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.
Highlights
The ubiquitin ligase Signaling Mothers Against Decapentaplegic (Smad) ubiquitination regulatory factor-1 (Smurf1) negatively regulates bone morphogenetic protein (BMP) pathway by ubiquitinating certain signal components for degradation
We developed a series of tests to select bio-active compounds, and identified two compounds which stabilize Smad1/5 protein level via inhibiting Smad ubiquitination regulatory factor-1 (Smurf1)-mediated ubiquitination of Smad1/5 when the cells were stimulated by recombinant human BMP-2 (rhBMP-2)
D211 coordinates the side chains of Arg[243], this interaction is different from what pS210 conducts and subsequently leads to a weakened contact between pS214 site and the WW1 domain (Figure 1b)
Summary
The ubiquitin ligase Smad ubiquitination regulatory factor-1 (Smurf1) negatively regulates bone morphogenetic protein (BMP) pathway by ubiquitinating certain signal components for degradation It can be an eligible pharmacological target for increasing BMP signal responsiveness. Our selected hits could reserve the protein level of Smad1/5 from degradation by interrupting Smurf1-Smad1/5 interaction and inhibiting Smurf[1] mediated ubiquitination of Smad1/5 These compounds increased BMP-2 signal responsiveness and the expression of certain downstream genes, enhanced the osteoblastic activity of myoblasts and osteoblasts. Expression of BMP signaling downstream genes and ALP activity in both mouse myoblasts and osteoblasts were increased after administrated those two compounds with a low dose These results suggest that targeting Smurf[1] for inhibition is an accessible method to increase BMP signaling responsiveness. Our study established a bio-active compounds selection approach including both in silico virtual screening and experimental assays, which is a proof of principle to discover small molecule compounds that antagonize HECT type ubiquitin ligase and design drugs to increase the cellular signal transduction efficiency
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