A stapled peptide for inhibiting osteoclast differentiation and its preparation method and application

Abstract

The invention relates to a stapled peptide for inhibiting osteoclast differentiation, a preparation method thereof and an application thereof. Using an amino resin as a carrier, according to the amino acid sequence of the template FRATtide: Ac-DPHRLLQQLVLSGNLIKEAVRRLHSR-NH2 in a DIC-Oxime condensation system, a peptide chain is synthesized by the Fmoc solid-phase synthesis method. During this process, on the basis of retaining key amino acid residues, S5 is used to replace the original amino acid at a specific position. After the linear peptide linked to the resin undergoes olefin metathesis reaction cyclization in a dichloroethane solution of Grubbs I reagent, it is cleaved from the resin to obtain the target double stapled peptide. The method of the invention is simple and feasible, with high purity and high yield. Further experiments confirm that the double stapled peptide of the invention can significantly inhibit osteoclast differentiation and has potential application value in the treatment of related diseases such as osteoporosis.

Description

Technical Field

[0001] The present invention relates to the field of polypeptide drugs, and in particular to a stapled peptide for inhibiting osteoclast differentiation, and a preparation method and application thereof. Background Art

[0002] Osteoporosis is a bone disease characterized by decreased bone mass, changes in bone tissue microstructure, increased bone brittleness, decreased bone strength, and easy fractures. Its pathophysiological basis is the imbalance between bone resorption and bone formation, which is regulated by osteoblasts that promote bone formation and osteoclasts that promote bone resorption. Currently, the most widely used anti-osteoporosis drugs in clinical practice mainly include small molecule drugs such as bisphosphonates that inhibit osteoclast differentiation and teriparatide that promotes bone formation. In order to improve the selectivity of therapeutic drugs and reduce side effects, currently, regulating important signaling pathways related to osteoblast and osteoclast differentiation through exogenous molecules has become one of the hot topics in osteoporosis research.

[0003] The inventors of the present application noticed from the numerous information disclosed in the prior art that studies have shown that glycogen synthase kinase 3β (GSK3β) can promote the transfer of the key osteoclast factor NFATc1 from the nucleus to the cytoplasm, thereby inhibiting the differentiation of osteoclasts; there are also literature reports that FRATtide and phosphorylated GSK3β can form a stable complex crystal structure; and there is a mutual conversion relationship between GSK3β and phosphorylated GSK3β. Therefore, the inventors of the present application speculate that specifically targeting phosphorylated GSK3β and then inhibiting phosphorylated GSK3β can inhibit the expression of GSK3β to a certain extent, thereby achieving negative regulation of osteoclasts.

[0004] The interaction between proteins in organisms plays a vital role in the life process. It is an effective strategy to regulate the protein-protein interaction interface through artificially synthesized molecules. It has been widely used in medicinal chemistry for disease intervention. Among them, artificially synthesized peptides are an important means to regulate the interaction between proteins. However, due to problems such as low stability and poor membrane permeability, the current clinical application of peptide drugs is greatly limited. The use of all-carbon skeletons to form side chain cyclization structures to modify peptides to stabilize the active conformation of α-helical peptides, namely stapled peptides, has become the most direct and effective method to overcome this defect.

[0005] At present, the prior art discloses some polypeptides with the activity of inhibiting osteoclast differentiation. For example, patent document CN109111506A discloses a polypeptide for treating osteoporosis, whose amino acid sequence is: Asp-Ser-Ser)6-(D-Tyr)-Asn-(D-Trp)-Asn-Ser-Phe-(azaGly)-Leu-Arg(Me)-Phe-NH2((AspSerSer)6-Kp-10, and proves that (AspSerSer)6-Kp-10 has a good bone targeting effect, can effectively inhibit osteoclast differentiation, and treat osteoporosis caused by ovarian removal. For example, patent document CN109251 242A, discloses a polypeptide that not only has the same or similar function as natural interleukin-3, but also has excellent skin permeability, and can inhibit the activation and nuclear transcription of nuclear factor κB by inhibiting the nuclear factor κB receptor activator ligand-nuclear factor κB receptor activator signaling pathway, and inhibit the expression of tartrate-resistant acid phosphatase, cathepsin K or type 1 or type 2 tumor necrosis factor receptor induced by nuclear factor κB receptor activator ligand and inflammatory cytokines, thereby inhibiting osteoclast differentiation in a concentration-dependent manner. However, there is no research on the effect of FRATtide or its stapled peptides on osteoclast differentiation. Summary of the invention

[0006] The purpose of the present invention is to solve the problems in the prior art, provide a stapled peptide for inhibiting osteoclast differentiation, provide the use of the stapled peptide and provide a method for preparing the stapled peptide.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] A stapled peptide, wherein the stapled peptide uses Ac-DPHRLLQQLVLSGNLIKEAVRRLHSR-NH2 as a peptide chain template, wherein 3 H , 7 Q , 19 A and 23 L It was replaced by S5 and cyclized twice.

[0009] To achieve the above second purpose, the technical solution adopted by the present invention is:

[0010] The stapled peptide is used in preparing medicine for treating osteoporosis.

[0011] The stapled peptide is used in preparing a reagent for inhibiting osteoclast differentiation.

[0012] To achieve the third purpose, the technical solution adopted by the present invention is:

[0013] The preparation method of the stapling peptide comprises the following steps:

[0014] (1) coupling the first amino acid at the C-terminus to the solid phase carrier under the action of a condensing agent;

[0015] (2) removing the Fmoc protecting group on the amino acid using a deprotection reagent;

[0016] (3) Connecting the next amino acid under the action of a condensing agent;

[0017] (4) Repeating the deprotection-coupling operation to synthesize a peptide chain according to the amino acid sequence; wherein the cyclization site is replaced by S5 to replace the amino acid at position i and i+4 respectively;

[0018] (5) Under the action of a cyclizing agent, the S5 amino acids at positions i and i+4 undergo olefin metathesis reaction to cyclize the peptide chain;

[0019] (6) Repeating the deprotection-coupling operation to synthesize a peptide chain according to the amino acid sequence; wherein the cyclization site is replaced by S5 to replace the amino acid at position i and i+4 respectively;

[0020] (7) the last amino acid is deprotected and then acetylated;

[0021] (8) Under the action of a cyclizing agent, the S5 amino acids at positions i and i+4 undergo olefin metathesis reaction to cyclize the peptide chain for the second time;

[0022] (9) Use a cleavage reagent to cut the peptide chain from the carrier, and purify the corresponding stapled peptide.

[0023] As a preferred example of the present invention, the purification method adopted in step (9) is reverse HPLC, and the conditions are as follows: chromatographic column: YMC-Pack ODS-AQ column; mobile phase: mobile phase A is 0.1% TFA / water, mobile phase B is 0.1% TFA / acetonitrile; gradient elution program: 40% B elution 0-5min, 40% B-60% B, 5-60min; flow rate is 15ml / min, injection volume is 5ml, and detection wavelength is 214nm.

[0024] As another preferred embodiment of the present invention, the condensing agent used in step (1) is a DIC-Oxyme condensation system, the activating agent is DIC, and NMP is used as the solvent.

[0025] More preferably, in step (1), the ratio of amino acid, oxygen and DIC is 1:1:1:6 (mol / mol / mol / ml) or 1:0.9:0.9:6 (mol / mol / mol / ml).

[0026] As another preferred embodiment of the present invention, during the solid phase synthesis in step (1), the sample loading amount of the resin is 0.3 mmol / g.

[0027] As another preferred embodiment of the present invention, the temperature of the coupling reaction in step (1) is 50-60°C, more preferably 55°C; the time of the coupling reaction is 20-30 min, more preferably 20 min.

[0028] As another preferred embodiment of the present invention, in step (2), the deprotection reagent is a mixed solution of Oxyme, piperidine and DMF in a ratio of 71:2:4 (m / v / v).

[0029] As another preferred embodiment of the present invention, in step (2), the Fmoc protection is removed by using a protective reagent for 5 minutes and then for 5 minutes again; the reaction temperature for removing the Fmoc group is 20-30°C, more preferably 25°C.

[0030] As another preferred example of the present invention, the reaction time of the first amino acid connected after S5 is 1 hour and the reaction is repeated once under the same conditions before proceeding to the next step.

[0031] As another preferred embodiment of the present invention, in step (7), the acetylation reagent used is a mixture of pyridine and acetic anhydride, and the feed ratio is 1:1 (v / v).

[0032] As another preferred embodiment of the present invention, the acetylation in step (7) is carried out by reacting the resin in an acetylation reagent for 20 minutes; the reaction temperature is 20-30°C, more preferably 25°C.

[0033] As another preferred embodiment of the present invention, the cyclizing agent in steps (5) and (8) is a solution of Grubbs I reagent in dichloroethane, and the feed ratio is resin loading: Grubbs I reagent: dichloroethane = 0.3:58:6 (mmol / mg / ml).

[0034] As another preferred embodiment of the present invention, the cyclization in steps (5) and (8) is performed by shaking the resin in the cyclization reagent twice, each time for 2 hours; the reaction temperature is 20-30°C, more preferably 25°C.

[0035] As another preferred embodiment of the present invention, in step (9), the cleavage reagent is a mixed solution of TIPS, H2O and TFA in a volume ratio of 2.5:2.5:95; the volume mass ratio of the cleavage reagent to the linear peptide is 1:10 mL / mg.

[0036] As another preferred embodiment of the present invention, in step (9), the cutting temperature is 20-30°C, more preferably 25°C; and the cutting time is 4 hours.

[0037] The beneficial effects of the present invention are:

[0038] 1. Based on rich research experience, the inventors of this application realized that FRATtide may have the effect of inhibiting osteoclast differentiation, and further designed and synthesized a double-stapled peptide. Experiments have confirmed that it can significantly inhibit osteoclast differentiation and has potential application value in the treatment of osteoporosis and other related diseases.

[0039] 2. The present invention uses amino resin as a carrier, and synthesizes a peptide chain in a DIC-Oxime condensation system according to the template FRATtide: Ac-DPHRLLQQLVLSGNLIKEAVRRLHSR-NH2 amino acid sequence by Fmoc solid phase synthesis. On the basis of retaining the key amino acid residues, the original amino acid is replaced by S5 at a specific position, and the linear peptide is connected to the resin. After olefin metathesis reaction and cyclization in a dichloroethane solution of Grubbs I reagent, the target stapled peptide is cut off from the resin, and the obtained compound is purified and characterized and analyzed by HPLC and MS spectra. The method is simple and easy, and the obtained stapled peptide has a purity of more than 98% and a high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is a schematic diagram of the stapled peptide of the present invention;

[0041] Figure 2 The synthetic route of the stapled peptide of the present invention is as follows;

[0042] Figure 3 The HPLC and mass spectra of the purified target compound;

[0043] Figure 4 This is a test result diagram of the cell experiment in Example 3;

[0044] Figure 5 The figure is a test result diagram of the animal experiment (subcutaneous injection) in Example 3;

[0045] Figure 6 This is a test result diagram of the animal experiment (oral administration) in Example 3. DETAILED DESCRIPTION

[0046] In order to enable those skilled in the art to better understand the scheme of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and implementation methods.

[0047] The present invention designs and synthesizes a stapled peptide according to the amino acid sequence of template FRATtide: Ac-DPHRLLQQLVLSGNLIKEAVRRLHSR-NH2 (SEQ ID NO: 1). Figure 1 shown.

[0048] In the following embodiments, the abbreviations involved are explained as follows:

[0049] Fmoc: fluorenylmethoxycarbonyl

[0050] DCM: dichloromethane

[0051] DCE: dichloroethane

[0052] DMF: N,N-dimethylformamide

[0053] Oxyme: Ethyl Cyanoglyoxylate-2-Oxime

[0054] DIC: N,N-diisopropylcarbodiimide

[0055] NMP: N-methylpyrrolidone

[0056] S5:2-amino-2-methylhept-6-enoic acid

[0057] TFA: trifluoroacetic acid

[0058] TIPs: Triisopropylsilane

[0059] Grubbs I: phenylmethylenebis(tricyclohexylphosphine)ruthenium dichloride

[0060] The sources of experimental materials involved are as follows:

[0061] Amino acids and amino resins were purchased from Shanghai Jier Biochemical Co., Ltd.; N-methylpyrrolidone (NMP), N,N-diisopropylcarbodiimide (DIC), ethyl cyanoglyoxylate-2-oxime, trifluoroacetic acid (TFA), and acetonitrile (chromatographic grade) were purchased from Beijing Bailingwei Technology Co., Ltd.; N,N-dimethylformamide (DMF), anhydrous ether, dichloromethane (DCM), dichloroethane (DCE), piperidine, and phenol were all of analytical grade and purchased from Sinopharm Chemical Reagent Beijing Co., Ltd.

[0062] Example 1. Preparation of stapled peptides that inhibit osteoclast differentiation

[0063] 1. Synthesis of stapled peptides

[0064] like Figure 2 As shown:

[0065] (1) Preparation of Compound 1

[0066] Take 500 mg of amino resin (the sample loading is 0.30 mmol·g -1 ) was added into a solid phase synthesis reaction tube, soaked in DCM for 20 min to allow the resin to fully swell, and then drained for later use.

[0067] Add 20% piperidine-DMF solution (0.1 M Oxyme) until the resin is completely submerged, shake at 25°C for 5 min×2 to remove Fmoc on the resin, and wash the resin with DCM and DMF for 3 times each.

[0068] (2) Preparation of Compound 2

[0069] The first amino acid in the sequence (1 mmol), Oxyme (142 mg, 1 mmol) and DIC (155.0 μL, 1 mmol) were mixed and dissolved in 6 ml of NMP, added to the resin and shaken at 60°C for 20 min (one amino acid after S5 was reacted for 1 h, and the reaction was repeated once), and the resin was washed with DCM and DMF three times each.

[0070] (3) Preparation of Compound 3

[0071] Repeat steps (1) and (2). According to the peptide sequence, Fmoc amino acid (1 mmol), Oxyme (142 mg) and DIC (155 μl) were mixed in 6 ml NMP, added to the resin, and shaken at 60°C for 20 min. Deprotection → condensation → deprotection were repeated. After the first two S5 special amino acids were connected, a dichloroethane solution (6 ml) of Grubbs I (58 mg) reagent was added and shaken at 25°C for two times, each time for 2 h. After the reaction was completed, it was washed three times with DCM and DMF in turn to complete the first cyclization.

[0072] (4) Preparation of Compound 4

[0073] Repeat deprotection → condensation → deprotection. After the last amino acid is deprotected, add 6 ml of a mixture of pyridine: acetic anhydride (1:1) and shake at 25°C for 20 min. Wash the resin with DCM, DMF, and anhydrous ether for 3 times each, and then dry the resin in vacuo.

[0074] (5) Preparation of Compound 5

[0075] After the resin is completely dried, add a dichloroethane solution (6 ml) of Grubbs I (58 mg) reagent and shake the reaction twice at 25°C for 2 h each time to complete the secondary cyclization. After the reaction is completed, wash the resin with DCM, DMF, and anhydrous ether for 3 times each, and vacuum dry the resin.

[0076] (6) Preparation of target compound

[0077] The resin was washed and dried, and 15 mL of TIPS:H2O:TFA = 2.5:2.5:95 (V / V / V) was added, and the mixture was shaken at room temperature for 4 h, filtered, and the resin was washed with a small amount of TFA, and the filtrate was collected. The excess TFA was blown away by argon bubbling, and the precipitation was centrifuged into icy ether, and the supernatant was discarded. The mixture was washed repeatedly with icy ether and centrifuged three times, and the crude staple peptide was obtained by argon drying.

[0078] 2. Purification of target stapled peptide

[0079] The crude peptide was dissolved in acetonitrile and water and purified by preparative RP-HPLC. The separation conditions were as follows:

[0080] Instrument: Pre-HPLC SD-1VARIAN high performance liquid chromatograph;

[0081] Chromatographic column: YMC-Pack ODS-AQ (250×20mml.D, S-5μm, 12nm);

[0082] Mobile phase: Mobile phase A is an aqueous solution with a volume fraction of 0.1% TFA, and mobile phase B is an acetonitrile solution with a volume fraction of 0.1% TFA;

[0083] Steps and parameters: 40% B elution for 0-5 min, 40% B-60% B, 5-60 min; flow rate of 15 ml / min, injection volume of 5 ml, detection wavelength of 214 nm.

[0084] Example 2. Identification and structural analysis of products

[0085] The product obtained in step 2 was identified by HPLC and structurally analyzed by HR-Q-TOF-MS (high resolution matrix-assisted laser desorption ionization time-of-flight mass spectrometry), and the chromatographic mobile phases were acetonitrile and water. Mobile phase A was an aqueous solution with a volume fraction of 0.1% TFA, and mobile phase B was an acetonitrile solution with a volume fraction of 0.1% TFA, with gradient elution (0-5 min, mobile phase B: 5%; 5-30 min, mobile phase B: 5%-90%); flow rate 15.0 mL·min -1 ; Detection wavelength 214nm and 254nm, injection volume 20μl. The peak time was consistent with the crude product, and the purity of the stapled peptide prepared by this method was >98% ( Figure 3 ). The results of HR-ESI-MS mass spectrometry analysis are as follows Figure 3 shown.

[0086] Example 3. Experiment on the inhibition of osteoclast activity by stapled peptides

[0087] 1. Experimental methods:

[0088] (I) Cell experiments

[0089] 1. Cytotoxicity test, using CCK-8 test method. BMM cells were cultured at 5x 10 3 The cells were inoculated into 96-well plates at a density of 100 μL of complete medium per well. The cells were cultured at 37°C and 5% CO2 for 24 h. FRT0 and FRNC-1 were administered at a concentration gradient of 0.31, 0.62, 1.25, 5, 10 and 20 μM. After 48 h of culture, 10 μL of CCK-8 solution was added to each well and incubated at 37°C for 1 hour. The absorbance at 450 nm was measured using an ELISA reader.

[0090] 2. Trap staining: cells isolated from femoral and tibia bone marrow were cultured in M-CSF and RANKL induction medium (10% FBS, 1% penicillin / streptomycin, 30 ng / mL M-CSF, 50 ng / mL RANKL). BMMs were then seeded into 96-well plates at a density of 8 x 10 per well. 3 The cells were cultured in an induction medium containing different concentrations of FRT0 and FRNC-1 (0, 0.625, 1.25 and 2.5 μM) and TRAP staining was performed.

[0091] 3. Bone resorption determination: 8 x 10 3 osteoclasts were cultured in each well. There were no RANKL, RANKL (50 ng / mL), RANKL (50 ng / mL) plus 1.25 μM and 2.5 μM FRNC-1, and RANKL (50 ng / mL) plus 1.25 μM and 2.5 μM FRT0.

[0092] 4. F-actin staining, culture cells for 24 hours until the density reaches 50%, wash twice with PBS at 37°C, fix with 4% paraformaldehyde solution dissolved in PBS for 10 minutes, wash 2-3 times with PBS, 10 minutes each time. Dehydrate with acetone for 5 minutes, wash 2-3 times with PBS, 10 minutes each time. Add the prepared TRITC-labeled phalloidin working solution to each well and incubate at room temperature for 30 minutes. Wash 3 times with PBS, 5 minutes each time. Add anti-fluorescence quencher containing DAPI. Observe with a fluorescence microscope and take pictures.

[0093] 5. Protein extraction and Western blot were performed, and the six groups of experiments in 3 were still carried out to measure the changes in p-GSK expression.

[0094] BMM cells were seeded in 6-well plates containing 10% FBS and 1% penicillin / streptomycin medium at a density of 5X105 cells / well. The cells were cultured for 24h until the density reached 80%, and RANKL (50ng / mL) was added to 5 wells to stimulate the cells for 1 hour. In order to determine the effect of FRNC-1 on p-GSK, 1.25μM, 2.5μM FRNC-1 and 1.25μM, 2.5μM FRT0 were added on the basis of RANKL and incubated for 1 day. Total protein was extracted from the cultured cells using lysis buffer. The lysate was centrifuged at 12,000g for 15 minutes, and the supernatant was collected. The proteins were separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked in blocking solution at room temperature for 15 minutes, then incubated with the primary antibody at 4°C overnight, and then incubated with the secondary antibody at room temperature for 2 hours. Protein bands were visualized using a LAS-4000 scientific imaging system (Fujifilm, Tokyo, Japan).

[0095] (II) Animal experiments

[0096] The animal experiment is divided into two parts: subcutaneous injection experiment and oral administration experiment

[0097] Subcutaneous injection experiment

[0098] 1. Experimental animal modeling: 7-8 week old female C57BL / 6 mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. Grouping: 51 C57BL / 6 mice were randomly divided into 6 groups, namely: sham operation group (6 mice), ovariectomy group (13 mice), FRNC-1 intervention group (divided into 10 mg group 7 mice, 2 mg group 9 mice), FRT0 control peptide group (divided into 10 mg group 7 mice, 2 mg group 9 mice).

[0099] Ovariectomized group: The ovaries of mice were removed by surgical operation.

[0100] Sham operation group: The operation was the same as that of the ovariectomized group except that the mice's ovaries were not removed.

[0101] FRNC-1 intervention group: After bilateral ovariectomy, the mice were given subcutaneous injection of 10mg / 2mg FRNC-1 every other day.

[0102] FRT0 control peptide group: After bilateral ovariectomy of mice, 10 mg / 2 mg of FRT0 was subcutaneously injected into the mice every other day.

[0103] 2. Sample processing

[0104] After 8 weeks of feeding, the mice were killed, and the femurs of both hind limbs and visceral tissues (heart, liver, spleen, lung, and kidney) of the mice were collected.

[0105] (1) The left hind femur of the mouse was fixed with 4% paraformaldehyde solution, decalcified with EDTA, embedded in paraffin, and sliced ​​(4 mm). The distal metaphysis of the femur was observed under a microscope and photographed.

[0106] (2) The femur of the right hind limb of the mouse was fixed with 4% paraformaldehyde solution and subjected to Micro-CT examination. The distal femur of the mouse was scanned using a SKYSCAN 1176 micro CT machine to construct two-dimensional and three-dimensional images of the distal femoral epiphysis of the mouse. The number of femoral trabeculae (Tb.N), the ratio of bone surface area to tissue volume (BS / TV), and the relative bone volume (BV / TV) were analyzed using the software provided by the CT machine to obtain quantitative results.

[0107] (3) The fixed visceral tissues were embedded in paraffin and sliced ​​(4 mm), stained with hematoxylin-eosin (HE staining), and the mouse viscera were observed under a microscope and photographed.

[0108] Oral administration experiment

[0109] 1. Experimental animal modeling: 7-8 week old female C57BL / 6 mice were purchased from the Experimental Animal Center of the Second Military Medical University. Grouping: 43 C57BL / 6 mice were randomly divided into 6 groups, namely: sham operation group, ovariectomy group, FRNC-1 intervention group, and FRT0 control peptide group. The intervention group and control peptide group included a high-dose group (10 mg) and a low-dose group (2 mg). There were 8 mice in the control group and 7 mice in the other groups.

[0110] Ovariectomized group: The ovaries of mice were removed by surgical operation.

[0111] Sham operation group: The operation was the same as that of the ovariectomized group except that the mice's ovaries were not removed.

[0112] FRNC-1 intervention group: After bilateral ovariectomy of mice, the mice were given 10mg / 2mg FRNC-1 orally (gavage) once every other day.

[0113] FRT0 control peptide group: After bilateral ovariectomy of mice, 10 mg / 2 mg of FRT0 was orally administered (gavage) once every other day.

[0114] 2. After 8 weeks of feeding, the mice were killed and the femurs of both hind limbs and visceral tissues (heart, liver, spleen, lung, and kidney) were taken.

[0115] (1) The mouse femur was fixed with paraformaldehyde solution, decalcified with EDTA, embedded in paraffin, and sliced ​​(4 mm). The femur was stained with hematoxylin-eosin (HE). The distal metaphysis of the mouse femur was observed under a microscope and photographed.

[0116] (2) The mouse femur was fixed with paraformaldehyde solution and subjected to Micro-CT examination. The distal femur of the mouse was scanned by Micro-CT using a SKYSCAN 1176 micro CT machine to construct two-dimensional and three-dimensional images of the distal femoral epiphysis of the mouse. The number of femoral trabeculae, the ratio of bone surface area to tissue volume, relative bone volume, bone mineral density, and the number of trabecular connections were analyzed using the CT machine's built-in software to obtain quantitative results.

[0117] (3) The fixed visceral tissues were embedded in paraffin and sliced ​​(4 mm), stained with hematoxylin-eosin (HE staining), and the mouse viscera were observed under a microscope and photographed.

[0118] 2. Experimental Results

[0119] Figure 4 A is the result of CCK-8 experiment. The results showed that when the concentration of FRNC-1 was less than or equal to 2.5 μM, it was non-toxic to cells, so 2.5 μM was selected as the highest intervention dose in subsequent experiments. Figure 4 B is the result of TRAP staining, which shows that FRNC-1 has a significant inhibitory effect on the formation of osteoclasts at 1.25μM and 2.5μM, while its straight peptide chain FRT0 has no obvious effect. Figure 4 C is the bone resorption. Compared with no drug addition and FRT0, FRNC-1 has a significant inhibitory effect on bone resorption, and there is a dose-dependent relationship. Figure 4 D is the immunofluorescence image result of F-actin and DAPI. The actin ring is considered to be an important characteristic of the osteoclast resorption process. It can be found that at 2.5μM FRNC-1, compared with no drug addition and FRT0, the formation of actin belt can be significantly reduced. Figure 4 E FRNC-1 significantly inhibited the phosphorylation of GSK, which is a protein related to Akt-GSK3β-NFATc1 signaling transduction. As the concentration of FRNC-1 increased, the expression of pGSK was inhibited.

[0120] Figure 5 The results of the subcutaneous injection experiment of FRNC-1 in mice. Figure 5A is HE staining of the distal femoral epiphysis of mice 8 weeks later. The results showed that the number of trabeculae in the ovariectomized group was significantly reduced compared with the sham operation group, and the distance between trabeculae increased, showing osteoporosis; after FRNC-1 intervention, the number of trabeculae in the distal femoral epiphysis of mice was significantly greater than that in the ovariectomized group. Figure 5 B. The statistical results of trabecular bone area also showed bone loss after ovariectomy. After FRNC-1 intervention, bone loss was alleviated, and the high-dose group was better than the low-dose group. Figure 5 C is the two-dimensional and three-dimensional Micro-CT structure of the distal femur of mice after 8 weeks. The picture shows that the number of trabeculae in the ovariectomized group of mice is significantly reduced compared with the normal group. After FRNC-1 intervention, the number of trabeculae in the distal femur of mice is significantly more than that in the ovariectomized group. Similarly, after FRT0 intervention, the number of trabeculae is also more than that in the ovariectomized group. Figure 5 D is the use of computer software to analyze the distal femur, and the number of trabeculae (Tb.N), the ratio of bone surface area to tissue volume (BS / TV), and the relative bone volume or bone volume fraction (BV / TV) were counted. It was found that the FRNC-1 group of mice was significantly better than the ovariectomized group and the control group, but not as good as the sham operation group, and the difference was statistically significant (p<0.05).

[0121] Figure 6 These are the results of oral administration of FRNC-1 in mice. Figure 6 A is HE staining of the distal femoral metaphysis section of mice 8 weeks later. The picture shows that the number of trabeculae in the ovariectomized group of mice was significantly reduced compared with the sham operation group, and the distance between trabeculae increased, showing osteoporosis; after administration of FRNC-1, the number of trabeculae in the distal femoral metaphysis of mice was significantly more than that in the ovariectomized group; while the number of trabeculae in the distal femoral metaphysis of mice in the control peptide FRT0 group did not increase significantly compared with the ovariectomized group. Figure 6 B. The statistical results of trabecular bone area also showed bone loss after ovariectomy, and bone loss was alleviated after FRNC-1 intervention. Figure 6 C is the two-dimensional and three-dimensional Micro-CT structure of the distal femur of mice after 8 weeks. The picture shows that the number of trabeculae in the ovariectomized group of mice is significantly reduced compared with the sham operation group. After FRNC-1 intervention, the number of trabeculae in the distal femur of mice is significantly more than that in the ovariectomized group and the control peptide group (FRT0 group). Figure 6 D is the use of computer software to analyze the distal femur, and the number of trabeculae (Tb.N), the ratio of bone surface area to tissue volume (BS / TV), and the relative bone volume or bone volume fraction (BV / TV) were counted. It was found that the FRNC-1 group of mice was significantly better than the ovariectomized group and the control group, but not as good as the sham operation group, and the difference was statistically significant (p<0.05).

[0122] 3. Results and Discussion

[0123] Postmenopausal osteoporosis in women is a primary osteoporosis, which is related to increased osteoclast differentiation and decreased osteogenesis after decreased estrogen levels. The ovariectomized mouse model simulates human menopause and shows significant osteoporosis 8 weeks after ovariectomy, mainly manifested by a significant decrease in the number of trabeculae and a decrease in trabecular area. Current studies have shown that the occurrence of osteoporosis is related to chronic inflammation of the bones. Chronic inflammation causes excessive activation of osteoclasts, resulting in significant bone loss.

[0124] Actin ring is considered to be an important feature of osteoclast resorption process, among which Akt-GSK3β-NFATc1 signaling-related protein GSK is closely related to the overactivation of osteoclasts. As an inhibitor of GSK activation, FRNC-1 significantly reduced the loss of bone mass in ovariectomized mice, which was manifested by the increase of trabecular number, the ratio of bone surface area to tissue volume, and relative bone volume or bone volume fraction.

[0125] The above examples show that the present invention successfully prepared a double-stapled peptide based on FRATtide, and proved that the stapled peptide can significantly inhibit the differentiation of osteoclasts and has good application prospects.

[0126] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A stapler peptide, characterized in that The stapled peptide uses Ac-DPHRLLQQLVLSGNLIKEAVRRLHSR-NH2 as a peptide chain template, wherein 3 H , 7 Q Replaced by S5 and cyclized, 19 A ,twenty three L replaced by S5 and cyclized; S5: 2-amino-2-methylhept-6-enoic acid.

2. A use of the stapler peptide as claimed in claim 1, characterized in that: The stapled peptide is used in preparing medicine for treating osteoporosis.

3. A use of the stapler peptide as claimed in claim 1, characterized in that: The stapled peptide is used for preparing an agent for inhibiting osteoclast differentiation.

4. A method for preparing the stapled peptide according to claim 1, characterized in that: The following steps are involved: (1) Under the action of a condensing agent, the first amino acid at the C-terminus is coupled to a solid phase carrier; (2) using a deprotection reagent to remove the Fmoc protecting group on the amino acid; (3) Connect the next amino acid under the action of a condensing agent; (4) Repeating the deprotection-coupling operation to synthesize a peptide chain according to the amino acid sequence; wherein the cyclization site is replaced by S5 to replace the amino acid at position i and i+4 respectively; (5) Under the action of a cyclizing agent, the S5 amino acids at positions i and i+4 undergo olefin metathesis reaction to cyclize the peptide chain; (6) Repeating the deprotection-coupling operation to synthesize a peptide chain according to the amino acid sequence; wherein the cyclization site is replaced by S5 to replace the amino acid at position i and i+4 respectively; (7) The last amino acid is deprotected and then acetylated; (8) Under the action of a cyclizing agent, the S5 amino acids at positions i and i+4 undergo olefin metathesis reaction to cyclize the peptide chain for the second time; (9) Use a cleavage reagent to cut the peptide chain from the carrier and purify the corresponding stapled peptide.

5. The method for preparing the stapled peptide according to claim 4, characterized in that: The purification method used in step (9) is reverse HPLC, and the conditions are as follows: Chromatographic column: YMC-Pack ODS-AQ column; Mobile phase: Mobile phase A was 0.1% TFA / water, mobile phase B was 0.1% TFA / acetonitrile; gradient elution program: 40% B elution 0~5min, 40% B~60% B, 5~60min; flow rate was 15 ml / min, injection volume was 5 ml, and detection wavelength was 214 nm.

6. The method for preparing the stapled peptide according to claim 4, characterized in that: The condensation agent used in step (1) is a DIC-Oxyme condensation system, the activator is DIC, and NMP is used as the solvent.

7. The method for preparing the stapled peptide according to claim 4, characterized in that: In step (2), the deprotection reagent is a mixed solution of oxyme, piperidine and DMF in a ratio of 71:2:4 (m / v / v).

8. The method for preparing the stapled peptide according to claim 4, characterized in that: In step (7), the acetylation reagent is a mixture of pyridine and acetic anhydride, and the feed ratio is 1:1 (v / v).

9. The method for preparing the stapled peptide according to claim 4, characterized in that: In step (5) and step (8), the cyclizing agent is a solution of Grubbs I reagent in dichloroethane, and the feed ratio is resin loading: Grubbs I reagent: dichloroethane = 0.3:58:6 (mmol / mg / ml).

10. The method for preparing the stapled peptide according to claim 4, characterized in that: In step (9), the cleavage reagent is a mixed solution of TIPS, H2O and TFA in a volume ratio of 2.5:2.5:95, and the volume mass ratio of the cleavage reagent to the linear peptide is 1:10 mL / mg.

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