KR101947340B1 - A method for extending half-life of FSH alpha - Google Patents

Abstract

The present invention relates to a method for increasing the half-life of follicle stimulating hormone a, including replacing one or more lysine residues present in the amino acid sequence of follicle stimulating hormone a, or to a follicle stimulating hormone a having an increased half-life, The residue-substituted follicle stimulating hormone a remains in the human body for a long time and has excellent therapeutic effect.

Description

A method for increasing the half-life of FSH [alpha]

The present invention relates to a method for increasing the half-life of a protein or (poly) peptide by substituting at least one amino acid residue of the protein or (poly) peptide. Further, the present invention relates to a protein or (poly) peptide having an increased half-life produced by such a method.

Intracellular proteolysis occurs through two pathways: lysosomes and proteasomes. The lysosomal pathway, which degrades 10-20% of the protein, lacks substrate specificity and elaborate temporal regulation. In other words, it is a process of decomposing mostly extracellular or membrane proteins as the cell surface proteins incorporated into cells by endocytosis are degraded in lysosomes. However, in order for proteins to be selectively degraded in eukaryotic cells, a process in which ubiquitin is bound to a target protein by a ubiquitin-binding enzyme to form a polyubiquitin chain, which is recognized and degraded by proteasome, that is, ubiquitin- The ubiquitin-proteasome pathway (UPP). More than 80% to 90% of eukaryotic proteins are degraded through this process, and the ubiquitin-proteasome pathway regulates protein degradation and protein homeostasis by regulating protein degradation in eukaryotic cells.

Ubiquitin is a highly conserved 76 amino acid protein that is present in almost all eukaryotic cells, of which 6, 11, 27, 29, 33, 48 and 63 amino acid residues are lysine (Lysine, Lys, K) 48 and 63 play a major role in the formation of the poly ubiquitin chain. The ubiquitination of ubiquitin involves a series of enzymes (E1, E2, E3), which are degraded by the ATP-dependent protease complex 26S proteasome. The ubiquitin-proteasome pathway involves two separate, sequential processes, the first of which is the process of labeling multiple ubiquitin molecules with a covalent bond to the substrate, and the second is that the protein labeled by ubiquitin is a 26S pro It is a process that is decomposed by a teasome complex. The binding of ubiquitin and substrate takes place via an isopeptide bond between the lysine residue of the substrate molecule and the glycine at the C-terminal of ubiquitin, and the ubiquitin-activating enzyme E1, the ubiquitin-binding enzyme E2 and the ubiquitin ligase E3 The formation of thiol esters between ubiquitin and enzymes. E1 (ubiquitin-activating enzyme) activates ubiquitin by ATP-dependent reaction. E2 (ubiquitin-conjugating enzyme) takes ubiquitin activated from E1 to cysteine residue in ubiquitin-conjugated domain and transfers it to E3 ligase or directly to substrate protein. The E3 enzyme also catalyzes stable isopeptide binding between the lysine residue of the substrate protein and the glycine residue of ubiquitin. Another ubiquitin may be linked to the C-terminal lysine residue of the ubiquitin bound to the substrate protein. When this process is repeated and several ubiquitin molecules are ligated to the substrate protein in a branched form to form a polyubiquitin chain, 26S proteasome and is selectively degraded.

On the other hand, various kinds of proteins and (poly) peptides having a therapeutic effect in vivo are known. Such a protein or (poly) peptide having a therapeutic effect in vivo can be used as a therapeutic agent for the treatment of diseases such as growth hormone releasing hormone (GHRH), growth hormone releasing peptide, interferons, interferon-α or interferon-β, interferon receptors, colony stimulating factors (CSFs), glucagon-like peptides, interleukins, interleukin receptors, Human interleukin binding proteins, cytokine binding proteins, G-protein-coupled receptors, human growth hormone (hGH), and the like. Macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A (prot ein A), allergy inhibitor, cell necrosis glycoproteins, G-protein-coupled receptor, immunotoxin, lymphotoxin, tumor necrosis Tumor necrosis factor, tumor suppressors, metastasis growth factor, alpha-1 antitrypsin, albumin, alpha-lactalbumin, Apolipoprotein-E, erythropoietin, highly glycosylated erythropoietin, angiopoietins, hemoglobin, thrombin, and the like. Thrombin receptor activating peptide, thrombomodulin, factor VII, factor VIIa, factor VIII, factor IX, factor IX, Factor XIII, A plasminogen activating factor, a urokinase, a streptokinase, a hirudin, a protein C, a C-reactive protein, a renin inhibitor renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epithelial growth factor, , Epidermal growth factor, angiostatin, angiotensin, bone growth factor, bone stimulating protein, calcitonin, insulin, art Atriopeptin, a cartilage inducing factor, a fibrin-binding peptide, elcatonin, a connective tissue activating factor, A tissue factor pathway inhibitor, a follicle stimulating hormone, a luteinizing hormone, a luteinizing hormone releasing hormone, a nerve growth factor, a parathyroid hormone parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, glucagon, cholecytokinin, cholesterol, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone, autotaxin, lactoferrin lactoferrin, myostatin, receptors, receptor antagonists, cell surface antigens, ns, virus derived vaccine antigens, monoclonal antibodies, polyclonal antibodies, and antibody fragments.

Follicle-stimulating hormone α (FSHα) is a glycoprotein polypeptide in the form of a 35.5 kDa polypeptide. Two polypeptides, alpha and beta, are heterodimers It is one of gonadotrophin that promotes and maintains female follicle growth and male spermatogenesis. It is synthesized and secreted in the gonadal cells of the anterior pituitary gland, and regulates the reproductive process, development, growth, pubertal maturation (Annu Rev Biochem., 50: 465-495, 1981; Proc Natl Acad Sci USA., 109 (31): 12491-12496, 2012). In general, follicle stimulating hormone (IVF) is used to induce ovarian hyperstimulation during in vitro fertilization (IVF). In the case of solid tumors, FSH receptors have been reported to be highly expressed in tumor vascular endothelium and are involved in the regeneration of neovascularization, and thus can be used as anticancer therapy when FSH and receptor antagonists are developed (N Engl J Med., 363 ): 1621-1630, 2010).

The present invention aims to provide a method for increasing the half-life of a protein.

The present invention also aims to provide a protein having one or more lysine residues substituted in the amino acid sequence, wherein the protein has an increased half-life.

It is also an object of the present invention to provide a pharmaceutical composition comprising a protein having an increased half-life.

In order to achieve the above object, the present invention provides a method for increasing the half-life of a protein, including replacing one or more lysine residues present in the amino acid sequence of the protein.

In the present invention, lysine residues of proteins may be substituted with conservative amino acids. In the present invention, "conservative amino acid substitution" means that an amino acid residue is substituted by another amino acid residue having a similar side chain, e.g., a charge or hydrophobic chemical character. In general, conservative amino acid substitutions do not substantially change the functional properties of the protein. Examples of amino acid groups having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chain: serine and threonine; 3) Amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) Basic side chains: lysine, arginine and histidine; 6) Acid side chains: aspartate and glutamate 7) Sulfur-containing side chains: include cysteine and methionine.

In the present invention, the lysine residue of the protein may be substituted with arginine or histidine containing a basic side chain, preferably arginine residue.

According to the present invention, a protein in which at least one lysine residue present in the amino acid sequence of a protein is substituted with arginine may have an increased half-life and may remain in the body for a long time.

Figure 1 shows the structure of an FSH-a expression vector.
Figure 2 shows the FSH-alpha gene size and PCR results.
Figure 3 shows the expression of FSH-a protein through plasmid in HEK-293T cells.
Figure 4 shows the degradation pathway of FSH-a through ubiquitination assay.
Figure 5 shows the degree of ubiquitination of FSH-alpha in which lysine residues were substituted with arginine as compared to the wild type.
Figure 6 shows the half-life change of FSH-alpha after treatment with the protein synthesis inhibitor cycloheximide (CHX).
Figure 7 shows the results for effects such as JAK-STAT, PI3K-AKT and MAPK / ERK signal induction.

In one embodiment of the invention, the protein is FSH-a. At least one of the 75th, 99th and 115th lysine residues from the N-terminus in the FSH-alpha amino acid sequence represented by SEQ ID NO: 1 is substituted with an arginine residue. Accordingly, there is provided a pharmaceutical composition for the treatment of infertility and / or solid cancer, which has increased half-life, FSH for inducing ovarian hyperstimulation containing the same, and /

In the present invention, site-directed mutagenesis was used to replace the lysine residue present in the amino acid sequence of the protein with the arginine (R) residue. In this method, a primer is prepared using a DNA sequence that induces a specific mutation, and then a PCR is carried out under specific conditions to prepare a plasmid DNA in which a specific amino acid residue is substituted.

In the present invention, the target protein was transfected into the cell line by immunoprecipitation analysis and precipitated to confirm the degree of ubiquitination. As a result of treatment with the MG132 (proteasome inhibitor) reagent, the degree of ubiquitination was increased, - proteasome degradation pathway.

In the present invention, the pharmaceutical composition may be delivered into the body by various routes including oral, transcutaneous, subcutaneous, intravenous or intramuscular administration and may be administered as a syringe formulation . In addition, the pharmaceutical composition of the present invention can be formulated according to methods well known to those skilled in the art, such as rapid release, delayed release or slow release after administration according to the method. The formulations may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, Aerosol, soft and hard gelatin capsules, sterile injectable solutions, sterile packaged powders, and the like. Suitable carriers, excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, mannitol, xylitol, erythritol, maltitol, carbohydrates, Gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearate, and mineral oil. . In addition, the formulations can additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives, and the like. have.

In the present invention, the singular forms include plural forms unless expressly stated otherwise. Furthermore, terms such as "comprising" in the present invention are interpreted to have a similar meaning to "including". In the present invention, " physiologically active (poly) peptide or protein " means a (poly) peptide or protein which exhibits biological activity useful when administered to a mammal including a human.

Hereinafter, the present invention will be described in more detail based on examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: FSH -α protein Ubiquitination  Analysis and confirmation of increased half-life and confirmation of intracellular signaling

1. As an expression vector Cloning  And protein expression confirmation

(1) expression vector Cloning

The FSH-α DNA amplification product by PCR and pcDNA3-myc (5.6 kb) were ligated with restriction enzymes BamHI and XhoI and then ligated (FIG. 1, FSH-α amino acid sequence: ), And the result was confirmed by agarose gel electrophoresis after restriction enzyme digestion (Fig. 2). Also, the underlined and bolded parts on the nucleotide sequence of Figure 1 are part of the primer set used for confirmation of the cloned site through polymerase chain reaction, and the results are shown in the agarose gel electrophoresis (Fig. 2). The polymerase chain reaction conditions were as follows; The initial denaturation was carried out at 94 ° C for 3 minutes, followed by repeated cycles of 25 cycles of denaturation at 94 ° C for 30 seconds, annealing at 56 ° C for 30 seconds, and extension at 72 ° C for 1 minute, Thereafter, reaction was carried out at 72 ° C for 10 minutes. In order to confirm whether the DNA thus prepared properly expressed in the protein, myc present in the pcDNA3-myc vector shown in the map of Fig. 1 was Western blotted using an anti-myc (9E10, Santa Cruz Biotechnology, sc- Lt; / RTI > expression. The myc-binding FSH-alpha protein was well expressed and the quantitation was loaded through the blot identified with actin (Figure 3).

(2) Lysine  (Lysine, K) Residue  substitution

The lysine residue was substituted with arginine using site-directed mutagenesis and the primer (FSH-α K75R FP 5'-ATGTTGGTCCAAAGGAACGTCACC-3 '(SEQ. (SEQ ID NO: 2), RP 5'-GGTGACGTTCCTTTGGACCAACAT-3 '(SEQ ID No. 3), FSH-α K99R FP 5'- ATGGGGGGTTTCAGAGTGGAGAAC-3' (SEQ ID No. 4), RP 5'- GTTCTCCACTCTGAAACCCCCCAT- (SEQ < RTI ID = 0.0 > No. 6), < / RTI > RP 5'-CTCGAGTTAAGATCTGTGATAATAACA-3 ' Three plasmid DNAs were prepared (pcDNA3-myc-FSH-α) and the lysine residues were substituted with arginine (K → R) (Table 1).

Lysine (K) residue position The FSH-alpha constructs in which Lysine (K) was substituted with Arginine (R) 75 pcDNA3-myc-FSH-alpha (K75R) 99 pcDNA3-myc-FSH-alpha (K99R) 115 pcDNA3-myc-FSH-a (K115R)

2. In vivo Ubiquitination  analysis

HEK 293T cells were infected with a plasmid encoding pcDNA3-myc-FSH-alpha WT and pMT123-HA-ubiquitin DNA. To confirm the ubiquitination process, 3 μg of pcDNA3-myc-FSH-α WT and 1 μg of pMT123-HA-ubiquitin DNA were co-transfected into the cells. After 24 hours, MG132 (proteasome inhibitor, 5 μg / Ml, Sigma-Aldrich) for 6 hours, followed by immunoprecipitation analysis (FIG. 4). In order to compare the degree of ubiquitination between WT and the substituent, pcDNA3-myc-FSH-α WT, pcDNA3-myc-FSH-α substituent (K75R), pcDNA3-myc-FSH-α substituent (K99R) 3 μg of each FSH-α substituent (K115R) was co-transfected with 1 μg of pMT123-HA-ubiquitin DNA in HEK 293T cells (ATCC, CRL-3216) and subjected to immunoprecipitation analysis after 24 hours (Fig. 5).

Samples obtained for immunoprecipitation were dissolved in lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8 and 1 mM PMSF (phenylmethanesulfonyl fluoride) and mixed with anti-myc (9E10) And incubated overnight at 4 ° C. Immunoprecipitates were separated by reaction with protein A / G beads (Santa Cruz Biotechnology) at 4 ° C. for 2 hours and then washed twice with lysis buffer. The protein samples were mixed with 2X SDS buffer, heated at 100 ° C for 7 minutes, and separated by SDS-PAGE. The separated proteins were transferred to a PVDF membrane and then subjected to anti-myc (9E10, Santa Cruz Biotechnology, blocking solution containing anti-beta-actin (sc-40), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti- beta -actin (Santa Cruz Biotechnology, labeled antibody to mouse IgG (H + L), KPL, 074-1806) using an ECL system Western blot detection kit, ABfrontier, Seoul, Korea).

As a result, when immunoprecipitation was carried out with anti-myc (9E10, Santa Cruz Biotechnology, sc-40), ubiquitin binding to pcDNA3-myc-FSH-α WT resulted in polyubiquitination, The band appeared thick (Fig. 4, lanes 3 and 4). In addition, treatment with MG132 (protease inhibitor, 5 / / ml) for 6 hours resulted in an increase in polyubiquitin formation and a darker band in which ubiquitin was detected (Fig. 4, lane 4). In the case of pcDNA3-myc-FSH-alpha substituent (K75R), pcDNA3-myc-FSH-alpha substituent (K99R) and pcDNA3-myc-FSH-alpha substituent (K115R) FSH-alpha substituent (K75R), pcDNA3-myc-FSH-alpha substituent (K99R) and pcDNA3-myc-FSH-alpha substituent (K115R) failed to bind ubiquitin, suggesting that ubiquitin was detected less 3 to 5). These results show that FSH-α binds to ubiquitin and is degraded by polyubiquitination through ubiquitin-proteasome system.

3. Protein production inhibitors cycloheximide  ( CHX )On by FSH Determination of half-life of -α

3 μg each of pcDNA3-myc-FSH-α WT, pcDNA3-myc-FSH-α substituent (K75R), pcDNA3-myc-FSH-α substituent (K99R) and pcDNA3-myc-FSH-α substituent (K115R) And transfected. After 48 h of transfection, the protein production inhibitor cycloheximide (CHX) (Sigma-Aldrich) (100 μg / ml) was treated and half-life was measured over 30 min, 60 min and 90 min. As a result, it was confirmed that decomposition of human FSH-α was inhibited. The half-life of human FSH-α is 45 minutes whereas the half-life of human pcDNA3-myc-FSH-alpha substituent (K99R) is longer than 90 minutes and the result is shown graphically (Fig. 6).

4. Intracellular FSH and FSH  Identification of signaling by substituents

FSH signaling has been reported to increase intracellular cAMP, thereby regulating the phosphorylation of transcription factors such as CREBP when PKA is activated, and is also associated with Erk and PI3K / AKT signaling (Front Endocrinol (Lausanne), 6 : 142, 2015; Biochem J., 473 (11): 1483-1501, 2016).

In this example, signal transduction by FSH-alpha and FSH-alpha substituents was observed in the cells. 3 μg each of pcDNA3-myc-FSH-α WT, pcDNA3-myc-FSH-α substituent (K75R), pcDNA3-myc-FSH-α substituent (K99R) and pcDNA3-myc-FSH-α substituent (K115R) To infect HeLa cells. After 2 days of infection, proteins were extracted from the cells, quantified, and Western blotting was carried out to confirm intracellular signal transduction. Proteins isolated from HeLa cells infected with pcDNA3-myc-FSH-alpha substituent (K75R), pcDNA3-myc-FSH-alpha substituent (K99R) and pcDNA3-myc-FSH-alpha substituent (K115R) (Santa Cruz Biotechnology, sc-21876), anti-phospho-STAT3 (Y705), anti- , cell signaling 9131S), anti-AKT (H-136, Santa Cruz Biotechnology, sc-8312), anti-phospho-AKT (S473, cell signaling 9271S), anti-Erk1 / 2 (9B3, Abfrontier LF-MAO134) Blocking solution containing anti-phospho-Erk1 / 2 (Thr202 / Tyr204, Abfrontier LF-PA0090) and anti-beta-actin (Santa Cruz Biotechnology, sc- 47778) at a weight ratio of 1: 1000 to 1: (ECL) system using a second antibody, anti-rabbit (goat anti-rabbit IgG-HRP, Santa Cruz Biotechnology, (Western blot detection kit, AB Frontier, Seoul, Korea). As a result, pcDNA3-myc-FSH-α substituent (K75R), pcDNA3-myc-FSH-α substituent (K99R) and pcDNA3-myc-FSH-α substituent (K115R) Showed the same or increased phospho-AKT signaling as WT (Fig. 7).

According to the present invention, a protein or (poly) peptide with an increased half-life is provided. Accordingly, the present invention relates to a protein or (poly) peptide which can be used as a therapeutic agent, and may be usefully used in the pharmaceutical industry.

&Lt; 110 > UbiProtein. Corp <120> A method for extending half-life of FSH alpha <130> UBPRN17P-0006 <160> 7 <170> KoPatentin 3.0 <210> 1 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Follicle-stimulating hormone alpha <400> 1 Met Asp Tyr Tyr Arg Lys Tyr Ala Ile Phe Leu Val Thr Leu Ser   1 5 10 15 Val Phe Leu His Val Leu His Ser Ala Pro Asp Val Gln Asp Cys Pro              20 25 30 Glu Cys Thr Leu Gln Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro          35 40 45 Ile Leu Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro      50 55 60 Leu Arg Ser Lys Lys Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu  65 70 75 80 Ser Thr Cys Cys Val Ala Lys Ser Tyr Asn Arg Val Thr Val Met Gly                  85 90 95 Gly Phe Lys Val Glu Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr             100 105 110 Tyr His Lys Ser         115 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 atgttggtcc aaaggaacgt cacc 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 ggtgacgttc ctttggacca acat 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 atggggggtt tcagagtgga gaac 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 gttctccact ctgaaacccc ccat 24 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 tgttattatc acagatctta actcgag 27 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 ctcgagttaa gatctgtgat aataaca 27

Claims (4)

Having an increased half-life, wherein any one of the lysine residues at positions 99 and 115 from the N-terminus of the follicle-stimulating hormone alpha (FSH alpha) having the amino acid sequence of SEQ ID NO: 1 is substituted with arginine Follicle stimulating hormone α.
delete (a) a promoter; And (b) an expression vector comprising a nucleotide sequence encoding the follicle stimulating hormone a of claim 1, wherein the promoter and the nucleotide sequence are operatively linked. A host cell comprising the expression vector of claim 3.

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