CN102181418B - Structure of coxsackie virus A16-3C protease and application thereof - Google Patents
Structure of coxsackie virus A16-3C protease and application thereof Download PDFInfo
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Abstract
The invention discloses a structure of a coxsackie virus A16-3C protease and an application thereof, which belong to the field of RNA (Ribose Nucleic Acid) virus proteins. The invention also discloses an application of using a 3C protease and a substrate binding groove in drug design. The protein provided by the invention has a special substrate binding groove; a novel anti-virus CVA16 3C protease drug can be designed according to the space structure of the substrate binding groove so as to obtain a more specific inhibitor with better effect for the virus CVA16 3C protease; therefore, a potential alternative drug is provided for clinically treating hand-foot-and-mouth diseases and very high application values are obtained.
Description
Technical field
The present invention relates to a kind of structure and application thereof of HRV 3CP, especially a kind of coxsackie virus A 16 (CVA16) HRV 3CP with unique Binding Capacity groove.
Background technology
Coxsackie virus A 16 (CVA16) is modal the one of pathogenic former of Hand-Food-Mouth Disease that cause.In recent years worldwide, especially the eruption and prevalence of East Asia Region (China's Mainland, Taiwan, Japan etc.), caused huge lives and properties and financial loss to hand foot mouth disease.Still there is no at present effective prevention and treatment means.
The special proteolytic enzyme of virus of CVA16 coding is HRV 3CP.This proteolytic enzyme can be processed into functional protein protomer by the polyprotein precursor of encoding viral, thus virus copy with life cycle in play an important role.And lacking the homologous protein of this proteolytic enzyme in the mankind, the compound that therefore can suppress this protease activity can effectively suppress copying of virus, and then becomes one of method for the treatment of hand foot mouth disease.At present, specificity is still deficient for the inhibitor molecules of CVA16 virus HRV 3CP.
Utilize the crystalline structure of high-resolution viral HRV 3CP, searching is the small molecules of specific binding with it, or known compound is carried out take structure as basic transformation and then obtains active higher compound, has become the effective means of drug screening.The crystalline structure that also there is no at present CVA16 virus HRV 3CP.
In the HRV 3CP that crystalline structure is arranged of having reported at present, with similarity on CVA16 virus HRV 3CP sequence the highest be the HRV 3CP of rhinovirus (HRV) and Coxsackie B virus 3 (CVB3), sequence similarity degree to each other also only has 50% left and right.Correspondingly, although these three kinds of viruses all belong to the Picornaviridae enterovirus, belong to, HRV belongs to human rhinovirus's kind, and CVB3 belongs to mankind enterovirus B kind, and CVA16 is attributed to mankind enterovirus A kind.May there is larger difference between the CVA16-3C proteolytic enzyme structure that the HRV 3CP structure of HRV or CVB3 virus of therefore take is built as basic mould and real structure, also make the screening specificity be difficult to realize for the inhibitor molecules of CVA16 virus HRV 3CP.
Therefore obtaining activity, highly purified CVA16-3C proteolytic enzyme are arranged in a large number, and then resolve its crystalline structure, thereby rely on for follow-up inhibitor screening provides structure, is current urgent problem.
Summary of the invention
The technical problem to be solved in the present invention is to provide the structure of a kind of coxsackie virus A 16 (CVA16) HRV 3CP.
Described HRV 3CP is as (a) or (b) or protein (c):
(a) protein formed by the amino acid shown in SEQ ID NO.1,
(b) by (a) the derivative protein of the aminoacid sequence in (a) through replacing, lack or adding an amino acid or several amino acid and there is the HRV 3CP activity,
(c) with (a) in the aminoacid sequence overall similarity more than 85%, and at R39, K130, H24, Q19, the derivative protein by (a) that six position upper amino acids such as E107 and H108 are fully conservative.
Another technical problem that the present invention will solve is to provide a kind of described HRV 3CP structure application in medicinal design, the especially application in SARS drug design.
Another technical problem that the present invention will solve is to provide a kind of Binding Capacity groove of described proteolytic enzyme.
Described Binding Capacity groove amino acid composition and spatial arrangement are as shown in SEQ ID NO.2 and Fig. 5 D, 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 is to provide a kind of described Binding Capacity groove application in medicinal design, the especially application in SARS drug design.
The present invention has obtained the crystalline structure of coxsackie virus A 16 (CVA16) HRV 3CP first, on the basis of crystalline structure, find the singularity of its Binding Capacity groove, for take the micromolecular inhibitor design that this engagement groove is target spot, provide architecture basics.
The accompanying drawing explanation
The molecular sieve collection of illustrative plates of Fig. 1 CVA16 HRV 3CP
Black line is the 280nm absorption value, and red line is the 260nm absorption value, and both ratio is in 1.5 left and right, shows in target protein not have nucleic acid to pollute, and goes out the place, peak in figure and respectively collects sample and further analyze with SDS-PAGE for No. 4-16.
Fig. 2 CVA16 HRV 3CP SDS-PAGE collection of illustrative plates
No. 4-16 collection sample in the corresponding molecular sieve collection of illustrative plates of each swimming lane,
mmean low molecular weight protein (LMWP) Marker.
The external activity of Fig. 3 CVA16 HRV 3CP detects
A: substrate, B: substrate+CVA16-3C-C147A, C: substrate+CVA16-3C.
The crystalline structure of Fig. 4 CVA16-3C proteolytic enzyme.
The Binding Capacity groove of Fig. 5 CVA16-3C proteolytic enzyme.
A: substrate polypeptide (sequence: the composite structure C of the composite structure B:AG7088+CVA16-3C of FAGLRQAVTQ)+CVA16-3C: Binding Capacity groove D: Binding Capacity slot space structure.
Fig. 6 AG7088 structural formula
A-E represents respectively P1 ', P1, P2, P3, each group of P4.
Fig. 7 CVA16-3C proteolytic enzyme and HRV-3C proteolytic enzyme S2 site are relatively
The S2 site of A:CVA16-3C, the S2 site of B:HRV-3C.
Fig. 8 CVA16-3C proteolytic enzyme S1 ' site
The composite structure of Fig. 9 CVA16-3C+AG7088 and with the comparison of HRV-3C+AG7088 structure.
Embodiment
the acquisition of embodiment 1 CVA16 HRV 3CP
Our successful vivoexpression the HRV 3CP of CVA16 virus, and obtain high purity, activated zymoprotein by affinity chromatography and molecular exclusion chromatography.
CVA16 HRV 3CP aminoacid sequence as shown in SEQ ID NO.1, sequence shown in SEQ ID NO.1 through replacement, lack or add an amino acid or several amino acid and have the HRV 3CP activity by the derivative protein of SEQ ID NO.1 aminoacid sequence.
The sequence SEQ ID NO.1 of protein:
GPSLDFALSLLRRNIRQVQTDQGHFTMLGVRDRLAILPRHSQPGKTIWVEHKLINVLDAVELVDEQGVNLELTLVTLDTNEKFRDVTKFIPETITGASDATLIINTEHMPSMFVPVGDVVQYGFLNLSGKPTHRTMMYNFPTKAGQCGGVVTSVGKIIGIHIGGNGRQGFCAGLKRGYFASEQ
1. plasmid construction:
Our will the encode DNA fragmentation of HRV 3CP is manually complete synthesis, with Nde1 and Xho1 restriction enzyme site, be connected in the pET-21a carrier, and the encoding sequence of insertion 6 histidine-tagged (hexa-His-tag) after the encode fragment of zymoprotein, then be translation stop codon, so after inducible protein is expressed, can obtain one and hold histidine-tagged recombinant protein with C-, be beneficial to follow-up protein purification.This DNA fragmentation complete sequence is as follows:
catatgGGTCCGTCTCTGGACTTCGCGCTGTCTCTGCTGCGTCGTAACATCCGTCAGGTTCAGACCGACCAAGGACATTTTACTATGTTAGGAGTGCGAGATCGCCTAGCTATTTTGCCACGCCACTCGCAACCAGGAAAAACTATTTGGGTGGAGCACAAGTTAATCAATGTATTGGATGCTGTTGAATTGGTGGATGAGCAAGGTGTAAACTTGGAACTCACACTAGTAACCTTGGACACCAACGAAAAGTTTAGGGATGTCACCAAGTTTATTCCAGAGACGATCACCGGGGCAAGCGACGCAACCTTGATCATCAACACTGAGCACATGCCCTCAATGTTCGTTCCAGTGGGCGATGTTGTACAATATGGGTTTCTAAACCTCAGCGGTAAGCCCACACACCGAACCATGATGTACAATTTCCCCACAAAGGCAGGACAGTGTGGAGGTGTGGTCACCTCAGTCGGCAAGATTATTGGAATTCATATCGGTGGGAATGGTCGCCAAGGTTTCTGTGCTGGACTGAAGAGAGGTTACTTTGCCAGTGAACAGCACCACCACCACCACCACTGActcgag
The aminoacid sequence of this albumen is as follows:
MGPSLDFALSLLRRNIRQVQTDQGHFTMLGVRDRLAILPRHSQPGKTIWVEHKLINVLDAVELVDEQGVNLELTLVTLDTNEKFRDVTKFIPETITGASDATLIINTEHMPSMFVPVGDVVQYGFLNLSGKPTHRTMMYNFPTKAGQCGGVVTSVGKIIGIHIGGNGRQGFCAGLKRGYFASEQHHHHHH
When in above-mentioned aminoacid sequence, first amino acid M is for translation, initiator codon is introduced, and the hexahistidine tag of C-end is also 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 affect the activity of this proteolytic enzyme.
This fragment obtains by the mode of complete sequence gene synthetic (Takara company), and lowercase means respectively Nde1 and Xho1 restriction enzyme site.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 built the active centre mutant recombinant plasmid of this virus HRV 3CP, the codon that the codon mutation that is about to 147 halfcystines of proteins encoded enzyme (Cys) is 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 structure of mutant recombinant plasmid.
2. protein expression and purifying:
At first by recombinant plasmid transformed E.coli BL21 (DE3) competent cell, resulting transformant clone picking is transferred in appropriate LB (adding ammonia benzyl mycin to 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 appropriate 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 rear supernatant liquor and Ni-NTA resin (GE) are combined to 2h in 4 ℃ of vibrations.Collect resin, and wash resin with the PBS solution of 20 times of resin volumes, to remove foreign protein.Process and wash resin with buffer A (50mM HEPES, pH6.5,2.5M NaCl), to remove non-specific binding to the RNA pollutent on target protein.Finally target protein is eluted from resin with buffer B (50mM HEPES, pH6.5,150mM NaCl, 200mM imidazole), and with the evaporating pipe that 10K holds back (10K cutoff), elutriant is concentrated into to 3ml.By the protein solution after concentrated 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.The molecular sieve collection of illustrative plates of typical CVA16-3C albumen and SDS-PAGE analysis chart are respectively as shown in Figures 1 and 2.
The active Cys that CVA16-3C-C147A albumen is CVA16-3C albumen sports the mutant protein of Ala, and the expression and purification technical scheme of this albumen is with above-mentioned CVA16-3C albumen.This mutain no longer has the nicking activity of substrate polypeptide.
3. external activity detects:
By substrate polypeptide, (sequence is: H for we
2n-FAGLRQAVTQGFPTEL-COOH) be dissolved in DMSO the polypeptide mother liquor that to be mixed with final concentration be 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.Then use HPLC, utilize the cut situation of C18 reversed-phase column detection substrate polypeptide, the absorption value under monitoring 215nm.The results are shown in Figure 3, after polypeptide self, and polypeptide as seen and CVA16-3C-C147A are hatched altogether, all only have a substrate polypeptide peak, and, after polypeptide and CVA16-3C hatch altogether, substrate is cut into two product polypeptide, corresponding substrate polypeptide peak disappears, and two product polypeptide peak occurred.
the structure of embodiment 2 CVA16 HRV 3CPs
1. the crystallization condition of protein:
We adopt sessile drop method (hang-drop vapor-diffusion), by condition optimizing, have obtained the measured crystal of diffraction matter,
The albumen that is used for crystallization prepares liquid and is protein concn 10mg/ml.Condition is as follows:
CVA16-3C crystallization condition: 0.1M BIS-TRIS pH 5.5,0.1M ammonium acetate and 17% w/v PEG 10000
The complex crystallization condition of CVA16-3C-C147A and FAGLRQAVTQ polypeptide: 0.1 M HEPES pH7.5,0.2M lithium sulfate and 25% w/v PEG 3350, the molar concentration rate of protein and polypeptide is 1:5.
The complex crystallization condition of CVA16-3C and AG7088: 0.1 M sodium acetate pH4.6,0.1M magnesium chloride 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.It is template that structure elucidation be take the HRV 3CP structure of CVB3 virus, adopts molecular replacement technique, utilizes that Coot is manual takes mould, utilizes Refmac5 (CCP4 suite) and Phenix.refine refine.
3. crystalline structure
CVA16-3C forms (Fig. 4 A) by two structural domains (domain), and it (is aI-gI by 7 beta-pleated sheets that the main body of each structural domain is one in domain1; Comprise aII-cII and fII-iII at domain2) the beta sheet bucket that forms, the core that these Folding buckets are main body further is exposed to surperficial a-spiral (a-helices A-D) and random loop structure is surrounded, and jointly forms typical Chymotrypsin sample (chymotrypsin-like) folding.In domain2, also have β-ribbon (β-ribbon) structure formed by dII, eII pleated sheet, relevant with the identification of substrate polypeptide.The active centre of CVA16-3C is by His40, and Glu71 and Cys147 form, and three amino acid are positioned at the slit of domain1 and domain2 formation, and spatially the next-door neighbour, form typical catalysis triplet.
The surface charge substep of CVA16-3C also has notable feature.At the offside of active centre catalysis triplet, a continuous strong positive charge step region (Fig. 4 B) is arranged.This zone is comprised of KFRDV and two amino acid motifs of VGK (motif).The positive surface charge character presented with two motifs is corresponding, both this HRV 3CP and RNA in conjunction with activity in play an important role, and then the copying of impact virus.Therefore can block HRV 3CP RNA and estimate also can effectively suppress copying of virus in conjunction with active antibody or small-molecule drug,, fully blank for the medicine of this target spot at present.
the Binding Capacity groove of embodiment 3 CVA16-3C proteolytic enzyme
The nomenclature in each site of HRV 3CP Binding Capacity groove: usually we to take cut peptide bond on substrate polypeptide be reference point, amino acid left is called after P1 successively, P2, P3, P4 ..Pn, called after P1 ' successively to the right, P2 ', P3 ', P4 ' ... Pn'; The part of holding these polypeptide amino acids on proteolytic enzyme is corresponding called after site S1, S2, and S3, S4 ... Sn, S1 ', S2 ', S3 ', S4 ' ... Sn'.
Composite structure by substrate polypeptide and CVA16-3C, and the composite structure of compd A G7088 and CVA16-3C, we are determined Binding Capacity groove (Fig. 5) conservative in proteolytic enzyme, in Fig. 5 A, rod shape model is substrate polypeptide, in Fig. 5 B, rod shape model is compd A G7088, both are incorporated in the same channel form structure on proteolytic enzyme surface, and this groove is Binding Capacity groove (seeing Fig. 5 C).By structural analysis, we have found the amino acid that forms this Binding Capacity groove, and it consists of SEQ ID NO.2, comprises altogether 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; Its spatial arrangement as shown in Figure 5 D.
According to the above naming rule to each site of Binding Capacity groove, in substrate polypeptide, each amino acid, corresponding to P1-P8 amino acid, holds accordingly these amino acid whose proteolytic enzyme parts and corresponds to the S1-S8 site.By structural analysis, we find that the P7-Leu of substrate polypeptide and P8-Gly are exposed in solvent fully, with any amino acid of zymoprotein molecule, do not interact, therefore, by the composite structure of substrate polypeptide and CVA16-3C, we can determine the S1-S6 site of CVA16-3C protease substrate engagement groove.
And AG7088(Fig. 6) be the specificity micromolecular inhibitor of a rhinovirus (HRV) HRV 3CP.This inhibitor can be described as a polypeptide analog be comprised of many groups, by the P1 position
β-lactam nucleus, the phenyl ring that the fluorine of P2 replaces, P3 L-Ala, the different imidazole group of P4 and P1 ' position
a, β-the unsaturated ester chain forms.In Fig. 6, A-E represents respectively P1 ', P1, P2, P3, each group of P4., according to the above-mentioned nomenclature to each site of Binding Capacity groove, in the AG7088 resolved at us accordingly and the structure of CVA16-3C mixture, hold the P1 ' of AG7088 on proteolytic enzyme, P1, P2, P3, the part of five groups such as P4 corresponds to respectively S1 ', S1, S2, S3 and S4 site.By structure alignment, show, the S1-S4 substrate binding site of the CVA16-3C limited by AG7088, with the S1-S4 substrate binding site of the above-mentioned CVA16-3C proteolytic enzyme limited by substrate polypeptide be on all four.To sum up, we have determined S1 ' and S1-S6 site (the seeing Fig. 5 C) of CVA16-3C protease substrate engagement groove.
Further, we compare the composite structure of the composite structure of AG7088+CVA16-3C and AG7088+HRV-3C, have found the special property that CVA16-3C has at this S2 and S1 ' two substrate binding sites:
The S2 site of CVA16-3C is by R39, H40, and L127, S128, the amino acid such as K130 and I162 form, and show as a semi-enclosed groove shape structure; HRV-3C is wide-open (Fig. 7) in this site.The S2 site that Fig. 7 A is CVA16-3C, the S2 site that Fig. 7 B is HRV-3C.Why both there will be such difference, are that K130 and R39 are long side chain amino acid because in CVA16-3C, 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, makes its S2 site be opened fully.And other amino acid that form the S2 site in two proteolytic enzyme, be guard or be the amino acid whose exchange of congeniality.
By the P1 ' group of AG7088, we are determined the S1 ' substrate binding site (Fig. 8) of CVA16-3C, and this site is in a side by G145, and the main chain group of Q146 and C147 forms; At opposite side, by the side-chain benzene ring of F25 and the backbone carbonyl oxygen of H24, formed.With the S1 ' site of HRV-3C, compare, the volume in the S1 ' site of CVA16-3C significantly diminishes.In figure, dark color represents CVA16-3C, and light color represents HRV-3C, the P1 ' group of " P1 ' " sign AG7088.In Fig. 8 A, can see, in CVA16-3C and HRV-3C, each amino acid or the atomic radical that form both S1 ' sites all can extraordinaryly coincide together; But for 24 amino acids, in CVA16-3C, the amino acid whose backbone carbonyl oxygen atom of its H24 has been moved upward 0.9 than the backbone carbonyl oxygen of the Methionin K24 in HRV-3C, and this has greatly dwindled the volume in the S1 ' site of CVA16-3C.Further the analysis showed that, cause reason that the H24 amino acid backbone ketonic oxygen of CVA16-3C moves up be due to 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 Binding Capacity groove of CVA16-3C.This engagement groove amino acid forms and comprises altogether 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; Its spatial arrangement as shown in Figure 5 D.We have further determined S1 ' and the S1-S6 site of CVA16-3C Binding Capacity groove, prove the peculiar property of this Binding Capacity groove in S2 and S1 ' site, its S2 binding site is a semi-enclosed groove shape structure, and its S1 ' binding site will significantly be less than the S1 ' binding site of other HRV 3CP (as HRV-3C).Further we have also determined the amino acid that causes S2 and S1 ' binding site singularity, comprise R39, K130, H24, Q19, E107 and H108.
The Binding Capacity groove of CVA16-3C the singularity in S2 and S1 ' site for inhibitor in conjunction with exerting an influence.We take the binding pattern of AG7088 and proteolytic enzyme is example.AG7088 is the irreversible micromolecular inhibitor for rhinovirus (HRV) HRV 3CP design at first, and as far back as 1999, its composite structure with HRV-3C proteolytic enzyme just was in the news.We compare rear discovery by the composite structure of the composite structure of CVA16-3C+AG7088 and HRV-3C+AG7088, and this compound is after CVA16-3C is combined, and its P2 and P1 ' group show maximum inadaptability, see Fig. 9:
In Fig. 9, the club model is AG7088, and light color represents the conformation of this compound in CVA16-3C, and dark color represents the conformation of this compound in HRV-3C.In CVA16-3C, the P2 group of AG7088 has moved up approximately 1.7, and this is that S2 site due to CVA16-3C is closed at far-end, and therefore, the P2 group of this compound can not well be embedded in this S2 site.On the other hand, in CVA16-3C, P1 ' the group of AG7088 is upturned significantly, this is because the S1 ' site volumes of CVA16-3C significantly is 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 CVA16-3C, can only take the conformation of perk.
By said structure analysis and experimental demonstration, we find that the Binding Capacity groove of CVA16-3C has the singularity of self, and these features can affect the combination of small molecules AG7088 for CVA16-3C proteolytic enzyme, these find that for take the micromolecular inhibitor design that the Binding Capacity groove is target spot be very crucial.And these constitutional featuress (S2 and S1 ' site) are to be determined by a few amino acid, comprise Q19, H24, R39, E107, H108 and K130.Therefore, with the sequence of CVA16-3C, compare, the replacement of different aminoacids occurs on one or several position, but as long as the global similarity (sequence identity) of protein sequence is more than 85%, and fully conservative at above-mentioned six locational amino acid, this HRV 3CP will show and the on all four Binding Capacity groove of above-mentioned CVA16-3C proteolytic enzyme and the special property on S2 and S1 ' site so.The concrete preparation method of the HRV 3CP that this amino acid is undergone mutation, and activity identification is with above-mentioned technical scheme.
At present, for the micromolecular inhibitor of HRV 3CP, design has all obtained success in rhinovirus (HRV) and sars coronavirus (SARS-CoV), is all that to take the Binding Capacity groove of proteolytic enzyme be target.In rhinovirus, AG7088 can high affine, special being attached in viral HRV 3CP, thereby suppress copying of virus and reach the purpose ［ 1 ］ for the treatment of flu (common cold).This compound is exactly that to take the crystalline structure of HRV-3C and Binding Capacity groove thereof be basis, and design obtains.
SARS-CoV and the atypical pneumonia that causes thereof were at flared in 2003, and 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 copying of virus, and therefore becomes the desirable target of inhibitor screening.By resolving the crystalline structure of SARS-CoV HRV 3CP, people have also found a conservative Binding Capacity groove, for this engagement groove, take structure as basis, pass through computer auxiliaring means, design has obtained a series of micromolecular inhibitor, all shows strong protease inhibiting activity in vitro with on cell levels, and can effectively suppress virus replication ［ 2-6 ］.
Visible, by resolving the crystalline structure of HRV 3CP, identify its substrate binding site, and then design to obtain the inhibitor molecules that avidity is high, specificity is good be practicable.Herein, we have reported the HRV 3CP structure of CVA16 virus for the first time, found the Binding Capacity district of this proteolytic enzyme, and pointed out the special property on its S2 and S1 ' site, being found to be of these constitutional featuress be take the micromolecular inhibitor design that this engagement groove is target provides architecture basics.
To sum up, the CVA16-3C structure that we report herein, and the Binding Capacity groove that has special property at S2 and S1 ' substrate binding site, for architecture basics has been established in small-molecule drug design based on this; Simultaneously also for meet above-mentioned sequence signature (on sequence with the CVA16-3C sequence global similarity of report herein more than 85%, simultaneously at R39, K130, H24, Q19, fully conservative on six positions such as E107 and H108) the small-molecule drug design of HRV 3CP established architecture basics.
reference:
Although the present invention with preferred embodiment openly as above; but it is not in order to limit 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 mechanism of coxsackie virus A 16-3 C protease and application thereof
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 183
<212> PRT
<213 > Coxsackie virus 16 types (Coxsachievirus 16)
<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 Ile Leu Pro Arg His Ser Gln Pro Gly Lys Thr Ile Trp
35 40 45
Val Glu His Lys Leu Ile Asn Val Leu Asp Ala Val Glu Leu Val Asp
50 55 60
Glu Gln Gly Val Asn Leu Glu Leu Thr Leu Val Thr Leu Asp Thr Asn
65 70 75 80
Glu Lys Phe Arg Asp Val Thr Lys Phe Ile Pro Glu Thr Ile Thr Gly
85 90 95
Ala Ser Asp Ala Thr Leu Ile 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 Ile 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
Gly Tyr Phe Ala Ser Glu Gln
180
<210> 2
<211> 25
<212> PRT
<213 > Coxsackie virus 16 types (Coxsachievirus 16)
<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 aaggacattt tactatgtta ggagtgcgag atcgcctagc tattttgcca 120
cgccactcgc aaccaggaaa aactatttgg gtggagcaca agttaatcaa tgtattggat 180
gctgttgaat tggtggatga gcaaggtgta aacttggaac tcacactagt aaccttggac 240
accaacgaaa agtttaggga tgtcaccaag tttattccag agacgatcac cggggcaagc 300
gacgcaacct tgatcatcaa cactgagcac atgccctcaa tgttcgttcc agtgggcgat 360
gttgtacaat atgggtttct aaacctcagc ggtaagccca cacaccgaac catgatgtac 420
aatttcccca caaaggcagg acagtgtgga ggtgtggtca cctcagtcgg caagattatt 480
ggaattcata tcggtgggaa tggtcgccaa ggtttctgtg ctggactgaa gagaggttac 540
tttgccagtg aacagcacca ccaccaccac cactgactcg ag 582
<210> 4
<211> 190
<212> PRT
<213 > Coxsackie virus 16 types (Coxsachievirus 16)
<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 Ile Leu Pro Arg His Ser Gln Pro Gly Lys Thr Ile
35 40 45
Trp Val Glu His Lys Leu Ile Asn Val Leu Asp Ala Val Glu Leu Val
50 55 60
Asp Glu Gln Gly Val Asn Leu Glu Leu Thr Leu Val Thr Leu Asp Thr
65 70 75 80
Asn Glu Lys Phe Arg Asp Val Thr Lys Phe Ile Pro Glu Thr Ile Thr
85 90 95
Gly Ala Ser Asp Ala Thr Leu Ile 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 Ile 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 Gly 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
Phe Ala Gly Leu Arg Gln Ala Val Thr Gln Gly Phe Pro Thr Glu Leu
1 5 10 15
<210> 8
<211> 10
<212> PRT
<213 > artificial synthesized sequence
<400> 8
Phe Ala Gly Leu Arg Gln Ala Val Thr Gln
1 5 10
Claims (1)
1. a coxsackie virus A 16 HRV 3CP, is characterized in that the amino acid of described proteolytic enzyme is as shown in SEQ ID NO.1.
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| CN 201110066257 CN102181418B (en) | 2011-03-18 | 2011-03-18 | Structure of coxsackie virus A16-3C protease and application thereof |
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| WO2016050199A1 (en) * | 2014-09-30 | 2016-04-07 | 三峡大学 | Medicament design pocket of ornithine decarboxylase and application of medicament design pocket |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101322715A (en) * | 2008-07-25 | 2008-12-17 | 中国科学院昆明植物研究所 | Anti-coxsackie medicament composition and medicine use thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101322715A (en) * | 2008-07-25 | 2008-12-17 | 中国科学院昆明植物研究所 | Anti-coxsackie medicament composition and medicine use thereof |
Non-Patent Citations (2)
| Title |
|---|
| Kuo CJ et al.Design,Synthesis,and evaluation of 3C protease inhibitors as anti-enterovirus 71 agents.《Bioorganic & Medical Chemistry》.2008,第16卷(第15期),7388-7398. * |
| Yong Zhang et al.Molecular Evidence of Persistent Epidemic and Evolution of Subgenotype B1 Coxsackievirus A16-Associated Hand,Foot, and Mouth Disease in China.《JOURNAL OF CLINICAL MICROBIOLOGY》.2010,第48卷(第2期),619-622. * |
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