CN114288292B - Application of aromatic compound as activator of nerve Cong Su protein-B2 in preparation of medicine for treating osteoporosis - Google Patents
Application of aromatic compound as activator of nerve Cong Su protein-B2 in preparation of medicine for treating osteoporosis Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4406—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 3, e.g. zimeldine
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- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
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- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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Abstract
The invention discloses an application of an aromatic compound serving as an activator of a nerve Cong Su protein-B2 in preparation of a medicament for treating osteoporosis. The aromatic compound is preferably a compound shown in a formula II, and the compound shown in the formula II can obviously increase the expression level of Plexin-B2 protein and genes and activate downstream signal paths of the Plexin-B2 protein and genes, and can also obviously promote the generation of osteoblasts and inhibit the generation of the osteoclasts, so that the bone mass is obviously increased, the development of osteoporosis is delayed, a research basis and a theoretical basis are provided for developing medicaments for treating OP, and the aromatic compound can be applied to the preparation of medicaments for preventing and/or treating the osteoporosis.
Description
Technical Field
The invention belongs to the technical field of medicine and biochemistry, and particularly relates to an application of an aromatic compound serving as an activator of nerve Cong Su protein-B2 (Plexin-B2) in preparation of a medicament for treating osteoporosis.
Background
Osteoporosis (OP) is one of the most common metabolic diseases of bone and is characterized by reduced bone mass, increased bone fragility due to destruction of bone microstructure, and increased risk of fracture. OP mainly includes senile osteoporosis and postmenopausal osteoporosis, and the high-frequency population is the elderly and postmenopausal women. In China, the first few of common diseases and frequently encountered diseases of OP are jumped, the incidence rate rises year by year, the total incidence rate is 12.4%, the incidence rate of the aged over 65 years is over 50%, wherein the incidence rate of fracture is close to one third, the risks of disability and total cause death of the aged, especially the aged females, are obviously increased, and heavy economic and medical burden is brought to society.
Currently, clinical anti-osteoporosis drugs mainly include basic supplements (e.g., calcium agents, vitamin D), drugs for inhibiting bone resorption (e.g., bisphosphates), and drugs for promoting bone formation (e.g., parathyroid hormone). From the current treatment situation, the problems of general treatment effect, high long-term treatment cost, certain side effects, poor patient compliance and the like exist, so that the development of effective anti-osteoporosis medicines is urgently needed.
The nerve Cong Su protein (Plexins) is a widely distributed family of transmembrane receptor proteins, and the nerve Cong Su protein-B2 (Plexin-B2) is a member of the Plexin-B subfamily, and no relevant report has been found so far regarding the use of Plexin-B2 activators for the preparation of a medicament for the treatment of osteoporosis.
Disclosure of Invention
The invention aims to solve the technical problem of unsatisfactory treatment effect of the existing medicament for treating osteoporosis, and provides an aromatic compound serving as an activator of nerve Cong Su protein-B2 and application thereof in preparation of the medicament for treating osteoporosis. The invention can provide a research basis and a theoretical basis for developing a new medicament for treating osteoporosis.
The specific technical scheme provided by the invention is as follows.
The invention provides an application of an aromatic compound as an activator of a nerve Cong Su protein-B2, wherein the aromatic compound is one or more of a compound shown in a formula I, a compound shown in a formula II and a compound shown in a formula III;
the invention also provides application of an aromatic compound in preparing a medicament for preventing and/or treating diseases related to the nerve Cong Su protein-B2, wherein the aromatic compound is one or more of a compound shown in a formula I, a compound shown in a formula II and a compound shown in a formula III.
The invention also provides application of the aromatic compound in preparing a medicament for preventing and/or treating osteoporosis, wherein the aromatic compound activator is one or more of a compound shown in a formula I, a compound shown in a formula II and a compound shown in a formula III;
in the present invention, the aromatic compound is preferably the compound represented by formula I, the compound represented by formula II, or the compound represented by formula III, for example, the compound represented by formula II.
In the invention, K784-9621 (shown as a compound in a formula II) is used as one of small molecule activators of Plexin-B2, and can obviously increase the expression level of Plexin-B2 protein and gene and activate downstream signal channels.
In the invention, L087-0246 (shown as a compound shown as a formula I), K784-9621 (shown as a compound shown as a formula II) and G856-6766 (shown as a compound shown as a formula III) can inhibit the expression level of an osteoclast gene CTSK-mRNA.
The invention also provides application of an aromatic compound as a gene Runx2, sp7 and Alp expression promoter, wherein the aromatic compound is one or more of the compound shown in the formula I, the compound shown in the formula II and the compound shown in the formula III.
In the present invention, the aromatic compound is preferably the compound represented by formula I, the compound represented by formula II, or the compound represented by formula III, for example, the compound represented by formula II.
The invention also provides application of an aromatic compound as an expression inhibitor of genes NFATc1, rank and CTSK, wherein the aromatic compound is one or more of the compound shown in the formula I, the compound shown in the formula II and the compound shown in the formula III.
In the present invention, the aromatic compound is preferably the compound represented by formula I, the compound represented by formula II, or the compound represented by formula III, for example, the compound represented by formula II.
The invention also provides application of the aromatic compound in preparing a medicament for preventing and/or treating postmenopausal osteoporosis, wherein the aromatic compound is shown in a formula II.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that:
in the invention, L087-0246 (shown as a compound shown as a formula I), K784-9621 (shown as a compound shown as a formula II) and G856-6766 (shown as a compound shown as a formula III) can inhibit the expression level of an osteoclast gene CTSK-mRNA. In particular, K784-9621 of the present invention can inhibit the formation of osteoclasts and promote the formation of osteoblasts, thereby increasing the bone mass and delaying the development of osteoporosis. Specifically:
(1) The K784-9621 can obviously promote the generation of in vitro osteoblasts, and the expression of the osteogenesis related genes Runx2, sp7 and Alp of the K784-9621 stem prognosis is obviously increased compared with that of a control group;
(2) The K784-9621 can obviously inhibit the generation of in vitro osteoclasts, and the expression of the osteoclast related genes NFATc1, rank and CTSK after the K784-9621 stem prognosis is obviously reduced compared with a control group;
(3) K784-9621 improves bone mass, increases bone volume fraction (BV/TV), bone trabecular number (Tb.N), bone trabecular thickness (Tb.Th) and bone formation rate, and decreases bone trabecular separation (Tb.Sp);
(4) K784-9621 increases OCN + Is capable of reducing Trap + Is an osteoclast of (a).
Drawings
FIG. 1A is a block diagram of a small molecule compound of example 1;
FIG. 1B is a schematic diagram of qPCR results of the effect of each small molecule compound on the expression level of the osteoclast gene CTSK;
FIG. 2 is a schematic representation of the effect of K784-9621 on the Plexin-B2 protein; wherein, FIG. 2A is a schematic diagram showing the results of K784-9621 promoting expression of Plexin-B2 protein; FIG. 2B is a graph of a statistical analysis of the promotion of expression of the Plexin-B2 protein by K784-9621;
FIG. 3 is a schematic diagram showing the qPCR results of K784-9621 for promoting expression of the Plexin-B2 gene;
FIG. 4 is a graph showing the qPCR results of K784-9621 for promoting the expression of osteogenic related genes;
FIG. 5 is a schematic representation of the effect of K784-9621 on osteoclasts; FIG. 5A is a schematic representation of K784-9621 staining with anti-tartrate acid phosphatase (Trap) inhibiting osteoclast formation; FIG. 5B Trap Positive (Trap + ) A statistical analysis chart of osteoclast number; FIG. 5C is a graph showing the qPCR results of K784-9621 inhibiting the expression of an osteoclast-associated gene;
FIG. 6 is a graph showing the effect of K784-9621 on bone mass and bone morphology parameters in osteoporotic mice; wherein, FIG. 6A is a three-dimensional reconstruction of bone scan of postmenopausal osteoporosis mice after ovariectomy by intraperitoneal injection of K784-9621; FIG. 6B is a schematic diagram of micro-CT bone parameter analysis of mice under different treatment conditions;
FIG. 7 is a graph showing the effect of K784-9621 on the number of osteoblasts positive for OCN in osteoporotic mice; wherein FIG. 7A is a schematic representation of mouse Osteocalcin (OCN) staining under different treatment conditions, arrows indicate OCN positivity (OCN) + ) Is a cell of (a); FIG. 7B shows the OCN of mice under different treatment conditions + A statistical analysis chart of cell number;
FIG. 8 is a graph showing the effect of K784-9621 on the number of osteoclasts positive for the Trap of an osteoporosis mouse; FIG. 8A is a schematic representation of mouse Trap staining under different treatment conditions, with arrows indicating Trap positivity (Trap + ) Is a cell of (a); FIG. 8B shows the mouse Trap positivity (Trap + ) Statistical analysis of cell number.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
Example 1a small molecule compound capable of binding with Plexin-B2 was obtained by computer drug-assisted design screening, and a small molecule compound having an effect on the expression level of the osteoclast gene CTSK was screened;
1. preparing 10mg/ml stock solution: (1) the small molecular compounds (shown in Table 1) are obtained through computer drug aided design screening and are purchased from chemdiv company and centrifuged at 1000r/min for 5mins; (2) in an ultra-clean workbench, 100ul of DMSO is sucked and added into 1mg of powder, and the powder is repeatedly blown and evenly mixed; (3) packaging the mixed liquid, and storing at-20deg.C.
2. Preparing 10ug/ml working solution: (1) taking the split-packed storage solution, and rewarming at room temperature; (2) working fluid was prepared in a ratio of 1ul stock solution per 1ml of medium for subsequent cell experiments.
3. The mononuclear cells of the bone marrow of a mouse are obtained by 1X 10 ≡ 5 Cell/well density was inoculated into alpha-MEM medium containing macrophage colony stimulating factor (M-CSF, 30 ng/ml), nuclear factor κB receptor activating factor ligand (Rankl, 100 ng/ml), 10% fetal bovine serum, 1% double antibody, and divided into Control group (Control) (not administered group) and each small molecule compound treatment group (structure of each compound is shown in FIG. 1A), liquid was changed every 3 days, and after differentiation culture for 9 days, trap staining was performed to detect the number of osteoclasts; cell RNA was extracted after 7-8 days of differentiation culture, and the expression level of the osteoclast gene CTSK-mRNA was detected by qPCR (qPCR specific experimental method reference: chang-Jun Li, et al Long noncoding RNA Bmncr regulates mesenchymal stem cell fate during skeletal imaging. J Clin Invest,2018,128 (12): 5251-5266.).
Referring to fig. 1B, table 1, each value is expressed as the mean ± SD of three assays. P <0.05, P <0.01, P <0.001 compared to Control group.
TABLE 1
Group of | Relative expression levels of CTSK |
Control | 20.000±2.843 |
L087-0246 | 11.200±5.432* |
G639-1239 | 23.981±7.250 |
1109-0033 | 14.520±4.744 |
K784-9621 | 3.943±1.347** |
K781-0814 | 14.406±0.379 |
G717-1369 | 18.382±2.935 |
D678-0039 | 16.458±4.076 |
T630-1975 | 17.133±5.219 |
G856-6766 | 12.559±0.254* |
The effect of 9 compounds shown in fig. 1A on the expression level of the osteoclast gene CTSK is shown in table 1, fig. 1B. This result demonstrates that K784-9621 has the most pronounced inhibitory effect on the osteoclast gene CTSK.
Example 2K784-9621 can significantly promote expression of Plexin-B2, promote osteoblast formation and inhibit osteoclast formation
(1) The specific experimental procedure for detecting Plexin-B2 protein and gene expression is as follows:
the mononuclear cells of the bone marrow of a mouse are obtained by 1X 10 ≡ 5 Cell/well density was inoculated in alpha-MEM medium containing macrophage colony stimulating factor (M-CSF, 30 ng/ml), 10% fetal bovine serum, 1% diabody, and divided into Control group (Control), K784-9621 treatment group (K784-9621). After 48 hours of K784-9621 intervention, cellular proteins and RNAs were extracted and the expression levels of the Plexin-B2 proteins and genes were detected by WB and q-PCR, respectively. (specific experimental methods for WB reference: chang-Jun Li, et al Long noncoding RNA Bmncr regulates mesenchymal stem cell fate during skeletal, ing. J Clin Invest,2018,128 (12): 5251-5266.).
For specific results, see fig. 2 and 3, tables 2 and 3, each numerical value is expressed as the mean ± SD of three assays. P <0.05, P <0.01, P <0.001 compared to Control group.
TABLE 2
Group of | Plexin-B2 relative expression level |
Control | 0.442±0.051 |
K784-9621 | 0.990±0.175* |
From FIGS. 2A and 2B, it can be seen that K784-9621 significantly increased the level of Plexin-B2 protein expression compared to the DMSO-treated Control group.
TABLE 3 Table 3
Group of | Plexin-B2 relative expression level |
Control | 1.000±0.095 |
K784-9621 | 1.534±0.057** |
As can be seen from FIG. 3, K784-9621 significantly increased the gene expression level of Plexin-B2 compared to the Control group.
(2) The specific experimental procedure for detecting the expression level of the osteogenic related gene is as follows:
according to a cell density of 5X 10 3 /cm 2 Mesenchymal Stem Cells (BMSCs) were inoculated into an alpha-MEM medium containing 0.1mM dexamethasone, 10mM beta-glycerophosphate, 50mM ascorbic acid-2-phosphate, and 10% fetal bovine serum, and divided into a Control group (Control) and a K784-9621-treated group (K784-9621), and after a single liquid change every 3 days, cellular RNAs were extracted and the expression level of the osteogenic related gene was detected by qPCR after 6 days of differentiation culture.
For specific results, see fig. 4, table 4, each value is expressed as mean ± SD of three assays. P <0.05, P <0.01, P <0.001 compared to Control group.
TABLE 4 Table 4
Group of | Runx2 relative expression level | Sp7 relative expression level | Relative expression level of Alp |
Control | 1.059±0.073 | 1.039±0.101 | 1.000±0.067 |
K784-9621 | 1.722±0.085** | 1.880±0.020** | 1.898±0.042*** |
As can be seen from FIG. 4 and Table 4, the expression levels of mRNA of the osteogenic related genes Runx2, sp7 and Alp after the treatment with K784-9621 were significantly increased as compared with the control group. This result demonstrates that K784-9621 can promote differentiation of BMSCs into osteoblasts.
(3) The specific experimental procedure for detecting the expression level of the osteoclast-associated gene is as follows:
the mononuclear cells of the bone marrow of a mouse are obtained by 1X 10 ≡ 5 Cell/well density seeded on macrophage colony stimulating factor (M-CSF, 30 ng/ml), nuclear factor kappa B receptor activating factorLigand (Rankl, 100 ng/ml), 10% fetal bovine serum and 1% double antibody in alpha-MEM culture medium are divided into a Control group (Control) and a K784-9621 treatment group (K784-9621), liquid is changed once every 3 days, and after differentiation culture for 9 days, trap staining is carried out to detect the number of osteoclasts; cell RNA was extracted after differentiation culture for 7-8 days, and the expression level of the osteoclast-associated gene was detected by qPCR.
Referring to fig. 5, table 5, each value is expressed as the mean ± SD of three assays. P <0.05, P <0.01, P <0.001 compared to Control group.
TABLE 5
From fig. 5A and 5B, table 5 shows that the number of osteoclasts was significantly reduced after the stem prognosis of K784-9621 compared to the control group.
TABLE 6
Group of | Relative expression level of NFATc1 | Rank relative expression level | CTSK relative expression level |
Control | 1.000±0.133 | 1.000±0.124 | 1.000±0.113 |
K784-9621 | 0.517±0.032** | 0.444±0.054** | 0.143±0.054** |
As can be seen from FIG. 5C and Table 6, the expression level of mRNA of the osteoclast-associated genes NFATc1, rank, CTSK was significantly reduced after the treatment with K784-9621, as compared with the control group. This result suggests that K784-9621 can inhibit osteoclastogenesis.
Example 3K 784-9621 can increase bone mass in postmenopausal osteoporosis mice by promoting bone formation and inhibiting bone resorption
Postmenopausal osteoporosis (PMO) is a metabolic bone disease that results in significant loss of bone mass due to estrogen decline, and is a high turnover type of osteoporosis that is characterized by a significant increase in osteoclastic production, a slight increase in osteoblastic cells, and eventually a significant decrease in bone mass due to greater bone resorption than bone formation. Since we observed earlier that K784-9621 can significantly inhibit osteoclastogenesis and promote osteoblastogenesis in vitro, we further hypothesize whether it has an improving effect on bone mass reduction in postmenopausal osteoporosis. Adaptively feeding C57BL/C mice for 1 week, dividing into a sham operation +velicle group (sham), an ovariectomy +velicle group (OVX) and an ovariectomy +K784-9621 group (K784-9621), injecting velicle or K784-9621 (70 mg/kg once a day) into the abdominal cavity after 1 week, weighing the mice in each group after 7 weeks of injection, taking blood from the apex of the heart, leaving a bilateral femur, fixing one femur with formaldehyde, placing the fixed femur in a sample tube for CT scanning, and then carrying out micro-CT analysis and three-dimensional reconstruction to observe bone mass conditions; the other femur was decalcified in 10% EDTA decalcification solution for 3 weeks, paraffin-embedded, and sectioned, and then stained with OCN and Trap, respectively.
For specific results, see FIGS. 6-8, tables 7-9, each value is expressed as the mean+ -SD of three assays. P <0.05, < P <0.01, < P <0.001, compared to sham group; compared to OVX group, #p <0.05, #p <0.01, #p <0.001.
TABLE 7
From the three-dimensional reconstruction of the bone scan of fig. 6A, it can be seen that the OVX group showed significantly reduced bone mass after ovariectomy compared to sham group, while the K784-9621 treated mice showed significantly increased bone mass compared to OVX group. As can be seen from the mir-CT analysis parameters of FIG. 6B, the bone volume fraction (BV/TV), the number of trabeculae (Tb.N), and the thickness of trabeculae (Tb.Th) were all significantly reduced in the OVX group compared to the sham group, while the trabeculae separation (Tb.Sp) was significantly increased, and K784-9621 improved the bone mass parameters described above in the ovariectomized mice. This result demonstrates that K784-9621 can improve bone mass loss in postmenopausal osteoporosis mice.
TABLE 8
Group of | OCN + Osteoblasts (N/mm) |
sham | 8.723±0.861 |
OVX | 8.690±0.813 |
K784-9621 | 10.616±0.556## |
As can be seen from FIGS. 7A and 7B, the OVX group OCN is compared with the sham group + Is not obvious; k784-9621 significantly increased the number of osteoblasts compared to the OVX group. This result suggests that K784-9621 may promote osteoblast formation in vivo.
TABLE 9
Group of | Trap + Is an osteoclast (N/mm) |
sham | 3.682±0.189 |
OVX | 8.392±0.587*** |
K784-9621 | 6.226±0.731### |
As can be seen from FIGS. 8A and 8B, the OVX group Trap compares with the sham group + Is significantly increased; k784-9621 significantly reduced the number of osteoclasts compared to the OVX group. This result demonstrates that K784-9621 can inhibit osteoclastogenesis in vivo.
The above results together demonstrate that K784-9621 can promote osteoblast formation by inhibiting osteoclast formation, thereby increasing bone mass in postmenopausal osteoporosis mice.
In the foregoing, the protection scope of the present invention is not limited to the preferred embodiments of the present invention, and any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention disclosed in the present invention fall within the protection of the present invention.
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