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Small-molecule inhibitor for bone formation negative regulatory factor Smurf1

A sensitizer, myoblast precursor cell technology, applied in the field of small molecule inhibitors, can solve problems such as high dosage requirements, difficult purification, and inability to achieve large-scale production, and achieve the effects of improving expression and signal response.

Active Publication Date: 2014-03-26
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the difficulty of purification and high dosage requirements, it is impossible to achieve large-scale production and it is difficult to reduce the cost of treatment

Method used

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  • Small-molecule inhibitor for bone formation negative regulatory factor Smurf1
  • Small-molecule inhibitor for bone formation negative regulatory factor Smurf1
  • Small-molecule inhibitor for bone formation negative regulatory factor Smurf1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1. Stability test of the compound represented by formula I to Smad1 / 5

[0049] (1) This example proves that: after adding rhBMP-2 (PEPROTECH Asia, Item No.: 120-02) to cells with a final concentration of 50ng / ml to activate the BMP pathway, adding the compound represented by formula I can prevent Smurf1 from activating the Degradation of state Smad1 / 5. The specific experimental process and experimental results are as follows:

[0050] The compound of formula I was added to mouse myoblast / osteoblast precursor C2C12 cells (purchased from the Cell Center of Chinese Academy of Medical Sciences) at two concentrations of 0.2 μM and 2 μM, and rhBMP-2 with a final concentration of 50 ng / ml was added at the same time. , 8 hours later, Western Blot detection (the primary antibody of Smad1 / 5 was purchased from Abcam Company, Cat. No.: ab75273), the obtained electropherogram is as follows figure 1 As shown, it can be known that the compound represented by formula I can st...

Embodiment 2

[0054] Example 2. Detection of the ubiquitination level of Smad1 / 5 by the compound represented by formula I

[0055] The compound represented by formula I was added to mouse myoblast / osteoblast precursor C2C12 cells at a concentration of 2 μM, and 50 ng / ml of rhBMP-2 and 20 μM of proteasome inhibitor MG132 were added at the same time to a final concentration of 50 ng / ml for 8 hours. After the cells were collected, the cells were lysed by sonication (0.3% power, sonicated for 1 sec, sonicated for 1.5 sec, and lysed for 2 min), and the lysate was lysed with Smad1 / 5 and ProteinA / G PLUS Agarose beads (Santa Cruz company product number: sc-2003). Co-immunoprecipitation was performed.

[0056] By Western Blot detection, it was found that the ubiquitination level of Smad1 / 5 was significantly enhanced after the addition of rhBMP-2 stimulation, while the ubiquitination level of Smad1 / 5 was greatly reduced after the addition of the compound of formula I, as shown in Figure 5 As shown,...

Embodiment 3

[0057] Example 3. Cellular osteogenic activity of the compound represented by formula I

[0058] (1) Three pairs of real-time quantitative PCR primers were designed to detect: 1. Alkaline phosphatase (ALP), 2. Osteocalcin and 3. Type I collagen expression. 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 all downstream target genes of the BMP pathway, and their increased expression can indirectly reflect the enhanced osteogenic ability of cells.

[0063] The compound represented by formula I was added to mouse myoblast / osteoblast precursor...

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Abstract

The invention discloses a small-molecule inhibitor for a bone formation negative regulatory factor Smurf1. The compound shown as a formula I can be used in preparation of medicines for promoting bone formation and preparation of a BMP sensitizing agent. The invention also provides one of the applications of the compound shown as the formula I: (1) preparation of products for improving osteogenic potential of cells, wherein the cells are specifically myogenic precursor cells or osteogenic precursor cells; (2) preparation of products for improving alkaline phosphatase, osteocalcin and / or type I collagen mRNA levels in cells, wherein the cells are specifically myogenic precursor cells or osteogenic precursor cells; (3) preparation of products for improving activity of alkaline phosphatase in myogenic precursor cells or osteogenic precursor cells or osteoblasts; (4) preparation of products for inhibiting degradation of an Smad1 / 5 protein under the condition of BMP pathway activation; and (5) preparation of products for reducing ubiquitination level of the Smad1 / 5 protein under the condition of BMP pathway activation.

Description

technical field [0001] The present invention relates to a small molecule inhibitor of Smurf1, a negative regulator of bone formation. Background technique [0002] Osteoporosis has become one of the common diseases that seriously affects the health of the elderly in my country. Statistics in 2010 further show that my country has entered an aging society. 12%, about 20% of postmenopausal women over the age of forty suffer from varying degrees of osteoporosis. The degree of bone regeneration after fracture in middle-aged and elderly people has also become an important factor affecting their normal life. [0003] Adults maintain bone renewal and maintenance through two coupled processes of bone resorption and bone formation after bone maturation. Among them, osteoblasts mediate bone formation and osteoclasts mediate bone resorption. At present, clinically used osteoporosis drugs such as alendronate, estrogen receptor modulators, active vitamin D, estrogen replacement therapy,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/496A61P19/10A61P19/08
Inventor 张令强曹宇邢桂春汪呈梁超
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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