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High-throughput screening for modulators of ACVR1 transcription: discovery of potential therapeutics for fibrodysplasia ossificans progressiva

View Article: PubMed Central - PubMed

ABSTRACT

The ACVR1 gene encodes a type I receptor of bone morphogenetic proteins (BMPs). Activating mutations in ACVR1 are responsible for fibrodysplasia ossificans progressiva (FOP), a rare disease characterized by congenital toe malformation and progressive heterotopic endochondral ossification leading to severe and cumulative disability. Until now, no therapy has been available to prevent soft-tissue swelling (flare-ups) that trigger the ossification process. With the aim of finding a new therapeutic strategy for FOP, we developed a high-throughput screening (HTS) assay to identify inhibitors of ACVR1 gene expression among drugs already approved for the therapy of other diseases. The screening, based on an ACVR1 promoter assay, was followed by an in vitro and in vivo test to validate and characterize candidate molecules. Among compounds that modulate the ACVR1 promoter activity, we selected the one showing the highest inhibitory effect, dipyridamole, a drug that is currently used as a platelet anti-aggregant. The inhibitory effect was detectable on ACVR1 gene expression, on the whole Smad-dependent BMP signaling pathway, and on chondrogenic and osteogenic differentiation processes by in vitro cellular assays. Moreover, dipyridamole reduced the process of heterotopic bone formation in vivo. Our drug repositioning strategy has led to the identification of dipyridamole as a possible therapeutic tool for the treatment of FOP. Furthermore, our study has also defined a pipeline of assays that will be useful for the evaluation of other pharmacological inhibitors of heterotopic ossification.

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Cellular assays of Dipy treatment. (A) Dose-response curve of Dipy on the luciferase reporter gene controlled by the promoter region of ACVR1 in ATDC5 cells (Pr2.9-Luc). The ratio of luciferase (Luc)/fluorescence (Fluo) was normalized to that obtained with DMSO [untreated (Un); value 100]. Bar graph represents the mean and s.d. of three independent experiments. §P<0.001. (B) Time course of Dipy treatment in ATDC5 Pr2.9-Luc clones. The ratio of luciferase/fluorescence was normalized to that obtained with DMSO (Un; value 100) for each time point. (C) Effect of Dipy on the expression of ACVR1 mRNA in native ATDC5 and C2C12 cells. Values were normalized on GAPDH and β2M and compared to expression level measured in cells treated with DMSO (Un). Bar graphs represent the mean and s.d. of at least three experiments, *P<0.01, §P<0.001, ns, non-significant.
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DMM023929F1: Cellular assays of Dipy treatment. (A) Dose-response curve of Dipy on the luciferase reporter gene controlled by the promoter region of ACVR1 in ATDC5 cells (Pr2.9-Luc). The ratio of luciferase (Luc)/fluorescence (Fluo) was normalized to that obtained with DMSO [untreated (Un); value 100]. Bar graph represents the mean and s.d. of three independent experiments. §P<0.001. (B) Time course of Dipy treatment in ATDC5 Pr2.9-Luc clones. The ratio of luciferase/fluorescence was normalized to that obtained with DMSO (Un; value 100) for each time point. (C) Effect of Dipy on the expression of ACVR1 mRNA in native ATDC5 and C2C12 cells. Values were normalized on GAPDH and β2M and compared to expression level measured in cells treated with DMSO (Un). Bar graphs represent the mean and s.d. of at least three experiments, *P<0.01, §P<0.001, ns, non-significant.

Mentions: Dipy showed a dose-dependent suppression of the luciferase activity driven by the ACVR1 promoter, with the strongest effect at 50 µM (Fig. 1A). The inhibition was detectable after 6 h of treatment for the highest dose, further increasing at 24 h (Fig. 1B). Normalization of the luciferase activity and monitoring of cell viability were obtained as described for the primary screening.Fig. 1.


High-throughput screening for modulators of ACVR1 transcription: discovery of potential therapeutics for fibrodysplasia ossificans progressiva
Cellular assays of Dipy treatment. (A) Dose-response curve of Dipy on the luciferase reporter gene controlled by the promoter region of ACVR1 in ATDC5 cells (Pr2.9-Luc). The ratio of luciferase (Luc)/fluorescence (Fluo) was normalized to that obtained with DMSO [untreated (Un); value 100]. Bar graph represents the mean and s.d. of three independent experiments. §P<0.001. (B) Time course of Dipy treatment in ATDC5 Pr2.9-Luc clones. The ratio of luciferase/fluorescence was normalized to that obtained with DMSO (Un; value 100) for each time point. (C) Effect of Dipy on the expression of ACVR1 mRNA in native ATDC5 and C2C12 cells. Values were normalized on GAPDH and β2M and compared to expression level measured in cells treated with DMSO (Un). Bar graphs represent the mean and s.d. of at least three experiments, *P<0.01, §P<0.001, ns, non-significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920148&req=5

DMM023929F1: Cellular assays of Dipy treatment. (A) Dose-response curve of Dipy on the luciferase reporter gene controlled by the promoter region of ACVR1 in ATDC5 cells (Pr2.9-Luc). The ratio of luciferase (Luc)/fluorescence (Fluo) was normalized to that obtained with DMSO [untreated (Un); value 100]. Bar graph represents the mean and s.d. of three independent experiments. §P<0.001. (B) Time course of Dipy treatment in ATDC5 Pr2.9-Luc clones. The ratio of luciferase/fluorescence was normalized to that obtained with DMSO (Un; value 100) for each time point. (C) Effect of Dipy on the expression of ACVR1 mRNA in native ATDC5 and C2C12 cells. Values were normalized on GAPDH and β2M and compared to expression level measured in cells treated with DMSO (Un). Bar graphs represent the mean and s.d. of at least three experiments, *P<0.01, §P<0.001, ns, non-significant.
Mentions: Dipy showed a dose-dependent suppression of the luciferase activity driven by the ACVR1 promoter, with the strongest effect at 50 µM (Fig. 1A). The inhibition was detectable after 6 h of treatment for the highest dose, further increasing at 24 h (Fig. 1B). Normalization of the luciferase activity and monitoring of cell viability were obtained as described for the primary screening.Fig. 1.

View Article: PubMed Central - PubMed

ABSTRACT

The ACVR1 gene encodes a type I receptor of bone morphogenetic proteins (BMPs). Activating mutations in ACVR1 are responsible for fibrodysplasia ossificans progressiva (FOP), a rare disease characterized by congenital toe malformation and progressive heterotopic endochondral ossification leading to severe and cumulative disability. Until now, no therapy has been available to prevent soft-tissue swelling (flare-ups) that trigger the ossification process. With the aim of finding a new therapeutic strategy for FOP, we developed a high-throughput screening (HTS) assay to identify inhibitors of ACVR1 gene expression among drugs already approved for the therapy of other diseases. The screening, based on an ACVR1 promoter assay, was followed by an in vitro and in vivo test to validate and characterize candidate molecules. Among compounds that modulate the ACVR1 promoter activity, we selected the one showing the highest inhibitory effect, dipyridamole, a drug that is currently used as a platelet anti-aggregant. The inhibitory effect was detectable on ACVR1 gene expression, on the whole Smad-dependent BMP signaling pathway, and on chondrogenic and osteogenic differentiation processes by in vitro cellular assays. Moreover, dipyridamole reduced the process of heterotopic bone formation in vivo. Our drug repositioning strategy has led to the identification of dipyridamole as a possible therapeutic tool for the treatment of FOP. Furthermore, our study has also defined a pipeline of assays that will be useful for the evaluation of other pharmacological inhibitors of heterotopic ossification.

No MeSH data available.