CN102199586A - Structure and application of Enterovirus 71 3C protease - Google Patents
Structure and application of Enterovirus 71 3C protease Download PDFInfo
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Abstract
The invention discloses a structure and application of an Enterovirus 71 (EV71) 3C protease and belongs to the field of RNA virus protein. The invention also discloses an application of the 3C protease and a substrate binding groove thereof in drug design. The protein provided by the invention has a special substrate binding groove and new EV71 virus 3C protease drugs are designed according to the spatial structure of the substrate binding groove, thus the inhibitors aiming at the EV71 virus 3C protease, which are more specific and have better effect, can be obtained, potential drug candidates can be provided for the clinical treatment of hand, foot and mouth disease and the EV71 virus 3C protease has very high application value.
Description
Technical field
The present invention relates to a kind of structure and application thereof of 3C proteolytic enzyme, especially a kind of enterovirus 71 (EV71) 3C proteolytic enzyme with unique substrate engagement groove.
Background technology
Enterovirus 71 (EV71) is modal the one of pathogenic former of Hand-Food-Mouth Disease that cause.EV71 also can cause central nervous system infection, causes aseptic meningitis, acute slowness paralysis etc., even dead.Hand foot mouth disease and EV71 infect in recent years worldwide, and especially the eruption and prevalence of East Asia Region (China's Mainland, Taiwan, Japan etc.) has caused huge lives and properties and financial loss.Still there are not effective prevention and treatment means at present.
The special proteolytic enzyme of virus of EV71 coding is in 3C proteolytic enzyme.This proteolytic enzyme can be processed into functional protein protomer with the polyprotein precursor of encoding viral, thus virus duplicate with life cycle in bringing into play important effect.And in the mankind, lacking the homologous protein of this proteolytic enzyme, the compound that therefore can suppress this protease activity can effectively suppress duplicating of virus, and then becomes one of method of treatment hand foot mouth disease.At present, specificity is still deficient at the inhibitor molecules of EV71 virus 3C proteolytic enzyme.
Utilize the crystalline structure of high-resolution viral 3C proteolytic enzyme, searching is specificity bonded small molecules with it, perhaps carries out based on the transformation of structure to known compound and then obtains active higher compound, has become the effective means of drug screening.The crystalline structure that does not also have at present EV71 virus 3C proteolytic enzyme.
In the 3C proteolytic enzyme of having reported at present that crystalline structure is arranged, with similarity on the EV71 virus 3C proteolytic enzyme sequence the highest be the 3C proteolytic enzyme of rhinovirus (HRV) and Coxsackie B virus 3 (CVB3), sequence similarity degree to each other also has only about 50%.Correspondingly, belong to though these three kinds of viruses all belong to the Picornaviridae enterovirus, HRV belongs to human rhinovirus's kind, and CVB3 belongs to human enterovirus B kind, and EV71 belongs to human enterovirus A kind.Therefore be that basic mould is built between EV71-3C proteolytic enzyme structure and real structure and may be had bigger difference with the 3C proteolytic enzyme structure of HRV or CVB3 virus, also make the screening specificity be difficult to realize at the inhibitor molecules of EV71 virus 3C proteolytic enzyme.
Therefore obtaining has activity, highly purified EV71-3C proteolytic enzyme in a large number, and then resolves its crystalline structure, thereby relies on for follow-up inhibitor screening provides structure, is present urgent problem.
Summary of the invention
The technical problem to be solved in the present invention provides the structure of a kind of enterovirus 71 (EV71) 3C proteolytic enzyme.
Described 3C proteolytic enzyme is as (a) or (b) or protein (c):
(a) protein of forming by the amino acid shown in the SEQ ID NO.1
(b) aminoacid sequence in (a) through replacement, lack or add an amino acid or several amino acid and have the 3C protease activity by (a) deutero-protein
(c) with (a) in aminoacid sequence global similarity (identity) more than 85%, and at R39, K130, H24, Q19, six position upper amino acids such as E107 and H108 conservative fully by (a) deutero-protein.
Another technical problem that the present invention will solve provides a kind of described 3C proteolytic enzyme structure application in medicinal design, the especially application in SARS drug design.
Another technical problem that the present invention will solve provides a kind of substrate engagement groove of described proteolytic enzyme.
Described substrate engagement groove amino acid composition and spatial arrangement are shown in SEQ ID NO.2 and Fig. 5 C, and its S2 site is semi-enclosed groove shape structure, and the volume in S1 ' site significantly diminishes.
Another technical problem that the present invention will solve provides a kind of described substrate engagement groove application in medicinal design, the especially application in SARS drug design.
The present invention has obtained the crystalline structure of enterovirus 71 (EV71) 3C proteolytic enzyme first, on the basis of crystalline structure, has found the singularity of its substrate engagement groove, for being that the micromolecular inhibitor design of target spot provides architecture basics with this engagement groove.
Description of drawings
The molecular sieve collection of illustrative plates of Fig. 1 EV71 3C proteolytic enzyme
Black line is the 280nm absorption value, and red line is the 260nm absorption value, and both ratio is about 1.5, and showing does not have the nucleic acid class to pollute in the target protein, goes out the place, peak among the figure and respectively collects sample 1-10 number and further analyze with SDS-PAGE.
Fig. 2 EV71 3C proteolytic enzyme SDS-PAGE collection of illustrative plates
Collect sample 1-10 number in the corresponding molecular sieve collection of illustrative plates of each swimming lane,
MExpression low molecular weight protein (LMWP) Marker.
The external activity of Fig. 3 EV71 3C proteolytic enzyme detects
A: substrate, B: substrate+EV71-3C-C147A, C: substrate+EV71-3C.
The crystalline structure of Fig. 4 EV71-3C proteolytic enzyme.
The substrate engagement groove of Fig. 5 EV71-3C proteolytic enzyme
The composite structure B of A:AG7088+EV71-3C: substrate engagement groove C: substrate engagement groove space structure.
Fig. 6 AG7088 structural formula
A-E represents P1 ' respectively, P1, P2, P3, each group of P4.
Fig. 7 EV71-3C proteolytic enzyme and HRV-3C proteolytic enzyme S2 site are relatively
The S2 site of A:EV71-3C, the S2 site of B:HRV-3C.
Fig. 8 EV71-3C proteolytic enzyme S1 ' site.
The composite structure of Fig. 9 EV71-3C+AG7088 and with the comparison of HRV-3C+AG7088 structure.
Embodiment
The acquisition of embodiment 1 EV71 3C proteolytic enzyme
Our successful vivoexpression the 3C proteolytic enzyme of EV71 virus, and obtain high purity, activated zymoprotein by affinity chromatography and molecular exclusion chromatography.
EV71 3C proteolytic enzyme aminoacid sequence shown in SEQ ID NO.1, sequence shown in the SEQ ID NO.1 through replacement, lack or add an amino acid or several amino acid and have the 3C protease activity by SEQ ID NO.1 aminoacid sequence deutero-protein.
Proteinic sequence SEQ ID NO.1:
GPSLDFALSLLRRNIRQVQTDQGHFTMLGVRDRLAVLPRHSQPGKTIWIEHKLVNVLDAVELVDEQGVNLELTLITLDTNEKFRDITKFIPENISTASDA
1. plasmid construction:
Our will the encode dna fragmentation of 3C proteolytic enzyme is manually complete synthesis, be connected in the pET-21a carrier with Nde1 and Xho1 restriction enzyme site, and the encoding sequence of insertion 6 histidine-tagged (hexa-His-tag) behind the encode fragment of zymoprotein, be translation stop codon then, so then after inducible protein is expressed, can obtain one and have the histidine-tagged recombinant protein of C-end, be beneficial to follow-up protein purification.This dna fragmentation complete sequence is as follows:
catatgGGTCCGTCTCTGGACTTCGCGCTGTCTCTGCTGCGTCGTAACATCCGTCAGGTTCAGACCGACCAGGGGCATTTCACCATGTTGGGTGTTAGGG ATCGCTTAGCAGTCCTCCCACGCCACTCACAACCCGGCAAAACTATTTGGATTGAGCACAAACTCGTGAACGTCCTTGATGCAGTTGAGTTAGTGGATGA GCAAGGAGTCAACTTGGAATTAACCCTCATCACTCTTGACACCAACGAAAAGTTTAGGGACATCACCAAATTCATTCCAGAAAATATTAGCACTGCCAGT GATGCCACCCTAGTGATCAACACGGAGCACATGCCCTCAATGTTTGTCCCGGTGGGTGACGTTGTGCAGTATGGCTTCTTGAATCTCAGTGGTAAGCCCA CCCATCGCACCATGATGTACAACTTTCCTACTAAAGCAGGACAGTGTGGGGGAGTGGTGACATCTGTTGGGAAGGTTATCGGTATTCACATTGGTGGCAA
This proteic aminoacid sequence is as follows:
MGPSLDFALSLLRRNIRQVQTDQGHFTMLGVRDRLAVLPRHSQPGKTIWIEHKLVNVLDAVELVDEQGVNLELTLITLDTNEKFRDITKFIPENISTASDATLVINTEHMPSMFVPVGDVVQYGFLNLSGKPTHRTMMYNFPTKAGQCGGVVTSVGKVIGIHIGGNGRQGFCAGLKRSYFASEQHHHHHH
Initiator codon was introduced when first amino acid M was for translation in the above-mentioned aminoacid sequence, and the hexahistidine tag of C-end also is the extra amino acid of introducing, and is to facilitate for subsequent purification.Our follow-up external activity experimental results show that these extra amino acid do not influence this protease activities.
This fragment obtains by the mode of complete sequence gene synthetic (Takara company), and lowercase is represented Nde1 and Xho1 restriction enzyme site respectively.After being built into the pET-21a carrier, recombinant plasmid confirms that by direct dna sequencing the external source fragment of inserting is entirely true.
In addition, we have also made up should virus 3C protease activities center mutant recombinant plasmid, and the codon mutation that is about to 147 halfcystines of proteins encoded enzyme (Cys) is the codon of L-Ala (Ala).Use Phusion Site-mutagenesis Kit (NEB) test kit, utilize following primer Forward-primer:5 '-GCAGGACAGGCTGGGGGAGTG-3 ' and Reverse-primer:5 '-CACTCCCCCAGCCTGTCCTGC-3 ' to realize the mutant construction of recombinant plasmid.
2. protein expression and purifying:
At first with recombinant plasmid transformed E.coli BL21 (DE3) competent cell, resulting transformant clone picking is transferred in an amount of LB (adding ammonia benzyl mycin to the final concentration 100ug/ml) substratum, 37 ℃ are cultured to OD600 is 0.8, add IPTG to final concentration be 0.2mM, induce 5h for 37 ℃.Centrifugal collect thalline after, with an amount of PBS (137mM NaCl, 2.7mM KCl, 10mM Na
2HPO
4, 2mM KH
2PO
4, pH7.4) the outstanding bacterium of solution.Gained bacteria suspension ultrasonic method cracking thalline, residual and other particulate contamination of the centrifugal removal of low-temperature and high-speed bacterium.Then centrifugal back supernatant liquor is combined 2h with Ni-NTA resin (GE) in 4 ℃ of vibrations.Collect resin, and wash resin, to remove foreign protein with the PBS solution of 20 times of resin volumes.Handle and washing resin with buffer A (50mM HEPES, pH6.5,2.5M NaCl), to remove the RNA pollutent of non-specific combination to the target protein.At last target protein is eluted from resin with buffer B (50mM HEPES, pH6.5,150mM NaCl, 200mM imidazole), and elutriant is concentrated into 3ml with the evaporating pipe that 10K holds back (10K cutoff).With the protein solution after concentrating further with the molecular exclusion chromatography purifying, use AKTA-purifier (GE) and superdex200 Hiload 16/60 pillar (GE), use bufferC (50mM HEPES, pH6.5,150mM NaCl, 1mM EDTA, 5mM DTT), monitor the ultraviolet absorption value of 260nm and 280nm simultaneously, collect target protein, and identify purity of protein by SDS-PAGE.Typical proteic molecular sieve collection of illustrative plates of EV71-3C and SDS-PAGE analysis chart are respectively as shown in Figures 1 and 2.
EV71-3C-C147A albumen is the mutant protein that the proteic active Cys of EV71-3C sports Ala, and this proteic expression and purification technical scheme is with above-mentioned EV71-3C albumen.This mutain no longer has the nicking activity of substrate polypeptide.
3. external activity detects:
(sequence is: H with substrate polypeptide for we
2N-LWLDEEAMEQGVSDYI-COOH) be dissolved among the DMSO, being mixed with final concentration is the polypeptide mother liquor of 50mM.The buffer that vitro enzyme is tested conscientiously is 50mM HEPES, pH6.5,150mM NaCl, 1mM EDTA, 2mM DTT, 10% glycerol.It is 5uM that enzyme is tested the zymoprotein final concentration conscientiously, and the polypeptide final concentration is 250uM, 25 ℃ of reaction 2h.Use HPLC then, utilize the C18 reversed-phase column to detect the situation that substrate polypeptide is cut, the absorption value under the monitoring 215nm.The results are shown in Figure 3, after as seen polypeptide self, and polypeptide and EV71-3C-C147A are hatched altogether, all have only a substrate polypeptide peak, and after polypeptide and EV71-3C hatched altogether, substrate was cut into two product polypeptide, corresponding substrate polypeptide peak significantly diminishes, and two product polypeptide peak occurred.
The structure of embodiment 2 EV71 3C proteolytic enzyme
1. proteinic crystallization condition:
We adopt sessile drop method (hang-drop vapor-diffusion), have obtained the measured crystal of diffraction matter by condition optimizing, are used for crystalline protein Preparation liquid and are protein concn 10mg/ml.Condition is as follows:
The crystallization condition of EV71-3C-C147A: 0.1 M Tris-HCl (pH8.5), 20mM lithium sulfate and 25% (w/v) PEG 5000 MME; This proteic crystal growth also needs the KPVLRTA polypeptide of 8mM as additive.
The complex crystallization condition of EV71-3C and AG7088: 0.1 M sodium acetate (pH4.6), 0.2M ammonium sulfate and 25% (w/v) PEG 4000, the molar concentration rate of protein and compound is 1:3.
2. data gathering and structure elucidation:
Indoor X-ray diffraction instrument (in-house Rigaku MicroMax007 rotating-anode X-ray generator) is used in data gathering.Data processing is used HKL2000.Structure elucidation is a template with the 3C proteolytic enzyme structure of CVB3 virus, adopts molecular replacement technique, utilizes Coot to take mould by hand, utilizes Refmac5 (CCP4 suite) and Phenix.refine refine.
3. crystalline structure
EV71-3C-C147A forms (Fig. 4 A) by two structural domains (domain), and it (is aI-gI in domain1 by 7 beta-pleated sheets that the main body of each structural domain all is one; In domain2, comprise aII-cII and fII-iII) the beta sheet bucket that forms, these Folding buckets are surrounded by alpha-helix (α-helices A-D) and random loop structure that the core of main body further is exposed to the surface, constitute typical Chymotrypsin sample (chymotrypsin-like) jointly and fold.In domain2, also have a β-ribbon that forms by dII, eII pleated sheet (structure of β-ribbon), relevant with the identification of substrate polypeptide.The active centre of EV71-3C is by His40, and the catalysis triplet that Glu71 and Cys147 constituted is formed.In this structure, Cys147 is sported Ala, and three amino acid His40, Glu71 and Ala147 spatially still show as the arrangement mode of typical catalysis triplet, is positioned at the slit that domain1 and domain2 form.
The surface charge of EV71-3C-C147A distributes and also has notable feature.At the offside of active centre catalysis triplet, the strong positive charge of a successive step region (Fig. 4 B) is arranged.This zone is made up of KFRDI and two amino acid motifs of VGK (motif).Corresponding with two positive surface charge character that motif presented, the both is bringing into play important effect at this 3C proteolytic enzyme with combining of RNA in the activity, and then viral the duplicating of influence.Therefore can block 3C albumen ribozyme and estimate also can effectively suppress duplicating of virus in conjunction with active antibody or small-molecule drug, blank fully at the medicine of this target spot at present.
The substrate engagement groove of embodiment 3 EV71-3C proteolytic enzyme
The nomenclature in each site of 3C protease substrate engagement groove: we are reference point with the peptide bond that is cut on the substrate polypeptide usually, and amino acid left is called after P1 successively, P2, P3, P4 ..Pn, called after P1 ' successively to the right, P2 ', P3 ', P4 ' ... Pn'; The then corresponding called after site S1 of part that holds these polypeptide amino acids on the proteolytic enzyme, S2, S3, S4 ... Sn, S1 ', S2 ', S3 ', S4 ' ... Sn'.
By compd A G7088 and the proteic composite structure of EV71-3C, we are determined the substrate engagement groove (Fig. 5) of EV71-3C proteolytic enzyme, and Fig. 5 A toffee rod shape model is AG7088; AG7088 is incorporated in the channel form structure on proteolytic enzyme surface, and this groove is the substrate engagement groove (seeing Fig. 5 B) of EV71-3C proteolytic enzyme.By structural analysis, we have found the amino acid that constitutes this substrate engagement groove, and it consists of SEQ ID NO.2, comprises H24 altogether, F25, R39, H40, E71, Y122, F124, L125, N126, L127, S128, K130, T142, K143, A144, G145, Q146, C147, H161, I162, G163, G164, N165, G166, F170; Its spatial arrangement is shown in Fig. 5 C.
AG7088(Fig. 6) is the specificity micromolecular inhibitor of a rhinovirus (HRV) 3C proteolytic enzyme.This inhibitor can be described as a polypeptide analog of being made up of many groups, by the P1 position
β-Lactam nucleus, the phenyl ring that the fluorine of P2 replaces, P3 L-Ala, different imidazole group of P4 and P1 ' position
A, β-The unsaturated ester chain constitutes.A-E represents P1 ' respectively among Fig. 6, P1, P2, P3, each group of P4.According to above-mentioned nomenclature to each site of substrate engagement groove, accordingly in the structure of AG7088 that we resolved and EV71-3C mixture, the P1 ' that holds AG7088 on the proteolytic enzyme, P1, P2, P3, the part of five groups such as P4 then corresponds to S1 ' respectively, S1, S2, S3 and S4 site (seeing Fig. 5 B).
Further, we compare the composite structure of AG7088+EV71-3C and the composite structure of AG7088+HRV-3C, have found that EV71-3C is in this S2 and two special propertys that substrate binding site had of S1 ':
The S2 site of EV71-3C is by R39, H40, and L127, S128, amino acid such as K130 and I162 are formed, and show as a semi-enclosed groove shape structure; HRV-3C then is wide-open (Fig. 7) in this site.Fig. 7 A is the S2 site of EV71-3C, and Fig. 7 B is the S2 site of HRV-3C.Why both such difference can occur, are that K130 and R39 are long side chain amino acid because in EV71-3C, therefore its long side chain seals the S2 site, and in HRV-3C, corresponding amino acid is N130 and T39, its side chain is all very little, and it is open fully to make that its S2 site is able to.And other amino acid that constitute the S2 site in two proteolytic enzyme be guard or congeniality amino acid exchange.
By the P1 ' group of AG7088, we are determined the S1 ' substrate binding site (Fig. 8) of EV71-3C, and by G145, the main chain group of Q146 and C147 constitutes in a side in this site; Then formed at opposite side by the side-chain benzene ring of F25 and the backbone carbonyl oxygen of H24.Compare with the S1 ' site of HRV-3C, the volume in the S1 ' site of EV71-3C significantly diminishes.The dark EV71-3C that represent among the figure, light color is represented HRV-3C, and " P1 ' " identifies the P1 ' group of AG7088.Can see among Fig. 8 A that in EV71-3C and HRV-3C, each amino acid or the atomic radical that constitute both S1 ' sites all can extraordinaryly coincide together; But for 24 amino acids, the amino acid whose backbone carbonyl oxygen atom of its H24 has been moved upward 0.85 than the backbone carbonyl oxygen of the Methionin K24 among the HRV-3C in EV71-3C, and this has greatly dwindled the volume in the S1 ' site of EV71-3C.Further analysis revealed, the reason that causes the H24 amino acid backbone ketonic oxygen of EV71-3C to move up be since its side-chain radical can with Q19, E107 and H108 interact, and then its conformation is stablized; And in HRV-3C, the amino acid of corresponding position is T19, N107 and Q108, these amino acid all can't interact with K24 (Fig. 8 B).
To sum up, we have found the substrate engagement groove of EV71-3C.This engagement groove amino acid is formed and is comprised H24, F25, R39, H40, E71, Y122, F124, L125, N126, L127, S128, K130, T142, K143, A144, G145, Q146, C147, H161, I162, G163, G164, N165, G166, F170 altogether; Its spatial arrangement is shown in Fig. 5 C.We have further determined the S1 ' and the S1-S4 site of EV71-3C substrate engagement groove, prove the peculiar property of this substrate engagement groove in S2 and S1 ' site, its S2 binding site is a semi-enclosed groove shape structure, and its S1 ' binding site is wanted significantly the S1 ' binding site less than other 3C proteolytic enzyme (as HRV-3C).Further we have also determined to cause the amino acid of S2 and S1 ' binding site singularity, comprise R39, K130, H24, Q19, E107 and H108.
The substrate engagement groove of EV71-3C can exert an influence in the combination for inhibitor of the singularity in S2 and S1 ' site.We are example with the binding pattern of AG7088 and proteolytic enzyme.AG7088 is the irreversible micromolecular inhibitor at rhinovirus (HRV) 3C proteolytic enzyme design at first, and as far back as 1999, its composite structure with HRV-3C proteolytic enzyme just was in the news.We find the composite structure comparison back of the composite structure of EV71-3C+AG7088 and HRV-3C+AG7088, and this compound is with after EV71-3C combines, and its P2 and P1 ' group show the inadaptability of maximum, see Fig. 9:
The club model is AG7088 among Fig. 9, and light color is represented the conformation of this compound among the EV71-3C, the dark conformation of representing this compound among the HRV-3C.In EV71-3C, it is about 2.3 that the P2 group of AG7088 has moved up, and this is that therefore, the P2 group of this compound can not well be embedded in this S2 site because the S2 site of EV71-3C is closed at far-end.On the other hand, in EV71-3C, the P1 ' group of AG7088 is upturned significantly, this be because the S1 ' site volumes of EV71-3C significantly less than the S1 ' site of HRV-3C, therefore P1 ' the group of this compound is difficult to lie low in the S1 ' site of EV71-3C, can only take the conformation of perk.
By said structure analysis and experimental demonstration, we find that the substrate engagement groove of EV71-3C has its own particularity, and these features can influence the combination of small molecules AG7088 for EV71-3C proteolytic enzyme, and these find that for the micromolecular inhibitor design that with the substrate engagement groove is target spot be very crucial.And these constitutional featuress (S2 and S1 ' site) are determined by a few amino acid, comprise Q19, H24, R39, E107, H108 and K130.Therefore, compare with the sequence of EV71-3C, the replacement of different aminoacids takes place on one or several position, but as long as the global similarity (sequence identity) of protein sequence is more than 85%, and conservative fully at above-mentioned six locational amino acid, this 3C proteolytic enzyme will show and on all four substrate engagement groove of above-mentioned EV71-3C proteolytic enzyme and the special property on S2 and S1 ' site so.The concrete preparation method of the 3C proteolytic enzyme that this amino acid is undergone mutation, and activity identification is with above-mentioned technical scheme.
At present, design has all obtained success in rhinovirus (HRV) and sars coronavirus (SARS-CoV) at the micromolecular inhibitor of 3C proteolytic enzyme, all is that the substrate engagement groove with proteolytic enzyme is a target.In rhinovirus, AG7088 can high affine, the special 3C proteolytic enzyme that is attached to virus in, thereby suppress duplicating of virus and reach the purpose ［ 1 ］ of treatment flu (common cold).This compound is that basic design obtains with crystalline structure and the substrate engagement groove thereof of HRV-3C.
SARS-CoV and the atypical pneumonia outburst suddenly in 2003 that causes thereof, its high case fatality rate and high infectivity were once once causing world's fear.This virus 3C sample proteolytic enzyme of also encoding is being brought into play irreplaceable vital role in the duplicating of virus, therefore become the desirable target of inhibitor screening.By resolving the crystalline structure of SARS-CoV 3C proteolytic enzyme, people have also found a conservative substrate engagement groove, at this engagement groove, based on structure, pass through computer auxiliaring means, design has obtained a series of micromolecular inhibitor, all shows strong protease inhibiting activity on external and cell levels, and can effectively suppress virus replication ［ 2-6 ］.
As seen,, identify its substrate binding site, and then design obtains the avidity height, inhibitor molecules that specificity is good is practicable by resolving the crystalline structure of 3C proteolytic enzyme.Herein, we have reported the 3C proteolytic enzyme structure of EV71 virus for the first time, found the substrate land of this proteolytic enzyme, and pointed out the special property on its S2 and the S1 ' site, these constitutional featuress to be found to be with this engagement groove be that the micromolecular inhibitor design of target provides architecture basics.
To sum up, the EV71-3C structure that we report herein, and the substrate engagement groove that has special property at S2 and S1 ' substrate binding site are for architecture basics has been established in small-molecule drug design based on this; Simultaneously also for satisfy above-mentioned sequence signature (on the sequence with the EV71-3C sequence global similarity of report herein more than 85%, simultaneously at R39, K130, H24, Q19, conservative fully on six positions such as E107 and H108) the small-molecule drug design of 3C proteolytic enzyme established architecture basics.
Reference:
Though the present invention with preferred embodiment openly as above; but it is not in order to qualification the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, so protection scope of the present invention should be with being as the criterion that claims were defined.
Sequence table
?
＜110〉Institute of Microorganism, Academia Sinica
＜120〉a kind of enterovirus 71 type 3C proteolytic enzyme and its production and application
<160> 8
<170> PatentIn?version?3.3
<210> 1
<211> 183
<212> PRT
＜213〉enterovirns type 71 (Enterovirus 71)
<400> 1
Gly?Pro?Ser?Leu?Asp?Phe?Ala?Leu?Ser?Leu?Leu?Arg?Arg?Asn?Ile?Arg
1 5 10 15
Gln?Val?Gln?Thr?Asp?Gln?Gly?His?Phe?Thr?Met?Leu?Gly?Val?Arg?Asp
20 25 30
Arg?Leu?Ala?Val?Leu?Pro?Arg?His?Ser?Gln?Pro?Gly?Lys?Thr?Ile?Trp
35 40 45
Ile?Glu?His?Lys?Leu?Val?Asn?Val?Leu?Asp?Ala?Val?Glu?Leu?Val?Asp
50 55 60
Glu?Gln?Gly?Val?Asn?Leu?Glu?Leu?Thr?Leu?Ile?Thr?Leu?Asp?Thr?Asn
65 70 75 80
Glu?Lys?Phe?Arg?Asp?Ile?Thr?Lys?Phe?Ile?Pro?Glu?Asn?Ile?Ser?Thr
85 90 95
Ala?Ser?Asp?Ala?Thr?Leu?Val?Ile?Asn?Thr?Glu?His?Met?Pro?Ser?Met
100 105 110
Phe?Val?Pro?Val?Gly?Asp?Val?Val?Gln?Tyr?Gly?Phe?Leu?Asn?Leu?Ser
115 120 125
Gly?Lys?Pro?Thr?His?Arg?Thr?Met?Met?Tyr?Asn?Phe?Pro?Thr?Lys?Ala
130 135 140
Gly?Gln?Cys?Gly?Gly?Val?Val?Thr?Ser?Val?Gly?Lys?Val?Ile?Gly?Ile
145 150 155 160
His?Ile?Gly?Gly?Asn?Gly?Arg?Gln?Gly?Phe?Cys?Ala?Gly?Leu?Lys?Arg
165 170 175
Ser?Tyr?Phe?Ala?Ser?Glu?Gln
180
<210> 2
<211> 25
<212> PRT
＜213〉enterovirns type 71 (Enterovirus 71)
<400> 2
His?Phe?Arg?His?Glu?Tyr?Phe?Leu?Asn?Leu?Ser?Lys?Thr?Lys?Ala?Gly
1 5 10 15
Gln?Cys?His?Ile?Gly?Gly?Asn?Gly?Phe
20 25
<210> 3
<211> 582
<212> DNA
＜213〉artificial synthesized sequence
<400> 3
catatgggtc?cgtctctgga?cttcgcgctg?tctctgctgc?gtcgtaacat?ccgtcaggtt 60
cagaccgacc?aggggcattt?caccatgttg?ggtgttaggg?atcgcttagc?agtcctccca 120
cgccactcac?aacccggcaa?aactatttgg?attgagcaca?aactcgtgaa?cgtccttgat 180
gcagttgagt?tagtggatga?gcaaggagtc?aacttggaat?taaccctcat?cactcttgac 240
accaacgaaa?agtttaggga?catcaccaaa?ttcattccag?aaaatattag?cactgccagt 300
gatgccaccc?tagtgatcaa?cacggagcac?atgccctcaa?tgtttgtccc?ggtgggtgac 360
gttgtgcagt?atggcttctt?gaatctcagt?ggtaagccca?cccatcgcac?catgatgtac 420
aactttccta?ctaaagcagg?acagtgtggg?ggagtggtga?catctgttgg?gaaggttatc 480
ggtattcaca?ttggtggcaa?tggcagacag?ggtttttgcg?cgggtctcaa?aaggagttac 540
tttgctagtg?aacaacacca?ccaccaccac?cactgactcg?ag 582
<210> 4
<211> 190
<212> PRT
＜213〉enterovirns type 71 (Enterovirus 71)
<400> 4
Met?Gly?Pro?Ser?Leu?Asp?Phe?Ala?Leu?Ser?Leu?Leu?Arg?Arg?Asn?Ile
1 5 10 15
Arg?Gln?Val?Gln?Thr?Asp?Gln?Gly?His?Phe?Thr?Met?Leu?Gly?Val?Arg
20 25 30
Asp?Arg?Leu?Ala?Val?Leu?Pro?Arg?His?Ser?Gln?Pro?Gly?Lys?Thr?Ile
35 40 45
Trp?Ile?Glu?His?Lys?Leu?Val?Asn?Val?Leu?Asp?Ala?Val?Glu?Leu?Val
50 55 60
Asp?Glu?Gln?Gly?Val?Asn?Leu?Glu?Leu?Thr?Leu?Ile?Thr?Leu?Asp?Thr
65 70 75 80
Asn?Glu?Lys?Phe?Arg?Asp?Ile?Thr?Lys?Phe?Ile?Pro?Glu?Asn?Ile?Ser
85 90 95
Thr?Ala?Ser?Asp?Ala?Thr?Leu?Val?Ile?Asn?Thr?Glu?His?Met?Pro?Ser
100 105 110
Met?Phe?Val?Pro?Val?Gly?Asp?Val?Val?Gln?Tyr?Gly?Phe?Leu?Asn?Leu
115 120 125
Ser?Gly?Lys?Pro?Thr?His?Arg?Thr?Met?Met?Tyr?Asn?Phe?Pro?Thr?Lys
130 135 140
Ala?Gly?Gln?Cys?Gly?Gly?Val?Val?Thr?Ser?Val?Gly?Lys?Val?Ile?Gly
145 150 155 160
Ile?His?Ile?Gly?Gly?Asn?Gly?Arg?Gln?Gly?Phe?Cys?Ala?Gly?Leu?Lys
165 170 175
Arg?Ser?Tyr?Phe?Ala?Ser?Glu?Gln?His?His?His?His?His?His
180 185 190
<210> 5
<211> 21
<212> DNA
＜213〉artificial synthesized sequence
<400> 5
gcaggacagg?ctgggggagt?g 21
<210> 6
<211> 21
<212> DNA
＜213〉artificial synthesized sequence
<400> 6
cactccccca?gcctgtcctg?c 21
<210> 7
<211> 16
<212> PRT
＜213〉artificial synthesized sequence
<400> 7
Leu?Trp?Leu?Asp?Glu?Glu?Ala?Met?Glu?Gln?Gly?Val?Ser?Asp?Tyr?Ile
1 5 10 15
<210> 8
<211> 7
<212> PRT
＜213〉artificial synthesized sequence
<400> 8
Lys?Pro?Val?Leu?Arg?Thr?Ala
1 5
Claims (7)
1. the structure of enterovirus 71 type 3C proteolytic enzyme is characterized in that its amino acid is formed as (a) or (b) or protein (c):
(a) protein of forming by the amino acid shown in the SEQ ID NO.1;
(b) aminoacid sequence in (a) through replacement, lack or add an amino acid or several amino acid and have the 3C protease activity by (a) deutero-protein;
(c) with (a) in the aminoacid sequence overall similarity more than 85%, and at R39, K130, H24, Q19, six position upper amino acids such as E107 and H108 conservative fully by (a) deutero-protein.
2. the application of the structure of the described proteolytic enzyme of claim 1 in medicinal design.
3. the described application of claim 2 is characterized in that being applied to the molecular designing of medicine.
4. the substrate engagement groove of the described proteolytic enzyme of claim 1 comprises S2 and S1 ' binding site, it is characterized in that its aminoacid sequence is shown in SEQ ID NO.2.
5. according to the described substrate engagement groove of claim 4, its feature is semi-enclosed groove shape structure in the S2 site, and the volume in S1 ' site significantly diminishes.
6. the application of the described substrate engagement groove of claim 4 in medicinal design.
7. the described application of claim 6 is characterized in that being applied to the molecular designing of medicine.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851268A (en) * | 2012-09-25 | 2013-01-02 | 厦门大学 | Crystal structure of enterovirus 71 3C protease and application thereof |
| CN104419712A (en) * | 2013-08-20 | 2015-03-18 | 中国人民解放军军事医学科学院微生物流行病研究所 | Visible recombinant virus of human enterovirus 71 and application thereof |
| CN105067574A (en) * | 2015-06-18 | 2015-11-18 | 山东省医学科学院基础医学研究所 | EV71 3C protease action target point detection kit and EV71 3C protease action target point detection method |
| CN105255995A (en) * | 2015-11-23 | 2016-01-20 | 江苏康缘药业股份有限公司 | Hand-foot-and-mouth disease resistant drug activity detection method and kit |
| WO2016050199A1 (en) * | 2014-09-30 | 2016-04-07 | 三峡大学 | Medicament design pocket of ornithine decarboxylase and application of medicament design pocket |
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| CN101928728A (en) * | 2009-06-18 | 2010-12-29 | 胡育诚 | Method for preparing enterovirus virus-like particle and application thereof |
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| CN101928728A (en) * | 2009-06-18 | 2010-12-29 | 胡育诚 | Method for preparing enterovirus virus-like particle and application thereof |
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| 《GenBanK》 20090121 Zhang,X.等 登录号：ACL97381 全文 1 , * |
| 《GenBanK》 20101219 Lei，X.等人 登录号：ADT71698 全文 1 , * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851268A (en) * | 2012-09-25 | 2013-01-02 | 厦门大学 | Crystal structure of enterovirus 71 3C protease and application thereof |
| CN102851268B (en) * | 2012-09-25 | 2014-12-10 | 厦门大学 | Crystal structure of enterovirus 71 3C protease and application thereof |
| CN104419712A (en) * | 2013-08-20 | 2015-03-18 | 中国人民解放军军事医学科学院微生物流行病研究所 | Visible recombinant virus of human enterovirus 71 and application thereof |
| WO2016050199A1 (en) * | 2014-09-30 | 2016-04-07 | 三峡大学 | Medicament design pocket of ornithine decarboxylase and application of medicament design pocket |
| CN105067574A (en) * | 2015-06-18 | 2015-11-18 | 山东省医学科学院基础医学研究所 | EV71 3C protease action target point detection kit and EV71 3C protease action target point detection method |
| CN105067574B (en) * | 2015-06-18 | 2017-11-14 | 山东省医学科学院基础医学研究所 | EV71 HRV 3CP action target spot detection kits and detection method |
| CN105255995A (en) * | 2015-11-23 | 2016-01-20 | 江苏康缘药业股份有限公司 | Hand-foot-and-mouth disease resistant drug activity detection method and kit |
| CN105255995B (en) * | 2015-11-23 | 2019-02-05 | 江苏康缘药业股份有限公司 | A kind of anti-hand-foot-and-mouth-disease pharmaceutical activity detection method and its kit |
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| CN102199586B (en) | 2013-04-17 |
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