CN103951731A - Collagen-integrin alpha2beta1 interacted polypeptide inhibitors and screening method thereof - Google Patents
Collagen-integrin alpha2beta1 interacted polypeptide inhibitors and screening method thereof Download PDFInfo
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Abstract
The invention discloses collagen-integrin alpha2beta1 interacted polypeptide inhibitors and a screening method thereof. The collagen-integrin alpha2beta1 interacted polypeptide inhibitors provided for the first time are LWWNSYY, LRWNSPY and LYWNSGY. According to a molecular mechanics-Poisson-Boltzmann solvent accessible surface area analysis method, the interaction between collagen short peptide and integrin alpha2beta1 on the surface of blood platelet is analyzed, so as to obtain a key residue with affinity interaction; therefore, a collagen-integrin alpha2beta1 interacted polypeptide inhibitor library can be constructed, the polypeptide inhibitors having high affinity to collagen can be obtained by screening polypeptides through molecular docking, root mean square deviation comparison and molecular dynamic simulation. Isothermal titration calorimetry experiments and blood platelet adsorption experiments proved that the obtained polypeptide inhibitors can inhibit the bonding between the collagen and integrin alpha2beta1, thus being potential high-efficiency thrombus inhibitors.
Description
Technical field
The design and rational flow process that the present invention relates to collagen protein-beta 2 integrin alpha 2 β 1 interactional peptide inhibitors builds and obtains novel polypeptide inhibitor, and the computer mould belonging in biotechnology fits protein interface behavioral study field.
Background technology
Thrombosis is due to vessel wall generation pathology, and blood vessel endothelium undertissue thing is exposed in blood plasma, brings out blood aggegation and causes the process of angiemphraxis, causes the ischemic necrosis of organ, tissue, causes cardiovascular disorder, as apoplexy and myocardial infarction.According to World Health Organization's statistics, die from every year the number of cardiovascular disorder far above cancer, occupy first of all kinds of causes of the death.Wherein, the death and the deformity that cause due to myocardial infarction, cerebral infarction occupy very big ratio, and national economy and social development are caused to very big negative impact.Therefore, developing thrombus prevention and cure medicine evident in efficacy, side effect is little has become the focus of medical circle research, significant.
Thrombosis is by the common complex process regulating of many factors, comprises that thrombocyte is in the initial adherence at damaged vessel wall place, hematoblastic stable absorption and three significant process of platelet aggregation.At present, medicine mostly on the market is the anti-platelet aggregation medicinal for during platelet aggregation, for example, and acetylsalicylic acid (Aspirin), ridogrel (Ridogrel), Ticlopidine (Ticlopidine) etc.The mechanism of action of this class medicine is that the key enzyme in irreversible inhibition thrombocyte born of the same parents is lived, thereby make thrombocyte in quiescent condition, suppress thrombotic object to reach, but also destroy normal blood coagulation and anticoagulant balance of human body simultaneously, exist and cause extensive hemorrhage risk, have larger side effect.Studies have reported that at present, the overexpression of beta 2 integrin alpha 2 β 1 will cause the formation of thrombus, only can the mild prolonged bleeding time and suppress that it expresses, can't cause the problem of serious haemophilia.Therefore, stablize by suppressing thrombocyte the formation that 2 β 1 of beta 2 integrin alpha in adhesion process and the combination of collagen protein suppress thrombus, contribute to reduce hemorrhage risk on a large scale.
Integrin is a kind of transmembrane protein that is positioned at cell surface, can by with the interaction of extracellular matrix, reach the effect that regulating cell transfer, absorption and signal transmit.Wherein, beta 2 integrin alpha 2 β 1 play very important effect for thrombocyte in the stable adhesion on collagen protein surface in thrombosis.The people such as Emsely probe into the microtexture of α 2A structural domain by crystalline diffraction.Result shows, beta 2 integrin alpha 2 β 1 are α 2A structural domain with collagen protein calmodulin binding domain CaM, it is arranged between second and the 3rd β-pleated sheet structure (β-sheet) of β spiral (β-propeller) structure of the α subunit of beta 2 integrin alpha 2 β 1, centered by the laminated structure of 5 antiparallel β-pleated sheet structures, around, round 7 α spirals (α-helix) structure, residue N154, Y157 and H258 wherein play a key effect in integrin is combined with collagen protein.
The ligand i collagen type of beta 2 integrin alpha 2 β 1 is made up of two α 1 chains and α 2 chains, and wherein three α chains are cross-linked to form triple-helix structure, and this structure is proved and is extensively present in the patch that atherosclerotic patient causes blood vessel infarct.At present, GFOGER(O: oxyproline) aminoacid sequence is to be proved to be the integrin binding α 2 β 1 the highest sites of affinity on type i collagen albumen, metal ion binding site (the Metal ion-dependent adhesion site of the Glutamatergic in sequence and α 2A structural domain, MIDAS) chimeric melts combine, and then form regular octahedron structure with amino residue D151, S153, S155, T221 and the D254 at MIDAS core place; And arginine can be combined by hydrogen bond action with the D219 of MIDAS site.In addition, the phenylalanine on collagen protein can be combined with integrin hydrophobic region by its hydrophobic interaction.
As can be seen here, the amino-acid residue being associated when the specific binding region of beta 2 integrin alpha 2 β 1 on collagen protein and two-way interaction are mainly illustrated in forefathers' research work.But, on collagen protein and integrin bonding interface, be not that all amino-acid residues all play promoter action to the combination of the two, in conjunction with producing the further investigation of still needing of the Key residues of decisive role and keying action power and regulation and control factor.Therefore, from molecular level disclose the keying action power of collagen protein and beta 2 integrin alpha 2 β 1 combinations thrombosis and regulation and control factor also by this development of new inhibitor for the exploitation important in inhibiting of antithrombotic reagent and wide application prospect.
Molecular dynamics (MD) simulation is widely used in the research of protein interface behavior, the especially research of the cohesive process between molecule.Conventional molecular dynamics simulation software mainly contains GROMACS, NAMD, AMBER and CHARMM etc.Not only can obtain the various character of simulated system by molecular dynamics simulation binding molecule mechanics-Poisson-Boltzmann Solvent accessible surface analytical procedure, comprise interaction force between conformation, energy, kinetic property and aglucon-target protein etc., amino-acid residue on can also clear elaboration bonding interface is in conjunction with being favourable or unfavorable, and assesses each amino acid whose Relative Contribution and significance level.
Molecular docking is by geometric match and can be flux matched and the process of mutual identification between two molecules.It is that aglucon molecule is placed on to target protein binding site place that molecular docking is calculated, and evaluates the combination quality of aglucon and target protein, and provide the best combination conformation of two molecules according to the principle of how much complementations, energy complement and chemical environment complementations.With regard to its principle, molecular docking is a kind of Direct Method of Design based on acceptor.Along with the continuous renewal of rapid growth and the small molecules database of albumin crystal structural information, molecular docking has become the most important method based in structure design in recent years.Popular software has DOCK, Autodock and FlexX etc.
Adopt Rational design method, can reduce costs by combined strategy reasonable in design.For example, adopt the fast but limited method preliminary screening of precision of computing velocity such as molecular docking etc. to adopt thereafter the calculated amount such as MD simulation greatly but the screening of more accurate method, and adopt experimental technique more consuming time and that cost is high to verify at stage in early stage.
Summary of the invention
The object of the invention is to propose for design and rational and the screening of the interactional novel polypeptide inhibitor of collagen protein-beta 2 integrin alpha 2 β 1, and gained peptide inhibitor is to propose first and empirical tests is effective.
The interactional peptide inhibitor of collagen protein-beta 2 integrin alpha 2 β 1 that the present invention proposes is first: LWWNSYY, LRWNSPY and LYWNSGY.
The interactional peptide inhibitor screening method of collagen protein-beta 2 integrin alpha 2 β 1 of the present invention is, peptide storehouse for collagen protein and the interactional novel polypeptide inhibitor of beta 2 integrin alpha 2 β 1 builds, sequence is LXXNSXY, and wherein X represents any of 20 kinds of common amino acids.
Utilize the calculating of molecular mechanics-Poisson-Boltzmann Solvent accessible surface free energy and decomposition method to determine that the Key residues of being combined with collagen protein in beta 2 integrin alpha 2 β 1 is Y157, N154, S155 and L220.
Peptide library construction basis is 4 Key residues: Y157, N154, S155 and L220 in beta 2 integrin alpha 2 β 1.
In the scope in peptide storehouse, utilize amino acid localization method to build candidate's peptide molecule storehouse.
Sieve again by molecular docking screening, root-mean-square deviation comparison and molecular dynamics simulation, build the screening for collagen protein and the interactional novel polypeptide inhibitor of beta 2 integrin alpha 2 β 1.
Utilize Vina molecular docking software that the peptide inhibitor in peptide inhibitor storehouse is docked with collagen segment successively, choose the peptide inhibitor lower than-6.5 kcal/mol in conjunction with free energy, amount to 177.
Utilize g_rms program that GROMACS molecular simulation software carries to calculate Vina and dock in 177 peptide inhibitors obtaining and beta 2 integrin alpha 2 β 1 root-mean-square deviation between Key residues accordingly, select 25 peptide inhibitors to carry out next step research.
According to binding site and the comparison of original combination model, 25 peptide inhibitors are continued to screening, choose 8 similar peptide inhibitors of binding site and carry out next step MD modeling effort.
8 peptide inhibitors and collagen segment that screening obtains carry out MD simulation, again screen peptide inhibitor, obtain in simulated time yardstick can with the peptide inhibitor of collagen segment stable bond, join probability P
bindbe distributed in 0.50～1.00 scope; The combination that filters out LWWNSYY, LRWNSPY and LYWNSGY can be compared with efficient peptide inhibitor little and that join probability is larger.
The interactional peptide inhibitor of collagen protein-beta 2 integrin alpha 2 β 1 that the present invention proposes is first: LWWNSYY, LRWNSPY and LYWNSGY.According to molecular mechanics-Poisson-Boltzmann Solvent accessible surface analytical procedure, resolve collagen protein small peptide (GPO)
2gFOGER (GPO)
3beta 2 integrin alpha 2 β 1 interaction details with platelet surface, obtain the Key residues with collagen protein small peptide in beta 2 integrin alpha 2 β 1 with higher affinity interaction, build by this collagen protein and the beta 2 integrin alpha interactional peptide inhibitor of 2 β 1 storehouse, utilize molecular docking, root-mean-square deviation comparison and molecular dynamics simulation screening candidate polypeptide, obtain the peptide inhibitor with collagen protein with high-affinity.Testing by isothermal titration calorimetric subsequently and thrombocyte adsorption experiment confirms that gained peptide inhibitor can suppress the combination between collagen protein and beta 2 integrin alpha 2 β 1, is potential efficient depressor of thrombus.
Brief description of the drawings
The design and rational schema of Fig. 1 collagen protein-beta 2 integrin alpha 2 β 1 interactional peptide inhibitors
The thermodynamical coordinate figure of Fig. 2 isothermal titration calorimetric experiment
Fig. 3 thrombocyte adsorption experiment design sketch.Add the mensuration of the platelet solution light absorption value (600 nm) of different samples
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in detail, and this embodiment does not limit the present invention in any way for explaining.
The peptide storehouse that the present invention is directed to the interactional novel polypeptide inhibitor of collagen protein-beta 2 integrin alpha 2 β 1 builds, and construction basis is 4 Key residues: Y157, N154, S155 and L220 in beta 2 integrin alpha 2 β 1; Its characteristic sequence is LXXNSXY, and wherein X represents any of 20 kinds of common amino acids.
Novel polypeptide inhibitor of the present invention; The application of Rational design method, is on the basis in above-mentioned peptide storehouse, utilizes amino acid localization method to build candidate's peptide inhibitor storehouse:
Peptide inhibitor in candidate's peptide storehouse is sieved again by molecular docking screening, root-mean-square deviation comparison and molecular dynamics simulation, build design and rational and screening process for collagen protein and the interactional novel polypeptide inhibitor of beta 2 integrin alpha 2 β 1, obtain and there is the peptide inhibitor of high-affinity taking LWWNSYY as representative with collagen protein, and testing and thrombocyte adsorption experiment confirms that gained peptide inhibitor can suppress the combination between collagen protein and beta 2 integrin alpha 2 β 1 by isothermal titration calorimetric, is potential efficient depressor of thrombus.
It is emphasized that all peptide inhibitors (8000) that comprise in above-mentioned peptide storehouse, all likely block in theory the combination of collagen protein and beta 2 integrin alpha 2 β 1; Screen and MD sieves again by molecular docking, can screen with collagen protein and there is the peptide inhibitor compared with high-affinity, effectively dwindle candidate's peptide inhibitor quantity and be convenient to subsequent experimental checking; Through screening successively the peptide inhibitor finally obtaining, be to have compared with the peptide inhibitor of the maximum probability of high-affinity with collagen protein.Although being developed so far, computer modeling technique is constantly tending towards ripe and perfect, but can't reach and the on all four level of practical situation the interactional prediction between biomolecules, therefore molecular simulation software just inevitably has various limitation (as utilized different molecular simulation softwares or adopting different parameters just may produce different results), can only accomplish to be tending towards as much as possible approaching with practical situation.So when application molecular simulation software screens, there will be two kinds of situations: the one, the peptide inhibitor that screening obtains is that the hit rate of effective inhibitor constantly increases with screening number of times; The 2nd, leak the peptide inhibitor that can be used as effective inhibitor sieve, that exclude with screening increased frequency.So, do not get rid of the combination that all the other peptide inhibitors in peptide inhibitor storehouse also may effectively suppress collagen protein and beta 2 integrin alpha 2 β 1.
In the present invention, design and rational, screening and the experimental verification flow process of collagen protein-beta 2 integrin alpha 2 β 1 binding inhibitors comprise (Fig. 1) as follows:
1. utilize molecular mechanics-Poisson-Boltzmann Solvent accessible surface (Molecular Mechanics-Poisson-Boltzmann Surface Area, MM-PBSA) free energy calculating and decomposition method determine that the Key residues of being combined with collagen protein in beta 2 integrin alpha 2 β 1 is Y157, N154, S155 and L220, calculate the distance between corresponding C and N end between residue between two according to the conformation of Key residues and relative position, according to inserting amino acid no object principle, calculate the amino-acid residue number that should insert therebetween, build by this collagen protein-beta 2 integrin alpha interactional peptide inhibitor of 2 β 1 storehouse, its characteristic sequence be LXXNSXY(wherein X represent any of 20 kinds of common amino acids), utilize shell script to call charmm and build peptide inhibitor storehouse, comprise altogether 8000 sequences, see the peptide sequence table in the peptide inhibitor storehouse of specification sheets, each sequence comprises 4 Key residues.
2. utilize Vina molecular docking software that the peptide inhibitor in peptide inhibitor storehouse is docked with collagen segment successively, choose the peptide inhibitor lower than-6.5 kcal/mol in conjunction with free energy (marking mark), amount to 177.Utilize g_rms program that GROMACS molecular simulation software carries to calculate Vina and dock the root-mean-square deviation (RMSD) between corresponding Key residues in 177 peptide inhibitors obtaining and beta 2 integrin alpha 2 β 1, relatively Vina docks the conformational change of the Key residues in rear peptide inhibitor accordingly.RMSD value is less, and in the docking conformation of the Key residues that expression peptide inhibitor comprises and α 2 β 1, focus residue conformation is more approaching.Result shows, RMSD value is mainly distributed between 0.3～0.6 nm, selects 25 peptide inhibitors of RMSD<0.4 nm to carry out next step research.Here it should be noted that, the RMSD value standard of screening peptide inhibitor also can be carried out suitably change according to practical situation, person skilled under similar research background and situation, can with reference to the present invention arrange suitable RMSD value standard make the peptide inhibitor number that obtains of screening moderate be unlikely to again to leak screen out too much potential inhibitor.And then according to binding site and the comparison of original combination model, last round of screening is obtained to 25 peptide inhibitors continuation screenings, choose 8 similar peptide inhibitors of binding site and carry out next step MD modeling effort.
3. step 2 is screened 8 peptide inhibitors and the collagen segment that obtain and is carried out MD simulation, again screen peptide inhibitor, obtain in simulated time yardstick can with the peptide inhibitor of collagen segment stable bond, these peptide inhibitors likely have higher affinity interaction with collagen protein.In MD simulation, find the combination energy E of 8 all candidates peptide inhibitor
mDbe all negative value, illustrate that 8 peptide inhibitors can be combined in collagen tablet section surface, and join probability P
bindbe distributed in 0.50～1.00 scope.Wherein, the combination of LRWNSPY, LYWNSGY and LWWNSYY can, compared with little and join probability is larger, be possible efficient peptide inhibitor.By these 3 peptide inhibitors of MD simplation verification to the interactional inhibition of collagen protein-beta 2 integrin alpha 2 β 1.Research shows, thereby 3 candidate's peptide inhibitors can be combined with collagen protein the combination of inhibition of integrins α 2 β 1 and collagen protein, but inhibition degree is different.LWWNSYY energy quick adsorption covers the binding site of beta 2 integrin alpha 2 β 1 to collagen protein surface, thereby inhibition is the strongest; LRWNSPY inhibition is taken second place; The inhibition of LYWNSGY is the most weak in 3.
Embodiment 1: the foundation in collagen protein-beta 2 integrin alpha 2 β 1 interaction analytic and peptide inhibitor storehouse
First use MM-PBSA method to calculate the combination free energy of α 2A structural domain mixture (PDB ID:1DZI) in collagen protein-beta 2 integrin alpha 2 β 1.Then, adopt a kind of free energy decomposition method based on MM-PBSA resolve the molecule mechanism of collagen protein and α 2A structural domain high-affinity and analyze each residue in collagen protein-α 2A structural domain mixture and, to the contribution in conjunction with free energy, determine thus its interactional Key residues.Adopt the standard of <-2.5 kcal/mol to identify the residue larger to free energy contribution, find to only have Y157 in α 2A, N154, these 4 residues of S155 and R288 meet above requirement.Residue L220's is-1.9 kcal/mol to mixture free energy contribution, is slightly greater than-2.5kcal/mol, so but follow Y157 closely and N154 is also added in model construction for the contribution of cohesive process.And because R288 is less and away from all the other 4 Key residues for the free energy contribution in mixture and cohesive process, therefore leave out R288 to reduce the calculated amount of follow-up screening, improve screening efficiency.The affine combination model LXXNSXY(that the molecule mechanism obtaining based on above parsing and Key residues build collagen protein wherein X represents any in 20 kinds of common amino acids).
Embodiment 2: peptide inhibitor docks with collagen segment
There is stronger interaction because single residue and collagen segment do not necessarily represent by stronger affinity interaction the peptide inhibitor and the collagen segment that are made up of residue, therefore continue with Vina, the whole peptide inhibitors in peptide inhibitor storehouse to be docked successively with collagen segment.Docking result shows that all peptide inhibitors can be combined with collagen segment, and the prediction of all peptide inhibitors in conjunction with free energy between-7.2～-3.7 kcal/mol.Choose the peptide inhibitor lower than-6.5 kcal/mol in conjunction with free energy, amount to 177.
In order to give full play to the affinity interaction between Key residues and collagen protein, will find the docking conformation of the Key residues in peptide inhibitor and the peptide inhibitor that in α 2A, corresponding focus residue conformation is consistent, such peptide inhibitor can be imitated α 2A and the efficient combination of collagen segment as far as possible.In order to analyze in polypeptide in Key residues and α 2A the difference of conformation between corresponding residue, the g_rms program that this research and utilization GROMACS molecular simulation software carries is calculated Vina and dock in 177 peptide inhibitors obtaining and α 2A the RMSD between Key residues accordingly.RMSD value is less, and the docking conformation of the Key residues that expression peptide inhibitor comprises is got over the conformation close to corresponding Key residues in α 2A.Result shows, RMSD value is distributed between 0.3～0.6 nm.Select 25 peptide inhibitors of RMSD<0.4 nm to carry out next step research.By in conjunction with conformational analysis, whether unanimously with the binding site of beta 2 integrin alpha 2 β 1 on collagen protein investigate the binding site of peptide inhibitor, obtain 8 peptide inhibitors with identical combination site and do further molecular dynamics simulation and analyze the affinity between itself and collagen protein.
Embodiment 3: the screening of peptide inhibitor and the checking of the molecular dynamics simulation of inhibition thereof
MD simulation is the very effective instrument of research albumen dynamic behavior, and albumen fast internal motion, slower conformational change and folding process can be applied MD simulation and studies.For the further interaction between checking collagen protein-peptide inhibitor, not only to consider its static structure (utilizing molecular docking), also to study its dynamic behaviour.Thereby utilize above-mentioned 8 multidate informations that candidate's peptide inhibitor is combined with collagen segment of MD modeling effort.
All MD simulations all adopt GROMACS 4.5.3 software package, select the G43a1 field of force, utilize pdb2gmx order to be converted into the special gro structure of GROMACS the pdb coordinate structure of 8 candidate's peptide inhibitors; Utilize editconf order that peptide inhibitor and collagen protein are placed in to 10 × 7 × 5.5 nm
3rectangular box center; Then add normal saline solution and add the required ionic species of equilibrium system net charge and quantity in simulation box with genbox order, wherein water molecules adopts TIP3P model; Be then thereby that structure and topological file generate tpr file with grompp order by the parameter integration of indicating in mdp file; Carry out afterwards energy minimization, the interatomic collision in removal system and incorrect geometric configuration; Next carry out canonical (NVT) the assemblage MD simulation of 10 ns with mdrun order.
MD simulates discovery, the combination free energy E of 8 candidate's peptide inhibitors
mDbe all negative value, illustrate that 8 peptide inhibitors can be combined in collagen tablet section surface, and join probability P
bindbe distributed in 0.50～1.00 scope.Choose in conjunction with can be compared with little and join probability is larger 3 peptide inhibitor: LRWNSPY, LYWNSGY and LWWNSYY carry out follow-up inhibition MD checking.
3 peptide inhibitors that screen above acquisition are joined respectively and comprised in collagen protein and α 2A structural domain simulation box separated from one another.By test α 2A structural domain and the combination situation of collagen protein under 50 ns times, come intuitively the inhibition of filter out 3 peptide inhibitors to be assessed.In simulation, the molecule ratio with collagen protein/α 2A structural domain of peptide inhibitor is made as 5:1.Analog result shows, thereby 3 candidate's peptide inhibitors can be combined with collagen protein and be suppressed the keying action of α 2A structural domain-collagen protein.
Embodiment 4: isothermal titration calorimetric experimental verification
Collagen segment (GPO)
2gFOGER (GPO)
3(containing the affine site GFOGER of beta 2 integrin alpha 2 β 1) entrusts Qiang Yao bio tech ltd (Shanghai) to synthesize, and product is through high performance liquid chromatography (HPLC) purifying, and purity is 95.13%.Peptide inhibitor LWWNSYY entrusts the biochemical company limited of gill (Shanghai) to synthesize, and product is through high performance liquid chromatography (HPLC) purifying, and purity is 95.70%.
Adopt the isothermal titration calorimeter (ITC) of the VP model of Microcal company to carry out isothermal titration calorimetric experiment.In isothermal titration calorimetric experiment, drip the collagen segment that concentration is 1.2 mM in sample pool, in titration pin, adding concentration is the peptide inhibitor LWWNSYY of 100 μ M.All peptide Duan Jun are dissolved in 25%(v/v) ethylene glycol solution, and adding the processing of bleeding through 20 minutes before sample pool/titration pin.Design parameter in experiment is as follows: it is 8 μ L that every, titration pin splashes into volume; Drip altogether 30; Between every two, the timed interval is 500 s; Baseline is set as 20 μ cal/s; Speed setting is 307 rpm; Sample pool Temperature Setting is 10 DEG C.Remove the ethylene glycol solution of collagen protein by titration and measure the dilution enthalpy of peptide inhibitor, and deduct at follow-up Data processing.By peptide inhibitor and the titration experiments of collagen fragment obtain the thermodynamical coordinate of cohesive process.Data are utilized MicroCal Origin (Version 7.0) and the combination model processing of single site, thereby obtain dissociation constant, enthalpy change, Entropy Changes and the gibbs free energy change (Fig. 2) of reaction.
Result shows, the enthalpy change Δ H of reaction is 1.45 ± 0.17 kcal/mol, Entropy Changes T Δ S is 8.55 ± 0.01 kcal/mol, the two be all on the occasion of, the cohesive process that shows peptide inhibitor LWWNSYY and collagen protein is driven by entropy, hydrophobic interaction is the main drive of cohesive process, matches with analog result.Gibbs free energy change Δ G value is-7.10 ± 0.17 kcal/mol, shows that peptide inhibitor LWWNSYY and collagen protein can spontaneous combinations.Dissociation constant K
dnumerical value be 3.34 ± 1.22 μ M, show that the keying action of the two belongs to strong combination.
Therefore, ITC experiment confirms the spontaneous surface that is combined in collagen protein of peptide inhibitor LWWNSYY energy, is the potential interactional highly efficient depressor of collagen protein-beta 2 integrin alpha 2 β 1.
Embodiment 5: thrombocyte adsorption experiment checking
The Sheep Whole Blood that contains 0.4% Sodium Citrate anti-freezing is bought and is obtained from Guangzhou Rui Te Bioisystech Co., Ltd, at 4 DEG C, preserves.Sheep Whole Blood 180 g at 4 DEG C obtain and are rich in hematoblastic blood plasma (platelet-rich plasma, PRP) for centrifugal 20 minutes.Collagen segment is dissolved in phosphate buffered saline buffer (PBS, 80 g/L NaCl, 2.0 g/L KCl, 14.4 g/L Na
2hPO
4, 2.4 g/L KH
2pO
4, 1.9 g/L MgCl
2; PH 7.4) acquisition 0.2 mM collagen solution.
Thrombocyte is adsorbed in after collagen protein by beta 2 integrin alpha 2 β 1 on film surface, causes platelet aggregation, causes thrombocyte sedimentation, thereby reduces the platelet counts in solution.And light absorption value under platelet counts and 600 nm wavelength in solution presents positive correlation, therefore reduce and investigate thrombocyte and collagen protein adsorbs situation by measuring the light absorption value at 600 nm places.
For measuring thrombocyte adsorpting aggregation, by 900 μ L PRP warm bath 5 minutes at 22 DEG C in advance, add subsequently the collagen solution of 100 μ L, at 22 DEG C, cultivate after 25 minutes, measure the light absorption value at 600 nm places.
For the restraining effect of checking peptide inhibitor LWWNSYY, by be dissolved in PBS 0.3 mM LWWNSYY and collagen protein by volume 1:1 mixes, at 4 DEG C, hatch the collagen protein that acquisition is sealed 12 hours.The sealing collagen solution of 100 μ L joins in the PRP of the 900 μ L that temperature was bathed in advance, cultivates after 25 minutes at 22 DEG C, measures the light absorption value at 600 nm places.
100 μ L PBS join in the PRP of the 900 μ L that temperature was bathed in advance, cultivate after 25 minutes at 22 DEG C, measure the light absorption value at 600 nm places, as blank.
Result shows (Fig. 3), the PRP that is only added with collagen segment has significantly and declines at the light absorption value (0.046 ± 0.004) at 600 nm places compared with blank group (0.144 ± 0.001), after thrombocyte absorption collagen protein is described, assemble sedimentation, cause the platelet counts in solution significantly to reduce.And the collagen protein being sealed by peptide inhibitor LWWNSYY adds rear light absorption value close with blank, illustrate that peptide inhibitor well covers the binding site of beta 2 integrin alpha 2 β 1 on collagen protein, cause collagen protein cannot adsorb thrombocyte, thereby anticoagulant sedimentation, therefore the platelet cell number in solution is without considerable change.
Therefore, thrombocyte adsorption experiment shows, the interaction of the peptide inhibitor LWWNSYY fine inhibition collagen protein of energy and beta 2 integrin alpha 2 β 1, thus affect follow-up thrombosis, be an efficient depressor of thrombus.Two other LYWNSGY filtering out and LWWNSYY inhibitor show through thrombocyte adsorption experiment, the interaction of the fine inhibition collagen protein of energy and beta 2 integrin alpha 2 β 1, thus affect follow-up thrombosis, be efficient depressor of thrombus.Here just do not repeat one by one.
The present invention proposes design and rational and the screening of the interactional novel polypeptide inhibitor of collagen protein-beta 2 integrin alpha 2 β 1, and test and thrombocyte adsorption experiment confirms that gained peptide inhibitor can suppress the combination between collagen protein and beta 2 integrin alpha 2 β 1 by isothermal titration calorimetric, be potential depressor of thrombus.In addition, what be worth proposition is, it on whole 8000 sequencing theories in peptide inhibitor storehouse, is likely all the interactional effective inhibitor of collagen protein-beta 2 integrin alpha 2 β 1, but due to the restriction of time manpower and materials, the present invention can not carry out experimental verification successively by whole peptide inhibitors, and the best peptide inhibitor of effect that can only obtain final screening carries out Experimental Characterization.But according to acquired experimental data, show that the peptide inhibitor in peptide storehouse is the interactional effective candidate inhibitor of collagen protein-beta 2 integrin alpha 2 β 1.
Claims (10)
1. the interactional peptide inhibitor of collagen protein-beta 2 integrin alpha 2 β 1 is characterized by: LWWNSYY, LRWNSPY and LYWNSGY.
2. the interactional peptide inhibitor screening method of collagen protein-beta 2 integrin alpha 2 β 1 of claim 1, it is characterized in that building for the peptide storehouse of collagen protein and the interactional novel polypeptide inhibitor of beta 2 integrin alpha 2 β 1, sequence is LXXNSXY, and wherein X represents any of 20 kinds of common amino acids.
3. method as claimed in claim 2, is characterized in that utilizing molecular mechanics-Poisson-Boltzmann Solvent accessible surface free energy to calculate and decomposition method determines that the Key residues of being combined with collagen protein in beta 2 integrin alpha 2 β 1 is Y157, N154, S155 and L220.
4. method as claimed in claim 2, is characterized in that peptide library construction basis is 4 Key residues in beta 2 integrin alpha 2 β 1:
Y157, N154, S155 and L220.
5. the method as described in claim 2 or 4, is characterized in that: in the scope in peptide storehouse, utilize amino acid localization method to build candidate's peptide molecule storehouse.
6. method as claimed in claim 2, is characterized in that sieving again by molecular docking screening, root-mean-square deviation comparison and molecular dynamics simulation, builds the screening for collagen protein and the interactional novel polypeptide inhibitor of beta 2 integrin alpha 2 β 1.
7. method as claimed in claim 6, it is characterized in that utilizing Vina molecular docking software that the peptide inhibitor in peptide inhibitor storehouse is docked with collagen segment successively, choose the peptide inhibitor lower than-6.5 kcal/mol in conjunction with free energy, amount to 177.
8. method as claimed in claim 6, it is characterized in that utilizing g_rms program that GROMACS molecular simulation software carries to calculate Vina and dock in 177 peptide inhibitors obtaining and beta 2 integrin alpha 2 β 1 root-mean-square deviation between Key residues accordingly, select 25 peptide inhibitors to study.
9. method as claimed in claim 6, is characterized in that, according to binding site and the comparison of original combination model, 25 peptide inhibitors are continued to screening, chooses 8 similar peptide inhibitors of binding site and carries out MD modeling effort.
10. method as claimed in claim 6, it is characterized in that 8 peptide inhibitors and collagen segment that screening obtains carry out MD simulation, again screen peptide inhibitor, obtain in simulated time yardstick can with the peptide inhibitor of collagen segment stable bond, join probability P
bindbe distributed in 0.50～1.00 scope; The combination that filters out LWWNSYY, LRWNSPY and LYWNSGY can be little and efficient peptide inhibitor that join probability is large.
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| CN104800857A (en) * | 2015-04-14 | 2015-07-29 | 天津大学 | Nano inhibitor for inhibiting mutual action of collagen and integrin alpha2beta1 in forming process of thrombus, preparation method and application |
| CN109456385A (en) * | 2018-12-13 | 2019-03-12 | 无限极（中国）有限公司 | A method of screening has anti-ageing active polypeptide |
| CN110010206A (en) * | 2019-03-28 | 2019-07-12 | 华南理工大学 | A kind of emulation regulation method of albumen absorption behavior on titanium dioxide surface |
| CN111613275A (en) * | 2020-05-26 | 2020-09-01 | 中国海洋大学 | Drug molecular dynamics result analysis method based on rmsd multi-feature |
| CN112034184A (en) * | 2020-09-11 | 2020-12-04 | 中国水产科学研究院黄海水产研究所 | Auxiliary screening method of protein interaction blocking polypeptide |
| CN114907444A (en) * | 2022-05-10 | 2022-08-16 | 天津大学 | Collagen-targeted thrombus inhibitor and preparation method thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104800857A (en) * | 2015-04-14 | 2015-07-29 | 天津大学 | Nano inhibitor for inhibiting mutual action of collagen and integrin alpha2beta1 in forming process of thrombus, preparation method and application |
| CN104800857B (en) * | 2015-04-14 | 2018-06-19 | 天津大学 | For the nanometer inhibitor and preparation method and application of 2 β 1 of collagen-beta 2 integrin alpha in thrombosis interactions |
| CN109456385A (en) * | 2018-12-13 | 2019-03-12 | 无限极（中国）有限公司 | A method of screening has anti-ageing active polypeptide |
| CN110010206A (en) * | 2019-03-28 | 2019-07-12 | 华南理工大学 | A kind of emulation regulation method of albumen absorption behavior on titanium dioxide surface |
| CN111613275A (en) * | 2020-05-26 | 2020-09-01 | 中国海洋大学 | Drug molecular dynamics result analysis method based on rmsd multi-feature |
| CN112034184A (en) * | 2020-09-11 | 2020-12-04 | 中国水产科学研究院黄海水产研究所 | Auxiliary screening method of protein interaction blocking polypeptide |
| CN114907444A (en) * | 2022-05-10 | 2022-08-16 | 天津大学 | Collagen-targeted thrombus inhibitor and preparation method thereof |
| CN114907444B (en) * | 2022-05-10 | 2024-01-26 | 天津大学 | Collagen-targeted thrombus inhibitor and preparation method thereof |
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