A competitive inhibitor and its application

Abstract

The present application relates to the technical field of biomedicine, and particularly relates to a competitive inhibitor and application thereof, the competitive inhibitor can specifically bind to PGK1, and has a competitive inhibition effect on the combination of PGK1 and PD-1. The present application finds a new mechanism of PGK1 in mediating the activation of PD-1 without relying on the extracellular ligand of PD-1. The competitive inhibitor can directly target PGK1, and block the interaction between PGK1 and PD-1, so as to avoid the activation of PD-1 by PGK1 as much as possible. The competitive inhibitor provided by the present application has a promising prospect as an effective solution to the problem of PD-1 monoclonal antibody resistance.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a competitive inhibitor and its application. Background Technology

[0002] PD-1 (programmed death protein 1) is a classic immunosuppressive receptor, highly expressed in tumor-infiltrating CD8+ T cells. Classical theory posits that PD-L1 is the most important extracellular ligand of PD-1, frequently highly expressed in tumor cells and antigen-presenting cells. Both PD-1 and PD-L1 are type I transmembrane proteins that recognize and bind to each other through their respective extracellular domains. Several monoclonal antibody drugs targeting either PD-1 or the extracellular domain of PD-L1 have been developed, including nivolumab and pembrolizumab targeting the PD-1 extracellular domain, and atezolizumab, durvalumab, and avelumab targeting the PD-L1 extracellular domain. These monoclonal antibodies can relieve the immunosuppressive effect of the PD-1 / PD-L1 axis by disrupting the PD-1-PD-L1 binding pathway, preventing CD8+ T cell dysfunction and exhaustion. These drugs have been approved for first-line treatment of tumors such as melanoma, non-small cell lung cancer, kidney cancer, Hodgkin's lymphoma, and liver cancer.

[0003] Monoclonal antibodies targeting PD-1 / PD-L1 have enhanced the body's anti-tumor immunity to some extent, improving the quality of life and survival prospects of cancer patients. However, regrettably, only a small percentage of patients truly benefit from them. For example, melanoma is one of the solid tumors with the highest response rate to PD-1 / PD-L1 monoclonal antibodies, but the response rate of melanoma patients to the PD-1 monoclonal antibody nivolumab is only about 40%, with 60% of these patients exhibiting primary resistance. Furthermore, even among patients who respond to PD-1 monoclonal antibodies, the majority eventually relapse and gradually develop secondary resistance.

[0004] In fact, tumors are divided into "hot" tumors with high PD-1 expression levels, such as melanoma, liver cancer, and non-small cell lung cancer, and "cold" tumors with low PD-1 expression levels, such as pancreatic cancer. Current clinical guidelines primarily recommend PD-1 monoclonal antibodies for "hot" tumors. While there is no shortage of PD-1 monoclonal antibody targets in tumors with high PD-1 expression, their primary resistance rates to PD-1 / PD-L1 monoclonal antibodies remain high. For example, the response rate to the PD-1 monoclonal antibody pembrolizumab in non-small cell lung cancer (NSCLC) patients is approximately 19.4%, while the response rate to nivolumab is approximately 19-20%. In liver cancer patients, the response rate to PD-1 monoclonal antibodies is only about 15-20%. These data indicate that despite the high PD-1 expression in these tumors, their response levels to PD-1 monoclonal antibodies are indeed concerning.

[0005] Currently, clinical practice often employs various approaches, such as combining PD-1 monoclonal antibodies with chemotherapy, radiotherapy, PD-L1 monoclonal antibodies, and targeted therapy, in an attempt to improve the high resistance rate of PD-1 monoclonal antibodies. Although combination therapy has improved the response rate of clinical oncology patients to some extent, it has also brought many problems: increased adverse reactions, higher treatment costs, drug resistance issues, and uncertain long-term effects. Summary of the Invention

[0006] The purpose of this invention is to provide a competitive inhibitor and its application, which can block the PGK1-PD-1 pathway, thereby minimizing the activation of PD-1 by PGK1. PGK1-mediated PD-1 activation is a novel PD-1 activation mode independent of the PD-1 extracellular ligand. This non-classical activation pathway may be an important reason for PD-1 monoclonal antibody resistance in the prior art. Therefore, this competitive inhibitor helps to overcome the problem of PD-1 monoclonal antibody resistance.

[0007] Therefore, in a first aspect, the present invention provides the use of a competitive inhibitor in the preparation of a medicament for treating cancer, said competitive inhibitor specifically binding to PGK1 and competitively inhibiting the binding of PGK1 and PD-1.

[0008] In some embodiments, the competitive inhibitor does not reduce the glycolytic activity of PGK1.

[0009] In some embodiments, the competitive inhibitor inhibits the dephosphorylation of serine at position 203 of PGK1.

[0010] In some embodiments, the competitive inhibitor comprises a compound selected from those of Formula I, their salts, or solvates thereof:

[0011]

[0012] In some embodiments, the cancer includes at least one selected from the group consisting of: melanoma, lung cancer, colorectal cancer, liver cancer, brain tumor, bladder cancer, stomach cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, head and neck cancer, cervical cancer, endometrial cancer, kidney cancer, esophageal cancer, gallbladder cancer, non-Hodgkin's lymphoma, prostate cancer, thyroid cancer, female reproductive tract cancer, lymphoma, bone cancer, skin cancer, and testicular cancer.

[0013] In some embodiments, the formulation of the drug includes any one of the following: injection, tablet, capsule, aerosol, suppository, film, controlled-release or sustained-release formulation, or nanoformulation.

[0014] In a second aspect, the present invention provides the use of a competitive inhibitor in the preparation of a drug that inhibits PD-1 phosphorylation, said competitive inhibitor specifically binding to PGK1 and competitively inhibiting the binding of PGK1 and PD-1.

[0015] In some embodiments, the competitive inhibitor does not reduce the glycolytic activity of PGK1.

[0016] In some embodiments, the competitive inhibitor inhibits the dephosphorylation of serine at position 203 of PGK1.

[0017] In some embodiments, the competitive inhibitor comprises a compound selected from those shown in Formula I, their salts, or their solvates.

[0018] In some embodiments, the formulation of the drug includes any one of the following: injection, tablet, capsule, aerosol, suppository, film, controlled-release or sustained-release formulation, or nanoformulation.

[0019] A third aspect of the invention provides a pharmaceutical composition comprising a compound of Formula I and pharmaceutically acceptable excipients.

[0020] In some embodiments, the pharmaceutical composition further includes an antibody or an antigen-binding fragment thereof; the antibody specifically binds to the extracellular domain of PD-1 or PD-L1.

[0021] In some embodiments, the antibody is a monoclonal antibody.

[0022] In some embodiments, the antibody comprises any one or more combinations selected from the group consisting of nivolumab, pembrolizumab, atezolizumab, durvalumab, and avelumab, etc.

[0023] In some embodiments, the pharmaceutical composition is formulated as any one of injections, tablets, capsules, aerosols, suppositories, films, controlled-release or sustained-release formulations, or nanoformulations.

[0024] Compared with the prior art, the beneficial effects of the present invention include:

[0025] (1) This invention provides the application of a competitive inhibitor in the preparation of a drug for treating cancer. The competitive inhibitor works mainly based on the following principle: by competitively inhibiting the binding of PGK1 and PD-1, it significantly blocks the PGK1-PD-1 signaling pathway and avoids the activation of PD-1 by PGK1 as much as possible.

[0026] (2) In the solution provided by this invention, the aforementioned competitive inhibitor can be used alone or in combination with drugs such as PD-1 or PD-L1 monoclonal antibodies. When used in combination, it has a significantly better therapeutic effect. This invention has verified the above-mentioned effects through animal experiments.

[0027] (3) This invention discovers a novel mechanism by which PGK1 mediates PD-1 activation without relying on PD-1 extracellular ligands. The aforementioned competitive inhibitors can directly target PGK1 and block its interaction with PD-1. Therefore, the competitive inhibitors provided by this invention show promise as an effective solution to the problem of PD-1 monoclonal antibody resistance.

[0028] (4) This invention provides a small molecule inhibitor as shown in Formula I. Compared with the combined use of multiple expensive monoclonal antibody drugs, the small molecule drug has a lower production cost and is more economical to use. This can significantly reduce treatment costs and improve the accessibility and availability of treatment.

[0029] (5) The technical solution provided by this invention can improve long-term efficacy. The competitive inhibitor of this invention, through a novel mechanism of action, can stably inhibit the non-classical activation pathway of PD-1 for a long period. In animal tumor models, the competitive inhibitor of this invention can effectively prevent CD8+ T cells from reaching terminal exhaustion, ensuring a sustained and effective anti-tumor immune response for a long time, and mice exhibit strong tolerance. It is expected to demonstrate better efficacy and safety in long-term clinical treatment.

[0030] (6) The technical solution provided by this invention has therapeutic specificity and low toxicity. The competitive inhibitor provided by this invention is specifically targeted at the specific interaction site between PGK1 and PD-1, which has high specificity, reduces interference with other pathways, has low cytotoxicity, and improves the precision and efficacy of treatment. Attached Figure Description

[0031] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. In the drawings:

[0032] Figure 1 Results of the experiment to detect the interaction between PGK1 and PD-1 using protein immunoprecipitation assay;

[0033] Figure 2 Experimental results of detecting the interaction between PGK1 and PD-1 using the GST-pull-down experiment;

[0034] Figure 3 : Binding curve of PGK1 with the small molecule inhibitor E465;

[0035] Figure 4 Curve showing the competitive inhibition of PGK1-PD-1 binding by the small molecule inhibitor E465;

[0036] Figure 5 In vitro phosphorylation of PD-1 by PGK1, phosphorylation of PD-1 by PGK1 in HEK393T, and the effect of small molecule inhibitors on PD-1 phosphorylation;

[0037] Figure 6 Figure: Results of flow cytometry analysis of the effect of the small molecule inhibitor E465 on the terminal exhaustion of CD8+ T cells in an in vitro CD8+ T cell exhaustion model.

[0038] Figure 7 Effects of the small molecule inhibitor E465 on the expression of exhaustion markers and cytokine secretion in an in vitro CD8+ T cell exhaustion model;

[0039] Figure 8 Effects of the small molecule inhibitor E465 on PGK1 glycolytic enzyme activity and cell survival in CD8+ T cells;

[0040] Figure 9 Effects of the small molecule inhibitor E465 on tumor growth in a subcutaneous injection model of B16-OVA and LLC1;

[0041] Figure 10 Effects of small molecule inhibitors on exhaustion and cytokine secretion of locally infiltrating CD8+ T cells in the B16-OVA tumor model. Detailed Implementation

[0042] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

[0043] PGK1, short for phosphoglycerate kinase 1, is an important kinase in cellular glycolysis. Recent studies have found that PGK1 can not only reversibly catalyze the conversion of ADP and 1,3-bisphosphoglycerate into ATP and 3-phosphoglycerate as a glycolytic enzyme, but also act as a protein kinase to phosphorylate other proteins, such as Beclin1 and PDHK1, thereby regulating various cellular physiological activities. However, there are currently no reports on the mechanism by which PGK1 can directly act on PD-1, nor are there any drug studies targeting this interaction.

[0044] This invention discovers that the glycolytic enzyme PGK1 can mediate PD-1 activation and lead CD8+ T cells to terminal exhaustion in a way that does not depend on PD-1 extracellular ligands. This is a novel, non-classical PD-1 activation pathway and a key mechanism leading to resistance to traditional treatment strategies that only target PD-1 extracellular signals.

[0045] Specifically, this invention has found that PGK1 and PD-1 have a direct interaction, and phosphorylation of serine at position 203 in PGK1 can regulate this interaction (this serine at position 203 refers to the 203rd amino acid in human PGK1, whose amino acid sequence can be found in the NCBI database NP_000282, along the direction from the amino terminus to the carboxyl terminus). Dephosphorylation of serine at position 203 will enhance the PGK1-PD-1 interaction.

[0046] Targeting the key amino acid residues in PGK1 that interact with PD-1, this invention has screened and obtained small molecule drugs that can effectively block PGK1-mediated PD-1 activation and downstream T cell terminal exhaustion signals. Simultaneously, it avoids the toxicity problems of traditional PGK1 inhibitors.

[0047] In existing technologies, small molecule drugs targeting PGK1 primarily target the ATP-binding region of PGK1, nonspecifically inhibiting PGK1 glycolytic activity and producing cytotoxicity. In some embodiments of this invention, a small molecule inhibitor targeting PGK1, E465, is provided. This inhibitor can inhibit the PGK1-PD-1 interaction without affecting PGK1 glycolytic enzyme activity, exhibiting stronger specificity. Furthermore, E465 did not cause significant toxicity in cell and animal models.

[0048] The structural formula of the small molecule inhibitor E465 obtained by screening in this invention is shown in Formula I.

[0049]

[0050] The small molecule inhibitor E465 is available commercially from ChemDiv, catalog number E465-0645.

[0051] After screening and obtaining inhibitors of PGK1-PD-1 interaction, this invention conducted experiments on their effects on PD-1 function, CD8+ T cell terminal exhaustion, PGK1 glycolytic activity, cell survival, and their efficacy in melanoma, lung cancer, colorectal cancer, and liver cancer-bearing mice, and obtained the following results:

[0052] (1) In vitro experiments:

[0053] A) Protein immunoprecipitation (Co-IP) and GST-pull-down experiments showed that PGK1 can directly interact with PD-1. Using S203A or S203D point mutations to simulate PGK1, continuous dephosphorylation and continuous phosphorylation at the S203 position can significantly change the interaction strength between PGK1 and PD-1.

[0054] B) Surface plasmon resonance experiments demonstrate that PGK1 and the small molecule inhibitor E465 can interact, and that the small molecule inhibitor E465 can compete for the interaction between PGK1 and PD-1.

[0055] C) Protein immunoprecipitation (Co-IP) and in vitro phosphorylation experiments demonstrated that PGK1 can phosphorylate PD-1, while the small molecule inhibitor E465 can significantly inhibit the interaction between PGK1 and PD-1 and the phosphorylation of PD-1.

[0056] D) In ​​vitro induction of CD8+ T cell terminal exhaustion results showed that the small molecule inhibitor E465 could inhibit the promoting effect of lactate on CD8+ T cell terminal exhaustion and salvage its ability to secrete cytokines.

[0057] E) The addition of the small molecule inhibitor E465 during the in vitro culture of CD8+ T cells did not significantly interfere with the glycolytic activity of PGK1 and cell survival.

[0058] (2) In vivo experiments:

[0059] A) Results from subcutaneous injection tumor models of B16-OVA (melanoma) and LLC1 (lung cancer) showed that the single use of the small molecule inhibitor E465 significantly inhibited the growth of subcutaneous tumors, with tumor growth curves and final tumor size / weight showing significant differences compared to the control group. Furthermore, combined use with PD-1 monoclonal antibodies significantly improved PD-1 antibody resistance and further enhanced the therapeutic effect of PD-1 monoclonal antibodies.

[0060] B) Analysis of tumor-infiltrating CD8+ T cells revealed that the small molecule inhibitor E465 could significantly rescue CD8+ T cells from terminal exhaustion and promote their secretion of more cytokines.

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0062] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0063] Example 1: Interaction between PGK1 and PD-1

[0064] This embodiment experimentally analyzes the interaction between PGK1 and PD-1. The S203A point mutation was used to simulate the persistent dephosphorylation state at the S203 position of PGK1, and the S203D point mutation was used to simulate the persistent phosphorylation state at the S203 position of PGK1. Specific experiments included:

[0065] 1. Protein co-precipitation (Co-IP) experiment

[0066] In the HEK293T cell line, exogenous plasmids were transfected with PEI to simultaneously express PD-1 and PGK1. The following experimental groups were set up, and the specific proteins expressed in each group were as follows:

[0067] Group 1, untagged PD-1, wild-type PGK1 with Flag tag;

[0068] Group 2, untagged PD-1, PGK1 with a Flag tag and an S203A mutation;

[0069] Group 3: unlabeled PD-1, PGK1 with a Flag tag and the S203D mutation.

[0070] A control group was also set up:

[0071] Control group 1, expressing only unlabeled PD-1;

[0072] Control group 2 expressed only wild-type PGK1 with the Flag tag;

[0073] Control group 3, expressing only PGK1 with the Flag tag and the S203A mutation.

[0074] Control group 4 expresses only PGK1 with the Flag tag and the S203D mutation.

[0075] Twenty-four hours after transfection, cell samples were collected, and PGK1 and its interacting PD-1 were immunoprecipitated using Flag antibody, followed by Western blot analysis. The experimental results are as follows: Figure 1 As shown, according to Figure 1 It is known that PGK1 can interact with PD-1; and phosphorylation at position 203 of PGK1 can regulate this interaction, while dephosphorylation at position 203 will enhance the PGK1-PD-1 interaction.

[0076] 2. GST-pull-down experiment

[0077] After expression in host cells, the following proteins were obtained through protein purification: GST-tagged protein, GST-tagged PGK1 protein, and His-tagged PD-1 protein. 1 μg of each of the following proteins was added to 1×PBS and incubated overnight at 4°C: GST-tagged protein, GST-tagged PGK1 protein, and His-tagged PD-1 protein. The next day, GST, GST-PGK1, and their interacting PD-1 protein were enriched using glutathione purification resin. Western blot analysis was used to detect the interaction between PGK1 and PD-1.

[0078] Experimental results are as follows Figure 2 As shown, according to Figure 2 It is known that PGK1 can interact with PD-1.

[0079] Example 2: Small molecule inhibitors significantly compete for PGK1 binding to PD-1

[0080] This embodiment analyzes the binding interaction between PGK1 and the small molecule inhibitor E465, and the competitive effect of the small molecule inhibitor E465 on the binding of PGK1-PD-1. Specific experiments include:

[0081] 1. PGK1 was used for surface plasmon resonance (SPR) experiments using the Biacore 8K high-throughput molecular interaction analysis platform. Each target was immobilized on the flow cell of a CM5 sensor chip (GE Healthcare) via amine coupling. Briefly, PGK1 was diluted to 50 μg / ml in 10 mM pH 4.5 acetate. The protein solutions were then injected separately onto the carboxyl-modified sensor surface to form amine bonds. The PGK1 immobilization level was approximately 10,000 RU. Binding analysis was performed at 25 °C and a flow rate of 30 μl / min. Electrophoresis buffer (1×PBS, 0.05% Tween 20 and 5% dimethyl sulfoxide, pH 7.4) was used at indicated gradient concentrations (specifically, the concentrations for E465 are shown in the image). Figure 3 (As shown) the recovered drug was run on each target. An empty flow cell without any immobilized proteins was used as a subtraction reference. Binding curves were analyzed using the kinetic binding model provided by Biacore evaluation software.

[0082] The analysis results combined with the curves are as follows: Figure 3 As shown, this indicates that PGK1 has a good binding effect with the small molecule inhibitor E465.

[0083] 2. As described in section 1, immobilize the PGK1 protein onto the sensor surface. The PGK1 immobilization level is approximately 10,000 RU. Binding analysis is performed at 25°C and a flow rate of 30 μl / min. After competitive binding of the PGK1 protein to the sensor surface for 30 minutes at room temperature with a final concentration of 25 μM E465 or an equal volume of DMSO added to the electrophoresis buffer (same as in section 1), electrophoresis buffer containing a final concentration of 1 μM PD1 protein is added. An empty flow cell without any immobilized protein is used as a subtraction reference. The binding kinetics of PGK1 and PD-1 after the addition of E465 or an equal volume of DMSO are analyzed using the kinetic binding model provided by Biacore evaluation software.

[0084] Combining the curves and analysis results, as follows Figure 4 As shown, the small molecule inhibitor E465 can significantly compete for the binding of PGK1 and PD-1.

[0085] Example 3: Inhibitory effect of small molecule inhibitors on PD-1 activation levels

[0086] The phosphorylation level at position Y248 of PD-1 is a recognized indicator of PD-1 activation. This study experimentally analyzed the effect of the small molecule inhibitor E465 on the PD-1 activation level. Specifically, this included:

[0087] In the HEK293T cell line, a PD-1 plasmid with a Flag tag at the C-terminus was transfected with PEI. 24 hours after transfection, 10 μM of the small molecule inhibitor E465 was added, or an equal volume of DMSO was used for an additional 24 hours of treatment. Cell samples were then collected, and PD-1 protein was enriched using immunoprecipitation. The phosphorylation level at PD-1 Y248 was then detected. Experimental results are as follows: Figure 5 As shown, the small molecule inhibitor E465 can significantly inhibit the phosphorylation of PD-1, indicating that the small molecule inhibitor E465 can inhibit the activation level of PD-1.

[0088] Example 4: Effects of Small Molecule Inhibitors on CD8+ T Cell Exhaustion

[0089] Naive CD8+ T cells were isolated from the spleen and lymph nodes of OT1 mice and induced to exhaust the cells by continuous stimulation with 100 ng / mL OVA for 5 days daily. Simultaneously, the cells were treated with 10 mM lactate, 10 μM small molecule inhibitor E465, or an equal volume of DMSO. The expression of exhaustion markers and cytokine secretion of CD8+ T cells were detected on days 5 and 8, respectively.

[0090] Flow cytometry results as follows Figure 6 As shown, the relevant statistical results are as follows: Figure 7 As shown above, the results indicate that the small molecule inhibitor E465 can significantly inhibit the promoting effect of lactate on T cell terminal exhaustion and significantly rescue cytokine secretion.

[0091] Example 5: Effects of small molecule inhibitors on PGK1 glycolytic activity and cell survival

[0092] Naive CD8+ T cells were isolated from the spleen and lymph nodes of OT1 mice. CD8+ T cells were activated with 100 ng / mL OVA, and simultaneously treated with a final concentration of 10 μM of the small molecule inhibitor E465 or an equal volume of DMSO. After 24 h, the glycolytic activity of PGK1 in CD8+ T cells (Bioss, AK509U) and apoptosis (Elabscience, E-CK-A238) were detected using commercially available kits.

[0093] Experimental results are as follows Figure 8 As shown, the small molecule inhibitor E465 did not significantly alter the activity of PGK1 glycolytic enzyme or cell survival in CD8+ T cells.

[0094] Example 6: Effects of Small Molecule Inhibitors on Tumor Growth

[0095] Female C57BL / 6 mice aged 6-8 weeks were selected for the experiment. B16-OVA and LLC1 cells in the logarithmic growth phase were digested, washed twice with PBS, counted, and then resuspended in PBS to adjust the cell suspension concentration to 1×10⁻⁶. 7 / ml. Tumor cells were subcutaneously inoculated into the lateral rib area of ​​mice, 100 μL of cell suspension per mouse, containing 1×10⁶ cells / ml. 6 Tumor cells were used. Day 0 was the day of tumor cell inoculation. The treatment group received intraperitoneal injections of the small molecule inhibitor E465 5 mg / kg / day or an equal volume of PBS on Days 1, 3, 5, and 7, respectively. Days 8, 11, and 14 after tumor cell inoculation, 200 μg / animal of PD-1 monoclonal antibody was administered intraperitoneally. Starting 7 days after tumor cell inoculation, the long diameter (L) and short diameter (S) of the tumor were measured daily using calipers. The tumor size was calculated using the formula L×S×S×0.5, and a tumor growth curve was plotted. The experimental results are as follows: Figure 9 As shown.

[0096] On Day 20, mice were euthanized, and tumor tissue was isolated, minced, and digested at room temperature for 1 hour using a digester containing 1 mg / ml collagenase D and 100 μg / mL DNase 1. The resulting cell suspension was then ground through a 200-mesh sieve, and tumor-infiltrating immune cells were enriched using Ficol lymphocyte separation medium. The immune cell samples were divided into two aliquots. One aliquot was stained for CD45, CD3, CD8, PD-1, and Tim3; CD8+ T cells positive for both PD-1 and Tim3 were identified as terminally exhausted CD8+ T cells. The other aliquot was resuspended in 1640 medium containing 100 ng / mL PMA (phorbol ester), 1 μg / mL iomycin, and 1 μg / mL BFA (brevidin A). After 6 hours, the cells were harvested and stained for CD45, CD3, CD8, IFNg, and Granzyme B. The proportion of terminally exhausted and cytokine-secreting positive cells among CD8+ T cells was calculated. The experimental results are as follows: Figure 10 As shown.

[0097] according to Figure 9 It is evident that small molecule inhibitors significantly inhibit tumor growth in subcutaneously injected B16-OVA and LLC1 tumor models, thereby enhancing the therapeutic effect of PD-1 monoclonal antibodies.

[0098] according to Figure 10 It is known that small molecule inhibitors can significantly inhibit the terminal exhaustion of tumor-infiltrating CD8+ T cells and promote their cytokine secretion.

[0099] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Use of a competitive inhibitor for the preparation of a medicament for the treatment of cancer, characterized in that, The competitive inhibitor can specifically bind to PGK1 and has a competitive inhibition effect on the binding of PGK1 and PD-1; the competitive inhibitor is a compound selected from the compounds shown in Formula I or a salt thereof: ； The cancer includes at least one selected from the group consisting of melanoma, lung cancer, colorectal cancer, liver cancer, brain tumor, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, head and neck cancer, cervical cancer, endometrial cancer, kidney cancer, esophageal cancer, gallbladder cancer, non-Hodgkin's lymphoma, prostate cancer, thyroid cancer, female genital tract cancer, lymphoma, bone cancer, skin cancer, and testicular cancer.

2. Use according to claim 1, wherein The competitive inhibitor has no reduction effect on the glycolytic activity of PGK1.

3. Use according to claim 1 or 2, characterized in that, The competitive inhibitor inhibits the dephosphorylation of the serine at position 203 of PGK1.

4. The use according to claim 1, wherein The preparation form of the drug includes any one of injection, tablet, capsule, aerosol, suppository, film, controlled or sustained release preparation, or nano preparation.

5. A pharmaceutical composition, characterized by, The compound includes the compound shown in Formula I or a salt thereof, and pharmaceutically acceptable excipients.

6. The pharmaceutical composition of claim 5, wherein The pharmaceutical composition further includes an antibody or an antigen binding fragment thereof; the antibody specifically binds to the extracellular domain of PD-1 or PD-L1.

7. The pharmaceutical composition of claim 6, wherein The antibody is a monoclonal antibody.

8. The pharmaceutical composition of claim 6, wherein The antibody includes any one or a combination of two or more selected from the group consisting of nivolumab, pembrolizumab, atezolizumab, durvalumab, and avelumab.

9. The pharmaceutical composition according to claim 5, wherein The preparation form of the pharmaceutical composition includes any one of injection, tablet, capsule, aerosol, suppository, film, controlled or sustained release preparation, or nano preparation.

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