Eureka AIR delivers breakthrough ideas for toughest innovation challenges, trusted by R&D personnel around the world.

Crystal structure of enterovirus 71 type 2A protease and application of crystal structure in drug design

A technology of enterovirus and protease, applied in the field of crystal structure of protease, can solve the problems of complex transmission route, no enterovirus 71 drug, strong infectivity, etc., and achieve far-reaching effects

Inactive Publication Date: 2013-05-08
XIAMEN UNIV +1
View PDF2 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Hand, foot and mouth disease has the characteristics of high epidemic intensity, strong infectivity, and complicated transmission routes. So far, there is no specific anti-enterovirus 71 drug

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Crystal structure of enterovirus 71 type 2A protease and application of crystal structure in drug design
  • Crystal structure of enterovirus 71 type 2A protease and application of crystal structure in drug design
  • Crystal structure of enterovirus 71 type 2A protease and application of crystal structure in drug design

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Expression and purification of enterovirus 71 type 2A protease

[0038]Based on the E2004104-TW-CDC virus strain of enterovirus 71 (EV71), the DNA sequence encoding EV712A protease was artificially synthesized. A single point mutation was performed on the DNA sequence by PCR experiment, and the cysteine ​​(Cys) at the 110th position was mutated to alanine (Ala), as shown in SEQ ID NO.2. Afterwards, the mutated DNA sequence was inserted into the pGEX-4T-1 vector to construct a recombinant plasmid capable of expressing 2A protease with a nitrogen-terminal glutathione S-transferase (GST) tag.

[0039] Transform the recombinant plasmid into Escherichia coli pLysS competent cells, pick the obtained transformant clones and transfer them to appropriate amount of LB medium (adding chloramphenicol and ampicillin to the final concentration of 50 μg / ml respectively), culture at 37°C When the absorbance at 600 nm is 0.6, add isopropyl-β-D-thiogalactopyranoside (IPTG) ind...

Embodiment 2

[0040] Example 2 Crystallization and structural analysis of enterovirus 71 type 2A protease

[0041] 2.1 Crystallization conditions

[0042] Crystallization was carried out by the hanging drop method. The volume ratio of the protein solution in the crystallization droplet to the crystallization reagent was 1:1, and the protein concentration in the protein solution was 10mg / ml. Crystals grew after three days. The crystallization condition was 0.1M hydroxyethylpiperazine B Sulfuric acid (HEPES) pH 7.5, 20% isopropanol, 10% polyethylene glycol 4000 (PEG4000), crystals were stored at 16°C.

[0043] In order to obtain the complex formed by the 2A protease and the small molecular compound, the protease and the small molecular compound were mixed in an ice bath at a molar ratio of 1:5 for 2 hours. The co-crystallization conditions of protease and small molecule compound are the same as when no compound is added.

[0044] 2.2 Data collection and structure analysis

[0045] Data col...

Embodiment 3

[0049] The substrate binding groove of embodiment 3 enterovirus 71 type 2A protease

[0050] 2A The nomenclature of the protease active site: take the cleaved peptide bond on the substrate polypeptide as the reference point, and the amino acids to the left are named P1, P2, P3, ... P N , named P1', P2', P3', ...P in sequence to the right N ’; The part of the protease that accommodates these polypeptide amino acids is correspondingly named as the site S1, S2, S3...S N and S1', S2', S3'...S N '.

[0051] The substrate-binding groove of enterovirus type 2A protease is long enough to accommodate P1-P5 amino acid residues. If the catalytic triad is placed on the right, the lower end of the substrate-binding groove is formed by a carbon-terminal domain with an active residue, Cys110, located below the edge of the narrow region of the substrate-binding groove . The nitrogen-terminal domain with active residues His21 and Asp39 is at the upper right of the substrate-binding groove...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a crystal structure of enterovirus 71 type 2A protease and application of the crystal structure in drug design, relating to a crystal structure of protease. The invention provides the crystal structure of enterovirus 71 type 2A protease, a substrate-binding groove, application of the substrate-binding groove in drug molecule design, a compound capable of interacting with the substrate-binding groove and a compound structure formed through interaction between the compound and the substrate-binding groove. The crystal structure is composed of a nitrogen-terminal structural domain and a carbon-terminal structural domain, and the substrate-binding groove is provided with a catalytic active center consisting of a catalytic triad composed of 21th-locus histidine, 39 th-locus asparaginic acid and 110 th-locus cysteine; and the substrate-binding groove comprises application of S4, S3, S2, S1, S1', S2' and S3' loci in drug molecule design. Four small molecule compounds are capable of interacting with the substrate-binding groove of the enterovirus 71type 2A protease.

Description

technical field [0001] The invention relates to a crystal structure of protease, in particular to a crystal structure of enterovirus 71 type 2A protease and its application in drug design. Background technique [0002] Enterovirus 71 belongs to the Picornaviridae family and is one of the most common pathogens of HFMD. In addition, it can also cause a variety of nervous system-related diseases such as sporopharyngitis, aseptic meningitis, encephalitis, and polio-like paralytic diseases, which may be accompanied by severe central nervous system complications or neurological disorders. Inflammatory pulmonary edema. [0003] Hand, foot and mouth disease has the characteristics of high epidemic intensity, strong infectivity, and complicated transmission routes. So far, there is no specific anti-enterovirus 71 drug. [0004] The gene of enterovirus 71 encodes a virus-specific 2A protease, which plays an important role in the replication and life cycle of the virus. It can proce...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50G06F19/16C07F9/40C07F9/572C07D401/14C12R1/93
Inventor 林天伟黎健蔡期湑李宁柳立婕
Owner XIAMEN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com