CN111012895B - Application of inhibitor SC-3 of Cyr61/CCN1 protein epitope polypeptide - Google Patents
Application of inhibitor SC-3 of Cyr61/CCN1 protein epitope polypeptide Download PDFInfo
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
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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
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
Abstract
The invention discloses a Cyr61/CCN1 protein epitope polypeptide and application thereof in preparing a medicament for detecting or treating rheumatoid arthritis synovial cell hyperplasia, psoriasis epidermal cell hyperplasia and inflammatory diseases, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO.1 and SEQ ID NO. 2. The invention also discloses a specific anti-Cyr 61/CCN1 monoclonal antibody combined with the Cyr61/CCN1 protein epitope polypeptide and a Cyr61/CCN1 protein epitope polypeptide inhibitor. The Cyr61/CCN1 protein epitope polypeptide and related new drugs thereof have wide application prospects.
Description
The application is a divisional application of Chinese invention patent application with the application number of 201410152740.7 (the application number of divisional application: 201610897943.8, named as "Cyr61/CCN1 protein epitope polypeptide and inhibitor thereof, monoclonal antibody and application thereof"), named as "Cyr61/CCN1 protein epitope polypeptide and inhibitor thereof, monoclonal antibody and application thereof", filed 4, 16, 2014, the parent application requires priority of Chinese invention patent application with the application date of 2014, 02, 08, the application number of 201410045817.0, named as "Cyr61/CCN1 protein epitope polypeptide and inhibitor thereof, monoclonal antibody and application thereof".
Technical Field
The invention belongs to the field of medicines, and relates to Cyr61/CCN1 protein epitope polypeptide and application thereof in preparation of medicines for detecting or treating Rheumatoid Arthritis (RA) synovial cell (FLS) hyperplasia, psoriasis epidermal cell hyperplasia and inflammatory diseases.
Background
Cyr61 (cysteine-rich 61), also known as CCN1, is a secreted protein of 381 amino acid residues in relative molecular mass of 42kDa, and the earliest cloned Cyr61/CCN1 was discovered by Lau et al in 1985 when stimulating mouse BALB/c 3T3 fibroblasts with serum or platelet-derived growth factor (PDGF), and was named Cyr61/CCN1 because of the rich cysteine (containing 10% cysteine residues). Cyr61/CCN1 has obvious mitogenic activity and chemotaxis, can induce fibroblast and epidermal cell to proliferate and secrete extracellular matrix, participate in regulating cell proliferation, differentiation and embryonic development and formation, and is a basic protein for life activities.
In the binding reaction of antigen and antibody, the site where the antibody participates in the binding is called para (paratope) of the antibody, and the site where the antigen (Cyr 61/CCN1 protein) participates in the binding is called epitope (epitope) of the antigen. Epitopes are the basis for protein antigenicity. The determination of the antigen epitope (small molecule peptide segment) has important significance for defining the action site of the protein, namely the small molecule peptide segment, further designing and adopting the small molecule peptide segment to simulate the action of the protein, or designing a small molecule compound capable of simulating the peptide segment to replace the action of the protein or the peptide molecule, or designing the small molecule compound to block the action of the protein, and the determination is also one of the development and preparation methods of novel medicaments popular in the world at present.
Disclosure of Invention
The invention provides Cyr61/CCN1 protein epitope polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1 and is GLECNFG.
The invention also provides Cyr61/CCN1 protein epitope polypeptide which is a cyclic peptide segment shown as SEQ ID NO.2, namely, a cysteine is added at the N end of the peptide segment of SEQ ID NO.1, thereby forming a cyclic peptide segment 'C-G LECNF G' shown as SEQ ID NO. 2.
The Cyr61/CCN1 protein epitope polypeptide is positioned at 75-81 th amino acid of Cyr61/CCN1 protein, can be combined with specificity anti-Cyr 61/CCN1 monoclonal antibody 093G9 (KD is 10-7) and neutralizes the functions of Cyr61/CCN1 in vitro and in vivo, and can be artificially synthesized into a cyclic peptide segment C-G LECNF G when a C is added at the N end.
In the invention, the Cyr61/CCN1 protein epitope polypeptide can be prepared by hydrolyzing protease, and the Cyr61/CCN1 protein epitope polypeptide can also be prepared by synthesizing by a conventional solid phase polypeptide synthesis method or a genetic engineering technology.
In the invention, the full-length sequence (GenBank: M _ 001554) of human Cyr61/CCN1 protein obtained by gene library search is shown as SEQ ID NO.3 as follows:
wherein the sequence in the black box is a signal peptide of Cyr61/CCN1 protein.
The invention also provides a specific anti-Cyr 61/CCN1 monoclonal antibody, and the specific anti-Cyr 61/CCN1 monoclonal antibody can be combined with the Cyr61/CCN1 protein epitope polypeptide.
The invention also provides an inhibitor of the Cyr61/CCN1 protein epitope polypeptide, which can be combined with the Cyr61/CCN1 protein epitope polypeptide, can be combined with the GLECNFG ring structure and can block the mediated biological function.
The inhibitor of the Cyr61/CCN1 protein epitope polypeptide comprises a small molecular compound which can block the biological function of the Cyr61/CCN1 protein epitope polypeptide. Preferably, the small molecule compounds SC1-SC15 are shown in the embodiment of the invention. Specifically, a small molecular compound with the biological function of simulating and inhibiting the peptide fragment can be designed according to the conformational epitope of the peptide fragment of the Cyr61/CCN1 protein epitope polypeptide. The small molecular compound capable of blocking the polypeptide peptide segment has the effect of inhibiting the molecular pathobiology of Cyr61/CCN1, and has important application in further developing into a lead compound and preparing medicaments for resisting synovium hyperplasia and bone destruction, resisting Cyr61/CCN1 high-expression breast cancer cell proliferation, resisting epidermal cell hyperproliferation and resisting fibrosis (such as hepatic fibrosis). The inhibitor can be combined with a GLECNFG cyclic structure and can block pathological effects (including causing inflammation, causing fibroblast proliferation, causing proliferation and metastasis of Cyr61/CCN1 high-expression breast cancer cells, causing proliferation and activation of epidermal cells and the like) mediated by the GLECNFG cyclic structure.
Previous studies in the present invention have shown that IL-17 is capable of upregulating Cyr61/CCN1 expression, whereas upregulating Cyr61/CCN1 expression promotes synovial cell hyperproliferation by binding to the integrin receptor AvB5, which is indicated by synovial cells, and can promote the synthesis of metalloproteinase 3 and metalloproteinase 9 and thus be involved in cartilage and bone destruction. The research of the invention shows that Cyr61/CCN1 protein can directly promote synovial cells to produce IL-6, thereby participating in the generation and development of inflammation. Furthermore, the research of the invention shows that Cyr61/CCN1 can also promote the human epidermal cell proliferation and participate in the psoriasis. In the animal experiment, cyr61/CCN1 is also found to mediate the occurrence of sicca syndrome and proliferative inflammatory bowel disease. Experiments prove that the protein expression can be inhibited by neutralizing the anti-Cyr 61/CCN1 monoclonal antibody, so that the synovial cell proliferation and IL-6 production can be inhibited, and the clinical symptoms, tissue destruction and inflammation of arthritis in a CIA model can be alleviated; inhibiting epidermal cell proliferation, relieving skin thickening and scale generation of psoriasis-like mice, and relieving inflammation and clinical symptoms of xerosis syndrome and proliferative enteropathy animal model.
The monoclonal antibody capable of binding Cyr61/CCN1 protein is prepared by the invention. These antibodies show differences in function due to the epitopes bound by these different monoclonal antibodies, i.e., different parts of the Cyr61/CCN1 protein.
The invention also provides application of the Cyr61/CCN1 protein epitope polypeptide in preparing medicaments for detecting and/or treating inflammatory diseases. Preferably, the inflammatory disease comprises rheumatic diseases, rheumatoid arthritis, psoriasis. Preferably, the inflammatory disease comprises sjogren's syndrome. Preferably, the inflammatory disease comprises a proliferative inflammatory bowel disease.
The invention also provides application of the Cyr61/CCN1 protein epitope polypeptide in preparing a medicament for resisting rheumatoid arthritis synovial cell hyperplasia. The embodiment of the invention shows that the monoclonal antibody 093G9 capable of specifically neutralizing the epitope can inhibit synovial cell proliferation of the rheumatoid arthritis and animal models thereof to inhibit epidermal cell proliferation of psoriasis and animal models thereof, inhibit symptoms and inflammation of the sicca syndrome animal models and inhibit inflammation and symptoms of the proliferative inflammatory bowel disease animal models in vivo and in vitro.
The invention also provides application of the Cyr61/CCN1 protein epitope polypeptide in preparing a medicament for inhibiting high expression of breast cancer cells. The embodiment of the invention shows that the monoclonal antibody 093G9 capable of specifically neutralizing the Cyr61/CCN1 protein epitope can inhibit proliferation, migration and invasion of breast cancer cells highly expressing Cyr61/CCN1 protein in vivo and in vitro, and can inhibit the tumor growth and lymphatic metastasis of the type in an animal model (in vivo).
The invention also provides application of the Cyr61/CCN1 protein epitope polypeptide in preparing anti-fibrosis drugs.
The invention also provides application of the Cyr61/CCN1 protein epitope polypeptide in preparing anti-psoriasis medicaments. The embodiment of the invention shows that the monoclonal antibody 093G9 capable of specifically neutralizing the epitope can inhibit epidermal cell proliferation, skin thickening and scale generation of psoriasis-like mice in vivo and in vitro.
The invention also provides application of the inhibitor of the Cyr61/CCN1 protein epitope polypeptide in preparing a medicament for treating rheumatic diseases.
Functional analysis of the epitope on Cyr61/CCN1 protein of the present invention showed that the cyclic peptide of the recognition peptide fragment of antibody 093G9 has the effect of stimulating the in vitro proliferation of the rheumatoid arthritis synovial cells, while the recognition epitope of control antibody 096B7 does not have such a property, as shown in FIG. 1.
The obtained antibody recognition epitope is artificially synthesized into peptide fragments, and the peptide fragments are added into cell culture to observe the biological characteristics of the peptide fragments. The result shows that certain peptide segments can inhibit the proliferation of synovial cells and Cyr61/CCN1 high-expression breast cancer cell strains in vitro, and the peptide segments have the function of simulating anti-Cyr 61/CCN1 monoclonal antibodies. Therefore, the peptide fragments and the compound micromolecules capable of being combined with the peptide fragments have the effect of inhibiting Cyr61/CCN1 protein and have pharmaceutical potential.
The invention also has the beneficial effects that the application of the Cyr61/CCN1 protein epitope polypeptide in researching and developing new medicaments for treating rheumatoid arthritis, psoriasis, sicca syndrome and proliferative inflammatory bowel disease is also included. The incidence of rheumatoid arthritis is as high as 0.4-1% in China; the incidence rate of psoriasis in Chinese is up to 0.3-0.5%, the incidence rate in caucasians is higher (for example, the incidence rate in the United states is 1-3%, and the incidence rate in Norway is up to 7%), and the Cyr61/CCN1 protein epitope polypeptide and the new medicine developed according to the epitope have wide application prospects.
The significance of the epitope of the peptide fragment G LECNF G (114, cyclic peptide is 111) of the invention is as follows: according to "Trends Biochem sci.2008 October;33 (10): 461-473 "report sequence: amino acids 75-81 of IGFBP region of CCN family molecule sequence contain three conservative amino acids of G L C.
Of the above, 7 amino acids in the colored portion are conserved in the molecule (CCN 1-6) of this family, however, 093G9 binds only to Cyr61/CCN1. Although the G LECNF G is located only on Cyr61/CCN1 and is a specific recognition epitope of 093G9, the conserved sequence G-L-C is present in all CCN families. According to the results of the experiment that 093G9 binds not to the linear peptide G LECNFG, but only to the cyclic peptide C G LECNF G, it is suggested that although the conformational epitope formed by the cyclic peptide C-G-L-C may be located in the CCN family of molecules, G LECNF G is a specific epitope with pathogenic effect.
Biological significance of the epitopes of the invention include:
the cyclic peptide 111 can stimulate in vitro proliferation of synovial cells and Cyr61/CCN1 high-expression breast cancer MDA-MB-231 cells of RA patients, and therefore, the peptide fragment is a specific pathogenic epitope of Cyr61/CCN1 molecules.
The monoclonal antibody 093G9 can specifically recognize an epitope in a peptide fragment (IGFBP) of Cyr61/CCN1, and the antibody 093G9 blocking the epitope has the effects of blocking synovial cells of Cyr61/CCN1, MDA-MB-231 cells with high expression of breast cancer and proliferation of other fibroblasts.
According to the conformational epitope of the peptide fragment, a chemical small molecule (SC) with the functions of simulating and inhibiting the biology of the peptide fragment can be designed. Wherein, the chemical small molecule (SC) capable of blocking the peptide segment has the function of inhibiting the molecular pathology and biology of Cyr61/CCN1. The compound has important application in further developing into a lead compound and preparing medicaments for resisting synovium hyperplasia and bone destruction, resisting Cyr61/CCN1 high-expression breast cancer cell proliferation and resisting fibrosis (such as hepatic fibrosis).
As Cyr61/CCN1 protein is rich in cysteine and easy to form disulfide bonds, the expression is very difficult by adopting a genetic engineering method, and the peptide segment is easy to artificially synthesize, so that the protein can replace Cyr61/CCN1 molecular immunity to obtain antiserum for resisting the functional epitope or prepare a corresponding monoclonal antibody for further clinical detection and application in treatment.
The present invention is directed to protecting the sequence of polypeptide 111, the steric structure (including linear and cyclic structures) that may be generated by the sequence, and small molecule compounds that bind thereto and produce the same effects (e.g., promoting synovial cell proliferation, producing antibodies after immunization that can inhibit collagen-induced arthritis (CIA) mouse inflammation, etc.) or directly inhibit the function of these peptide fragments.
Drawings
FIG. 1 shows a schematic diagram of the simulated conformation and chemical structure of a polypeptide of the present invention, wherein (A) shows a linear conformation of the polypeptide 114; (B) polypeptide 111 in a cyclic conformation; (C) represents the chemical structure of the polypeptide 111.
FIG. 2 shows the results of an experiment in which the polypeptide of the present invention stimulates the proliferation of RA synovial cells in vitro.
FIG. 3 shows the results of an experiment in which the polypeptide of the present invention stimulates MDA-MB-231 cell proliferation.
FIG. 4 shows the inhibitory effect of the small molecule compounds of the present invention on peptide fragment-stimulated synovial cell proliferation, wherein (A) shows the significant inhibition of peptide fragment 111-stimulated synovial cell proliferation by 15 chemical small molecules (SC 1-15), respectively; (B) Shows that 15 chemical small molecules (SC 1-15) respectively have obvious inhibition on the synovial cell proliferation stimulated by Cyr61/CCN1 protein.
FIG. 5 shows the inhibitory effect of the small molecule compound of the present invention on the proliferation of the peptide-stimulated breast cancer cell line MDA-MB-231, wherein (A) shows that 15 chemical small molecules (SC 1-15) significantly inhibit the in vitro proliferation of the peptide-111-stimulated breast cancer cell line MDA-MB-231; (B) Shows that 15 chemical small molecules (SC 1-15) respectively inhibit the in vitro proliferation of a breast cancer cell strain MDA-MB-231 stimulated by Cyr61/CCN1 protein.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
Example 1 screening of the epitope polypeptide of Cyr61/CCN1 protein of the present invention
In the invention, the immunoblotting method is adopted for screening: an improved traditional method for determining the epitope is adopted, namely a series of small peptide fragments of Cyr61/CCN1 are expressed in a segmented mode through genetic engineering, and then the 2 strains of antibodies are used for carrying out Western Blot (Western Blot) hybridization to screen binding epitopes, so that the peptide fragments capable of being combined with the 2 strains of Cyr61/CCN1 monoclonal antibodies are obtained.
According to the existing research results, cyr61/CCN1 protein is divided into small peptide segments with the length of about 25 amino acid segments, then primers are designed to clone the genes of the segments into escherichia coli, the genes are induced and expressed after expression sequencing is correct, the genes are transferred to a nitrocellulose membrane after PAGE electrophoresis, and then a mouse anti-human Cyr61/CCN1 specific monoclonal antibody 09369 (CGMCC No) is respectively used.3351) And a mouse anti-human Cyr61/CCN1 specific monoclonal antibody 096B7 (CGMCC No).3299)Hybridization and color development were performed, and binding was observed. Binding is positive, otherwise negative. Thereby determining the binding epitope of the two antibodies. Screening of antibodies by the above method09369 binds to an epitope located at amino acids 75-81 of Cyr61/CCN 1: cyr61/CCN1 75-81 */(&1) *. The position of this epitope in the Cyr61/CCN1 sequence (in bold):
example 2 Synthesis of Cyr61/CCN1 protein epitope Polypeptides
In the embodiment, the Cyr61/CCN1 protein epitope polypeptide is synthesized by Shanghai Qiangyao company. The basic principle is a full-automatic solid-phase synthesis method. The basic process is as follows: based on Fmoc chemical synthesis, the carboxyl of C-terminal amino acid of target polypeptide to be synthesized is firstly connected with insoluble macromolecular resin in a covalent bond mode, then the amino group of the amino acid is used as the starting point of polypeptide synthesis, and reacts with activated carboxyl of other amino acid to form peptide bond, and the process is repeated continuously, so that the polypeptide can be obtained. The synthesized polypeptide is purified by HPLC, and the purity is over 95 percent. According to the binding epitope obtained by screening, respectively synthesizing linear polypeptide and cyclic polypeptide, wherein the synthesized polypeptides are respectively: a linear polypeptide (No. 114) having the sequence GLECNFG; cyclic polypeptide (No. 111) having the sequence C GLECNFG.
Example 3 conformation and chemical Structure of epitope polypeptide of Cyr61/CCN1 protein
And fitting the Cyr61/CCN1 protein epitope peptide fragment in a pure water solution by adopting Cassion molecular simulation computer software to obtain the most likely conformation formed in an in-vivo solution environment and obtain a molecular structure with a three-dimensional observation effect. The three-dimensional conformation of the Cyr61/CCN1 protein epitope polypeptide shown in figures 1 (a) and (b), wherein figure 1 (a) shows the polypeptide 114 as a linear conformation; FIG. 1 (b) shows polypeptide 111 in a cyclic conformation; FIG. 1 (c) shows the chemical structure of the polypeptide 111.
Example 4 Cyr61/CCN1 protein epitope Polypeptides stimulate synovial cells to proliferate in vitro
Culture and proliferation experiment of rheumatoid arthritis synovial cells
Clinical samples: all samples from the study were obtained from RA patients with clinical orthopedic knee replacement or synovectomy, and diagnosis in all cases met international diagnostic criteria. The patient synovial tissue was used for in vitro cell culture. Clinical samples used in this study were informed of the patients.
Preparing synoviocytes and primary culture: obtaining synovial tissue aseptically, washing with PBS, and shearing into 1mm × 1mm × 1mm pieces with aseptic surgical scissors; digesting with collagenase I or collagenase II with 2-3 times of volume and 0.5mg/ml at 37 ℃ for 2 hours; after filtration through a 200 mesh gauze and centrifugation to remove the supernatant, the cells were resuspended in DMEM medium and plated in a petri dish at 37 ℃ and 5% CO 2 And (4) culturing. Then when FLS grows to be more than 80%, digesting and passaging. Observed under a microscope, the RA FLS of 3-5 generations grows rapidly, and the cell morphology tends to a long spindle shape, is regularly arranged and is consistent in orientation. The above-mentioned molecules were found to be negative in expression by flow cytometry after labeling with specific lymphocyte differentiation antigens such as CD3, CD14, CD19, CD11C and the like (< 2% CD1Ib, < 2% CD14+, < 1% CD3+, and < 5% CD19 +), indicating that the cultured cells were homogeneous FLS with little lymphocyte contamination.
Proliferation assay of synovial cells: taking FLS in logarithmic growth phase, digesting with 0.25% trypsin, and adjusting cell concentration to 1 × 10 4 Cells/ml, inoculated in 96-well cell culture plates, and added with the peptide fragments (final peptide fragment concentration: 2.5ug/ml,5.0ug/ml 10 ug/ml) at different concentrations. Cyr61/CCN1 intact protein was also used as a positive control (Proptech, inc., USA, san Diego). Culture medium controls were also provided. The peptide fragment or pure Cyr61/CCN1 protein was co-cultured with FLS, 1. Mu. Ci of 3H (per well) was added 16 hours before the end of the culture, the cells were collected, and proliferation was detected by beta scintillation.
Type II collagen-induced mouse arthritis (CIA) model experiment:
the peptide fragment immunization experiment for preventing the CIA mice from generating arthritis: the peptide fragment and CFA are completely mixed, DBA1 mice are immunized subcutaneously on the back for 3 times: for the first time: peptide + CFA, second and third: peptide + IFA (incomplete adjuvant), immunization dose: 100ug of peptide/mouse, 2-3 points of back immunization, 1 month interval each time, 7 days after the third immunization, tail blood collection, and ELISA for detecting whether antiserum is generated; meanwhile, CII100ug and the same amount of IFA are used for immunizing mice, and the morbidity, morbidity speed and inflammation intensity of the mice are observed.
Peptide fragment treatment or shock of CIA mice: namely, a CIA mouse model is prepared firstly, and the peptide segment is used for mucosal administration when the inflammation reaches 4 minutes. The method comprises the following steps: the peptide fragment was 2500ug/ml,10ul/mouse, and the final concentration was 25ug/mouse/day. Solvent was used as a negative control.
The peptide segments of the Cyr61/CCN1 protein epitope polypeptide are respectively added into synovial cells of a Fengguan primary culture, and the stimulation capability of the peptide segments on the proliferation of the synovial cells is detected after 48 hours, and the experimental result shows that the peptide segment 111 can obviously stimulate the proliferation of the synovial cells and is dose-dependent, as shown in figure 2. The experimental result shows that the peptide segment 111 positioned at 75-81 positions of Cyr61/CCN1 can form a ring conformation in aqueous solution to simulate the effect of Cyr61/CCN1 protein on stimulating the proliferation of synovial cells.
Example 5 Cyr61/CCN1 protein epitope polypeptide stimulates the in vitro proliferation of Cyr61/CCN1 high expression breast cancer cell strain (MDA-MB-231)
Will be 1 × 10 4 And (3) inoculating the MDA-MB-231 cells into a 96-well plate, adding peptide fragments with different concentrations (2.5 ug/ml, 5ug/ml and 10 ug/ml), adding 3H-TdR after 4 hours, continuously incubating for 16 hours, collecting the cells, and detecting a c.p.m. value by a Beta scintillator for judging the cell proliferation state. As shown in FIG. 3, peptide fragment 111 has a significant effect of stimulating cell proliferation.
Example 6 screening to obtain a Small Compound (SC 1-15) that binds to and inhibits the Activity of Cyclic peptide 111
Designing a chemical small molecule (SC) with the functions of simulating and inhibiting the biological function of the peptide fragment according to the conformational epitope (cyclic polypeptide C GLECNFG, number 111) of the peptide fragment, wherein the chemical small molecule (SC) capable of blocking the peptide fragment has the function of inhibiting the molecular pathobiology of Cyr61/CCN1. The compound has important application in further developing into a lead compound and preparing medicaments for resisting synovium hyperplasia and bone destruction, resisting Cyr61/CCN1 high-expression breast cancer cell proliferation and resisting fibrosis (such as hepatic fibrosis). The invention adopts a virtual screening method commonly used in the development of new drugs, and screens out chemical molecules with possible therapeutic effect by a calculation method. At present, the application is more extensive, and the three-dimensional high-resolution structure of the target is obtained by using an X-ray diffraction or Nuclear Magnetic Resonance (NMR) experiment on the target of the drug or using a biological information structure prediction method, and then virtual screening is carried out.
In this embodiment, the successful virtual drug screening includes: (1) a sufficiently large chemical space (chemical space), (2) an accurate scoring function (scoring function), and (3) a search algorithm (searching algorithm) with high efficiency. Chemical space, i.e. the chemical database used; in a broad sense, the chemical space includes the more likely conformation (conformation) of the ligand (ligand) and receptor; the scoring function is designed mainly by searching a Protein database (Protein database, http:// www.pdb.org) for a ligand-receptor combination (complex) with better resolution, applying energy calculation and a statistical method such as multivariate linear regression to establish an accurate mathematical function, and quickly and effectively presuming whether an unknown ligand can be stably bound to a given receptor, even the binding strength (binding affinity) or the binding free energy (binding free energy). The scoring function is based on the complex energy changes in translation, rotation, or conformation changes of the ligand molecules relative to the receptor, and finds the binding mode with the best scoring value.
In this embodiment, on the basis of the research that the target cyclic polypeptide C GLECNFG (number 111) can effectively simulate the biological effect of Cyr61/CCN1 protein, lead compounds targeting cyclic peptide 111 with structural entities are obtained by screening, and these compounds with small molecular structures can bind to cyclic peptide 111 and inhibit the activity thereof. According to the chemical structure of the cyclic polypeptide 111, 15 small-molecule compounds SC capable of binding with the cyclic polypeptide are screened by the method, and the structures and names of the compounds SC-1 to SC-15 are shown in the following table 1. The structure and the synthesis method of the small molecular compound SC are the prior art.
Example 7 Effect of chemical Small molecules (SC) 1-15 capable of binding to peptide fragment 111 on in vitro inhibition of synovial cell proliferation stimulated by Cyclic peptide 111
Clinical samples, synovial cell preparations, and primary cultures were the same as those described in example 4.
Proliferation assay of synovial cells: taking FLS in logarithmic growth phase, digesting with 0.25% trypsin, and adjusting cell concentration to 1 × 10 4 Cells/ml were plated in 96 well cell culture plates and 10ug/ml of peptide fragment 111 and 2.5ug/ml of Cyr61/CCN1 protein (Proptech, san Diego, USA) were added. Then, 10. Mu.M of the small chemical molecules SC-1 to SC-15 selected by the method of example 6, as shown in Table 1 below, were added, and the mixture was co-cultured for 48 hours. 1 mu Ci of 3H (each well) is added 16 hours before the culture is finished, cells are collected, proliferation is detected by a beta liquid scintillation counter, the experimental result is shown in figure 4, and 15 chemical small molecules (SC-1 to SC-15) respectively have obvious inhibition effect on the proliferation of synovial cells stimulated by peptide fragments 111 (shown in figure 4A) and Cyr61/CCN1 protein (shown in figure 4B).
Example 8 Effect of the Small chemical molecules 1-15 binding to peptide fragment 111 in vitro inhibition of proliferation of Breast cancer cell line MDA-MB-231 stimulated by cyclopeptide 111 and Cyr61/CCN1
Will be 1 × 10 4 Each MDA-MB-231 cell was seeded into a 96-well plate, and 10ug/ml of peptide fragment 111 and 2.5ug/ml of Cyr61/CCN1 protein (Proptech, inc., san Diego, USA) were added. Then 10 μ M of chemical small molecules SC-1 to SC-15 obtained by screening according to the method of example 6 were added, as detailed in table 1 below, 1 μ Ci of 3H (per well) was added after co-culturing for 4 hours, incubation was continued for 16 hours, cells were collected, and c.p.m. values were detected by Beta scintillator for judging cell proliferation status. The experimental result is shown in fig. 5, and the 15 chemical small molecules (SC 1-15) have obvious inhibition effect on the in vitro proliferation of the breast cancer cell strain MDA-MB-231 stimulated by the peptide fragment 111 (shown in fig. 5A) and the Cyr61/CCN1 protein (shown in fig. 5B).
<110> Texas diagnostic System (Shanghai) Co., ltd
Application of SC-3 inhibitor of <120> Cyr61/CCN1 protein epitope polypeptide
<160> 3
<210> 1
<211> 7
<212> PRT
<213> race (Homo sapiens)
<400> 1
GLECNFG
<210> 2
<211> 8
<212> PRT
<213> race (Homo sapiens)
<400> 2
C-G LECNF G
<210> 3
<211> 77
<212> PRT
<213> ethnic species (Homo sapiens)
<400> 3
MSSRI ARALA LVVTL LHLTR LALST CPAAC HCPLE APKCA PGVGL VRDGC GCCKV CAKQL NEDCS KTQPC DHTKG LECNF GASST ALKGI CRAQS EGRPC EYNSR IYQNG ESFQP NCKHQ CTCID GAVGC IPLCP QELSL PNLGC PNPRL VKVTG QCCEE WVCDE DSIKD PMEDQ DGLLG KELGF DASEV ELTRN NELIA VGKGS SLKRL PVFGM EPRIL YNPLQ GQKCI VQTTS WSQCS KTCGT GISTR VTNDN PECRL VKETR ICEVR PCGQP VYSSL KKGKK CSKTK KSPEP VRFTY AGCLS VKKYR PKYCG SCVDG RCCTP QLTRT VKMRF RCEDG ETFSK NVMMI QSCKC NYNCP HANEA AFPFY RLFND IHKFR D
Claims (2)
- The application of an inhibitor of Cyr61/CCN1 protein epitope polypeptide in preparing a medicament for resisting the proliferation of rheumatoid arthritis synovial cells is characterized in that the inhibitor of Cyr61/CCN1 protein epitope polypeptide is:。
- the application of an inhibitor of Cyr61/CCN1 protein epitope polypeptide in preparing a medicament for inhibiting breast cancer cell proliferation is characterized in that the inhibitor of Cyr61/CCN1 protein epitope polypeptide is:。
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| CN201410152740.7A Active CN103923174B (en) | 2014-02-08 | 2014-04-16 | Cyr61/CCN1 protein antigenic epitope polypeptide, inhibitor and application thereof as well as monoclonal antibody |
| CN201610897943.8A Active CN106492188B (en) | 2014-02-08 | 2014-04-16 | Cyr61/CCN1 protein epitope polypeptide, inhibitor and monoclonal antibody thereof, and application thereof |
| CN201911090995.4A Active CN111068040B (en) | 2014-02-08 | 2014-04-16 | Application of inhibitor SC-4-SC-15 of Cyr61/CCN1 protein epitope polypeptide |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101492495A (en) * | 2009-02-24 | 2009-07-29 | 中国农业科学院哈尔滨兽医研究所 | A group of antigen epitope polypeptide and uses thereof |
| CN101709088A (en) * | 2009-12-04 | 2010-05-19 | 上海市免疫学研究所 | Monoclonal antibody for resisting Cyr61 protein and application thereof |
| WO2011054315A1 (en) * | 2009-11-06 | 2011-05-12 | 上海市免疫学研究所 | Use of cyr61 protein for preparing medicine |
| WO2013049830A2 (en) * | 2011-09-30 | 2013-04-04 | The Washington University | Tip-1 and grp-78 binding peptides and method of identifying peptide receptors |
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| US5326751A (en) * | 1992-06-26 | 1994-07-05 | Arizona Board Of Regents On Behalf Of The University Of Arizona | Enkephalin analogs |
| IL121134A0 (en) * | 1997-06-22 | 1997-11-20 | Yeda Res & Dev | Peptides and antiallergic compositions comprising them |
| TW201102086A (en) * | 2009-06-04 | 2011-01-16 | Hoffmann La Roche | Antibodies against human CCN1 and uses thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101492495A (en) * | 2009-02-24 | 2009-07-29 | 中国农业科学院哈尔滨兽医研究所 | A group of antigen epitope polypeptide and uses thereof |
| WO2011054315A1 (en) * | 2009-11-06 | 2011-05-12 | 上海市免疫学研究所 | Use of cyr61 protein for preparing medicine |
| CN101709088A (en) * | 2009-12-04 | 2010-05-19 | 上海市免疫学研究所 | Monoclonal antibody for resisting Cyr61 protein and application thereof |
| WO2013049830A2 (en) * | 2011-09-30 | 2013-04-04 | The Washington University | Tip-1 and grp-78 binding peptides and method of identifying peptide receptors |
Also Published As
| Publication number | Publication date |
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| CN103923174B (en) | 2017-01-11 |
| CN111068040A (en) | 2020-04-28 |
| CN106492188B (en) | 2019-12-20 |
| CN111012895A (en) | 2020-04-17 |
| CN111150832B (en) | 2023-03-07 |
| CN111068040B (en) | 2023-03-07 |
| CN106492188A (en) | 2017-03-15 |
| CN111150832A (en) | 2020-05-15 |
| CN103923174A (en) | 2014-07-16 |
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