KR101769911B1 - Skp2 inhibitor as a novel anti-cancer agent - Google Patents

Abstract

The present invention relates to novel peptoid compounds which exhibit anticancer effects by inhibiting Skp2 / p300 protein interaction and uses thereof.

Description

As a novel anticancer agent, a Skp2 inhibitor (Skp2 inhibitor as a novel anti-cancer agent)

The present invention relates to novel peptoid compounds which exhibit anticancer effects by inhibiting Skp2 / p300 protein interaction and uses thereof.

Skp2 induces cancer development through two pathways (Figure 1).

The first pathway is the regulation of cell cycle regulatory protein degradation as part of the SCF ^Skp2 ubiquitin ligase. SCF ^Skp2 is an E3 ubiquitin ^ligating complex that regulates the degradation of cell cycle regulatory proteins such as p27 and p21. Skp2 is an F-box protein that constitutes the SCF ^Skp2 complex. It ^activates these cell cycle regulating proteins to ubiquitinate them. Such poly-ubiquitinated proteins are recognized and decomposed by 26S proteasome. Skp2 is overexpressed in various cancer cells and inhibits the expression of tumor suppressor proteins, thereby promoting cancer progression and metastasis. Thus, inhibiting Skp2 can be an effective anti-cancer strategy. Thus, there have been various attempts to develop Skp2 inhibitors, such as inhibiting Skp1 / Skp2 interactions (CH Chan, et al. Cell 2013, 154 , 556-568), inhibiting the interaction between p27 and Skp2 (Q. Chen, et al. Blood 2008, 111, 4690-4699), or by inhibiting Skp2 / Cksl interaction (L. Wu, (Ungermannova, et al., J. Biomol. Screen. 2013, 18, 910-920) have been developed.

The second pathway that Skp2 has on cancer development is to block p53-mediated apoptosis by inhibiting the interaction between p53 and p300. In other words, Skp2 binds to p300 and inhibits p300-mediated p53 acetylation by inhibiting the interaction between p300, a transactivator with acetyl transferase activity, and p53, a cancer-inhibiting protein. Since p53 acetylation affects the stabilization and activity of p53, Skp2 acts as a negative regulator of p53-mediated transcription and apoptosis, and this activity is mediated by ubiquitin-mediated protein degradation of Skp2 Is also referred to as the "non-proteolytic activity" of Skp2. Therefore, inhibition of Skp2 / p300 protein interactions can activate p53 and thus be used as an anti-cancer strategy.

However, until now, there has been no report on a technique for developing an Skp2 inhibitor that inhibits Skp2-p300 interaction and providing it as a cancer treatment agent.

C. H. Chan, et al. Cell, 154, 556-568 (2013) L. Wu, et al. Chem. Biol. 19, 1515-1524 (2012) Q. Chen, et al. Blood, 111, 4690-4699 (2008) D. Ungermannova, et al. J. Biomol. Screen. 18, 910-920 (2013)

Accordingly, the present invention provides a novel peptide compound which binds to Skp2 and inhibits Skp2 / p300 interaction. The novel peptoid compound of the present invention inhibits Skp2 / p300 interaction without affecting the proteolytic activity of Skp2, inducing p300-mediated p53 acetylation and activation, thereby inducing p53-mediated cell death and cancer cell death And can exhibit anticancer effects.

As one example, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof.

As another example, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof.

As another example, the present invention provides a method of treating cancer, comprising administering to the individual an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

Skp2 is a part of the SCF ^Skp2 ubiquitin ligase that not only regulates the degradation of cell cycle regulatory proteins but also blocks p53-mediated apoptosis by inhibiting p53 binding to p300, thus playing an important role in cancer development And is overexpressed in various cancers. The present invention provides novel peptoid compounds which bind Skp2 and inhibit the Skp2 / p300 interaction. The novel peptoid compounds of the present invention inhibit Skp2's interaction with p300 without affecting the proteolytic activity of Skp2 as inhibitors of the non-proteolytic activity of Skp2, Induce death. Therefore, unlike the conventional Skp2 inhibitors, the compounds of the present invention exhibit an activity of inhibiting Skp2 / p300 interaction, and can induce p53-mediated cell death and cell death in cancer cells, thereby exhibiting anticancer effects.

Accordingly, in one aspect, the invention relates to a compound of formula I or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

In this embodiment, the compound of formula 1 is referred to as "M1 ".

The compounds according to the invention can also form pharmaceutically acceptable salts. Such "pharmaceutically acceptable salts" include those acids which form non-toxic acid addition salts containing a pharmaceutically acceptable anion, for example inorganic acids such as sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid and the like; Organic carboxylic acids such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid and the like; Methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, and the like. In addition, pharmaceutically acceptable base addition salts include alkali metal or alkaline earth metal salts formed by, for example, lithium, sodium, potassium, calcium, magnesium and the like; Amino acid salts such as lysine, arginine and guanidine; Organic salts such as dicyclohexylamine, N-methyl-D-glucamine, tris (hydroxymethyl) methylamine, diethanolamine, choline, triethylamine and the like. The compound according to the present invention can be converted into its salt by a conventional method, and the preparation of the salt can be easily carried out by those skilled in the art based on the structure of the above formulas without further explanation.

In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer, which comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In another aspect, the invention relates to a method of treating cancer, comprising administering to the individual an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

As used herein, an effective amount refers to an amount of an effective ingredient capable of inducing a delay in the clinical symptom of cancer or an amount of an effective ingredient sufficient to inhibit, ameliorate, alleviate or treat cancer in an individual, And can be determined experimentally within the normal capability range.

The cancer includes all cancers in which the development, progression or metastasis of cancer is induced by overexpression of Skp2 or Skp2 / p300 protein interactions. The cancer may be a solid cancer or a blood cancer. The present invention relates to a method for the treatment and prophylaxis of cervical cancer, osteosarcoma, chronic or acute leukemia, lung cancer, peritoneal cancer, skin cancer, melanoma, rectal cancer, cholangiocarcinoma, esophageal cancer, small bowel cancer, endocrine cancer, But are not limited to, pancreatic cancer, glioma, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colon cancer, endometrial or uterine cancer, salivary cancer, kidney cancer, prostate cancer, mucin cancer, thyroid cancer, or head and neck cancer .

The subject may be an animal, preferably a mammal, particularly an animal, including a human, and may be an animal-derived cell, tissue, organs, and the like. The subject may be a patient requiring treatment.

The composition may further comprise an appropriate carrier, excipient and diluent commonly used in the production of a pharmaceutical composition. The composition may be formulated into an oral form such as a powder, granule, tablet, capsule, suspension, emulsion, syrup or aerosol Formulation, external preparation, suppository or sterile injection solution, and the like.

When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may include at least one excipient and / or lubricant. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

The preferred dosage of the composition varies depending on the condition and the weight of the patient, the degree of the disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. For a more preferable effect, the dose of the composition of the present invention is preferably 0.1 mg / kg to 20 mg / kg per day based on the active ingredient, but is not limited thereto. The administration may be carried out once a day or divided into several doses. The compositions of the present invention may be administered to a mammal, including a human, in various ways. All modes of administration may be expected, for example, by oral, intravenous, intramuscular, subcutaneous injection, and the like. The pharmaceutical dosage form of the composition of the present invention may be used in the form of a pharmaceutically acceptable salt of the active ingredient, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable combination.

The present invention provides novel peptoid compounds exhibiting anticancer effects by inhibiting Skp2 / p300 protein interaction, and is useful for the treatment of various cancerous diseases in which the development, progression or metastasis of cancer is induced by overexpression of Skp2 or interaction between Skp2 / . ≪ / RTI >

In the figures herein, the compound of formula (1) is referred to as "M1 ".
Fig. 1 (a) is a schematic representation of the function of Skp2 promoting ubiquitin-mediated protein degradation as a member of the SCF ^Skp2 ubiquitin ^ligating complex, (b) Skp2 inhibits the interaction between p53 and p300, Which is a function of inhibiting the human body.
2 (a) shows the process of constructing the cyclic peptide compound library, (b) shows the chemical structure of the primary amine used in the monomer building block, and (c) shows the ring opening and cleavage reaction .
3 is a schematic representation of an on-bead screening process for screening compounds that bind directly to the Skp1-Skp2 complex from a cyclic peptoid compound library.
Fig. 4 shows the result of mass spectrometry for the selected compound M1, (a) showing the MS result and (b) showing the MS / MS result.
Figure 5 is a schematic representation of the synthesis of fluorescently labeled compounds M1-FL and N1-FL.
(A) is a compound M1 and structure, and fluorescence-labeled compound of N1 M1-FL, and shows the structure of a N1-FL, (b) is the compound M1 K _D value for Skp1-Skp2 dimer of Figure 6 is combined with 3.85 μM One N1 indicates no binding.
7 shows MALDI-TOF mass spectrometry results of M1-FL and N1-FL.
(B) shows the result of cross reaction between Skp1-Skp2 dimer and M1-BD or N1-BD, (c) shows the result of cross-linking analysis between Skp1-Skp2 dimer and M1-BD.
9 shows MALDI-TOF mass spectrometry results of M1-BD and N1-BD.
10 shows the LC / MS analysis results of the purified M1.
Figure 11 shows the LC / MS analysis results of purified N1.
Fig. 12 (a) shows the results of western blotting in which the effect of M1 on the expression level of p21 and p27 in HeLa cells was observed, and Fig. 12 (b) shows the effect of M1 on p53 and acetylation in ADR (Adriamycin) (c) shows the results of alpha screen analysis confirming that M1 interferes with the Skp2 / p300 protein interaction. The error bars represent standard deviations from three independent experiments. (d) is an in vitro binding assay that shows the effect of M1 on the expression level of FLAG-tagged Skp2 and Myc-tagged p300. (e) shows that M1 in HeLa cells expresses Skp2 The results of western blot confirmed the effect.
13 (a) shows Western blotting results showing that M1 affects the expression level of p21 and p27 in SJSA-1 cells, (b) shows that M1 in adriamycin-treated SJSA- (C) shows Western blotting results confirming the effect of N1 (negative control) on the expression level of p21 and p27 in HeLa cells, and , and (d) show Western blotting results confirming the effect of N1 on the expression level of p53 in ADR (Adriamycin) -treated HeLa cells.
FIG. 14 (a) is a graphical illustration of competitive fluorescence anisotropy for Skp1-Skp2 / Cksl / fluorescently labeled p27 peptide (FAM-NAGSVEQ-pT-PKKPGLRRRQT) complex, 27 peptides of Skp1-Skp2-Cks1. (c) shows the concentration dependent curves of M1 and the unlabeled p27 peptide (NAGSVEQ-pT-PKKPGLRRRQT) in the fluorescence anisotropic direct binding assay for Skp1-Skp2-Cksl of FAM-27 peptide. The p27 peptide showed low micromolar inhibition ( K _i = 2.68 ± 0.57 μM), whereas M1 did not. The error bars represent standard deviations from two independent experiments.
15 (a) shows the results of the Caspase-Glo 3/7 assay kit for the effect of M1 on apoptosis in HeLa cells and WS-1 cells, (b) And the effect on cell viability is shown. The error bars represent standard deviations from two independent experiments.
Figure 16 shows the results of analysis of the effect of negative control N1 on cell viability in various cells. The error bars represent standard deviations from two independent experiments.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Experimental material

TentaGel S NH ₂ resin (90 μm, 0.29 mmol / g) was purchased from Rapp Polymere. Rink amide MBHA resin (0.96 mmol / g) was purchased from Novabiochem. Liquid chromatograph-mass spectrometer (LC-MS) analysis was performed using a C18 reversed-phase column (kinetex, 2 μm, 4.6 mm × 50 mm) on an Agilent 1200 LC / MS system (Agilent Technology). The solvent A was 95% water, 5% methanol, 0.01% TFA, and the solvent B was 0.8 ml / min for 2 minutes in a gradient elution of 90% A solvent and 14 minutes in 100% Methanol, 0.01% TFA). HPLC purification was performed using a C18 reverse phase column (Agilent Technology, 5 [mu] m, 25 mm x 125 mm) on an Agilent 1120 Compact LC system (Agilent Technology). A linear gradient was used from 10% B solvent to 100% B solvent while varying the solvent composition every 40 minutes. MALDI-TOF MS was performed using an ABI 4800 and ABI 5800 mass spectrometer (Applied Biosystems) and α-cyano-4-hydroxycinnamic acid was used as the substrate.

Example  2. Ring type Peptoid  Building a library with a 5-mer structure

Of TentaGel S NH ₂ resin solid (1 g, 0.35 mmol) was treated for 2 hours at DMF. To the resin was added 3- ((2-chloro-4-methoxyphenyl) propane-1-carboxylic acid in the presence of HOBt (5 equiv), HBTU (5 equiv) and DIPEA (10 equiv) in DMF Fmoc-amino) -3- (2-nitrophenyl) propanoic acid or Fmoc-6-aminohexanoic acid (5 equiv) and reacted at room temperature. After stirring for 2 h, the reaction mixture was removed and washed with DMF (3x), CH ₂ Cl ₂ (2x), MeOH (2x) and DMF (3x). DMF (2 x 10 min) was treated with 20% piperidine to remove the Fmoc group. Fmoc-Cys (Mmt) -OH (5 equiv) was added using the same peptide coupling conditions. After Fmoc deprotection, the resulting amine was bromoacetylated by treatment with 1 M bromoacetic acid (20 equiv) and 1 M DIC (20 equiv) in DMF at room temperature. The beads were equally divided into eleven 2-mL syringe reactors and 11 different amines were treated for amine substitution in each section. After washing the beads, all the beads were randomized into one vessel. This process was repeated until a 5 mer linear peptide library was synthesized. In the final step of peptide synthesis, cyanuric chloride (5 equiv) and DIPEA (6 equiv) were treated with anhydrous THF to make the N-terminus of the peptide to the triazine form and reacted overnight at room temperature. Then, 2% trifluoroacetic acid (TFA) and 5% triisopropylsilane (TIS) were treated with CH ₂ Cl ₂ (2 mL) to remove the Mmt group of the cysteine residues and reacted for 30 minutes. For the macrocyclization reaction, the peptides containing triazine attached to the beads were reacted with 10% DIPEA in DMF overnight at room temperature. In order to substitute methoxyethylamino groups for the remaining chloride groups in the triazine of the cyclized peptoids, methoxyethylamine (20 equiv.) Was treated in the presence of DIPEA (20 equiv.) In DMF and reacted overnight at 65 ° C. The beads with the final product attached thereto were treated with 95% TFA, reacted at room temperature for 2 hours, and neutralized with 10% DIPEA in DMF. As a result, in theory, it could be building a library containing cyclic peptoid compound 161,051 of the total species (= 11 ⁵⁾ (Fig. 2).

Example  3. Protein purification / Biotinylation  ( Biotinylation )

The screening target, Skp2 protein, was isolated and purified in the form of a protein duplex of Skp2? N-Skp1? (Deletion construct of human Skp1-Skp2 complex) through methods known in the literature [ Methods Enzymol . 2005, 398 , 125-142). In order to biotinylate the purified Skp2ΔN-Skp1ΔΔ protein, EZ-Link NHS-PEG4-Biotin labeling kit in 0.1 M PBS (phosphate-buffered saline, pH 7.2) (Thermo Scientific) for 2 hours at < RTI ID = 0.0 > 0 C. < / RTI > The labeled proteins were separated by dialysis with 1x PBS (phosphate-buffered saline, pH 7.2) buffer. The degree of labeling was determined using a Pierce Biotin Quantitation Kit (Thermo Scientific) (1.05 biotin per protein). Proteins such as Cks1 (amino acid residues of 5-73), Myc-p300 (aa 1514-1922) and His-p300 (aa 1514-1922th amino acid) were also purified as known in the literature.

Example  4. High efficiency Bead phase  High-throughput on-bead screening

In order to screen for cyclic peptoids binding to the target protein (Skp2ΔN-Skp1ΔΔ complex), three levels of affinity-based on-bead screening were performed, including alkaline phosphatase-based detection after two steps of magnetic separation 3). 250 mg (about 196,000 beads) of a library containing compounds bound to beads to prevent nonspecific binding prior to screening for the cyclic peptoid library against the target protein (Skp2ΔN-Skp1ΔΔ complex) was diluted 1 × TBS-T 0.1% gelatin and 2% BSA). The library beads were then incubated with streptavidin coupled Dynabeads (Life Technologies) for 1 hour at room temperature to remove beads associated with streptavidin. The remaining beads were incubated for 18 hours at 4 ° C with 500 nM of biotinylated Skp2ΔN-Skp1ΔΔ (biotinylated Skp2ΔN-Skp1ΔΔ). Proteins that did not form a bond were washed out and treated with streptavidin-coupled Dynabeads, and only positive beads were selected by magnetic separation. In the primary screening, hundreds of beads were screened. Selected beads were heated at 1% SDS to remove bound proteins. After washing, the beads were again incubated with 100 nM of biotinylated Skp2? N-Skp1? For 12 hours at 4 占 폚. The unbound protein was removed by washing the beads with 1 x TBS-T, and the positive beads were pulled with a strong magnet. From the secondary screening using magnetic separation, 20 beads were obtained as potential candidate compounds. After removing the bound protein, the beads were treated with 100 nM of biotinylated Skp2? N-Skp1? At 4 占 폚 for 4 hours and labeled with streptavidin-conjugated alkaline phosphatase. The beads were treated with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) to colorimetric detect alkaline phosphatase. The most blue colored beads were obtained as candidate compounds. The beads were treated with mCBPA (10 equiv.) And sodium bicarbonate (15 equiv) for 1 hour and 2M methoxyethylamine (20 equiv) in NMP to convert the cyclic peptoids of the selected beads to linear peptoids (Scheme S1). After UV irradiation (360 nm, rt, 1 hour), linearized peptoids derived from beads were analyzed by MS / MS. As a result, the M1 compound was selected as an effective compound (Fig. 4).

Example  5. Synthesis of fluorescently labeled compounds (M1-FL and N1-FL)

Rink amide MBHA resin (100 mg, 75 μmol) was treated with DMF (2 mL) for 1 hour. 20% piperidine in DMF (2 x 10 min) was treated to remove the Fmoc protecting group. Next, Fmoc-Lys (Alloc) -OH (5 equiv) and Fmoc-Abu-OH (5 equiv) were coupled under the same peptide coupling conditions. In the same manner as described for library synthesis, a cyclic peptide structure was synthesized (Figure 5). Pd (PPh ₃ ) ₄ (0.2 equiv) and PhSiH ₃ (10 equiv) in anhydrous CH ₂ Cl ₂ (1 mL) were treated for 2 hours to deprotonate Alloc and then the resulting Lys NH ₂ on the residue was coupled with 5,6-carboxyfluorescein (5 equiv). For cleavage, 1 mL of cut cocktail (95% TFA, 2.5% TIS and 2.5% water) was reacted against the beads at room temperature for 2 hours. The crude product was purified by HPLC (Fig. 6 (a), Fig. 7).

Example  6. M1- BD  And N1- BD's  synthesis

Biotinylated and DOPA-conjugated analogs of M1 and N1 were synthesized on a Rink amide MBHA resin by a procedure similar to M1-FL and N1-FL synthesis. Using peptide coupling conditions, the cyclic peptide intermediate 6 (FIG. 5) were coupled with Fmoc-Glu (biotinyl-PEG) -OH and Fmoc-DOPA (acetonide) -OH. After removing the Fmoc protecting group, acetic anhydride (6.75 equiv) and DIPEA (6.75 equiv) in DMF were reacted for 2 hours to acetylate the resulting amine group. To the beads, 1 mL of cut cocktail (95% TFA, 2.5% TIS and 2.5% water) was reacted at room temperature for 2 hours. The crude product was purified by HPLC. The purity and identity of M1-BD and N1-BD were analyzed by LC / MS (Fig. 8 (a), Fig. 9).

Example  7. Synthesis of M1 and N1

M1 and N1 were synthesized on a Rink amide MBHA resin by a procedure similar to library synthesis. In this case, Fmoc-Cys (Mmt) -OH (5 equiv) was first coupled via peptide coupling conditions. After removal of the Fmoc protecting group, the cyclic peptoid was synthesized. The final product was cleaved from the beads and purified by HPLC. The purity and identity of the compounds were analyzed by LC / MS (FIG. 10, FIG. 11).

Example  8. Fluorescence anisotropy

8-1. M1 and Skp1 - Of Skp2  Direct coupling

Fluorescently labeled compound M1-FL or N1-FL (40 nM each) was diluted in buffer solution (50 mM Tris, pH 7.5, 100 mM NaCl, 0.01%) to a final volume of 50 μL in a black Costar 384- tween-20) was used. And incubated with various concentrations of the Skp2? N-Skp1? Protein complex at room temperature for 30 minutes. Fluorescence anisotropy was measured using an EnVision multilabel reader (Perkin Elmer) to confirm the binding force. At this time, the excitation wavelength was set to 485 nm, and the emission was measured at 535 nm. K _D GraphPad Prism® v5.0 (San Diego, Calif.) Software. Y = Bmax x X / (K _D + X). The R squared value is 0.9877.

8-2. p27 Peptides and Skp1 - Skp2 - Cks1 and  Direct coupling

The fluorescence-labeled p27 peptide at the N-terminus (FAM-NAGSVEQ-pT-PKKPGLRRRQT) was purchased from Peptron. The binding force was measured using the fluorescently labeled compound p27 peptide (40 nM) in the same manner as above. The R squared value is 0.9858.

Example  9. Competition assay

The fluorescently labeled compound p27 peptide (40 nM) was added to a buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 0.01% tween-20) so that the final volume of the solution was 50 μL and 750 nM of Skp2ΔN-Skp1ΔΔ-Cks1 Protein complex. ≪ / RTI > After 30 minutes, various concentrations of M1 or unlabeled p27 peptide (NAGSVEQ-pT-PKKPGLRRRQT) were added. After further incubation for 45 min, the fluorescence anisotropy values were measured at 485 nm for excitation with an EnVision multilabel reader (Perkin Elmer) and at 535 nm for emission. The IC ₅₀ values of the unlabeled p27 peptide from fluorescence anisotropy analysis were obtained using linear regression and the following equation using GraphPad Prism v5.0 software:

Y = Bottom + (Top - Bottom) / (1 + 10 ^{X- LogIC50} )

The K _i values were calculated using the following equation:

K _i  = I ₅₀ / (L ₅₀ / K _D  + P ₀ / K _D  +1)

In this formula,

I- ₅₀ : inhibitor concentration indicating 50% inhibition,

L ₅₀ : a labeled ligand exhibiting 50% inhibition,

P ₀ : protein concentration indicating 0% inhibition, and

K _D : dissociation constant of protein-ligand complex (Anal. Biochem. 2004, 332, 261-273).

Example  10. Chemical cross-linking experiments

The cross-linking reaction was carried out in ½ Nuclear Extract Buffer (1/2 NEB) (10 mM HEPES, pH 7.5, 10% glycerol, 50 mM KCl, 6.25 mM MgCl 2, 0.1 mM EDTA, 1 mM DTT) . 1 μM of Skp2ΔN-Skp1ΔΔ and 0.5 μM of M1-BD or N1-BD reaction mixture was incubated at room temperature for 10 minutes and then NaIO ₄ adjusted to a final concentration of 5 mM was added. After 1 minute of incubation, the cross-linking reaction was stopped with a 6x SDS-PAGE loading buffer. Streptavidin-HRP or IRDye-Streptavidin (Li-COR). For competitive chemical cross-linking reactions, unlabeled M1 was added to the reaction mixture increasing the concentration (Fig. 8 (a)).

Example  11. Cell culture and Western Blat

Hela cells were cultured in DMEM medium containing 10% FBS, and SJSA-1 was cultured in RPMI 1640 medium containing 10% FBS. Cells were treated with the indicated concentrations of M1, N1 or DMSO for 24 hours. At this time, adriamycin (ADR) (0.5 μg / ml) was not added or added. The cells were then lysed in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM DTT, protease inhibitor cocktail tablet). After washing twice with PBS, cell lysates were separated on SDS-PAGE and collected for western blotting. The following antibodies were used: anti-p21 antibody (Santa Cruz), anti-p27 antibody (BD transduction laboratories), anti-actin antibody (Santa Cruz), anti- Cell signaling, and anti-skp2 antibody (Invitrogen).

Example  12. Alpha screen  analysis( AlphaScreen  assay)

Alpha screen analysis was performed on an OptiPlate-384 plate (Perkin Elmer). Purified GST-Skp2ΔN-Skp1ΔΔ protein (500 nM) and His-p300 (aa 1514-1922) protein (500 nM) were added to an AlphaLISA universal lysis buffer (Perkin Elmer) to a final volume of 15 μL. Next, M1, N1 or DMSO of the specified concentration was added, and the mixture was incubated at room temperature for 1 hour. Then, 5 μL of glutathione donor beads (Perkin Elmer) was added at about 0.02 mg / well. After 30 min incubation, Nickel chelate acceptor beads (Perkin Elmer) were added to the reaction mixture at 0.02 mg / ml / well and incubated for further 3 h in the presence of darkness. The energy transfer between the donor bead and the donor bead was measured at 615 nm with an EnVision multilabel reader (Perkin Elmer).

Example  13. In vitro binding assay

HEK293T cells were transfected with myc-p300 (1514-1922) expression plasmid DNA for 24 hours and lysed in c-myc lysis buffer (50 mM Tris, pH 8.0, 120 mM NaCl, 1% NP-40, 1 mM DTT) . Total lysates were incubated overnight at 4 ° C with anti-c-Myc agarose affinity gel (Sigma-Aldrich), and then lysed in lysis buffer and IPP150 buffer (100 mM Tris, pH 8.0, 150 mM NaCl, 0.1% NP- mM DTT). HEK293T cells were introduced into the FLAG-Skp2 expression plasmid for 24 hours and dissolved in FLAG lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% triton-x100). FLAG-Skp2 was purified from the total lysate using anti-FLAG M2 affinity gel (Sigma-Aldrich) according to the manufacturer's instructions and dialyzed against IPP150 buffer. For the binding reaction, 100 nM of FLAG-Skp was added to myc-p300 (1514-1922) conjugated to myc beads at room temperature for 1 hour under M1 of defined concentration. The beads were thoroughly washed with IPP150 buffer, and the precipitated proteins were subjected to Western blotting using anti-FLAG M2 (Sigma Aldrich) and anti-myc (Santa Cruz, Millipore) antibodies.

Example  14. Cell viability assay and Caspase  Caspase assay

For cell viability analysis, 1 × 10 ⁴ cells were plated in 96-well plates for 24 hours, washed with PBS, and treated with various concentrations of M1, N1 or DMSO in Opti-MEM medium for 24 hours . Cell viability was assayed using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay kit (Promega) according to the manufacturer's instructions. For apoptosis analysis, 5 × 10 ³ cells / well were plated on a white-walled 96-well plate for 24 hours, washed with PBS, and incubated in Opti-MEM medium with various concentrations of M1, N1 or DMSO was treated for 12 hours. Caspase 3/7 activity was measured using the Caspase-Glo 3/7 Assay kit (Promega) according to the manufacturer's instructions.

Experiment result

From the total 161,051 peptoid libraries constructed by the present inventors, cyclic peptoids binding to the target protein (Skp2ΔN-Skp1ΔΔ, a deletion construct of the human Skp1-Skp2 complex) were identified through affinity-based on-bead screening Were selected, and as a result, the M1 compound was selected as the final effective compound (hit compound). The structure of the M1 compound is represented by the following formula (1).

[Chemical Formula 1]

First, to evaluate the binding affinity of M1 through fluorescence anisotropy analysis, a fluorescently labeled derivative M1-FL and a negative control N1-FL were synthesized and purified by HPLC. M1-FL was bound to Skp2ΔN-Skp1ΔΔ in a concentration-dependent manner and the K _D value was 3.85 μM, whereas the negative control N1-FL did not show fluorescence (FIGS. 6 and 7). This supports that the backbone structure only minimally affects the binding affinity of < RTI ID = 0.0 > M1 < / RTI > for Skp2? N-Skp1?.

To investigate whether M1 directly binds to Skp1 or Skp2, dihydroxyphenylalanine (DOPA) -mediated chemical cross-linking experiments were performed. Biotinylated and were synthesized DOPA- combined derivative M1-BD was added (Fig. 8 (a), Fig. 9), NaIO _4, and incubated for 1 minutes with Skp2ΔN-Skp1ΔΔ. Analysis by gel electrophoresis and Western blotting using Streptavidin-HRP showed that the cross-linked product had a molecular weight of 36 kDa and exactly the same as Skp2, but no band corresponding to Skp1 (FIG. 8 (b)). These results support that M1 binds only to Skp2 and not to Skp1. In addition, no significant band was observed in the cross-linking reaction using the negative control N1-BD, indicating that the direct link between Skp2 and M1 is due to cross-linking and not by the influence of DOPA, biotin or linker. When M1 was added to the cross-linking reaction between M1-BD and Skp1-Skp2, the cross-linked product decreased in a concentration dependent manner (Fig. 8 (c)). This supports the binding of M1-BD and M1 to the same binding site of Skp2.

The above fluorescence anisotropy analysis and cross-linking experiments show that M1 is a direct Skp2-binding ligand and can act as an inhibitor of protein degradation mediated by Skp2 to increase cellular levels of substrate proteins such as p27 and p21 .

To investigate the effect of M1 on cancer cells, the HeLa cervical cancer cell line or SJSA-1 cell line was treated by M1 concentration and the cellular level of p27 and p21 was evaluated by Western blotting. As a result, it was confirmed that M1 increased the level of p21 in a dose dependent manner, but had no significant effect on p27 level. On the other hand, N1 did not affect its cellular level for both p21 and p27 (Fig. 12 (a), Fig. 13 (a) (c)). Because p27 and p21 are substrate proteins recognized by Skp2, if M1 inhibits Skp2-mediated protein degradation like the previously reported Skp2 inhibitors, then both p27 and p21 proteins should be increased, We also investigated whether M1 affects the proteolytic activity of Skp2, since we only increased p27 in spite of binding.

In vitro competition assay, Skp1-Skp2 / Cks1 / fluorescently labeled p27 peptide (FAM-NAGSVEQ-pT-PKKPGLRRRQT) complex was incubated with increasing concentrations of M1 followed by fluorescence anisotropy , It was concluded that M1 did not affect the proteolytic activity of Skp2 but binds to a specific surface region of Skp2 since no change in anisotropy was observed as in the cell test results (Figure 14) .

Therefore, the inventors hypothesized that M1 targets the p-300 binding site of Skp2 rather than the substrate binding site of Skp2, thereby inhibiting the Skp2 / p300 interaction. In addition, considering that p300 binds to p53 and induces activation through the acetylation of p53 and that p21 is a major transcription target of p53, M1 promotes p53-mediated transcription without affecting the level of p27 To increase the cellular level of p21. To test this hypothesis, we evaluated whether M1 affects the non-proteolytic function of Skp2 associated with p53 acetylation and stabilization. To this end, HeLa cells or SJSA-1 cells were incubated with various concentrations of M1 or N1 in the presence of ADR (Adriamycin). ADR is a DNA-damaging agent that increases the basal level of p53. Since Skp2 inhibits the activation of p53 induced by DNA damage, if M1 inhibits Skp2 activity, p53 levels will be increased by M1 treatment. Analysis of cell lysates by western blot revealed that M1 treatment significantly increased p53 level as well as acetylated p53, while N1 had no effect (Fig. 12 (b), Fig. 13 (b) (d). These results support that M1 inhibits Skp2 / p300 protein interaction and induces p300-mediated p53 acetylation.

We confirmed that M1 interferes with Skp2 / p300 protein interaction through AlphaScreen assay (amplified luminescent proximity homogeneous assay screen). Alpha screen analysis is a bead-based assay that can detect whether protein-protein interactions occur through specific binding when two proteins are in close proximity. GST-Skp2 and His-tagged p300 (where p300 is comprised of the 1514th-1929th amino acid, which contains the essential motif that binds to Skp2). Various concentrations of M1 or N1 were added and incubated for 1 hour. After the addition of glutathione donor beads and nickel chelate acceptor beads, the effect of each compound on the Skp2 / p300 interaction was evaluated by measuring the energy transfer between two beads. As a result, it was confirmed that M1 interferes with the interaction between Skp2 and p300 because M1 decreased the signal in a concentration-dependent manner (Fig. 12 (c)). Furthermore, in vitro binding assay using purified FLAG-tagged Skp2 and Myc-tagged p300 also confirmed that Western blot inhibits binding of Skp2 to p300 (Fig. 12 (d)). ). These results support that M1 inhibits Skp2 / p300 protein interaction.

In addition, since it is known that Skp2 stability is regulated by p300-mediated acetylation, it has been confirmed whether M1 decreases the cellular level of Skp2 by interfering with the Skp2 / p300 interaction. As a result, it was confirmed that M1 significantly decreased the level of Skp2 in a concentration-dependent manner in HeLa cells (Fig. 12 (e)). These results support that M1 inhibits the interaction between Skp2 and p300.

Considering that p53 plays an important role in apoptosis-mediated apoptosis through caspase activation, we examined whether M1 promotes p53-mediated apoptosis. HeLa cells or WS-1 normal human fibroblasts were treated with various concentrations of M1 and the cytotoxic activity was measured by the Caspase-Glo 3/7 assay kit. As a result, as expected, M1 induced caspase activity in HeLa cells but not WS-1 cells (Fig. 15 (a)). These results indicate that M1 can selectively act on toxic or malignant cells. In order to confirm the selective cytotoxicity of such M1, cell viability assay was performed on various cells. Cells were treated with various concentrations of M1, and cell viability was assessed by MTS cell proliferation assay. As a result, it was confirmed that M1 inhibited growth of cancer cells (SJSA-1, Jurkat T, HeLa cells) in a concentration-dependent manner, but did not affect normal cells, WS-1 cells. N1 showed no cell growth inhibitory effect (Fig. 15 (b), Fig. 16).

Taken together, M1 is an inhibitor of the non-proteolytic activity of Skp2, preventing Skp2 from interacting with p300, leading to p300-mediated p53 stabilization and apoptosis. Therefore, unlike the conventional Skp2 inhibitors, M1 exhibits an activity of inhibiting Skp2 / p300 interaction, and can induce p53-mediated cell death and cell death in cancer cells, thereby exhibiting anticancer effects.

Claims (5)

Claims 1. Compounds of the general formula (I) or a pharmaceutically acceptable salt thereof.
[Chemical Formula 1]

A pharmaceutical composition for the prophylaxis or treatment of cancer, comprising a compound of the formula (1) or a pharmaceutically acceptable salt thereof.
[Chemical Formula 1]

3. The pharmaceutical composition according to claim 2, wherein the composition further comprises a pharmaceutically acceptable carrier.
3. The pharmaceutical composition according to claim 2, wherein the cancer is cancer in which the development, progression or metastasis of cancer is induced by overexpression of Skp2 or interaction between Skp2 and p300 proteins.
The method of claim 2, wherein the cancer is selected from the group consisting of osteosarcoma, chronic or acute leukemia, lung cancer, peritoneal cancer, skin cancer, cholangiocarcinoma, esophageal cancer, small bowel cancer, endocrine adenocarcinoma, papillary cancer, adrenal cancer, urethral cancer, lymphoma, gastric cancer, pancreatic cancer, Wherein the composition is an ovarian cancer, a liver cancer, a bladder cancer, a breast cancer, a colon cancer, a uterine cancer, an salivary cancer, a kidney cancer, a prostate cancer, a mucin cancer, a thyroid cancer or a head and neck cancer.

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