Telomerase inhibitor and pharmaceutical composition

Abstract

To provide a telomerase inhibitor.SOLUTION: The telomerase inhibitor includes a Sabin protein or a protein having an amino acid sequence in which 1 to 5 amino acids are inserted, deleted, substituted and / or added in the amino acid sequence of the Sabin protein and exhibiting mono-ADP-ribosyltransferase activity.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to telomerase inhibitors and pharmaceutical compositions. [Background technology]

[0002] The 3' ends of eukaryotic chromosomes contain telomeres, which contain repeated sequences of the amino acid GGTTAG, and play an important role in stabilizing chromosomes. In normal cells, telomeres shorten with each cell division, and when they reach a certain length, cell division ceases, which is thought to determine the cell's lifespan. Therefore, normal cells do not proliferate indefinitely. In contrast, in cancer cells, a reverse transcriptase called telomerase acts to extend telomeres, preventing proliferation. While telomerase is not expressed in most normal cells, it is expressed in approximately 90% of human cancer cells. Therefore, telomerase inhibitors are considered promising targets for anticancer drugs. Many telomerase inhibitors have been identified to date, including oligonucleotides and related compounds, G-quartet binding molecules, and natural products.

[0003] The Scabin protein, discovered in Streptomyces scabies, a pathogen of potato scab, is known to have mono-ADP-ribosyltransferase (mART) activity. Scabin proteins convert the amino group at the 2nd position of guanine bases (G) in DNA to ADP-ribosylated (see, for example, Non-Patent Document 1). Research on Pierisin, a protein with similar activity, has revealed that mART activity is associated with the induction of apoptosis (see, for example, Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] J. Biol. Chem. (2016) 11198-215. [Non-patent document 2] PNAS (1999) 10608-10613. Summary of the Invention [Problem to be solved by the invention]

[0005] One aspect of the present invention aims to provide a telomerase inhibitor. [Means for solving the problem]

[0006] A first aspect is a telomerase inhibitor comprising at least one selected from the group consisting of a Scabin protein and a Scabin-like protein that exhibits mono-ADP-ribosyltransferase activity and has 80% or more homology with the amino acid sequence of the Scabin protein. In one aspect, the Scabin-like protein may include a protein that has an amino acid sequence in which 1 to 10 amino acids are inserted, deleted, substituted, and / or added in the amino acid sequence of the Scabin protein and exhibits mono-ADP-ribosyltransferase activity. A second aspect is a pharmaceutical composition comprising the telomerase inhibitor and used for treating tumors. [Effects of the Invention]

[0007] According to one aspect of the present invention, a telomerase inhibitor can be provided. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows the results of ADP-ribosylation of single-stranded DNA by Scabin. [Figure 2A] FIG. 1 is a schematic diagram showing the structure of the double-stranded DNA used in the evaluation. [Figure 2B] FIG. 1 shows the results of ADP-ribosylation of double-stranded DNA by Scabin. [Figure 3] FIG. 1 shows the effect of ADP-ribosylation of single-stranded DNA on the DNA elongation reaction of double-stranded DNA by reverse transcriptase. [Figure 4] FIG. 1 shows the effect of ADP-ribosylation of double-stranded DNA on the DNA elongation reaction of double-stranded DNA by reverse transcriptase. [Figure 5] FIG. 1 shows the effect of ADP-ribosylation of single-stranded DNA on the DNA elongation reaction of double-stranded DNA by reverse transcriptase. [Figure 6] FIG. 1 shows the effect of ADP-ribosylation of double-stranded DNA on the DNA elongation reaction of double-stranded DNA by reverse transcriptase. [Figure 7A] FIG. 1 shows the expression of Scabin in Hela cells. [Figure 7B] FIG. 1 shows the expression of Scabin in U2OS cells. [Figure 8] FIG. 1 shows the survival rate of Hela cells in which Scabin was expressed. [Figure 9] FIG. 1 shows the survival rate of U2OS cells in which Scabin was expressed. DETAILED DESCRIPTION OF THE INVENTION

[0009] As used herein, the term "process" refers not only to an independent process, but also to processes that cannot be clearly distinguished from other processes, as long as the intended purpose of the process is achieved. Furthermore, the content of each component in a composition refers to the total amount of the multiple substances present in the composition, unless otherwise specified, if the composition contains multiple substances corresponding to each component. Furthermore, the upper and lower limits of the numerical ranges described herein can be arbitrarily selected and combined from the numerical values ​​exemplified as numerical ranges. Hereinafter, embodiments of the present invention will be described with reference to the drawings. However, the embodiments shown below are examples of telomerase inhibitors intended to embody the technical concept of the present invention, and the present invention is not limited to the telomerase inhibitors shown below.

[0010] telomerase inhibitors The telomerase inhibitor comprises at least one member selected from the group consisting of Scabin protein and Scabin-like proteins that exhibit mono-ADP-ribosyltransferase activity and have 80% or more homology with the amino acid sequence of Scabin protein.

[0011] The Scabin protein has an amino acid sequence consisting of 208 amino acid residues (SEQ ID NO: 1) and is a 22 kDa single-domain protein. The Scabin protein has mono-ADP-ribosyltransferase (mART) activity. In addition to the ADP-ribosyltransferase activity, the Scabin protein also has the ability to convert nicotinamide adenine nucleotides (NAD + ) glycohydrolase activity, and NAD + Scabin cleaves α-amino acid to nicotinamide and adenosine diphosphate (ADP)-ribose, and then adds ADP-ribose to the substrate. The mART activity exhibited by Scabin proteins ADP-ribosylates the amino group at position 2 of guanine residues in DNA. In addition to being able to ADP-ribosylate guanine residues at the 3' end of DNA, Scabin proteins can also ADP-ribosylate guanine residues other than the 3' end of single-stranded DNA that contains guanine residues other than the 3' end. Furthermore, in double-stranded DNA, they can ADP-ribosylate only the 3' end guanine residue.

[0012] [Table 1]

[0013] Telomeres, located at the 3' end of chromosomes, are composed of a six-residue repeat sequence of GGTTAG. The synthesis of telomere repeat sequences involves the use of a reverse transcriptase called telomerase, which has a template RNA with the repeat sequence as its complementary strand. Telomerase complementarily binds to the 3' end of telomeric DNA, then synthesizes the GGTTAG repeat sequence by reverse transcription, thereby extending the telomeric DNA. Scabin proteins are thought to inhibit the reverse transcription activity of telomerase by ADP-ribosylating the guanine residue at the 3' end of telomeric DNA.

[0014] The telomerase inhibitor may include a Scabin-like protein that exhibits mono-ADP-ribosyltransferase activity and has 80% or more homology to the amino acid sequence of Scabin. The homology of the Scabin-like protein to the amino acid sequence of Scabin may be preferably 85% or more, 90% or more, or 95% or more. Protein homology can be calculated, for example, by BLAST.

[0015] Scabin-like proteins may include proteins that have an amino acid sequence in which 1 to 10 amino acids have been inserted, deleted, substituted, and / or added in the amino acid sequence of Scabin protein and that exhibit mono-ADP-ribosyltransferase activity. The number of inserted, deleted, substituted, and / or added amino acids in the Scabin-like protein may preferably be 1 to 8, 1 to 6, or 1 to 5.

[0016] Scabin proteins or Scabin-like proteins can be obtained by commonly used methods, for example, by inserting cDNA encoding a Scabin protein into an appropriate expression vector and expressing it.

[0017] Pharmaceutical Composition The pharmaceutical composition contains a telomerase inhibitor including a Scabin protein or a Scabin-like protein and is used for treating tumors. By including a telomerase inhibitor in the pharmaceutical composition, apoptosis can be induced in tumor cells. Here, tumor treatment refers to any treatment administered to tumors, including, for example, treatment, improvement, inhibition of progression (prevention of worsening), prevention, and alleviation of symptoms caused by tumors.

[0018] Pharmaceutical compositions can be administered parenterally or orally depending on their dosage form. Examples of dosage forms for parenteral administration include injections, drip infusions, eye drops, nasal preparations, and pulmonary preparations. Examples of dosage forms for oral administration include solid preparations such as tablets, capsules, granules, powders, lozenges, syrups, emulsions, and suspensions, as well as liquid and semi-liquid preparations.

[0019] Any pharmaceutically acceptable carrier can be used as long as it is a carrier commonly used in pharmaceutical formulations. Examples of carriers include excipients, disintegrants, binders, fluidizing agents, lubricants, etc. in solid preparations, and solvents, solubilizing agents, suspending agents, isotonicity agents, buffers or pH adjusters, soothing agents, etc. in liquid preparations. If necessary, the preparation may further contain additives such as preservatives, antioxidants, colorants, sweeteners, refreshing agents or flavoring agents, antifoaming agents, thickeners, etc.

[0020] The content of the active ingredient in the pharmaceutical composition varies depending on the dosage form, dosage, etc., but is, for example, 0.1 to 20% by weight, or 0.1 to 10% by weight of the total composition. The dosage of the pharmaceutical composition is selected appropriately depending on the subject, disease, symptoms, dosage form, administration route, etc. For example, when orally administered to an adult cancer patient, the dosage is usually about 0.1 to 500 mg, or about 0.5 to 100 mg, of the active ingredient per day, and can be administered once or in divided doses.

[0021] Methods for treating diseases The method for treating a disease comprises administering an effective amount of the pharmaceutical composition to a subject. The disease to be treated, details of the pharmaceutical composition, and the method of administration are as described above. The subject to be treated is, for example, a mammal, including a human. The subject to be treated may also be a non-human animal.

[0022] In other aspects, the present invention also includes the use of a telomerase inhibitor in the manufacture of a pharmaceutical composition for use in treating a tumor, the use of a telomerase inhibitor in treating a tumor, and a telomerase inhibitor for use in treating a tumor. [Example]

[0023] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0024] Example Preparation of Scabin Protein Scabin cDNA (Δ29; SEQ ID NO: 2) was prepared for integration by deleting 29 amino acid residues from the N-terminus of Scabin cDNA and adding His-Tag etc. Scabin cDNA (Δ29; SEQ ID NO: 2) and pCold TM pCold II (vector; Takara Bio) was treated with the restriction enzymes BamH I and EcoR I. 10 μL of Scabin cDNA (10 ng / μL), 1 μL of BamH I (12 units / μL), 5 μL of 10x CutSmart, and 34 μL of dH2O were mixed. TM The mixture was mixed with 500 ng / μL of BamH I (12 units / μL), 5 μL of 10x CutSmart, and 43 μL of dH2O. The resulting solution was incubated at 37°C for 2 hours, after which 1 μL of EcoR I (20 units / μL) was added to each solution and further incubated at 37°C for 1 hour. After 1 hour, the mixture was incubated with NucleoSpin (R) DNA purification was performed using Gel and PCR Clean-up (Mach-Ley-Nagel). Two volumes of Binding Buffer NTI (Mach-Ley-Nagel) were added to the reaction mixture, which was then transferred to a spin column. The mixture was then centrifuged (11,000 × g, 1 min) and the filtrate was discarded. 700 μL of Wash Buffer NT3 (Mach-Ley-Nagel) was added, and the mixture was centrifuged (11,000 × g, 1 min) and the filtrate was discarded. This procedure was repeated twice, and the column was then centrifuged again (11,000 × g, 1 min). The column was then placed in a new 1.5 mL tube. 15 μL of NE Buffer (New England BioLabs) preheated to 70°C was added, and the mixture was incubated at room temperature for 5 minutes. The column was then centrifuged (11,000 × g, 1 min) to obtain the DNA solution.

[0025] pCold TM To mix the DNA solution at a ratio of II (vector):Scabin cDNA (insert) = 1:5 (mol), 0.65 μL of restriction enzyme-treated Scabin cDNA (6.3 ng / μL) and restriction enzyme-treated pCold TM An equal volume (10.65 μL) of Ligation mix (manufactured by Takara Bio Inc.) was added thereto, and the ligation reaction was carried out at 16°C for 1 hour to produce pColdII-Scabin_WT.

[0026] [Table 2]

[0027] Cultivation of pColdII-Scabin_WT / DH5α DH5α competent cells (ThermoFisher Scientific) were thawed on ice, and 25 μL of DH5α and 2 μL of ligation solution (pColdII-Scabin_WT) were mixed and vortexed for 1 second. After 5 minutes of incubation on ice, the mixture was incubated at 42°C for 45 seconds and vortexed for 1 second. 2 μL and 20 μL of the mixture were then plated onto two LB plates (+Ampicillin) and cultured overnight at 37°C. After incubation, a single colony was picked, added to 1 mL of LB solution (+Ampicillin), and cultured overnight at 37°C with shaking.

[0028] QIAprep (R) DNA purification using the Miniprep Kit After the shaking culture, the solution was transferred to a 1.5 mL tube and centrifuged (7400 rpm, 3 min) to collect the precipitate. Then, 250 μL of P1 Buffer (Qiagen) was added, and the precipitate was dissolved by vortexing. After dissolution, 250 μL of P2 Buffer (Qiagen) was added and mixed by inversion 4 to 5 times. Next, 350 μL of N3 Buffer (Qiagen) was added, and the mixture was mixed by inversion 4 to 5 times. After centrifugation (14000 rpm, 5 min), the supernatant was collected in a spin column. The column was centrifuged once (8000 rpm, 1 min), and the filtrate was discarded. Then, 500 μL of PB Buffer (Qiagen) was added to the spin column, and the column was centrifuged (8000 rpm, 1 min), and the filtrate was discarded. Then, 750 μL of PE Buffer (Qiagen) was added, and the mixture was centrifuged (8000 rpm, 1 min). The filtrate was discarded. This procedure was repeated twice, and the mixture was centrifuged again (8000 rpm, 1 min). The column was then placed in a new 1.5 mL tube, and 50 μL of EB Buffer (Qiagen) was added. The mixture was then incubated for 5 minutes. The column was then centrifuged (10000 rpm, 1 min) to obtain a plasmid solution. Sequence analysis confirmed that the desired plasmid had been produced.

[0029] BL21(DE3) transformation competent cells (ThermoFisher Scientific) were thawed on ice for 10 minutes, and 1 μL of pColdII-Scabin_WT (100 pg / μL) was added. The mixture was gently mixed 4-5 times and incubated on ice for 30 minutes. Next, 950 μL of SOC (ThermoFisher Scientific) was added and cultured with shaking at 37°C for 1 hour. Five and 50 μL of the culture were then plated onto two LB plates (plus ampicillin) and cultured overnight at 37°C. Eight colonies were picked, added to 100 mL of 2xYT solution (tryptone 16 g, yeast extract 10 g, NaCl 5 g / L), and cultured overnight with shaking at 37°C.

[0030] After the shaking culture, 100 mL of the solution was added to 1 L of LB solution and cultured at 37°C with shaking until an OD600 of 0.8 was reached. Then, 1 mL of 1 M IPTG (final 1 mM) was added, and the mixture was shaken overnight at 16°C. After shaking, the cells were collected by centrifugation (4000 rpm, 15 min, 4°C). The cells were lysed in 20 mL of NTN500+ (50 mM Tris-HCl, 500 mM NaCl, 1% NP-40, 1 mM DTT, 0.5 mM PMSF, 1× protease inhibitor (EDTA-free)) and sonicated (On-5 sec / Off-5 sec, 1 min × 5). The mixture was then centrifuged (15000 rpm, 30 min, 4°C) and the supernatant was collected. The resulting supernatant was mixed with 500 μL of Ni Sepharose 6 Fast Flow (Cyteva) and incubated at 4°C for 1 hour. After incubation, the Ni Sepharose 6 Fast Flow was collected and washed five times with 1 mL of Wash Buffer (50 mM Tris-HCl, 500 mM NaCl, 40 mM imidazole). Then, 1 mL of Elution Buffer (50 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole) was added, and the mixture was incubated on ice for 5 minutes. The mixture was then centrifuged (900 × g, 1 min) and the supernatant was collected. This process was repeated five times.

[0031] MonoQ was used with AKTA pure (Cytiva, Tokyo, Japan). TM Purification was performed using a column. Elution was performed using a stepwise method, and a peak was observed at 200 nM NaCl, which was confirmed by SDS-PAGE. The desired Scabin protein was thus obtained.

[0032] Evaluation 1: ADP-ribosylation of ssDNA As model single-stranded DNA (ssDNA), Tel-mimic17 (SEQ ID NO: 3) having multiple guanine residues and 3'G-DNA (SEQ ID NO: 4) having a guanine residue only at the 3' end were obtained from FASMAC Co., Ltd., and (γ- 325'-end phosphorylation using P-ATP and T4 kinase 32 P labeled.

[0033] [Table 3]

[0034] 1 μL of 10× Scabin buffer was added to the labeled ssDNA and Scabin, and the mixture was diluted to 10 μL with Milli-Q water to prepare reaction mixtures with a final ssDNA concentration of 20 nM and final Scabin concentrations of 0 nM, 0.5 nM, 1 nM, 2.5 nM, and 5 nM. The composition of the 10× Scabin buffer was 200 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 2.5 mM NAD. + It was decided.

[0035] The reaction mixture was incubated at 25°C for 60 minutes. The reaction was then stopped by adding 10 μL of 2× Loading dye. The mixture was then incubated at 95°C for 5 minutes and analyzed by 12.5% ​​urea gel electrophoresis (running conditions: 200 V, 60 min). The gel was dried and detected using a Fuji FLA-7000 phosphorimager. The results are shown in Figure 1. The composition of the 2× Loading dye was 98% formamide, 20 mM ethylenediaminetetraacetic acid (EDTA), 0.5% bromophenol blue, and 0.5% xylene cyanol.

[0036] Figure 1 shows a Scabin concentration-dependent band shift. This indicates that ADP-ribosylation occurs when there is a guanine residue at the 3' end. Furthermore, in the case of the substrate Tel-mimic17, which contains multiple guanine bases, multiple bands shift upward, indicating that ADP-ribosylation occurs at multiple guanine residues.

[0037] Evaluation 2: ADP-ribosylation of dsDNA Ten μL of 0.2 μM labeled Tel-mimic17 was mixed with 10 μL of 0.2 μM RNA9DNA12 (SEQ ID NO: 5), and the mixture was incubated at 95°C for 5 minutes and then at room temperature for 1 hour to prepare 0.2 μM labeled double-stranded DNA (dsDNA). RNA9DNA12 was obtained from FASMAC. In the RNA9DNA12 sequence listed in Table 3, uppercase letters indicate RNA and lowercase letters indicate DNA. The sequence of the double-stranded DNA is shown in Figure 2A. The underlined portions in Figure 2A indicate DNA.

[0038] As shown in Figure 2A, it is thought that RNA9DNA12 forms an overhang on the 3' side of Tel-mimic17, forming a telomere-like structure.

[0039] 2 μL of 10× Scabin buffer was added to the labeled dsDNA and Scabin, and the mixture was diluted to 20 μL with Milli-Q water to prepare reaction mixtures with a final dsDNA concentration of 20 nM and final Scabin concentrations of 0 nM, 0.5 nM, 1 nM, 2.5 nM, 5 nM, 10 nM, and 20 nM.

[0040] The prepared reaction mixture was incubated at 25°C for 60 minutes. The reaction was then stopped by adding 2x loading dye (10 μL). The mixture was then incubated at 95°C for 5 minutes and analyzed by 12.5% ​​urea gel electrophoresis (running conditions: 200 V, 60 min). The results are shown in Figure 2B.

[0041] Figure 2B shows a Scabin concentration-dependent band shift, indicating that Scabin induces ADP-ribosylation modification only on the 3'-terminal guanine base.

[0042] Evaluation 3: Effect of ADP-ribosylation of Scabin on DNA elongation reaction 1 The labeled single-stranded DNA was ADP-ribosylated, and then annealed to form double-stranded DNA, and the effect on the DNA elongation reaction by reverse transcriptase was evaluated.

[0043] Labeled Tel-mimic17 and Scabin were mixed with 1 μL of 10x Scabin buffer and diluted to 20 μL with Milli-Q water to prepare reaction mixtures with final ssDNA concentrations of 20 nM and Scabin concentrations of 0 nM, 0.5 nM, 1 nM, 2.5 nM, 5 nM, 10 nM, and 20 nM. The reaction mixtures were incubated at 25°C for 60 minutes. Protein activity was then stopped by incubation at 95°C for 5 minutes. To the 10 μL reaction mixture, 2 μL of 0.2 μM DNA9RNA12 was then added and incubated at room temperature for 1 hour to prepare double-stranded DNA.

[0044] 1 μL of Milli-Q water, 4 μL of 2 mM dNTP Solution (TaKaRa), and 0.2 μM labeled double-stranded DNA were added and incubated at 65°C for 5 minutes to linearize the DNA structure, followed by ice cooling. 4 μL of 5x RT buffer and 1 μL of M-MLV Reverse Transcriptase (Nippon Gene) were added to make a 20 μL solution, which was then incubated at 42°C for 1 hour. The extension reaction was then stopped by incubating at 70°C for 15 minutes. The composition of the 5x RT buffer was 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, and 10 mM DTT.

[0045] 20 μL of 2× Loading dye was added, and the mixture was analyzed by 12.5% ​​urea gel electrophoresis. The results are shown in Figure 3.

[0046] 3, it can be seen that the band shift decreases in a Scabin concentration-dependent manner, indicating that the DNA polymerase elongation reaction is inhibited in a Scabin concentration-dependent manner.

[0047] Evaluation 4: Effect of ADP-ribosylation of Scabin on DNA elongation reaction 2 The labeled double-stranded DNA was subjected to ADP-ribosylation, and the effect on DNA elongation was evaluated as follows.

[0048] DNA9RNA12 was added to the labeled Tel-mimic17 and incubated at room temperature for 1 hour to prepare double-stranded DNA. A reaction mixture was prepared in the same manner as in Evaluation 3, except that the prepared double-stranded DNA was used. The reaction mixture was incubated at 25°C for 60 minutes. The protein activity was then stopped by incubating at 95°C for 5 minutes.

[0049] 1 μL of Milli-Q water, 4 μL of 2 mM dNTP solution (TaKaRa), and 0.2 μM of labeled double-stranded DNA were added and incubated at 65°C for 5 minutes to linearize the DNA structure. The mixture was then cooled on ice. 4 μL of 5x RT buffer and 1 μL of M-MLV Reverse Transcriptase (Nippon Gene) were added to make a 20 μL solution, which was then incubated at 42°C for 1 hour. The extension reaction was then stopped by incubating at 70°C for 15 minutes.

[0050] 20 μL of 2× Loading dye was added, and the mixture was analyzed by 12.5% ​​urea gel electrophoresis. The results are shown in Figure 4.

[0051] As can be seen from FIG. 4, ADP-ribosylated bands were observed in a Scabin concentration-dependent manner, indicating that the elongation reaction of DNA polymerase was inhibited in a Scabin concentration-dependent manner.

[0052] Evaluation 5: Effect of ADP-ribosylation of Scabin on DNA elongation reaction 3 Labeled single-stranded DNA was subjected to ADP-ribosylation, and the results were compared in the presence and absence of DNA polymerase.

[0053] 1 μL of 10x Scabin buffer was added to 0.2 μM of labeled Tel-mimic17 and Scabin, and the mixture was diluted to 20 μL with Milli-Q water to prepare reaction mixtures with final Tel-mimic17 concentrations of 20 nM and final Scabin concentrations of 0 nM, 0.5 nM, 1 nM, 2.5 nM, and 5 nM.

[0054] The reaction mixture was incubated at 25°C for 60 minutes. Protein activity was stopped by incubation at 95°C for 5 minutes. 2 μL of 0.2 μM DNA9RNA12 was then added to 10 μL of the reaction mixture, and the mixture was incubated at room temperature for 1 hour to allow annealing and obtain double-stranded DNA.

[0055] The resulting double-stranded DNA was mixed with 1 or 2 μL of Milli-Q water and 4 μL of dNTP Solution (2 mM; TaKaRa) and incubated at 65°C for 5 minutes to linearize the DNA structure. The mixture was then cooled on ice. 4 μL of 5x RT buffer and 1 μL of M-MLV Reverse Transcriptase (Nippon Gene) were added or not to make a total volume of 20 μL, and the mixture was incubated at 42°C for 1 hour. The extension reaction was then stopped by incubating at 70°C for 15 minutes.

[0056] 20 μL of 2× Loading dye was added, and the mixture was analyzed by 12.5% ​​urea gel electrophoresis. The results are shown in Figure 5.

[0057] Figure 5 confirms that the amount of elongated DNA decreased and the amount of ADP-ribosylated DNA increased in a Scabin concentration-dependent manner. Similar to Evaluation 3, this result indicates that ADP-ribosylation by Scabin inhibits the DNA elongation reaction.

[0058] Evaluation 6 Effect of ADP-ribosylation of Scabin on DNA elongation reaction 4 ADP-ribosylation of labeled double-stranded DNA was performed, and the results were compared in the presence and absence of DNA polymerase.

[0059] DNA9RNA12 was added to the labeled Tel-mimic17 and incubated at room temperature for 1 hour to prepare double-stranded DNA. The prepared double-stranded DNA was used to prepare a reaction mixture as in Evaluation 3. The reaction mixture was incubated at 25°C for 60 minutes. Protein activity was then stopped by incubation at 95°C for 5 minutes.

[0060] The resulting reaction mixture was mixed with 1 or 2 μL of Milli-Q water and 4 μL of 2 mM dNTP solution (TaKaRa) and incubated at 65°C for 5 minutes to linearize the DNA structure. The mixture was then cooled on ice. 4 μL of 5x RT buffer and 1 μL of M-MLV Reverse Transcriptase (Nippon Gene) were added or not to make a total volume of 20 μL, and the mixture was incubated at 42°C for 1 hour. The extension reaction was then stopped by incubating at 70°C for 15 minutes.

[0061] 20 μL of 2× Loading dye was added, and the mixture was analyzed by 12.5% ​​urea gel electrophoresis. The results are shown in Figure 6.

[0062] Figure 6 confirms that the amount of elongated DNA decreased and the amount of ADP-ribosylated DNA increased in a Scabin concentration-dependent manner. Similar to Evaluation 4, this result is thought to indicate that ADP-ribosylation by Scabin can inhibit telomerase at telomere repeat sequences.

[0063] Reference example Construction of pEGFP-Scabin(WT) To express Scabin in cells, a cell expression vector was constructed as follows: PrimeSTAR (R) 25 μL of MAX DNA Polymerase (manufactured by Takara Bio) (x2), 1.5 μL of 10 μM Scabin Forward Primer (SEQ ID NO: 6), 1.5 μL of 10 μM Scabin Reverse Primer (SEQ ID NO: 7), 1 μL of pColdII-Scabin_WT (403 pg / μL), and 21 μL of dH2O were mixed. Then, PCR (98°C - 10 sec, 55°C - 15 sec, 72°C - 5 sec x 35 cycles) was performed. After the reaction, the mixture was diluted with NucleoSpin (R)DNA purification was performed using Gel and PCR Clean-up (Takara Bio). Two volumes of NTI were added to the reaction solution, transferred to a spin column, and centrifuged (11,000 × g, 1 min). The filtrate was discarded. 700 μL of NT3 was added, and the mixture was centrifuged (11,000 × g, 1 min). The filtrate was discarded. This procedure was repeated twice, followed by centrifugation (11,000 × g, 1 min). The column was placed in a 1.5 mL tube, 15 μL of 70°C NE Buffer was added, and the mixture was incubated at room temperature for 5 minutes. The mixture was then centrifuged (11,000 × g, 1 min) to obtain a DNA solution.

[0064] [Table 4]

[0065] The resulting DNA solution and pEGFP-c1 (Takara Bio) were treated with two restriction enzymes, BamHI and EcoRI, respectively, and then mixed and ligated in a ratio of pEGFP-c1 (Vector):cDNA (Insert) = 1:5 to incorporate Scabin cDNA into pEGFP-c1.

[0066] DH5α was thawed on ice, and 25 μL of DH5α and 2 μL of ligation solution were mixed and vortexed for 1 second. After 5 minutes on ice, the mixture was incubated at 42°C for 45 seconds. After 2 minutes on ice, 250 μL of SOC was added and the mixture was shaken at 37°C for 1 hour. After shaking, 2 μL and 20 μL of the mixture were plated onto two LB plates (with Kanamycin) and cultured overnight at 37°C. After incubation, a single colony was picked, added to 1 mL of LB solution (with Kanamycin), and cultured overnight at 37°C with shaking.

[0067] After the shaking culture, the solution was transferred to a 1.5 mL tube and centrifuged (7400 rpm, 3 min) to collect the precipitate. Then, 250 μL of P1 Buffer was added and the precipitate was dissolved using a vortex. After dissolution, 250 μL of P2 Buffer was added and mixed by inverting 4 to 5 times. Next, 350 μL of N3 Buffer was added and mixed by inverting 4 to 5 times. After centrifugation (14000 rpm, 5 min), the supernatant was collected in a spin column. The column was centrifuged once (8000 rpm, 1 min) and the filtrate was discarded. Next, 500 μL of PB Buffer was added to the spin column and centrifuged (8000 rpm, 1 min) and the filtrate was discarded. Finally, 750 μL of PE Buffer was added and centrifuged (8000 rpm, 1 min) and the filtrate was discarded. This procedure was repeated twice, followed by centrifugation (8,000 rpm, 1 min). The column was placed in a new 1.5 mL tube, 50 μL of EB Buffer was added, and the mixture was incubated for 5 minutes. The column was then centrifuged (10,000 rpm, 1 min) to obtain the plasmid solution (pEGFP-Scabin_WT). Sequence analysis confirmed that the desired plasmid had been produced.

[0068] Construction of pEGFP-Scabin (Y129A and W155A) To compare the mutant with WT, we constructed a cell expression vector for the mutant. (R) 25 μL of MAX DNA Polymerase (x2), 1 μL of 10 μM Forward Primer (Y129A or W155A), 1 μL of 10 μM Reverse Primer (Y129A or W155A), 22 μL of dH2O, and 1 μL of pEGFP-Scabin_WT (500 pg / μL) were mixed and subjected to mutagenesis PCR (98°C - 10 sec, 55°C - 15 sec, 72°C - 30 sec x 30 cycles) to obtain DNA solutions.

[0069] [Table 5]

[0070] The resulting DNA solution was inserted into pEGFP-c1 (Takara Bio) and transfected into DH5α to obtain pEGFP-Scabin(Y129A) and pEGFP-Scabin(W155A). Sequence analysis of the resulting pEGFP-Scabin(Y129A) and pEGFP-Scabin(W155A) confirmed that they contained the Y129A and W155A mutations, respectively.

[0071] Expression in human cells Scabin was overexpressed in human cells (HeLa and U2OS) as follows, and expression was confirmed by Western blotting using cell suspensions obtained during the process. (R) Add 2.0 x 10 HeLa or U2OS cell solution to a 6-well cell culture plate. 5 The cells were seeded at 1000 cells / well into a total of 4 wells and cultured overnight at 37°C. The cultured cells were transfected with one of four types of DNA: pEGFP (without Scabin), pEGFP-Scabin_WT, pEGFP-Scabin (Y129A), or pEGFP-Scabin (W155A). The cells were washed with PBS, and 2 mL of medium was added. Afterwards, 4 μg of each type of DNA (2 μg / well × 2 wells) was prepared for transfection into HeLa cells and U2OS cells, and the cells were incubated in Opti-MEM (R) At the same time, one type of DNA was mixed with 200 μL of jetPEI. (R) 12 μL (3x the amount of DNA) and Opti-MEM (R) 200 μL of the prepared DNA solution, jetPEI, and 200 μL of the prepared DNA solution were mixed and stirred by inversion and vortexing. (R) 200 μL of the solution was mixed and vortexed for 5 seconds. After incubation for 10 to 15 minutes, 200 μL was added to each well. The plate was gently rotated to mix the medium, and the plate was incubated overnight at 37°C.

[0072] After overnight incubation, expression was confirmed using a fluorescence microscope. After washing with PBS, the cells were detached from the bottom of the plate with trypsin + EDTA and suspended in medium. The suspension was transferred to a 50 mL tube, and the cell number was measured using a cell counter. Based on the measured cell number, the cells were re-seeded on the plate. For HeLa cells, 0.03 × 10 4 cells / mL (total volume 8 mL), and 0.06 × 10 for U2OS cells 4 cells / mL (total volume 8 mL) or 0.12 × 10 4 The cell suspension was diluted to 100 cells / mL (total volume 8 mL).

[0073] Western blotting was performed using the obtained cell suspension. The results are shown in Figures 7A and 7B. As shown in Figures 7A and 7B, it was confirmed that the target protein was expressed.

[0074] Cell viability was assessed after overexpression of Scabin in human cells (HeLa and U2OS). The antibiotic G418 was added to the diluted cell suspension at 400 μg / mL for HeLa cells and 40 μg / mL for U2OS cells. The cells were seeded at 600 cells / well and incubated at 37°C for one week. After washing with PBS, the cells were stained with a staining solution (50% EtOH, 7% acetic acid, Coomassie Brilliant Blue 3 g / L) at room temperature for one hour. After rinsing with RO water and drying, the cells were imaged using a ChemiDoc (Bio-Rad Laboratories, Hercules, CA, USA). The image data were analyzed using ImageJ to obtain quantitative data. One-way ANOVA was used for statistical analysis. The results are shown in Figures 8 and 9.

[0075] The results for both cell types confirmed a significant difference in viability between cells expressing WT Scabin and those expressing GFP alone (Fig. 8 and Fig. 9). This suggests that expression of WT Scabin is cytotoxic and reduces cell viability. Furthermore, when we examined the differences between WT and Y129A, WT and W155A, and Y129A and W155A, no significant differences were observed in HeLa cells, but only between WT and Y129A in U2OS cells. This suggests that there is little difference in the effects of differences in Scabin activity in either HeLa or U2OS cells. However, the significant difference between WT and Y129A in U2OS cells suggests that the toxicity is due to the strong DNA-binding ability of Y129A, in addition to the ADP-ribosylation activity of Scabin, which binds to DNA.

Claims

1. A telomerase inhibitor comprising at least one member selected from the group consisting of a Scabin protein and a Scabin-like protein that exhibits mono-ADP ribosyltransferase activity and has 80% or more homology with the amino acid sequence of the Scabin protein.

2. The telomerase inhibitor according to claim 1, wherein the Scabin-like protein includes a protein having an amino acid sequence in which 1 to 10 amino acids are inserted, deleted, substituted and / or added in the amino acid sequence of the Scabin protein, and exhibiting mono-ADP ribosyltransferase activity.

3. A pharmaceutical composition for use in treating tumors, comprising the telomerase inhibitor of claim 1 or 2.

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