CN101824084A - Targeting antiglioma protein and application thereof - Google Patents

Abstract

The invention provides targeting antiglioma protein, wherein on the basis of the approaches (inhabiting the activity of a chloride channel CLC and MMP-2) of experimentally approved scorpion chloride channel neurotoxin (Cltx and BmK CT) in treating brain glioma, an SWISS-MODEL server is utilized for carrying out the homology modeling of a protein atom structure for the BmK CT, a ZDOCK server is utilized for carrying out the atom structure modeling of a complex of chloride channel toxin and a receptor, a model with stable structure is screened in candidate models, energy minimization and molecular kinetic analysis are carried out, and a novel protein molecular sequence and a model which are optimally designed by using a chloride channel neurotoxin space structure as a framework and have high selectivity and high specificity for antiglioma cells are constructed through analyzing the binding energy and the hydrogen bonding times of the Cltx and the BmK CT. The protein can be used for preparing a medicament for treating glioma.

Description

Targeting antiglioma protein and application thereof

Technical field

The present invention relates to protein amino acid sequence, specifically is a kind of targeting antiglioma protein and the application of this protein in preparation treatment neuroglia tumor medicine based on scorpion chloride channel toxin.

Background technology

See that on the whole the present stage mankind still are in the exploratory stage for the treatment of malignant tumour.Monoclonal antibody medicine and gene therapy are still difficult as people's will, but the development of studying with gene biological of finishing along with the Human Genome Project, human more understandings and assurance to tumor invasion mechanism and correlation properties, the individual special catabolic cancer treatment method that discovery brought of the specific antigens that cancer cells is more, more stable will make monoclonal antibody medicine and gene therapy become the main trend of oncotherapy.

2003, U.S. Sonthrimer finds that isolating a kind of catilan Cltx can suppress the neuroglial cytoma growth effectively from Israel's scorpion toxin, and the infiltration and the mechanism of Cltx anti-glioma cell explored, confirmed that at glioma cell film surface expression, the matrix metalloproteinase-2 (MMP-2) that plays an important role in tumour cell diffusion be the bind receptor of Cltx, Cltx can be by suppressing the diffusion of tumour cell in conjunction with MMP-2.Chinese scholar finds to have a kind of neurotoxin BmK CT in buthus martensii Karscs in research buthus martensii Karscs toxin process, infer that according to the high sequence homology of itself and Cltx its function class is similar to Cltx.

This seminar is consulting on the basis of relative literature, has carried out the research of BmK CT albumen anti-glioma, has obtained progress preferably.We at first utilized the bypass method synthetic improvement BmK CT gene, realized that it efficiently expresses in intestinal bacteria, and this gene order is granted national inventing patent (a kind of scorpion chloride channel neurotoxin gene-rBmK CTa of synthetic, the patent No.: 200410012544.6,2009 years).Secondly, detect reorganization scorpion toxin rBmKCTa to the human glioma cell SHG-44 of cultivation and the influence of normal mice neurogliocyte, confirm that it has specific effect to neuroglial cytoma with mtt assay.And, utilize full cell patch tongs technology to prove conclusively the electrophysiological property of rBmK CTa, be the chloride channel inhibitor.At last, adopt radioactivity ¹³¹This toxin of I mark, foundation moves the nude mice animal model of growing the neuroglial cytoma that the people is arranged, followed the trail of the trend of this toxin in animal model, confirmed rBmK CTa be a kind of molecular weight little, can pass the blood brain and organize barrier, and can be in vivo and the pharmaceutical protein (bioactivity research of buthus martensii Karscs chloride channel toxin of the acceptor target position specific combination of tumor tissues, the doctorate paper, Yin Litian, 2009).

Above analytical proof, Cltx and BmK CT can be used as the active drug of potential mark and treatment glioma brain tumour and develop.But as the small molecular protein medicine, the research of this class catilan and acceptor (chloride channel protein and MMP-2 albumen) molecular mechanism of action does not still have report at present, illustrated and have only when the interactional molecule mechanism between them, just can help us further to utilize and transform this proteinoid, strengthen the specific effect of itself and target cell.The present invention utilizes protein structure database PDB, SwissModel server by the exploitation of Switzerland information biology institute, ZDOCK server by the research and development of U.S. BOSTON university, associated biomolecule information science resources such as Biopolymer and Discover3 software package among the Insight II of Accelrys company, at protein structure and function prediction, the interaction of protein molecule and dynamic molecular are simulated this experiment bottleneck problem, carried out the molecular computing modeling effort of catilan and acceptor interaction, make up the 3 d structure model of important molecule incident in this process, by optimizing the part amino acid sites, made up the active drug molecular model and the sequence that have more target, the research and development of novel anti neuroglia tumor medicine have been had important theory be worth and application prospect.

Summary of the invention

The object of the present invention is to provide a kind of targeting antiglioma protein and the application of this protein in preparation treatment neuroglia tumor medicine based on scorpion chloride channel toxin.

A kind of targeting antiglioma protein provided by the invention, aminoacid sequence are SEQ ID No.5.

This targeting antiglioma protein preparation method comprises the steps:

(1) (derives from Israel scorpion (Leiurus quinquestriatus) with protein C ltx, its aminoacid sequence is seen SEQ IDNo.1) and insect toxin 15A (PDB numbering: 1SIS) structure that is provided is the basis, carry out the homology modeling of BmK CT protein (derive from buthus martensii Karscs (Buthus matensii Karsch), its aminoacid sequence is seen SEQ ID No.2) atomic structure and do energy minimization and molecular dynamics optimization;

(2) utilize V.3.7 software of DeepView, adopt the Coulomb computation schema, calculate the electrostatic potential of Cltx, BmK CT protein surface;

(3) the X-ray crystallographic structural analysis result of the ligand binding domain of the receptor protein that provides according to the PDB storehouse, be chloride channel PROTEIN C lC-2C end cytoplasmic region and MMP-2 PROTEIN C end structure, with above-mentioned operational pattern, calculate its surface electrostatic gesture, and analyze and can get, very likely there is electrostatic attraction between Cltx/BmK CT and its acceptor CLC-2C end and the MMP-2C end molecule;

(4) the atomic structure mould that utilizes the ZDOCK server to carry out Cltx/BmK CT and acceptor ClC-2C and MMP-2C mixture is built, and ZDOCK result done energy minimization and analysis on Molecular Dynamics, final every kind of butt joint complex body obtains 10 candidate's models, obtains the total energy of each model;

(5) frequency of each amino acid and acceptor formation hydrogen bond in statistics Cltx, BmK CT and acceptor ClC-2, the MMP-2 combination model;

(6), design respectively at ClC-2,4 new protein amino acid sequences that the MMP-2 joint efficiency is the highest according to the result of (5):

CCL1(SEQ?ID?No.3)：

MCMPCFTTDHQMARKCRECCGGKGRC FGPQCLCR

CCL2(SEQ?ID?No.4)：

MCMPCFTTDHQMARKCRECCGGKGRC YGPQCLCR

CMM1(SEQ?ID?No.5)：

MCGPCFTTDH QMARKCRECCGGKGRCYGPQCLCR

CMM2(SEQ?ID?No.6)：

MCGPCFTTDH NMARKCRECCGGKGRCYGPQCLCR

(7) detect new protein sequence and receptors bind parameter, can 4 indexs sign and estimate newly-designed protein sequence from mixture hydrogen bond quantity, the electrostatic interaction of the mixture total energy of energy minimum, average total energy, energy minimum;

(8), determine that finally CMM1 (SEQ ID No.5) is the targeting antiglioma protein sequence according to more little stable more, many more stable more, the big more stable more principles of static potential energy absolute value of hydrogen bonded quantity of complex body total energy.

CMM1 provided by the invention (SEQ ID No.5) targeting antiglioma protein confirms to improve propagation and the diffusion that suppresses neuroglial cytoma by computer simulation, can use in preparation treatment neuroglia tumor medicine.

Description of drawings

Fig. 1: BmK CT albumen space atomic diagram (A) and space secondary structure Pareto diagram (B)

The electrostatic potential of Fig. 2: Cltx, BmK CT protein surface

Fig. 3: chloride channel PROTEIN C lC-2C end cytoplasmic region and MMP-2C end structure and surface electrostatic gesture thereof

Each amino acid and acceptor form the frequency of hydrogen bond in Fig. 4: Cltx, BmK CT and acceptor ClC-2C, the MMP-2C combination model

Fig. 5: design at ClC-2C, new protein amino acid sequence that the MMP-2C joint efficiency is the highest

Position (A) and Gly3 the position (B) in CMM1 three-dimensional structure of Fig. 6: Met3 in the CCL2 three-dimensional structure

Embodiment

Embodiment 1

1, the homology modeling of PROTEIN B mK CT atomic structure: scorpion toxin Cltx has the solution space conformation of resolving by NMR accordingly in the protein structure proximity database (pdb) proximity (the PBD numbering: 1CHL), and BmK CT does not have.Therefore, need homology modeling server by Swiss Model, based on homologous protein Cltx and insect toxin 15A (PDB numbering: the 1SIS) structure that is provided, BmK CT albumen and template are carried out sequence alignment, the operational method and the energy minimization principle of utilization atom mechanics, with the formwork structure is fundamental construction BmK CT space atomic structure model, and the Discover3 software package obtains BmK CT albumen space atomic diagram (Figure 1A) and space secondary structure Pareto diagram (Figure 1B) after doing energy minimization and analysis on Molecular Dynamics in Insight II.The parameter that the Discover3 software package is done energy minimization among the Insight II is set to:

Water box, temperature 298K, maximum step time (Max Step) be that 3000 times, the final degree of convergence (Final Convergence) are 0.001, conjugation method (Conjugate-Method) is the Polak-Ribiere method; The analysis on Molecular Dynamics parameter is set to: temperature 298K, step-length (Time Step) are to be 1fs, working time (Run Time) 150ps.White expression carbon atom C, sky-blue are that hydrogen atom H, avy blue are nitrogen-atoms N, the red Sauerstoffatom O of being among Figure 1A, yellow expression hydrogen bond.

2, utilize (the Swiss Institute of Bioinformatics of Switzerland bioinformation association, SIB) DeepView that provides is software V.3.7, adopt the Coulomb computation schema, with ± 1.8kT/e is a standard, calculates the electrostatic potential (see figure 2) of Cltx, BmK CT protein surface.

Shown in Fig. 2 A, Cltx is made of a α-helix and two antiparallel β-sheet, and this tertiary structure with present known short chain scorpion toxin is consistent.The surface electrostatic potential analysis shows that the Cltx surface arrangement a large amount of positive potentials, and the basic aminoacids in the hint molecule and the positive potential of generation play a decisive role to itself and receptors bind.This phenomenon is in BmK CT (Fig. 2 B) equally.

3, the X-ray crystallographic structural analysis result of the ligand binding domain of the receptor protein that provides according to the PDB storehouse, be chloride channel PROTEIN C lC-2C end cytoplasmic region and MMP-2C end structure, with above-mentioned operational pattern, calculate its surface electrostatic gesture (see figure 3), the electrostatic force that produces by electric field analyzes these two acceptors and interactional possibility takes place the chloride channel toxin.

The cytoplasmic region of ClC-2 can be divided into the class glutaredoxin structural domain of two structural domain: N ends and the structural domain (Fig. 3 A) of whole α of being-helix that C holds.Tart " foot loop " district that is positioned at the C end is the peculiar structure of people ClCs family.From Fig. 3 B, because the existence of 6 acidic residues is arranged, foot loop district is strong negative potential; And be positioned at N and C end intermediary groove positively charged gesture, show that these two sites may be the zones with its ligand interaction.

MMP-2C end regions (MMP-2C, Thr ⁴⁵⁸-Cys ⁶⁶⁰) be a compact form (Fig. 3 C) of forming by the albumen of 4 foliated lamellar β propeller samples, 4 middle holes that surround a channel-like of lamella, Ca ²⁺And Na-Cl ^-To passing through.The surface electrostatic potential analysis shows that negative potential mainly is distributed in (Fig. 3 D) on I, II, the IV lamella.

As above analyze, very likely have electrostatic attraction between Cltx/BmK CT and its acceptor ClC-2C end and the MMP-2C end molecule.

4, the atomic structure mould that utilizes the ZDOCK server of U.S. BOSTON university to carry out Cltx/BmK CT and acceptor ClC-2C end and MMP-2C end mixture is built.Utilize that Biopolymer and Discover3 software package do energy minimization and analysis on Molecular Dynamics to ZDOCK result among the Insight II, do optimization with method of steepest descent,

Do 300 step conjugate gradient energy minimizations in the water box, having done step-length at 298K is 1fs, the analysis on Molecular Dynamics (MD) that amounts to 5000fs, all operations under CVFF (consistent-valence force field) field of force function of above process.Final every kind of butt joint complex body obtains 10 candidate's models.List the total energy of every kind of butt joint complex body in the table 1.

The total energy of 10 candidate's models of table 1 butt joint complex body ^*

The mixture title	??1	??2	??3	??4	??5	??6	??7	??8	??9	??10
											??Cltx-ClC-2C	??8827.708	??8787.235	??8824.385	??8936.501	??8891.536	??8852.775	??8712.011	??8870.135	??8733.026	??8796.467
??BmK?CT??-ClC-2C	??8675.524	??8818.053	??8710.359	??8802.362	??8786.077	??8830.029	??8723.958	??8784.826	??8661.587	??8734.667
											??Cltx-MMP-2C	??8867.521	??8810.024	??8794.646	??8835.639	??8781.633	??8855.765	??8795.532	??8704.685	??8867.731	??8854.332
??BmK?CT-??MMP-2C	??8753.638	??8721.404	??——	??8802.211	??8791.389	??8845.689	??8661.025	??8815.227	??8801.841	??8751.346

( ^*Total energy is kinetic energy and potential energy sum, and unit is kcal/mol;---expression is owing to the compatibling problem between software causes calculating the result.)

5, the frequency (see figure 4) of each amino acid and acceptor formation hydrogen bond in statistics Cltx, BmK CT and acceptor ClC-2C, the MMP-2C combination model.

6,, design respectively at ClC-2C, 4 new protein amino acid sequence (see figure 5)s that the MMP-2C joint efficiency is the highest according to the result of Fig. 4.

7, detect new protein sequence and receptors bind parameter: utilize ZDOCK server, Insight II software package that newly-designed protein sequence is done with ClC-2C, MMP-2C to dock, energy minimization and analysis on Molecular Dynamics, sign and estimate newly-designed protein sequence (seeing Table 2,3,4) from mixture hydrogen bond quantity, these a few indexs of electrostatic interaction energy of the mixture total energy of energy minimum, average total energy, energy minimum.

Table 2 has been listed new design protein sequence docks 10 candidate's models of complex body with acceptor total energy; Table 3 listed in Cltx/BmK CT before and after the sequence optimisation combine on average total energy and energy minimum with the ClC-2C end two complex bodys join, the comparison of hydrogen bond position, quantity and the electrostatic interaction energy of acceptor interaction; Table 4 listed in Cltx/BmK CT before and after the sequence optimisation combine on average total energy and energy minimum with the MMP-2C end two complex bodys join, the comparison of hydrogen bond position, quantity and the electrostatic interaction energy of acceptor interaction.

The new design of table 2 protein sequence docks the total energy of 10 candidate's models of complex body with acceptor ^*

The mixture title	??1	??2	??3	??4	??5	??6	??7	??8	??9	??10
											??CCL1-??ClC-2C	??8692.494	??8711.141	??8634.207	??8623.221	??8755.685	??8707.374	??8782.786	??8717.297	??8711.211	??8774.209
??CCL2-??ClC-2C	??8691.543	??8782.683	??8712.545	??8635.346	??8607.804	??8677.652	??8719.005	??8713.295	??8788.054	??8639.399
											??CMM1??-MMP-2C	??8691.614	??8711.593	??8673.626	??8550.030	??8675.108	??8691.341	??8635.214	??8694.564	??8641.591	??8675.535
??CMM2??-MMP-2C	??8665.936	??8630.769	??8701.186	??8607.310	??——	??8621.717	??8626.319	??8655.476	??8574.467	??8523.503

( ^*Total energy is kinetic energy and potential energy sum, and unit is kcal/mol;---expression is owing to the compatibling problem between software causes calculating the result.)

Before and after table 3 sequence optimisation Cltx/BmK CT combine with ClC-2C in two complex bodys of on average total energy and energy minimum join, the comparison (wherein power unit is kcal/mol) of hydrogen bond position, quantity and the electrostatic interaction energy of acceptor interaction

Before and after table 4 sequence optimisation Cltx/BmK CT combine with MMP-2C in two complex bodys of on average total energy and energy minimum join, the comparison (wherein power unit is kcal/mol) of hydrogen bond position, quantity and the electrostatic interaction energy of acceptor interaction

8, according to more little stable more, many more stable more, the big more stable more principles of static potential energy absolute value of hydrogen bonded quantity of complex body total energy, filtering out following 2 sequences is optimal sequence:

CCL2(SEQ?ID?No.4)：

MC MPCFTTDHQMARKCRECCGGKGRCYGPQCLCR

CMM1(SEQ?ID?No.5)：

MC GPCFTTDHQMARKCRECCGGKGRCYGPQCLCR

By sequence as can be known, the difference of these two sequences only is the 3rd amino acids difference, from the structure (see figure 6), square frame is depicted as Met3 in the CCL2 three-dimensional structure of Fig. 6 A, square frame is depicted as Gly3 in the CMM1 three-dimensional structure of Fig. 6 B, these two amino-acid residues all are positioned at the position that the N end does not form secondary structure, influence to its three-dimensional structure is also little, so in conjunction with other index, on function, can effectively suppress the neuroglial cytoma migration from it and consider, determine that the CMM1 sequence is final a kind of targeting antiglioma protein sequence based on scorpion chloride channel toxin.

A kind of targeting antiglioma protein sequence based on scorpion chloride channel toxin provided by the invention confirms to improve propagation and the diffusion that suppresses neuroglial cytoma by computer simulation.

SEQUENCE?LISTING

＜110〉University Of Shanxi

＜120〉targeting antiglioma protein and application thereof

<130>.

<160>6

<170>PatentIn?version?3.5

 

<210>1

<211>36

<212>PRT

<213>Leiurus?quinquestriatus

<400>1

Met?Cys?Met?Pro?Cys?Phe?Thr?Thr?Asp?His?Gln?Met?Ala?Arg?Lys?Cys

1???????????????5???????????????????10??????????????????15

Asp?Asp?Cys?Cys?Gly?Gly?Lys?Gly?Arg?Gly?Lys?Cys?Tyr?Gly?Pro?Gln

20??????????????????25??????????????????30

Cys?Leu?Cys?Arg

35

 

<210>2

<211>36

<212>PRT

<213>Buthus?matensii?Karsch

<400>2

Met?Cys?Gly?Pro?Cys?Phe?Thr?Thr?Asp?Ala?Asn?Met?Ala?Arg?Lys?Cys

1???????????????5???????????????????10??????????????????15

Arg?Glu?Cys?Cys?Gly?Gly?Ile?Gly?Lys?Cys?Phe?Gly?Pro?Gln?Cys?Leu

20??????????????????25??????????????????30

Cys?Asn?Arg?Ile

35

 

<210>3

<211>34

<212>PRT

<213>Aritifical

<400>3

Met?Cys?Met?Pro?Cys?Phe?Thr?Thr?Asp?His?Gln?Met?Ala?Arg?Lys?Cys

1???????????????????5???????????????10??????????????????15

Arg?Glu?Cys?Cys?Gly?Gly?Lys?Gly?Arg?Cys?Phe?Gly?Pro?Gln?Cys?Leu

20??????????????????25??????????????????30

Cys?Arg

 

<210>4

<211>34

<212>PRT

<213>Aritifical

<400>4

Met?Cys?Met?Pro?Cys?Phe?Thr?Thr?Asp?His?Gln?Met?Ala?Arg?Lys?Cys

1???????????????5???????????????????10??????????????????15

Arg?Glu?Cys?Cys?Gly?Gly?Lys?Gly?Arg?Cys?Tyr?Gly?Pro?Gln?Cys?Leu

20??????????????????25??????????????????30

Cys?Arg

 

<210>5

<211>34

<212>PRT

<213>Aritifical

<400>5

Met?Cys?Gly?Pro?Cys?Phe?Thr?Thr?Asp?His?Gln?Met?Ala?Arg?Lys?Cys

1???????????????5???????????????????10??????????????????15

Arg?Glu?Cys?Cys?Gly?Gly?Lys?Gly?Arg?Cys?Tyr?Gly?Pro?Gln?Cys?Leu

20??????????????????25??????????????????30

Cys?Arg

 

<210>6

<211>34

<212>PRT

<213>Aritifical

<400>6

Met?Cys?Gly?Pro?Cys?Phe?Thr?Thr?Asp?His?Asn?Met?Ala?Arg?Lys?Cys

1???????????????5???????????????????10??????????????????15

Arg?Glu?Cys?Cys?Gly?Gly?Lys?Gly?Arg?Cys?Tyr?Gly?Pro?Gln?Cys?Leu

20??????????????????25??????????????????30

Cys?Arg

Claims (2)

1. targeting antiglioma protein, its aminoacid sequence is SEQ ID No.5.

2. the application of targeting antiglioma protein as claimed in claim 1 in preparation treatment neuroglia tumor medicine.

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