PROTAC compound targeting Foxp3 and application of PROTAC compound in tumor immunotherapy

Abstract

The invention relates to a PROTAC compound targeting Foxp3 and application of the PROTAC compound in tumor immunotherapy, and particularly discloses a compound for immunotherapy, the compound is a conjugate of P60 peptide and VHL ligand, and the compound has a structural formula as shown in the following formula I. The invention further discloses a preparation method of the compound. The invention also discloses a method for hydrolyzing the Foxp3 by using a proteolysis pathway. The method for hydrolyzing the Foxp3 comprises a step of performing contact culture on the compound and cells containing the Foxp3. In addition, the invention also provides application of the compound in preparation of a reagent for a marker molecule Foxp3 for proteolysis of Tregs in cells and application of the compound in preparation of drugs for treating tumors and autoimmune diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a PROTAC compound targeting Foxp3 and its use in tumor immunotherapy. Background Technology

[0002] Regulation of T-reg cells (Tregs) plays a crucial role in the treatment of autoimmune diseases, tumors, and transplantation immunity. While Tregs can suppress autoimmune diseases and maintain immune homeostasis, this process can also lead to immune escape by some tumor cells. Therefore, understanding and controlling the function of Tregs is of great significance for the development of immunotherapy and drug research. Studies on tumors and autoimmune diseases have shown that Tregs influence immune responses, often manifesting as abnormalities in their number and function. Reports indicate that inhibiting the immunomodulatory function of Tregs can balance the body's immune response, enhance the body's ability to clear pathogens, and inhibit immune escape by tumor cells. Research on the immunomodulatory function of inhibiting Tregs can provide new approaches and strategies for immunotherapy, making it a significant area of ​​research.

[0003] Foxp3 is a marker molecule for Tregs and belongs to the forkhead box transcription factor family. Since its discovery, Foxp3 has been considered a key factor in immune tolerance. Foxp3 is crucial for balancing the immune system, acting as a transcriptional regulator to directly or indirectly regulate gene activity to modulate Treg function. Generally, Foxp3 can balance and stabilize the immune system, suppressing autoimmune diseases. Disruption of Foxp3 leads to the loss of Treg immunosuppressive function. Therefore, regulating Foxp3 in tumors and autoimmune diseases offers a new direction for controlling Treg function.

[0004] P60 is a 15-amino acid peptide discovered in phage display libraries that can bind to Foxp3, inhibiting its nuclear translocation and attenuating Foxp3-mediated inhibition of NFKB and NFAT function. However, high doses (approximately 100 μM) of the P60 peptide are required to inhibit Foxp3 function in HEK293T cells, which may lead to high toxicity. Unlike traditional drugs that only inhibit or activate protein function, PROTAC (PROteolysis TAgeting Chimeras) technology achieves more direct protein regulation by promoting the degradation of target proteins. PROTACs utilize bifunctional molecules to link target proteins to specific E3 ubiquitin ligases, inducing ubiquitination of the target protein, which is then degraded by the proteasome. In recent years, the design and use of PROTACs have attracted widespread attention due to their unique advantages, such as lower dosage and less resistance.

[0005] In 2010, Casares et al. discovered P60 using a phage library. Through experiments with CF-labeled peptides, they found that P60 can enter cells as a CPP and reduces the nuclear translocation of Foxp3, thereby inhibiting Treg activity. In subsequent experiments, they found that P60 reduced the inhibitory effect of FOXP3 on NF-κB and NFAT activity, suggesting that the binding of P60 to FOXP3 may affect regions interacting with these transcription factors. These data are consistent with P60's ability to upregulate IL-2 mRNA expression on Tregs, and the interaction between NFAT and FOXP3 has demonstrated the suppression of P60 expression. In summary, through a series of experiments, they determined that the P60 peptide can enter cells, bind to FOXP3, reduce its nuclear translocation, and inhibit its ability to downregulate the activity of transcription factors NF-κB and NFAT. P60 can inhibit the immunosuppressive activity of mouse and human Tregs and enhance effector T cell stimulation in vitro. Furthermore, and most importantly, P60 can enhance the immunogenicity of cancer and viral vaccines.

[0006] In 2016, addressing the challenges posed by the high concentrations required for P60 peptide therapy, the group selected an aptamer capable of binding to CD28, which is highly expressed in Treg cells. By hybridizing the P60 peptide to the CD28 aptamer, they obtained the CD28Apt-P60 construct. This method allowed them to significantly reduce the therapeutic dose required to inhibit Foxp3. However, this approach lowers the affinity of the P60 peptide for Foxp3; despite this, the effect was still much better than that of the P60 peptide alone. In their in vivo tumor experiments, the CD28Apt-P60 construct required hundreds of times less dose than the P60 peptide. In conclusion, they successfully reduced the dosage of drug required to inhibit Foxp3 using this method, providing a framework for future experimental developments. Summary of the Invention

[0007] To address the limitations of current methods for inhibiting Foxp3, such as their reliance on single methods and high drug dosages, this invention offers a novel therapeutic approach. One aspect of this invention provides a compound for immunotherapy, said compound being a conjugate of a P60 peptide and a VHL ligand, having the structural formula shown in Formula I:

[0008]

[0009] Another aspect of the present invention provides a method for preparing the compound as described above, the method comprising the following steps:

[0010] S1) N3-(CH2)5-PEG2-COOH is obtained by reacting NH2-PEG2-COOH and N3-(CH2)5-NHS;

[0011] S2) VHL ligand and N3-C5-PEG2-COOH are condensed together under the action of a condensing agent to form an amide bond to obtain VHL-PEG2-(CH2)5-N3;

[0012] S3) The alkynyl-P60 peptide and VHL-PEG2-(CH2)5-N3 were reacted under the conditions of CuSO4·5H2O and NaVc to obtain the compound shown in Formula I.

[0013] The VHL ligand structure is as follows: The structural formula of alkynyl-P60 peptide is as follows:

[0014]

[0015] Furthermore, the reaction conditions in step S1) are that the reaction takes place in an anhydrous organic solvent, and the organic solvent contains an alkaline agent.

[0016] Furthermore, the alkaline agent is triethylamine.

[0017] Furthermore, in step S2), the condensing agent is a combination of HATU and DIEA.

[0018] Furthermore, steps S1), S2), and S3) also include a step of purifying the synthesized compound.

[0019] Further, the purification method in step S3) is as follows: the crude peptide is purified by semi-preparative HPLC; the purification column specifications are C18, 5μm, 21.2 x 250 mm; the crude product is dissolved in a mixed solution of acetonitrile and water and the insoluble matter is removed by filter membrane; the purification method is as follows: 4.0 mL injection volume, elution with a gradient of 10%-100% MeCN / H2O containing 0.1% HCl for 40 min, the eluent of the pure component is collected, and the pure product is obtained by lyophilization.

[0020] Another aspect of the present invention provides a method for hydrolyzing Foxp3 using a protease hydrolysis pathway, the method comprising the step of contacting and culturing the compound described above with cells containing Foxp3.

[0021] Furthermore, the concentration of the compound is less than 10 μM, preferably less than 5 μM, and more preferably 1-3 μM.

[0022] In another aspect, the present invention provides the use of the above-described compounds in the preparation of a reagent for the enzymatic hydrolysis of Tregs by Foxp3 in cells.

[0023] In another aspect, the present invention provides the use of the above-mentioned compounds in the preparation of medicaments for treating tumors and autoimmune diseases.

[0024] Beneficial effects

[0025] The compounds provided by this invention can be used to degrade Foxp3 via the proteasome pathway at low doses. In other words, this invention provides a novel pathway and method for controlling Foxp3. Attached Figure Description

[0026] Figure 1 The HRMS spectrum of compound P60-L1-VHL is shown.

[0027] Figure 2 The HRMS spectrum of compound P60-L2-VHL is shown.

[0028] Figure 3 The HRMS spectrum of compound P60-L3-VHL is shown.

[0029] Figure 4The image shows immunofluorescence patterns of P60-L1-VHL, P60-L2-VHL, and P60-L3-VHL from Example 4. In the image, L1, L2, and L3 represent the results of P60-L1-VHL, P60-L2-VHL, and P60-L3-VHL, respectively.

[0030] Figure 5 This is an immunofluorescence image of P60-L3-VHL and P60 in Example 4. In the image, L3 represents the result of P60-L3-VHL.

[0031] Figure 6 This is a protein blotting experiment diagram of P60-L1-VHL, P60-L2-VHL, and P60-L3-VHL from Example 5. In the diagram, L1, L2, and L3 represent the results of P60-L1-VHL, P60-L2-VHL, and P60-L3-VHL, respectively.

[0032] Figure 7 This is a protein imprinting experiment of P60-L3-VHL under the influence of MG132 in Example 5. In the figure, L3 represents the result of P60-L3-VHL.

[0033] Figure 8 This is a graph showing the elapsed and protein imprinting analysis of the inhibitory effect of P60-L3-VHL on Foxp3 in T cells. In the graph, L3 represents the result of P60-L3-VHL. Detailed Implementation

[0034] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below, but should not be construed as limiting the scope of the present invention.

[0035] Example 1: Synthesis of P60-L1-VHL

[0036] Step a. Dissolve VHL ligand (1.0 equiv) and 6-azidohexanoic acid (1.2 equiv) in N,N-dimethylformamide (DMF) (1.0 mL), then add HATU (1.2 equiv) and DIEA (3.0 equiv). React at room temperature for 2 h. Purify by semi-preparative HPLC. Lyophilize to obtain a white powder of VHL-azidohexanoic acid.

[0037] Step b. Alkyne-P60 (1.0 equiv), VHL-azidoacetic acid (2.0 equiv), CuSO4·5H2O (0.6 equiv), and NaVc (4.0 equiv) were dissolved in 1 mL of DMF:H2O (1:1, v / v). The mixture was then reacted at room temperature for 2 h. Purification was performed by semi-preparative HPLC. After lyophilization, a white powder of P60-L1-VHL was obtained.

[0038] Purification: The crude peptide product was purified by semi-preparative HPLC. The purification column specifications were C18, 5 μm, 21.2 x 250 mm. The crude product was dissolved in a mixture of acetonitrile and water, and insoluble matter was removed by filtration. Purification method: 4.0 mL injection volume, eluted with a gradient of 10%-100% MeCN / H2O (0.1% HCl) for 40 min, the eluent of the pure fraction was collected, and lyophilized to obtain the pure product.

[0039]

[0040] (a)HATU(1.2eq)and DIEA(3.0eq)in DMF,2h.(b)CuSO4·5H2O(0.6eq)and NaVc(4eq)in DMF,2h.

[0041] Example 2: Synthesis of P60-L2-VHL

[0042] Step a. Dissolve VHL ligand (1.0 equiv) and N3-PEG2 (1.2 equiv) in N,N-dimethylformamide (DMF) (1.0 ml), then add HATU (1.2 equiv) and DIEA (3.0 equiv). React at room temperature for 2 h. Purify by semi-preparative HPLC. Lyophilize to obtain a white powder of VHL-PEG2.

[0043] Step b. Alkyne-P60 (1.0 equiv), VHL-PEG2 (2.0 equiv), CuSO4·5H2O (0.6 equiv), and NaVc (4.0 equiv) were dissolved in 1 mL of DMF:H2O (1:1, v / v) solution. The mixture was then reacted at room temperature for 2 h. Purification was performed by semi-preparative HPLC. After lyophilization, a white powder of P60-L2-VHL was obtained.

[0044] Purification: The crude peptide product was purified by semi-preparative HPLC. The purification column specifications were C18, 5 μm, 21.2 x 250 mm. The crude product was dissolved in a mixture of acetonitrile and water, and insoluble matter was removed by filtration. Purification method: 4.0 mL injection volume, eluted with a gradient of 10%-100% MeCN / H2O (0.1% HCl) for 40 min, the eluent of the pure fraction was collected, and lyophilized to obtain the pure product.

[0045]

[0046] (a)HATU(1.2eq)and DIEA(3.0eq)in DMF,2h.(b)CuSO4·5H2O(0.6eq)and NaVc(4eq)in DMF,2h.

[0047] Example 3: Synthesis of P60-L3-VHL

[0048] Step a. Dissolve NH2-PEG2-COOH (1.5 equiv), N3-C5-NHS (1.0 equiv), and Et3N (1.5 equiv) in anhydrous dichloromethane (DCM) (2.0 ml) and react at room temperature for 2 h. Purify by semi-preparative HPLC. Lyophilize to obtain a white powder of N3-C5-PEG2-COOH.

[0049] Step b. Dissolve VHL ligand (1.0 equiv) and N3-C5-PEG2-COOH (1.5 equiv) in N,N-dimethylformamide (DMF) (1.0 ml), then add HATU (1.2 equiv) and DIEA (3.0 equiv). React at room temperature for 2 h. Purify by semi-preparative HPLC. Lyophilize to obtain a white powder of VHL-PEG2-C5.

[0050] Step c. Alkyne-P60 (1.0 equiv), VHL-PEG2-C5 (3.0 equiv), CuSO4·5H2O (0.6 equiv), and NaVc (4.0 equiv) were dissolved in 1 mL of DMF:H2O (1:1, v / v) solution. The mixture was then reacted at room temperature for 2 h. Purification was performed by semi-preparative HPLC. After lyophilization, a white powder, P60-L3-VHL, was obtained.

[0051] Purification: The crude peptide product was purified by semi-preparative HPLC. The purification column specifications were C18, 5 μm, 21.2 x 250 mm. The crude product was dissolved in a mixture of acetonitrile and water, and insoluble matter was removed by filtration. Purification method: 4.0 mL injection volume, eluted with a gradient of 10%-100% MeCN / H2O (0.1% HCl) for 40 min, the eluent of the pure fraction was collected, and lyophilized to obtain the pure product.

[0052]

[0053] (a) Et3N (1.5eq) in DCM, 2h. (b) HATU (1.2eq) and DIEA (3.0eq) in DMF, 2h. (c) CuSO4·5H2O (0.6eq) and NaVc (4eq) in DMF, 2h.

[0054] Example 4 Immunofluorescence assay

[0055] Foxp3-expressing HEK293T cells and HeLa cells were placed in DMEM medium containing 10% fetal bovine serum (Gibco) and 1% Pen-Strep solution (Gibco). The cells were cultured in an incubator at 37°C and 5% CO2.

[0056] HEK293T and HeLa cells were fixed with 4% paraformaldehyde (PFA) for 15 min, washed three times with PBS (Cytiva) for 5 min each time. Cells were blocked with 10% bovine serum albumin for 1 h, permeable with 0.5% Triton X-100, and incubated overnight at 4°C with anti-Foxp3 antibody (Invitgen, 1:400). Cells were then washed three times with PBS (Cytiva) for 5 min each time, and incubated with anti-MICE-555 (Invitgen) at room temperature in the dark for 1 h. Cell nuclei were stained with DAPI (Invitgen) for 15 min. Cells were then washed three times with PBS for 5 min each time. After treating HeLa cells with 1 μm of P60-L1-VHL, P60-L2-VHL, and P60-L3-VHL for 24 h, typical confocal microscopic images of HeLa cells were observed using a confocal microscope. HEK293T cells were treated with different concentrations of P60-L3-VHL and P60, and typical confocal microscopic images of HEK293T cells were observed under a confocal microscope. Immunofluorescence assays showed that P60-L1-VHL and P60-L2-VHL had poor degradation effects, while P60-L3-VHL had a significant degradation effect, and at low doses, it had a similar effect to high doses of P60.

[0057] Example 5: Western blotting test experiment

[0058] HEK293T cells were cultured in 24-well plates (Nest Biotech) at a density of 70%–80%. Foxp3 plasmid was transfected with transfection reagent. To determine the optimal concentrations of P60-L1-VHL, P60-L2-VHL, and P60-L3-VHL, the plasmid was diluted with DMEM to different doses and then co-incubated with the cells.

[0059] To validate the degradation pathway, MG132 (10 μM) was added, and cells were cultured at 37°C for 4 h. Cells were then lysed, and proteins were separated using 10% sodium dodecyl sulfate-PAGE, followed by transfer to a PVDF membrane. A 5% milk-dissolved TBST membrane was then used as a blocking membrane, followed by incubation with primary antibody and HRP-labeled secondary antibody sequentially. Protein blot bands were visualized using electrochemiluminescence (ECL) protein blot substrate (Millipoore).

[0060] Experimental results show that ( Figure 6-7 The poor degradation effects of P60-L1-VHL and P60-L2-VHL are consistent with the results of immunofluorescence experiments. P60-L3-VHL has a better degradation effect, and the degradation pathway test experiment shows that P60-L3-VHL hydrolyzes Foxp3 through proteolytic hydrolysis.

[0061] Example 6: Foxp3 Inhibition Experiment in T Cells

[0062] CD62LHigh / CD25-T cells were isolated from the spleen and lymph nodes of 8-week-old female C57BL / 6 mice. Under Treg activation conditions, 1×10^6 naïve CD4+ T cells were induced with IL-2 (100 U / mL), transforming growth factor (TGF-β), and CD3 and CD28 antibodies (2 and 1 μg / mL, respectively). TGF-β-induced Tregs were treated with different doses of the P60-L3-VHL compound. Fluorescence activated cell classification (FACS) assays were performed at 48 h and 72 h. Furthermore, Western blot analysis was performed at 72 h. The results showed that P60-L3-VHL had a dose-dependent inhibitory effect on CD4+ T cells isolated from mouse tissues after in vitro Treg activation. These results suggest that L3 may affect immune differentiation by inhibiting Foxp3 expression in T cells.

[0063] Experiments have demonstrated that the preparation method of P60-L3-VHL and its series of compounds in this invention is feasible, and the experimental conditions are reproducible with stable yields. Immunofluorescence experiments showed that the compound can achieve the same effect at lower doses as at high concentrations of P60. In the degradation pathway test, the proteasome inhibitor (MG132) was used. The experimental results showed that the expression of Foxp3 was significantly increased in cells treated with MG132, indicating that the degradation of Foxp3 by this compound is via the proteasome pathway. Experiments using cells isolated from mice further confirmed the degradation effect of L3 on Foxp3.

Claims

1. A compound for immunotherapy, characterized in that, The compound is a conjugate of P60 peptide and VHL ligand, and has the structural formula shown in Formula I below:

2. The method for preparing the compound according to claim 1, characterized in that, The preparation method includes the following steps: S1) N3-(CH2)5-PEG2-COOH is obtained by reacting NH2-PEG2-COOH and N3-(CH2)5-NHS; S2) VHL ligand and N3-C5-PEG2-COOH are condensed together under the action of a condensing agent to form an amide bond to obtain VHL-PEG2-(CH2)5-N3; S3) The alkynyl-P60 peptide and VHL-PEG2-(CH2)5-N3 were reacted under the conditions of CuSO4·5H2O and NaVc to obtain the compound shown in Formula I. The VHL ligand structure is as follows: The structural formula of alkynyl-P60 peptide is as follows:

3. The method for preparing the compound according to claim 1, characterized in that, The reaction conditions in step S1) are that the reaction is carried out in an anhydrous organic solvent, and the organic solvent contains an alkaline agent.

4. The method for preparing the compound according to claim 3, characterized in that, The alkaline agent is triethylamine.

5. The method for preparing the compound according to claim 1, characterized in that, In step S2), the condensing agent is a combination of HATU and DIEA.

6. The method for preparing the compound according to claim 1, characterized in that, Steps S1), S2), and S3) also include a step of purifying the synthesized compound.

7. The method for preparing the compound according to claim 1, characterized in that, The purification method in step S3) is as follows: the crude product peptide is purified by semi-preparative HPLC; the purification column specifications are C18, 5μm, 21.2 x 250 mm; the crude product is dissolved in a mixed solution of acetonitrile and water and the insoluble matter is removed by filter membrane; the purification method is as follows: 4.0 mL injection volume, elution with a gradient of 10%-100% MeCN / H2O containing 0.1% HCl for 40 min, the eluent of the pure component is collected, and the pure product is obtained by lyophilization.

8. A method for hydrolyzing Foxp3 using a protease hydrolysis pathway, the method comprising the step of contacting and culturing the compound described above with cells containing Foxp3.

9. Use of the compound of claim 1 in the preparation of a reagent for the enzymatic hydrolysis of Tregs by Foxp3 in cells.

10. Use of the compound of claim 1 in the preparation of a medicament for treating tumors and autoimmune diseases.

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