CN106074488A - Benzene methoxycarbonyl group L glutamic acid is in the purposes preparing BLyS antagonist - Google Patents
Benzene methoxycarbonyl group L glutamic acid is in the purposes preparing BLyS antagonist Download PDFInfo
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- CN106074488A CN106074488A CN201610408919.3A CN201610408919A CN106074488A CN 106074488 A CN106074488 A CN 106074488A CN 201610408919 A CN201610408919 A CN 201610408919A CN 106074488 A CN106074488 A CN 106074488A
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Abstract
The invention discloses benzene methoxycarbonyl group L glutamic acid and preparing the purposes of BLyS antagonist, competitive ELISA test result indicate that: benzene methoxycarbonyl group L glutamic acid can suppress the combination of BLyS Yu TACI Fc.Inhibitory action is proportionate with the concentration of benzene methoxycarbonyl group L glutamic acid.Benzene methoxycarbonyl group L glutamic acid can suppress the combination of BLyS Yu BCMA Fc；Inhibitory action is proportionate with the concentration of benzene methoxycarbonyl group L glutamic acid.Benzene methoxycarbonyl group L glutamic acid can be as potential BLyS small molecular antagonists.
Description
Technical field
The invention belongs to biomedicine field, relate to the benzene methoxycarbonyl group-Pidolidone purposes as BLyS antagonist.
Background technology
Bone-marrow-derived lymphocyte stimulating factor (B lymphocyte stimulator, BLyS), is also called B cell activity factor (B
Cell activating factor belonging to the TNF family, BAFF), it is a member in TNF family.By
Mononuclear cell and macrophage persistently synthesize, secrete.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 withering of mature B cell
Die.If knocking out BAFF, the mature B cell in Mice Body lacks completely.Owing to BLyS sends out in bone-marrow-derived lymphocyte activation, propagation
Wave important function, and the humoral immunization that the antibody that bone-marrow-derived lymphocyte produces is mediated is at numerous autoimmune diseasees having been found that
In the middle of be in Central Position.It is now recognized that the overexpression that BLyS is in vivo and some autoimmune disease such as systematicness erythema
The generation of the courses of disease such as lupus (SLE), rheumatoid arthritis (RA), develop closely related.Therefore, to block BLyS merit biology
Can be strategy, inquire into BLyS and suppress the autoimmune disease such as antagonist for treating systemic lupus erythematosus (sle) and rheumatoid arthritis
In the carrying out that research with B cell tumor disease is the most like a raging fire.At present, the research for the inhibitor of BLyS is main
Concentrate on to develop and there is BLyS Decoy receptors (decoy receptor), anti-BLyS antibody and the antagonistic peptide neutralizing BLyS effect
[1-3].In March, 2011 U.S. FDA approval by Human Genome Sciences Inc. (Human Genome Sciences) and Ge Lanshi-
Anti-BLyS antibody BENLYSTA (Belimumab) the treatment systemic red of SmithKline (GlaxoSmithKline) company cooperative research and development
Yabbi skin ulcer.This is over 50 years, and FDA ratifies to treat the medicine of such disease first.But protein inhibitor has as medicine
Having certain limitation, such as originating, less and isolated and purified difficulty is big and medicine stability is poor, additionally there are oral
Bioavailability relatively low, be primarily due to internal various enzyme high to the degradability of polypeptide drug, thus this class can be caused
The effect half-life of medicine is greatly shortened.Therefore, exploitation micromolecular compound has important as the research of BLyS inhibitor
Realistic meaning.At present, not yet there is the benzene methoxycarbonyl group-Pidolidone 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 benzene methoxycarbonyl group-Pidolidone is short of money at preparation BLyS
The purposes of anti-agent.
Technical scheme is summarized as follows:
Benzene methoxycarbonyl group-Pidolidone is preparing the purposes of BLyS antagonist
Benzene methoxycarbonyl group-Pidolidone is hereinafter simply referred to as CBz-L-glutamic acid.
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: CBz-L-aminoglutaric acid concentration can suppress 1.26 ± 3.79% when 1mg/mL
The combination of BLyS Yu TACI-Fc；The combination of 59.35 ± 11.87%BLyS and TACI-Fc can be suppressed when 3mg/mL；?
The combination of 74.13 ± 6.78%BLyS and TACI-Fc can be suppressed during 5mg/mL.Inhibitory action and the concentration of CBz-L-glutamic acid
It is proportionate.
The micromolecular compound (benzoic acid) of non-binding BAFF, i.e. negative control, the interaction to BLyS with TACI does not has
There is obvious inhibiting effect and there is no dose-dependence.The inhibitory action of proof CBz-L-glutamic 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: CBz-L-aminoglutaric acid concentration can suppress 5.63 ± 0.88% when 1mg/mL
The combination of BLyS Yu BCMA-Fc；The combination of 56.70 ± 7.90%BLyS and BCMA-Fc can be suppressed when 3mg/mL；At 5mg/
The combination of 82.80 ± 3.98%BLyS and BCMA-Fc can be suppressed during mL.The concentration of inhibitory action and CBz-L-glutamic acid is in just
Relevant.
Negative control compound (benzoic acid) does not has obvious inhibiting effect to the interaction of BLyS Yu BCMA and does not has agent
Amount dependence.The inhibitory action of proof CBz-L-glutamic acid is special and effective.
Accompanying drawing explanation
Fig. 1 is the Key residues of BLyS bind receptor ,+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 special
Property calmodulin binding domain CaM.
Fig. 3 is virtual screening flow chart.
Fig. 4 is CBz-L-glutamic acid (benzene methoxycarbonyl group-Pidolidone) structural formula.
Fig. 5 is the pattern of CBz-L-glutamic acid and the interaction of BLyS.
Fig. 6 is that CBz-L-glutamic acid suppresses the TACI combination to BLyS.* represents p < 0.01；* represent 0.01 < p <
0.05。
Fig. 7 is that CBz-L-glutamic 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 have been subject to
The possible pattern of body interaction and key amino acid.We pass through molecular dynamics simulation in previous work, based on BLyS
With the crystal structure of its receptor BCMA, by the Interactions Mode of computer simulation BLyS Yu its three natural receptors, probe into
It participates in conservative combination and the selective Key residues of affinity.On this basis, utilize " key-lock-principle ", for pass
Key calmodulin binding domain CaM carries out commercialization chemical small molecule data base and carries out molecule virtual screening, it was predicted that obtain a series of having potential pressing down
The micromolecular compound made, it is thus achieved that its entity also carries out active determination in vitro discovery, binary acid compounds dibenzoyl
Tartaric acid has certain suppression BLyS and combines the effect of TACI and BCMA.The new drug gateway drug of potential applicability in clinical practice is had for exploitation
Thing has established experimentation basis.
Below in conjunction with specific embodiment, the present invention is further illustrated.Embodiments of the invention are to make this area
Technical staff can implement, but the present invention is not imposed any restrictions.Disclosing of common agents therein is to make this
The technical staff in field preferably implements 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 puts forth energy again. human soluble bone-marrow-derived lymphocyte stimulates
The clone of factor gene and the expression [J] in escherichia coli. cell and molecular immunology magazine, 2006, (2) SEQ ID
Shown in NO.8.
TACI-Fc albumen: Ji Lijun, white Wu Ren figure is refined, Chai Lin, Sun Jian .TACI-Fc fusion protein gene constructed, former
Nuclear expression and Biological Activity Identification [J]. biotechnology, shown in 2013, (3) .SEQ ID NO.9
BCMA-Fc albumen: Sun Jian, Feng Jiannan, Linzhou, Li Yan, Shen is put forth energy again. and computer-aided design solubility BLyS is subject to
Body, eBCMA-Fc fusion protein and the expression in escherichia coli thereof. Chinese biological chemistry and molecular biosciences journal, 2008,24
(2) shown in SEQ ID NO.9.
Fc albumen: Wu Zhen .BCMA-Fc is as the research [D] of potential drug. University Of Tianjin, 2012SEQ ID NO.10 institute
Show.
It is coated liquid: weigh 33.6g sodium bicarbonate and 63.6g natrium carbonicum calcinatum, put it in beaker, inwardly add
600ml distilled water, stirring all dissolves 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.In room
Temperature is lower to be preserved.
The preparation of 5% defatted milk powder: weigh and be dissolved under defatted milk powder 1g, stirring in 10ml 0.01M PBS, adds PBS fixed
Hold 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% peroxides before use
Change hydrogen.
1%TMB liquid storage: weigh and be completely dissolved in 80ml DMSO under 1g TMB, stirring, add DMSO and be settled to 100ml,
It is 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, add 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: from Protein Date Bank Protein Data Bank (http://www.rcsb.org/
Pdb) BLyS protein structure (PDB ID:1OQD and 1OQE) is downloaded in.Use the computational methods such as molecular dynamics, from molecular level
On probe into the interaction mechanism of BLyS and its natural receptor (BCMA, TACI, BR3).According to each amino acid residue to combination
Energy contribution, determines its Key residues.Fig. 1 and Fig. 2 respectively illustrates the pivotal role residue of BLyS target spot natural receptor, with
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, its main source
In two approach: the little molecule in natural products database or commercial little molecular database, seminar synthesizes the seriation obtained
Compound.Commercial little molecular database refers mainly to lark prestige (J&K), AlfaAesar (Alfa-Aesar), Sigma-Aldrich
(Sigma-Aldrich) three Reagent Company websites obtain.Three-dimensional small molecule structure is hydrogenated with, adds after electric charge processes, will
File preserves into pdbqt file format, carries out further molecular Docking Study.
Virtual screening: by AutoDock 4.2 software, by little molecules all in data base and region, BLyS active center
Carry out screening docking to calculate.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 compound and make with BLyS
Best conformation, and predict its binding pattern.Analyzing docking result, the little molecular prediction according to being given combines score value, investigates
Compound is combined the matching degree at center with BLyS, and with key amino acid mutual relation situation, filter out and potential there is BLyS
The little molecule candidate compound of affinity.Score value is the biggest, and representation compound is the highest with the affinity of BLyS, i.e. compound may have
Preferably affinity.Select the compound that overall merit is optimal, it is thus achieved that chemical entities, and carry out ELISA experiment.Fig. 3 retouches
State the flow chart of virtual screening.
Embodiment 2: computer analyzes small molecular antagonists and BLyS binding pattern.
Utilize computer molecular docking method, inquire into CBz-L-glutamic acid (structure sees Fig. 4) in conjunction with Chimera software
With BLyS Study on Molecular Mechanism, analyze its critical amino acid residues being mutually distinguishable with BLyS and key interactions (Fig. 5).
Find that a phenyl ring of CBz-L-glutamic acid is inserted into the conservative hydrophobic pocket of BLyS, formed with His69, Leu70 and Ile92
Hydrophobic interaction, and form π-cation effect with Arg124 and Arg90 residue side chains, common strengthen CBz-L-glutamic acid with
The combination of BLyS；Carboxyl on two side chains forms salt bridge with Arg90；In oxygen on benzyloxycarbonyl group and aminoacid carboxyl
Key residues Arg124 of ketonic oxygen and BLyS form interaction of hydrogen bond, OH Yu the Y65 formation hydrogen bond in aminoacid carboxyl.
Embodiment 3: competitive ELISA analysis of compounds combines the inhibitory action of TACI/BCMA albumen for BLyS.
If compound CBz-L-glutamic acid can be with TACI/BCMA competition binding BLyS active pocket, the then change added
Compound CBz-L-glutamic acid can suppress BLyS to combine TACI/BCMA；On the contrary, if compound CBz-L-glutamic acid does not combines
BLyS, does not the most exist and competes with TACI/BCMA.Then add compound CBz-L-glutamic acid BLyS is combined TACI/BCMA not have
Have an impact.Therefore, tested by Competitive assays ELISA, test little molecule CBz-L-glutamic acid and block BLyS and receptor TACI
The effect combined with BCMA.
First, ELISA method is the ELISA that BLyS with TACI-Fc/BCMA-Fc is combined routinely.Determine Competitive assays
ELISA experiment uses the concentration of TACI-Fc/BCMA-Fc.Finally determine that concentration is 10 μ g/mL.The tool of Competitive assays ELISA
Body operating procedure 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.By dilution
BLyS liquid 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) wash plate, then the CBz-L-glutamic acid aqueous solution of TACI-Fc or BCMA-Fc of 10g/mL with different volumes is mixed
Closing, prepare the mixed solution that micromolecular compound CBz-L-glutamic acid final concentration gradient is 0,1,3 and 5mg/mL, every hole adds 50
Microlitre.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 interaction of compound competition BLyS Yu TACI-Fc/BCMS-Fc
Inhibition calculate 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. benzene methoxycarbonyl group-Pidolidone is preparing the purposes of BLyS antagonist.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004113363A2 (en) * | 2003-06-19 | 2004-12-29 | The Nottingham Trent University | Dipeptide transglutaminase inhibitors and methods of using the same |
| CN101969920A (en) * | 2008-03-12 | 2011-02-09 | 株式会社资生堂 | Parakeratosis inhibitor, pore shrinking agent or agent for preventing or improving rough skin, and composition for external use on the skin containing the same |
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- 2016-06-08 CN CN201610408919.3A patent/CN106074488B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004113363A2 (en) * | 2003-06-19 | 2004-12-29 | The Nottingham Trent University | Dipeptide transglutaminase inhibitors and methods of using the same |
| CN101969920A (en) * | 2008-03-12 | 2011-02-09 | 株式会社资生堂 | Parakeratosis inhibitor, pore shrinking agent or agent for preventing or improving rough skin, and composition for external use on the skin containing the same |
Non-Patent Citations (2)
| Title |
|---|
| JIAN SUN ET AL.: "BAFF-targeting therapy, a promising strategy for treating autoimmune diseases", 《EUROPEAN JOURNAL OF PHARMACOLOGY》 * |
| XAVIER MARIETTE: "The B cell: a new therapeutic target in rheumatoid arthritis and other autoimmune diseases", 《JOINT BONE SPINE》 * |
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