Selective small molecule compounds increase BMP-2 responsiveness by inhibiting Smurf1-mediated Smad1/5 degradation

Abstract

The ubiquitin ligase Smad ubiquitination regulatory factor-1 (Smurf1) negatively regulates bone morphogenetic protein (BMP) pathway by ubiquitinating certain signal components for degradation. Thus, it can be an eligible pharmacological target for increasing BMP signal responsiveness. We established a strategy to discover small molecule compounds that block the WW1 domain of Smurf1 from interacting with Smad1/5 by structure based virtual screening, molecular experimental examination and cytological efficacy evaluation. Our selected hits could reserve the protein level of Smad1/5 from degradation by interrupting Smurf1-Smad1/5 interaction and inhibiting Smurf1 mediated ubiquitination of Smad1/5. Further, these compounds increased BMP-2 signal responsiveness and the expression of certain downstream genes, enhanced the osteoblastic activity of myoblasts and osteoblasts. Our work indicates targeting Smurf1 for inhibition could be an accessible strategy to discover BMP-sensitizers that might be applied in future clinical treatments of bone disorders such as osteopenia.

Figure 1. Binding pocket definition and in silico screening.

(a) Refined structure of the Smurf1 WW1 domain bound to the diphosphorylated (pS210/pS214) region of the Smad1 linker. Key residues in Smad1 (black) and Smurf1 (purple) are labeled. (b) Refined structure of the Smurf1 WW1 domain bound to the monophosphorylated (pS214) region of the Smad1 linker. Key residues in Smad1 (black) and Smurf1 (purple) are labeled. (c) Receptor model of the Smurf1 WW1 domain (multiple colored) and diphosphorylated (pS210/pS214) region of the Smad1 linker (yellow). Key residues important for interactions are highlighted, and the region encompassed in those residues was defined as the binding pocket. (d) Workflow of in silico screening.

Figure 2. Primary selection of potential bio-active compounds.

(a) Determination of the efficacy of various selected compounds of enhancing BMP induced ALP activity. Compounds were added at a concentration of 2 μM, while rhBMP-2 was used at 50 ng/ml. The compound concentration for screening was selected empirically. In DMSO controls, cells were treated with DMSO (0.01%) instead of compounds (same for the following figures). Data points were determined in triplicate and showed with the mean ± SD (*: p < 0.05, t-test). (b) Chemical structures of A01 and A17.

Figure 3. Selective compounds stabilize Smad1/5 protein level.

(a) Selective compounds stabilized Smad1/5 protein level. C2C12 cells were treated A01 and A17 at incremental concentrations, while rhBMP-2 was used at 50 ng/ml. For the zero concentration, cells were treated with DMSO (0.01%) instead of compounds (same for the following figures). GAPDH were used as loading controls. Note that cropped blots are shown here. Left graph shows densitometry levels of percent Smad1/5 (normalized by GAPDH and compared with vehicle groups) and was fitted by GraphPad Prism 5. (b) Selective compounds prolonged Smad1/5 protein decay. C2C12 cells were treated with cycloheximide (CHX, 10 μg/ml), rhBMP-2 (50 ng/ml) and selected compounds (2 μM). Cells were collected at different time points. GAPDH were used as loading controls. Note that cropped blots are shown here. Left graph shows densitometry levels of percent Smad1/5 (normalized by GAPDH) and was fitted by GraphPad Prism 5. (c) Selective compounds had insignificant impact on Smad1 and Smad5 mRNA levels. C2C12 cells were treated A01 and A17 at 2 μM, while rhBMP-2 was used at 50 ng/ml. Data points were determined in triplicate and showed with the mean ± SD (NS: p > 0.05, t-test). (d) Selective compounds stabilized Smad1 phosphorylation (S206) level. C2C12 cells were treated A01 and A17 at 2 μM and 10 μM, while rhBMP-2 was used at 50 ng/ml. GAPDH were used as loading controls. Note that cropped blots are shown here. (e) Detection of Smad1/5 protein level following selective compounds or proteasome inhibitor treatments. C2C12 cells were treated with A01 and A17 at 2 μM or MG132 at 20 μM, while rhBMP-2 was absent or present (used at 50 ng/ml). GAPDH were used as loading controls. Note that cropped blots are shown here.

Figure 4. Selective compounds impair Smurf1-mediated Smad1/5 degradation.

(a) Selective compounds impeded the ubiquitination of Smad1/5. C2C12 cells were treated A01 and A17 at 2 μM, while MG132 and rhBMP-2 were used at 20 μM and 50 ng/ml. GAPDH were used as loading controls. Note that cropped blots are shown here. (b) Selective compounds blocked Smurf1-Smad1 interaction. 293T cells were co-transfected Flag-empty vector and Myc-Smad1 (lane 1) or Flag-Smurf1-CA and Myc-Smad1 (lane 2–4) plasmids. For inhibitors administration, cells were treated A01 and A17 at 2 μM. Note that cropped blots are shown here. (c) Selective compounds had unnoted effects on Smad1/5 without Smurf1. Smurf1 was knocked down in C2C12 cells (lane1: control siRNA, lane2–4: mouse Smurf1 siRNA). For inhibitors administration, cells were treated A01 and A17 at 2 μM, while rhBMP-2 was used at 50 ng/ml. GAPDH were used as loading controls. Note that cropped blots are shown here. (d) Selective compounds impaired Smurf1-Smad1 but not Smurf1-E2s interaction. Prokaryotic expressed proteins were purified and employed in GST-pull down. A01 and A17 were used at 10 μM. Note that cropped blots are shown here. (e) Effects of selective compounds on Smurf1 substrates and TGF-β pathway components. C2C12 cells were treated A01 and A17 at 2 μM, while rhBMP-2 was used at 50 ng/ml. GAPDH were used as loading controls. Note that cropped blots are shown here.

Figure 5. Selective compounds enhance BMP-2 signaling responsiveness.

(a) Selective compounds increased BMP-2 responsible reporter gene activity. Data points were determined in triplicate and showed with the mean ± SD (*: p < 0.05, t-test). (b) Selective compounds increased ALP, osteocalcin and type I collagen (α1) expression in mouse myoblasts. C2C12 cells were treated A01 and A17 at 2 μM, while rhBMP-2 was used at 50 ng/ml. Data points were determined in triplicate and showed with the mean ± SD (*: p < 0.05, t-test). (c) Selective compounds increased ALP, osteocalcin and type I collagen (α1) expressions in mouse pre-osteoblasts. MC3T3-E1 cells were treated A01 and A17 at 2 μM, while rhBMP-2 was used at 50 ng/ml. Data points were determined in triplicate and showed with the mean ± SD (*: p < 0.05, t-test).

Figure 6. Selective compounds potentiate BMP-2 induced osteoblastic activity.

(a) Selective compounds increased BMP-2 induced ALP activity in mouse myoblasts. C2C12 cells were treated A01 and A17 at 2 μM, while rhBMP-2 was used at 50 ng/ml. Data points were determined in triplicate and showed with the mean ± SD (*: p < 0.05, t-test). (b) Selective compounds increased BMP-2 induced ALP activity in mouse pre-osteoblasts. MC3T3-E1 cells were treated A01 and A17 at 2 μM, while rhBMP-2 was used at 50 ng/ml. Data points were determined in triplicate and showed with the mean ± SD (*: p < 0.05, t-test). (c) Selective compounds enhanced BMP-2 induced intracellular Ca²⁺ accumulation. C2C12 cells were treated A01 and A17 at 10 μM, while rhBMP-2 was used at 100 ng/ml. Data points were determined in triplicate and showed with the mean ± SD (*: p < 0.05, t-test). (d) Selective compounds mildly facilitated BMP-2 induced cell proliferation. C2C12 cells were treated A01 and A17 at 10 μM, while rhBMP-2 was used at 100 ng/ml. Data points were determined in triplicate and showed with the mean ± SD. (e) Selective compounds potentiated BMP-2 induced ALP enrichment. C2C12 cells were treated A01 and A17 at 10 μM, while rhBMP-2 was used at 100 ng/ml. (f) ALP staining results under microscope. Scale bar: 100 μm.

Figure 7. Predicted binding modes of selective compounds to the defined pocket.

(a) Predicted binding modes of A01 with the defined pocket. Important residues in the pocket were labeled in black. The distances (angstrom) of hydrogen bonds donors and receptors were noted in numerical values and red dotted lines. (b) Predicted binding modes of A17 with the defined pocket. Important residues in the pocket were labeled in black. The distances (angstrom) of hydrogen bonds donors and receptors were noted in numerical values and red dotted lines.