Application of indole-3-pyruvic acid in preparation of blocker of phosphatase and kinase compound interacting with striatum

Abstract

The invention discloses an application of indole-3-pyruvic acid in preparation of an STRIPAK blocking agent interacting with striatum, the inventor accidentally discovers that the indole-3-pyruvic acid (IPyA) is combined with striatum protein (STRN3) of STRIPAK to prevent the STRIPAK from being combined with serine / threonine protein phosphatase (PP2A), so that the structure of the STRIPAK is destroyed, PP2A in the compound is inactivated, and the STRIPAK blocking agent interacts with striatum. According to the present invention, with the application of PP2A, the dephosphorylation of PP2A on a variety of kinases such as AMPK, PI3K and the like on the downstream of PP2A can be weakened, the tumor cell proliferation can be ultimately inhibited, the tumor cell killing ability of killer T cells (CD8 + T cells and Tc) can be enhanced, the treatment effect of the immune checkpoint inhibitor (ICI) in tumor treatment can be improved, and the two have the synergistic effect;

Description

Technical field:

[0001] The present invention relates to the use of indole-3-pyruvic acid in preparing a striatum-interacting phosphatase and kinase complex (STRIPAK) blocker. Background technology:

[0002] The striatum-interacting phosphatase and kinase complex (STRIPAK) is an evolutionarily highly conserved, ultra-large molecular complex that includes GCK family kinases including protein phosphatase 2A (PP2A), MST1 / 2, and striatins family members (STRNs) as auxiliary scaffold proteins. The core of STRIPAK is composed of GCK family kinases including PP2A and MST1 / 2, among which PP2A is composed of three subunits: structural subunit (A subunit, PP2Aa), catalytic subunit (C subunit, PP2Ac) and regulatory subunit (B″′ subunit, including STRN1, STRN3, STRN4). In the mammalian STRIPAK complex, the core of the complex (PP2A-STRNs) composed of protein phosphatase 2A and striatins family members as auxiliary scaffold proteins can recruit kinase molecules through scaffold proteins to assemble into a complete supramolecular complex to exert regulatory functions. STRIPAK can participate in the regulation of various cell biological processes such as cell proliferation, vesicle transport, and cycle, thereby playing an important role in tissue and organ development, homeostasis maintenance, and immune regulation. A large number of studies have found that after the formation of the STRIPAK complex, its key component PP2A plays a key role in protein phosphorylation and dephosphorylation by dephosphorylating downstream Carma1 protein, MST1 / 2 kinase, STK23 kinase, MINK1 kinase, etc., thereby achieving inhibition. STRIPAK complex participates in the occurrence, development and metastasis of tumors through Hippo-dependent or -independent modes, and plays a key role in the acquisition of drug resistance by tumor cells, providing a theoretical basis for anti-tumor strategies targeting this complex. For example, STRIPAK can not only promote YAP activation and the occurrence and development of gastric cancer by negatively regulating MST1 / 2 kinase, but also lead to gastric cancer cells to tolerate radiotherapy, chemotherapy and PARP inhibitor anti-tumor strategies. At present, the anti-tumor strategy targeting STRIPAK complex mainly focuses on breaking the binding of STRN3-PP2A, but there are no clinical research reports on related drugs, and there are no marketed anti-tumor drugs targeting this target. STRIPAK plays an important role in tumor progression and the formation of tumor immunosuppressive microenvironment, but there are no effective small molecule inhibitors.

[0003] Tryptophan (Trp) is an essential amino acid that the human body cannot synthesize by itself and is also one of the building blocks of DNA. Indole-3-pyruvic acid (IPyA) is one of the important metabolites of Trp in the metabolism of intestinal microorganisms. It exists naturally in the human body. The inventors previously discovered the use of indole-3-pyruvic acid (IPyA) or its pharmaceutical salt in the preparation of drugs for the treatment of breast cancer. Summary of the invention:

[0004] The purpose of the present invention is to provide an application of indole-3-pyruvic acid in preparing a striatum-interacting phosphatase and kinase complex (STRIPAK) blocker.

[0005] The inventors unexpectedly discovered that indole-3-pyruvate (IPyA) binds to the striatin (STRN3) of STRIPAK, hindering its binding to serine / threonine protein phosphatase 2A (PP2A), thereby destroying the structure of STRIPAK, inactivating PP2A in the complex, and weakening the dephosphorylation of PP2A on multiple downstream kinases such as AMPK and PI3K, ultimately inhibiting tumor cell proliferation and enhancing cytotoxic T cells (CD 8+ The tumor cell killing ability of T cells (Tc) is enhanced, and the efficacy of immune checkpoint inhibitors (ICI) in tumor treatment is improved. The two have a synergistic effect.

[0006] Therefore, the present application protects the use of indole-3-pyruvic acid in the preparation of striatum-interacting phosphatase and kinase complex (STRIPAK) inhibitors.

[0007] The present invention also protects a composition comprising indole-3-pyruvic acid and an immune checkpoint inhibitor (ICI).

[0008] The immune checkpoint inhibitors (ICI) include inhibitors targeting the PD-1 / PD-L1 pathway and the CTLA-4 / B27 pathway. For example, the inhibitor targeting the PD-1 pathway is an αPD-1 antibody.

[0009] The present invention also protects the use of indole-3-pyruvic acid and an immune checkpoint inhibitor in combination in the preparation of an adjuvant therapeutic drug for breast cancer therapy.

[0010] Preferably, the breast cancer therapy adjuvant therapeutic drug contains pharmaceutically acceptable excipients and is prepared into various medically acceptable dosage forms, including oral dosage forms and intravenous dosage forms.

[0011] Since indole-3-pyruvate (IPyA) is a metabolite of tryptophan (Trp), it is inferred that oral supplementation of tryptophan (Trp) can also help the host produce more IPyA, which acts on the STRIPAK complex. Therefore, the present invention also protects the use of tryptophan and immune checkpoint inhibitors in combination in the preparation of adjuvant therapeutic drugs for breast cancer treatment.

[0012] The beneficial effects of the present invention are as follows: IPyA can bind to the striatin (STRN3) of STRIPAK, hindering its binding to serine / threonine protein phosphatase (PP2A), thereby destroying the structure of STRIPAK, inactivating PP2A in the complex, weakening the dephosphorylation of PP2A on its downstream kinases such as AMPK and PI3K, and ultimately inhibiting tumor cell proliferation, while strengthening the cytotoxic T cells (CD 8+ The tumor cell killing ability of T cells (Tc) is enhanced, and the efficacy of immune checkpoint inhibitors (ICI) in tumor treatment is improved. The two have a synergistic effect. Description of the drawings:

[0013] Figure 1 The results of analyzing the specific binding target protein of IPyA by the drug affinity reaction targeted stability technology (DARTS) in Example 1; wherein, A, electrophoresis bands of the samples; B, volcano plot of proteins whose mass spectrometry peak intensity ratios significantly changed after binding with IPyA; C, top 8 proteins in mass spectrometry peak intensity ratios after binding with IPyA.

[0014] Figure 2 In Example 2, the surface plasmon resonance technology (SPR) was used to analyze the binding kinetic curve and analysis results of IPyA and STRN3.

[0015] Figure 3 In Example 3, a cell thermal shift assay (CETSA) was used to analyze the effect of IPyA on the thermal stability of STRN3.

[0016] Figure 4 This is Example 4, the effect of indole-3-pyruvate (IPyA) on STRN3-PP2A binding and AMPK phosphorylation in tumor cells, wherein A, the effect of IPyA on STRN3-PP2A binding in tumor cells; B, the effect of IPyA on AMPK phosphorylation in tumor cells.

[0017] Figure 5 Example 5 Indole-3-pyruvic acid (IPyA) on CD 8+ STRN3-PP2A binding, PI3K phosphorylation, and granzyme B secretion in T cells, among which A and IPyA have an effect on CD 8+Effect of STRN3-PP2A binding in T cells; B, IPyA on CD 8+ Effects of PI3K phosphorylation and granzyme B secretion in T cells.

[0018] Figure 6 This is the effect of indole-3-pyruvic acid (IPyA) combined with αPD1 antibody on tumor growth in breast cancer-bearing animals in Example 6. Among them, A, tumor picture; B, tumor weight; C, tumor volume change curve; D, tumor weight reduction ratio.

[0019] Figure 7 This is the effect of indole-3-pyruvic acid (IPyA) combined with αPD1 antibody on Tc cells and their granzyme B secretion in tumors of breast cancer-bearing animals in Example 6. Among them, A, representative pictures of flow cytometry analysis; B, proportion of Tc in tumor-infiltrating lymphocytes; C, comparison of granzyme B expression in tumor-infiltrating Tc; D, increase ratio of granzyme B expression in tumor-infiltrating Tc. Specific implementation method:

[0020] The following is a further description of the present invention, rather than a limitation of the present invention.

[0021] The cell lines and their culture methods used in the following examples are as follows.

[0022] Mouse breast cancer cell line 4T1, mouse colorectal cancer cell line MC38, and gastric cancer cell line MFC were purchased from the cell bank of the Typical Culture Committee of the Chinese Academy of Sciences. The above cells were cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (Biological Industries) and 1% penicillin / streptomycin (Gibco). The above culture medium containing specific additives is hereinafter referred to as complete medium. All cell lines were cultured in a 5% CO2, 37°C constant temperature incubator.

[0023] Example 1: Specific binding analysis of indole-3-pyruvic acid (IPyA) and striatin (STRN3) of STRIPAK

[0024] Drug Affinity Responsive Target Stability (DARTS) was used to analyze the specific binding target protein of IPyA. Take a certain amount of 4T1 cells, add lysis buffer (WB and IP special lysis buffer, add 1× cocktail protease inhibitor), lyse at 4℃ for 2h, and take out and vortex every half hour. Centrifuge the protein extract at 13000rpm for 20min at 4℃, and transfer the supernatant to a new EP tube. Centrifuge the protein extract again at 13000rpm for 5min at 4℃, transfer the supernatant to a new EP tube, and determine the concentration by BCA method. Divide the quantified protein lysate into two components, add 100μM IPyA and an equal volume of DMSO reagent to each group, and incubate at room temperature for 1h. Then incubate the above lysate with Pronase (1:300) at room temperature for 30min, add 20× protease inhibitor solution, and mix thoroughly. The obtained samples were subjected to SDS-PAGE, and the gel strips of each lane were collected for decolorization, reduction alkylation, enzymatic hydrolysis (Trypsin), extraction of enzymatic peptides, and desalting. After vacuum drying, the obtained samples were re-dissolved in 0.1% formic acid aqueous solution, and equal volume samples were taken for loading. EASY-nLC1200 (Thermo Scientific, USA) was used for separation, using an analytical column (C18, 1.9m, 75m×15cm) at a flow rate of 300nL / min. The mass spectrometer was Q-Exactive HF system (Thermo Scientific, USA). The tandem mass spectrometry detection adopted the data dependent acquisition (DDA) mode. The full scan resolution was 60,000 (FWHM), and the parent ion scanning range was: 350-1500m / z. The automatic gain control value (AGC) was set to 3e6, the scanning window was 1.6m / z, and the maximum ion injection time was 20ms. In HCD fragmentation mode, the collision energy was set to 25%. The resolution of the secondary mass spectrometer was set to 15000, the AGC was set to 1e5, the maximum ion injection time was 40ms, and the dynamic exclusion time was set to 30s.

[0025] The results of IPyA binding protein screening are as follows Figure 1 The electrophoresis bands of each group of samples are shown in Figure 1 As shown in A. According to mass spectrometry analysis, there are 74 proteins with significantly increased mass spectrometry peak intensity ratios in the IPyA-treated histone samples ( Figure 1 B), among which STRN3 has the highest ratio (IPyA_VS_con_Ratio=40, Figure 1 (C), indicating that STRN3 is the specific binding target of IPyA.

[0026] Example 2: Kinetic parameter analysis of indole-3-pyruvic acid (IPyA) binding to STRN3

[0027] Surface Plasmon Resonance (SPR) was used to analyze the kinetic parameters of IPyA binding to STRN3. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, 400mM) and N-hydroxysuccinimide (NHS, 100mM) were used to treat the CM5 chip for 240s to activate it; 0.05mg / mL STRN3 protein solution was injected at a flow rate of 10μL / min, and the capture time was 420s, so that the maximum coupling response value was 5700RU. 50μM was used as the highest analytical concentration of IPyA, and six analytical concentrations were set for doubling dilution. The analytical buffer was PBS (containing 5% DMSO and 0.2% Tween 20, pH7.4), the flow rate was set to 30μL / min, the binding time was 90s, and the dissociation time was 200s. The experiment was run for a total of 7 cycles, and the response signal was based on the analysis time as the horizontal axis and the response value as the vertical axis. The obtained data were fitted by analysis software, and the fitting model adopted was 1:1Binding binding model to determine its affinity constant and binding and dissociation characteristics.

[0028] The binding kinetics curve and analysis results of IPyA and STRN3 are shown in Figure 2 The affinity constant of IPyA and STRN3 is 16.1 μM, indicating that IPyA has a strong binding affinity with STRN3.

[0029] Example 3: Effect of indole-3-pyruvic acid (IPyA) on the thermal stability of STRN3

[0030] The effect of IPyA on the thermal stability of STRN3 was analyzed by cellular thermal shift assay (CETSA). A drug-treated group and a blank control group were set up, and a total of 5 temperature points (50°C, 53°C, 56°C, 59°C, and 62°C) were tested. Three replicates were set for each temperature point, and each sample was inoculated into a 10 cm culture dish at 1×106 cells / dish. 4T1 cells were taken and inoculated according to the above scheme and adhered overnight. The drug-treated group was given IPyA 250μM, and the blank control group was given an equal volume of DMSO. After incubation for 3 hours, the cells were digested with trypsin, centrifuged, washed twice with PBS containing protease inhibitors and phosphatase inhibitors, and transferred to 0.2mL PCR tubes, 50μL per tube, and placed on ice. Heat for 5 minutes under the above temperature conditions on a PCR thermal cycler, take out immediately after heating, and quickly cool on ice for 3 minutes. Then freeze in liquid nitrogen. The sample was frozen with liquid nitrogen for 30 seconds, then taken out, thawed at room temperature, vortexed evenly, and then frozen with liquid nitrogen, repeated 3-5 times to ensure that the sample was fully lysed. The sample was transferred to a new 1.5mL centrifuge tube, centrifuged at 4℃20000g for 20min, and the supernatant was taken. After centrifugation, the supernatant was transferred to a new centrifuge tube, and the obtained samples were compared with the difference in STRN3 protein residue at each temperature point according to the conventional Western blotting method.

[0031] The results of the analysis of the effect of IPyA on the thermal stability of STRN3 are shown in the Appendix. Figure 3 IPyA can make STRN3 more stable at 50-56°C, indicating that IPyA has a strong binding force with STRN3.

[0032] Example 4: Effect of indole-3-pyruvate (IPyA) on STRN3-PP2A binding and AMPK phosphorylation in tumor cells

[0033] Take 4T1 cells, adjust the cell concentration, and inoculate them in a 24-well plate (1.5×10 6cell / mL, total volume of each well 1mL), and cultured overnight with complete RPMI1640 medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin). On the next day, discard the original culture medium of each well, wash the cells twice with PBS, and replace the above culture medium with Opti-MEM medium containing 1% penicillin-streptomycin. Take a small amount of Opti-MEM Medium, plasmid and Lipo8000 (S173 is paired with P155, V173 is paired with V155), add 50ng / well to the corresponding group of cells, and incubate for 4 to 6 hours. Then wash the cells twice with PBS, add the corresponding drug-containing culture medium (without dual antibodies, the final concentration of IPyA is 250μM), and continue to culture for 48 hours. The mean fluorescence intensity (Median Fluorescence Intensity, MFI) of the FITC channel of each group was detected by flow cytometry, and the relative mean fluorescence intensity (Relative MFI) was calculated: MFI 转染细胞 / MFI 非转染细胞 The plasmids used were as follows: S173 was a plasmid expressing the N-terminal 173 amino acids of YFP protein and STRN3 protein; P155 was a plasmid expressing the C-terminal 155 amino acids of YFP protein and PP2A protein; V173 was an empty plasmid expressing the N-terminal 173 amino acids of YFP protein; V155 was an empty plasmid expressing the C-terminal 155 amino acids of YFP protein.

[0034] Another 4T1 cell was inoculated into a 100 mm cell culture dish (170,000 cells / dish), and complete medium containing IPyA (500 μM) was added and cultured for 48 h (the control without IPyA was used, i.e. Figure 4 0μM in the medium), discard the culture medium, wash twice with pre-cooled PBS, add protein lysis buffer containing phosphatase inhibitors and protease inhibitors, and extract total protein according to the corresponding steps in the instructions. After the total protein concentration was quantified by microspectrophotometry, a consistent amount of total protein was taken from each sample and mixed with the loading buffer and denatured (100℃, 10min). The obtained samples were compared for AMPK and p-AMPK expression differences according to conventional Western blotting methods.

[0035] Effects of IPyA on STRN3-PP2A binding and AMPK phosphorylation in tumor cells Figure 4 IPyA can significantly reduce the Relative MFI of 4T1 cells carrying S173-P155 plasmid, suggesting that IPyA can significantly disrupt STRN3-PP2A binding ( Figure 4 Meanwhile, IPyA can significantly increase the phosphorylation level of AMPK in 4T1 cells ( Figure 4(B) This indicates that IPyA can enhance the phosphorylation of AMPK by disrupting the combined STRN3-PP2A binding and weakening its dephosphorylation effect on downstream proteins.

[0036] Example 5: Effect of indole-3-pyruvic acid (IPyA) on CD 8+ Effects of STRN3-PP2A binding, PI3K phosphorylation, and granzyme B secretion in T cells

[0037] Female Balb / c mice (6-7 weeks old, 18-20 g) were taken, the spleen was removed under sterile conditions, chopped in PBS, and gently ground on a sterile cell filter with a pore size of 70 μm. The filtrate was centrifuged (340×g, 5 min), and the red blood cells were removed with red blood cell lysis buffer, and then washed twice with pre-cooled PBS to obtain spleen lymphocytes. The obtained cells were resuspended in Opti-MEM Medium culture medium and the cell concentration was adjusted to 6×10 6 cell / mL, inoculated in a 96-well cell culture plate. Referring to Example 4, add Opti-MEM Medium containing the corresponding plasmid and incubate for 4-6 hours (without dual antibody); then directly add 100 μL of the corresponding inactivated serum-containing medium (without dual antibody, the final concentration of IPyA is 250 μM), supplement with inactivated serum, and continue to culture for 48 hours. The obtained samples were used to detect the CD 8+ T cells (CD 3+ CD 8+ , Tc) ratio, and the median fluorescence intensity (MFI) of the FITC channel, and calculate the relative mean fluorescence intensity (Relative MFI): MFI 转染细胞 / MFI 非转染细胞 .

[0038] Collect the obtained lymphocytes, resuspend them in complete RPMI1640 medium, and adjust the cell concentration to 6×10 6 cell / mL, inoculated in a 96-well cell culture plate. Add 100 μL of drug-containing culture medium (final concentration of IPyA is 250 μM), culture for 48 hours, and detect the Tc ratio, as well as the expression changes of granzyme B (Granzyme B) and phosphorylated PI3K (p-PI3K) by flow cytometry. Granzyme B is expressed as the percentage of positive cells, and p-PI3K is expressed as the mean fluorescence intensity (MFI).

[0039] IPyA versus CD 8+Effects of STRN3-PP2A binding, PI3K phosphorylation, and granzyme B secretion in T cells Figure 5 IPyA can significantly reduce the CD carrying S173-P155 plasmid 8+ The relative median fluorescence intensity (MFI) of T cells indicated that IPyA could significantly disrupt STRN3-PP2A binding ( Figure 5 Meanwhile, IPyA can significantly increase the phosphorylation of PI3K and the secretion of granzyme B in 4T1 cells ( Figure 5 (B), indicating that IPyA can enhance the phosphorylation of PI3K and its granzyme B secretion by disrupting the combined STRN3-PP2A binding and weakening its dephosphorylation effect on downstream proteins.

[0040] Example 6 Effect of indole-3-pyruvic acid (IPyA) combined with immune checkpoint inhibitor αPD-1 antibody on tumor growth in breast cancer bearing animals

[0041] Female Balb / c mice (6-8 weeks old) were subcutaneously injected with 4T1 cells (8×10 4 cells / mouse). The tumor volume grew to about 20 mm 3At the same time, the tumor-bearing mice were randomly divided into 6 groups according to tumor volume: Control group (Ctrl group), IPyA group, αPD-1 group, "IPyA+αPD-1" group, "IPyA+αPD-1+αCD8" group, and "IPyA+αPD-1+αCD8Iso" group, with 6 mice in each group. Drugs were administered continuously for 21 days from the day of grouping. The specific dosing regimen is shown in Table 1. In the Control group (Ctrl group), αPD-1 antibody isotype control (IgG2a) was intraperitoneally injected at 500 μg / kg once a week; the IPyA group was intraperitoneally gavaged with IPyA 120 mg / kg once a day; the αPD-1 group was intraperitoneally injected with αPD-1 antibody 500 μg / kg once a week; the "IPyA+αPD-1" group was intraperitoneally gavaged with IPyA 120 mg / kg once a day, and αPD-1 antibody was intraperitoneally injected at the same time, once a week; the "IPyA+αPD-1+αCD8" group was intraperitoneally gavaged with IPyA 120 mg / kg once a day, and αPD-1 antibody was intraperitoneally injected at the same time, once a week. The mice were given IPyA 120 mg / kg, once a day, αPD-1 antibody was intraperitoneally injected at 500 μg / kg, once a week, and αCD8 antibody was intraperitoneally injected at 500 μg / kg, once a week. The mice were given IPyA 120 mg / kg, once a day, αPD-1 antibody was intraperitoneally injected at 500 μg / kg, once a week, and αCD8 antibody isotype control (IgG2b), 500 μg / kg, was intraperitoneally injected once a week.

[0042] During the administration period, the tumor size and body weight changes were detected twice a week. On the 22nd day, the tumors of mice in each group were dissected, the tumor weight was weighed, and photos were taken. After obtaining the tumor tissues of each group, lymphocytes were extracted from the tumor tissues, and the secretion of granzyme B by Tc cells was analyzed by flow cytometry.

[0043] Table 1 Grouping and administration instructions for Example 6

[0044]

[0045] Effects of IPyA on tumor growth in breast cancer-bearing animals Figure 6 , Figure 7 shown. Figure 6 The results showed that IPyA combined with αPD1 antibody could significantly slow down the growth rate of 4T1 breast cancer tumors and ultimately reduce tumor weight ( Figure 6 A~C), and the tumor weight reduction ratios when IPyA and αPD-1 antibody were used alone were 19.9% ​​and 16.0%, respectively, while the tumor weight reduction ratio after combined use was 36.0%, which was greater than the sum of the reduction ratios when the two drugs were used alone, indicating that the anti-tumor effect of combined use is significantly better than that of the two drugs used alone.

[0046] Figure 7 The results showed that IPyA could increase the proportion of Tc cells in tumor tissue and promote their Granzyme B secretion ( Figure 7 At the same time, when IPyA and αPD-1 antibody were used alone, the increase in the expression of granzyme B in tumor-infiltrating Tc was 0.7% and -6.1%, respectively, while after combined use, the ratio was 56.8%, which was greater than the sum of the increase ratios when the two drugs were used alone, indicating that the effect of combined use in improving the tumor killing ability of Tc was significantly better than that of the two drugs used alone.

Claims

1. Use of indole-3-pyruvic acid in the preparation of inhibitors of phosphatases and kinase complexes interacting with the striatum.

2. A composition comprising indole-3-pyruvate and an immune checkpoint inhibitor.

3. The composition according to claim 2, characterized in that The immune checkpoint inhibitors include inhibitors targeting the PD-1 / PD-L1 pathway and the CTLA-4 / B27 pathway.

4. The composition according to claim 2, characterized in that The immune checkpoint inhibitor is an αPD-1 antibody.

5. Application of indole-3-pyruvate or tryptophan in combination with immune checkpoint inhibitors in the preparation of adjuvant therapeutic drugs for breast cancer treatment.

6. The use according to claim 5, characterized in that: The breast cancer therapy adjuvant treatment medicine contains pharmaceutically acceptable excipients and is prepared into various medically acceptable dosage forms.

7. The use according to claim 6, characterized in that: The dosage forms include oral dosage forms and intravenous dosage forms.

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