A small-molecule inhibitor of the negative regulator of bone formation SMURF1
A technology of osteoblasts and myoblast precursor cells, which can be applied to medical preparations containing active ingredients, organic active ingredients, bone diseases, etc., can solve the problems of high dosage requirements, difficult purification, and difficulty in reducing treatment costs, etc. To achieve the effect of improving expression and improving signal response
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Embodiment 1
[0048] Embodiment 1, the stability test of the compound shown in formula I to Smad1 / 5
[0049] (1) This example proves that after adding rhBMP-2 (PEPROTECH Asia company, catalog number: 120-02) with a final concentration of 50ng / ml to cells to activate the BMP pathway, adding the compound represented by formula I can prevent Smurf1 from activating State of degradation of Smad1 / 5. The specific experimental process and experimental results are as follows:
[0050] The compound represented by formula I was added to the mouse myogenic / osteogenic precursor C2C12 cells (purchased from the Cell Center of the Chinese Academy of Medical Sciences) at two concentrations of 0.2 μM and 2 μM, and rhBMP-2 was added at a final concentration of 50 ng / ml After 8 hours, Western Blot detection was carried out (the primary antibody of Smad1 / 5 was purchased from Abcam, product number: ab75273), and the electrophoresis obtained was as follows figure 1 As shown, it can be known that the compound re...
Embodiment 2
[0054] Example 2, detection of the ubiquitination level of Smad1 / 5 by the compound shown in formula I
[0055] The compound represented by formula I was added to the mouse myogenic / osteogenic precursor C2C12 cells at a concentration of 2 μM, and at the same time, 50 ng / ml of rhBMP-2 and 20 μM proteasome inhibitor MG132 were added at a final concentration of 50 ng / ml for 8 hours After collecting the cells, sonicate the cells (0.3% power, sonicate for 1 second, pause for 1.5 seconds, and lyse for 2 minutes), and use Smad1 / 5 and ProteinA / G PLUS Agarose beads (Santa Cruz company product number: sc-2003) to lyse the lysate Perform co-immunoprecipitation.
[0056] By Western Blot detection, it was found that after adding rhBMP-2 stimulation, the ubiquitination level of Smad1 / 5 was significantly enhanced, and after adding the compound shown in formula I, this ubiquitination was greatly reduced, as shown in Figure 5 As shown, this shows that the compound shown in formula I stabilize...
Embodiment 3
[0057] The cellular osteogenic activity of the compound shown in embodiment 3, formula I
[0058] (1) Three pairs of primers for real-time quantitative PCR were designed to detect the expression of: 1. Alkaline phosphatase (ALP), 2. Osteocalcin and 3. Type I collagen. The primer pairs are as follows:
[0059] The primer pair for detecting alkaline phosphatase is: upstream: 5'-CACGCGATGCAACACCACTCAGG-3', downstream: 5'-GCATGTCCCCGGGCTCAAAGA-3';
[0060] The primer pair for detecting osteocalcin is: Upstream: 5'-ACCCTGGCTGCGCTCTGTCTCT-3' Downstream: 5'-GATGCGTTTGTAGGCGGTCTTCA-3';
[0061] The primer pair for detecting type I collagen is: upstream: 5'-TCGGGCCTGCTGGTGTTCGTG-3', downstream: 5'-TGGGCGCGGCTGTATGAGTTCTTC-3'.
[0062] The above three genes are the downstream target genes of the BMP pathway, and the increase of their expression can indirectly reflect the enhancement of the osteogenic ability of cells.
[0063] The compound represented by formula I was added to the mou...
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