Application of PKC [beta] inhibitor 1 as NID1 inhibitor

Abstract

The invention discloses application of a PKC beta inhibitor 1 as an NID1 inhibitor, and relates to the technical field of biological medicines. The invention provides an application of a PKC [beta] inhibitor 1 as an NID1 inhibitor in preparation of antitumor drugs. By constructing a systematic screening and verification system, it is determined for the first time that the PKC beta inhibitor 1 plays a significant anti-tumor activity by efficiently blocking the interaction between NID1 and integrin alpha5beta1, and an innovative treatment strategy is provided for targeting tumor stem cells and reversing drug resistance.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, specifically to the application of PKCβ inhibitor 1 as an NID1 inhibitor. Background Technology

[0002] Nestin 1 (Nidogen-1, NID1) is a basement membrane-specific glycoprotein primarily involved in the assembly and stabilization of the extracellular matrix. Through interactions with components such as laminin and type IV collagen, it plays a crucial role in the formation and function of the basement membrane, and is associated with tissue development, cell adhesion, and signal transduction. Studies have shown that NID1 is highly expressed in various tumor types and is closely related to poor patient prognosis. However, the specific molecular mechanisms by which NID1 participates in tumor progression have not been systematically elucidated. Although some studies have suggested a possible association between NID1 and members of the integrin family, the ability of NID1 to physically bind to specific integrin subtypes, the biological function of this interaction, and its specific role in maintaining tumor stemness remain unclear in current technology. This knowledge gap limits the development and application of targeted therapies based on this signaling pathway.

[0003] Existing technologies include several peptide or antibody inhibitors designed to block the binding of integrins to their ligands. However, these inhibitors have broad targets and are not specifically designed for the protein-protein interaction between NID1 and integrins, thus failing to achieve precise intervention and potentially causing unnecessary toxic side effects due to off-target effects.

[0004] PKCβ Inhibitor 1 is a highly potent PKCβ inhibitor that inhibits human PKCβ II (PKCβ2), thereby suppressing tumor cell proliferation and inhibiting cell cycle progression in 2F7 and BCBL-1 cells. In the prior art, PKCβ Inhibitor 1 is clearly defined as an inhibitor of the protein kinase Cβ (PKCβ) signaling pathway, and all its known uses are based on its inhibition of PKC kinase activity. The prior art has never disclosed or suggested that this compound has the function of directly binding to and inhibiting the NID1 protein, nor has it addressed any potential applications in disrupting NID1-integrin interactions. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of PKCβ inhibitor 1 as an NID1 inhibitor.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is: the application of PKCβ inhibitor 1 as an NID1 inhibitor in the preparation of drugs for anti-tumor purposes.

[0007] By constructing a systematic screening and verification system, the inventors of this application have for the first time clarified that PKCβ inhibitor 1 exerts significant anti-tumor activity by efficiently blocking the interaction between NID1 and integrin α5β1, providing an innovative treatment strategy for targeting tumor stem cells and reversing drug resistance.

[0008] As a preferred embodiment of the application described in this invention, the CAS number of the PKCβ inhibitor 1 is 257879-35-9.

[0009] As a preferred embodiment of the application described in this invention, the PKCβ inhibitor 1 exerts its anti-tumor effect by blocking the interaction between NID1 and integrin α5β1.

[0010] Preferably, the core region of the interaction between NID1 and integrin α5β1 is the EGF-like domain and G2 domain in the NID1 protein.

[0011] Through extensive experimental verification, the inventors of this application have determined that the EGF-like domain and G2 domain in the NID1 protein are the core regions for binding to integrin α5β1, providing a precise molecular basis for subsequent targeted intervention.

[0012] In a preferred embodiment of the application described in this invention, the drug exerts its anti-tumor effect by inhibiting tumor cell proliferation, weakening tumor cell migration, or inhibiting the stemness of tumor stem cells.

[0013] In a preferred embodiment of the application described in this invention, the tumor includes lung cancer.

[0014] This invention also provides the use of PKCβ inhibitor 1 as an NID1 inhibitor in the preparation of drugs to reverse tumor chemotherapy resistance.

[0015] This invention provides multi-level evidence, from in vitro cell proliferation, maintenance of tumor stem cell stemness and reversal of drug resistance to in vivo animal model verification, to fully demonstrate that by inhibiting the interaction between NID1 and integrin α5β1, it is possible to effectively reverse chemotherapy and targeted therapy resistance, exert a significant anti-tumor effect, and have good in vivo safety.

[0016] This invention also provides the application of PKCβ inhibitor 1 as an inhibitor of the interaction between NID1 and integrin α5β1.

[0017] The inventors of this application have discovered that PKCβ inhibitor 1 can act as a direct inhibitor of NID1, specifically disrupting the interaction between NID1 and integrin α5β1 by binding to NID1 with high affinity.

[0018] The present invention also provides an NID1 inhibitor, wherein the inhibitor includes PKCβ inhibitor 1.

[0019] The present invention also provides an antitumor pharmaceutical composition comprising a PKCβ inhibitor 1, a pharmaceutically acceptable carrier, and an anticancer drug.

[0020] As a preferred embodiment of the pharmaceutical composition of the present invention, the anticancer drug includes cisplatin and osimertinib.

[0021] The beneficial effects of this invention are as follows: This invention provides the application of PKCβ inhibitor 1 as an NID1 inhibitor. This invention reveals for the first time that there is a direct interaction between NID1 and integrin α5β1, and that this interaction is a key molecular event in maintaining the stemness of tumor stem cells, filling a gap in the understanding of this specific signaling pathway in the field of tumor biology. This invention discovers that PKCβ inhibitor 1 can specifically disrupt the interaction between NID1 and integrin α5β1 by directly binding to the NID1 protein (surface plasmon resonance experiments show that its affinity reaches the nanomolar level). Based on this direct targeting mechanism, PKCβ inhibitor 1 achieves precise blocking of the NID1-integrin α5β1 specific signaling pathway. Compared with traditional broad-spectrum inhibitors, this strategy theoretically exhibits higher targeting, potentially possessing better therapeutic effects and lower risk of toxic side effects. This invention also discovers that PKCβ inhibitor 1 effectively reverses chemotherapy and targeted therapy resistance by inhibiting the NID1-integrin α5β1 interaction, exerting a significant anti-tumor effect with good in vivo safety, thus uncovering new pharmaceutical value for PKCβ inhibitor 1. Attached Figure Description

[0022] Figure 1 The interaction between NID1 and integrin α5β1 is characterized, where A shows the expression level of NID1 and the expression of the constituent subunits ITGA5 and ITGB1 of integrin α5β1; B shows the survival analysis of lung cancer patients with high expression of ITGA5 and ITGB1; C shows the SDS-PAGE gel electrophoresis image of the precipitate; and D shows the structural analysis and image rendering of the interaction between NID1 and integrin α5β1.

[0023] Figure 2 For screening and molecular docking analysis of candidate compounds targeting NID1, A is a heatmap of the top 32 NID1 candidate inhibitor compounds screened using cMap, with color bars and blocks representing similarity scores; B is a comparison of NID1 expression levels using the COMPARE tool based on the NCI60 project; C is a comparison of GI50 values ​​of PKCβ-inhibitor1 in cancer cell lines using the COMPARE tool based on the NCI60 project; C is the mean NID1 expression or the mean log10 (GI50) value.

[0024] Figure 3The three-dimensional structure of NID1 obtained from protein homology modeling shows the NID1 drug-binding pocket on the left and the two-dimensional structure of the drug, the amino acid residues involved, molecular forces, and molecular spatial distances on the right. The visualization analysis was completed using Discovery Studio software.

[0025] Figure 4 The binding kinetics of PKCβ inhibitor 1 and NID1 were measured using SPR, and the response values ​​are expressed in response units (RU).

[0026] Figure 5 This study investigates the mechanism by which PKCβ inhibitor 1 inhibits lung cancer progression by blocking the NID1-integrin α5β1 interaction. A shows Western blot analysis of PKCβ inhibitor 1 inhibiting the uptake of CAF-EV-derived NID1 by A549 cells; B shows Western blot validation of NID1 protein uptake in HCC827 cells; C shows the MTT assay for the inhibitory effect of PKCβ inhibitor 1 on A549 cell proliferation; D shows the MTT assay for the inhibitory effect of PKCβ inhibitor 1 on HCC827 cell proliferation; E shows bright-field images of tumor spheroid formation (scale bar: 100 μm); F shows statistical analysis of tumor spheroid number and diameter; G shows crystal violet staining images from the Transwell migration assay (A549: 100 μm, HCC827: 200 μm). (μm); H represents statistical analysis of the number of migrating cells; I represents the representative atlas of the ALDH⁺ / CD44⁺ stem cell population detected by flow cytometry; J represents the statistical analysis of the ALDH⁺ / CD44⁺ cell proportion; K represents the dose-response curve and IC50 value of Cisplatin treatment for A549 cells; L represents the dose-response curve and IC50 value of Osimertinib treatment for HCC827 cells. Data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0027] Figure 6 This study investigated the effect of PKCβ inhibitor 1 on NID1-mediated tumor growth in vivo. Figure A shows a schematic diagram of a subcutaneous xenograft model of LLC-1 cells in C57BL / 6 mice; Figure B shows tumor images in each group after day 18 of treatment; experimental groups included: control group, EV-NID1 treatment group, and EV-NID1 combined with PKCβ inhibitor 1 treatment group; Figure C shows a statistical analysis of tumor weight (*p<0.05); Figure D shows the mouse body weight change curve during treatment, with ns representing no significant difference; Figure E shows the dynamic curve of xenograft growth (****p<0.0001). All data are expressed as mean ± standard deviation (n=2), and statistical analysis was performed using one-way ANOVA and Tukey's post-hoc test. Detailed Implementation

[0028] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Unless otherwise specified, the various raw materials, reaction equipment, testing equipment, and testing methods used in the following embodiments are all known in the art.

[0029] Example 1: Interaction between NID1 and integrin α5β1 This embodiment systematically elucidates the direct interaction and biological function of NID1 and integrin α5β1 in lung cancer through a multi-level experimental system. The specific experimental methods are as follows: 1. Bioinformatics analysis was performed on clinical lung adenocarcinoma samples based on the TIMER2.0 database. Statistical analysis of 515 clinical samples showed that the expression level of NID1 was significantly positively correlated with the expression of the constituent subunits ITGA5 and ITGB1 of integrin α5β1. Figure 1 A). Further survival analysis, by tracking the clinical prognostic data of patients (the aforementioned 515 clinical samples), revealed that lung cancer patients with high expression of both ITGA5 and ITGB1 had significantly shorter overall survival, suggesting that this interaction has important clinical significance. Figure 1 B).

[0030] 2. Establishment of a cell-level interaction verification model: A549 lung cancer cells were treated with extracellular vesicles rich in NID1 protein. The specific experimental steps are as follows: A549 cells were placed in RPMI medium containing 10% fetal bovine serum and 1% penicillin-streptomycin and cultured at 37℃ and 5% CO2. When the cells grew to about 70% confluence, the medium was replaced with extracellular vesicle-free medium. The experimental group was incubated with extracellular vesicles derived from cancer-associated fibroblasts (CAFs) that overexpressed NID1 (CAF-EV-NID1) at a working concentration of 20 μg / mL (based on total vesicle protein) for 24 hours; the control group was incubated with equal amounts of control vesicles (CAF-EV-Ctrl) or PBS.

[0031] Next, immunoprecipitation was used to verify protein-protein interactions. Treated cells were collected and lysed on ice for 30 minutes using RIPA lysis buffer containing a protease inhibitor, followed by centrifugation at 12,000 × g for 15 minutes at 4°C. The supernatant was collected as the total protein sample. An equal volume of protein lysis buffer was added, along with a specific primary antibody against NID1, and the mixture was gently incubated overnight at 4°C. The following day, Protein A / G agarose beads, pre-equilibrated with lysis buffer, were added, and the mixture was incubated at 4°C for another 4 hours. The agarose bead-antibody-antigen complex was collected by centrifugation, and the beads were washed five times with pre-chilled lysis buffer. Finally, 1× SDS loading buffer was added, and the mixture was boiled for 10 minutes to elute the bound proteins.

[0032] The eluted products were then analyzed by Western blot. The procedure was as follows: after separation by 10% SDS-PAGE gel electrophoresis, proteins were transferred to a PVDF membrane using a wet transfer method. The membrane was blocked for 1 hour at room temperature using TBST solution containing 5% skim milk powder. Subsequently, the membrane was incubated overnight at 4°C with specific primary antibodies against NID1 and integrin α5, respectively. After washing with TBST, the corresponding horseradish peroxidase-labeled secondary antibodies were added, and the membrane was incubated for 1 hour at room temperature. After thorough washing, the membrane was developed using enhanced chemiluminescence (ECL) substrate. The results showed that specific bands for both NID1 and integrin α5 were detected in the immunoprecipitate products of the experimental group (CAF-EV-NID1 treatment) (see [link to relevant documentation]). Figure 1 The control group showed that NID1 was detected in the control group (C), while only NID1 was detected in the control group. This result provides strong experimental evidence that NID1 can specifically bind to the integrin α5β1 receptor on the cell surface.

[0033] 3. Further computational biology methods were employed for structural studies: High-precision three-dimensional structural models of NID1 and integrin α5 were obtained using the AlphaFold protein structure prediction system. The binding process between the two was then simulated using Lightdock molecular docking software. Through thousands of conformational samplings and energy optimizations, the three most stable binding conformations were selected. Finally, structural analysis and image rendering were performed using PyMOL molecular visualization software, precisely locating the binding interface between NID1 and integrin α5, which is mainly concentrated in two key regions of the NID1 molecule: the EGF-like domain (191-900 amino acids) and the G2 domain (901-1200 amino acids). Figure 1 D).

[0034] Example 2: Inhibitor Screening and Validation Based on the Interaction between NID1 and Integrin α5β1 This embodiment uses experiments to screen for inhibitory compounds that inhibit the interaction between NID1 and integrin α5β1. The specific experimental methods are as follows: 1. Cellular models of NID1 gene knockdown (shNID1-1 and shNID1-2) were constructed using genetic engineering techniques. The specific steps are as follows: Two lentiviral vectors targeting different NID1 target sequences (shNID1-1 and shNID1-2) were designed and constructed, with a non-targeting shRNA (shCtrl) as a control. These lentiviral vectors were transduced into target cell lines, and stable NID1 knockdown cell lines were obtained after puromycin selection. The knockdown efficiency of NID1 at the mRNA and protein levels was verified using quantitative real-time PCR (qRT-PCR) and Western blot techniques. Fifty-four genes with significantly altered expression were identified through genome-wide expression profiling, constructing an NID1 functional profile. Subsequently, large-scale computational comparative analysis was performed using the cMap (connectivity map) database, which contains gene expression data from thousands of small molecule treatments. The results showed that 10 small molecule compounds could induce gene expression changes similar to NID1 knockdown, suggesting that these compounds may mimic the inhibitory effect of NID1 function. To further validate the specificity of these compounds, correlation analysis was conducted using the NCI COMPARE analysis tool in a database covering 60 cancer cell lines. The results showed that the drug sensitivity of PKCβ inhibitor 1 (measured by the GI50 concentration that inhibits 50% cell growth) was significantly positively correlated with the basal expression level of NID1 (see [link to analysis]). Figure 2 This finding suggests that tumor cells with high NID1 expression are more sensitive to PKCβ inhibitor 1, supporting the compound's anti-tumor effect by acting on NID1-related pathways.

[0035] 2. In-depth molecular docking analysis was conducted using computational biology methods. First, a high-precision three-dimensional structural model of the NID1 protein was obtained using the AlphaFold 3.0 artificial intelligence system, and the complete molecular structure of PKCβ inhibitor 1 was retrieved from the PubChem database. Large-scale computational simulations were performed using the professional molecular simulation software PyMol 2.5 and Pyrx 9.5. Through thousands of conformational samplings and energy optimizations, the most stable binding mode between PKCβ inhibitor 1 and the NID1 protein was finally determined. The calculation results show that PKCβ inhibitor 1 can precisely embed into the specific binding pocket on the surface of the NID1 protein (see...). Figure 3 This finding reveals from a structural biology perspective that PKCβ inhibitor 1 may interfere with the interaction between NID1 and integrin α5β1 through steric hindrance or conformational regulation mechanisms.

[0036] 3. To verify the accuracy of the calculated predictions, the interaction between PKCβ inhibitor 1 and NID1 was monitored using surface plasmon resonance (SPR) technology with a BIAcore T200 (Cytiva). The experiment was conducted at 25°C in a low molecular weight, multi-cycle mode. Biotin-labeled NID1 protein was immobilized on the surface of a CM5 chip (approximately 2000 RU). Subsequently, different concentrations (6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM) of PKCβ inhibitor 1 were passed through the chip, and real-time response curves (RU) were recorded. All experiments were performed in a pH 7.4 buffer system. The binding affinity (equilibrium dissociation constant KD value) for each interaction pair was calculated using the BIAcore T200 evaluation software (Cytiva). Didoxigenin was used as a negative control to exclude non-specific binding. SPR results showed that PKCβ inhibitor 1 could directly bind to NID1, with a kinetically fitted KD value of 179 nM (…). Figure 4 This nanomolar affinity indicates a highly specific and strong interaction between the two.

[0037] 4. To verify the functional blocking ability of PKCβ inhibitor 1 on the interaction between NID1 and integrin α5β1 (NID1-integrin α5β1 interaction), Western blot analysis revealed that this compound effectively inhibited the uptake of NID1 protein in CAF-EV-NID1-treated lung cancer cells. Figure 5 (AB). Further, the inhibitory effect of this compound on cell proliferation was evaluated using the MTT assay. The specific steps were as follows: A549 and HCC827 cells in logarithmic growth phase were seeded in 96-well plates. After cell attachment, the medium was replaced with culture medium containing different concentrations of PKCβ inhibitor 1, and the treatment continued for 72 hours. 10 μL of MTT solution (5 mg / mL) was added to each well, and after culturing for another 4 hours, the supernatant was discarded, and 100 μL of DMSO was added to dissolve the formazan crystals. The absorbance was measured at 490 nm using a microplate reader. A dose-response curve was plotted based on cell viability, and the half-maximal inhibitory concentration (IC50) was calculated using a logistic model. 50 The results showed that PKCβ inhibitor 1 could inhibit lung cancer cell proliferation in a concentration-dependent manner (A549 IC50). 50 = 38.2 ± 0.36 nM; HCC827 IC 50 = 23.9 ± 1.33 nM)( Figure 5 CD). In functional studies, this compound significantly inhibited the ability of tumor spheroids to form (CD). Figure 5 EF), weakening cell migration ( Figure 5 GH) and reduce the proportion of ALDH⁺ / CD44⁺ stem cell-like cells ( Figure 5 (IJ). Furthermore, PKCβ inhibitor 1 effectively reversed chemotherapy resistance induced by CAF-EV-NID1 interaction via NID1-integrin α5β1, and significantly reduced the IC50 of cisplatin and osimertinib. 50 value( Figure 5 These results systematically confirm the function of PKCβ inhibitor 1 in inhibiting tumor malignancy by blocking the NID1-integrin α5β1 interaction.

[0038] Example 3: In vivo experimental verification of the inhibitory effect of PKCβ inhibitor 1 on tumorigenesis mediated by NID1-integrin α5β1 interaction. like Figure 6 As shown in Figure A, an LLC-1 cell xenograft model was established in C57BL / 6 mice. Four days after tumor formation, the experimental group mice received NID1-overexpressing EV (tail vein injection, 20 μg / time, every 3 days) combined with PKCβ inhibitor 1 (1 mg / kg, intraperitoneal injection every other day). After 18 days, PKCβ inhibitor 1 significantly inhibited tumor growth, and no significant change in mouse body weight was observed. Figure 6 (BD), indicating no significant toxicity. Tumor growth curves showed that the tumor volume in the PKCβ inhibitor group was significantly smaller than that in the EV-NID1 group, indicating that this inhibitor effectively inhibited the EV-NID1-mediated pro-tumorigenesis by blocking the NID1-integrin α5β1 interaction. Figure 6 E).

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Use of PKC beta inhibitor 1 as NID1 inhibitor in the preparation of a medicament for anti-tumor.

2. Use according to claim 1, characterized in that, The CAS number of the PKC beta inhibitor 1 is 257879-35-9.

3. Use according to claim 1, characterized in that, The PKC beta inhibitor 1 exerts anti-tumor effect by blocking the interaction of NID1 with integrin alpha5beta1.

4. Use according to claim 1, characterized in that, The medicament exerts anti-tumor effect by inhibiting tumor cell proliferation, attenuating tumor cell migration or inhibiting the stemness of tumor stem cells.

5. The use according to claim 1, characterized in that, The tumor includes lung cancer.

6. Use of PKC beta inhibitor 1 as NID1 inhibitor in the preparation of a medicament for reversing tumor chemotherapy resistance.

7. Use of PKC beta inhibitor 1 as an inhibitor of the interaction of NID1 with integrin alpha5beta1.

8. A NID1 inhibitor, characterized in that, The inhibitor includes PKC beta inhibitor 1.

9. An antitumor pharmaceutical composition, characterized by, The pharmaceutical composition includes PKC beta inhibitor 1, a pharmaceutically acceptable carrier and an anticancer drug.

10. The pharmaceutical composition of claim 9, wherein, The anticancer drug includes cisplatin, osimertinib.

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