CN105919989A - Application of (2R, 3R)-dibenzoyltartaric acid to preparation of BLyS (B lymphocyte stimulator) antagonist - Google Patents
Application of (2R, 3R)-dibenzoyltartaric acid to preparation of BLyS (B lymphocyte stimulator) antagonist Download PDFInfo
- Publication number
- CN105919989A CN105919989A CN201610410544.4A CN201610410544A CN105919989A CN 105919989 A CN105919989 A CN 105919989A CN 201610410544 A CN201610410544 A CN 201610410544A CN 105919989 A CN105919989 A CN 105919989A
- Authority
- CN
- China
- Prior art keywords
- blys
- dibenzoyltartaric acid
- taci
- bcma
- tartaric acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
Landscapes
- Health & Medical Sciences (AREA)
- Emergency Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses application of (2R, 3R)-dibenzoyltartaric acid to preparation of BLyS (B lymphocyte stimulator) antagonist. According to competitive ELISA (enzyme-linked immuno sorbent assay) experimental results, the (2R, 3R)-dibenzoyltartaric acid is capable of inhibiting combination of BLyS and TACI (transmembrane activator and CAML interactor)-Fc, and the inhibition effect is positively related to concentration of the (2R, 3R)-dibenzoyltartaric acid; the (2R, 3R)-dibenzoyltartaric acid is capable of inhibiting combination of BLyS and BCMA (B cell mature antigen)-Fc, and the inhibition effect is positively related to concentration of the (2R, 3R)-dibenzoyltartaric acid. Therefore, the (2R, 3R)-dibenzoyltartaric acid can serve as potential BLyS micromolecular antagonists.
Description
Technical field
The invention belongs to biomedicine field, relate to (2R, 3R)-dibenzoyl tartaric acid in the purposes preparing BLyS antagonist.
Background technology
Bone-marrow-derived lymphocyte stimulating factor (B lymphocyte stimulator, BLyS), it is also called B cell activity factor (B cell activating factor belonging to the TNF family, BAFF), is a member in TNF family.Persistently synthesized by mononuclear cell and macrophage, secreted.BLyS can be in conjunction with the three of B cell surface kind receptor, B cell maturation antigen (B cell mature antigen, BCMA), cross-film activator and CAML conjugate (Transmembrane activator and CAML-interactor, TACI) and BAFF-R 3 (BR3).After receptor combines BAFF, reduce the apoptosis of mature B cell.If knocking out BAFF, the mature B cell in Mice Body lacks completely.Owing to BLyS plays a significant role in bone-marrow-derived lymphocyte activation, propagation, and the humoral immunization that the antibody that bone-marrow-derived lymphocyte produces is mediated is in Central Position in the middle of numerous autoimmune diseasees having been found that.It is now recognized that the overexpression that BLyS is in vivo and the generation of the courses of disease such as some autoimmune disease such as systemic lupus erythematosus (sle) (SLE), rheumatoid arthritis (RA), develop closely related.Therefore, to block BLyS biological function as strategy, inquire in the carrying out that the research of the BLyS suppression autoimmune disease such as antagonist for treating systemic lupus erythematosus (sle) and rheumatoid arthritis and B cell tumor disease is the most like a raging fire.At present, the research for the inhibitor of BLyS is concentrated mainly on to develop and has BLyS Decoy receptors (decoy receptor), anti-BLyS antibody and the antagonistic peptide [1-3] neutralizing BLyS effect.In March, 2011, U.S. FDA was ratified by Human Genome Sciences Inc. (Human Genome Sciences) and anti-BLyS antibody BENLYSTA (Belimumab) systemic lupus erythematosus of Ge Lanshi-SmithKline (GlaxoSmithKline) company cooperative research and development.This is over 50 years, and FDA ratifies to treat the medicine of such disease first.But protein inhibitor has certain limitation as medicine, such as originating, less and isolated and purified difficulty is big and medicine stability is poor, additionally there are oral bioavailability relatively low, it is primarily due to internal various enzyme high to the degradability of polypeptide drug, thus the effect half-life of this class medicine can be caused to be greatly shortened.Therefore, exploitation micromolecular compound has important practical significance as the research of BLyS inhibitor.At present, not yet there is (2R, the 3R)-dibenzoyl tartaric acid report in the purposes preparing BLyS antagonist.
1.Jian Sun* (Sun Jian), Zhou Lin, Jiannan Feng, Yan Li, Beifen Shen. (2008) BAFF-targeting therapy, a promising strategy for treating autoimmune diseases.Eur J Pharmacol 597:1 5.
2.Yacong Zhao, Xiafei Hao, Jiannan Feng, Beifen Shen, Jing Wei* (Wei Jing), Jian Sun* (Sun Jian) (2015), The comparison of BLyS-binding peptides from phage display library and computer-aided design on BLyS TACI interaction.Int Immunopharmacol, 24:219 223.
3.Xiafei Hao, Yanfeng Zhu, Chang Zheng, Xuegang Fu, Jiannan Feng, Beifen Shen, Jing Wei* (Wei Jing), Jian Sun* (Sun Jian) .A comparison of biological activity of B lymphocyte stimulator (BLyS)
antagonist peptibodies and the elucidation of possible BLyS binding sites.Protein&Peptide Letters,2016,23,17-23
4.Xuegang Fu, Liyan Xuan, Yuzhe Wang, Jing Wei* (Wei Jing), Jian Sun* (Sun Jian) .Molecular mechanism of the affinity interactions between BAFF and its peptides by molecular simulations.Protein&Peptide Letters, 2015,22,992-999.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that (2R, 3R)-dibenzoyl tartaric acid is preparing the purposes of BLyS antagonist pharmaceuticals.
Technical scheme is summarized as follows:
(2R, 3R)-dibenzoyl tartaric acid is preparing the purposes of BLyS antagonist.
Advantages of the present invention:
1. TACI is combined for BLyS and there is the effect of significantly inhibiting
Competitive ELISA test result indicate that: (2R, 3R)-dibenzoyl tartaric acid concentration can suppress the combination of 2.71 ± 2.51%BLyS and TACI-Fc when 1mg/mL；The combination of 12.36 ± 6.38%BLyS and TACI-Fc can be suppressed when 3mg/mL；The combination of 79.68 ± 2.68%BLyS and TACI-Fc can be suppressed when 5mg/mL.Inhibitory action is proportionate with the concentration of (2R, 3R)-dibenzoyl tartaric acid inhibitor.
The micromolecular compound (benzoic acid) of non-binding BAFF, i.e. negative control, the interaction to BLyS Yu TACI does not has obvious inhibiting effect and does not has dose-dependence.The inhibitory action of proof (2R, 3R)-dibenzoyl tartaric acid is special and effective.
2. BCMA is combined for BLyS and there is the effect of significantly inhibiting
Competitive ELISA test result indicate that: (2R, 3R)-dibenzoyl tartaric acid concentration can suppress the combination of 5.59 ± 2.01%BLyS and BCMA-Fc when 1mg/mL；The combination of 8.91 ± 4.36%BLyS and BCMA-Fc can be suppressed when 3mg/mL；The combination of 83.72 ± 0.89%BLyS and BCMA-Fc can be suppressed when 5mg/mL.Inhibitory action is proportionate with the concentration of (2R, 3R)-dibenzoyl tartaric acid.
Negative control compound benzoic acid does not has obvious inhibiting effect to the interaction of BLyS Yu BCMA and does not has dose-dependence.The inhibitory action of proof (2R, 3R)-dibenzoyl tartaric acid is special and effective.
Accompanying drawing explanation
Fig. 1 is the Key residues of BLyS bind receptor, wherein+represent Key residues.
Fig. 2 is molecular surface and the Key residues of BLyS binding pocket, and D1 represents conservative calmodulin binding domain CaM, and D2 represents specific binding area.
Fig. 3 is virtual screening flow chart.
Fig. 4 is (2R, 3R)-dibenzoyl tartaric acid structural formula.
Fig. 5 is the pattern of (2R, 3R)-dibenzoyl tartaric acid and the interaction of BLyS.
Fig. 6 is that (2R, 3R)-dibenzoyl tartaric acid suppresses the TACI combination to BLyS.* represents p < 0.01；Asterisk is not had to represent p > 0.05.
Fig. 7 is that (2R, 3R)-dibenzoyl tartaric acid suppresses the BCMA combination to BLyS.* represents p < 0.01.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further illustrated.Numerous researchs have elaborated BLyS and the possible pattern of acceptor interaction and key amino acid.We pass through molecular dynamics simulation in previous work, crystal structure based on BLyS He its receptor BCMA, by the Interactions Mode of computer simulation BLyS Yu its three natural receptors, probe into it and participate in conservative combination and the selective Key residues of affinity.On this basis, utilize " key-lock-principle ", carry out commercialization chemical small molecule data base for territory, critical binding domain and carry out molecule virtual screening, prediction obtains a series of micromolecular compound with potential inhibitory action, obtain its entity and carry out active determination in vitro discovery, binary acid compounds (2R, 3R)-dibenzoyl tartaric acid has certain suppression BLyS and combines the effect of TACI and BCMA.The new drug lead drug of potential applicability in clinical practice is had to establish experimentation basis for exploitation.
Below in conjunction with specific embodiment, the present invention is further illustrated.Embodiments of the invention are to enable those skilled in the art to implement, but do not impose any restrictions the present invention.Disclosing of common agents therein is to make those skilled in the art preferably implement the present invention, but does not impose any restrictions the present invention.
BLyS albumen: Sun Jian, Li Yan, Feng Jiannan, Sun Yingxun, Hu Meiru, Shen is put forth energy again,. the clone of human soluble bone-marrow-derived lymphocyte stimulating factor gene and the expression [J] in escherichia coli. cell and molecular immunology magazine, shown in 2006, (2) SEQ ID NO.8.
TACI-Fc albumen: Ji Lijun, white Wu Ren figure is refined, gene constructed, the prokaryotic expression of Chai Lin, Sun Jian .TACI-Fc fusion protein and Biological Activity Identification [J]. biotechnology, and shown in 2013, (3) .SEQ ID NO.9
BCMA-Fc albumen: Sun Jian, Feng Jiannan, Linzhou, Li Yan, Shen is put forth energy again. computer-aided design solubility BAFF-R, eBCMA-Fc fusion protein and the expression in escherichia coli thereof. and Chinese biological chemistry and molecular biosciences journal, shown in 2008,24 (2) SEQ ID NO.9.
Fc albumen: Wu Zhen .BCMA-Fc is as the research [D] of potential drug. University Of Tianjin, shown in 2012SEQ ID NO.10
Being coated liquid: weigh 33.6g sodium bicarbonate and 63.6g natrium carbonicum calcinatum, put it in beaker, inwardly add 600ml distilled water, stirring is all dissolved to solute, adds in distilled water constant volume to 1L.Place it in and preserve at 4 DEG C.
The preparation of PBST: taking in 1 × PBS that 250 μ l tween 20s add 500ml, mix homogeneously is to merging completely.At room temperature preserve.
The preparation of 5% defatted milk powder: weigh and be dissolved under defatted milk powder 1g, stirring in 10ml 0.01M PBS, add PBS and be settled to 20ml, in 4 DEG C of preservations
TMB nitrite ion: take 4.95ml substrate buffer solution, 0.05ml 1%TMB liquid storage, adds 5 μ l 30% hydrogen peroxide before use.
1%TMB liquid storage: weigh and be completely dissolved in 80ml DMSO under 1g TMB, stirring, add DMSO and be settled to 100ml, be stored in 4 DEG C.
Substrate buffer solution (pH 5.0): take 24.3ml 0.1M citric acid, 25.7ml 0.2M disodium hydrogen phosphate, adds about 40ml water, adjusts pH to 5.0, adds water and be settled to 100ml, be stored in room temperature.TMB working solution (now with the current).
Embodiment 1: by computer virtual sifting technology, filters out BLyS and is correlated with micromolecular compound.
Protein structural information obtains: download BLyS protein structure (PDB ID:1OQD and 1OQE) from Protein Date Bank Protein Data Bank (http://www.rcsb.org/pdb).Use the computational methods such as molecular dynamics, from molecular level, probe into the interaction mechanism of BLyS and its natural receptor (BCMA, TACI, BR3).According to each amino acid residue to combining energy contribution, determine its Key residues.Fig. 1 and Fig. 2 respectively illustrates the pivotal role residue of BLyS target spot natural receptor, and the binding pocket of BLyS target spot and the relevant information of Key residues.
The acquisition of little molecular database: little molecular database contains nearly ten thousand different compounds of structure, it is mainly derived from two approach: the little molecule in natural products database or commercial little molecular database, seminar synthesizes the series compound obtained.Commercial little molecular database refers mainly to lark prestige (J&K), AlfaAesar (Alfa-Aesar), three Reagent Company websites of Sigma-Aldrich (Sigma-Aldrich) obtain.Three-dimensional small molecule structure is hydrogenated with, adds after electric charge processes, file is preserved into pdbqt file format, carries out further molecular Docking Study.
Little molecules all in data base are carried out screening and dock calculating by virtual screening: by AutoDock 4.2 software with region, BLyS active center.During molecular docking, three-dimensional small molecule structure data are placed at the avtive spot of BLyS one by one, by continuing to optimize the position of small molecule model, conformation, the dihedral angle of the rotatable key of intramolecule, find the best conformation of compound and BLyS effect, and predict its binding pattern.Analyze docking result, combine score value according to the little molecular prediction provided, investigate compound and be combined the matching degree at center with BLyS, and with key amino acid mutual relation situation, filter out the potential little molecule candidate compound with BLyS affinity.Score value is the biggest, and representation compound is the highest with the affinity of BLyS, i.e. compound may have preferable affinity.Select the compound that overall merit is optimal, it is thus achieved that chemical entities, and carry out ELISA experiment.Fig. 3 describes the flow chart of virtual screening.
Embodiment 2: computer analyzes small molecular antagonists and BLyS binding pattern.
Utilize computer molecular docking method, (2R has been inquired in conjunction with Chimera software, 3R)-dibenzoyl tartaric acid (structural formula sees Fig. 4) and BLyS Study on Molecular Mechanism, analyze its critical amino acid residues being mutually distinguishable with BLyS and key interactions (Fig. 5).Find (2R, one phenyl ring of 3R)-dibenzoyl tartaric acid is inserted into the conservative hydrophobic pocket of BLyS, with His69, Leu70 and Ile92 forms hydrophobic interaction, and form π-cation effect with Arg124 and Arg90 residue side chains, the common combination strengthening little molecule (2R, 3R)-dibenzoyl tartaric acid and BLyS；Two carboxyls and Key residues Arg124 of BLyS and Arg90 form stable salt bridge effect；The carbonyl of another one benzyl position forms interaction of hydrogen bond with Arg124.
Embodiment 3: competitive ELISA analysis of compounds combines the inhibitory action of TACI/BCMA albumen for BLyS.
If compound (2R, 3R)-dibenzoyl tartaric acid can suppress BLyS to combine TACI/BCMA with TACI/BCMA competition binding BLyS active pocket, the then compound added (2R, 3R)-dibenzoyl tartaric acid；On the contrary, if compound (2R, 3R)-dibenzoyl tartaric acid does not combine BLyS, the most do not exist and compete with TACI/BCMA.Then add that compound (2R, 3R)-dibenzoyl tartaric acid combines TACI/BCMA to BLyS not to be affected.Therefore, tested by Competitive assays ELISA, test little molecule (2R, 3R)-dibenzoyl tartaric acid and block the effect that BLyS and receptor TACI and BCMA is combined.
First, ELISA method is the ELISA that BLyS with TACI-Fc/BCMA-Fc is combined routinely.Determine the concentration using TACI-Fc/BCMA-Fc in Competitive assays ELISA experiment.Finally determine that concentration is 10 μ g/mL.The concrete operation step of Competitive assays ELISA is as follows:
A) it is coated: the BLyS of 20g/mL is diluted by 1:1 with being coated liquid, prepares the BLyS liquid of 10 μ g/mL.The BLyS liquid of dilution is coated 96 orifice plates by 50 microlitres/hole.4 DEG C overnight.
B), after washing plate, the defatted milk powder room temperature with 5% is closed 2 hours.
C) plate is washed, again by the (2R of TACI-Fc or BCMA-Fc of 10g/mL Yu different volumes, 3R)-dibenzoyl tartaric acid aqueous solution, prepare micromolecular compound (2R, 3R)-dibenzoyl tartaric acid final concentration gradient is the mixed solution of 0,1,3 and 5mg/mL, and every hole adds 50 microlitres.37 DEG C, incubation 1 hour.Do not add the mixed solution of small molecule solution as blank group.Benzoic acid is negative control compound.
D) wash plate, add HRP labelling goat-anti people 2 anti-(by specification dilution).
E) TMB chromogenic reagent is added after washing plate.
F) terminate reaction, survey the OD value of 450 nanometers.The inhibition of the interaction of compound competition BLyS Yu TACI-Fc/BCMS-Fc calculates with following formula: suppression ratio %=[(blank group OD450-experimental group OD450)/blank group OD450] * 100%.(suppression ratio is shown in Fig. 6 and Fig. 7).
Claims (1)
1. (2R, 3R)-dibenzoyl tartaric acid is preparing the purposes of BLyS antagonist.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610410544.4A CN105919989B (en) | 2016-06-08 | 2016-06-08 | (2R, 3R)-dibenzoyl tartaric acid is in the purposes for preparing BLyS antagonist |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610410544.4A CN105919989B (en) | 2016-06-08 | 2016-06-08 | (2R, 3R)-dibenzoyl tartaric acid is in the purposes for preparing BLyS antagonist |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105919989A true CN105919989A (en) | 2016-09-07 |
| CN105919989B CN105919989B (en) | 2019-01-18 |
Family
ID=56833620
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610410544.4A Expired - Fee Related CN105919989B (en) | 2016-06-08 | 2016-06-08 | (2R, 3R)-dibenzoyl tartaric acid is in the purposes for preparing BLyS antagonist |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105919989B (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103936833B (en) * | 2014-04-18 | 2016-02-10 | 天津大学 | BLyS antagonistic peptide, containing the plasmid of TC-Fc antigen-4 fusion protein gene and TC-Fc fusion rotein |
-
2016
- 2016-06-08 CN CN201610410544.4A patent/CN105919989B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN105919989B (en) | 2019-01-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ma et al. | Druggable orthosteric and allosteric hot spots to target protein-protein interactions | |
| Cheong et al. | The Significance of 2-Furyl Ring Substitution with a 2-(para-substituted) Aryl Group in a New Series of Pyrazolo-triazolo-pyrimidines as Potent and Highly Selective hA3 Adenosine Receptors Antagonists: New Insights into Structure− Affinity Relationship and Receptor− Antagonist Recognition | |
| Kurczab et al. | Amino acid hot spots of halogen bonding: a combined theoretical and experimental case study of the 5-HT7 receptor | |
| Arviv et al. | Folding of multidomain proteins: Biophysical consequences of tethering even in apparently independent folding | |
| Almerico et al. | A3 adenosine receptor: Homology modeling and 3D-QSAR studies | |
| Planesas et al. | Studying the binding interactions of allosteric agonists and antagonists of the CXCR4 receptor | |
| Magnani et al. | Electronic sculpting of ligand-GPCR subtype selectivity: the case of angiotensin II | |
| Fulton et al. | Conformationally constrained peptides target the allosteric kinase dimer interface and inhibit EGFR activation | |
| Fassi et al. | Oxidation state dependent conformational changes of HMGB1 regulate the formation of the CXCL12/HMGB1 heterocomplex | |
| Mungalpara et al. | Rational design of conformationally constrained cyclopentapeptide antagonists for CXC chemokine receptor 4 (CXCR4) | |
| Yoshida et al. | Peptide-to-small molecule: a pharmacophore-guided small molecule lead generation strategy from high-affinity macrocyclic peptides | |
| Preti et al. | Design, synthesis, and biological evaluation of novel 2-((2-(4-(substituted) phenylpiperazin-1-yl) ethyl) amino)-5′-N-ethylcarboxamidoadenosines as potent and selective agonists of the A2A adenosine receptor | |
| CN103936833B (en) | BLyS antagonistic peptide, containing the plasmid of TC-Fc antigen-4 fusion protein gene and TC-Fc fusion rotein | |
| CN105919989A (en) | Application of (2R, 3R)-dibenzoyltartaric acid to preparation of BLyS (B lymphocyte stimulator) antagonist | |
| Baraldi et al. | Water-soluble pyrazolo [4, 3-e][1, 2, 4] triazolo [1, 5-c] pyrimidines as human A3 adenosine receptor antagonists | |
| CN106074488B (en) | Benzene methoxycarbonyl group-Pidolidone is in the purposes for preparing BLyS antagonists | |
| Trifonov et al. | Structurally simple, readily available peptidomimetic 1-benzyl-5-methyl-4-(n-octylamino) pyrimidin-2 (1 H)-one exhibited efficient cardioprotection in a myocardial ischemia (MI) mouse model | |
| CN106074476A (en) | Palmoxiric acid is preparing the purposes of BLyS antagonist | |
| Liu et al. | Rational design of EGFR dimerization-disrupting peptides: A new strategy to combat drug resistance in targeted lung cancer therapy | |
| Zhao et al. | Molecular electrostatic potential and volume-aided drug design based on the isoindolinone-containing cyclopeptide S-PK6 | |
| Kubota et al. | Chemogenetic approach using Ni (II) complex–agonist conjugates allows selective activation of class A G-protein-coupled receptors | |
| Tanaka et al. | Template-assisted and self-activating clicked peptide as a synthetic mimic of the SH2 domain | |
| Rathmann et al. | Ligand-mimicking receptor variant discloses binding and activation mode of prolactin-releasing peptide | |
| Jarmuła et al. | Crystal structures of nematode (parasitic T. spiralis and free living C. elegans), compared to mammalian, thymidylate synthases (TS). Molecular docking and molecular dynamics simulations in search for nematode-specific inhibitors of TS | |
| Chen et al. | Integrated in silico–in vitro rational design of oncogenic EGFR-derived specific monoclonal antibody-binding peptide mimotopes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190118 Termination date: 20210608 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |