CN103655556A - Anti-inflammatory application of small molecule compound AG-4 - Google Patents
Anti-inflammatory application of small molecule compound AG-4 Download PDFInfo
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Abstract
The invention discloses anti-inflammatory application of a small molecule compound AG-4, and application in preparing anti-inflammatory drug. The small molecule compound AG-4 is screened through a candidate drug screening platform combining structural biology, information biology and biological activity determination. The small molecule compound AG-4 can realize specific binding with CD147 protein to inhibit inflammatory cell chemotaxis and secretion of chemotaxis; the invention is also suitable for preparing medicines for treating CD147 protein high expression related diseases (such as rheumatoid arthritis).
Description
Technical field
The invention belongs to biotechnology and medical domain, be specifically related to the inflammatory applications of micromolecular compound AG-4.
Background technology
Rheumatoid arthritis (rheumatoid arthritis, RA) is a kind of the most common chronic inflammatory joint disease, global sickness rate 1%, and China RA patient has exceeded 5,000,000 people.RA patient's disability rate is high, in initial 2 years of morbidity, approximately has 75% patient to occur joint damage, and patient's posterior joint distortion in 20 years of 80% is disabled, and part patient's disease progression is rapid, even causes death.Therefore, the treatment of RA is the focus that medical circle is paid close attention to always.The pathogenesis of RA is also indefinite at present, has therefore limited the progress for the treatment of means.
CD147 molecule, is a kind of glycoprotein of wide expression, belongs to a member of immunoglobulin superfamily.CD147 wide expression, in various types of cells, especially at tumor cell high expressed, is a focus molecule of malignant tumor research.Recent study is found, the expression of CD147 rising in RA patient's synovial membrane, the equal high expressed CD147 of T cell of the Monocytes/Macrophages in RA peripheral blood in patients, synovial fluid, neutrophilic granulocyte and activation.The cyclophilin A (CypA) of RA inflammation joint high expressed can be by assembling in the focus of joint with the T cell of CD147 interaction chemotactic Monocytes/Macrophages, neutrophilic granulocyte and activation, and CD147 antibody can be blocked chemotaxis.Monocytes/Macrophages, neutrophilic granulocyte become respectively fiber-like synovial cell (RA FLS) to cultivate altogether with Patients With Rheumatoid Arthritis, can promote FLS emiocytosis MMPs, thereby promote cartilage, bone erosion.RA animal model experiment result shows, CypA can increase the weight of RA synovial membrane inflammation, and the treatment of CD147 monoclonal antibody can be alleviated synovial membrane inflammation.Research is discovery also, and the treatment of CD147 monoclonal antibody can suppress the secretion of matrix metalloproteinase (MMPs), alleviates the invasion and attack of RA synovial membrane to cartilage.More than research prompting, CD147 has participated in RA pathogenesis, and CD147 may become a novel targets of research RA medicine.Development along with structure biology and bioinformatics, area of computer aided drug screening technology based on protein three-dimensional structure arises at the historic moment, thereby realize docking and screening of protein and compound, both shortened the cycle of conventional medicament research and development, again can intensifier target tropism, greatly improved patent medicine efficiency.This technology has obtained abundant approval and extensive use at present.
Although the Drug therapy development of RA in recent years, the treatment level of RA is increased, but use at present traditional DMARDs medicine, and normal and other drug is while combining use, has 25% to 50% still state of an illness activity of RA patient, the aggravation of carrying out property of destruction of joint.Biological targeting preparation, especially inhibitors of tumor necrosis factor-alpha has obtained success on clinical treatment, but still have quite a few RA patient to resist that TNF fails to respond to any medical treatment or poor effect, and have quite serious untoward reaction, as infected and malignant tumor etc.Therefore, exploitation is still focus and the trend of RA treatment for the medicine of novel targets.Research shows, CD147 is a potential novel targets of RA treatment.At present, both at home and abroad still without any the report of the anti-inflammatory compound of targeting CD147.
Summary of the invention:
The object of the present invention is to provide the inflammatory applications of micromolecular compound AG-4, this micromolecular compound AG-4 can be used in and prepares anti-inflammatory drug.
For achieving the above object, the technical solution used in the present invention is:
The application of micromolecular compound AG-4 in preparing anti-inflammatory drug, wherein, the structural formula of described micromolecular compound AG-4 as the formula (1),
Described anti-inflammatory drug be can with the protein bound medicine of CD147.
Described anti-inflammatory drug is the medicine that can be combined with the inflammatory cell of high expressed CD147 albumen.
Described anti-inflammatory drug is the medicine that can targeting suppresses CD147 biological activity of albumen.
Described anti-inflammatory drug comprises the medicine that can suppress the medicine of inflammatory cell chemotactic and/or can suppress the secretion of inflammatory cell gelatinase.
Described anti-inflammatory drug comprises the medicine of the inflammatory cell chemotactic that can suppress cyclophilin A induction.
Described anti-inflammatory drug comprises the medicine that can suppress the secretion of cell gelatinase, and described cell is the cell obtaining inflammatory cell becomes fiber-like synovial cell to cultivate altogether with Patients With Rheumatoid Arthritis after.
Described gelatinase is MMP-9 and MMP-2.
Described inflammatory cell comprises Monocytes/Macrophages and neutrophilic granulocyte.
Described Monocytes/Macrophages is Peripheral Blood of Patients With Rheumatoid Arthritis mononuclear cell, and described neutrophilic granulocyte is Peripheral Blood of Patients With Rheumatoid Arthritis neutrophilic granulocyte.
With respect to prior art, beneficial effect of the present invention is:
The invention provides the application of micromolecular compound AG-4 in preparing anti-inflammatory drug.The drug candidate Screening Platform that the present invention combines by structure biology, bioinformatics and biologic activity checking, filters out this micromolecular compound of AG-4, can be applied to antiinflammatory field.
Further, micromolecular compound AG-4 provided by the invention is the antiinflammatory micromolecular compound that the first CD147 of take albumen that the structural design for CD147 albumen goes out is target spot, after AG-4 is combined with CD147 protein-specific, the biologic activity that can efficient targeting suppresses CD147 protein molecular, can be as the inhibitor of CD147 albumen, thereby for the preparation of anti-inflammatory drug, medicine in particular for the relevant inflammatory diseasess such as rheumatoid arthritis of preparation treatment CD147 albumen high expressed, there is the effect that can suppress inflammatory cell chemotactic and gelatinase secretion, especially have and can suppress the Monocytes/Macrophages of CypA induction and the effect of neutrophilic granulocyte chemotactic, and suppress Monocytes/Macrophages or neutrophilic granulocyte respectively with the effect of RA FLS co-cultured cell gelatinase secretion.
The affinity constant of micromolecular compound AG-4 provided by the invention and CD147 albumen is 10
-4mol/L.In patient's RA peripheral blood lymphocytes or neutrophilic granulocyte, add respectively after micromolecular compound AG-4, statistical result showed can suppress patient's RA peripheral blood lymphocytes and neutrophilic granulocyte chemotactic after adding micromolecular compound AG-4.After peripheral blood lymphocytes or neutrophilic granulocyte are cultivated altogether with RA FLS respectively, add wherein micromolecular compound AG-4, statistical result showed adds the secretion that can suppress co-cultured cell gelatinase after micromolecular compound AG-4.
Accompanying drawing explanation:
Fig. 1 is the statistical result block diagram that micromolecular compound AG-4 suppresses Peripheral Blood of Patients With Rheumatoid Arthritis monocyte chemotactic, wherein a is the experimental group of patient's RA peripheral blood lymphocytes, b for to add the experimental group after CypA in patient's RA peripheral blood lymphocytes, c for to add the experimental group after CypA and AG-4 in patient's RA peripheral blood lymphocytes, and d for to add the experimental group after CypA and DMSO in patient's RA peripheral blood lymphocytes.
Fig. 2 is the statistical result block diagram that micromolecular compound AG-4 suppresses Peripheral Blood of Patients With Rheumatoid Arthritis neutrophilic granulocyte chemotactic, wherein a is the experimental group of patient's RA peripheral blood neutrophil, b for to add the experimental group after CypA in patient's RA peripheral blood neutrophil, c for to add the experimental group after CypA and AG-4 in patient's RA peripheral blood neutrophil, and d for to add the experimental group after CypA and DMSO in patient's RA peripheral blood neutrophil.
Fig. 3 is the statistical result block diagram that micromolecular compound AG-4 suppresses patient's RA peripheral blood lymphocytes and the secretion of RA FLS co-cultured cell gelatinase, wherein a is the experimental group of patient's RA peripheral blood lymphocytes and RA FLS co-cultured cell, b for to add the experimental group after AG-4 in patient's RA peripheral blood lymphocytes and RA FLS co-cultured cell, and c for to add the experimental group after DMSO in patient's RA peripheral blood lymphocytes and RA FLS co-cultured cell.
Fig. 4 is the statistical result block diagram that micromolecular compound AG-4 suppresses patient's RA peripheral blood neutrophil and the secretion of RA FLS co-cultured cell gelatinase, wherein a is the experimental group of patient's RA peripheral blood neutrophil and RA FLS co-cultured cell, b for to add the experimental group after AG-4 in patient's RA peripheral blood neutrophil and RA FLS co-cultured cell, and c for to add the experimental group after DMSO in patient's RA peripheral blood neutrophil and RA FLS co-cultured cell.
The specific embodiment:
Below in conjunction with accompanying drawing and specific experiment result, the present invention is described in further detail.
Early-stage Study finds that the CD147 of Patients With Rheumatoid Arthritis Monocytes/Macrophages, neutrophils surface high expressed can promote FLS emiocytosis MMPs, thereby promote cartilage, bone erosion, CD147 can also be by interacting and promote inflammatory cell to the gathering of joint focus with CypA, exacerbate inflammation, therefore points out CD147 to participate in the pathogenesis of rheumatoid arthritis.And successfully with Hepatoma m Abs HAb18, screen people's cDNA library from hepatocellular carcinoma in early-stage Study, clone the cDNA fragment of coding HAb18G.Inquiry GenBank, finds HAb18G encoding gene open reading frame and people CD147 numberator height homology, is a newcomer of CD147 family, and successfully parses three-dimensional crystalline structure (the PDB numbering: 3B5H) of HAb18G/CD147 protein molecular extracellular fragment.Therefore, the present invention adopts the virtual screening method combining based on bioinformatics and Computer-Aided Drug Design technology to screen.First, method based on bioinformatics, appliance computer software Discovery Studio2.5 is to CD147 extracellular fragment three-dimensional crystalline structure model (PDB numbering: 3B5H, CD147 is identical with the three-dimensional crystalline structure model of HAb18G/CD147 extracellular fragment) carry out conformational analysis, finally determine at the N of a monomer of 3B5H end and set up Pharmacophore Model.According to the Pharmacophore Model having established, on medicine virtual screening platform, to screening purchased from Dutch Specs compound library (approximately 300,000 compounds), according to Optimum configuration with in conjunction with marking, select 1000 compounds that mark is higher.Continue application Discovery Studio2.5 software, 1000 of primary election compounds are analyzed, by further structure optimization, cluster analysis and perusal, remove after unnecessary analogue compounds and obvious irrational compound, finally choose 100 candidate compounds.Use subsequently external protein interaction system to detect the affinity of micromolecular compound and CD147 albumen, by external chemotactic experiment and the experiment of gelatin zymogram, these compounds are carried out to multiple sieve again, its function is screened, the novel anti-inflammatory micromolecular compound that final acquisition structural formula label is as the formula (1) AG-4, its English name is N-{4-[9-(4-{[(1, 3-dioxo-2-phenyl-2, 3-dihydro-1H-isoindol-5-yl) carbonyl] amino}phe nyl)-9H-fluoren-9-yl] phenyl}-1, 3-dioxo-2-phenyl-5-isoindolinecarboxamide.Micromolecular compound AG-4 can be as the inhibitor of CD147 albumen, through protein interaction system test, this micromolecular compound can be combined with CD147 protein-specific, and can suppress Monocytes/Macrophages, neutrophilic granulocyte chemotactic and suppress Monocytes/Macrophages, neutrophilic granulocyte respectively with the secretion of RA FLS co-cultured cell gelatinase.
1, the external combination test of micromolecular compound AG-4 and target CD147 albumen:
For the AG-4(selecting purchased from Dutch Specs company), adopt ProteOn XPR36 (BIO-RAD company) protein interaction system to detect the affinity size of this micromolecular compound and CD147 albumen, result confirms that AG-4 and CD147 can be in conjunction with, and its affinity constant is 10
-4mol/L.
2, micromolecular compound AG-4 suppresses the experiment of inflammatory cell chemotactic:
Separated patient's RA peripheral blood lymphocytes, neutrophilic granulocyte carry out chemotactic experiment.
Separating monocytic cell from peripheral blood: anticoagulant tube is collected patient's RA peripheral blood, and the refrigerator that is placed in 4 ℃ is to be separated.The PBS that adds equal-volume pre-cooling in every duplicate samples, fully blow even sample, add the anti-immunomagnetic beads that selects of mononuclear cell of Dynal Biotech, mix, in 4 ℃, hatch 30min, then test tube is placed in to magnet stand and carries out cell separation, room temperature is placed 3min, by vitro liquid assimilating is clean, with twice of PBS washed cell, then from magnet stand, take off test tube, add RPMI-1640 complete medium, re-suspended cell.
The separation of the neutrophilic granulocyte in peripheral blood: get acute stage RA peripheral blood in patients, use the dilution of equal-volume Hank ' s liquid, first in centrifuge tube, add granulocyte separating medium (Tianjin TBD company), slowly add 2 times of samples after volume dilution, centrifugal 20min under 2000rpm, GCL in the middle of carefully drawing with capillary tube, centrifugal 10min under 2000rpm again, then add erythrocyte cracked liquid to dissolve remaining erythrocyte, then under 1500rpm centrifugal 5min, repeat 3 times.Cell precipitation is resuspended, stand-by by RPMI-1640 culture medium.
Separated mononuclear cell or neutrophilic granulocyte is resuspended by the serum-free RPMI-1640 culture medium of the BSA containing 2% mass concentration, and counting.Get 1 * 10
5individual cell adds filter membrane (the 5 μ m) top of chemotactic cell, the corresponding compd A G-4 that adds of while, and lower chamber adds CypA.Each sample is done three multiple holes.At 37 ℃, CO
2volumetric concentration is to cultivate 90min in 5% incubator.Film is immersed to methanol solution and fix 3 minutes, violet staining, 5 visuals field are chosen in every hole at random, calculate the cell number at the film back side under high power microscope, and calculate chemotactic index, chemotactic index=processed group cell number/control wells cell number, result is expressed as mean value ± SD, adopts t check to carry out statistical analysis, and Fig. 1 and Fig. 2 are shown in statistical result, AG-4 need be dissolved in DMSO(dimethyl sulfoxide owing to adding AG-4) in, the d group in Fig. 1 and Fig. 2 is in order to get rid of the impact of solvent DMSO.* in Fig. 1 and Fig. 2 represents p < 0.05, illustrates with b group and compares, and result has significant difference.Result shows, micromolecular compound AG-4 can significantly suppress patient's RA peripheral blood lymphocytes that CypA induces, the chemotactic of neutrophilic granulocyte.
3, micromolecular compound AG-4 suppresses the experiment of cell gelatinase secretion:
The separation and Culture of RA FLS: collect RA patient's synovial tissue, reject fatty tissue wherein and blood vessel etc., fully shred into 1-2mm under aseptic condition
3fritter, then add wherein the collagenase solution of 4mg/mL, in 37 ℃ of incubators, digest 1-2 hour.Adding mass concentration is 0.25% trypsinization 30min again, and metal mesh filter, filtrate is at the hyclone DMEM(100U/mL penicillin containing 10% volumetric concentration, 100mg/L streptomycin) in culture medium, in 37 ℃, CO
2volumetric concentration is to cultivate 2 hours in 5% incubator, and then attached cell is not transferred in new culture bottle and continues to cultivate, and after 24h, inhales and abandons adherent not yet cell, and adding volumetric concentration is that 10% DMEM continues to cultivate.Until Growth of Cells, to incorporating period, in 1:3 ratio, go down to posterity, by drain cell art, identify its surface marker.The successful synovial cell of visible separation is the characteristic of adherent growth, along with the increase of going down to posterity, important molecule CD90 as fibroblast-like cells surface marker, its the positive expression rate increases along with the increase of going down to posterity, after passing to for the 3rd generation, its expression rate is all greater than 98%, further confirms that the successful cell of former culture is RA FLS.Select the RA FLS in 3-5 generation to use as further research.
Separated patient's RA peripheral blood lymphocytes or neutrophilic granulocyte and RA FLS cell is resuspended by serum-free RPMI-1640 culture medium, cell suspension is added in 96 orifice plates, every hole adds respectively 1 * 10
5individual RAFLS cell and 1 * 10
5individual mononuclear cell or neutrophilic granulocyte.In respective aperture, add compd A G-4, and blank hole is set respectively, add serum-free medium to 100 μ L.Cultivate after 20 hours, collect serum-free culture supernatant, low-speed centrifugal (200 * g) is removed after cell debris, is placed at the temperature of 4 ℃ standby.The concentrated glue that the preparation mass concentration separation gel that is 8% and mass concentration are 5%, in separation gel, adding whole mass concentration is 0.1% gelatin solution.Sample thief and 6 * non-reduced sample-loading buffer mixes, every hole loading 15 μ L.Do electrophoresis experiment, swimming voltage is: concentrated glue 90V, separation gel 120V.After electrophoresis finishes, gel is moved into mass concentration and be renaturation in 2.5% Triton X-100 solution, on shaking table, eluting is 45 minutes 2 times.Gel is placed in to gelatinase incubation buffer, at 37 ℃, hatches 12-16 hour.The dyeing liquor 4h that dyes for gel, the 1-2h that decolours in destaining solution, to contrast occur obviously, negative staining band clearly, with after distilled water rinsing, observe, film making.With gel imaging system, take pictures, and it is quantitative with Smartview software, to carry out density scan, adopts t check to carry out statistical analysis.The results are shown in Figure 3 and Fig. 4, AG-4 need be dissolved in DMSO(dimethyl sulfoxide owing to adding AG-4) in, the c group in Fig. 3 and Fig. 4 is in order to get rid of the impact of solvent DMSO.* in Fig. 3 and Fig. 4 represents p < 0.05, and * * represents p < 0.01, illustrates with a group and compares, and statistical result has significant difference.Result shows, micromolecular compound AG-4 can significantly suppress the secretion of cell gelatinase MMP-9 and MMP-2, and this cell is that patient's RA peripheral blood lymphocytes or neutrophilic granulocyte become fiber-like synovial cell with RA FLS(rheumatoid arthritis) common cultured cells.
Micromolecular compound AG-4 provided by the invention has good penetrability, and toxic and side effects is little, simple in structure, the advantage such as is easy to synthesize, and can be used in the medicine of preparation treatment and CD147 albumen high expressed related inflammatory disease (as inflammatory diseasess such as rheumatoid arthritis).AG-4 has the effect that suppresses inflammatory cell chemotactic and gelatinase secretion, and wherein, inflammatory cell is the inflammatory cell of high expressed CD147, is Monocytes/Macrophages and neutrophilic granulocyte.Micromolecular compound AG-4 can, at the selectively targeted CD147 molecule of protein level, suppress its biologic activity, thereby reach antiphlogistic effects.
Claims (10)
1. the application of micromolecular compound AG-4 in preparing anti-inflammatory drug, wherein, the structural formula of described micromolecular compound AG-4 as the formula (1),
2. application as claimed in claim 1, is characterized in that: described anti-inflammatory drug for can with the protein bound medicine of CD147.
3. application as claimed in claim 1, is characterized in that: the medicine of described anti-inflammatory drug for being combined with the inflammatory cell of high expressed CD147 albumen.
4. application as claimed in claim 1, is characterized in that: described anti-inflammatory drug is for can targeting suppressing the medicine of CD147 biological activity of albumen.
5. application as claimed in claim 1, is characterized in that: described anti-inflammatory drug comprises the medicine that can suppress the medicine of inflammatory cell chemotactic and/or can suppress the secretion of inflammatory cell gelatinase.
6. application as claimed in claim 1, is characterized in that: described anti-inflammatory drug comprises the medicine of the inflammatory cell chemotactic that can suppress cyclophilin A induction.
7. application as claimed in claim 1, is characterized in that: described anti-inflammatory drug comprises the medicine that can suppress the secretion of cell gelatinase, and described cell is that inflammatory cell becomes fiber-like synovial cell cultured cells altogether with Patients With Rheumatoid Arthritis.
8. the application as described in claim 5 or 7, is characterized in that: described gelatinase is MMP-9 and MMP-2.
9. the application as described in any one in claim 3,5,6 or 7, is characterized in that: described inflammatory cell comprises Monocytes/Macrophages and neutrophilic granulocyte.
10. application as claimed in claim 9, is characterized in that: described Monocytes/Macrophages is Peripheral Blood of Patients With Rheumatoid Arthritis mononuclear cell, and described neutrophilic granulocyte is Peripheral Blood of Patients With Rheumatoid Arthritis neutrophilic granulocyte.
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| CN108701171A (en) * | 2015-10-22 | 2018-10-23 | 马古苏托科技大学 | In pharmacophore, the Compounds and methods for by inhibiting that there is application in CYP17A1 and CYP19A1 treating cancers |
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| CN1720999A (en) * | 2005-06-03 | 2006-01-18 | 陈志南 | Hab18G/CD147 molecule small segment interfering RNA medicine and application thereof |
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Non-Patent Citations (2)
| Title |
|---|
| PING ZHU ET AL.: "CD147 overexpression on synoviocytes in rheumatoid arthritis enhances matrix metalloproteinase production and invasiveness of synoviocytes", 《ARTHRITIS RESEARCH & THERAPY》 * |
| WEIJIA DONG ET AL.: "The differential expressions of 78-kDa glucose-regulated protein of infiltrating plasma cells in peripheral joints with the histopathological variants of rheumatoid synovitis", 《ARTHRITIS RESEARCH & THERAPY》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108701171A (en) * | 2015-10-22 | 2018-10-23 | 马古苏托科技大学 | In pharmacophore, the Compounds and methods for by inhibiting that there is application in CYP17A1 and CYP19A1 treating cancers |
| CN108701171B (en) * | 2015-10-22 | 2022-06-10 | 马古苏托科技大学 | Pharmacophores, compounds and methods having application in the treatment of cancer by inhibition of CYP17a1 and CYP19a1 |
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Effective date of registration: 20160128 Address after: 710032 Changle West Road, Shaanxi, China, No. 169, No. Patentee after: The Fourth Military Medical University of the Chinese People's Liberation Army Address before: 710032 research center of cell engineering, The Fourth Military Medical University, 169 West Changle Road, Xi'an, Shaanxi Patentee before: Chen Zhinan |