KR101607282B1 - Secretory APE1/Ref-1 Fusion Protein and Pharmaceutical Composition Containing the Protein - Google Patents

Abstract

The present invention relates to a novel redox regulating protein-1 capable of releasing the redox regulatory protein-1 from the cell so as to be able to effectively act on an extracellular inflammation-inducing receptor which is a new target of the redox regulatory protein- Protein-1 fusion protein (PPT-LS-3xFLAG-APE1 / Ref-1) of SEQ ID NO: 1, and more particularly to a pharmaceutical composition containing the fusion protein And a pharmaceutical composition containing the same as an active ingredient.

Description

[0001] The present invention relates to a free redox-regulating protein-1 and an anti-inflammatory pharmaceutical composition containing the same.

The present invention relates to a novel redox-regulating protein-1 capable of exerting redox-regulating protein-1 in the form of a free-form redox regulator Protein-1 fusion protein and a pharmaceutical composition containing the same as an active ingredient.

Redox regulatory protein-1 (APE1 / Ref-1) is the main protein in the cell that protects against oxidative stress. Typically, it plays an important role in regulating the repair or repair of damaged DNA and regulating the redox of transcription factors to maintain normal expression of genes in cells.

The redox regulatory protein-1 is composed of 318 amino acids. Nuclear localization signal (NLS) and nuclear export signal (NES) exist in the N-terminus region to regulate intracellular location . In normal conditions, the redox regulatory protein-1 is mainly located in the nucleus, but it is reported to be transferred to the cytoplasm by stimulation of intracellular damage such as oxidative stress. In accordance with environmental changes such as intracellular redox state, -1 is known to appear.

Overexpression of APE1 / Ref-1 in vascular endothelial cells inhibits the production of nitric oxide and inhibits the production of reactive oxygen species and suppresses the expression of vascular cell adhesion molecule-1 (VCAM-1) It is known that adhesion of mononuclear cells to vascular endothelial cells is also inhibited. With this in mind, the present inventors have disclosed a cell-permeable APE1 / Ref-1 fusion protein for treating an anti-inflammatory disease useful for the treatment of vascular diseases, in Korean Patent No. 10-0877151.

Among the various proteins in the cell, there are proteins functioning excreted out of the cell according to need. It is known that high mobility group protein-1 (HMG-1) protein is acetylated at the specific site of the nuclear locus signal region and excreted into the cell. It binds to the receptor that induces the inflammation that is exposed outside of the cell and produces inflammatory cytokine secretion Has been reported.

Recently, it has also been reported that APE1 / Ref-1 is released outside the cell under certain conditions. That is, it has been reported that acetylation of APE1 / Ref-1 by trichostatin A, a histone deacetylase inhibitor, induces a change in the position of APE1 / Ref-1, Biophys Res Commun., 2013 Jun 7; 435 (3): 403-7). It was also confirmed that APE1 / Ref-1 is released into the blood in an animal model of sepsis (Biochem Biophys Res Commun. 2013 Jun 14; 435 (4): 621-6). Japanese Patent Registration No. 10-1251222 reports the increase in the concentration of APE1 / Ref-1 in serum of bladder cancer and the measurement of APE1 / Ref-1 concentration from blood collected from a patient to diagnose bladder cancer . From these reports, APE1 / Ref-1 normally performs anti-inflammatory function through excessive ROS inhibition in cells, but it can be used for specific conditions in which APE1 / Ref-1 protein is released such as sepsis or cancer The extracellular Is expected to play a role as a new action target.

In order to examine the function of the redox-regulating protein-1 released from the cell as a new mechanism of action of the redox-regulating protein-1, the inventors of the present invention have developed plasmid and adenoviral-form free redox protein -1, and the produced free redox protein-1 was effective in inhibiting inflammation and acting as an anti-inflammatory therapeutic agent, thus completing the present invention.

Patent No. 10-0877151 Patent No. 10-1251222

Biochem Biophys Res Commun. 2013 Jun 7; 435 (3): 403-7 Biochem Biophys Res Commun. 2013 Jun 14; 435 (4): 621-6

The present invention confirms that the redox regulatory protein-1 released from the cell as a new mechanism of action has a remarkable effect on the degradation of the inflammatory indicator substance, and the new fusion protein capable of efficiently releasing the redox regulatory protein- And to provide the above objects.

It is still another object of the present invention to provide a gene encoding the fusion protein and an expression vector containing the gene for expression of the fusion protein.

It is still another object of the present invention to provide a composition for the treatment of inflammatory diseases containing the fusion protein as an active ingredient.

In order to accomplish the above object, the present invention relates to a free redox-regulated protein-1 fusion protein (PPT-LS-3xFLAG-APE1 / Ref-1) of SEQ. The present invention is characterized in that the Reduced Regulatory Protein-1 can be released to the outside of the cell so as to be able to effectively act on the extracellular inflammation-inducing receptor, which is a new target of the Reduced Regulatory Protein-1 having anti-inflammatory activity.

The present invention also relates to a gene encoding the fusion protein. It is particularly preferred that the gene has the sequence of SEQ ID NO: 2.

As a result of overexpression of the fusion protein according to the present invention by overexpressing adenovirus expression vector in macrophages of vascular endothelial cells and mouse, it was confirmed that the fusion protein was increased in proportion to the infection concentration of adenovirus in the cell culture medium. In addition, it was confirmed that the overexpression of the fusion protein effectively reduced the inflammatory markers VCAM-1, iNOS and COX-2 in each cell, and thus the anti-inflammatory effect of the fusion protein was excellent.

Accordingly, the present invention provides a pharmaceutical composition comprising the free redox protein-1 fusion protein as an active ingredient.

The fusion protein may be provided not only in the form of a separated and purified fusion protein, but also in the form of a gene encoding a fusion protein. The gene includes DNA, RNA or PNA, preferably a gene represented by the sequence of SEQ ID NO: 2.

The gene may be introduced into the target cell in an expression form by infection or transduction in the form contained in the expression vector by a variety of methods known in the art. In particular, the expression vector is preferably an adenovirus vector though it is not limited thereto. Adenoviral vectors can carry large quantities of DNA fragments and infect very high reverse transcription-free cells.

The pharmaceutical composition may be formulated in the form of a skin preparation, oral, intravenous, and intramuscular injection formulated with carriers usually accepted in the pharmaceutical field. It can also be used as an anti-inflammatory agent for inflammation of the skin, or as an oral preparation for capsules or tablets, for treating or preventing inflammatory diseases. The pharmaceutical composition containing the fusion protein as an active ingredient can be applied to all kinds of therapeutic methods such as the repair of active oxygen and genetic factors, the therapeutic agent for vascular inflammation, the sepsis, the inflammation caused by cancer, It can be used as a treatment for my genetic diseases and as a new disease treatment.

The pharmaceutical composition according to the present invention can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. Effective dose levels are well known to those skilled in the art, including the type of disease, severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, Can be determined according to the element. The composition of the present invention may be administered alone or in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art

As described above, according to the fusion protein of the present invention, since the redox regulatory protein-1 is efficiently released from the cell, the inflammatory disease can be effectively inhibited by controlling the redox reaction of the extracellular inflammation-inducing receptor, which is a new action target.

Figure 1 shows the cleavage map of the PPT-LS-3xFLAG-APE1 / Ref-1 expression vector.
FIG. 2 shows cleavage map of PPT-LS-3xFLAG-APE1 / Ref-1 adenovirus expression vector.
Fig. 3 shows the sequence of a free redox-regulatory protein-1 fusion protein and a gene encoding the same.
FIG. 4 is a photograph of the results of immunoprecipitation and Western blot analysis showing changes in the expression amount of VCAM-1 by overexpression of free redox-regulatory protein-1 fusion protein in inflammation-induced vascular endothelial cells.
FIG. 5 is a graph of quantitation of free redox-regulated protein-1 fusion protein released from cells in vascular endothelial cells.
FIG. 6 is a photograph of the results of immunoprecipitation and Western blot analysis showing changes in the expression levels of iNOS and COX-2 by overexpression of the free redox regulatory protein-1 fusion protein in inflammation-induced mouse macrophages.
FIG. 7 is a graph of quantitation of free redox regulatory protein-1 fusion protein released from cells in mouse macrophages.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the drawings and the embodiments are only illustrative of the contents and scope of the technical idea of the present invention, and the technical scope of the present invention is not limited or changed. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the technical idea of the present invention based on these examples.

Example

Example 1: Preparation and infection of a free redox-regulating protein-1 expression vector

For the overexpression of free redox-regulatory protein-1 containing extracellular excretory signals, three FLAG nucleotide sequences and extracellular emission signals, PPT-LS (Sigma-Aldrich, USA) PP-LS-3xFLAG-APE1 / Ref-1 was prepared. The pENTR-TOPO-CMV-LS-3xFLAG-APE1 / Ref-1 gene was recombined using the pENTR-TOPO-CMV vector (Invitrogen, USA) as a gateway vector containing the attL site for cloning into the adenovirus vector.

Specifically, to prepare PPT-LS-3xFLAG-APE1 / Ref-1 containing the extracellular emission signal PPT-LS, the full length APE1 / Ref-1 cDNA (NM_001641) (Biochem Biophys (SEQ ID NO: 3) in which ATG, which is an initiation codon, was added to the HindIII site together with HindIII site, and TGA which is a termination codon was added to the end with EcoRI And cloned into the p3xFLAG-CMV-8 vector using the reverse primer of SEQ ID NO: 4. The PCR reaction was carried out using high performance DNA polymerase according to the manufacturer's manual (Intron, Korea). PCR was carried out at 94 ° C, 58 ° C and 72 ° C for 25 cycles.

SEQ ID NO: 3: 5'-CCA AGC TTA TGC CGA AGC GTG GGA A-3 '

SEQ ID NO: 4: 5'-GGA ATT CCGTCA CAG TGC TAG GTA TA-3 '

The p3xFLAG-CMV-8 / APE1 / Ref-1 (FIG. 1) was constructed using the PPI-LS- The 3xFLAG-APE1 / Ref-1 gene was amplified and isolated. The amplified PPT-LS-3xFLAG-APE1 / Ref-1 gene was recombined into the pENTR-TOPO-CMV vector to produce the pENTR-TOPO-CMV / PPT-LS-3xFLAG-APE1 / Ref-1 vector.

SEQ ID NO: 5: 5'-AAG GAT CCG CCA CCA TGT CTG CAC TTC TGA TCC-3 '

SEQ ID NO: 6: 5'-AAC TCG AGT CAC AGT GCT AGG TAT AGG G-3 '

All recombinant DNA was sequenced to identify sequences.

TOPO-CMV vector (Invitrogen, USA) was reacted with two vectors, pENTR-TOPO-CMV / PPT-LS-3xFLAG-APE1 / Ref-1 and pAd / PL- PL-PPT-LS-3xFLAG-APE1 / Ref-1 by genetically recombining the recombinant PPT-LS-3xFLAG-APE1 / Ref-1 gene into the adenoviral vector pAd / Respectively.

10 μg of Ad / PL-PPT-LS-3xFLAG-APE1 / Ref-1 plasmid in the form of plasmid was ligated with PacI enzyme (New England Biolabs, UK) to construct adenovirus form of PPT- LS-3xFLAG-APE1 / To obtain cDNA. 1.6 μg of the obtained Ad / PL-PPT-LS-3xFLAG-APE1 / Ref-1 cDNA was transfected into HEK293A (LifeTechnologies, USA) cells using Lipofectamine ^™ 2000 (Invitrogen, USA) reagent according to the manufacturer's instructions. When more than 80% of the cytopathic effect is observed after transfection, the cells are collected and purified by a cesium chloride gradient centrifugation to purify the adenovirus expressing PPT-LS-3xFLAG-APE1 / Ref-1 and stored at -80 ° C Respectively.

1 and 2 are cleavage maps of PPT-LS-3xFLAG-APE1 / Ref-1 and an adenovirus expression vector, respectively, and FIG. 3 is a map showing the DNA sequence of PPT-LS-3xFLAG-APE1 / Ref- Sequence.

Example 2 Identification of Inflammation Inhibitory Function by Fusion Protein in Vascular Endothelial Cells

(VCAM-1) expression induced by Tumor necrosis factor-alpha (TNF-α) in cultured vascular endothelial cells (HUVECs).

HUVEC cultured cells (Lonza, USA) diluted 2 × 10 ⁵ / ml were dispensed in 2-well plates in 6-well plates and cultured in a 37 ° C water-fed incubator for 18 hours. Thereafter, the cells were infected with the adenovirus purified in Example 1 (50 or 100 MOI) and cultured at 37 DEG C for an additional 24 hours. TNF-α (15 ng / ml) was treated 24 hours after viral infection and cultured for 12 hours. The same experiment was carried out for the control group in which TNF-α was not treated or β-galactosidase in the form of adenovirus was infected.

In order to confirm the expression of VCAM-1 in the cells, the cells were treated with RIPA buffer to dissolve the cells, and VCAM-1 expression was confirmed by Western blotting after quantification of intracellular proteins using Bradford protein assay . In order to detect PPT-LS-3xFLAG-APE1 / Ref-1 protein liberated with the cell culture medium, 12 hours after treatment with TNF-α, the cell culture medium was transferred to a new tube and centrifuged at 3000 rpm for 3 minutes Respectively. After immunization with 1 μg of APE1 / Ref-1 antibody in the supernatant, immunoprecipitation was carried out with agarose A / G (Santa Cruz Biotechnology, USA), and the resultant product was analyzed by Western blot using PPT-LS-3xFLAG- APE1 / Ref-1. Further, in order to confirm the amount of PPT-LS-3xFLAG-APE1 / Ref-1 protein liberated from the cell, the cell culture broth was taken and cultured according to the method described in the registered patent 10-1251222 And quantified using an ELISA kit.

FIG. 4 shows the results of immunoprecipitation and Western blot analysis. The expression of VCAM-1, which is a protein expressed by inflammation, was induced by TNF-α. In the adenovirus-treated group, PPT-LS- The expression of 3xFLAG-APE1 / Ref-1 protein was markedly decreased by the increase of VCAM-1 protein.

In HUVEC, the amount of free redox-regulating protein-1 secreted by adenovirus into the cell was quantitated from the cell culture medium using the ELISA kit according to the method described in Patent No. 10-1251222, and the results are shown in FIG. 5 Respectively. According to FIG. 5, when adenovirus was treated at a concentration of 50 or 100 MOI, extracellular fusion proteins of about 150-300 ng / ml were released and the secreted amount was proportional to the infection concentration.

Example 3: Confirmation of inflammation inhibition by fusion proteins in macrophages

The effect of free redox regulatory protein-1 on LPS-induced inflammation in mouse macrophage Raw264.7 cells was investigated.

Raw 264.7 cells (ATCC, USA) diluted 2 × 10 ⁵ / ml were dispensed into 6 wells in 2 ml portions and cultured in a 37 ° C water-fed incubator for 18 hours. Thereafter, the cells were infected with the adenovirus purified in Example 1 (50 or 100 MOI) and cultured at 37 DEG C for an additional 24 hours. After 24 hours of virus infection, LPS (300 ng / ml) was treated and cultured for 12 hours. For the control, the same experiment was carried out for the group that did not treat LPS or infected with β-galactosidase in the form of adenovirus.

To examine the expression of iNOS and COX-2 in the cells, LPS-treated cells were lysed with RIPA buffer, and the intracellular proteins were quantitated by bradford protein assay. Western blotting was performed to detect iNOS and COX-2 . To detect PPT-LS-3xFLAG-APE1 / Ref-1 protein liberated by cell culture, 12 hours after the LPS treatment, the cell culture medium was transferred to a new tube and centrifuged at 3000 rpm for 3 minutes to remove suspension of the cell culture medium . Immunoprecipitation was carried out with 1 μg of APE1 / Ref-1 antibody, followed by immunoprecipitation with agarose A / G (Santa Cruz Biotechnology, USA), and the resultant PPT-LS-3xFLAG -APE1 / Ref-1. The amount of PPT-LS-3xFLAG-APE1 / Ref-1 protein liberated from the cell was quantified by the method described in Example 2.

Fig. 6 shows the results of immunoprecipitation and Western blot analysis. The expression of iNOS and COX-2, which are proteins expressed upon inflammation, was induced by LPS. In the treated group of adenovirus, PPT-LS- The expression of 3xFLAG-APE1 / Ref-1 protein was significantly decreased in iNOS and COX-2.

As shown in FIG. 7, the degree of secretion of free redox-regulating protein-1 by adenovirus in Raw264.7 cells was quantitated by ELISA from the cell culture medium. As a result, adenovirus was detected in 50 or 100 MOI , The amount of fusion protein of 100 ~ 200ng / ml was excreted and the secreted amount was proportional to the infection concentration.

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Secretory APE1 / Ref-1 Fusion Protein and Pharmaceutical          Composition Containing the Protein <130> P1214-605 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 356 <212> PRT <213> Artificial Sequence <220> PPT-LS-3xFLAG-APE1 / Ref-1 fused protein <400> 1 Met Ser Ala Leu Leu Ile Leu Ala Leu Val Gly Ala Ala Val Ala Asp   1 5 10 15 Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys              20 25 30 Asp Asp Asp Lys Leu Met Pro Lys Arg Gly Lys Lys Gly Ala Val          35 40 45 Ala Glu Asp Gly Asp Glu Leu Arg Thr Glu Pro Glu Ala Lys Lys Ser      50 55 60 Lys Thr Ala Ala Lys Lys Asn Asp Lys Glu Ala Ala Gly Glu Gly Pro  65 70 75 80 Ala Leu Tyr Glu Asp Pro Pro Asp Gln Lys Thr Ser Pro Ser Gly Lys                  85 90 95 Pro Ala Thr Leu Lys Ile Cys Ser Trp Asn Val Asp Gly Leu Arg Ala             100 105 110 Trp Ile Lys Lys Lys Gly Leu Asp Trp Val Lys Glu Glu Ala Pro Asp         115 120 125 Ile Leu Cys Leu Gln Glu Thr Lys Cys Ser Glu Asn Lys Leu Pro Ala     130 135 140 Glu Leu Gln Glu Leu Pro Gly Leu Ser His Gln Tyr Trp Ser Ala Pro 145 150 155 160 Ser Asp Lys Glu Gly Tyr Ser Gly Val Gly Leu Leu Ser Arg Gln Cys                 165 170 175 Pro Leu Lys Val Ser Tyr Gly Ile Gly Asp Glu Glu His Asp Gln Glu             180 185 190 Gly Arg Val Ile Val Ala Glu Phe Asp Ser Phe Val Leu Val Thr Ala         195 200 205 Tyr Val Pro Asn Ala Gly Arg Gly Leu Val Arg Leu Glu Tyr Arg Gln     210 215 220 Arg Trp Asp Glu Ala Phe Arg Lys Phe Leu Lys Gly Leu Ala Ser Arg 225 230 235 240 Lys Pro Leu Val Leu Cys Gly Asp Leu Asn Val Ala His Glu Glu Ile                 245 250 255 Asp Leu Arg Asn Pro Lys Gly Asn Lys Lys Asn Ala Gly Phe Thr Pro             260 265 270 Gln Glu Arg Gln Gly Phe Gly Glu Leu Leu Gln Ala Val Pro Leu Ala         275 280 285 Asp Ser Phe Arg His Leu Tyr Pro Asn Thr Pro Tyr Ala Tyr Thr Phe     290 295 300 Trp Thr Tyr Met Met Asn Ala Arg Ser Lys Asn Val Gly Trp Arg Leu 305 310 315 320 Asp Tyr Phe Leu Leu Ser His Ser Leu Leu Pro Ala Leu Cys Asp Ser                 325 330 335 Lys Ile Arg Ser Lys Ala Leu Gly Ser Asp His Cys Pro Ile Thr Leu             340 345 350 Tyr Leu Ala Leu         355 <210> 2 <211> 1071 <212> DNA <213> Artificial Sequence <220> <223> DNA encoding PPT-LS-3xFLAG-APE1 / Ref-1 fused protein <400> 2 atgtctgcac ttctgatcct agctcttgtt ggagctgcag ttgctgacta caaagaccat 60 gacggtgatt ataaagatca tgacatcgat tacaaggatg acgatgacaa gcttatgccg 120 aagcgtggga aaaagggagc ggtggcggaa gacggggatg agctcaggac agagccagag 180 gccaagaaga gtaagacggc cgcaaagaaa aatgacaaag aggcagcagg agagggccca 240 gccctgtatg aggacccccc agatcagaaa acctcaccca gtggcaaacc tgccacactc 300 aagatctgct cttggaatgt ggatgggctt cgagcctgga ttaagaagaa aggattagat 360 tgggtaaagg aagaagcccc agatatactg tgccttcaag agaccaaatg ttcagagaac 420 aaactaccag ctgaacttca ggagctgcct ggactctctc atcaatactg gtcagctcct 480 tcggacaagg aagggtacag tggcgtgggc ctgctttccc gccagtgccc actcaaagtt 540 tcttacggca taggcgatga ggagcatgat caggaaggcc gggtgattgt ggctgaattt 600 gactcgtttg tgctggtaac agcatatgta cctaatgcag gccgaggtct ggtacgactg 660 ggtaccggc agcgctggga tgaagccttt cgcaagttcc tgaagggcct ggcttcccga 720 aagccccttg tgctgtgtgg agacctcaat gtggcacatg aagaaattga ccttcgcaac 780 cccaagggga acaaaaagaa tgctggcttc acgccacaag agcgccaagg cttcggggaa 840 ttactgcagg ctgtgccact ggctgacagc tttaggcacc tctaccccaa cacaccctat 900 gcctacacct tttggactta tatgatgaat gctcgatcca agaatgttgg ttggcgcctt 960 gattactttt tgttgtccca ctctctgtta cctgcattgt gtgacagcaa gatccgttcc 1020 aaggccctcg gcagtgatca ctgtcctatc accctatacc tagcactgtg a 1071 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ccaagcttat gccgaagcgt gggaa 25 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ggaattccgt cacagtgcta ggtata 26 <210> 5 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 aaggatccgc caccatgtct gcacttctga tcc 33 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 aactcgagtc acagtgctag gtataggg 28

Claims (11)

Free redox regulatory protein-1 fusion protein of SEQ ID NO: 1.
A gene encoding the free redox-regulatory protein-1 fusion protein of claim 1.
3. The method of claim 2,
A gene having the sequence of SEQ ID NO: 2.
An expression vector comprising the gene of claim 2 which expresses the free-form redox regulatory protein-1 fusion protein of claim 1.
5. The method of claim 4,
Wherein the expression vector is an adenovirus expression vector.
A pharmaceutical composition for treating an inflammatory disease containing the free-form redox regulatory protein-1 fusion protein of claim 1 as an active ingredient.
The method according to claim 6,
Wherein the fusion protein is provided in the form of a gene encoding the fusion protein.
8. The method of claim 7,
Wherein the gene encoding the fusion protein is contained in an expression vector.
9. The method of claim 8,
Wherein said expression vector is an adenoviral vector.
10. The method according to any one of claims 6 to 9,
Wherein the inflammatory disease is sepsis or an inflammatory disease caused by cancer.
10. The method according to any one of claims 6 to 9,
Wherein the composition is formulated into a skin preparation, an oral preparation, an intravenous injection or an intramuscular injection.

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