The acetyltransferase ARD1 inhibitor D753-0266 and its application in the treatment of colorectal cancer

Abstract

This invention discloses an acetyltransferase ARD1 inhibitor, D753-0266, and its application in the treatment of colorectal cancer, belonging to the field of biomedical technology. Through molecular dynamics simulations and binding energy calculations, it was found that the binding energy of D753-0266 to NAA10 can reach -68.12 kcal / mol. In vitro experiments show that D753-0266 can directly bind to and stabilize the ARD1 protein, downregulate its expression level, and dose-dependently inhibit the proliferation of colorectal cancer cells, while significantly inhibiting cell migration and colony formation. In vivo experiments show that D753-0266 effectively inhibits tumor growth in a mouse xenograft model and is well-tolerated. This inhibitor fills a research gap in the field of ARD1 targeting and can be used to prepare colorectal cancer therapeutic drugs, possessing high specificity and clinical translational potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an acetyltransferase ARD1 inhibitor D753-0266 and its application in the treatment of colorectal cancer. Background Technology

[0002] Acetyltransferase ARD1 (also known as NAA10) is the catalytic subunit of the NatA complex, mediating N-terminal acetylation modification of proteins and regulating protein stability, localization, and function. Studies have shown that ARD1 is highly expressed in a variety of tumors. (References: [1] Yang H, Li Q, Niu J, et al. microRNA-342-5p and miR-608 inhibit colon cancer tumorigenesis by targeting NAA10 [J]. Oncotarget, 2016, 7(3): 2709-20.; [2] Jiang B, Ren T, Dong B, et al. Peptide mimic isolated by autoantibody reveals human arrest defective 1 overexpression is associated with poor prognosis forcolon cancer patients [J]. Am J Pathol, 2010, 177(3): 1095-103.; [3] Fang, X, Lee, YH, Jang, JH, et al. ARD1 stabilizes NRF2 through direct interaction and promotes colon cancer progression. LIFE SCI. 2022; 313 121217. doi: (10.1016 / j.lfs.2022.121217), particularly in colorectal cancer, it promotes cell proliferation, invasion and metastasis, and this molecule has the potential to be a therapeutic target.

[0003] However, in current technologies, most small molecule inhibitors used in clinical applications or research target HDAC or other acetyltransferases, and there are no reported small molecule inhibitors specifically for ARD1. Simultaneously, there is a lack of compounds that can bind to ARD1 and effectively inhibit its enzymatic activity, hindering the development of targeted therapies for ARD1. Drug development targeting ARD1 remains a technological gap, and existing methods cannot effectively validate the clinical translational potential of ARD1 as a drug target. Based on these issues, developing a molecule that can target and bind to ARD1 while simultaneously effectively inhibiting its acetyltransferase activity has become an urgent need.

[0004] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a small molecule compound that can directly bind to and inhibit ARD1, and to verify its application value in the treatment of colorectal cancer.

[0006] To achieve the above objectives, the present invention provides an acetyltransferase ARD1 inhibitor, D753-0266, the structural formula of which is shown in Formula 1: Formula 1.

[0007] The inhibitor D753-0266 provided by this invention can be used to prepare its pharmaceutically acceptable salt.

[0008] The present invention also provides a pharmaceutical composition comprising the above-described inhibitor D753-0266 and a pharmaceutically acceptable carrier or excipient, and may further comprise a pharmaceutically acceptable salt prepared from the above-described inhibitor.

[0009] The inhibitor D753-0266 or its pharmaceutical composition provided by the present invention can be used to prepare drugs that inhibit the activity of acetyltransferase ARD1.

[0010] The inhibitor D753-0266 or its pharmaceutical composition provided by the present invention can be used in the preparation of medicaments for the prevention and / or treatment of tumors. Preferably, it can be used in the preparation of medicaments for the prevention and / or treatment of colorectal cancer, which can inhibit the proliferation, migration and clonal formation of colorectal cancer cells.

[0011] The present invention also provides a kit for the prevention and / or treatment of colorectal cancer, comprising the above-described inhibitor D753-0266 or a pharmaceutical composition thereof.

[0012] The inhibitor D753-0266 provided by this invention can be used to prepare standards or probes for the detection of ARD1 protein levels.

[0013] The present invention has the following advantages:

[0014] This invention is the first to discover and validate the small molecule compound D753-0266 that targets ARD1, filling a research gap in this field.

[0015] The D753-0266 disclosed in this invention can effectively inhibit ARD1 function, significantly inhibit the proliferation and metastasis of colorectal cancer cells, and show good anti-tumor effects in animal models.

[0016] Compared with existing broad-spectrum inhibitors, the D753-0266 disclosed in this invention has the advantages of strong target specificity and significant inhibitory effect, and has the potential for clinical translation. Attached Figure Description

[0017] Figure 1 The diagram shows the structural formula of candidate compound D753-0266 and its molecular docking with NAA10 (ARD1). Figure 1 In this text, A represents the chemical structural formula of candidate compound D753-0266. Figure 1 B in the diagram represents a schematic representation of molecular docking.

[0018] Figure 2 The inhibitory effect of D753-0266 on the activity of colorectal cancer cells (HCT116, RKO) and normal colonic epithelial cells (CCD) and its IC50 value. 50 CCK8 detection results for the curve.

[0019] Figure 3 This study describes the changes in ARD1 protein expression levels in colorectal cancer cells after treatment with compound D753-0266 of this invention.

[0020] Figure 4 CETSA experimental results were used to verify the direct binding and stability of D753-0266 with ARD1.

[0021] Figure 5 The results show the evaluation of the binding activity of compound D753-0266 of this invention with the ARD1 protein, wherein... Figure 5 In the figure, A represents the signal intensity of the specific binding between D753-0266 and NAA10 protein over time. Figure 5 In the figure, B represents the signal intensity of the specific binding of different concentrations of D753-0266 to the NAA10 protein.

[0022] Figure 6 The Transwell and colony formation assays show the inhibitory effect of D753-0266 on the migration and proliferation of colorectal cancer cells. Figure 6In the figure, A represents the effect of D753-0266 on the transmembrane migration of HCT116 and RKO cells. Figure 6 B in the figure represents the effect of different concentrations of D753-0266 on the number of clones formed in HCT116 and RKO cells.

[0023] Figure 7 These are the experimental results of an in vivo xenograft model; among them, Figure 7 In this context, A represents the drug administration schedule during model construction. Figure 7 In the curve, B represents the tumor volume change curve; Figure 7 C in the image represents a photograph of the actual tumor tissue. Figure 7 In this context, D represents the statistical result of tumor weight. Figure 7 E in the figure represents the change in body weight of the model mice during the drug administration process. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0026] This technical approach utilizes structural biology-guided drug design, combined with high-throughput screening and fragment conjugation strategies, to identify small molecule compounds that specifically bind to the ARD1 catalytic pocket. These molecules must possess high affinity, good selectivity, and cell permeability, enabling them to stably inhibit ARD1-mediated substrate acetylation in in vitro and in vivo models, thereby blocking tumor growth and metastasis-related signaling pathways. This provides a crucial tool for validating ARD1 as an anticancer target.

[0027] Example 1: Screening of small molecule compounds for ARD1-targeted inhibition

[0028] 1. Virtual screening and molecular dynamics simulation

[0029] (1) This technical solution utilizes structural biology-guided drug design, combined with virtual screening and molecular docking strategies, to identify small molecule compounds that specifically bind to the ARD1 catalytic pocket. ARD1, as a component of the NAA10 / NAA15 complex, has a catalytic region capable of binding acetyl-CoA (ACO) and undergoing acetylation modification on lysine residues. Based on this, the selected NAA10 crystal structure is from the Protein Data Bank (PDB ID: 6PPL, https: / / www.rcsb.org / structure / 6PPL). This structure has a resolution of 2.5 Å and clearly demonstrates the binding mode of NAA10 with the cofactor Acetyl-CoA. The catalytic site is essentially the Acetyl-CoA binding pocket. This pocket includes conserved catalytic residues (such as Glu24, Tyr139, etc.), which are key regions for the NAA10 / ARD1 acetylation reaction.

[0030] (2) During molecular docking, the acetyl-CoA binding pocket of NAA10 was selected as the docking target region (gridcenter coordinates: -15.585, -5.624, 15.02; XYZ dimensions: 46 × 38 × 56, spacing=0.375A). The docking was performed using AutoDock Vina software (version 1.1.2, http: / / vina.scripps.edu / ), and the compound libraries were Biodiversity_Library, Natural_Product_Like_Library, and Pharm_Dversity_Library provided by ChemDiv, with 27,659, 17,656, and 47,490 compounds, respectively.

[0031] (3) After screening each library, compounds with binding energies below -11.7 kcal / mol were extracted. Further screening was conducted based on Lipinski's Rule of Five and the feasibility of commercial synthesis and purchase. The final retained compounds were selected as candidate molecules. A 10 ns molecular dynamics simulation was performed to assess binding stability using RMSD values ​​and to evaluate binding affinity by calculating the binding free energy between the compound and NAA10 using GMX_MMPBSA.

[0032] (4) The molecular dynamics simulation results are shown in Table 1. It can be seen that, except for lig2108, all candidate compounds can stably bind to NAA10. The binding energy calculation results show that the binding energy of substrate ACO to NAA10 is -67.89 kcal / mol. Only the binding energy of candidate compound D753-0266 is better than that of NAA10 (-68.12 kcal / mol).

[0033] Table 1. Evaluation results of molecular dynamics simulations

[0034]

[0035] Example 2: In vitro validation of D753-0266

[0036] 1. Molecular docking analysis of candidate compound D753-0266 with NAA10 (ARD1)

[0037] To verify the potential binding ability of the screened candidate compound D753-0266 to NAA10 (ARD1), molecular docking was used to predict the binding site. Using molecular docking software such as Autodock, the three-dimensional structure of D753-0266 was docked with the active pocket of the NAA10 (ARD1) protein for analysis, obtaining the binding energy and binding site.

[0038] The results are as follows Figure 1 As shown, where, Figure 1 In the diagram, A represents the chemical structure of D753-0266, and B represents the molecular docking configuration. It can be seen that D753-0266 can form a stable bond with the active site of NAA10 (ARD1), maintaining the stability of the complex primarily through hydrogen bonding and hydrophobic interactions. This suggests that D753-0266 may exert its biological effects by directly binding to ARD1.

[0039] 2. Inhibitory effect of D753-0266 on the viability of colorectal cancer cells

[0040] The inhibitory effects of compound D753-0266 on colorectal cancer cells HCT116 and RKO, and normal colonic epithelial cells CCD, were detected using the CCK8 assay. Compound D753-0266 was dissolved in DMSO and diluted with cell culture medium to prepare working solutions of different concentrations, ensuring that the final treatment solution contained 0.1% DMSO by volume.

[0041] HCT116, RKO, and CCD cells in logarithmic growth phase were digested and prepared into single-cell suspensions, which were then seeded at a specific density into 96-well plates and cultured until adherent. Different concentrations of D753-0266 solution were added to each well in the experimental groups, while an equal volume of DMSO was added to the control group. Cells were cultured for 1, 2, 3, and 4 days. At each time point, 10 μL of fresh culture medium containing CCK8 reagent was added to each well, and the cells were cultured for another hour. The absorbance of each well was measured at 450 nm using a microplate reader. The relative viability of cells in each experimental group was calculated, with the control group considered as 100% viability.

[0042] Experimental results are as follows Figure 2 As shown: D753-0266 exhibited dose-dependent inhibitory effects on colorectal cancer cells HCT116 and RKO, with an IC50 value of [missing information]. 50 The values ​​were 1.4 μM and 2.1 μM, respectively, while the effect on normal CCD cells was weaker, IC50... 50 The concentration was 9.2 μM. The results indicate that D753-0266 has a good selective inhibitory effect on colorectal cancer cells.

[0043] 3. Western blot analysis of the effect of D753-0266 on ARD1 protein expression in colorectal cancer cells.

[0044] To investigate the regulatory effect of compound D753-0266 on ARD1 protein expression in colorectal cancer cells, HCT116 and RKO cells were selected as research subjects. Cells in the logarithmic growth phase were seeded in culture dishes. After cell attachment, different concentrations of D753-0266 (0 μM, 1 μM, 2 μM) were added. After a certain period of culture, cells were collected, lysed, and total protein was extracted. Protein quantification was performed using the BCA method. Equal amounts of protein were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane.

[0045] The membrane was then blocked with TBST buffer containing 5% skim milk powder for 2 h, followed by incubation at 4°C overnight with ARD1 primary antibody and internal control β-actin primary antibody. The next day, the membrane was washed with TBST, and incubated at room temperature for 1 h with HRP-labeled secondary antibody. After development with ECL chemiluminescence reagent, the signal was detected and photographed.

[0046] Experimental results are as follows Figure 3 As shown, compared with the control group, the expression level of ARD1 protein in HCT116 and RKO cells was significantly decreased after treatment with D753-0266, and the decrease showed a dose-dependent trend. The results indicate that D753-0266 can effectively downregulate ARD1 protein expression in colorectal cancer cells.

[0047] 4. CETSA assay to detect the effect of D753-0266 on the thermostability of ARD1 protein

[0048] To verify the binding of D753-0266 to ARD1 protein and its effect on protein stability, a cell heat transfer assay (CETSA) was performed. HCT116 cells in logarithmic growth phase were divided into experimental and control groups. The experimental group was treated with D753-0266, while the control group was treated with an equal volume of DMSO. After treatment for a certain period, cells were collected, lysed, and then heated at different temperatures (42 ℃, 44 ℃, 46 ℃, 48 ℃, 50 ℃, 52 ℃, 54 ℃, 56 ℃) to induce protein denaturation.

[0049] After heating, the sample was immediately cooled in an ice bath, followed by centrifugation to remove the precipitate, and the supernatant was collected. The extracted soluble protein was quantified using the BCA method, and equal amounts of protein were subjected to SDS-PAGE electrophoresis and transferred to a membrane. The residual level of ARD1 protein was detected by Western blot, with β-actin used as an internal control.

[0050] Experimental results are as follows Figure 4 As shown, in the DMSO control group, ARD1 protein levels gradually decreased with increasing heating temperature; however, in the D753-0266 treatment group, the ARD1 protein signal remained at a high level even under high temperature conditions. These results indicate that D753-0266 can significantly improve the thermal stability of ARD1 protein, suggesting that it can directly bind to ARD1 and enhance its conformational stability.

[0051] 5. Evaluation of the binding activity of the representative compound D753-0266 of the present invention on NAA10 protein.

[0052] The binding of small molecule D753-0266 to NAA10 protein was determined using SPR (surface plasmon resonance detection). The experiment was performed on a Biacore S200 instrument. NAA10 protein was immobilized on the surface of a CM5 chip via amino-coupling. The coupling buffer was sodium acetate buffer (pH=4.5), and the coupling conditions were 50 μg / mL, 10 μL / min, and 120 s. The run buffer was 1×HBSET (containing 150 mM NaCl) + 3% DMSO. D753-0266 was used as the analyte, and different concentrations were injected in a gradient. The kinetic detection conditions were: flow rate 30 μL / min, binding for 90 s, and dissociation for 180 s.

[0053] Experimental results are as follows Figure 5As shown, under injection of different concentrations of D753-0266, the binding signal (RU value) increases with increasing concentration, indicating that D753-0266 can specifically bind to the NAA10 protein (see...). Figure 5 According to equilibrium analysis (Steady-state fitting), the affinity constant (KD) between compound D753-0266 and NAA10 was calculated to be 91.85 μM (see A). Figure 5 (B in the text). The results indicate a detectable direct binding between D753-0266 and the NAA10 protein.

[0054] 6. Transwell assay to detect the inhibitory effect of D753-0266 on the migration of colorectal cancer cells.

[0055] HCT116 and RKO cells were selected as research subjects, and the effect of compound D753-0266 on cell migration was detected using Transwell assays. Cells were digested and prepared into single-cell suspensions, seeded in the upper chamber of a Transwell assay, and treated with serum-free medium and either DMSO or different concentrations of D753-0266 (2 μM). Medium containing 10% FBS was added to the lower chamber as a chemokine. After 24 h of culture, unmigrated cells in the upper chamber were gently wiped away with a cotton swab, washed with PBS, fixed with 4% paraformaldehyde for 10 min, stained with crystal violet for 30 min, washed with PBS to remove excess stain, and air-dried. Multiple fields of view were randomly selected for photographing and cell counting.

[0056] Experimental results are as follows Figure 6 As shown in Figure A, compared with the DMSO control, D753-0266 (2 μM) significantly reduced the number of HCT116 and RKO cells that migrated across the membrane, suggesting that it has a significant inhibitory effect on the migration of colorectal cancer cells.

[0057] 7. Clonogenic assay to detect the inhibitory effect of D753-0266 on the proliferation of colorectal cancer cells.

[0058] After digestion and counting, HCT116 and RKO cells were seeded at approximately 800 cells / well in 6-well plates, and cultured with different concentrations of D753-0266 (1 μM, 2 μM), with an equal volume of DMSO as a control. Cells were cultured for 10–14 days until visible colony formation was observed. After removing the culture medium, the cells were washed 1–2 times with PBS, fixed with 4% paraformaldehyde for 10–15 min, and then stained with crystal violet for 20–30 min. After removing the stain, the cells were rinsed with PBS and air-dried. The number of colonies was then observed and counted; a cluster containing more than 50 cells was defined as one colony.

[0059] Experimental results are as follows Figure 6As shown in Figure B, the number of clones formed by HCT116 and RKO cells decreased significantly with increasing D753-0266 concentration. These results indicate that D753-0266 can significantly inhibit the proliferation and colony formation ability of colorectal cancer cells.

[0060] 8. Evaluation of the inhibitory activity of D753-0266 on the growth of colorectal cancer xenografts in nude mice

[0061] To evaluate the in vivo antitumor effect of compound D753-0266, HCT116 cells in logarithmic growth phase were harvested, digested, collected, and counted. Cells were counted at a rate of 1 × 10⁻⁶ cells per cell. 6 Single-cell suspensions were prepared from individual cells, centrifuged to remove the supernatant, and resuspended in pre-cooled 1×PBS. Subsequently, 50 μL of the suspension was subcutaneously injected into the right axilla of BALB / c nude mice using an insulin injection needle. Ten days after inoculation, when the tumor volume reached approximately 50–60 mm... 3 Mice were randomly divided into three groups of five each: Vehicle, D753-0266 20 mg / kg, and D753-0266 40 mg / kg.

[0062] During the experiment, the drug injection solution (10% DMSO or drug + 30% PEG300 + 5% Tween80 + 30% physiological saline) was prepared at a volume of 100 μL / mouse and administered intraperitoneally every 3 days. Mouse weight was recorded during the experiment, and the long and short diameters of the tumor were measured using calipers. Tumor volume was calculated using the formula V = 0.5 × length × width. 2 Calculations. On day 28 of the experiment, mouse tumor tissue was removed, weighed, photographed, and analyzed.

[0063] Experimental results are as follows Figure 7 As shown: Figure 7 In this context, A represents the dosing schedule. Figure 7 B in the study showed that, compared with the Vehicle control, D753-0266 (20 mg / kg and 40 mg / kg) significantly inhibited the growth of xenografts in mice, and the tumor volume was significantly reduced. Figure 7 C in the image represents the actual tumor tissue that was dissected. Figure 7 In the figure, D represents the tumor weight statistical result; the tumor weight in the D753-0266 treatment group was significantly lower than that in the control group. Furthermore, Figure 7 E in the figure showed no significant difference in mouse body weight among the groups during the experiment (grouping same). Figure 7 The value of D in the figure is: v = Vehicle, s = 20 mg / kg, b = 40 mg / kg, indicating that D753-0266 has no obvious toxicity to mice.

[0064] The animal experiments were conducted in accordance with the regulations of the International Committee for Assessment and Accreditation of Laboratory Animals (AAALAC). The health and behavioral status of the mice were monitored daily during the experiments, and no abnormalities were found.

[0065] The results showed that D753-0266 could significantly inhibit the growth of colorectal cancer xenografts in vivo without adversely affecting the weight of animals, suggesting that it has good in vivo anticancer activity and safety.

[0066] In summary, compound D753-0266 of this invention not only downregulates the expression level of ARD1 protein in colorectal cancer cells and improves their thermal stability, but also significantly inhibits the proliferation, migration, and colony formation of colorectal cancer cells. Furthermore, in a nude mouse xenograft model of colorectal cancer, D753-0266 exhibits a significant dose-dependent inhibitory effect on tumor growth without adversely affecting mouse body weight, demonstrating good in vivo safety. These results indicate that D753-0266 is a promising candidate compound for the treatment of colorectal cancer.

[0067] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. The use of an acetyltransferase ARD1 inhibitor, D753-0266, in the preparation of a drug for treating colorectal cancer, wherein, The structural formula of the inhibitor D753-0266 is shown in Formula 1: Formula 1.

2. The application according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable carriers or excipients.

Images