KR101997994B1 - High sensitive peptides for target receptor AXL, and the inhibiting agent of development of AXL in Zika virus entry - Google Patents

Abstract

The present invention relates to a peptide having a high specific and high binding capacity to a chicavirus receptor AXL protein, a chikavirus receptor AXL binding agent and an inhibitor using the same.
It is known that Zikagavirus is introduced into the body via a series of mechanisms after binding to AXL, known as a receptor, in the process of infection into host cells. The present invention is based on the fact that the receptor AXL, which plays an important role in this process, A peptide having selective binding ability, and a binding agent and an inhibitor for chikavirus receptor AXL using the same.
Which can be used for the diagnosis of zikavirus including inhibition of zikavirus into host cells.

Description

[0002] Peptides capable of high specificity and high selective binding to chicavirus receptor AXL, and AXL inhibitors using the same, and an inhibitory agent of development of AXL in Zika virus entry,

The present invention relates to peptides having high specificity and high selective binding ability to chicavirus receptor AXL and chicavirus AXL inhibitors using the same.

The peptide of the present invention not only can easily increase the target specificity to AXL, a zikavir receptor, through mutation generation and further modification, but also can obtain an inhibitor capable of preventing entry of zikavirus into a host cell.

The zika virus is transmitted through Egyptian forest mosquitoes with Zicca viruses and is associated with microcephaly in newborn infants who are infected with Zicca viruses. Symptoms are like mild dengue fever, are treated through rest, and can not be prevented by drugs or vaccines. Since there is no definitive study of the vaccine or prevention of Zikavirus, studies on the vaccine and prevention of Zikavirus have become very necessary (Hamel et al., J. Virol, 2015; Musso et al., Clin, Microbiolo. , 2014).

One in five patients infected with Zicca virus develops the information, which may be infected but not symptomatic, has severe redness with pain in the eye, and rash appears on the skin (Fonseca et al., Am. J. Trop. Med Hyg., 2014). There is a slight headache and pain on the joint side, and the infected patient has symptoms such as fever and rash, complex pain and eye congestion for a week. The risk of mortality in healthy adults is very low at 3%, but it is related to the microcephaly of newborn infants who are infected with Zicca virus.

Observation of the mechanism of neonatal microcephaly induced by Zicca virus has been shown to result in infection through the AXL protein receptor (OLAGNIER ET AL., Dna and Cell Biology, 2016).

Therefore, in order to develop an inhibitor of Zikavirus infection, the present inventors selected AXL-specific and selective binding-capable peptides using AXL protein receptors that have been proven to be infected with Zikavirus through various documents, Inhibitors of one zycavirus were found.

The present inventors recognized that zikavirus binds to AXL, a representative protein receptor, and is infected with a host cell through a series of mechanisms. By selecting peptides having high specificity and high binding ability to AXL receptors, And to elucidate the mechanism of Zikavirus AXL inhibition by further improvement.

As a result, biopanning using M13 bacteriophage, which is one of the molecular evolutionary methods, was performed to discover peptides composed of 12 amino acids of low molecular weight capable of selective binding to AXL, known as chikavirus receptor protein, and AXL inhibitor of chikavirus Development.

As a result, it is a main object of the present invention to find a peptide capable of high specific binding and highly selective binding to Zicavirus receptor AXL, and to provide a peptide containing essentially the amino acid constituting the peptide Peptide library and link it to a Zikavir receptor AXL inhibitor.

According to an aspect of the present invention,

The present invention provides a peptide having high specificity and high selective binding ability to AXL protein, which is a receptor protein known to bind when Zikagavirus is introduced into a host cell.

Herein, the peptide probe for the chikavirus receptor protein AXL is characterized in that it comprises a sequence comprising amino acid sequence listing 1 (AHNHTPIKQKYL).

Further, the amino acid constituting the peptide is characterized in that it is any one of peptide mimetics including D-form, L-form, peptoid monomer, and unnatural amino acid.

Also, the peptide is characterized by being a dimer, trimer, or multimer containing various functional groups at the N-terminus or C-terminus.

Also, the peptide N-terminal functional group includes free amine, acetylation, biotin or flurophore, and the C-terminal functional group includes free acid, amidation, biotin or flurophore.

The present invention also provides zicavirus receptor AXL binding agents and inhibitors comprising said peptides.

The present invention as described above will be further described.

The inventors have uncovered peptides through a fusion technique including a biopanning process using M13 bacteriophage to select a zikavir receptor AXL inhibitor, wherein the fused form technique has at least a systematic evolution of ligands exponential enrichment) is basically included.

The Zikavir receptor AXL-binding peptide of the present invention can be prepared by liquid-phase synthesis or solid-phase synthesis including basically attachment, protection, coupling, deprotection and cleavage processes.

The peptides prepared by the above method include at least 12 amino acids (D-type, L-type, peptide mimetic including peptoid, non-natural amino acid) composed of different amino acids.

In addition, the N-terminus or C-terminus of the peptide comprising at least 12 amino acids produced by the above method may contain various functional groups.

Also, functional groups of the peptides prepared by the above method may include free amine, acetylation, biotin, various flurophores, and C terminus may include free acid, amidation, biotin and various flurophores.

The present invention is directed to screening peptides with high specificity and high selectivity for Zicavirus receptor AXL and obtaining Zicavirus AXL inhibitors by using them to prevent zikavirus from entering the body, It is expected. In addition, it seems to be able to effectively prevent Zicca virus in the absence of vaccine or treatment for Zicca virus.

FIG. 1 shows the result of ELISA for comparing the binding ability to the target protein AXL using the bacteriophage containing the selected three peptides.
FIG. 2 shows the results of EIS performed to compare the binding ability to the target protein AXL using the bacteriophage containing the selected three peptides.
Fig. 3 shows the result of measuring the binding strength of AXL, a target protein, and bacteriophage (AXL R4 # 1) containing a selected peptide according to BSA concentration.
4 shows the result of calculation of the binding constant of the target protein AXL and the bacteriophage containing the selected peptide (AXL R4 # 1).
FIG. 5 shows the results of measurement of binding force against AXL and BSA proteins according to the concentration of the selected phage (AXL R4 # 1).
6 shows the result of measurement of binding force of bacteriophage (AXL R4 # 1) containing a selected peptide according to addition of Serum.

The present inventors selected a novel peptide having a high selective binding capacity to AXL through a biopanning process using M13 bacteriophage against a target protein using AXL protein receptor, which is known as a pathway of infection with Zikavirus.

The novel peptides discovered through the above method basically contain 12 amino acids, which may be dicentric, trinuclear, or multimeric, including various functional groups at the N-terminus or C-terminus.

The peptides selected in the present invention can be produced in the form of recombinant proteins in host cells. The host cell can be E. coli, and various tags of the target protein in the recombinant protein form can be bound to the N-terminus and the C-terminus.

And can be used to inhibit infection by blocking the infection of Zikavirus into host cells by studying or exploiting the interaction between the target protein AXL and the peptide.

Hereinafter, the present invention will be described in more detail by way of examples. It is to be understood, however, that these examples are for illustrative purposes only and that the scope of the present invention is not limited by these examples.

Example 1 : Target protein (AXL)

The target protein AXL used in the present invention was purchased from Abnova as a human recombinant protein and stored in a freezer at -80 ° C. until use.

Example 2: Detection of novel peptide library capable of highly specific and selective binding to AXL using M13 bacteriophage display

The bacteriophage random peptide library used in the present invention is Ph.D.-12 (New England BioLab), which is a gene terminator that produces pIII, a kind of coat protein in the genome of M13 bacteriophage Is composed of recombinant bacteriophage expressing hundreds of millions of different peptides obtained by artificially inserting gene sequences to express peptides of 12 random amino acid sequences and then infecting E. coli.

A total of four panning steps were performed. Polystyrene plates were used for the microwell used in this experiment.

First, the polystyrene plate was washed with TBS (Tris buffered saline) three times, 150 μL of AXL protein was added to the microwell, and the mixture was stirred at 150 rpm for 16 hours at 4 ° C. Then, 200 μL of 0.5% BSA was added to the microwell to block the portion where the AXL protein was not immobilized, and the mixture was stirred at 150 rpm for 1 hour at 4 ° C. The remaining AXL or BSA that had not been immobilized was washed with 0.1% Tween 20 in TBS (Tris buffered saline). Then, 100 μL of bacteriophage was added to the microwell and stirred at room temperature for 1 hour at 150 rpm. The unbound bacteriophage was washed with 0.1% Tween 20 in TBS (Tris buffered saline). Finally, the phage bound specifically to AXL was eluted in 100 μL of 0.2 M Glycin-HCl (pH 2.2) 15 [mu] L of Tris-HCl (pH 9.0) was added. Through the above procedure, three bacteriophage candidates binding to AXL were firstly selected and amino acid sequences were confirmed by DNA sequencing (Table 1).

 (Attached respectively to amino acid sequence listing 1 to 3) Selected candidates Amino acid sequence (N → C) AXL R4 # 1 AHNHTPIKQKYL AXL R4 # 4 ANTELALANRKH AXL R4 # 18 NWGVMPWIGATT

Example 3: Measurement of binding force of candidate bacteriophage containing novel peptide capable of highly specific and highly selective binding to AXL using enzyme immunoassay (ELISA)

Amplification was carried out in order to use the bacteriophage candidate group containing three peptides selected through a total of 4 times of biopanning for the ELISA experiment. Each phage stock was inoculated into LB medium with previously cultured E. coli ER2738 and cultured at 37 DEG C and 150 rpm for 4-5 hours. The supernatant was recovered by centrifugation, and then 20% PEG / 2.5M NaCl was added and left on ice for 1 hour. After centrifuging again, the precipitate was dissolved in TBS (Tris buffered saline) and subjected to phage titration to calculate the concentration of each phage (PFU / mL: Plaque forming units / mL).

ELISA was performed as follows to compare the binding strength of three phages and AXL, respectively. First, the polystyrene plate was washed with TBS (Tris buffered saline) three times, 150 μL of AXL protein was added to the microwell, and the mixture was stirred at 150 rpm for 16 hours at 4 ° C. Then, 200 μL of 0.5% BSA was added to the microwell to block the portion where the AXL protein was not immobilized, and the mixture was stirred at 150 rpm for 1 hour at 4 ° C. The remaining AXL or BSA that had not been immobilized was washed with 0.1% Tween 20 in TBS (Tris buffered saline). Then, 100 μL of bacteriophage was added to the microwell and stirred at room temperature for 1 hour at 150 rpm. Unbound bacteriophages were washed with 0.1% Tween 20 in TBS (Tris buffered saline) and OD values were measured and compared at 405 nm using M13-HRP antibody and ABTS. As a result, AXL R4 # 1 among the three phages was observed to exhibit a relatively high binding force (FIG. 1).

Example 4: Measurement of binding force of candidate bacteriophage containing novel peptide capable of highly specific and highly selective binding to AXL using an electrochemical analyzer (EIS)

Amplification was performed to use three bacteriophage candidates selected through 4 biopanning for EIS experiments. Each phage stock was inoculated into LB medium with previously cultured E. coli ER2738 and cultured at 37 DEG C and 150 rpm for 4-5 hours. The supernatant was recovered by centrifugation, and then 20% PEG / 2.5M NaCl was added and left on ice for 1 hour. After centrifuging again, the precipitate was dissolved in TBS (Tris buffered saline) and subjected to phage titration to calculate the concentration of each phage (PFU / mL: Plaque forming units / mL).

In order to compare the binding strength of three phages and AXL, EIS was performed as follows. First, Gold surface was treated with 50 mM cysteamine and incubated at 4 ° C for 16 hours. Unimmobilized cysteamine was washed with PBS (phosphate buffered saline), treated with 5% glutaraldehyde, and stirred at room temperature for 1 hour at 150 rpm. Unbound glutaraldehyde is washed with PBS (phosphate buffered saline) and the AXL protein is loaded onto cysteamine-glutaraldehyde-treated Gold to immobilize the AXL protein. The immobilized AXL protein was washed with PBS (phosphate buffered saline), and then 50 μL of the bacteriophage was placed on the Gold surface and stirred at 150 rpm for 1 hour at room temperature. The unbound bacteriophages were washed with PBS (phosphate buffered saline), and the binding ability of the three phages to AXL was examined through differences in impedance values at each step. As a result, it was observed that AXL R4 # 1 among the three phages exhibited a relatively high binding force (FIG. 2), in the same manner as in the ELISA Test.

As a result of comparing the binding force of AXL R4 # 1 according to the change of AXL and BSA concentration by the same EIS method, it was confirmed that the binding force of AXL R4 # 1 increases in proportion to the increase of AXL concentration. However, the AXL concentration was not significantly increased when the concentration was 20 μg / mL or more (FIG. 3). In addition, the K _d (binding constant) value was calculated from the results of AXL concentration experiments, and it was observed to have a binding constant of about 10 nM (FIG. 4).

As a result of comparing the binding strengths of AXL R4 # 1 phage to gold treated with the same concentration of AXL and BSA protein by the same EIS method as above, it was observed that the binding force was increased in proportion to the concentration (FIG. 5). This result shows that the selected phage AXL R4 # 1 specifically binds to AXL, and the 12 peptide sequence "AHNHTPIKQKYL" possessed by AXL R4 # 1 can be used for peptide-derived chikavirus receptor AXL inhibitors and the like Lt; / RTI >

As shown above, the peptide of the present invention can be highly specific and highly selectively bindable to AXL, which is a pathway of chikavirus infection, so that it can bind to a target biomarker protein precisely, thereby enabling inhibition of infection with Zikavirus.

Example 5: Influence of addition of serum on binding force of bacteriophage candidate group containing novel peptide capable of highly specific and highly selective binding to AXL

In order to investigate the change of high specific binding capacity of AXL in the condition of mixed serum sample (FBS: Fetal bovine serum) and AXL of bacteriophage AXL R4 # 1 selected as the final candidate group, .

First, Gold surface was treated with 50 mM cysteamine and incubated at 4 ° C for 16 hours. Unimmobilized cysteamine was washed with PBS (phosphate buffered saline), treated with 5% glutaraldehyde, and stirred at room temperature for 1 hour at 150 rpm. Unbound glutaraldehyde is washed with PBS (phosphate buffered saline) and the AXL protein is loaded onto cysteamine-glutaraldehyde-treated Gold to immobilize the AXL protein. The non-immobilized AXL protein was washed with PBS (phosphate buffered saline), then the bacteriophage AXL R4 # 1 was added to gold with various concentrations of FBS (serum 0.1%, 1%) and stirred at 150 rpm for 1 hour at room temperature The Washed with phosphate-buffered saline (PBS), and then the impedance values were compared.

As a result, as the concentration of serum added with the binding force of bacteriophage AXL R4 # 1, which contains a novel peptide capable of high specific and highly selective binding to AXL due to the addition of serum, is increased as compared with the case without addition of serum (Figure 6). This is because the unknown components in the serum act as factors that interfere with the binding of peptides through the interaction of AXL proteins. It is obvious to those of ordinary skill in the art that this problem can be achieved through redesign and functionalization of peptide probes through peptide engineering techniques.

<110> Industry-academic cooperation foundation Daegu Hanny University <120> High sensitive peptides for target receptor AXL, and the          inhibiting agent of development of AXL in Zika virus entry <130> ULA-1712 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 12 <212> PRT <213> bacteriophage <400> 1 Ala His Asn His Thr Pro Ile Lys Gln Lys Tyr Leu   1 5 10 <210> 2 <211> 12 <212> PRT <213> bacteriophage <400> 2 Ala Asn Thr Glu Leu Ala Leu Ala Asn Arg Lys His   1 5 10 <210> 3 <211> 12 <212> PRT <213> bacteriophage <400> 3 Asn Trp Gly Val Met Pro Trp Ile Gly Ala Thr Thr   1 5 10

Claims (7)

delete A peptide having a high specific and highly selective binding ability to a chicavirus receptor AXL protein, characterized in that it consists of the amino acid sequence of amino acid sequence listing 1 (AHNHTPIKQKYL). 3. The method of claim 2,
Wherein the peptide is any one of D-type, L-type, peptide mimetics including peptoid monomers, or unnatural amino acids. 3. The method of claim 2,
Wherein the peptide is a dimer, trimer or multimer comprising various functional groups at the N-terminus or C-terminus. 3. The method of claim 2,
The N-terminal functional group of the peptide includes free amine, acetylation, biotin or flurophore,
Wherein the C-terminal functional group comprises free acid, amidation, biotin or flurophore. A zikavir receptor AXL binding agent comprising a peptide according to any one of claims 2 to 5. A prophylactic agent for microcephaly comprising the peptide of any one of claims 2 to 5.

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