CN103655568B - A kind of micromolecular compound of promoting bone growing - Google Patents
A kind of micromolecular compound of promoting bone growing Download PDFInfo
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- CN103655568B CN103655568B CN201310646316.3A CN201310646316A CN103655568B CN 103655568 B CN103655568 B CN 103655568B CN 201310646316 A CN201310646316 A CN 201310646316A CN 103655568 B CN103655568 B CN 103655568B
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Abstract
The invention discloses a kind of micromolecular compound of promoting bone growing.Compound shown in formula I can be used for preparing the medicine of promoting bone growing and the sensitizer of preparation BMP; Present invention also offers following arbitrary described application of compound shown in formula I: (1) preparation improves the product of the osteogenic ability of cell; Described cell is specially into flesh precursor or bone precursor cells; (2) preparation improves the product of cell alkaline phosphatase, Bone Gla protein and/or I-type collagen mRNA level in-site; Described cell is specially into flesh precursor or bone precursor cells; (3) preparation improves into the product of flesh precursor or bone precursor cells or osteoblast activity change of Alkaline phosphatase; (4) product of the degraded of the Smad1/5 albumen in preparation suppression BMP Pathway Activation situation; (5) product of the ubiquitination level of the Smad1/5 albumen in preparation reduction BMP Pathway Activation situation.
Description
Technical field
The present invention relates to a kind of micromolecular compound of promoting bone growing.
Background technology
Osteoporosis (osteoporosis) has become one of commonly encountered diseases having a strong impact on China senior health and fitness, the statistics of 2010 show China further and enter aging society, the quantity of more than 60 years old old men has reached 1.67 hundred million, account for 12% of total population, the postmenopausal women of more than 40 years old of about 20% suffers from osteoporosis in various degree.And the osteanagenesis degree after middle-aged and elderly people fracture also becomes the key factor affecting its orthobiosis.
Adult, after bone development maturation, keeps renewal and the maintenance of bone by the process of bone resorption and bone formation two couplings.Wherein, osteoblast mediation bone formation, Osteoclasts mediate bone resorption.At present, osteoporosis therapy medicine such as fosamax, estrogenic agents, activated vitamin D, estrogen replacement therapy, calcitonin, the calcitriol etc. of Clinical practice are all suppress bone resorption based on the regulation and control for osteoclast, but cannot recover for the bone amount of having lost.Uniquely being ratified by U.S. FDA the medicine be used for by stimulating new bone formation to recover the bone amount of losing is exactly parathyroid hormone.But this medicine also have stimulated bone resorption while stimulation new bone formation.In view of this, Europe and the U.S. are strictly limited in 18 months and 2 years respectively to the useful life of parathyroid hormone, guarantee to make it be greater than the effect of bone resorption to osteoplastic effect at treatments period, reach the effect recovering bone amount.Cause one of reason of this situation to be also deep and not clear to the molecular mechanism of severe osteoporosis osteogenic ability reduction to the understanding of the regulatory mechanism of osteoblast differentiation/activation, deepening the understanding of the osteoblastic molecular mechanism of the regulation and control of participating is an urgent demand that current short bone synthesizes new drug Study on Transformation.
Bone morphogenetic protein (BMP) path is the important path that bone formation occurs, the people source BMP-2(rhBMP-2 of restructuring) obtain U.S. FDA certification, become the protein medicaments for rehabilitation after fracturing.But because purification difficulty is large, volume requirements is high, cannot realize large-scale production, be also difficult to reduce treatment cost.Therefore wish the activity of the negative regulatory factor by antagonism BMP path, the effect of signal path is strengthened, thus reach the object improving bone formation ability.
Smurf1 is the ubiquitin ligase of a HECT class, plays important negative regulation effect in BMP path.Research based on Smurf1 knock out mice finds, knock out the mainly lifting of mice bone amount of phenotype that Smurf1 causes, the special increase of bone formation and bone resorption does not have significant change, therefore under physiological condition, the function of Smurf1 mainly suppresses bone formation, lowers bone amount.Relevant mechanism is that Smurf1 can degrade key signal transduction Protein S mad1, the Smad5, protein kinase MEKK2 and transcription factor RunX2 etc. of BMP path, and mediates that Smurf1 identifies, the key structure territory of bound substrates albumen is the WW domain of mid-molecule.Because Smurf1 is a kind of ubiquitin ligase, Smurf1 plays and suppresses osteoplastic effect to be combined as ubiquitin ligase with it and to promote that degradation of substrates is closely related, so, suppress the combination of Smurf1 and substrate by micromolecular compound, block Smurf1 the ubiquitination of substrate is degraded, just can suppress the activity of Smurf1 thus promoting bone growing, namely the compound with this kind of effect can be used for the treatment of osteoporosis etc.
Summary of the invention
The object of this invention is to provide one and treat osteoporotic medicine.
The invention provides the application of compound shown in formula I in the medicine preparing promoting bone growing;
In above-mentioned application, described application is embodied in following 1)-3) in any one:
1), compound shown in formula I impels the mRNA level in-site of alkali phosphatase to raise;
2), compound shown in formula I impels the mRNA level in-site of Bone Gla protein to raise;
3), compound shown in formula I impels the mRNA level in-site of I-type collagen to raise.
Present invention also offers the application of compound shown in formula I in the sensitizer of preparation BMP;
In above-mentioned application, described application is embodied in following 1)-3) in any one:
1), compound shown in formula I impels the mRNA level in-site of alkali phosphatase to raise;
2), compound shown in formula I impels the mRNA level in-site of Bone Gla protein to raise;
3), compound shown in formula I impels the mRNA level in-site of I-type collagen to raise.
Present invention also offers following arbitrary described application of compound shown in formula I:
(1) preparation improves the product of the osteogenic ability of cell; Described cell is specially into flesh precursor or bone precursor cells;
(2) preparation improves the product of cell alkaline phosphatase, Bone Gla protein and/or I-type collagen mRNA level in-site; Described cell is specially into flesh precursor or bone precursor cells;
(3) preparation improves into the product of flesh precursor or bone precursor cells or osteoblast activity change of Alkaline phosphatase;
(4) product of the degraded of the Smad1/5 albumen in preparation suppression BMP Pathway Activation situation;
(5) product of the ubiquitination level of the Smad1/5 albumen in preparation reduction BMP Pathway Activation situation.
In above-mentioned application, in described (1), in described (2) neutralization described (3), described one-tenth flesh precursor is that mice becomes flesh precursor; Described bone precursor cells is mice bone precursor cells;
In described (3), described osteoblast is rat osteoblast.
In above-mentioned application, in described (2), described cell is the cell in BMP Pathway Activation situation;
In described (3), described one-tenth flesh precursor or bone precursor cells or osteoblast are the cell in BMP Pathway Activation situation.
The present invention still further provides a kind of medicine of promoting bone growing, and its active component is compound shown in formula I.
Invention further provides the sensitizer of a kind of BMP, its active component is compound shown in formula I.
Tool of the present invention has the following advantages:
1, block the combination of Smurf1 and Smad1/5, reduce Smurf1 to the degradation capability of Smad1/5.
2, play a role under BMP Pathway Activation state, can not effect be played under BMP path unactivated state.
3, effectively can improve the signal response of BMP path, improve the expression of bone formation marker gene.
Accompanying drawing explanation
Fig. 1 is the protein level of Smad1/5 in the embodiment of the present invention 1.
Fig. 2 is the mRNA level in-site of Smad1/5 in the embodiment of the present invention 1, and in figure, NS indicates without significant difference.
The concentration of compound shown in the protein level (Fig. 3 (a)) that Fig. 3 is Smad1/5 in the embodiment of the present invention 1 and formula I and the curve (Fig. 3 (b)) of protein level.
Fig. 4 is the protein level of Smad1/5, Smurf1 and MEKK2 in the embodiment of the present invention 1.
Fig. 5 is Smad1/5 ubiquitination level in the embodiment of the present invention 2.
Fig. 6 is the mRNA level in-site of BMP downstream alkaline phosphatase gene in the embodiment of the present invention 3.
Fig. 7 is the mRNA level in-site of BMP downstream Bone Gla protein gene in the embodiment of the present invention 3.
Fig. 8 is the mRNA level in-site of BMP downstream type i collagen gene in the embodiment of the present invention 3.
Fig. 9 is that the embodiment of the present invention 3 activity change of Alkaline phosphatase detects.
Detailed description of the invention
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Compound shown in formula I is purchased from Enamine company (article No.: Z203242870).
Shown in embodiment 1, formula I, compound is to the stability test of Smad1/5
(1), the present embodiment proves: add at cell the rhBMP-2(PEPROTECHAsia company that final concentration is 50ng/ml, article No.: 120-02) activate BMP path after, add the compound shown in formula I and Smurf1 can be stoped the degraded of the Smad1/5 of state of activation.Specific experiment process and experimental result as follows:
Compound shown in formula I is added mice by 0.2 μM with 2 μMs of two concentration to be become in flesh/bone precursor cells C2C12 cell (purchased from Chinese Academy of Medical Sciences's cell centre), add the rhBMP-2 that final concentration is 50ng/ml simultaneously, (primary antibodie of Smad1/5 is purchased from Abcam company to carry out WesternBlot detection after 8 hours, article No.: ab75273), the electrophoretogram obtained as shown in Figure 1, can learn, compound shown in formula I can stablize the protein content of Smad1/5.
(2), (primer: Smad1 upstream: 5 '-TCTGAAGTGGGCTTTCATCA-3 ', downstream: 5 '-TATGCCTGCCATCATTCTGA-3 ' is detected by PCR; Smad5 upstream: 5 '-TAGGCGGCATATTGGAAAAG-3 ', downstream: 5 '-CCAGAAGCTGAGCAAACTCC-3 '), detect the expression of Smad1/5, result as shown in Figure 2, can be learnt by figure, compound shown in formula I does not affect the mRNA level in-site of Smad1/5, illustrates that compound shown in formula I is by suppressing the degraded of Smad1/5 albumen to stablize its protein content.
(3), gradient experiment is carried out according to above-mentioned experimental procedure, the concentration of compound shown in formula I is set as successively 0.2 μM, 2 μMs, 10 μMs and 25 μMs, obtain electrophoretogram (concentration of compound shown in Fig. 3 (a) He formula I and the curve (Fig. 3 (b)) of protein level, can be learnt by this figure, it is dose-dependent that compound shown in formula I plays effect.
(4), compound shown in formula I is added mice by 2 μMs to be become in flesh/bone precursor cells C2C12 cell, set up rhBMP-2 two experimental grouies not adding rhBMP-2 He add 50ng/ml simultaneously, WesternBlot detection is carried out after 8 hours, find in the experimental group not having signal path to activate, Smad1/5 and MEKK2 as stream substrates is all unchanged, and Smad1/5 protein content after adding compound shown in formula I is stablized in the experimental group of Pathway Activation, the protein content of MEKK2 compares Smad1/5 not too large change, electrophoretogram as shown in Figure 4, prove that compound shown in formula I has specificity for the impact of Smurf1 ubiquitin ligase activity: the degraded that can suppress the Smad1/5 albumen in BMP Pathway Activation situation.
Shown in embodiment 2, formula I, compound detects the ubiquitination level of Smad1/5
Compound shown in formula I is added mice by 2 μMs of concentration to be become in flesh/bone precursor cells C2C12 cell, the final concentration simultaneously adding 50ng/ml is rhBMP-2 and the 20 μM of proteasome inhibitor MG132 of 50ng/ml, 8 h before harvest cells, ultrasonic degradation cell (0.3% power, ultrasonic 1 second, stop 1.5 seconds, copyrolysis 2 minutes), and with Smad1/5 and ProteinA/GPLUSAgarose pearl (SantaCruz company article No.: sc-2003), co-immunoprecipitation is carried out to lysate.
Detected by WesternBlot, find after adding rhBMP-2 and stimulating, the ubiquitination level of Smad1/5 obviously strengthens, and after adding compound shown in formula I, this ubiquitination significantly reduces, as shown in Figure 5, this illustrates that compound shown in formula I realizes by reducing its ubiquitination level the stable of Smad1/5 protein level.
The cell osteogenic activity of compound shown in embodiment 3, formula I
(1) design the primer of three pairs of real-time quantitative PCRs, be used for respectively detecting: 1, alkali phosphatase (ALP), 2, Bone Gla protein and 3, I-type collagen expression, wherein each primer pair is as follows:
The primer pair of detection of alkaline phosphatase is: upstream: 5 '-CACGCGATGCAACACCACTCAGG-3 ', downstream: 5 '-GCATGTCCCCGGGCTCAAAGA-3 ';
The primer pair detecting Bone Gla protein is: upstream: 5 '-ACCCTGGCTGCGCTCTGTCTCT-3 ' downstream: 5 '-GATGCGTTTGTAGGCGGTCTTCA-3 ';
The primer pair detecting I-type collagen is: upstream: 5 '-TCGGGCCTGCTGGTGTTCGTG-3 ', downstream: 5 '-TGGGCGCGGCTGTATGAGTTCTTC-3 '.
Above-mentioned three genes are all the downstream target gene of BMP path, their expressions increase the enhancing that indirectly can reflect cell osteogenic ability.
Compound shown in formula I is added mice by 0.5 μM of concentration become in flesh/bone precursor cells C2C12 cell, add the rhBMP-2 that final concentration is 50ng/ml, 12 h before harvest cells simultaneously, extract total serum IgE in cell, then carry out reverse transcription and obtain cDNA library.By the detection of real-time quantitative PCR, obtain Fig. 6, Fig. 7 and Fig. 8, can find out that compound shown in formula I can make the mRNA level in-site of ALP, Bone Gla protein and I-type collagen raise by these figure, illustrate that compound shown in formula I has the osteogenic ability that can raise cell.
(2) activity of alkali phosphatase is that osteogenic activity detects " goldstandard ".The change of Cellular alkaline phosphatase after the present embodiment uses the ALP test kit of Bio-Assay to detect to add compound shown in formula I and rhBMP-2 equally.
Compound shown in formula I is added mice by 0.5 μM of concentration to be become in flesh/bone precursor cells C2C12 cell and rat osteoblast ROS17/2.8 cell (purchased from Chinese Academy of Medical Sciences's cell centre), add the rhBMP-2 that final concentration is 50ng/ml simultaneously, 48 h before harvest cells, carry out non denatured cracking (using the TritonX-100 solution of 0.2%), ALP Activity determination is carried out to cell pyrolysis liquid, found that the active obviously rise of group ALP that the experimental group adding rhBMP-2 does not add relatively, and the group adding compound shown in formula I compares the rise that rhBMP-2 group has 25% ~ 40%, as shown in Figure 9.
As can be seen from above-mentioned, compound shown in formula I can raise Cellular alkaline phosphatase activity.
Claims (6)
1. the application of compound shown in formula I in the medicine preparing promoting bone growing;
2. the application of compound shown in formula I in the sensitizer of preparation BMP;
3. application according to claim 1 and 2, is characterized in that: described application shows as following 1)-3) in any one:
1), compound shown in formula I impels the mRNA level in-site of alkali phosphatase to raise;
2), compound shown in formula I impels the mRNA level in-site of Bone Gla protein to raise;
3), compound shown in formula I impels the mRNA level in-site of I-type collagen to raise.
4. following arbitrary described application of compound shown in formula I:
(1) preparation improves the product of the osteogenic ability of cell; Described cell is for becoming flesh precursor or bone precursor cells;
(2) preparation improves the product of cell alkaline phosphatase, Bone Gla protein and/or I-type collagen mRNA level in-site; Described cell is for becoming flesh precursor or bone precursor cells;
(3) preparation improves into the product of flesh precursor or bone precursor cells or osteoblast activity change of Alkaline phosphatase;
(4) product of the degraded of the Smad1/5 albumen in preparation suppression BMP Pathway Activation situation;
(5) product of the ubiquitination level of the Smad1/5 albumen in preparation reduction BMP Pathway Activation situation.
5. application according to claim 4, is characterized in that: in described (1), in described (2) neutralization described (3), described one-tenth flesh precursor is that mice becomes flesh precursor; Described bone precursor cells is mice bone precursor cells;
In described (3), described osteoblast is rat osteoblast.
6. the application according to claim 4 or 5, is characterized in that: in described (2), and described cell is the cell in BMP Pathway Activation situation;
In described (3), described one-tenth flesh precursor or bone precursor cells or osteoblast are the cell in BMP Pathway Activation situation.
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