The Identification To Small Molecule Inhibitor That Targeting Smurf1 HECT Domain In Cell Level

Background/Object: Osteoporosis is a common disease that can lead to bone fracture, and its characteristics is bone mass reduction and bone tissue microstructure damage. With the intensification of ageing of population,the patient of this disease become more and more, it result in serious economic burden to patient and society. The treatment of osteoporosis is not a few, but the main method is drug therapy, such as supplement calcium, estrogen replacement therapy, and so on. However, the curative effect of many drug to osteoporosis at present is not satisfactory. The reason is that these drug can increase bone density by inhibiting bone resorption, but not promoting bone formation.So it is extremely urgent that finding new target that can promote bone formation and development new drug that can stimulate bone formation. In order to explore new pathway that can promote bone formation, this work is screening small inhibitor that target to ubiquitin protein ligase Smurf1. It perhaps stimulate bone formation by promoting BMP pathway, and lay a foundation for next developing bone formation enhancers. This drug have potential that can recover the lost bone mass, notable increase bone density and reduce bone fracture.Methods: First of all, we obtained 24 candidate compounds in silico selection technology. Then we selected these candidate compounds by ALP activity test and Western Blot, and afterwards obtain two compound that is they are better, their respectively number is B06 and B75. In order to evaluated and confirmed compound B06 and B75 in cell level and molecule mechanism, next we conducted various test,such as ALP colouration test and WST-1 cell proliferation and cell toxicity text. Finally, we conducted molecule imitate butt joint between compound B06 or B75 and Ub combination region of Smurf1 HECT domain, then forecast their structure and preliminary analyzed their interaction.Results: Both the first and the second experiments demonstrated that: the total effect of compounds B06 and B75 is better, so they can be object to next research. The concentration grads experiment and half-time experiment indicated that: with the increase of the concentration of two compounds, the protein level of Smad1/5 first rise and then stability in general; after given compounds, with the time pass, the half-time of Smad1/5 is prolonged. The phosphorylation experiment and the effects of drug comparison experiment indicated that: compounds B06 and B75 can increse p-Smad1 protein level and promote signal transmission of this pathway. Moreover, the effect of compounds and MGA132 is very approximately when BMP signal is activated. ALP colouration and activity test indicated that: both B06 and B75 can increase ALP content and its activity in C2C12 cells. WST-1 cell proliferation text indicated that: the two compounds can promote C2C12 cells proliferation, and their toxicity is lower.Conclusion: In short we found that compounds B06 and B75 not only can increase or stabilise the protein level of Smad1/5 by inhibiting the combination between Smurf1 and Ub, and then promoting the signal transmission of BMP pathway, but also can obvious enhance the differentiation ability and proliferation ability of C2C12 cells. In addition, the relative information in silico selection and the analysis to molecule imitate butt joint picture indicated that the combination between aforesaid two compounds and Ub bond region of Smurf1 HECT domain are more tightness,and their structure butt joint is good. So compared other candidate compounds, the effect of B06 and B75 are better, and they have the potential of promoting bone formation.