WO2023108488A1 - Method for screening foxp3-inhibiting small molecule drugs - Google Patents

Abstract

Disclosed is a method for screening FOXP3-inhibiting small molecule drugs. By combining NanoBiT live-cell protein-protein interaction detection technology with an FDA-approved small molecule drug library, the method screens out small molecules capable of effectively inhibiting the interaction between FOXP3 and TRAF6; and the efficacy of the small molecules is validated by means of a perfect immunofluorescence staining technique, an in-vitro Treg functionality experiment and a tumor-bearing mouse model.

Description

A method for screening small molecule drugs inhibiting FOXP3 technical field

The invention belongs to the field of drug screening, and more specifically relates to a method for screening small molecule drugs inhibiting FOXP3.

Background technique

In the tumor microenvironment, Treg cells have the function of suppressing the immune response of other T cells and assisting tumor growth. At present, Treg mainly secretes immunosuppressive cytokines or molecules such as transforming growth factor-β (TGF-β), and highly expresses immunosuppressive receptors such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and program Sexual death receptor 1 (PD-1), which plays an immunosuppressive function.

At this stage, methods targeting immunosuppressive receptors or cytokines have achieved certain results in cancer treatment research, but there are still obvious deficiencies. For example, the objective response rate of Ipilimumab (CTLA-4 antibody) to patients with metastatic melanoma is only 10%-16%, while TGF-β inhibitors are not effective in clinical treatment and can cause cardiovascular side effects. The main reason for these problems is that the mechanism of immunosuppression in the tumor microenvironment is complex and cannot be effectively reversed by targeting a single target. Non-specific expression of the target leads to off-target toxicity.

As a drug target, transcription factors have the following development difficulties: transcription factors have the ability to bind to other proteins and DNA at the same time, and the abundant binding sites are difficult to simply inhibit; transcription factors lack enzymatic activity and binding sites for small molecule drugs ; Transcription factors are mainly distributed in the nucleus and are difficult to be recognized by macromolecular markers such as antibodies. It is worth noting that the protein function of FOXP3 is regulated by a variety of post-translational modifications, such as methylation, phosphorylation and ubiquitination, which have important regulatory significance for the stability, degradation and nuclear transport of FOXP3. Therefore, by regulating the post-translational modification of this protein, its function and stability can be changed indirectly.

The ubiquitin ligase TRAF6 needs to bind FOXP3 directly to guide the polyubiquitination of FOXP3 and promote its nuclear translocation. Knockdown of Traf6 in Treg decreased FOXP3 expression and significantly enhanced the ability of tumor-bearing mice to resist B16 melanoma and MC38 colon adenocarcinoma cells.

Contents of the invention

Aiming at the defects of related technologies, the purpose of the present invention is to screen small molecules capable of inhibiting the combination of TRAF6 and FOXP3 from the FDA-approved small molecule drug library, aiming to solve the problem that transcription factors are difficult to be easily inhibited and lack small molecule drugs as drug targets. The binding site is difficult to be recognized and inhibited by macromolecular markers such as antibodies.

To achieve the above object, one aspect of the present invention provides a method for screening small molecule drugs that inhibit FOXP3, comprising the following steps:

S1: construction of FOXP3 and TRAF6 plasmid vectors respectively marked by fusion of LgBit and SmBit;

S2: 293T cells were transfected by co-transfection liposomes to stably express the target protein;

S3: Harvest live cells and transfer them to a microwell plate for fluorescence detection, then add FDA-approved small molecules for preliminary screening on the microwell plate;

S4: Preliminary selection of small molecules that can hinder the combination of FOXP3 and TRAF6, taking the top 10.

Further, the plasmid vectors used are pBiT1.1 and pBiT2.1-N/C terminal vectors.

Further, the constructed plasmid vectors are pBit2.1-C Foxp3 vector and pBit1.1-N Traf6 vector.

Further, the constructed plasmid vector can also be pBit2.1-C Foxp3 vector and pBit1.1-C Traf6 vector, pBit2.1-N Foxp3 vector and pBit1.1-N Traf6 vector, pBit2.1-N Foxp3 vector and pBit1.1-C Traf6 vector, pBit1.1-C Foxp3 vector and pBit2.1-N Traf6 vector, pBit1.1-C Foxp3 vector and pBit2.1-C Traf6 vector, pBit1.1-N Foxp3 vector and pBit2. 1-N Traf6 vector, pBit1.1-N Foxp3 vector and pBit2.1-N Traf6 vector.

Further, the method also includes:

Step S5: Carry out in vivo experimental verification and in vitro experimental verification to verify the effect of the small molecule drug.

Another aspect of the present invention also provides a method for screening drugs that inhibit FOXP3 nuclear translocation, comprising the following specific steps:

S1: Use eBioscience or Miltenyi initial CD4+ sorting kit to obtain naive CD4+ T cells from the spleen and lymph nodes of C56BL/6 mice, which can be induced by adding 100U/ml IL-2 and 5ng/ml TGF-β Treg cell differentiation, inducible Treg (iTreg) expressing FOXP3 can be obtained after 72 hours, and the small molecules screened out in any one of claims 1-3 are added at the beginning or process of inducing cell differentiation, and different concentration gradients and different In the time experiment group, the cells can be harvested after 72 hours, and the cells are transferred to the glass slide using a cytospin machine, the cells are fixed with 4% paraformaldehyde, the cells are permeabilized with 0.5% Triton X 100, the cells are blocked with 1% BSA solution, and FITC The labeled antibody was stained with FOXP3 and DAPI dyes to mark the nuclei, and finally observed with a laser scanning confocal microscope to initially select inhibitory small molecules that can hinder the translocation of FOXP3 in the nucleus;

S2: Carry out in vitro and in vivo experiments to verify the effects of the initially screened small molecule drugs.

Further, the in vitro experimental verification specifically includes the following steps:

Foxp3-Yfp+Cre and C56BL/6 mouse spleen cells can be sorted by Sony MA900 flow cytometer to obtain CD4+YFP+ mouse Treg cells and CD4+CD25-CD69Llo initial T cells, and the Treg cells Co-cultured with the fluorescent dye CTV-labeled naive T cells for 72 hours, and analyzed the cells under the ThermoFisher Attune NxT flow cytometer, it can be observed that Treg significantly inhibits the differentiation of naive T cells. The small molecule screened in step S4 in claim 1 or step S1 in claim 4 is tested to see whether it can effectively inhibit the function of Treg.

Further, the in vivo experimental verification specifically includes the following steps:

B16 melanoma cells or MC38 colon cancer cells were inoculated subcutaneously in C56BL/6 mice, each mouse was inoculated with approximately ^1x105 cells, and a tumor-bearing mouse model could be established; the tumor size was measured every 3 days after 7 days of inoculation; After the diameter is greater than or equal to 1cm, after the injection of the inhibitory small molecule, continue to detect the tumor size every 3 days, a total of 5 times, and euthanize the mice 15 days after the administration, obtain the tumor tissue, and analyze it under the flow cytometer Cell types CD4, CD8, FOXP3 and cytokines in tumor tissue expressed IFN-γ, TNF-α, IL-17 levels, and the effects of small molecule drugs on tumor growth and microenvironment were analyzed.

Further, the verification of in vivo experiments can also be combined with CTLA-4 or PD-1 monoclonal antibodies to test the anti-cancer effect of combined drugs.

Compared with the prior art, the technical solution of this method can achieve the following beneficial effects:

(1) The present invention combines the FDA-approved small molecule drug library to screen small molecules that can effectively inhibit the interaction between FOXP3 and TRAF6, and through perfect immunofluorescence staining techniques, in vitro Treg functional experiments, and tumor-bearing mouse models to verify the efficacy of small molecules. Through the above technical solutions conceived by the present invention, compared with the prior art, since the small molecules that can inhibit the interaction between FOXP3 and TRAF6 are screened, it is possible to use FOXP3 as a drug target.

(2) The present invention uses FDA-approved small-molecule drugs when screening drugs, and combines old drugs with a fluorescence detection system to screen out effective small molecules. Compared with the existing technology, due to the re-screening of old drugs that have passed clinical trials and been marketed, they are applied to new target research, shortening the time of clinical research and expanding the application range of drugs, greatly improving the efficiency of drug development and reduced costs.

Description of drawings

Figure 1 is a schematic diagram of the principle of NanoBiT technology for screening small molecules that inhibit the binding of FOX3 and TRAF6;

Figure 2 is a schematic diagram of analyzing the distribution of FOXP3 in the nucleus by immunofluorescence staining to test the effect of small molecules;

Figure 3 is a schematic diagram of the effect of inhibiting small molecules on Treg function in the Treg proliferation inhibition experiment in vitro;

Figure 4 is a schematic diagram of testing the anticancer effect of small molecule drugs in tumor-bearing mice;

Fig. 5 is a schematic diagram showing the binding intensity of FOXP3/TRAF6 fusion protein with different fluorescence intensity of NanoLuc.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not constitute a conflict with each other.

The invention provides a small molecule system and method for screening the interaction between FOXP3 and TRAF6. Combined with the FDA-approved small molecule drug library, small molecules that can effectively inhibit the interaction between FOXP3 and TRAF6 were screened, and the efficacy of small molecules was verified by perfect immunofluorescence staining techniques, in vitro Treg functional experiments, and tumor-bearing mouse models . Compared with the prior art, because the screening is a small molecule that can inhibit the interaction between FOXP3 and TRAF6, it is possible to use FOXP3 as a drug target.

The embodiment of the present invention provides a method for screening drugs that inhibit the combination of FOXP3 and TRAF6, the method comprising the following steps:

S1: First construct the FOXP3 and TRAF6 plasmid vectors which are fusion-marked by LgBit and SmBit respectively. The plasmid vectors used are pBiT1.1 and pBiT2.1-N/C terminal vectors. Since the fusion site can be at the N-terminal and C-terminal of the target protein, a total of eight fusion proteins can be constructed and expressed. In the experiment, it was found that the fluorescence detection effect of the fusion protein expressed by pBit2.1-C Foxp3 and pBit1.1-N Traf6 was better (as shown in Figure 5).

S2: After constructing the plasmid vector combination, 293T cells can be transfected by co-transfection liposomes to stably express the target protein.

S3: After the living cells are harvested and transferred to a 96-well plate, the reaction substrate Furimazine is added for reaction, and then fluorescence detection can be performed. On this basis, add FDA-approved small molecules, which can be selected from Selleck's FDA drug library, and screened on 96 microwell plates.

S4: Preliminary selection of small molecules that can hinder the combination of FOXP3 and TRAF6, the top 10 can be selected.

S5: Carry out in vitro and in vivo experiments to verify the effects of the initially screened small molecule drugs. The specific method for in vitro experiment verification is as follows: Foxp3-Yfp+Cre and C56BL/6 mouse spleen cells can be sorted by Sony MA900 flow cytometry to obtain CD4+YFP+ mouse Treg cells and CD4+CD25- For CD69Llo naive T cells, Treg cells were co-cultured with naive T cells labeled with fluorescent dyes (such as CTV) for 72 hours, and the cells were analyzed under the ThermoFisher Attune NxT flow cytometer. It can be observed that Treg significantly inhibits the differentiation of naive T cells . Based on this experiment, the small molecule screened out by step S4 was added during the co-culture period to test whether it can effectively inhibit the function of Treg; the specific method of in vivo experiment verification was to inoculate B16 melanoma cells or MC38 colonic cells subcutaneously in C56BL/6 mice. For cancer cells, each mouse was inoculated with about ^1x105 cells to construct a tumor-bearing mouse model; the tumor size was measured every 3 days after 7 days of inoculation. After the tumor diameter is greater than or equal to 1cm, after the injection of the inhibitory small molecule, continue to detect the tumor size every 3 days, a total of 5 times, and euthanize the mice 15 days after the administration, obtain the tumor tissue, and analyze it in the flow cytometer Next, analyze the cell types (CD4, CD8, FOXP3) and cytokine expression (IFN-γ, TNF-α, IL-17) levels in tumor tissue, and analyze the effects of small molecule drugs on tumor growth and microenvironment. This experiment can be combined with CTLA-4 or PD-1 monoclonal antibody to test the anti-cancer effect of the combined drug.

The principle of the NanoBiT technology used in S1 can be seen in Figure 1.

A method for screening drugs that inhibit FOXP3 nuclear translocation, the method includes the following specific steps:

S1 Initial CD4+ T cells can be purified from the spleen and lymph nodes of C56BL/6 mice using eBioscience or Miltenyi initial CD4+ sorting kit, and Treg can be induced by adding 100U/ml IL-2 and 5ng/ml TGF-β After 72 hours of cell differentiation, inducible Treg (iTreg) expressing FOXP3 can be obtained. At the beginning or process of inducing cell differentiation, a small molecule screened in method S4 for screening drugs that inhibit the combination of FOXP3 and TRAF6 can be added, and different concentration gradients and different time experimental groups can be set up. After 72 hours, the cells can be harvested and centrifuged. A smear machine transfers the cells onto slides. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X 100, blocked with 1% BSA solution, FITC-labeled antibody stained with FOXP3 and DAPI dye-labeled nuclei, and finally observed with a laser scanning confocal microscope. Inhibitory small molecules of FOXP3 nuclear translocation. The technical process can be seen in Figure 2.

S2: Carry out in vitro and in vivo experiments to verify the effects of the initially screened small molecule drugs. The specific method for in vitro experiment verification is as follows: Foxp3-Yfp+Cre and C56BL/6 mouse spleen cells can be sorted by Sony MA900 flow cytometry to obtain CD4+YFP+ mouse Treg cells and CD4+CD25- For CD69Llo naive T cells, Treg cells were co-cultured with naive T cells labeled with fluorescent dyes (such as CTV) for 72 hours, and the cells were analyzed under the ThermoFisher Attune NxT flow cytometer. It can be observed that Treg significantly inhibits the differentiation of naive T cells . Based on this experiment, the small molecule screened out by step S4 was added during the co-culture period to test whether it can effectively inhibit the function of Treg; the specific method of in vivo experiment verification was to inoculate B16 melanoma cells or MC38 colonic cells subcutaneously in C56BL/6 mice. For cancer cells, each mouse is inoculated with about ^1x105 cells, and a tumor-bearing mouse model can be constructed. Tumor size was measured every 3 days 7 days after inoculation. After the tumor diameter is greater than or equal to 1cm, after the injection of the inhibitory small molecule, continue to detect the tumor size every 3 days, a total of 5 times, and euthanize the mice 15 days after the administration, obtain the tumor tissue, and analyze it in the flow cytometer Next, analyze the cell types (CD4, CD8, FOXP3) and cytokine expression (IFN-γ, TNF-α, IL-17) levels in tumor tissue, and analyze the effects of small molecule drugs on tumor growth and microenvironment. This experiment can be combined with CTLA-4 or PD-1 monoclonal antibody to test the anti-cancer effect of the combined drug.

It is easy for those skilled in the art to understand that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention, All should be included within the protection scope of the present invention.

Claims (10)

A method for screening small-molecule drugs that inhibit FOXP3, characterized in that the method can screen out small-molecule drugs that inhibit the combination of FOXP3 and TRAF6, and the method includes the following steps: S1: construction of FOXP3 and TRAF6 plasmid vectors respectively marked by fusion of LgBit and SmBit; S2: 293T cells were transfected by co-transfection liposomes to stably express the target protein; S3: Harvest live cells and transfer them to a microwell plate for fluorescence detection, then add FDA-approved small molecules for preliminary screening on the microwell plate; S4: Preliminary selection of small molecules that can hinder the combination of FOXP3 and TRAF6, taking the top 10. The method according to claim 1, characterized in that the plasmid vectors used are pBiT1.1 and pBiT2.1-N/C terminal vectors. The method according to claim 2, wherein the plasmid vector constructed is pBit2.1-C Foxp3 vector and pBit1.1-N Traf6 vector. The method according to claim 2, wherein the plasmid vector constructed can also be pBit2.1-C Foxp3 carrier and pBit1.1-C Traf6 carrier, pBit2.1-N Foxp3 carrier and pBit1.1-N Traf6 Vector, pBit2.1-N Foxp3 vector and pBit1.1-C Traf6 vector, pBit1.1-C Foxp3 vector and pBit2.1-N Traf6 vector, pBit1.1-C Foxp3 vector and pBit2.1-C Traf6 vector, pBit1.1-N Foxp3 vector and pBit2.1-N Traf6 vector, pBit1.1-N Foxp3 vector and pBit2.1-N Traf6 vector. The method according to any one of claims 1-4, further comprising: Step S5: Carry out in vivo experimental verification and in vitro experimental verification to verify the effect of the small molecule drug. A method for screening small-molecule drugs that inhibit FOXP3, through which small-molecule drugs that inhibit FOXP3 nuclear translocation can be screened out, characterized in that it includes the following specific steps: S1: Naive CD4+ T cells were purified from the spleen and lymph nodes of C56BL/6 mice using eBioscience or Miltenyi Naive CD4+ Sorting Kit, after adding 100 U/ml IL-2 and 5 ng/ml TGF-β Induce Treg cell differentiation, obtain inducible Treg expressing FOXP3 after 72 hours, add the small molecule screened out in any one of claims 1-4 at the beginning or process of inducing cell differentiation, set different concentration gradients and different time experimental groups , Harvest the cells after 72 hours, transfer the cells to slides using a cytospin, fix the cells with 4% paraformaldehyde, permeabilize the cells with 0.5% Triton X 100, block with 1% BSA solution, and stain FOXP3 with FITC-labeled antibody and DAPI dyes to mark the nuclei, and finally use a laser scanning confocal microscope to observe, and preliminarily select small inhibitory molecules that can hinder the translocation of FOXP3 in the nucleus; S2: Carry out in vitro and in vivo experiments to verify the effects of the initially screened small molecule drugs. The method according to claim 5 or claim 6, wherein said in vitro experimental verification specifically comprises the following steps: Foxp3-Yfp+Cre and C56BL/6 mouse spleen cells can be sorted by Sony MA900 flow cytometer to obtain CD4+YFP+ mouse Treg cells and CD4+CD25-CD69Llo initial T cells, and the Treg cells Co-cultured with fluorescent dye-labeled naive T cells for 72 hours, and analyzed the cells under the ThermoFisher Attune NxT flow cytometer, it can be observed that Treg significantly inhibits the differentiation of naive T cells. Based on this experiment, the right The small molecule screened in step S4 in claim 1 or step S1 in claim 6 is tested to see whether it can effectively inhibit the function of Treg. The method according to claim 5 or claim 6, wherein said in vivo experimental verification specifically comprises the following steps: B16 melanoma cells or MC38 colon cancer cells were inoculated subcutaneously in C56BL/6 mice, each mouse was inoculated with approximately ^1x105 cells, and a tumor-bearing mouse model was established; the tumor size was measured every 3 days after 7 days of inoculation; After ≥ 1 cm, after the injection of the inhibitory small molecule, continue to detect the size of the tumor every 3 days, a total of 5 times, euthanize the mice 15 days after the administration, obtain tumor tissue, and analyze the tumor under the flow cytometer Cell types CD4, CD8, FOXP3 and cytokines in tissues express IFN-γ, TNF-α, IL-17 levels, and analyze the effects of small molecule drugs on tumor growth and microenvironment. The method according to claim 6, characterized in that the verification of the in vivo experiment can also be combined with CTLA-4 or PD-1 monoclonal antibody to test the anticancer effect of the combined drug. The method of claim 7, wherein the fluorescent dye is CTV.

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