Application of PGG in preparation of Pygo1 protein inhibitor

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of PGG to preparation of a Pygo1 protein inhibitor. The invention provides a polyphenol small molecular skeleton capable of inhibiting Pygo1. The PGG is finally selected by researching the crystal structure of the Pygo1 protein, selecting a target area on the protein, and screening and comparing numerous compounds by taking a small molecule drug as a target. Through verification, the PGG and the Pygo1 protein jointly form relatively strong binding affinity through non-polar and polar effects, and the binding affinity of the PGG and the Pygo1-PHD region is-11.96 kcal / mol. Cell experiments further confirm that the PGG has a significant inhibition rate on gastric cancer cells, can significantly promote apoptosis of the gastric cancer cells, and can significantly inhibit proliferation of the gastric cancer cells, and IC50 of the PGG acting on the gastric cancer cells for 24 hours is 44.66 [mu] m.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to the application of PGG in the preparation of inhibitors of Pygo1 protein. Background Art

[0002] Pygo1 (Pygopus homolog 1) was first discovered in a fruit fly model in 2002. It is closely related to embryonic development and has a highly conserved sequence in vertebrates. Currently, the main reports on the correlation between Pygo1 and human diseases are as follows: (1) Pygo1 plays an important regulatory role in embryonic development; (2) Pygo1 overexpression is associated with pathological cardiac hypertrophy, which can lead to cardiac hypertrophy, decreased cardiac function and increased cell size; (3) Pygo1 is associated with non-alcoholic fatty degeneration; (4) High expression of Pygo1 is closely related to the poor prognosis of patients with non-small cell lung cancer. The pathogenic mechanism of Pygo1 is mainly through the Wnt / β-catenin signaling pathway to exert regulatory functions. Summary of the invention

[0003] The purpose of the present invention is to provide application of PGG in preparing inhibitors of Pygo1 protein.

[0004] Pentagalloylglucose(PGG), molecular formula: C 41 H 32 O 26 , also known as 1,2,3,4,6-Penta-O-galloyl-β-D-glucose; 1,2,3,4,6-penta-O-galloyl-β-D-glucose or 1,2,3,4,6-pentahydroxyphenylglucose, is a polyphenol compound with a complex structure. The pure product is white crystals with a melting point of >250°C and a boiling point of 1365.7°C. It is soluble in organic solvents such as methanol, ethanol, and DMSO. PGG participates in regulating multiple biological processes such as inflammation, anti-cancer, anti-oxidation, and metabolic regulation by acting on multiple targets, including NF-κB, PI3K / Akt, p53, AMPK, MMPs, VEGF, etc.

[0005] The structural formula of PGG is as follows:

[0006]

[0007] The present invention also provides an inhibitor of Pygo1 protein, wherein the main component of the inhibitor is PGG.

[0008] PGG interferes with the binding of Pygo1-PHD region to histone H3K4 2 / 3The interaction affects the transcription of the downstream pathway ITGB1 / CD47, significantly inhibiting the expression of ITGB1 and CD47. The present invention provides PGG as a target for Pygo1 and histone H3K4 2 / 3 Application of combined blocking agents.

[0009] Preferably, the inhibitor further comprises pharmaceutically acceptable excipients. The drug can be prepared into oral preparations or injections; the oral preparations include but are not limited to capsules, tablets, granules, oral liquids; the injections include but are not limited to sterile powder for injection, water injection, sodium chloride or glucose intravenous infusion.

[0010] In the above-mentioned medicine, the oral preparation includes additives, and the additives are selected from at least one of fillers, diluents, disintegrants, binders, lubricants, glidants, surfactants, solvents, flavoring agents, stabilizers, colorants, and preservatives.

[0011] The fillers or diluents include sugars such as lactose, sucrose, glucose, mannitol, sorbitol, and dextrin; starches such as starch, pregelatinized starch, α-starch, and dextrin; celluloses such as microcrystalline cellulose, gum arabic, fenugreek gum, and dextran; and inorganic salts such as calcium sulfate, calcium hydrogen phosphate, medicinal calcium carbonate, light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminosilicate.

[0012] The lubricant, glidant or anti-adhesive agent includes stearic acid; metal stearate such as calcium stearate or magnesium stearate; talc; colloidal silica; micro powder silica gel, hydrogenated vegetable oil; polyethylene glycol, lauryl sulfate such as sodium lauryl sulfate or magnesium lauryl sulfate; silicate such as silicic anhydride or silicate hydrate, etc.

[0013] The adhesive comprises distilled water, ethanol of different concentrations, starch slurry, hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, polyvinyl pyrrolidone, polyethylene glycol and compounds similar to the above excipients.

[0014] The disintegrants include cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium or cross-linked carboxymethyl cellulose sodium; cross-linked polyvinyl pyrrolidone; and chemically modified starch / cellulose, such as carboxymethyl starch or carboxymethyl starch sodium.

[0015] The surfactant includes Tween-80, sodium lauryl sulfate, sodium stearate sulfonate and the like.

[0016] The antioxidants include sodium bisulfite, sodium pyrosulfite, sodium sulfite, dried sodium sulfite, sodium thiosulfate, ascorbic acid, methionine (methionine), thiourea, phosphoric acid, citric acid and the like.

[0017] The preservatives or antibacterial agents include benzoic acid and sodium benzoate, sorbic acid, ethanol, parabens (parabens), benzalkonium bromide, o-phenylphenol, benzyl alcohol, phenylethyl alcohol, sodium propionate, sorbic acid, eucalyptus oil, cinnamon oil and peppermint oil.

[0018] The flavoring agents include sweeteners such as saccharin sodium, aspartame, syrup, stevioside, mannitol, sorbitol, mannose, galactose, maltose, fructose, glucose, sucrose, etc.; sour flavoring agents such as citric acid, malic acid or tartaric acid; and aromatics such as fennel oil, peppermint oil, menthol, peppermint water, cinnamon oil, lemon essence, lemon oil and spices of various flavors.

[0019] According to a specific embodiment of the present invention, the above inhibitor promotes apoptosis of gastric cancer cells, or the inhibitor inhibits proliferation of gastric cancer cells.

[0020] The method of using the above inhibitor includes administering an effective amount of the above drug to a subject, which can be administered orally, intravenously or transdermally to a patient in need of treatment.

[0021] Pharmaceutically effective amount refers to a reasonable benefit / risk ratio that can be obtained by applying drug treatment, and a quantity sufficient to treat the disease. The level of effective dose can be determined depending on some factors, including the patient's disease type, severity, activity of the drug, sensitivity to the drug, application time, route of administration, excretion rate, treatment cycle, drugs used simultaneously, and other factors known in the medical field. The drug of the present invention can be used as an independent therapeutic agent, or combined with other therapeutic agents. In addition, the composition of the present invention can be added with typical therapeutic agents continuously or simultaneously, and the composition can be applied once or multiple times. It is important to consider all the above factors and apply the minimum dose that can produce the maximum effect without side effects, and the dosage can be determined by the physician according to the patient's condition, age, etc.

[0022] Beneficial effects of the present invention:

[0023] The invention provides a natural polyphenol skeleton molecule capable of inhibiting Pygo1 protein.

[0024] The present invention studies the crystal structure of Pygo1 protein, selects the target region on the protein, takes small molecule drugs as the target, screens and compares many compounds, and finally selects PGG. It is verified that PGG forms a strong binding affinity with Pygo1 protein through non-polar and polar effects. The binding affinity of PGG to Pygo1-PHD region is -11.96kcal / mol, and it interferes with the binding of Pygo1 to histone H3K4. 2 / 3The interaction affects the transcription of the downstream pathway ITGB1 / CD47 and can serve as a regulator of ITGB1 and CD47 targets.

[0025] Cell experiments further confirmed that PGG has a significant inhibitory rate on gastric cancer cells, can significantly promote the apoptosis of gastric cancer cells, and can significantly inhibit the proliferation of gastric cancer cells. The IC50 of PGG acting on gastric cancer cells for 24 hours is 44.66μm. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 Schematic diagram showing the analysis of the Pygo1-PHD region structure, which contains two binding pockets, A and B.

[0028] Figure 2 Showing the inhibitory effects of 10 small molecules on gastric cancer cells.

[0029] Figure 3 The effect of PGG on the transcription of the ITGB1 / CD47 pathway is shown.

[0030] Figure 4 The binding mode of PGG to the Pygo1-PHD region is shown.

[0031] Figure 5 The 24h IC50 of PGG on HGC27 cells is shown.

[0032] Figure 6 Showing the inhibition rate of PGG on gastric cancer cell HGC27.

[0033] Figure 7 The 24h apoptosis rate of HGC27 cells treated with PGG was shown.

[0034] A is the flow cytometry image; B is the 24h apoptosis statistics of HGC27 cells.

[0035] Figure 8 The results showed that PGG has an inhibitory effect on the proliferation of gastric cancer cells HGC27. DETAILED DESCRIPTION

[0036] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0037] H3K4me 2 / 3 :H3K4me 2 and H3K4me 3 Refers to the dimethylation of histone H3 on the fourth lysine residue (K4) 2 ) and trimethylation (me 3 ) modifications. These modifications are an important form of epigenetics and have multiple biological functions. These modifications are related to gene activation and chromatin structure remodeling, and their abnormal regulation is related to the occurrence and development of various diseases (such as cancer).

[0038] BCL9 (B-cell lymphoma 9): is a protein related to cell signal transduction, especially the Wnt signaling pathway. BCL9 mainly acts as one of the regulators of the Wnt signaling pathway. The Wnt signaling pathway plays an important role in cell proliferation, differentiation, development, and stem cell maintenance. BCL9 helps form a transcriptional activation complex by binding to β-catenin, thereby enhancing the expression of Wnt target genes. BCL9 has been found to be upregulated in many types of cancer (such as colon cancer, breast cancer, lung cancer, etc.), suggesting its role in tumor occurrence and progression. As a factor that promotes tumor cell proliferation and survival, it may become a potential target for anti-cancer treatment.

[0039] Example 1

[0040] 1.1 Screening of candidate compounds

[0041] This example studies the crystal structure of Pygo1 protein (pdb ID: 2VPG). The PHD domain of Pygo1 protein is as follows Figure 1 As shown, region A has a distinct groove structure, while region B is relatively smooth. The PHD domain can simultaneously bind to H3K4me 2 / 3 It interacts with the HD1 domain of BCL9. Based on this structural feature, the screening strategy is to use the A region as the main target and small molecule drugs as the target, in order to screen out potential drug molecules that interact with the PYGO1 protein.

[0042] The present invention first uses a self-built anti-tumor small molecule database as a screening library. The software version (2020-2) was used for semi-flexible docking screening, and 94 lead compounds were screened out from a large number of compounds with binding affinity less than -5 kcal / mol.

[0043] Based on experience and in combination with the suggestions of TCM prescriptions for treating gastric cancer, the inventors focused on small molecules derived from astragalus, licorice, rhubarb, peony, etc., and the final small molecules verified were mainly the 10 species shown in Table 1.

[0044] Table 1 Compounds obtained by preliminary screening

[0045] Compound Name Binding affinity (kcal / mol) 1,2,3,4,6-Pentahydroxyphenylglucose (Pentagalloylglucose, PGG) -11.96 Sennoside A -7.70 Isoviolanthin -7.07 Rutin -6.99 Astragaloside II -6.17 Isoschaftoside -5.59 Schaftoside -5.45 Anthraglycoside B -5.63 Glycyram -5.63 Albiflorin -5.78

[0046] The above 10 small molecules have been preliminarily validated for their efficacy in gastric cancer cells. Figure 2 As shown, the validation results showed that only PGG had a significant inhibitory effect on gastric cancer cells.

[0047] Example 2 Verification of PGG Interference with Pygo1-PHD Region and Histone H3K4 2 / 3 Interaction affects the transcription of the downstream pathway ITGB1 / CD47

[0048] Different concentrations (0.0 μM, 20 μM, 40 μM) of compounds were applied to gastric cancer cell HGC27 for 24 hours, and the cells were collected for real-time fluorescence quantitative PCR detection. Total RNA was extracted from cells using TRIzol reagent (Invitrogen, New York). Reverse transcription was performed using PrimeScript containing gDNA removal enzyme. TM RT kit (Takara, Japan). For RT-qPCR analysis, SYBR Premix Ex Taq TM II (Tli RNaseH Plus, Takara, Japan), and the reaction was performed on the QuantStudio Dx Real-Time PCR System (Thermo Fisher, USA). GAPDH was used as a reference gene for normalization, and the relative expression level was calculated using 2 -ΔΔCt The primer sequences are shown in Table 2. The test results were analyzed and statistically analyzed using Graphpad.

[0049] Table 2 List of gene amplification sequences

[0050]

[0051]

[0052] like Figure 3As shown in the results, PGG can significantly inhibit the expression of ITGB1 and CD47, and the difference is statistically significant. Therefore, PGG can be used as a regulator of ITGB1 and CD47 targets.

[0053] The effects of the compounds in Table 1 on the transcription of ITGB1 / CD47 were verified by the same method. The results showed that other compounds did not have the effect of significantly inhibiting the expression of ITGB1 and CD47.

[0054] Example 3 Analysis of the action sites of compounds inhibiting the PYGO1 protein PHD region

[0055] Pygo1 (PDB ID: 2VPB) was downloaded from the PDB database as the target structure for molecular docking. The software's Prep Wiz module was used for preprocessing. The Pygo1 binding pocket was determined by reference. Ligand preparation: The small molecule structure of the self-built tumor small molecule library was processed by LigPrep, and the corresponding low-energy conformation was obtained using the MMFFs force field. Epik28 assigned ionization states and performed docking calculations at a pH of 7.0±2.0. Molecular docking was performed using Glide's extra precision method (standard precision, XP).

[0056] The binding modes of the compounds to the Pygo1-PHD region were visualized using Discovery Studio software.

[0057] like Figure 4 As described, the compound PGG forms a hydrophobic interaction with the Tyr379 and Ile357 residues of the Pygo1 protein; PGG forms hydrogen bonds with the residues Gln354, Asp355, Ala356, Arg369, Tyr379, Leu382, Thr383, Glu385, Ala386 and Ala388 of the PYGO1 protein, and PGG also forms a π-π stacking effect with the side chain of the PYGO1 protein residue Tyr379 through the terminal pyrogallol group. The above-mentioned non-polar and polar interactions together provide a strong binding affinity for the binding of PGG and Pygo1 protein (Affinity = -11.96 kcal / mol).

[0058] Therefore, based on the binding mode of PGG to the Pygo1-PHD region and the binding affinity to the Pygo1 protein, PGG was selected from the primary screening compounds in Table 1.

[0059] Example 4 Experimental verification of the effect of PGG on gastric cancer cell lines

[0060] (1) IC50 of PGG on gastric cancer cells HGC27

[0061] Different concentrations (0.0μM, 0.625μM, 1.25μM, 2.5μM, 5.0μM, 10μM, 20μM, 40μM, 80μM) of PGG were acted on gastric cancer cells HGC27 for 24 hours, cck8 was added, and incubated for 2 hours. The absorbance at 480nm was detected by ELISA, and Graphpad was used for analysis and statistics.

[0062] like Figure 5 As shown, the IC50 of PGG for 24 hours on gastric cancer cells is 44.66 μM.

[0063] (2) Inhibition rate of PGG on gastric cancer cell HGC27

[0064] Different concentrations (0.0μM, 0.625μM, 1.25μM, 2.5μM, 5.0μM, 10μM, 20μM, 40μM, 80μM) of PGG were acted on gastric cancer cells HGC27 for 24 hours, cck8 was added and incubated for 2 hours, the absorbance at 480nm was detected by ELISA, and Graphpad was used for analysis and statistics.

[0065] like Figure 6 As shown, compared with the control group, the average inhibition rate of PGG on gastric cancer cells at a concentration of 0.625 μM was less than 4%, the average inhibition rate of 1.25 μM was less than 10%, the average inhibition rate of 2.5 μM was less than 15%, the average inhibition rate of 5.0 μM was less than 21%, the average inhibition rate of 10 μM was less than 58%, the average inhibition rate of 20 μM was less than 72%, the average inhibition rate of 40 μM was 90%, and the average inhibition rate of 80 μM reached 94%, indicating that PGG has a significant inhibition rate on gastric cancer cells.

[0066] (3) Pro-apoptotic effect of PGG on gastric cancer cell HGC27:

[0067] Different concentrations (0.0μM, 10μM, 20μM, 40μM) of PGG were applied to gastric cancer cells HGC27 for 24 hours, and the apoptosis level was detected by flow cytometry, and analyzed and statistically performed using Graphpad.

[0068] like Figure 7 As shown, the average apoptosis-promoting rates of PGG at 10μM, 20μM, and 40μM were 4%, 7%, and 9%, respectively. The average apoptosis-promoting and necrosis rates of PGG at 10μM, 20μM, and 40μM were 9%, 15%, and 16%, respectively, indicating that PGG can significantly promote apoptosis and necrosis of gastric cancer cells.

[0069] (4) The inhibitory effect of PGG on the proliferation of gastric cancer cells HGC27

[0070] The monolayer cells in the logarithmic growth phase were routinely digested and centrifuged to collect the cell pellet. The cell pellet was resuspended, counted, and diluted to 1×10 3 Pieces / mL.

[0071] Inoculate 1000 cells per well in a six-well plate, add 1 mL of culture solution, wait until the cells adhere overnight and add drugs the next day, the drug concentrations are 0.0 μM, 5 μM, 10 μM, 20 μM. Culture in an incubator and let stand for 2-3 weeks. When visible clones appear in the culture dish, stop culturing, discard the culture solution, carefully wash with PBS 2-3 times, and discard the PBS. Add 1 ml of 4% paraformaldehyde to each well, fix for 15-30 minutes, and discard the fixative.

[0072] Add 1 mL of 0.1% crystal violet staining solution to each well and stain for 10-30 minutes. Slowly wash away the staining solution with running water, air dry, and take pictures.

[0073] like Figure 8 As shown, PGG at 20 μM can significantly inhibit the number of clones formed and the size of clone spots, indicating that PGG can significantly inhibit the proliferation of gastric cancer cells.

[0074] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. Application of PGG in the preparation of inhibitors of Pygo1 protein.

2. An inhibitor of Pygo1 protein, characterized in that The main component of the inhibitor is PGG.

3. The inhibitor according to claim 2, characterized in that The inhibitor further includes pharmaceutically acceptable excipients.

4. The inhibitor according to claim 3, characterized in that The pharmaceutically acceptable excipients include any one or more combinations of diluents, excipients, disintegrants, fillers, binders, lubricants, flavoring agents, surfactants, and stabilizers.

5. The inhibitor according to claim 4, characterized in that The inhibitor promotes apoptosis of gastric cancer cells, or the inhibitor inhibits proliferation of gastric cancer cells.

Images