Use of a small molecule compound YZL-51N in preparation of a SIRT7 selective inhibitor

Abstract

The application provides application of a small molecule compound YZL-51N in preparation of a SIRT7 selective inhibitor. The application finds that the small molecule compound YZL-51N can be used as a specific small molecule inhibitor of SIRT7. The application verifies that the compound YZL-51N can effectively inhibit SIRT7 deacetylase activity by using polypeptide deacetylation. The application also proves that YZL-51N has stronger interaction affinity with SIRT7 compared with other sirtuins family members by a BLI experiment method. The application verifies that the small molecule compound inserts into a pocket and interacts with hot spot amino acids by a molecular docking experiment. In a cell model, YZL-51N can also specifically inhibit SIRT7 and replenish the substrate. Under ionizing radiation stimulation, the compound can effectively inhibit a SIRT7-ATM cell signal pathway.

Description

Technical Field

[0001] This invention relates to the fields of novel small molecule compounds and pharmaceuticals, and particularly to the application of a small molecule compound, YZL-51N, in the preparation of a selective inhibitor of SIRT7. Background Technology

[0002] Silent information regulators (sirtuins) are a class of highly conserved NAD+ receptors. + SIRT7 is a histone deacetylase family protein. Among human-derived sirtuins, the gene sequence of SIRT7 was first reported in 2000. With ongoing research, researchers have discovered that SIRT7 is an important target for various cell biological activities, such as rDNA stabilization and rRNA maturation, DNA damage repair, genome stability, and cellular senescence. For example, abnormal SIRT7 function can cause some embryonic lethality, premature aging, and fatty liver. SIRT7 is also highly expressed in various malignant tumors, including colorectal cancer, pancreatic cancer, and breast cancer. SIRT7 also participates in important DNA damage repair processes in the cell nucleus. In the early stages of DNA damage, SIRT7 is recruited to the damage site via PARP1, and after deacetylation of the H3K18 site, it recruits 53BP1 to participate in non-homologous end joining repair. Simultaneously, SIRT7 can desuccinylate the H3K122 site, making the chromatin structure more compact. In the later stages of DNA damage repair, SIRT7 inactivates ATM by deacetylation, ensuring the completion of DNA damage repair. Therefore, developing novel small molecule inhibitors targeting SIRT7 and achieving effective chemical intervention are of significant biological importance.

[0003] However, existing small molecule inhibitors targeting SIRT7 still face numerous challenges. Due to a lack of suitable screening systems, detailed crystal structure analysis, and target selectivity studies, progress in SIRT7 inhibitor development remains slow. For example, although NAD... + The hydrolysis product NAM is used to inhibit SIRT7 activity, but its feedback inhibition mechanism allows NAM to simultaneously inhibit all sirtuins. Compound ID: 97491 was the first small molecule inhibitor of SIRT7 screened from a library of compounds with known structures; however, the compound lacks selectivity analysis validation and has a controversial catalytic substrate. Additionally, two previously reported small molecule inhibitors of SIRT7 cyclic peptides were found to also inhibit the deacetylation of SIRT1 and SIRT6. The discovery of the recently reported compounds 2800Z and 40569Z relied too heavily on computer-aided screening and lacked experimental validation of drug-target interactions.

[0004] Therefore, existing technologies still need improvement. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide an application of the small molecule compound YZL-51N in the preparation of SIRT7 selective inhibitors, aiming to solve the problem of insufficient specificity of SIRT7 inhibitors in the prior art.

[0006] The technical solution of the present invention is as follows:

[0007] The application of a small molecule compound YZL-51N in the preparation of a selective inhibitor of SIRT7, wherein the structural formula of the small molecule compound YZL-51N is as follows:

[0008]

[0009] In the aforementioned application, the small molecule compound YZL-51N specifically inhibits the deacetylase activity of SIRT7 in vitro.

[0010] In the aforementioned application, the small molecule compound YZL-51N specifically binds to the enzyme active site pocket of SIRT7.

[0011] In the aforementioned application, the amino acids in which the small molecule compound YZL-51N interacts with the enzyme active site pocket of SIRT7 via hydrogen bonding include one or more combinations of Asn297, Gly268, Leu298, Thr112, Asp118, Cys315, Gln299, Pro117, Lys314, Gly109, Val296, and Ser269.

[0012] In the aforementioned application, the small molecule compound YZL-51N selectively inhibits SIRT7 activity in cells and replenishes the catalytic substrate of SIRT7.

[0013] In the aforementioned application, the catalytic substrate of SIRT7 includes H3K18ac.

[0014] The application of a small molecule compound YZL-51N in screening SIRT7-targeting drugs, wherein the structural formula of the small molecule compound YZL-51N is as follows:

[0015]

[0016] In the aforementioned applications, the drug further includes a pharmaceutically acceptable carrier and / or excipient.

[0017] The application of a small molecule compound, YZL-51N, in studying the SIRT7-ATM molecular signaling pathway, wherein the structural formula of the small molecule compound YZL-51N is as follows:

[0018]

[0019] In the aforementioned application, the small molecule compound YZL-51N intervenes in the SIRT7-ATM molecular signaling pathway within cells.

[0020] Beneficial Effects: This invention provides the application of the small molecule compound YZL-51N in the preparation of a selective inhibitor of SIRT7. The invention utilizes biomembrane layer interference (BLI) experiments to detect a strong binding affinity between this small molecule compound and SIRT7. Molecular docking calculations show that this compound can bind to the active pocket of SIRT7 and its interacting amino acids. Furthermore, this invention also verifies in cells that the small molecule compound can selectively inhibit SIRT7 activity and replenish its catalytic substrate. Moreover, this compound can also interfere with the SIRT7-ATM molecular signaling pathway. Therefore, this invention provides for the first time the application of compound YZL-51N as a highly selective small molecule inhibitor of SIRT7. Attached Figure Description

[0021] Figure 1 The image shows the hydrogen nuclear magnetic resonance spectrum of compound YZL-51N in this embodiment of the invention.

[0022] Figure 2 The image shows the carbon NMR spectrum of compound YZL-51N in this embodiment of the invention.

[0023] Figure 3 This is a schematic diagram showing the dose-response effect of compound YZL-51N in inhibiting SIRT7-catalyzed deacetylation of fluorescent peptide substrates in an embodiment of the present invention.

[0024] Figure 4 This is a schematic diagram showing the dose-response effect of compound YZL-51N in inhibiting SIRT7-catalyzed deacetylation of non-fluorescent peptide substrates in an embodiment of the present invention.

[0025] Figure 5 This is a schematic diagram showing the selective catalytic activity of compound YZL-51N on SIRT7 enzyme in an embodiment of the present invention.

[0026] Figure 6 This is a schematic diagram showing the selective affinity of compound YZL-51N for SIRT7 in an embodiment of the present invention.

[0027] Figure 7This is a schematic diagram showing the results of the docking verification of the binding amino acid sites between compound YZL-51N and SIRT7 molecules in the embodiments of the present invention.

[0028] Figure 8 This diagram illustrates the results of the specific inhibition of SIRT7 by compound YZL-51N and the replacement of its substrate in a cell model, according to the present invention.

[0029] Figure 9 This is a schematic diagram showing the results of compound YZL-51N intervening in the SIRT7-ATM signaling pathway in an embodiment of the present invention. Detailed Implementation

[0030] This invention provides the application of the small molecule compound YZL-51N in the preparation of a selective inhibitor of SIRT7. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0031] This invention provides an application of the small molecule compound YZL-51N in the preparation of a selective inhibitor of SIRT7.

[0032] In some embodiments, the small molecule compound methyl(Z)-2,4-bis(3,4-dihydroxyphenyl)-4-oxobut-2-enoate is named Periplanol F(YZL-51N), with the following structural formula:

[0033]

[0034] In some embodiments, the small molecule compound YZL-51N specifically inhibits the deacetylation activity of SIRT7 in vitro. This invention has found that compound YZL-51N inhibits SIRT7-catalyzed deacetylation of fluorescent or non-fluorescent peptide substrates with a dose-dependent effect. Furthermore, YZL-51N inhibits SIRT7 deacetylation activity without affecting the enzymatic activity of other sirtuins family proteins.

[0035] In some embodiments, the small molecule compound YZL-51N specifically binds to the enzyme active site pocket of SIRT7. This invention has revealed that compound YZL-51N exhibits selective affinity for SIRT7. YZL-51N possesses good binding affinity to SIRT7 (K... D =3.3μM), which is higher than the binding force of YZL-51N to SIRT1 / 2 / 6.

[0036] Specifically, the amino acids in which the small molecule compound YZL-51N interacts with the enzyme active site pocket of SIRT7 via hydrogen bonding include one or more combinations of Asn297, Gly268, Leu298, Thr112, Asp118, Cys315, Gln299, Pro117, Lys314, Gly109, Val296, and Ser269.

[0037] This invention predicts the potential SIRT7 crystal structure (Uniprot Q9NRC8) using the AlphaFold protein structure website (https: / / alphafold.ebi.ac.uk / ). Then, ligand docking energies are calculated using GLIDE software, and simulations using PyMol software reveal the binding pocket and hotspot amino acids for YZL-51N with SIRT7. Based on the molecular docking results, YZL-51N can bind to the active site pocket of the SIRT7 enzyme, and the amino acids with hydrogen-bonded interactions include Asn297, Gly268, Leu298, Thr112, Asp118, Cys315, Gln299, Pro117, Lys314, Gly109, Val296, and Ser269.

[0038] In some embodiments, the small molecule compound YZL-51N selectively inhibits SIRT7 activity in cells and replenishes SIRT7's catalytic substrate.

[0039] Specifically, the catalytic substrate of SIRT7 includes H3K18ac.

[0040] This invention reveals that compound YZL-51N specifically inhibits SIRT7 deacetylase activity and replenishes its substrate in a cell model. The levels of H3K18ac, H3K9ac, H3K14ac, Histone 3, SIRT7, SIRT6, SIRT1, and Tubulin were examined. In HCT116 cells, compound YZL-51N inhibited SIRT7 enzyme activity and replenished H3K18ac levels without affecting H3K9ac and H3K14ac levels. Furthermore, YZL-51N itself did not affect the expression of SIRT7, SIRT6, and SIRT1. Moreover, in HT29 cells, YZL-51N exhibited a similar biological function of inhibiting SIRT7 enzyme activity. These results indicate that YZL-51N can effectively inhibit SIRT7 enzyme activity in a colon cancer cell model, thus replenishing H3K18ac levels.

[0041] This invention also provides an application of the small molecule compound YZL-51N in screening SIRT7-targeting drugs.

[0042] In some embodiments, the drug further includes a pharmaceutically acceptable carrier and / or excipient.

[0043] This invention also provides an application of the small molecule compound YZL-51N in the study of the SIRT7-ATM molecular signaling pathway.

[0044] In some embodiments, the small molecule compound YZL-51N interferes with the SIRT7-ATM molecular signaling pathway within cells.

[0045] This invention also provides an application of the small molecule compound YZL-51N in the preparation of small molecules or formulations targeting SIRT7.

[0046] This invention employs fluorescent peptide screening technology to initially screen for SIRT7 inhibitors, and determines their dose-activity relationship using parallel peptide assays with both fluorescent and non-fluorescent peptide substrates. The binding constant of the compound to SIRT7 is calculated using BLI experiments. Intracellular validation of the compound's inhibitory effect on SIRT7 activity and its influence on its substrate is conducted. Finally, the effect of YZL-51N on inhibiting SIRT7 and intervening in the SIRT7-ATM molecular pathway under IR irradiation-induced DNA damage is investigated. Ultimately, this invention discovers that the small molecule compound YZL-51N can act as a specific small molecule inhibitor of SIRT7. This invention utilizes peptide deacetylation to verify that compound YZL-51N can effectively inhibit SIRT7 deacetylases. This invention also demonstrates, using BLI experiments, that YZL-51N has a stronger affinity for SIRT7 compared to other sirtuins family members. This invention uses molecular docking experiments to confirm the small molecule compound's insertion into pockets and interaction hotspot amino acids. In cell models, YZL-51N can also specifically inhibit SIRT7 and replenish its substrate. Under ionizing radiation stimulation, this compound can effectively inhibit the SIRT7-ATM cell signaling pathway.

[0047] The application of the small molecule compound YZL-51N in the preparation of SIRT7 selective inhibitors is further explained below through specific embodiments:

[0048] Example 1

[0049] Extraction, separation, and structural identification of the compounds described in this embodiment:

[0050] 30 kg of American cockroaches were crushed and extracted three times by reflux with 70% ethanol for 2 hours each time. After filtration, the ethanol was recovered under reduced pressure to obtain an aqueous suspension. The suspension was then extracted three times with an equal volume of ethyl acetate to obtain 230 g of the ethyl acetate extract. The extract was then subjected to MCI gel CHP 20P column chromatography with methanol / water elution at gradients of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0. The fractions were combined by TLC analysis to obtain six fractions of Fractions AF. Fr.D 30g was separated by Sephadex LH-20 column chromatography, eluted with methanol, and TLC analysis showed that the fractions were combined to obtain four fractions, D1-D4. Fr.D2 10g was further separated by MCIgel CHP 20P column chromatography, eluted with methanol / water at gradients of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, and 100:0. TLC analysis showed that fractions with the same composition were combined to obtain Fractions. Eight fractions, D2.1-D2.8, were further analyzed by RP-18 column chromatography on 1.5g of Fr.D2.3 using methanol / water elution gradients of 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:65, 50:50, and 100:0. TLC analysis and combining of identical fractions yielded six components, D2.3.1-D2.3.6. 150mg of Fr.D2.3.5 was further purified by semi-preparative RP-18 HPLC using an acetonitrile-water solvent system with a composition of 15:85, yielding compound YZL-51N, weighing 1.5mg, with a retention time t0. R =15.5min.

[0051] The structure of the compound was identified as follows:

[0052]

[0053] Compound YZL-51N (Periplanol F), yellow solid, ESIMS: m / z 329 [M–H] – HRESIMS m / z:[M–H] – Calculated for C 17 H 14 O7,329,0667; Found 329.0664; 1H NMR (600MHz, CD3OD) δ7.54(dd,J=8.3,2.2,H-6,1H),7.46(d,J=2.2,H-2,1H),7.38(s,H-8,1H),7.05(d,J=2.3,H -2′,1H),7.01(dd,J=8.4,2.3,H-6′,1H),6.87(d,J=8.3,H-5,1H),6.83(d,J=8.4,H-5′,1H),3.90(s,OCH3,3H); 13 C NMR(150MHz,CD3OD)δ188.9(s,C-7),172.1(s,C-8′),152.7(s,C-3),149 .8(s,C-7′),149.0(s,C-4′),146.9(s,C-3′),146.8(s,C-4),131.2(s,C -1),126.8(s,C-1′),123.6(d,C-6),120.9(d,C-6′),118.9(d,C-8),116 .7(d,C-5′),116.1(d,C-2),116.0(d,C-5),114.9(d,C-2′),53.0(OCH3).

[0054] The proton NMR spectrum of the compound is shown below. Figure 1 As shown, the carbon spectrum is as follows Figure 2 As shown.

[0055] Example 2

[0056] Compound YZL-51N inhibits SIRT7-catalyzed deacetylation of fluorescent peptides and exhibits a dose-response effect.

[0057] In this embodiment, to detect the in vitro deacetylation reaction of SIRT7, a 50 μL reaction system included 50 ng / μL SIRT7 and 0.5 mM NAD. + 10 μM peptide ARTKQTARKSTGGKAPRK(MCA)QLAGGK, 0.1-100 μM YZL-51N, and buffer (10 mM Tris-HCl, 4 mM MgCl2, 0.2 mM DTT, and 10% Glycerol, pH 8.0) were mixed and incubated at 37°C for 90 minutes. Fluorescence signals at 260 nm excitation and 300-600 nm emission were detected using a Synergy H4 Hybrid Reader (Biotek). The IC50 was calculated by statistically analyzing the fluorescence signals at 260 nm excitation and 450 nm emission. 50 value.

[0058] The results are as follows Figure 3As shown in Figure A, compared to the control group, the fluorescence intensity signal decreased with increasing YZL-51N concentration, indicating a decrease in SIRT7 deacetylase activity. This demonstrates a negative correlation between YZL-51N concentration and SIRT7 activity. Figure B shows the IC50 of YZL-51N for SIRT7. 50 The value is 12.71 μM.

[0059] Example 3

[0060] Compound YZL-51N inhibits SIRT7-catalyzed deacetylation of non-fluorescent peptide substrates and exhibits a dose-dependent effect.

[0061] In this embodiment, dot hybridization was used to calculate the change in SIRT7 enzyme activity by statistically analyzing the deacetylation gray value of the peptide. The 50 μL reaction system contained 50 ng / μL SIRT7 and 0.5 mM NAD. + 10 μM Biotin-H3K18ac peptide, 3.12–50 μM YZL-51N, and the above reaction buffer were mixed and incubated at 37°C for 90 minutes. The sample was filtered through a spotter onto a PVDF membrane and then blocked with 5% skim milk for 2 hours. The PVDF membrane was incubated with H3K18ac antibody diluted 1:10,000 at room temperature for 2 hours, followed by incubation with goat anti-rabbit secondary antibody diluted 1:5,000 at room temperature for 1 hour. Finally, ECL substrate was added for immunofluorescence development, and the color was measured using a gel imaging system, with the band grayscale calculated.

[0062] The results are as follows Figure 4 As shown in Figure A, the content of SIRT7 is negatively correlated with the grayscale change of H3K18ac, indicating that SIRT7 can deacetylate the H3K18ac substrate. Figure B shows that YZL-51N effectively replenished the H3K18ac content by inhibiting SIRT7 activity, exhibiting a dose-response relationship.

[0063] Example 4

[0064] Compound YZL-51N exhibits selective catalytic activity for SIRT7 deacetylation.

[0065] In this embodiment, the content and purity of sirtuins were detected by Coomassie brilliant blue staining, and the deacetylation activity of YZL-51N on different sirtuins was detected by a sirtuins deacetylation assay kit.

[0066] The results are as follows Figure 5As shown in Figure A, the Coomassie Brilliant Blue results indicate good purity and content of sirtuins proteins. Figure B shows that YZL-51N inhibits SIRT7 deacetylase activity without affecting the enzyme activity of other sirtuins family proteins.

[0067] Example 5

[0068] Compound YZL-51N exhibits selective affinity for SIRT7.

[0069] In this embodiment, the affinity of compound YZL-51N for SIRT1 / 2 / 6 / 7 was compared and detected using the BLI method on an Octet Red 96 instrument. First, SIRT7 was immobilized on a streptavidin sensor. Different concentration gradients of YZL-51N were diluted with PBST buffer and flowed through the immobilized protein sensor, with binding times ranging from 120 to 600 s and a dissociation time of 300 s set for each. The kinetic parameters and affinity were calculated using a nonlinear global fitting method with Octet data analysis software.

[0070] The results are as follows Figure 6 As shown in Figure AD, YZL-51N and SIRT7 have good bonding strength (K). D =3.3μM), which is higher than the binding force of YZL-51N to SIRT1 / 2 / 6.

[0071] Example 6

[0072] The binding amino acid sites of compound YZL-51N were verified by docking with SIRT7 molecules.

[0073] In this embodiment, the potential SIRT7 crystal structure (Uniprot Q9NRC8) was predicted using the AlphaFold protein structure website (https: / / alphafold.ebi.ac.uk / ). Then, the ligand docking energy was calculated using GLIDE software, and simulations were performed using PyMol software to identify the pocket and hotspot amino acids for YZL-51N binding to SIRT7.

[0074] The results are as follows Figure 7 As shown. According to the molecular docking results, YZL-51N can bind to the active site pocket of the SIRT7 enzyme, and the amino acids with hydrogen bond interactions include Asn297, Gly268, Leu298, Thr112, Asp118, Cys315, Gln299, Pro117, Lys314, Gly109, Val296, Ser269, etc.

[0075] Example 7

[0076] In cell models, compound YZL-51N specifically inhibits SIRT7 deacetylase activity and recovers its substrate changes.

[0077] In this embodiment, colon cancer cells HCT116 and HT29 were respectively injected at 3×10⁻⁶. 5 The cells were seeded into 6-well plates and cultured for 24 hours. Different concentrations of compound YZL-51N (0, 10, 20, and 40 μM) were added and the cells were treated for 6 hours each. Whole-cell proteins were extracted by lysis with RIPA lysis buffer. The samples were then denatured by boiling in 5× Loading buffer. Western blotting was used to detect H3K18ac, H3K9ac, H3K14ac, Histone 3, SIRT7, SIRT6, SIRT1, and Tubulin using an antigen-antibody chemiluminescence assay.

[0078] The results are as follows Figure 8 As shown in Figure A, in HCT116 cells, compound YZL-51N can inhibit SIRT7 enzyme activity and replenish H3K18ac levels, while not affecting H3K9ac and H3K14ac levels. Furthermore, YZL-51N itself does not affect the expression of SIRT7, SIRT6, and SIRT1. As shown in Figure B, in HT29 cells, YZL-51N exhibits a similar biological function of inhibiting SIRT7 enzyme activity. These results indicate that YZL-51N can effectively inhibit SIRT7 enzyme activity in a colon cancer cell model, thus replenishing H3K18ac levels.

[0079] Example 8

[0080] Compound YZL-51N interferes with the SIRT7-ATM signaling pathway.

[0081] In this embodiment, HCT116 cells were treated with irradiation (IR) for different repair times, followed by lysis with RIPA lysis buffer. Whole protein was extracted and subjected to immunoprecipitation experiments to detect changes in ATM acetylation and phosphorylation. Cells were treated with YZL-51N and subjected to the same IR treatment to compare the changes produced by YZL-51N.

[0082] The results are as follows Figure 9 As shown. By Figure 9 It was found that after IR treatment of colon cancer cell damage, the levels of ATM phosphorylation and ATM acetylation increased. In the drug blank group, 8 hours after IR treatment, SIRT7 deacetylated ATM, thereby inactivating ATM. In the YZL-51N drug treatment group, YZL-51N inhibited the process of SIRT7 deacetylation of ATM, thereby intervening in DNA damage repair.

[0083] In summary, this invention provides the application of the small molecule compound YZL-51N in the preparation of a selective inhibitor of SIRT7. This invention discovers a novel selective small molecule inhibitor targeting SIRT7. This invention finds that compound YZL-51N can inhibit the deacetylation of fluorescent and non-fluorescent peptide substrates by SIRT7. Compound YZL-51N selectively binds to SIRT7 and inhibits SIRT7 deacetylase activity without affecting other sirtuins. Molecular docking verified that YZL-51N binds to the SIRT7 enzyme active pocket and interacts with the amino acid sites. This invention validated in a cell model that compound YZL-51N selectively inhibits SIRT7 deacetylase activity and affects its substrate changes. Therefore, this invention is the first to discover that a novel small molecule compound can serve as a selective inhibitor of SIRT7 and provides guidance for subsequent interventions in various SIRT7-regulated biological processes.

[0084] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. The application of a small molecule compound YZL-51N in the preparation of a selective inhibitor of SIRT7, characterized in that, The structural formula of the small molecule compound YZL-51N is as follows: ； The SIRT7 selective inhibitor is used to treat colon cancer.

2. The application according to claim 1, characterized in that, The small molecule compound YZL-51N specifically inhibits the deacetylase activity of SIRT7 in vitro.

3. The application according to claim 1, characterized in that, The small molecule compound YZL-51N specifically binds to the enzyme active site pocket of SIRT7.

4. The application according to claim 3, characterized in that, The small molecule compound YZL-51N has hydrogen-bonded interactions with the enzyme active site pocket of SIRT7, and includes one or more combinations of Asn297, Gly268, Leu298, Thr112, Asp118, Cys315, Gln299, Pro117, Lys314, Gly109, Val296, and Ser269.

5. The application according to claim 1, characterized in that, The small molecule compound YZL-51N selectively inhibits SIRT7 activity in cells and replenishes the catalytic substrate of SIRT7.

6. The application according to claim 5, characterized in that, The catalytic substrates of SIRT7 include H3K18ac.

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