WO2023108488A1 - Procédé de criblage de médicaments à petites molécules inhibiteurs de foxp3 - Google Patents

Procédé de criblage de médicaments à petites molécules inhibiteurs de foxp3 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
cells
vector
traf6
small molecule
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PCT/CN2021/138383
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English (en)
Chinese (zh)
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潘璠
李奕葵
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深圳先进技术研究院
中国科学院深圳理工大学(筹)
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Priority to PCT/CN2021/138383 priority Critical patent/WO2023108488A1/fr
Publication of WO2023108488A1 publication Critical patent/WO2023108488A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • 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

L'invention concerne un procédé de criblage de médicaments à petites molécules inhibiteurs de FOXP3. En combinant la technologie NanoBiT de détection des interactions protéine-protéine dans les cellules vivantes avec une banque de petites molécules approuvée par la FDA, le procédé permet de cribler les petites molécules capables d'inhiber efficacement l'interaction entre FOXP3 et TRAF6. L'efficacité des petites molécules est validée par une technique parfaite de coloration par immunofluorescence, une expérience de fonctionnalité des Treg in vitro et un modèle de souris porteuse de tumeur.
PCT/CN2021/138383 2021-12-15 2021-12-15 Procédé de criblage de médicaments à petites molécules inhibiteurs de foxp3 WO2023108488A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130122046A1 (en) * 2010-07-09 2013-05-16 Institut Pasteur Of Shanghai, Cas Regulatory factor of foxp3 and regulatory t cells and use thereof
US20140030218A1 (en) * 2011-01-05 2014-01-30 Imperial Innovations Limited Treatment And Screening
US20150190370A1 (en) * 2012-08-27 2015-07-09 Ludwig-Maximilians-Universität München Inhibitors of CD40-TRAF6 Interaction
CN108368535A (zh) * 2014-08-21 2018-08-03 欧陆迪斯卡沃爱克斯公司 用于测量配体与靶标蛋白质的结合和细胞接合的方法
CN111100902A (zh) * 2018-10-26 2020-05-05 复旦大学 一种基于sim靶点抗kshv/ebv相关肿瘤小分子化合物筛选方法和应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130122046A1 (en) * 2010-07-09 2013-05-16 Institut Pasteur Of Shanghai, Cas Regulatory factor of foxp3 and regulatory t cells and use thereof
US20140030218A1 (en) * 2011-01-05 2014-01-30 Imperial Innovations Limited Treatment And Screening
US20150190370A1 (en) * 2012-08-27 2015-07-09 Ludwig-Maximilians-Universität München Inhibitors of CD40-TRAF6 Interaction
CN108368535A (zh) * 2014-08-21 2018-08-03 欧陆迪斯卡沃爱克斯公司 用于测量配体与靶标蛋白质的结合和细胞接合的方法
CN111100902A (zh) * 2018-10-26 2020-05-05 复旦大学 一种基于sim靶点抗kshv/ebv相关肿瘤小分子化合物筛选方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NI XUHAO, KOU WEI, GU JIAN, WEI PING, WU XIAO, PENG HAO, TAO JINHUI, YAN WEI, YANG XIAOPING, LEBID ANDRIANA, PARK BENJAMIN V, CHEN: "TRAF6 directs FOXP3 localization and facilitates regulatory T-cell function through K63-linked ubiquitination", THE EMBO JOURNAL / EUROPEAN MOLECULAR BIOLOGY ORGANIZATION, IRL PRESS, OXFORD, vol. 38, no. 9, 2 May 2019 (2019-05-02), Oxford , XP093071959, ISSN: 0261-4189, DOI: 10.15252/embj.201899766 *

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