Application of the small molecule compound HY-P3485 in radiosensitization for non-small cell lung cancer

Abstract

This invention discloses the application of the small molecule compound HY-P3485 in radiosensitization of non-small cell lung cancer (NSCLC), belonging to the field of biomedical technology. The small molecule compound HY-P3485, a polypeptide with the amino acid sequence GAGGVGKSAL, was obtained through high-throughput screening. This compound specifically binds to the ADAM17 protein, spatially shielding its Asn264 glycosylation site, effectively blocking N-glycosylation modification and inducing ADAM17 degradation. Experiments demonstrate that HY-P3485 can significantly reduce the expression of ADAM17 on the surface of tumor cell membranes, reverse the radiotherapy-induced upregulation, and significantly enhance the radiosensitivity of NSCLC in vitro and in vivo, promoting tumor cell apoptosis and effectively inhibiting tumor growth. This invention provides a new target and intervention strategy for overcoming radiotherapy resistance in NSCLC.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of the small molecule compound HY-P3485 in radiosensitization of non-small cell lung cancer (NSCLC). Background Technology

[0002] The efficacy of NSCLC treatment is severely limited by radioresistance. ADAM17 (de-integrin-metalloproteinase 17, also known as TACE) is a key transmembrane proteolytic enzyme that plays a crucial role in tumorigenesis, development, and treatment resistance. Currently, the development of inhibitors targeting ADAM17 is one of the hottest topics in the field of anti-tumor drugs. N-glycosylation modification at the Asn264 (N264) site is a prerequisite for ADAM17 protein membrane localization and the exertion of enzymatic activity. Inhibiting the N-glycosylation modification of ADAM17 has become a potential strategy to enhance radiosensitivity. Existing ADAM17 inhibitors mostly target its metalloproteinase active site, lacking specificity and having significant toxic side effects. Currently, there are no reports of drugs that precisely intervene in the ADAM17 N-glycosylation pocket. Therefore, developing inhibitors that can specifically bind to this glycosylation site region has significant clinical value. Summary of the Invention

[0003] The purpose of this invention is to provide the application of the small molecule compound HY-P3485 in radiosensitization of NSCLC, thereby addressing the problems existing in the prior art. This invention targets ADAM17, using the small molecule compound HY-P3485 to specifically occupy its N-glycosylation pocket, blocking glycosylation modification at the Asn264 site, thereby inducing target protein degradation, reducing radiotherapy resistance, and thus sensitizing NSCLC radiotherapy. In tumor models, inhibiting ADAM17 N-glycosylation significantly enhances the anti-tumor response and significantly sensitizes radiotherapy, effectively inhibiting NSCLC progression. This provides a new target and intervention strategy for developing radiosensitizing drugs for NSCLC and overcoming radiotherapy resistance in NSCLC.

[0004] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of the small molecule compound HY-P3485 in the preparation of a drug that targets and inhibits N-glycosylation modification of ADAM17 protein. The small molecule compound HY-P3485 is a polypeptide with the amino acid sequence GAGGVGKSAL (SEQ ID NO.1).

[0005] This invention also provides the application of the small molecule compound HY-P3485 in the preparation of radiosensitizers for NSCLC, wherein the small molecule compound HY-P3485 is a polypeptide with the amino acid sequence GAGGVGKSAL.

[0006] Preferably, the small molecule compound HY-P3485 enhances the sensitivity of tumor cells to radiotherapy by reducing the expression level of ADAM17 protein on the surface of NSCLC cell membranes, reversing radiotherapy-induced upregulation of ADAM17.

[0007] The present invention also provides the use of the small molecule compound HY-P3485 in the preparation of a drug for treating NSCLC and / or reversing radiotherapy resistance in NSCLC, wherein the small molecule compound HY-P3485 is a polypeptide with the amino acid sequence GAGGVGKSAL.

[0008] Preferably, the small molecule compound HY-P3485 is administered in combination with radiotherapy.

[0009] The present invention also provides a composition comprising a therapeutically effective amount of a small molecule compound HY-P3485, and a pharmaceutically acceptable carrier, excipient, or diluent, wherein the small molecule compound HY-P3485 is a polypeptide with the amino acid sequence GAGGVGKSAL.

[0010] The present invention also provides the use of the composition in the preparation of radiosensitizers for NSCLC.

[0011] The present invention also provides the use of the described composition in the preparation of medicaments for treating NSCLC and / or reversing radiotherapy resistance in NSCLC.

[0012] Preferably, the composition is used in combination with radiotherapy.

[0013] In this embodiment of the invention, the small molecule compound HY-P3485 is used as an example to illustrate the above technical solution. This small molecule compound specifically binds to the N-glycosylation pocket region of the extracellular domain of the ADAM17 protein, blocking the modification of the Asn264 site by glycosyltransferase, thereby spatially shielding the Asn264 site and reducing the stability of the ADAM17 protein and its expression level on the membrane surface.

[0014] The specific binding refers to the interaction between the small molecule compound HY-P3485 and the Asn264 amino acid residue of the ADAM17 protein through hydrogen bonds or salt bridges.

[0015] In this invention, the small molecule compound HY-P3485 inhibits the in vitro and in vivo growth of NSCLC and promotes apoptosis by effectively blocking ADAM17 N-glycosylation modification. Targeting ADAM17 in combination with radiotherapy can effectively inhibit tumor growth and reverse radioresistance in vivo.

[0016] However, the present invention is not limited to the polypeptide form of the small molecule compound HY-P3485 itself, but can also be a pharmaceutically acceptable salt, stereoisomer or solvate thereof.

[0017] The present invention discloses the following technical effects: This invention utilizes surface plasmon resonance (SPR) and microthermophoresis (MST) to determine that the small molecule compound HY-P3485 binds to the ADAM17 protein with high affinity. Simultaneously, cell biology experiments revealed that HY-P3485 dose-dependently reduces ADAM17 protein expression on the surface of NSCLC cell membranes, reverses radiotherapy-induced ADAM17 upregulation, and significantly promotes radiotherapy-induced apoptosis. In vivo experiments confirmed that targeting ADAM17 N-glycosylation can reverse radiotherapy resistance in NSCLC and restore in vivo radiosensitivity.

[0018] The HY-P3485 of this invention effectively blocks ADAM17 N-glycosylation modification, inhibiting the growth of NSCLC in vivo and in vitro and promoting apoptosis. Targeting ADAM17 in combination with radiotherapy can reverse radiotherapy resistance in vivo and effectively inhibit tumor growth. Therefore, using the N-glycosylation pocket of ADAM17 as a drug target and employing the small molecule compound HY-P3485 for precise intervention can be used to develop novel targeted sensitizing drugs for the treatment of NSCLC, and provides a new target and intervention strategy for overcoming radiotherapy resistance in NSCLC. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The molecular structure of HY-P3485, a small molecule compound that inhibits ADAM17 N-glycosylation modification, provided by this invention; Figure 2 The image shows the SPR single-concentration screening results for the binding of HY-P3485 to ADAM17 protein. Figure 3 The graph shows the SPR multi-concentration detection results of HY-P3485 binding to ADAM17 protein; Figure 4 The figure shows the MST experimental results verifying the binding of HY-P3485 to ADAM17 protein; Figure 5The figure shows the effects of different concentration gradients of HY-P3485 on the expression level of ADAM17 protein on the membrane of NSCLC cell lines (A549 and H1299). In the figure, A represents the expression level of ADAM17 in A549 cell lines after treatment with different compounds; B represents the expression level of ADAM17 in A549 cell lines under different concentration gradients of HY-P3485; and C represents the expression levels of ADAM17 in HY-P3485 alone, radiotherapy alone (RT), and radiotherapy combined with HY-P3485. The expression of ADAM17 in the A549 cell line under HY-P3485 treatment; D shows the expression of ADAM17 in the H1299 cell line after treatment with different compounds; E shows the expression of ADAM17 in the H1299 cell line under different concentration gradients of HY-P3485; F shows the expression of ADAM17 in the H1299 cell line under treatment with HY-P3485 alone, radiotherapy alone (RT), and radiotherapy combined with drugs (RT+HY-P3485). Figure 6 The results show the effect of HY-P3485 on the apoptosis levels of A549 and H1299 cells after radiotherapy; where A represents the statistical results of apoptosis in the A549 cell line under different treatments; B is a representative flow cytometry graph of A; C represents the statistical results of apoptosis in the H1299 cell line under different treatments; and D is a representative flow cytometry graph of C. Figure 7 This is a graph validating the in vivo antitumor efficacy of HY-P3485 combined with radiotherapy in an animal subcutaneous tumor experiment. Among them, A is a gross image of subcutaneous tumors in different groups that were removed at the experimental endpoint; B is a growth curve of tumor volume in mice in different groups as a function of treatment time; and C is a graph showing the quantitative analysis results of subcutaneous tumor weight in different groups. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Example 1: Virtual screening and binding mode identification of candidate compounds based on structure Protein preparation: The 3D structure of ADAM17 was downloaded from the PDB database (PDBID: 2DDF). Hydrogenation and energy optimization of the protein were performed using the ProteinPreparationWizard module in Schrödinger software (OPLS2005 force field, RMSD 0.30 Å). A grid file (box size: 20 Å × 20 Å × 20 Å) was generated centered at Asn264 using the ReceptorGridGeneration module.

[0027] Compound preparation and molecular docking: The 2D structures of the small molecule compound library are processed using the LigPrep module for hydrogenation, energy optimization, etc., to output 3D structures. Virtual Screening Workflow is used for virtual screening, and the Glide module enables acceptor and ligand molecules to dock with each other through geometric and energy matching. The top 20 small molecule compounds with the highest molecular docking scores are selected as candidate molecules for further validation. These 20 small molecule compounds include HY-N2456 (Mogroside IV-E), HY-P3321 (Phytochelatin 4), HY-N6833 (Rebaudioside M), HY-P3485 (GAGGVGKSAL), HY-N0205 (Pulchinenoside C), HY-P5222 (Angio-S), HY-100563A (Cyclo(RGDyK)), HY-N5063 (Plantainoside D), HY-P1139 (Cortistatin-8), HY-F0003A (NADPH (tetracyclohexanamine)), HY-N2524 (Camelliaside A), and HY-N2531 (Notoginsenoside). Fc), HY-N6647 (Luteolin-7-rutinoside), HY-19936A (ACHN-975 (TFA)), HY-125531 (Dactylorhin A), HY-N9529 (Kaempferol 3-O-(2''-O-α-rhamnosyl-6''-O-malonyl-β-glucoside)), HY-N10592 (Herbacetin-3-sophoroside-8--glucoside), HY-N7948 (Isomaltotetraose), HY-N5042 (Forsythoside I), HY-B165 (Deferoxamine).

[0028] Example 2: Surface plasmon resonance (SPR) and micro-thermophoresis (MST) to verify compound affinity 1. SPR test: Protein immobilization was performed using the amino-coupled method; Protein-coupled buffer solution: 1.0×PBS-P+ (pH 7.4); Interaction buffer solution: 1.0×PBS-P+ (pH 7.4), 5% (v / v) DMSO; The single-concentration screening concentration for compounds was 50 μM.

[0029] 1.1 Protein Coupling (1) Place the running buffer (200 mL 1×PBS Buffer), water bottle, and waste bottle in the left and right trays respectively, and insert the corresponding inlet tubes.

[0030] (2) Hold the CM5 chip with the printed side facing up. Gently push the chip into the slot following the direction of the arrow on the chip, and finally close the chip.

[0031] (3) Activate chip channel 2 with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, GE Healthcare) and N-hydroxysuccinimide (NHS, GE Healthcare) at a flow rate of 10 μL / min.

[0032] (4) Dilute the ligand protein to 50 μg / mL with sodium acetate at pH 4.5 and immobilize the protein in channel 2 of the chip at a flow rate of 10 μL / min.

[0033] (5) Seal the channel with ethanolamine at a flow rate of 10 μL / min.

[0034] (6) Repeat steps (3)-(5) for channel 1 as a reference, except that protein-free acetate buffer is used in step (4).

[0035] 1.2 Protein-analyte interaction test 1.2.1 Solvent Correction Prepare a 5% DMSO concentration calibration curve by mixing 4.5% and 5.8% mother liquor according to Table 1.

[0036] Table 1. Solvent Correction Solution Preparation Table 1.2.2 Determination of analyte Take a 96-well plate, dilute the sample for single-concentration screening to 50 μM with PBS solution, and flow it at a rate of 10 μL / min for 150 s in each run. At the end of each flow, regenerate the chip with 10 mM glycine hydrochloride (pH 2.0) solution for 5 min, and repeat this process until all compounds have been measured.

[0037] In a single-concentration test, a 50 μM HY-P3485 solution flowing through the chip resulted in a binding response value (RU) of 36.58, far exceeding the positive decision threshold (RU20). (See attached image.) Figure 2In multi-concentration gradient assays (0.3125 μM to 10 μM, two-fold gradient), HY-P3485 and ADAM17 exhibited significant dose-dependent binding (see [link to assay]). Figure 3 The presence of a rapidly binding phase and a relatively stable dissociation phase indicates the formation of a stable complex.

[0038] 2. MST detection To eliminate the steric hindrance effect of solid-phase coupling, further verification was performed in a free solution state. Recombinant ADAM17 protein was specifically labeled with a fluorescent dye (RED-NHS), and incubated with HY-P3485 solution at 16 serial dilutions before analysis. The results showed that the binding curve exhibited a typical S-type dose-dependent response (see [link to data]). Figure 4 The fitting yielded an equilibrium dissociation constant (Kd) of 2.84 ± 0.22 μM, further confirming that HY-P3485 can bind ADAM17 strongly and specifically in a liquid environment.

[0039] Example 3: In vitro cell experiment on the inhibition of ADAM17 protein expression on membrane surface by HY-P3485 Experimental methods: A549 and H1299 cell lines were digested with trypsin to prepare single-cell suspensions. Flow cytometry antibodies (Anti-ADAM17 antibody [1F6], abcam, ab57484) were added and incubated in the dark for 30 minutes. After washing and resuspending, the expression of ADAM17 on the membrane surface was detected by flow cytometry.

[0040] Experimental results: Compared with the DMSO control group, HY-P3485, HY-N0205, HY-N2456, HY-N6833, HY-P3321, and HY-P5222 showed better inhibitory effects, with HY-P3485 exhibiting the most significant inhibitory effect (see...). Figure 5 (A and D in the middle). Further concentration gradient experiments showed that HY-P3485 exhibited a significant dose-dependent inhibitory effect, with the inhibitory effect stabilizing at 50 μM and 100 μM (see A and D). Figure 5 (B and E in the middle).

[0041] Reversal of expression under radiotherapy intervention: Cells were divided into a control group (DMSO), a single-drug group (HY-P3485), a radiotherapy-only group (RT), and a combination group (RT+HY-P3485). Radiotherapy alone led to a significant compensatory increase in ADAM17 expression on the membrane surface; however, in the combination treatment, HY-P3485 successfully reversed the radiotherapy-induced upregulation, suppressing it to levels far below those of the control group (see [link to study]). Figure 5 (C and F in the middle).

[0042] Example 4: Effect of HY-P3485 on apoptosis levels in NSCLC cells after radiotherapy Experimental methods: A549 and H1299 cells in good growth condition were seeded at 2000 cells / well in 6-well plates. Grouping was the same as in Example 3. After 24 hours, the radiotherapy group and the combined group received a 15 Gy dose of radiotherapy. Cells were collected, stained using Annexin V-APC and PI double staining, and apoptosis was detected by flow cytometry.

[0043] Experimental results: such as Figure 6 As shown, both HY-P3485 alone and radiotherapy alone can increase the apoptosis rate to some extent (p<0.001). When HY-P3485 is used in combination with radiotherapy, the apoptosis rate is further significantly increased, far exceeding that of the radiotherapy-only group (p<0.001). This indicates that HY-P3485 can significantly enhance radiotherapy-induced apoptosis and has excellent radiosensitization ability.

[0044] Example 5: Antitumor application and safety assessment of HY-P3485 combined with radiotherapy in vivo Tumor model establishment and grouping: 1×10 6 A subcutaneous tumor model was established by injecting CMT167 cells into the right hind limb of C57BL / 6 mice. The tumor volume was increased to approximately 100 mm². 3 At that time, the patients were randomly divided into: control group, single drug group (intraperitoneal injection of HY-P3485, 5mg / kg / day, for 3 consecutive days), radiotherapy group (single local irradiation of 15Gy), and combination group (pre-radiotherapy injection of HY-P3485 combined with radiotherapy).

[0045] In vivo efficacy assessment results: Continuous monitoring showed that the tumors in the control group grew rapidly; the single-drug group and the single-radiotherapy group could slow down growth to some extent; while the combination therapy group showed the most significant tumor growth inhibition effect, with its tumor volume being significantly smaller than that of the radiotherapy-only group (p<0.001, see...). Figure 7 (Middle B). The experimental endpoint was the weighing of the dissected tumor. The average tumor weight in the combined treatment group was significantly lower than that in the other groups (p<0.001, see...). Figure 7 The mice showed good tolerance to the drug (both A and C) during the experiment, and no obvious toxic side effects were observed. This fully demonstrates that targeting ADAM17 combined with radiotherapy can effectively overcome radioresistance and inhibit the growth of NSCLC in vivo.

[0046] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of the small molecule compound HY-P3485 in the preparation of drugs that target and inhibit N-glycosylation modification of ADAM17 protein, characterized in that, The small molecule compound HY-P3485 is a polypeptide with the amino acid sequence GAGGVGKSAL.

2. The application of the small molecule compound HY-P3485 in the preparation of radiosensitizers for non-small cell lung cancer, characterized in that, The small molecule compound HY-P3485 is a polypeptide with the amino acid sequence GAGGVGKSAL.

3. Use according to claim 2, wherein the compound is ###0002### The small molecule compound HY-P3485 reduces the expression level of ADAM17 protein on the cell membrane surface of non-small cell lung cancer cells, reverses radiotherapy-induced upregulation of ADAM17, and enhances the sensitivity of tumor cells to radiotherapy.

4. The use of the small molecule compound HY-P3485 in the preparation of a medicament for treating non-small cell lung cancer and / or reversing radiotherapy resistance of non-small cell lung cancer, characterized in that, The small molecule compound HY-P3485 is a polypeptide with the amino acid sequence GAGGVGKSAL.

5. The use according to claim 4, wherein the compound is ###0002### The small molecule compound HY-P3485 was used in combination with radiotherapy.

6. A composition characterized in that, The composition comprises a therapeutically effective amount of a small molecule compound HY-P3485, and a pharmaceutically acceptable carrier, excipient, or diluent, wherein the small molecule compound HY-P3485 is a polypeptide with the amino acid sequence GAGGVGKSAL.

7. The use of the composition according to claim 6 in the preparation of a radiosensitizer for non-small cell lung cancer.

8. Use of the composition of claim 6 in the preparation of a medicament for treating non-small cell lung cancer and / or reversing radiotherapy resistance in non-small cell lung cancer.

9. The application as described in claim 8, characterized in that, The composition is used in combination with radiotherapy.

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