CN117683094A - Sclerostin binding cyclic peptide and application thereof - Google Patents
Sclerostin binding cyclic peptide and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/64—Cyclic peptides containing only normal peptide links
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Rheumatology (AREA)
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Abstract
The invention provides a sclerostin binding cyclic peptide and application thereof. The structural formula of the sclerostin binding cyclic peptide is as follows D PX 1 X 2 QDYX 3 X 3 PX 4 X 5 L P, wherein D P and L p is cyclized through the dehydration condensation reaction of amino acid; x is X 1 Is phenylalanine, tyrosine or tryptophan; x is X 2 Serine, threonine, glutamine, aspartic acid, methionine or cysteine; x is X 3 Serine, threonine or cysteine; x is X 4 Glycine, alanine, proline, valine, leucine or isoleucine; x is X 5 Serine, threonine, arginine, lysine or histidine. The cyclic peptide can bind with nanomolar affinity to sclerostin, thereby blocking the binding of sclerostin to LRP-6, and shows excellent ability to promote bone density. Meanwhile, compared with monoclonal antibodies, the cyclic peptide has no immune risk and is more excellent in safety and economy. The sclerostin-binding cyclic peptide thus has potential for the treatment of osteoporosis related indications.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a sclerostin binding cyclic peptide and application thereof.
Background
In China, 50% of postmenopausal women and 60% of elderly people over 60 years old have low bone mass and damaged bone microstructure, so that systemic bone metabolic disorder diseases such as bone strength reduction, bone fragility increase, fracture and the like are easy to occur, namely osteoporosis (Eastell R.et al.Nat.Rev.Dis.Primers,2016,2,16069;Black DM.et al,N.Engl.J.Med.2016,374,354-262). According to epidemiological investigation, the incidence of osteoporosis in the people over 50 years old in our country is 19.2%, wherein women are 32.1% and men are 6.9%; the incidence of osteoporosis in the population over 65 years old is 32%, with 51.6% in women and 10.7% in men (chinese osteoporosis epidemic investigation results, national health commission, 2018).
Sclerostin is a secreted glycoprotein encoded by SOST gene, can regulate osteoblast synthesis and metabolism, and is a negative regulatory factor for bone formation. There are studies showing that low or lack of SOST gene expression can lead to sclerosteosis (Balemans et al, human mol. Genet.,2001,10,547-543;Brunkow et al, am. J. Human Genet.,2001,68,577-589). Sclerostin can bind to bone morphogenic protein BMP and low density lipoprotein receptor LRP5/6, thereby inhibiting the Wnt signaling pathway and controlling osteogenesis (Winkler et al EMBO J.,2003,22,6267-6276; li X.et al., J. Biol. Chem.2005,280, 19883-19887). Thus, sclerostin has become a biological target for the treatment of osteoporosis.
Romiuzumab is a humanized IgG2 antibody targeting sclerostin co-developed by Amgen and better than company, and is marketed in U.S. at 4 months of 2019 for use in the treatment of osteoporosis in high fracture risk menopausal women and high fracture risk men. Clinical experiments show that when the Romosozumab is singly used, the bone density of a patient can be increased by 5-13% after one year after being administered once a month, and the risk of vertebral fracture is reduced by 73%; romosozumab can increase patient bone density by 3-8% compared to alendronate sodium, and reduce the risk of vertebral fracture by 50% (Cosman F.et al, N.Engl. J.Med.2016,375,1532-1543;Saag KG etal, N.Engl. J.Med.2017,377,1417-1427;Langdahl BL,et al, lancet 2017,390,1585-1594). In addition, companies such as Lilly, novartis and Hengrui medicine respectively push out sclerostin monoclonal antibody medicines, and the hard bone monoclonal antibody medicines are in clinical stage 2-3 at present.
However, the use of these monoclonal antibodies carries the risk of immune responses such as acute allergic reactions, serological diseases, and the production of anti-antibodies. Taking the romisozumab as an example, common adverse events include arthralgia, headache, muscle spasm, peripheral edema, debilitation, neck pain, insomnia, skin paresthesia and the like. In addition, the Romosozumab specification has a black box warning that the drug may increase the risk of myocardial infarction, stroke, and cardiovascular death, and that patients with heart attacks or strokes cannot use the drug within one year. Compared with monoclonal antibodies, the polypeptide drug has lower immune risk and better safety. However, few effective polypeptide drugs are currently available for the treatment of osteoporosis.
Disclosure of Invention
In order to overcome the defects in the prior art, the present invention provides a sclerostin-binding cyclic peptide capable of binding to sclerostin with high affinity, thereby blocking the binding of sclerostin to the low density lipoprotein receptor LRP 6.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a sclerostin-binding cyclic peptide having the structural formula I:
D PX 1 X 2 QDYX 3 X 3 PX 4 X 5 L P(I)
wherein:
D p and L p by amino acid removalCarrying out cyclization by water condensation reaction;
X 1 is phenylalanine, tyrosine or tryptophan;
X 2 serine, threonine, glutamine, aspartic acid, methionine or cysteine;
X 3 serine, threonine or cysteine;
X 4 glycine, alanine, proline, valine, leucine or isoleucine;
X 5 serine, threonine, arginine, lysine or histidine.
Further, X 1 Is phenylalanine.
Further, X 2 Glutamine, aspartic acid or cysteine, preferably glutamine.
Further, X 3 Serine or threonine, preferably threonine.
Further, X 4 Valine, leucine or isoleucine, preferably valine.
Further, X 5 Serine or threonine, preferably threonine.
Further, the amino acid sequence of the sclerostin binding cyclic peptide is selected from one of SEQ ID NO. 1-4, preferably SEQ ID NO. 1.
In a second aspect, the present invention provides the use of a sclerostin-binding cyclic peptide as described above for the manufacture of a medicament for the treatment of osteoporosis, the medicament comprising a sclerostin-binding cyclic peptide as an effective active ingredient.
Further, the medicament also comprises a pharmaceutically acceptable carrier or excipient.
In a third aspect, the present invention provides a medicament or pharmaceutical composition for treating osteoporosis, comprising a sclerostin-binding cyclic peptide as an effective active ingredient.
Further, the above-mentioned medicament or pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.
Compared with the prior art, the invention has the following technical effects:
the sclerostin binding cyclic peptide provided by the invention can bind with sclerostin with nanomolar affinity, so that the binding of the sclerostin with LRP-6 is blocked. The affinity of the cyclic peptide P01 and the sclerostin is similar to that of the sclerostin monoclonal antibody Romosozumab, and the in vitro inhibition capability of the hard bone peptide P01 and the LRP-6 is slightly better than that of the sclerostin monoclonal antibody Romosozumab, and the cyclic peptide P01 shows excellent bone density improving capability in the in-vivo drug effect evaluation of mice. Meanwhile, compared with monoclonal antibodies, the cyclic peptide has no immune risk and is more excellent in safety and economy. The sclerostin-binding cyclic peptide thus has potential for the treatment of osteoporosis related indications.
Drawings
FIG. 1 shows the change in bone density of mice following treatment with cyclopeptide P01 and Romosozumab in one embodiment of the invention.
Detailed Description
The present invention will be described in detail and specifically by way of the following specific examples and drawings to provide a better understanding of the present invention, but the following examples do not limit the scope of the present invention.
The methods described in the examples are carried out using conventional methods, if not specified, and the reagents used are, if not specified, conventional commercially available reagents or reagents formulated by conventional methods.
Example 1
This example provides four groups of sclerostin-binding cyclic peptides having amino acid sequences as shown in Table 1 below, wherein D P and L p undergoes cyclization by dehydration condensation reaction of amino acid.
TABLE 1 amino acid sequence of sclerostin binding cyclic peptides
| Name of the name | Amino acid sequence | SEQ ID NO: |
| P01 | D PFQQDYTTPVS L P | 1 |
| P02 | D PYQQDYSSPVS L P | 2 |
| P03 | D PFNQDYSSPLS L P | 3 |
| P04 | D PYCQDYTTPIS L P | 4 |
EXAMPLE 2 sclerostin binding force test
The sclerostin-binding cyclic peptide P01-04 provided in example 1 was covalently coupled to a CM5 chip (Cat. BR-1000-12, GE), and human sclerostin (purified from the nucleic acid encoding SEQ ID NO: 5) and mouse sclerostin (purified from the nucleic acid encoding SEQ ID NO: 6) were passed through the CM5 chip under a series of concentration gradients, and the reaction signal was detected in real time using Biacore X100 and binding and dissociation curves were obtained, and after completion of each cycle dissociation, washed and regenerated with buffer A (0.01M HEPES ph 7.4,0.15M NaCl,3mM EDTA,0.005%v/v P). Experimental data were analyzed and fitted with BIAevaluation version 4.1.4.1 to obtain affinity values, the results are shown in table 2 below.
Flag-tagged human sclerostin DNA sequence:
ATGGACATGAGGGTGCCTGCCCAACTGCTGGGCCTGTTGTTGCTTTGGTTCCCCGGAAGCAGGTGCCTCATCACCACCATCATCAGGGCTGGCAGGCCTTCAAGAACGACGCAACCGAGATTATCCCCGAACTGGGCGAATAGGCAGACCCCCCCATCATGCCGAGCCCCCTCCAGAGCTGGAGAATAACAAGACGAGAACCCAGGTACGTGACCGATGGCCCCTGGCCGAACGCTATCAGGAGACAGGTACCGCGCTCAGAGAGTGCCAGTTGTAAGTGCAAGAGGCTGGGGGGACCAGGTTCCACAACCAGAGCGAGCTGAAGGACTTCGGCACCGAAGCAGCCAGGCCGCAGAAGGGCAGGAAGCCCAGGCCACGAGCACGATCCGCCAAAGCCAATCAGGCAGAGCTCGAGAATGCCTACTGA(SEQ ID NO:5)
flag-tagged mouse sclerostin DNA sequence:
ATGGACATGAGGGTGCCTGCCCAACGGTTCCCCGGAAGCAGGTGCCAGGGCTGGCAGGCCTTCAGGAACGACGCAACCGAGGTGATCCCCGGACTGGGCGAATATCCCGAGCCCCCTCCAGAGAATAACCAGACCATGAACAGGGCCGAGAACGGCGGCAGACACGACGCTAAAGGCGTGAGCGAGTACAGCTGCAGGGAGCTGCATTACACCAGGTTCCTGACCGATGGCCCCTGTAGGAGCGCCAAGCCCGTGACTGAACTGCCAGACTGCTGCCGAACGCTATCGGCAGGGTCAAGTGGTGGAGGCCCAACGGGTGTGCAGCGGCTGTGTCCTGGGGGCGCCGCTCCGATGACCAGGTTCCACAACCAGAGCGAGCTGAAGGGAGCAGGCCAGGAGCACGAGGAGCCAAAGCCAATCAGGCAGAGCTCGAGAATGCCTACGACTACAAGGATGACGACGACAAGTGA(SEQ ID NO:6)
TABLE 2 affinity of cyclic peptides for human and mouse sclerostin
| Testing cyclic peptides or antibodies | Human sclerostin K D (nM) | Mouse sclerostin K D (nM) |
| P01 | 3.15 | 4.50 |
| P02 | 57.4 | 71.6 |
| P03 | 105 | >300 |
| P04 | 87.2 | 118 |
| Romosozumab | 0.85 | 1.23 |
As can be seen from table 2, cyclic peptide P01 shows a higher affinity for human and mouse sclerostin, on the same order of magnitude as the antibody romisozumab. The outer loop peptides P02 and P04 also have good affinity for human and mouse sclerostin.
Example 3 evaluation of the ability of sclerostin-LRP-6 to block binding
50. Mu.g of human LRP-6-Fc fusion protein (R) was added to each well in a 96-well plate&D System, cat.1505-LRP-025) at 37 ℃ for 2 hours, biotinylated human sclerostin protein (Abbkine, cat.PRP100151) and test substance (cyclic peptide P01-04 and Romosozumab) were incubated at 37 ℃ for 2 hours, the reaction solution was discarded and the plate was washed, the mixture was added to the ELISA plate and left at 4 ℃ overnight, strepavidin-peroxidase polymer (Sigma, cat.S2438-250 UG) was added to the plate and incubated at 37 ℃ for 2 hours, TMB chromogenic substrate was added and incubated at room temperature for 10 minutes, the reaction was stopped, and the absorbance was read with an ELISA reader at 450nm and IC blocking binding of sclerostin to LRP-6 was calculated 50 Values.
TABLE 3 blocking Activity of cyclopeptide P01 for sclerostin binding to LRP-6
The results of the experiment are shown in Table 3, in which the blocking activity of the cyclic peptide P01 on the binding of sclerostin to LRP-6 is slightly better than that of monoclonal antibody Romosozumab.
EXAMPLE 4 evaluation of in vivo efficacy of cyclopeptide P01 mice
C57BL/6 female mice were ovariectomized, and the mice were given intravenous cyclopeptide P01 (10 mg/kg or 30 mg/kg) or Romosozumab (10 mg/kg) 2 times a week, euthanized for 4 weeks, bled and used for tibial bone mineral density, and percent change was calculated. The blank group was normal fed mice (n=5) after ovariectomy.
As shown in FIG. 1, the bone density of the mice was reduced by 14% 4 weeks after ovariectomy, the bone density of the mice was increased by 7% by 2 intravenous injections of 10mg/kg of Romosozumab per week, and the bone density of the mice was increased by 6% by 2 intravenous injections of 10mg/kg of cyclopeptide P01 per week, similar to Romosozumab, but the bone density of the mice was increased by 12% by 2 intravenous injections of 30mg/kg of cyclopeptide P01 per week.
In conclusion, the cyclopeptide P01 can remarkably improve bone density in a mouse body and has the potential of treating osteoporosis.
The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. It will be apparent to those skilled in the art that any equivalent modifications and substitutions of the present invention are intended to be within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.
Claims (10)
1. A sclerostin-binding cyclic peptide, characterized in that the structure of the sclerostin-binding cyclic peptide is represented by formula I: D PX 1 X 2 QDYX 3 X 3 PX 4 X 5 L P(I)
wherein:
D p and L p is cyclized through the dehydration condensation reaction of amino acid;
X 1 is phenylalanine, tyrosine or tryptophan;
X 2 serine, threonine, glutamine, aspartic acid, methionine or cysteine;
X 3 serine, threonine or cysteine;
X 4 glycine, alanine, proline, valine, leucine or isoleucine;
X 5 serine, threonine, arginine, lysine or histidine.
2. The sclerostin-binding cyclic peptide of claim 1, wherein X is 1 Is phenylalanine.
3. The sclerostin-binding cyclic peptide of claim 1, wherein X is 2 Glutamine, aspartic acid or cysteine, preferably glutamine.
4. The sclerostin-binding cyclic peptide of claim 1, wherein X is 3 Serine or threonine, preferably threonine.
5. The sclerostin-binding cyclic peptide of claim 1, wherein X is 4 Valine, leucine or isoleucine, preferably valine.
6. The sclerostin-binding cyclic peptide of claim 1, wherein X is 5 Serine or threonine, preferably threonine.
7. The sclerostin-binding cyclic peptide according to claim 1, characterized in that the amino acid sequence of the sclerostin-binding cyclic peptide is selected from one of SEQ ID No. 1-4, preferably SEQ ID No. 1.
8. Use of a sclerostin-binding cyclic peptide according to any one of claims 1-7 in the manufacture of a medicament for the treatment of osteoporosis, wherein the medicament comprises the sclerostin-binding cyclic peptide as an effective active ingredient.
9. The use of claim 8, wherein the medicament further comprises a pharmaceutically acceptable carrier or excipient.
10. A medicament or pharmaceutical composition for treating osteoporosis, comprising the sclerostin-binding cyclic peptide according to any one of claims 1-7 as an effective active ingredient.
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| CN120173069A (en) * | 2025-05-21 | 2025-06-20 | 湖南中晟全肽生物科技股份有限公司 | A polypeptide for inhibiting the binding of SOST and LRP6 and its application |
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| CN120173069A (en) * | 2025-05-21 | 2025-06-20 | 湖南中晟全肽生物科技股份有限公司 | A polypeptide for inhibiting the binding of SOST and LRP6 and its application |
| CN120173069B (en) * | 2025-05-21 | 2025-08-08 | 湖南中晟全肽生物科技股份有限公司 | A polypeptide for inhibiting the binding of SOST to LRP6 and its application |
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