Competitive inhibitor and application thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to a competitive inhibitor and application thereof, and the competitive inhibitor can be specifically combined with PGK1 and has a competitive inhibition effect on combination of PGK1 and PD-1. According to the present invention, the new mechanism of mediating PD-1 activation of the PGK1 without depending on the PD-1 extracellular ligand is found, and the competitive inhibitor can directly target the PGK1 and block the interaction between the PGK1 and the PD-1 so as to avoid the activation effect of the PGK1 on the PD-1 as much as possible. The competitive inhibitor provided by the invention has a prospect of being used as an effective solution for the problem of PD-1 monoclonal antibody drug resistance.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a competitive inhibitor and application thereof. Background Art

[0002] PD-1 (programmed death protein 1) is a classic immunosuppressive receptor that is highly expressed in tumor-infiltrating CD8+T cells. Classical theory holds that PD-L1 is the most important extracellular ligand of PD-1 and is often highly expressed in tumor cells and antigen-presenting cells. Both PD-1 and PD-L1 are type 1 transmembrane proteins that recognize and bind to each other through their respective extracellular domains. A variety of monoclonal antibody drugs targeting the extracellular domains of PD-1 or PD-L1 have been developed, including nivolumab and pembrolizumab targeting the extracellular domain of PD-1, and atezolizumab, durvalumab, and avelumab targeting the extracellular domain of PD-L1. These monoclonal antibodies can relieve the immunosuppressive effects of the PD-1 / PD-L1 axis by disrupting the PD-1-PD-L1 binding, thereby preventing CD8+T cell dysfunction and exhaustion. These drugs have been approved for the first-line treatment of tumors such as melanoma, non-small cell lung cancer, kidney cancer, Hodgkin lymphoma and liver cancer.

[0003] Monoclonal antibodies targeting PD-1 / PD-L1 enhance the body's anti-tumor immunity to a certain extent, and improve the quality of life and survival expectancy of cancer patients. However, unfortunately, only a small number of patients can truly benefit from it. 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%, of which 60% of patients show primary resistance. In addition, even among patients who respond to PD-1 monoclonal antibodies, most will 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, non-small cell lung cancer, etc., and "cold" tumors with low PD-1 expression levels, such as pancreatic cancer. In the current clinical guidelines, PD-1 monoclonal antibodies are mostly recommended for "hot" tumors. There is no shortage of targets for PD-1 monoclonal antibodies in tumors with high PD-1 expression, but their primary resistance to PD-1 / PD-L1 monoclonal antibodies is still very high. For example, the response rate of patients with non-small cell lung cancer (NSCLC) to PD-1 monoclonal antibody Pembrolizumab is about 19.4%, while the response rate to Nivolumab is about 19-20%. In patients with liver cancer, the response rate to PD-1 monoclonal antibodies is only about 15-20%. These data show that despite the high PD-1 expression in these tumors, their level of response to PD-1 monoclonal antibodies is indeed worrying.

[0005] At present, clinical trials have used PD-1 monoclonal antibodies in combination with chemotherapy, radiotherapy, PD-L1 monoclonal antibodies, and targeted therapy to try to improve the high drug resistance of PD-1 monoclonal antibodies. Although combined therapy has improved the response rate of clinical tumor patients to a certain extent, it has also brought many problems: increased adverse reactions, higher treatment costs, drug resistance problems, and uncertain long-term effects. Summary of the invention

[0006] The purpose of the present invention is to provide a competitive inhibitor and its application, which can block the PGK1-PD-1 pathway, thereby avoiding the activation of PGK1 on PD-1 as much as possible. PGK1-mediated PD1 activation is a new type of PD1 activation that does not depend on the extracellular ligand of PD-1. This non-classical activation pathway may be an important reason for the occurrence of PD-1 monoclonal antibody resistance in the prior art, so the competitive inhibitor helps to overcome the problem of PD-1 monoclonal antibody resistance.

[0007] To this end, in a first aspect, the present invention provides a use of a competitive inhibitor in the preparation of a drug for treating cancer, wherein the competitive inhibitor can specifically bind to PGK1 and has a competitive inhibitory effect on the binding of PGK1 and PD-1.

[0008] In some embodiments, the competitive inhibitor has no reducing effect on the glycolytic activity of PGK1.

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

[0010] In some embodiments, the competitive inhibitor comprises a compound selected from Formula I, a salt thereof, or a solvate thereof:

[0011]

[0012] In some embodiments, the cancer includes at least one selected from the following groups: 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 reproductive tract cancer, lymphoma, bone cancer, skin cancer and testicular cancer, etc.

[0013] In some embodiments, the drug is in the form of an injection, a tablet, a capsule, an aerosol, a suppository, a film, a controlled-release or sustained-release agent, or a nanoformulation.

[0014] In a second aspect, the present invention provides a use of a competitive inhibitor in the preparation of a drug for inhibiting PD-1 phosphorylation, wherein the competitive inhibitor can specifically bind to PGK1 and has a competitive inhibitory effect on the binding of PGK1 and PD-1.

[0015] In some embodiments, the competitive inhibitor has no reducing effect on the glycolytic activity of PGK1.

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

[0017] In some embodiments, the competitive inhibitor comprises a compound selected from Formula I, a salt thereof, or a solvate thereof.

[0018] In some embodiments, the drug is in the form of an injection, a tablet, a capsule, an aerosol, a suppository, a film, a controlled-release or sustained-release agent, or a nanoformulation.

[0019] The third aspect of the present invention provides a pharmaceutical composition comprising a compound of formula I and a pharmaceutically acceptable excipient.

[0020] In some embodiments, the pharmaceutical composition further comprises 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 includes any one or a combination of two or more selected from the following group: Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab and Avelumab, etc.

[0023] In some embodiments, the pharmaceutical composition is in the form of an injection, a tablet, a capsule, an aerosol, a suppository, a film, a controlled-release or sustained-release formulation, or any one of a nanoformulation.

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

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

[0026] (2) In the solution provided by the present invention, the above competitive inhibitor can be used alone or in combination with drugs such as monoclonal antibodies against PD-1 or PD-L1. When the two are used in combination, they have significantly better therapeutic effects. The present invention verifies the above effects through animal experiments.

[0027] (3) The present invention has discovered a new mechanism by which PGK1 mediates PD-1 activation independent of PD-1 extracellular ligands, and the above competitive inhibitors can directly target PGK1 and block its interaction with PD-1. Therefore, the competitive inhibitors provided by the present invention have the potential to be an effective solution to the problem of PD-1 monoclonal antibody resistance.

[0028] (4) The present invention provides a small molecule inhibitor as shown in Formula I, which has a lower production cost and more economical use cost than the combined use of multiple expensive monoclonal antibody drugs. This can significantly reduce the cost of treatment and improve the accessibility and popularity of treatment.

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

[0030] (6) The technical solution provided by the present invention has therapeutic specificity and low toxicity. The competitive inhibitor provided by the present invention specifically targets the specific interaction site between PGK1 and PD-1, has high specificity, reduces interference with other pathways, has low cytotoxicity, and improves the accuracy and effect of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Various other advantages and benefits will become apparent to those skilled in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only used for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. In the accompanying drawings:

[0032] Figure 1 :The experimental results of detecting the interaction between PGK1 and PD-1 by protein immunoprecipitation experiment;

[0033] Figure 2 :The experimental results of detecting the interaction between PGK1 and PD-1 by GST-pull down experiment;

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

[0035] Figure 4 :Competitive inhibition curve of the small molecule inhibitor E465 on the binding of PGK1 to PD-1;

[0036] Figure 5 :PGK1 phosphorylates PD-1 in vitro, PGK1 phosphorylates PD-1 in HEK393T cells, and the effects of small molecule inhibitors on PD-1 phosphorylation;

[0037] Figure 6 :The results of flow cytometry detection of the effect of small molecule inhibitor E465 on terminal CD8+T cell exhaustion in an in vitro CD8+T cell exhaustion model;

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

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

[0040] Fig. 9 :Effects of small molecule inhibitor E465 on tumor growth in B16-OVA and LLC1 subcutaneous injection tumor models;

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

[0042] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0043] PGK1 is the full name of phosphoglycerate kinase 1, which is one of the important kinases in the glycolytic metabolism of cells. Recent studies have found that PGK1 can not only act as a glycolytic enzyme to reversibly catalyze ADP and 1,3-diphosphoglycerate to generate ATP and 3-phosphoglycerate, but also act as a protein kinase to phosphorylate other proteins, such as Beclin1, PDHK1, etc., to regulate various cell physiological activities. However, there is no report on the mechanism of whether PGK1 can directly act on PD-1 in the existing technology, nor is there any drug research on this interaction.

[0044] The present invention discovered that the glycolytic enzyme PGK1 can mediate PD-1 activation and terminal exhaustion of CD8+T cells in a manner independent of PD-1 extracellular ligands. This is a novel non-classical PD-1 activation pathway and a key mechanism leading to drug resistance in traditional treatment strategies that only target PD-1 extracellular signals.

[0045] Specifically, the present invention has found that there is a direct interaction between PGK1 and PD-1, and the phosphorylation of serine 203 in PGK1 can regulate the interaction (the serine 203 refers to: PGK1 is human PGK1, and its amino acid sequence can be referred to in NCBI database NP_000282, the 203rd amino acid from the amino terminus to the carboxyl terminus). Dephosphorylation of serine 203 will enhance the PGK1-PD-1 interaction.

[0046] Targeting the above key amino acid residues in PGK1 that interact with PD-1, the present invention screened out small molecule drugs that can effectively block the PGK1-mediated PD-1 activation process and downstream T cell terminal exhaustion signals, while avoiding the toxicity problem of traditional PGK1 inhibitors.

[0047] In the prior art, small molecule drugs targeting PGK1 mainly target the ATP binding region of PGK1, which can non-specifically inhibit the glycolytic activity of PGK1 and produce cytotoxicity. In some embodiments of the present invention, a small molecule inhibitor E465 targeting PGK1 is provided, which can inhibit the interaction of PGK1-PD-1 without affecting the glycolytic enzyme activity of PGK1, and has a stronger specificity of action. Moreover, E465 did not cause obvious toxicity in cell models and animal models.

[0048] The structural formula of the small molecule inhibitor E465 screened by the present invention is shown in Formula I,

[0049]

[0050] The small molecule inhibitor E465 can be obtained commercially, manufacturer: ChemDiv, product number: E465-0645.

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

[0052] (1) In vitro experiments:

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

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

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

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

[0057] E) Addition of the small molecule inhibitor E465 during 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) The results of subcutaneous injection tumor models of B16-OVA (melanoma) and LLC1 (lung cancer) showed that the use of small molecule inhibitor E465 alone can significantly inhibit the growth of subcutaneous tumors, and its tumor growth curve and final tumor size / weight are significantly different from those of the control group. The combined use of PD-1 monoclonal antibody can significantly improve the resistance of PD-1 monoclonal antibody and further enhance the therapeutic effect of PD-1 monoclonal antibody.

[0060] B) Analysis of local tumor-infiltrating CD8+T cells found that the small molecule inhibitor E465 can significantly rescue the terminal exhaustion of CD8+T cells 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, the materials and reagents used in the following examples can be obtained from commercial sources.

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

[0064] This example analyzes the interaction between PGK1 and PD-1 through experiments. The S203A point mutation is used to simulate the continuous dephosphorylation state of PGK1 at S203, and the S203D point mutation is used to simulate the continuous phosphorylation state of PGK1 at S203. The specific experiments include:

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

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

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

[0068] Group 2, untagged PD-1, Flag-tagged PGK1 with S203A mutation;

[0069] Group 3, untagged PD-1, Flag-tagged PGK1 with S203D mutation.

[0070] At the same time, set up a control group:

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

[0072] Control group 2, expressing only wild-type PGK1 with a Flag tag;

[0073] Control group 3, expressing only Flag-tagged PGK1 with S203A mutation

[0074] Control group 4, only expressed Flag-tagged PGK1 with S203D mutation.

[0075] 24 hours after transfection, cell samples were collected, PGK1 and its interacting PD-1 were immunoprecipitated using Flag antibody, and then detected by western blot. Figure 1 As shown, according to Figure 1 It can be seen that PGK1 can interact with PD-1; and, phosphorylation at position 203 in PGK1 can regulate the interaction, and 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 by protein purification: GST tag, PGK1 with GST tag, and PD-1 with His tag. 1 μg of GST tag, PGK1 with GST tag, and PD-1 with His tag were added to 1×PBS, incubated overnight at 4°C, and glutathione purification resin was used to enrich GST, GST-PGK1, and PD-1 protein interacting with them the next day, and Western blot was used to detect the interaction between PGK1 and PD-1.

[0078] The experimental results are as follows Figure 2 As shown, according to Figure 2 It can be seen that PGK1 can interact with PD-1.

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

[0080] This example analyzes the binding effect of PGK1 and the small molecule inhibitor E465, as well as the competitive effect of the small molecule inhibitor E465 on the binding of PGK1-PD-1. The specific experiments include:

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

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

[0083] 2. Fix the PGK1 protein to the sensor surface as described in 1. The PGK1 fixation level is about 10,000RU. Perform binding analysis at 25°C and a flow rate of 30μl / min. Add E465 with a final concentration of 25μM or an equal volume of DMSO to the electrophoresis buffer (same as 1) to compete for binding to the PGK1 protein on the sensor surface at room temperature for 30 minutes, and then add electrophoresis buffer containing a final concentration of 1μM PD1 protein. Use an empty flow cell without any fixed protein as a subtraction reference. Use the kinetic binding model provided by the Biacore evaluation software to analyze the binding curve of PGK1 and PD-1 after adding E465 or an equal volume of DMSO.

[0084] Binding curve and analysis results are shown in Figure 4 As shown, the small molecule inhibitor E465 can significantly compete for the binding of PGK1 to PD-1.

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

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

[0087] HEK293T cells were transfected with PEI using a PD-1 plasmid with a Flag tag at the C-terminus. 10 μM of the small molecule inhibitor E465 was added 24 hours after transfection, or an equal volume of DMSO was treated for an additional 24 hours. Cell samples were then collected and PD-1 protein was enriched using the immunoprecipitation method, and the phosphorylation level of PD-1Y248 was detected. Figure 5 As shown, the small molecule inhibitor E465 can significantly inhibit the phosphorylation of PD-1, which indicates that the small molecule inhibitor E465 can inhibit the activation level of PD-1.

[0088] Example 4 Effect 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 CD8+T cell exhaustion was induced by adding 100 ng / mL OVA daily for 5 days. At the same time, a final concentration of 10 mM lactic acid, 10 μM small molecule inhibitor E465 or an equal volume of DMSO were added for treatment. The expression of CD8+T cell exhaustion markers and cytokine secretion were detected on D5 and D8, respectively.

[0090] The results of flow cytometry were as follows Figure 6 The relevant statistical results are shown in Figure 7 The above results show that the small molecule inhibitor E465 can significantly inhibit the effect of lactate on promoting 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 spleen and lymph nodes of OT1 mice, and 100 ng / mL OVA was added to activate CD8+T cells. The small molecule inhibitor E465 was added at a final concentration of 10 μM or an equal volume of DMSO for treatment. After 24 hours, commercial kits were used to detect the glycolytic activity of PGK1 in CD8+T cells (bioss, AK509U) and cell apoptosis (Elabscience, E-CK-A238).

[0093] The experimental results are as follows Figure 8 As shown, it can be seen that the small molecule inhibitor E465 does not significantly change the PGK1 glycolytic enzyme activity and cell survival of CD8+ T cells.

[0094] Example 6 Effects of small molecule inhibitors on tumor growth

[0095] 6-8 week old C57BL / 6 female mice were selected for the experiment. B16-OVA and LLC1 cells in the logarithmic growth phase were digested, washed twice with PBS, counted, and resuspended in PBS to adjust the cell suspension concentration to 1×10 7 / ml. Tumor cells were subcutaneously inoculated into the flank of mice, 100 μL of cell suspension per mouse, containing 1×10 6 cells. The day of tumor cell inoculation was defined as Day 0. The drug-treated group was intraperitoneally injected with 5 mg / kg / d of small molecule inhibitor E465 or an equal volume of PBS on Days 1, 3, 5, and 7 after tumor cell inoculation. 200 μg / mouse PD-1 monoclonal antibody was intraperitoneally injected on Days 8, 11, and 14 after tumor cell inoculation. Starting from day 7 after tumor cell inoculation, the long diameter (L) and short diameter (S) of the tumor were measured every day with a vernier caliper. The tumor size was calculated using the formula L×S×S×0.5, and the tumor growth curve was drawn. The experimental results are shown in the figure. Fig. 9 shown.

[0096] On Day 20, mice were euthanized, and the tumor tissues were separated and chopped with scissors. The tissues were digested at room temperature for 1 hour using a digestion medium containing 1 mg / ml collagenase D and 100 ug / mL DNase 1. After grinding into a cell suspension using a 200-mesh sieve, tumor-infiltrating immune cells were enriched using ficol lymphocyte separation solution. The immune cell samples were divided into two parts, one of which was stained for CD45, CD3, CD8, PD-1, and Tim3. The CD8+T cell samples that were both positive for PD-1 and Tim3 were judged as terminally exhausted CD8+T cells. The other part was resuspended in 1640 culture medium containing 100 ng / mL PMA (phorbol ester), 1 μg / mL ionomycin, and 1 μg / mL BFA (Brefeldin A). The cells were collected after 6 hours and stained for CD45, CD3, CD8, IFNg, and Granzyme B. The proportion of terminally exhausted and cytokine-secreting positive cells in CD8+T cells was counted separately. The experimental results are shown in the following table. Fig.10 shown.

[0097] according to Fig. 9 It can be seen that small molecule inhibitors significantly inhibited the growth of B16-OVA and LLC1 subcutaneous injection tumor models and enhanced the therapeutic effect of PD-1 monoclonal antibodies.

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

[0099] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. Use of a competitive inhibitor in the preparation of a drug for treating cancer, characterized in that: The competitive inhibitor can specifically bind to PGK1 and has a competitive inhibitory effect on the binding between PGK1 and PD-1.

2. The use according to claim 1, characterized in that The competitive inhibitor had no effect on reducing the glycolytic activity of PGK1.

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

4. The use according to claim 1 or 2, characterized in that: The competitive inhibitor includes a compound selected from the group consisting of a compound, a salt thereof, or a solvate thereof as shown in Formula I:

5. The use according to any one of claims 1 to 4, characterized in that: The cancer comprises 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; Preferably, the drug is in the form of an injection, tablet, capsule, aerosol, suppository, film, controlled-release or sustained-release agent, or nanoformulation.

6. Use of a competitive inhibitor in the preparation of a drug for inhibiting PD-1 phosphorylation, characterized in that: The competitive inhibitor can specifically bind to PGK1 and has a competitive inhibitory effect on the binding between PGK1 and PD-1.

7. The use according to claim 6, characterized in that The competitive inhibitor has no effect on reducing the glycolytic activity of PGK1; Preferably, the competitive inhibitor inhibits the dephosphorylation of serine 203 of PGK1; Preferably, the competitive inhibitor comprises a compound selected from the group consisting of a compound represented by Formula I, a salt thereof, or a solvate thereof: Preferably, the drug is in the form of an injection, tablet, capsule, aerosol, suppository, film, controlled-release or sustained-release agent, or nanoformulation.

8. A pharmaceutical composition, characterized in that Including the compound described in formula I, its salt or solvate, and pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition further comprises an antibody or an antigen-binding fragment thereof; the antibody specifically binds to the extracellular domain of PD-1 or PD-L1; Preferably, the antibody is a monoclonal antibody; Preferably, the antibody comprises any one or a combination of two or more selected from the following group: nivolumab, pembrolizumab, atezolizumab, durvalumab and avelumab.

10. The pharmaceutical composition according to claim 8 or 9, characterized in that The pharmaceutical composition may be in the form of an injection, tablet, capsule, aerosol, suppository, film, controlled-release or sustained-release preparation or nano-preparation.

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