WO2023108488A1 - 一种筛选抑制foxp3小分子药物的方法 - Google Patents

一种筛选抑制foxp3小分子药物的方法 Download PDF

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WO2023108488A1
WO2023108488A1 PCT/CN2021/138383 CN2021138383W WO2023108488A1 WO 2023108488 A1 WO2023108488 A1 WO 2023108488A1 CN 2021138383 W CN2021138383 W CN 2021138383W WO 2023108488 A1 WO2023108488 A1 WO 2023108488A1
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foxp3
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vector
traf6
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潘璠
李奕葵
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Shenzhen Institute of Advanced Technology of CAS
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  • the invention belongs to the field of drug screening, and more specifically relates to a method for screening small molecule drugs inhibiting FOXP3.
  • Treg cells have the function of suppressing the immune response of other T cells and assisting tumor growth.
  • Treg mainly secretes immunosuppressive cytokines or molecules such as transforming growth factor- ⁇ (TGF- ⁇ ), 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.
  • TGF- ⁇ transforming growth factor- ⁇
  • CTL-4 cytotoxic T lymphocyte-associated protein 4
  • PD-1 program Sexual death receptor 1
  • 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.
  • 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.
  • 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
  • S4 Preliminary selection of small molecules that can hinder the combination of FOXP3 and TRAF6, taking the top 10.
  • the plasmid vectors used are pBiT1.1 and pBiT2.1-N/C terminal vectors.
  • the constructed plasmid vectors are pBit2.1-C Foxp3 vector and pBit1.1-N Traf6 vector.
  • 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.
  • 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:
  • 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.
  • 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- ⁇ , TNF- ⁇ , IL-17 levels, and the effects of small molecule drugs on tumor growth and microenvironment were analyzed.
  • 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.
  • 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.
  • 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.
  • the invention provides a small molecule system and method for screening the interaction between FOXP3 and TRAF6.
  • 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 .
  • 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).
  • 293T cells After constructing the plasmid vector combination, 293T cells can be transfected by co-transfection liposomes to stably express the target protein.
  • 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 .
  • step S4 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.
  • 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.
  • the tumor diameter is greater than or equal to 1cm
  • 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- ⁇ , TNF- ⁇ , 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.
  • a method for screening drugs that inhibit FOXP3 nuclear translocation includes the following specific steps:
  • 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- ⁇ 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.
  • 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 .
  • step S4 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.
  • 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.
  • the tumor diameter is greater than or equal to 1cm
  • 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- ⁇ , TNF- ⁇ , 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.

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Abstract

本发明公开了一种筛选抑制FOXP3小分子药物的方法。本发明通过NanoBiT活细胞蛋白互作检测技术，结合FDA批准小分子药物库，对能有效抑制FOXP3和TRAF6相互作用的小分子进行筛选，并且通过完善的免疫荧光染色技术，体外Treg功能性实验，以及荷瘤小鼠模型验证了小分子的效力。

Description

一种筛选抑制FOXP3小分子药物的方法 技术领域
本发明属于药物筛选领域，更具体地，涉及一种筛选抑制FOXP3小分子药物的方法。
背景技术
在肿瘤微环境中，Treg细胞具有抑制其他T细胞免疫反应的功能，协助肿瘤生长。现阶段，Treg主要通过分泌免疫抑制性的细胞因子或分子例如转化生长因子-β(TGF-β)，及高表达免疫抑制受体例如细胞毒性T淋巴细胞相关蛋白4(CTLA-4)和程序性死亡受体1(PD-1)，发挥免疫抑制功能。
现阶段针对免疫抑制受体或者细胞因子的手段在癌症治疗研究中有一定的成效，但仍存在明显的不足。如Ipilimumab(CTLA-4抗体)对转移性黑素瘤病人的客观应答率仅有10％-16％，而TGF-β抑制剂在临床治疗效果不佳并可导致心血管毒副作用。造成这些问题的主要原因是：肿瘤微环境中免疫抑制的机制复杂，针对单一靶点无法有效逆转。靶点的非特异性表达导致了脱靶毒性。
转录因子作为药物靶点，有以下开发难点：转录因子同时具有与其他蛋白结合和与DNA结合的能力，丰富的结合位点难以简单抑制；转录因子缺乏酶活性以及与小分子药物结合的位点；转录因子主要分布在核内，难以被抗体等大分子标记识别。值得注意的是，FOXP3的蛋白功能由多种翻译后修饰所调控，如甲基化、磷酸化和泛素化等修饰，对FOXP3的稳定、降解和核内转运等有重要调控意义。因此，通过调控该蛋白的翻译后修饰，可以间接改变其功能和稳定性。
泛素连接酶TRAF6需要通过直接与FOXP3结合，从而引导FOXP3多聚泛素化，促进其核内转运。在Treg中敲除Traf6会降低FOXP3表达，并显著提升荷瘤小鼠抵抗B16黑色素瘤和MC38结肠腺癌细胞的能力。
发明内容
针对相关技术的缺陷，本发明的目的在于从FDA批准的小分子药物库中筛选出能够抑制TRAF6与FOXP3结合的小分子，旨在解决转录因子作为药物靶点具有难以简单抑制、缺乏小分子药物结合位点、难以被抗体等大分子标记识别抑制的问题。
为实现上述目的，本发明的一个方面提供了一种筛选抑制FOXP3小分子药物的方法，包括如下步骤：
S1:构建分别被LgBit和SmBit融合标记的FOXP3和TRAF6质粒载体；
S2:通过共转染脂质体转染293T细胞，使其稳定表达目的蛋白；
S3:收获活细胞并转移到微孔板后，进行荧光检测，再加入FDA批准的小分子,在微孔板上进行初步筛选；
S4:初步选取出能阻碍FOXP3和TRAF6结合的小分子，取前10种。
进一步地，采用的质粒载体是pBiT1.1和pBiT2.1-N/C端载体。
进一步地，构建的质粒载体是pBit2.1-C Foxp3载体和pBit1.1-N Traf6载体。
进一步地，构建的质粒载体还可以是pBit2.1-C Foxp3载体和pBit1.1-C Traf6载体、pBit2.1-N Foxp3载体和pBit1.1-N Traf6载体、pBit2.1-N Foxp3载体和pBit1.1-C Traf6载体、pBit1.1-C Foxp3载体和pBit2.1-N Traf6载体、pBit1.1-C Foxp3载体和pBit2.1-C Traf6载体、pBit1.1-N Foxp3载体和pBit2.1-N Traf6载体、pBit1.1-N Foxp3载体和pBit2.1-N Traf6载体。
进一步地，该方法还包括：
步骤S5：进行体内实验验证和体外实验验证，验证小分子药物的效果。
本发明的另一方面还提供了一种筛选抑制FOXP3核内转运的药物方法，包括如下具体步骤：
S1：使用eBioscience或Miltenyi初始CD4+分选试剂盒，从C56BL/6小鼠的脾脏和淋巴结中的纯化得到初始CD4+T细胞，加入100U/ml IL-2和5ng/ml TGF-β后可诱导Treg细胞分化，在72小时后可得到表达FOXP3的诱导型Treg(iTreg)，在诱导细胞分化开始或过程中加入权利要求1-3任意一项中筛选出来的小分子，设置不同浓度梯度和不同时间实验组，72小时后可收获细胞，并使用细胞离心涂片机将细胞转移到玻片上，使用4％多聚甲醛固定细胞，0.5％Triton X 100透化细胞，1％BSA溶液封闭，FITC标记抗体染FOXP3和DAPI染料标记细胞核，最后使用激光扫描共聚焦显微镜进行观测，初步选取出可以阻碍FOXP3核内转运的抑制小分子；
S2:对初步筛选出来的小分子药物效果，分别进行体外实验和体内实验的验证。
进一步地，所述体外实验验证具体包括如下步骤：
通过Sony MA900流式分选细胞仪可以对Foxp3-Yfp+Cre和C56BL/6小鼠脾脏细胞进行分选，得到CD4+YFP+的小鼠Treg细胞和CD4+CD25-CD69Llo初始T细胞，将Treg细胞与荧光染料CTV标记的初始T细胞进行共培养72小时，在ThermoFisher Attune NxT流式细胞分析仪下分析细胞，可以观测到Treg显著抑制初始T细胞的分化，基于此实验，在共培养期间加入通过权利要求1中S4步骤或权利要求4中S1步骤筛选出的小分子，检验其是否有效抑制Treg的功能。
进一步地，所述体内实验验证具体包括以下步骤：
在C56BL/6小鼠皮下接种B16黑色素瘤细胞或MC38结肠癌细胞，每只小鼠接种大约1x10 5个细胞，可构建荷瘤小鼠模型；在接种7天后每3天测量肿瘤大小；在肿瘤直径大于等于1cm后，注射抑制小分子给药后，继续每3天检测肿瘤大小，一共检测5次，给药后15天后对小鼠进行安乐死， 获取肿瘤组织，在流式细胞分析仪下分析肿瘤组织中细胞类别CD4，CD8，FOXP3和细胞因子表达IFN-γ，TNF-α，IL-17水平，分析小分子药物对肿瘤生长和微环境的影响。
进一步地，体内实验的验证还能联合CTLA-4或PD-1单克隆抗体测试联合用药的抗癌效果。
与现有技术相比，本方法的技术方案能够取得以下有益效果：
(1)本发明结合FDA批准小分子药物库，对能有效抑制FOXP3和TRAF6相互作用的小分子进行筛选，并且通过完善的免疫荧光染色技术，体外Treg功能性实验，以及荷瘤小鼠模型验证了小分子的效力。通过本发明所构思的以上技术方案，与现有技术相比，由于筛选的是能够抑制FOXP3和TRAF6相互作用的小分子，使FOXP3做为药物靶点成为可能。
(2)本发明在进行药物筛选时，采用的是FDA批准小分子药物，将旧药新用和荧光检测系统结合，筛选出有效的小分子。与现有技术相比，由于对已经通过临床试验并上市的老药，进行再筛选，应用于新的靶点研究，缩短临床研究时间和扩展药物的应用范围，大大地提高了药物开发的效率和降低了成本。
附图说明
图1是NanoBiT技术筛选抑制FOX3和TRAF6结合的小分子的原理示意图；
图2是免疫荧光染色技术分析FOXP3核内分布以检验抑制小分子作用的示意图；
图3是Treg体外抑制增殖实验研究抑制小分子对Treg功能的影响示意图；
图4是荷瘤小鼠检验小分子药物抗癌效果示意图；
图5是NanoLuc荧光强度表现不同FOXP3/TRAF6融合蛋白结合的强度示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。此外，下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
本发明提供了一种筛选抑制FOXP3和TRAF6相互作用的小分子系统和方法。结合FDA批准小分子药物库，对能有效抑制FOXP3和TRAF6相互作用的小分子进行筛选，并且通过完善的免疫荧光染色技术，体外Treg功能性实验，以及荷瘤小鼠模型验证了小分子的效力。与现有技术相比，由于筛选的是能够抑制FOXP3和TRAF6相互作用的小分子，使FOXP3做为药物靶点成为可能。
本发明实施例提供了一种筛选抑制FOXP3和TRAF6结合的药物方法，该方法包括如下步骤：
S1:首先构建了分别被LgBit和SmBit融合标记的FOXP3和TRAF6质粒载体。采用的质粒载体是pBiT1.1和pBiT2.1-N/C端载体。由于融合位点可在目的蛋白的N端和C端，因此一共可以构建表达八种融合蛋白。在实验中发现pBit2.1-C Foxp3和pBit1.1-N Traf6表达出的融合蛋白荧光检测效果较好(如图5所示)。
S2:在构建质粒载体组合后，可通过共转染脂质体转染293T细胞，使其稳定表达目的蛋白。
S3:收获活细胞并转移到96微孔板后，加入反应底物Furimazine进行反应后，便可进行荧光检测。在此基础上，加入FDA批准的小分子,该小分子可以选自Selleck的FDA药物库，在96微孔板上进行筛选。
S4:初步选取出可以阻碍FOXP3和TRAF6结合的小分子，可以取前10种。
S5:对初步筛选出来的小分子药物效果，分别进行体外实验和体内实验的验证。进行体外实验验证具体方法如下：通过Sony MA900流式分选细胞仪可以对Foxp3-Yfp+Cre和C56BL/6小鼠脾脏细胞进行分选，得到CD4+YFP+的小鼠Treg细胞和CD4+CD25-CD69Llo初始T细胞，将Treg细胞与荧光染料(如CTV)标记的初始T细胞进行共培养72小时，在ThermoFisher Attune NxT流式细胞分析仪下分析细胞，可以观测到Treg显著抑制初始T细胞的分化。基于此实验，在共培养期间加入通过S4步骤筛选出的小分子，检验其是否能有效抑制Treg的功能；进行体内实验验证具体方法是在C56BL/6小鼠皮下接种B16黑色素瘤细胞或MC38结肠癌细胞，每只小鼠接种约1x10 5个细胞，可构建荷瘤小鼠模型；在接种7天后每3天测量肿瘤大小。在肿瘤直径大于等于1cm后，注射抑制小分子给药后，继续每3天检测肿瘤大小，一共检测5次，给药后15天后对小鼠进行安乐死，获取肿瘤组织，在流式细胞分析仪下分析肿瘤组织中细胞类别(CD4，CD8，FOXP3)和细胞因子表达(IFN-γ，TNF-α，IL-17)水平，分析小分子药物对肿瘤生长和微环境的影响。该实验可联合CTLA-4或PD-1单克隆抗体测试联合用药的抗癌效果。
S1中用到的NanoBiT技术其原理可参见图1。
一种筛选抑制FOXP3核内转运的药物方法，该方法包括如下具体步骤：
S1使用eBioscience或Miltenyi初始CD4+分选试剂盒可从C56BL/6小鼠的脾脏和淋巴结中的纯化得到初始CD4+T细胞，加入100U/ml IL-2和5ng/ml TGF-β后可诱导Treg细胞分化，在72小时后可得到表达FOXP3的诱导型Treg(iTreg)。在诱导细胞分化开始或过程中可加入一种筛选抑制FOXP3和TRAF6结合的药物方法S4中筛选出来的小分子，设置不同浓度梯度和不同时间实验组，72小时后可收获细胞，并使用细胞离心涂片机将细胞转移到玻片上。使用4％多聚甲醛固定细胞，0.5％Triton X 100透化细胞，1％BSA溶液封闭，FITC标记抗体染FOXP3和DAPI染料标记细胞核，最 后使用激光扫描共聚焦显微镜进行观测，初步选取出可以阻碍FOXP3核内转运的抑制小分子。该技术流程可参见图2。
S2:对初步筛选出来的小分子药物效果，分别进行体外实验和体内实验的验证。进行体外实验验证具体方法如下：通过Sony MA900流式分选细胞仪可以对Foxp3-Yfp+Cre和C56BL/6小鼠脾脏细胞进行分选，得到CD4+YFP+的小鼠Treg细胞和CD4+CD25-CD69Llo初始T细胞，将Treg细胞与荧光染料(如CTV)标记的初始T细胞进行共培养72小时，在ThermoFisher Attune NxT流式细胞分析仪下分析细胞，可以观测到Treg显著抑制初始T细胞的分化。基于此实验，在共培养期间加入通过S4步骤筛选出的小分子，检验其是否能有效抑制Treg的功能；进行体内实验验证具体方法是在C56BL/6小鼠皮下接种B16黑色素瘤细胞或MC38结肠癌细胞，每只小鼠接种约1x10 5个细胞，可构建荷瘤小鼠模型。在接种7天后每3天测量肿瘤大小。在肿瘤直径大于等于1cm后，注射抑制小分子给药后，继续每3天检测肿瘤大小，一共检测5次，给药后15天后对小鼠进行安乐死，获取肿瘤组织，在流式细胞分析仪下分析肿瘤组织中细胞类别(CD4，CD8，FOXP3)和细胞因子表达(IFN-γ，TNF-α，IL-17)水平，分析小分子药物对肿瘤生长和微环境的影响。该实验可联合CTLA-4或PD-1单克隆抗体测试联合用药的抗癌效果。
本领域的技术人员容易理解，以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种筛选抑制FOXP3小分子药物的方法，其特征在于，通过该方法能够筛选出抑制FOXP3与TRAF6结合的小分子药物，该方法包括如下步骤：
    S1:构建分别被LgBit和SmBit融合标记的FOXP3和TRAF6质粒载体；
    S2:通过共转染脂质体转染293T细胞，使其稳定表达目的蛋白；
    S3:收获活细胞并转移到微孔板后，进行荧光检测，再加入FDA批准的小分子,在微孔板上进行初步筛选；
    S4:初步选取出能阻碍FOXP3和TRAF6结合的小分子，取前10种。
  2. 如权利要求1所述的方法，其特征在于，采用的质粒载体是pBiT1.1和pBiT2.1-N/C端载体。
  3. 如权利要求2所述的方法，其特征在于，构建的质粒载体是pBit2.1-C Foxp3载体和pBit1.1-N Traf6载体。
  4. 如权利要求2所述的方法，其特征在于，构建的质粒载体还可以是pBit2.1-C Foxp3载体和pBit1.1-C Traf6载体、pBit2.1-N Foxp3载体和pBit1.1-N Traf6载体、pBit2.1-N Foxp3载体和pBit1.1-C Traf6载体、pBit1.1-C Foxp3载体和pBit2.1-N Traf6载体、pBit1.1-C Foxp3载体和pBit2.1-C Traf6载体、pBit1.1-N Foxp3载体和pBit2.1-N Traf6载体、pBit1.1-N Foxp3载体和pBit2.1-N Traf6载体。
  5. 如权利要求1-4任意一项所述的方法，其特征在于，该方法还包括：
    步骤S5：进行体内实验验证和体外实验验证，验证小分子药物的效果。
  6. 一种筛选抑制FOXP3小分子药物的方法,通过该方法能够筛选出抑制FOXP3核内转运的小分子药物，其特征在于，包括如下具体步骤：
    S1：使用eBioscience或Miltenyi初始CD4+分选试剂盒，从C56BL/6小鼠的脾脏和淋巴结中的纯化得到初始CD4+T细胞，加入100 U/ml IL-2和5 ng/ml TGF-β后诱导Treg细胞分化，在72小时后得到表达FOXP3的 诱导型Treg，在诱导细胞分化开始或过程中加入权利要求1-4任意一项中筛选出来的小分子，设置不同浓度梯度和不同时间实验组，72小时后收获细胞，并使用细胞离心涂片机将细胞转移到玻片上，使用4％多聚甲醛固定细胞，0.5％Triton X 100透化细胞，1％BSA溶液封闭，FITC标记抗体染FOXP3和DAPI染料标记细胞核，最后使用激光扫描共聚焦显微镜进行观测，初步选取出可以阻碍FOXP3核内转运的抑制小分子；
    S2:对初步筛选出来的小分子药物效果，分别进行体外实验和体内实验的验证。
  7. 如权利要求5或权利要求6所述的方法，其特征在于，所述体外实验验证具体包括如下步骤：
    通过Sony MA900流式分选细胞仪可以对Foxp3-Yfp+Cre和C56BL/6小鼠脾脏细胞进行分选，得到CD4+YFP+的小鼠Treg细胞和CD4+CD25-CD69Llo初始T细胞，将Treg细胞与荧光染料标记的初始T细胞进行共培养72小时，在ThermoFisher Attune NxT流式细胞分析仪下分析细胞，可以观测到Treg显著抑制初始T细胞的分化，基于此实验，在共培养期间加入通过权利要求1中S4步骤或权利要求6中S1步骤筛选出的小分子，检验其是否有效抑制Treg的功能。
  8. 如权利要求5或权利要求6所述的方法，其特征在于，所述体内实验验证具体包括以下步骤：
    在C56BL/6小鼠皮下接种B16黑色素瘤细胞或MC38结肠癌细胞，每只小鼠接种大约1x10 5个细胞，构建荷瘤小鼠模型；在接种7天后每3天测量肿瘤大小；在肿瘤直径大于等于1cm后，注射抑制小分子给药后，继续每3天检测肿瘤大小，一共检测5次，给药后15天后对小鼠进行安乐死，获取肿瘤组织，在流式细胞分析仪下分析肿瘤组织中细胞类别CD4，CD8，FOXP3和细胞因子表达IFN-γ，TNF-α，IL-17水平，分析小分子药物对肿瘤生长和微环境的影响。
  9. 如权利要求6所述的方法，其特征在于，体内实验的验证还能联合CTLA-4或PD-1单克隆抗体测试联合用药的抗癌效果。
  10. 如权利要求7所述的方法，其特征在于，所述荧光染料为CTV。
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