inverse expression pattern with secreted frizzled-related protein 1, and its ability to modulate Wnt signaling in osteoblastic cells suggest that Ror2 may regulate bone formation.