Application of BFAR inhibitor iBFAR2 in tumor targeting

Abstract

The invention discloses an application of a BFAR inhibitor iBFAR2 in tumor targeting. Specifically, the invention discovers that the iBFAR2 small-molecule inhibitor can effectively inhibit the growth of malignant tumors, can generate a synergistic effect when being combined with a PD-1 inhibitor for use, and has an excellent inhibiting effect on malignant tumors.

Description

Technical Field

[0001] The present invention relates to the field of targeted therapy and tumor medicine, and in particular to the field of application of BFAR inhibitor iBFAR2 in targeted tumors. Background Art

[0002] Cancer is a major public health problem worldwide, placing a huge burden on society and the economy. Conventional cancer treatments include chemotherapy and radiotherapy for rapid tumor growth and surgery, which have limited benefits for the overall survival of cancer patients.

[0003] Tumor immunotherapy, especially the treatment targeting immune checkpoints, has become a new trend in tumor treatment and has broadened the development of the field of tumor treatment. As the first-line cell to kill tumor cells, CD8 + The infiltration degree and functional status of T cells in tumors are closely related to the response to immunotherapy and prognosis. In clinical practice, the application of PD-1 and CTLA4 monoclonal antibodies effectively improves the tumor infiltration of CD8 + The anti-tumor activity of T cells inhibits the development of tumors to a certain extent. However, immune checkpoint blockade therapy still has problems such as drug resistance and immune-related adverse reactions.

[0004] The JAK-STAT signaling pathway is a widely expressed signal transduction pathway in cells, and plays an important role in cell proliferation, apoptosis, invasion, migration, and immune response. In tumor cells, the JAK-STAT signaling pathway is a key signaling pathway that determines tumor growth and metastasis, and therefore plays a key regulatory role in the occurrence and development of tumors. On the other hand, the activation of the JAK-STAT signal is important for enhancing CD8 + T cell anti-tumor immune responses are also critical. Given this pervasive role of JAK-STAT signaling, targeting this pathway may both inhibit tumor growth and impair anti-tumor immunity, which may be the reason why JAK2 inhibitors have failed in clinical trials for the treatment of colorectal cancer and lung cancer.

[0005] Therefore, it is necessary to develop a method to specifically inhibit the JAK-STATs signaling pathway or activate CD8 + Small molecule drugs that target this pathway in T cells may be a promising strategy for the clinical treatment of tumors. Summary of the invention

[0006] The purpose of the present invention is to provide an active ingredient for treating tumors and a new use thereof in inhibiting tumors.

[0007] Specifically, the present invention provides iBFAR-2 as shown in Formula I, and its chiral monomer or composition for use in inhibiting tumors.

[0008] The first aspect of the present invention provides a use of iBFAR-2 or a pharmaceutically acceptable salt thereof as shown in Formula I.

[0009]

[0010] Used for preparing drugs for treating tumors.

[0011] In another preferred embodiment, the chiral monomer of the active ingredient is selected from the following group:

[0012] In another preferred embodiment, the use targets CD8 + BFAR, an E3 ubiquitin ligase involved in the anti-tumor function of T cells.

[0013] In another preferred embodiment, the iBFAR-2 inhibits the activity of E3 ubiquitin ligase BFAR.

[0014] In another preferred embodiment, the E3 ubiquitin ligase BFAR regulates the JAK2-STAT1 signaling pathway.

[0015] In another preferred embodiment, the iBFAR-2 or a pharmaceutically acceptable salt thereof is used in combination with a PD-1 inhibitor.

[0016] In another preferred embodiment, the PD-1 inhibitor includes PD-1 antibody and PD-L1 antibody.

[0017] In another preferred embodiment, the PD-1 inhibitor is selected from the following group: mouse PD-1 antibody (clone number: RMP1-14), mouse PD-1 antibody (clone number: 29F.1A12), mouse PD-1 antibody (clone number: J43), PD-1 and PD-L1 antibodies or inhibitors clinically used to treat tumors, or a combination thereof.

[0018] In another preferred embodiment, the PD-1 inhibitor is a mouse PD-1 antibody (clone number: J43).

[0019] In another preferred embodiment, the tumor is selected from the group consisting of lung cancer, bladder cancer, melanoma, breast cancer, prostate cancer, liver cancer, pancreatic cancer, digestive tract tumors, or a combination thereof.

[0020] In another preferred embodiment, the tumor is a tumor of an elderly patient.

[0021] The second aspect of the present invention provides a pharmaceutical composition, which contains:

[0022] (i) iBFAR-2 of formula I, or a chiral monomer thereof, or a combination of chiral monomers thereof, or a pharmaceutically acceptable salt thereof;

[0023]

[0024] (ii) PD-1 inhibitors;

[0025] and (iii) a pharmaceutically acceptable carrier.

[0026] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug for treating tumors.

[0027] In another preferred embodiment, the PD-1 inhibitor includes PD-1 antibody and PD-L1 antibody.

[0028] In another preferred embodiment, the PD-1 inhibitor is selected from the following group: mouse PD-1 antibody (clone number: RMP1-14), mouse PD-1 antibody (clone number: 29F.1A12), mouse PD-1 antibody (clone number: J43), various PD-1 and PD-L1 antibodies or inhibitors clinically used to treat tumors, or a combination thereof.

[0029] In another preferred embodiment, the PD-1 inhibitor is a mouse PD-1 antibody (clone number: J43).

[0030] In another preferred embodiment, the tumor includes but is not limited to lung cancer, bladder cancer, melanoma, breast cancer, prostate cancer, liver cancer, pancreatic cancer, digestive tract tumors, or a combination thereof.

[0031] In another preferred embodiment, the tumor is a tumor of an elderly patient.

[0032] In another preferred embodiment, the chiral monomer of iBFAR-2 is selected from the following group: or a combination thereof.

[0033] The third aspect of the present invention provides an in vitro non-therapeutic and non-diagnostic method for inhibiting tumor cell proliferation, comprising the steps of:

[0034]

[0035] iBFAR-2 shown in formula I, or a chiral monomer thereof, or a combination of chiral monomers thereof, or a pharmaceutically acceptable salt thereof is contacted with tumor cells to inhibit tumor cell proliferation.

[0036] In another preferred embodiment, iBFAR-2, or a chiral monomer thereof, or a combination of chiral monomers thereof, or a pharmaceutically acceptable salt thereof, shown in Formula I, inhibits the activity of E3 ubiquitin ligase BFAR, regulates the JAK2-STAT1 signaling pathway, and inhibits CD8 + T cells have anti-tumor function, thereby inhibiting tumor cell proliferation.

[0037] In another preferred embodiment, the tumor cells are selected from the group consisting of lung cancer cells, bladder cancer cells, melanoma cells, breast cancer cells, prostate cancer cells, liver cancer cells, pancreatic cancer cells, intestinal cancer cells, or a combination thereof.

[0038] In another preferred embodiment, the tumor cells are selected from the following group: mouse bladder cancer cell line MB49, mouse melanoma cell line B16-F10, mouse breast cancer cell lines py8119 and E0771, mouse pancreatic cancer cell line Pan02, mouse intestinal cancer cell line MC38, mouse prostate cancer cell line Myc-CaP, or a combination thereof.

[0039] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 BFAR was shown to be involved in the expression of CD8 + Knockout in T cells significantly inhibited tumor growth.

[0041] Figure 2 BFAR knockout showed enhanced remodeling of CD8 in the tumor microenvironment + Anti-tumor function of T cells.

[0042] Figure 3 showed that BFAR expression was associated with tumor infiltrating CD8 + The cytotoxic activity of T cells is negatively correlated with the proportion of their TRM cell subsets (tissue-resident memory T cells).

[0043] Figure 4 BFAR knockout promotes tumor CD8 + The killing ability of T cells.

[0044] Figure 5 BFAR was shown to regulate CD8 by targeting JAK2-STATs signaling pathway + Anti-tumor function of T cells.

[0045] Figure 6 It was shown that iBFAR2 small molecule inhibitors can promote CD8 + T cell activation.

[0046] Figure 7 It was shown that iBFAR-2 small molecule inhibitors can directly bind to the SAM domain of the BFAR protein.

[0047] Figure 8 Small molecule inhibitors of iBFAR2 have been shown to promote CD8 + Activation of JAK2-STAT1 signaling in T cells.

[0048] Fig. 9 It was shown that iBFAR2 small molecule inhibitors had no significant effect on the activation of the JAK2-STAT1 signaling pathway and proliferation of tumor cells.

[0049] Fig.10 iBFAR2 was shown to inhibit MB49 bladder cancer tumor growth in a dose-dependent manner.

[0050] Fig.11 Small molecule inhibitors of iBFAR2 have been shown to inhibit the growth of a variety of tumors.

[0051] Fig.12 It was shown that BFAR knockout or use of iBFAR2 small molecule inhibitors can increase the sensitivity of tumors to PD-1 blocking antibody treatment.

[0052] Fig.13 It showed that iBFAR2 small molecule inhibitors had no obvious side effects in vivo.

[0053] Fig.14 It showed that iBFAR2 small molecule inhibitors can significantly improve the CD8 + Anti-tumor function of T cells.

[0054] Fig.15 It was shown that BFAR had no obvious preference in expression in different cells and tissues in vivo. DETAILED DESCRIPTION

[0055] After extensive and in-depth research, the inventor unexpectedly discovered for the first time a strategy for inhibiting tumor growth. The inventor discovered that iBFAR2 small molecule inhibitors can effectively inhibit the growth of malignant tumors. The iBFAR2 small molecule inhibitor of the present invention and the PD-1 antibody combined therapy can produce a synergistic effect and have an excellent inhibitory effect on malignant tumors. The present invention was completed on this basis.

[0056] the term

[0057] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0058] As used herein, "active compound of the present invention", "active ingredient of the present invention", and "active compound for inhibiting male tumors of the present invention" are used interchangeably and refer to iBFAR-2 shown in Formula I, or a chiral monomer thereof, or a combination of chiral monomers thereof, or a pharmaceutically acceptable salt thereof.

[0059] As used herein, "a formulation of the present invention" refers to a formulation containing an active compound of the present invention.

[0060] As used herein, the term “comprise” or variations thereof such as “include” or “comprising”, etc., are understood to include the stated elements or components but do not exclude other elements or components.

[0061] As used herein, the term "prodrug" includes a class of compounds that may be biologically active or inactive, and when taken by appropriate methods, undergo metabolism or chemical reactions in the human body to be converted into a compound of iBFAR2, or a salt or solution composed of iBFAR2. The prodrug includes (but is not limited to) carboxylate, carbonate, phosphate, nitrate, sulfate, sulfone, sulfoxide, amino compound, carbamate, azo compound, phosphoramide, glucoside, ether, acetal and the like forms of the compound.

[0062] Ubiquitination

[0063] Ubiquitination refers to the process in which ubiquitin (a class of low molecular weight proteins) molecules, under the action of a series of special enzymes, classify proteins in cells, select target protein molecules from them, and specifically modify the target proteins. As a post-translational modification of proteins, ubiquitination modification is involved in almost all levels of cellular activities in eukaryotes. As a 76-amino acid protein, ubiquitin, after connecting to substrate proteins in different ways, will cause different signaling pathways and participate in important physiological functions such as autophagy, DNA damage response, cell cycle regulation, protein transport, and proteasome degradation of proteins. Ubiquitination modification can also affect the biological processes of immune cells, thereby affecting the anti-tumor effect of immune cells.

[0064] Active compounds and active ingredients of the invention

[0065] In the present invention, an active ingredient that can effectively inhibit tumors is provided. The active ingredient is selected from the following group:

[0066] Experiments have shown that the active ingredients of the present invention inhibit the activity of E3 ubiquitin ligase BFAR, regulate the JAK2-STAT1 signaling pathway, and inhibit CD8 + T cells can inhibit tumor proliferation and / or alleviate tumor-related diseases.

[0067] As used herein, "active compound of the present invention" and "active tumor-inhibiting compound of the present invention" are used interchangeably and refer to a compound with tumor-inhibiting activity, including iBFAR-2, or a chiral monomer thereof, or a combination of chiral monomers thereof, or a pharmaceutically acceptable salt thereof, or a combination thereof, or a crystal thereof, or a solvate thereof.

[0068] It should be understood that the active ingredients of the present invention include the active compounds of the present invention, or pharmaceutically acceptable salts, enantiomers, diastereomers or racemates thereof, or prodrugs thereof. It should be understood that the active ingredients of the present invention also include crystalline forms, amorphous compounds, and deuterated compounds of the active compounds of the present invention.

[0069] The "pharmaceutically acceptable salt" is a conventional non-toxic salt formed by the reaction of the active compound of the present invention with an inorganic acid or an organic acid. For example, conventional non-toxic salts can be prepared by the reaction of the active compound of the present invention with an inorganic acid or an organic acid, wherein the inorganic acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, aminosulfonic acid and phosphoric acid, and the organic acid includes citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalene disulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid and isethionic acid, etc.; or the active compound of the present invention forms an ester with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid or glutamic acid, and then forms a sodium salt, potassium salt, calcium salt, aluminum salt or ammonium salt with an inorganic base; or the active compound of the present invention forms an ester with lysine, arginine, or ornithine, and then forms a corresponding inorganic acid salt with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid or phosphoric acid, or a corresponding organic acid salt with formic acid, acetic acid, picric acid, methanesulfonic acid or ethanesulfonic acid.

[0070] The compounds of the present invention and their pharmaceutically acceptable salts may have isomers, such as optical isomers (including diastereomers and enantiomers), atropisomers, geometric isomers (cis-trans isomers), conformational isomers, tautomers and mixtures thereof, but are not limited thereto. These isomers are also included in the scope defined by the claims of the present invention.

[0071] In addition, the active ingredients of the present invention are particularly suitable for use in combination with other antiviral drugs. Representative other antiviral drugs include (but are not limited to): reverse transcriptase inhibitors, protease inhibitors, coreceptor antagonists, retroviral integrase inhibitors, viral adsorption inhibitors, specific viral transcription inhibitors, antibodies, or combinations thereof.

[0072] Pharmaceutical compositions and applications

[0073] The present invention also provides the use of the active ingredient of the present invention, or its or its pharmaceutically acceptable salt, or its prodrug, or its extract, or a mixture of one or more of its medicinal materials as an effective ingredient in the preparation of a drug for treating male tumor-related diseases.

[0074] The pharmaceutical composition provided by the present invention preferably contains an active ingredient in a weight ratio of 0.001-99wt%, and the preferred ratio is that the active compound of the present invention as the active ingredient accounts for 0.1wt% to 90wt% or 1wt% to 50wt% of the total weight, and the rest is a pharmaceutically acceptable carrier, diluent or solution or saline solution.

[0075] When necessary, the drug of the present invention may also include a pharmaceutically acceptable carrier, which includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0076] The compounds and pharmaceutical compositions provided by the present invention may be in various forms, such as tablets, capsules, powders, syrups, solutions, suspensions and aerosols, and may be present in suitable solid or liquid carriers or diluents and in suitable sterile devices for injection or instillation.

[0077] The various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the pharmaceutical field. The unit dosage of the preparation formula generally contains 0.05-400 mg of the active compound of the present invention, preferably, the unit dosage of the preparation formula contains 1 mg-500 mg of the active compound of the present invention.

[0078] The compounds and pharmaceutical compositions of the present invention can be used clinically in mammals, including humans and animals, and can be administered by oral, nasal, skin, lung or gastrointestinal tract routes. Injection is the most preferred. The most preferred daily dose is 0.01-1 mg / kg body weight, taken at one time, or 0.01-0.5 mg / kg body weight taken in divided doses. Regardless of the method of administration, the optimal dose for an individual should be determined based on the specific treatment. Usually, a small dose is started and the dose is gradually increased until the most suitable dose is found.

[0079] The drugs or inhibitors of the present invention can be administered in various ways, for example, by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediated methods such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue; or mixed or wrapped with other substances to introduce into the body. From the standpoint of easy administration, the preferred pharmaceutical composition is a liquid composition, especially an injection.

[0080] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0081] Material

[0082] BFAR CD8-KO Preparation of mice

[0083] BFAR flox The mice were constructed from the Shanghai South Model Organisms Research Center, and CD8a-cre mice were purchased from the Jackson Lab in the United States. flox BFAR can be obtained by mating mice with CD8a-cre mice flox / flox -CD8acre(BFAR CD8-KO ) mice, which can make the BFAR gene specific only in CD8 + Knockout in T cells.

[0084] Example 1 BFAR in CD8 + Knockout in T cells inhibits tumor growth

[0085] 1.1 Methods

[0086] Twelve pairs of 6-8 week old wild-type and BFARCD8-KO mice were prepared. Mouse melanoma cell line B16-F10 and bladder cancer cell line MB49 were used at 1 × 10 6After 7 days of tumor growth, the tumor size was recorded every other day and a growth curve was drawn. The observation was terminated 20-25 days after tumor inoculation. 2 The mice were sacrificed, and the tumor tissues were removed and weighed.

[0087] 1.2 Results

[0088] The results showed that CD8 + T cell BFAR knockout significantly inhibits the growth rate and size of subcutaneous tumors in melanoma and bladder cancer ( Figure 1 ).

[0089] Example 2 BFAR knockout remodels and enhances CD8 in the tumor microenvironment + Anti-tumor function of T cells

[0090] 2.1 Methods

[0091] Prepare 6 pairs of 6-8 week old wild type and BFAR CD8-KO Mouse, using mouse bladder cancer cell line MB49, 1×10 6 The subcutaneous transplant tumor model was established with 10 cells / cell. The observation was terminated 20-25 days after the tumor was implanted. High concentration CO 2 The mice were killed, the tumors were removed, digested into single cell suspensions, and lymphocytes were enriched and then stained by flow cytometry. + T cells were subjected to single-cell sequencing, and the single-cell sequencing results were further verified using flow cytometry analysis.

[0092] 2.2 Results

[0093] Single-cell sequencing analysis discovered BFAR CD8-KO Tumor-infiltrating CD8 + A higher proportion of tissue-resident CD8 T cells + T cells (TRM cells) Figure 2 (A).

[0094] Flow cytometry analysis also confirmed that BFAR CD8-KO Mouse CD69 + CD103 + The proportion and number of TRMs have increased significantly ( Figure 2 (middle B).

[0095] These results suggest that BFAR knockout can reshape CD8 + Anti-tumor function of T cells.

[0096] Example 3 BFAR expression and tumor infiltration IFNγ in lung cancer patients + CD8+ T cells and CD8 + TRM ratio is negatively correlated

[0097] 3.1 Methods

[0098] Fresh pathological specimens of lung cancer tissue were collected from patients, digested into single cell suspensions, and stained using flow cytometry. A portion of the suspensions were analyzed by flow cytometry to detect tumor CD8 + The expression of IFNγ and CD103 in T cells; the other part was flow cytometry sorted for tumor infiltrating CD8 + T cells, RNA was extracted and the expression level of BFAR was detected by real-time quantitative fluorescence PCR.

[0099] 3.2 Results

[0100] Correlation analysis results showed that CD8 + Gene expression of BFAR in T cells and tumor-infiltrating IFNγ + CD8 + The ratio of T cells to TRM cells is negatively correlated ( Figure 3 ).

[0101] Example 4 BFAR knockout can promote tumor CD8 + T cell killing ability

[0102] 4.1 Methods

[0103] Tumor infiltrating CD8 in wild-type and BFAR knockout mice using the R package AUCell + The gene set related to T cell killing was scored, and the expression of different related genes was analyzed and displayed, and verified by flow cytometry analysis.

[0104] 4.2 Results

[0105] Single-cell sequencing analysis showed that BFAR knockout tumors had high levels of CD8 + T cells have stronger cell killing activity, which is manifested by CD8 + The expression of multiple characteristic cell killing-related genes in T cells was higher, and the related cell killing scores were also higher ( Figure 4 (in AC).

[0106] Flow cytometric analysis further demonstrated that BFAR knockout significantly increased the expression of tumor lymph nodes and tumor-infiltrating CD8 + T cells expressed higher levels of IFNγ ( Figure 4 (middle D).

[0107] Example 5 BFAR regulates CD8 by targeting the JAK2-STATs signaling pathway+ Anti-tumor function of T cells

[0108] 5.1 Methods

[0109] Prepare 6 pairs of 6-8 week old wild-type, BFAR CD8-KO and BFAR / JAK2 CD8-KO Mouse, using mouse bladder cancer cell line MB49 at 1×10 6 The subcutaneous transplant tumor model was established with 10 cells / cell, and the tumor size was recorded every other day and a growth curve was drawn. The observation was terminated 20-25 days after tumor inoculation. High concentration CO 2 The mice were killed, the tumors were removed, digested into single cell suspensions, and lymphocytes were enriched and then stained by flow cytometry. + Expression of p-STAT1 in T cells. CD8-KO Mice, high CO concentration 2 The mice were killed, the spleen and lymph nodes were removed, ground and lysed, and CD8 + CD8 T cell negative selection kit + T cells were isolated by flow cytometry and CD44 lo CD62L hi Initial CD8 + T cells. 5×10 6 Each cell was treated with IFNγ (100 ng / ml) for 0 min, 5 min, and 10 min, and then the protein supernatant was collected and the activation of JAK-STAT1 downstream signal was detected by Western blotting.

[0110] 5.2 Results

[0111] The results of flow cytometry analysis showed that BFAR CD8-KO Mouse-derived tumor-infiltrating CD8 + The phosphorylation level of STAT1 in T cells increased significantly ( Figure 5 (A).

[0112] Western blot results showed that after knocking out BFAR, the phosphorylation of JAK2 was not affected, and the phosphorylation level of downstream STAT1 was significantly increased ( Figure 5 Middle B), suggesting that BFAR is CD8 + However, when both BFAR and JAK2 were knocked out, tumor growth results and flow cytometry results showed that BFAR knockout had an inhibitory effect on tumor growth and CD8 + The promotion of T cell killing was eliminated ( Figure 5 C and D).

[0113] The above results indicate that BFAR regulates the anti-tumor function of CD8+ T cells by targeting the JAK2-STAT1 signaling pathway.

[0114] Example 6 iBFAR2 small molecule inhibitors can promote CD8 + T cell activation

[0115] 6.1 Methods

[0116] To find chemical probes that inhibit BFAR activity and thus activate CD8 + T cells, the inventors performed a sequence-based deep learning chemical library screening to search for SAM domain inhibitors of BFAR protein. The inventors designed TransformerCPI2.0 software to predict compound-protein interactions using only protein sequence information ( Figure 6 A). After screening the non-drug-like compounds in the Chemdiv library (1474,607 compounds remaining), TransformerCPI2.0 was used to score the compounds, and the top 70,000 molecules were screened. Then, pan-assay interference compounds (PAINS) were screened and automatically clustered. After screening by the Lipinski rule, 4461 candidate data were finally obtained and manually screened. Finally, 53 candidate compounds were determined and the next step of experimental evaluation was carried out.

[0117] Prepare 6-8 week wild type and BFAR CD8-KO From mice, the spleen and lymph nodes were removed, ground, lysed, and CD8 + T cell negative selection kit to sort out the total CD8 + T cells were then sorted using flow cytometry to isolate CD44 lo CD62L hi Initial CD8 + T cells were divided into several groups and treated with different BFAR candidate inhibitors (1 μM) at 5×10 5 The cells were plated into 96-well plates and cell proliferation was detected after 72 hours of culture. After obtaining the target compound by comparing the cell proliferation ratio, the CD44 lo CD62L hi Initial CD8 + T cells were treated with DMSO or different concentrations of the target compound iBFAR2, with 5×10 5 The cells were plated into 96-well plates, and the cell proliferation and IFNγ expression levels were detected after 72 h of culture.

[0118] 6.2 Results

[0119] Flow cytometry analysis showed that among the 53 candidate compounds, iBFAR2 increased CD8 + T cells have the strongest ability to activate cells, showing similar functions to BFAR gene knockout ( Figure 6 B and C).

[0120] As the concentration gradient of iBFAR2 increased, it promoted CD8 + The proliferation and cell killing activity of T cells, and the initial concentration of its efficacy reached nM level ( Figure 6 (middle D).

[0121] Example 7 Surface Plasmon Resonance (SPR) experiments show that the small molecule compound iBFAR-2 can directly bind to the SAM domain of the BFAR protein

[0122] 7.1 Methods

[0123] The SPR experiment was performed using Biacore T200 (GE Healthcare) with HBS-EP (50 mM HEPES [pH 7.4], 150 mM NaCl) as the buffer. BFAR-SAM protein (aa166-260) was covalently coupled to the CM5 chip using a standard amino coupling procedure under 10 mM sodium acetate (pH 4.5). iBFAR-2 was diluted in HBS-EP buffer and injected at a flow rate of 30 μl / min for 120 seconds. The sample was dissociated for 180 seconds, and the response signal was recorded over time during the process. After the experiment, the experimental results were analyzed using Biacore T200 evaluation software (GE Healthcare) to calculate the binding affinity constant (K) of iBFAR-2 and BFAR-SAM protein. D value).

[0124] 7.2 Results

[0125] SPR experiments showed that the small molecule compound iBFAR-2 directly bound to the BFAR-SAM protein, and its binding affinity constant (K D value) was 7.95μM ( Figure 7 ).

[0126] Example 8 iBFAR2 small molecule inhibitors can promote CD8 + Activation of JAK2-STAT1 signaling in T cells

[0127] 8.1 Methods

[0128] Prepare 6-8 week old wild-type mice and place them in a high concentration CO 2 Mice were sacrificed, spleen and lymph nodes were removed, ground, lysed, and CD8 + T cell negative selection kit to sort out the total CD8 + T cells were then flow cytometry sorted for CD44 lo CD62L hi Initial CD8 + T cells were divided into two groups. They were pretreated with DMSO or iBFAR2 (1 μM) for 2 h, with 5×10 6 Each cell was grouped together and treated with IFNγ (100 ng / ml) for 0 min, 5 min, and 10 min, respectively. The protein supernatant was then collected and the activation of JAK2-STAT1 signaling was detected by Western blotting.

[0129] 8.2 Results

[0130] Western blot results showed that iBFAR2 small molecule inhibitors showed similar effects to BFAR gene knockout, that is, inhibitor treatment did not affect CD8 + Phosphorylation of JAK2 in T cells can significantly promote the activation of STAT1 signaling downstream of JAK2 ( Figure 8 ).

[0131] Example 9 iBFAR2 small molecule inhibitor has no significant effect on the activation of the JAK2-STAT1 signaling pathway and proliferation of tumor cells

[0132] 9.1 Methods

[0133] An appropriate amount of mouse bladder cancer cell line MB49 cells were prepared and divided into two groups, which were pretreated with DMSO or iBFAR2 (1 μM) for 2 h. 6 Each cell was treated with IFNγ (100 ng / ml) for 0 min, 5 min, and 10 min, and then the protein supernatant was collected and the activation of JAK2-STAT1 signal was detected by western blotting. In addition, tumor cells were treated with DMSO or iBFAR2 (1 μM) for 6 h, 12 h, 24 h, and 48 h, and then the cell proliferation was detected using the CCK-8 kit.

[0134] 9.2 Results

[0135] Western blot results showed that iBFAR2 small molecule inhibitors had no significant effect on the activation of the JAK2-STAT1 signaling pathway in tumor cells ( Fig. 9 (A).

[0136] The results of cell proliferation experiments showed that iBFAR2 small molecule inhibitors had no significant effect on the proliferation of tumor cells.

[0137] Example 10 iBFAR2 inhibits MB49 bladder cancer tumor growth in a dose-dependent manner

[0138] 10.1 Methods

[0139] Prepare 6-8 week old wild-type mice and use the mouse bladder cancer cell line MB49 at 1×10 6 The subcutaneous transplanted tumor model was established with 10 cells / mouse. The tumor-bearing mice were divided into four groups, with 8 mice in each group, and treated with saline, iBFAR2 (1 mg / kg), iBFAR2 (5 mg / kg), and iBFAR2 (10 mg / kg) respectively. The tumor growth and survival curve were observed and recorded every other day. The observation was terminated 25-30 days after the tumor was implanted. High concentration CO 2 The mice were sacrificed.

[0140] 10.2 Results

[0141] Subcutaneous transplant tumor model showed that iBFAR2 significantly inhibited the growth of MB49 transplant tumors and exhibited a dose-dependent inhibition of tumor growth ( Fig.10 ).

[0142] Example 11 iBFAR2 small molecule inhibitors can inhibit the growth of different tumors

[0143] 11.1 Methods

[0144] Prepare 6-8 week old mice and use mouse melanoma cell line B16-F10 and breast cancer cell line PY8119 at 1×10 6 The subcutaneous tumor model was constructed with 10 cells / mouse. The tumor-bearing mice were divided into two groups according to different tumors, with 8 mice in each group. They were treated with saline and iBFAR2 (10 mg / kg) respectively. The tumor size was recorded every other day after the tumor grew for 7 days and the growth curve was drawn. The observation was terminated after 20-25 days of tumor inoculation. High concentration CO 2 The mice were sacrificed.

[0145] 11.2 Results

[0146] The results of different subcutaneous transplanted tumor models in mice showed that iBFAR2 treatment significantly inhibited the growth of different transplanted tumors ( Fig.11 ).

[0147] Example 12 BFAR knockout or use of iBFAR2 small molecule inhibitors can increase tumor sensitivity to PD-1 blocking antibody treatment

[0148] 12.1 Methods

[0149] Prepare 6-8 week old mice and treat the insensitive prostate cancer cell line Myc-CaP with the mouse bladder cancer cell line MB49 or PD-1 antibody (clone number: J43) (InVivoPlus anti-mouse PD-1, BioXcell, BP0033-2) at a concentration of 1×10 6 The subcutaneous transplanted tumor model was established with 10 cells / mouse. The different tumor-bearing mice were divided into five groups or four groups, with 8 mice in each group. Wild-type mice or BFARCD8-KO mice were given saline, low-concentration PD-1 blocking antibody (αPD-1, 40 μg / mouse), high-concentration PD-1 blocking antibody (αPD-1, 200 μg / mouse), iBFAR2 (10 mg / kg), and low-concentration PD-1 blocking antibody-iBFAR2 combined treatment (iBFAR2 / αPD-1). After the tumor grew for 7 days, the tumor size was recorded every other day and the growth curve was drawn. The observation was terminated 20-25 days after the tumor was inoculated. High-concentration CO 2 The mice were sacrificed.

[0150] 12.2 Results

[0151] MB49 tumor growth results showed that BFAR knockout or the use of small molecule compounds iBFAR2 small molecule inhibitors can significantly inhibit the growth of transplanted tumors, and BFAR knockout or iBFAR2 combined with low-dose PD-1 blocking antibody groups have better therapeutic efficacy, indicating that targeting BFAR can improve the sensitivity of PD-1 blocking antibody treatment of tumors ( Fig.12 A and B).

[0152] Similarly, using a subcutaneous prostate cancer model that is insensitive to PD-1 antibody treatment, it was found that iBFAR2 could significantly inhibit Myc-CaP tumor growth and enhance the sensitivity of PD-1 blocking antibody treatment ( Fig.12 Middle C).

[0153] Example 13 iBFAR2 small molecule inhibitors have no obvious side effects in vivo

[0154] 13.1 Methods

[0155] Prepare 6-8 week old mice and use the mouse derived bladder cancer cell line MB49 at 1×10 6The subcutaneous transplanted tumor model was established with 10 cells / mouse. The tumor-bearing mice were divided into five groups, with 8 mice in each group. They were given saline, low-concentration PD-1 blocking antibody (αPD-1, 40 μg / mouse), high-concentration PD-1 blocking antibody (αPD-1, 200 μg / mouse), iBFAR2 (10 mg / kg), and low-concentration PD-1 blocking antibody-iBFAR2 combined treatment (iBFAR2 / αPD-1). After the tumor grew for 7 days, the tumor size was recorded every other day and the growth curve was drawn. The observation was terminated 20-25 days after the tumor was inoculated, and the high-concentration CO 2 The mice were killed, and different tissues including liver, kidney and lung were sampled for Q-PCR detection and HE staining, respectively.

[0156] 13.2 Results

[0157] The results of Q-PCR analysis and HE staining showed that high-dose PD-1 blocking antibody treatment did induce inflammation in multiple tissues and organs of tumor-bearing mice. However, when iBFAR2 was treated alone or in combination with low-dose PD-1 blocking antibody treatment, the tumor growth of tumor-bearing mice was inhibited, and no obvious inflammation occurred in the main tissues and organs ( Fig.13 ).

[0158] Example 14 iBFAR2 small molecule inhibitor can significantly improve CD8 + Anti-tumor function of T cells

[0159] 14.1 Methods

[0160] Prepare 6-8 weeks Rag1 - / - Immunodeficient mice were used to culture 1×10 6 Subcutaneous transplant tumor model was established by using Rag1 - / - The immunodeficient mice were divided into three groups, each with 8 mice, and adoptively transferred CD8 + T cells (8 mice) and CD8 + T cells (16 cells), the number is 5×10 6 cells / mouse. Adoptive transfer of CD8 + The mice with T cells were divided into two groups and treated with saline and iBFAR2 (20 mg / kg) respectively. After the tumors grew for 7 days, the tumor size was recorded every other day and the growth curve was drawn. The observation was terminated after 20-25 days of tumor inoculation. High concentration CO 2 The mice were sacrificed.

[0161] 14.2 Results

[0162] Tumor growth results showed that CD8 + The anti-tumor ability of T cells was significantly weakened, and the small molecule compound iBFAR2 small molecule inhibitor can significantly reverse the aging CD8 + Defective anti-tumor ability of T cells Fig.14 (A).

[0163] Flow cytometry analysis also confirmed that iBFAR2 small molecule inhibitors promoted CD8 + TRM and IFNγ + CD8 + T cell infiltration, inhibiting the growth of transplanted tumors ( Fig.14 B and C).

[0164] Example 15 BFAR has no obvious preference in expression in different cells and tissues in vivo

[0165] 15.1 Methods

[0166] The public databases HPA (Human Protein Atlas, https: / / www.proteinatlas.org / ) and GEPIA 2 (gene expression profiling interactive analysis, GEPIA 2 (cancer-pku.cn)) were used to query BFAR gene expression.

[0167] 15.2 Results

[0168] The database results showed that BFAR expression had no obvious specificity in different cells, and had no obvious preference in expression between tumors and normal tissues.

[0169] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Use of iBFAR-2 of formula I or a pharmaceutically acceptable salt thereof, It is characterized in that Used for preparing drugs for treating tumors.

2. The use according to claim 1, characterized in that The chiral monomer of iBFAR-2 is selected from the following group: or a combination thereof.

3. The use according to claim 1, characterized in that The iBFAR-2 inhibits the activity of the E3 ubiquitin ligase BFAR.

4. The use according to claim 3, characterized in that The E3 ubiquitin ligase BFAR regulates the JAK2-STAT1 signaling pathway.

5. The use according to claim 1, characterized in that The iBFAR-2 is used in combination with a PD-1 inhibitor.

6. The use according to claim 5, characterized in that The PD-1 inhibitors include PD-1 antibodies and PD-L1 antibodies.

7. The use according to claim 1, characterized in that The tumor is selected from the group consisting of lung cancer, bladder cancer, melanoma, breast cancer, prostate cancer, liver cancer, pancreatic cancer, digestive tract tumors, or a combination thereof.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition contains: (i) iBFAR-2 of formula I, or a chiral monomer thereof, or a combination of chiral monomers thereof, or a pharmaceutically acceptable salt thereof; (ii) PD-1 inhibitors; and (iii) a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that The PD-1 inhibitors include PD-1 antibodies and PD-L1 antibodies.

10. An in vitro non-therapeutic and non-diagnostic method for inhibiting tumor proliferation, characterized in that: Includes steps: Contacting iBFAR-2 represented by Formula I, or a chiral monomer thereof, or a combination of chiral monomers thereof, or a pharmaceutically acceptable salt thereof with tumor cells, thereby inhibiting tumor cell proliferation; In another preferred embodiment, iBFAR-2, or a chiral monomer thereof, or a combination of chiral monomers thereof, or a pharmaceutically acceptable salt thereof shown in Formula I inhibits the activity of E3 ubiquitin ligase BFAR, regulates the JAK2-STAT1 signaling pathway, and inhibits CD8 + T cells have anti-tumor function, thereby inhibiting tumor cell proliferation.

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