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CN118895121A - A kit and method for detecting or screening PARP2/PARP3 inhibitors - Google Patents

A kit and method for detecting or screening PARP2/PARP3 inhibitors Download PDF

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CN118895121A
CN118895121A CN202410948763.2A CN202410948763A CN118895121A CN 118895121 A CN118895121 A CN 118895121A CN 202410948763 A CN202410948763 A CN 202410948763A CN 118895121 A CN118895121 A CN 118895121A
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parp3
parp2
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曹东升
邓友超
魏闯
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Central South University
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    • C09K2211/1088Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom

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Abstract

The invention provides a kit and a method for detecting or screening PARP2/PARP3 inhibitors, wherein the kit comprises a fluorescent probe with a structural formula shown as A 4 or A 5 and PARP2 protein and/or PARP3 protein. According to the invention, the combination of the fluorescent probe and the PARP2/PARP3 protein is used as a substrate, then a compound to be screened competes with the fluorescent probe for the binding site of the PARP2/PARP3 protein, the fluorescence polarization value of a detection system is detected, the active compound shows a lower fluorescence polarization value, and the inactive compound shows a higher fluorescence polarization value. The kit and the corresponding method can simply and rapidly screen out a large amount of active PARP2/PARP3 inhibitors, and provide effective means and useful tools for discovering and developing lead compounds and effective medicaments for preventing and treating cancers.

Description

一种用于检测或筛选PARP2/PARP3抑制剂的试剂盒及方法A kit and method for detecting or screening PARP2/PARP3 inhibitors

技术领域Technical Field

本发明涉及生物医药技术领域,特别涉及一种用于检测或筛选PARP2/PARP3抑制剂的试剂盒及方法。The present invention relates to the field of biomedicine technology, and in particular to a kit and method for detecting or screening PARP2/PARP3 inhibitors.

背景技术Background Art

聚ADP核糖聚合酶(PARP)蛋白质超家族通过将NAD+合成的ADP~核糖基团转移至靶蛋白从而影响各种细胞过程,调节广泛的生物途径;在DNA损伤修复、维持基因组稳定性、调节信号通路等方面发挥着十分重要的作用。PARP2/PARP3是PARP家族中的成员,在DNA损伤修复、细胞凋亡、染色质重塑等生物学过程中发挥着不可替代作用。最近的研究表明,PARP2/PARP3在多种疾病中的过表达也引起了研究者的关注。PARP2还调节表观遗传、增殖和炎症过程,对精原细胞、胸腺和脂肪组织的发育很重要。PARP3则在癌症中的治疗优势揭示了其特异性,表明PARP3是治疗高度侵袭性乳腺癌的精准医学方法中一个突出的有益靶点。PARP3在乳腺癌中促进转化生长因子β(TGFβ)诱导的上皮间质转化(Epithelial-Mesenchymal Transition)及mTORC2讯息传递,使得肿瘤侵袭能力增强,肿瘤侵袭能力与PARP3高表达的关联性说明PARP3能作为高度侵袭性乳癌精准医学中一个重要的有益靶点。在此基础上,PARP3的抑制也揭示了通过增加有丝分裂缺陷和诱导细胞死亡,使转移性乳腺癌对长春花生物碱敏感的前景。与此相一致的是,在原发性胶质母细胞瘤活检中检测到PARP3水平上调。此外,在胶质母细胞瘤细胞模型中沉默PARP3的表达可以有效地增强放射治疗的细胞毒性影响,这表明PARP3可能是克服这些原发性恶性脑肿瘤放射耐药的潜在靶点。The poly ADP ribose polymerase (PARP) protein superfamily affects various cellular processes and regulates a wide range of biological pathways by transferring the ADP-ribose group synthesized by NAD + to target proteins; it plays a very important role in DNA damage repair, maintaining genome stability, and regulating signaling pathways. PARP2/PARP3 is a member of the PARP family and plays an irreplaceable role in biological processes such as DNA damage repair, cell apoptosis, and chromatin remodeling. Recent studies have shown that the overexpression of PARP2/PARP3 in a variety of diseases has also attracted the attention of researchers. PARP2 also regulates epigenetic, proliferation, and inflammatory processes, and is important for the development of spermatogonia, thymus, and adipose tissue. The therapeutic advantages of PARP3 in cancer reveal its specificity, indicating that PARP3 is a prominent and beneficial target in the precision medicine approach for the treatment of highly aggressive breast cancer. PARP3 promotes transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transition and mTORC2 signaling in breast cancer, which enhances tumor invasiveness. The correlation between tumor invasiveness and high PARP3 expression indicates that PARP3 can serve as an important and beneficial target in precision medicine for highly invasive breast cancer. On this basis, inhibition of PARP3 also reveals the prospect of sensitizing metastatic breast cancer to vinca alkaloids by increasing mitotic defects and inducing cell death. Consistent with this, upregulated PARP3 levels were detected in primary glioblastoma biopsies. In addition, silencing PARP3 expression in glioblastoma cell models can effectively enhance the cytotoxic effects of radiotherapy, suggesting that PARP3 may be a potential target for overcoming the radioresistance of these primary malignant brain tumors.

自2014年PARP1抑制剂奥拉帕尼问世以来,全球已上市六款PARP抑制剂,分别是奥拉帕尼、鲁卡帕利、尼拉帕利、他拉唑帕利、氟唑帕利和帕米帕利。此外,在过去的五年中,用于癌症治疗的PARP靶向调节剂经历了快速增长。除了被批准上市的PARP抑制剂外,许多PARP抑制剂正在临床试验中评估实体瘤的治疗,包括小细胞肺癌、前列腺癌、胰腺癌和胃癌。总之,PARP2/PARP3抑制剂是一类具有多种作用机制的靶向药物,在癌症治疗中显示出了广阔的应用前景。为了寻找有效的先导化合物,需要开发简单有效的体外测试PARP2/PARP3抑制剂活性的方法,实现药物筛选的快速、高效、微量化、自动化和规模化。Since the advent of the PARP1 inhibitor olaparib in 2014, six PARP inhibitors have been launched worldwide, namely olaparib, rucaparib, niraparib, talazoparib, fluzoparib and pamiparib. In addition, PARP targeted modulators for cancer treatment have experienced rapid growth in the past five years. In addition to the approved PARP inhibitors, many PARP inhibitors are being evaluated in clinical trials for the treatment of solid tumors, including small cell lung cancer, prostate cancer, pancreatic cancer and gastric cancer. In short, PARP2/PARP3 inhibitors are a class of targeted drugs with multiple mechanisms of action, showing broad application prospects in cancer treatment. In order to find effective lead compounds, it is necessary to develop simple and effective methods for testing the activity of PARP2/PARP3 inhibitors in vitro to achieve rapid, efficient, micro-quantified, automated and scalable drug screening.

发明内容Summary of the invention

基于此,本发明提供了一种荧光探针在制备用于检测或筛选PARP2/PARP3抑制剂的产品中的应用,所述荧光探针的结构式如A4或A5所示:Based on this, the present invention provides an application of a fluorescent probe in the preparation of a product for detecting or screening PARP2/PARP3 inhibitors, wherein the structural formula of the fluorescent probe is shown in A4 or A5 :

在其中一个实施例中,所述产品为试剂盒、检测芯片或检测试纸。In one embodiment, the product is a test kit, a detection chip or a detection test strip.

本发明还提供了一种用于检测或筛选PARP2/PARP3抑制剂的试剂盒,包括上述荧光探针,以及PARP2蛋白和PARP3蛋白中的至少一种。The present invention also provides a kit for detecting or screening PARP2/PARP3 inhibitors, comprising the above-mentioned fluorescent probe, and at least one of PARP2 protein and PARP3 protein.

在其中一个实施例中,试剂盒还包括DMSO和Tris缓冲液。In one embodiment, the kit further comprises DMSO and Tris buffer.

本发明还提供了一种检测或筛选PARP2/PARP3抑制剂的方法,包括以下步骤:The present invention also provides a method for detecting or screening PARP2/PARP3 inhibitors, comprising the following steps:

将上述荧光探针和PARP2或PARP3蛋白在缓冲液中进行孵育得到缓冲溶液A,然后检测荧光偏振值,得到标准偏振值;The fluorescent probe and PARP2 or PARP3 protein are incubated in a buffer to obtain a buffer solution A, and then the fluorescence polarization value is detected to obtain a standard polarization value;

在所述缓冲溶液A中加入待筛选化合物与所述荧光探针竞争PARP2或PARP3蛋白的结合位点,室温下避光孵育,然后检测荧光偏振值,得到目标偏振值;Adding the compound to be screened to the buffer solution A to compete with the fluorescent probe for the binding site of the PARP2 or PARP3 protein, incubating at room temperature in the dark, and then detecting the fluorescence polarization value to obtain the target polarization value;

根据所述标准偏振值和目标偏振值的大小,判断所述待筛选化合物是否具有PARP2或PARP3抑制活性。According to the size of the standard polarization value and the target polarization value, it is judged whether the compound to be screened has PARP2 or PARP3 inhibitory activity.

在其中一个实施例中,所述荧光探针的工作浓度为5~50nM,所述PARP2或PARP3蛋白的工作浓度为10~1000nM。In one embodiment, the working concentration of the fluorescent probe is 5 to 50 nM, and the working concentration of the PARP2 or PARP3 protein is 10 to 1000 nM.

在其中一个实施例中,包括以下步骤:配制不同浓度的待筛选化合物溶液与所述缓冲溶液A混合,室温下避光孵育,检测荧光偏振值,以检测的荧光偏振值为纵坐标,以所述待筛选化合物的浓度的对数值为横坐标,绘制所述待筛选化合物的抑制曲线,计算得到所述待筛选化合物的Ki值,从而量化所述待筛选化合物的PARP2或PARP3抑制活性。In one embodiment, the method comprises the following steps: preparing solutions of the compounds to be screened of different concentrations and mixing them with the buffer solution A, incubating them in the dark at room temperature, detecting the fluorescence polarization value, plotting the inhibition curve of the compounds to be screened with the detected fluorescence polarization value as the ordinate and the logarithm of the concentration of the compounds to be screened as the abscissa, and calculating the K i value of the compounds to be screened, thereby quantifying the PARP2 or PARP3 inhibitory activity of the compounds to be screened.

在其中一个实施例中,所述待筛选化合物溶液的最大浓度为10μM,以3倍浓度梯度稀释从而得到不同浓度的待筛选化合物溶液。In one embodiment, the maximum concentration of the solution of the compound to be screened is 10 μM, and the solution is diluted in a 3-fold concentration gradient to obtain solutions of the compound to be screened with different concentrations.

在其中一个实施例中,所述缓冲液含有50mM Tris HCl、150mM NaCl,pH为8。In one embodiment, the buffer contains 50 mM Tris HCl, 150 mM NaCl, and has a pH of 8.

在其中一个实施例中,所述PARP2或PARP3蛋白通过大肠杆菌原核表达的方法制备得到。In one embodiment, the PARP2 or PARP3 protein is prepared by prokaryotic expression in Escherichia coli.

本发明的上述方案具有如下的有益效果:The above solution of the present invention has the following beneficial effects:

本发明利用含有奥拉帕尼的部分结构且在其N端连接荧光团的荧光探针,其与PARP2/PARP3的相互作用受到抑制剂的影响时产生荧光偏振强度的变化,从而用于筛选PARP2/PARP3抑制剂。具体通过荧光探针和PARP2/PARP3蛋白结合成较大复合物,使其具有较大的荧光偏振值,然后用待筛选化合物竞争性置换荧光探针,使荧光偏振(mP)值减小。活性化合物在本筛选方法中表现出较低的mP值,非活性化合物则表现较高的mP值,以多功能酶标仪检测实验体系的mP值即可得知待筛选化合物的PARP2/PARP3抑制活性高低。The present invention utilizes a fluorescent probe containing a partial structure of olaparib and having a fluorophore connected to its N-terminus. When the interaction between the fluorescent probe and PARP2/PARP3 is affected by an inhibitor, a change in fluorescence polarization intensity is produced, thereby being used to screen PARP2/PARP3 inhibitors. Specifically, the fluorescent probe and PARP2/PARP3 protein are combined into a larger complex so that it has a larger fluorescence polarization value, and then the fluorescent probe is competitively replaced by the compound to be screened, so that the fluorescence polarization (mP) value is reduced. Active compounds show lower mP values in this screening method, while inactive compounds show higher mP values. The PARP2/PARP3 inhibitory activity of the compound to be screened can be determined by detecting the mP value of the experimental system with a multifunctional microplate reader.

本发明基于上述荧光探针的PARP2/PARP3抑制剂高通量筛选方法适用于以PARP2/PARP3为靶标的抗癌药物的快速筛选与药理活性的体外评价,且具有均相反应、操作简便、检测灵敏、成本低廉、稳定可靠等优点,在药物筛选中具有操作简便、筛选快速、稳定可靠、成本低廉等优势,非常适合大规模的药物高通量筛选,还可以与其他筛选模型联合使用,快速发现更有效的新PARP2/PARP3小分子抑制剂。The high-throughput screening method for PARP2/PARP3 inhibitors based on the fluorescent probe of the present invention is suitable for rapid screening of anticancer drugs targeting PARP2/PARP3 and in vitro evaluation of pharmacological activity, and has the advantages of homogeneous reaction, simple operation, sensitive detection, low cost, stability and reliability, etc. It has the advantages of simple operation, rapid screening, stability and reliability, low cost, etc. in drug screening, and is very suitable for large-scale high-throughput drug screening. It can also be used in combination with other screening models to quickly discover more effective new PARP2/PARP3 small molecule inhibitors.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1为本发明实施例1中制备PARP2蛋白所使用的原核表达载体的图谱;FIG1 is a diagram of a prokaryotic expression vector used to prepare PARP2 protein in Example 1 of the present invention;

图2为本发明实施例1中制备的PARP2蛋白的SDS-PAGE检测结果,其中UB为切掉后的纯PARP2蛋白;FIG2 is the SDS-PAGE detection result of the PARP2 protein prepared in Example 1 of the present invention, wherein UB is the pure PARP2 protein after cleavage;

图3为本发明实施例1中荧光探针A5的荧光强度随浓度变化图;FIG3 is a graph showing the fluorescence intensity of the fluorescent probe A5 as a function of concentration in Example 1 of the present invention;

图4为本发明实施例1中PARP2蛋白质与荧光探针A5结合的曲线图和结合强度Kd值;FIG4 is a curve diagram of the binding of PARP2 protein to fluorescent probe A5 and the binding strength Kd value in Example 1 of the present invention;

图5为本发明实施例1中验证阳性药物奥拉帕尼的抑制曲线图以及计算对PARP2抑制活性的Ki值(荧光探针A5);FIG5 is an inhibition curve diagram of Olaparib, a positive drug verified in Example 1 of the present invention, and a calculated K i value of PARP2 inhibition activity (fluorescent probe A 5 );

图6为本发明实施例2中制备PARP3蛋白所使用的原核表达载体的图谱;FIG6 is a diagram of the prokaryotic expression vector used to prepare the PARP3 protein in Example 2 of the present invention;

图7为本发明实施例2中制备的PARP3蛋白的SDS-PAGE检测结果,其中Elu及附近泳道为酶切后的纯PARP3蛋白;FIG7 is the SDS-PAGE detection result of the PARP3 protein prepared in Example 2 of the present invention, wherein Elu and the adjacent lanes are pure PARP3 proteins after enzyme cleavage;

图8为本发明实施例2中PARP3蛋白质与荧光探针A5结合的曲线图和结合强度Kd值;FIG8 is a curve diagram of the binding of PARP3 protein to fluorescent probe A5 and the binding strength Kd value in Example 2 of the present invention;

图9为本发明实施例2中验证阳性药物奥拉帕尼的抑制曲线图以及计算对PARP3抑制活性的Ki值(荧光探针A5)。FIG. 9 is an inhibition curve diagram of Olaparib, a positive drug verified in Example 2 of the present invention, and the calculated K i value of PARP3 inhibition activity (fluorescent probe A 5 ).

具体实施方式DETAILED DESCRIPTION

为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合附图及具体实施例进行详细描述。显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

此外,下面所描述的本发明不同实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互结合。实施例中采用的试剂和仪器如非特别说明,均为本领域常规选择。实施例中未注明具体条件的实验方法,按照常规条件,例如文献、书本中所述的条件或者生产厂家推荐的方法实现。In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. The reagents and instruments used in the examples are conventionally selected in the art unless otherwise specified. The experimental methods without specific conditions in the examples are carried out according to conventional conditions, such as the conditions described in the literature, books or methods recommended by the manufacturer.

本发明提供了荧光探针在制备用于检测或筛选PARP2/PARP3抑制剂的产品中的应用,荧光探针的结构式如A4或A5所示:The present invention provides the use of a fluorescent probe in the preparation of a product for detecting or screening PARP2/PARP3 inhibitors. The structural formula of the fluorescent probe is shown in A4 or A5 :

本发明通过荧光探针和PARP2/PARP3蛋白结合成较大复合物,使其具有较大的荧光偏振值,然后用待筛选化合物竞争性置换荧光探针,使荧光偏振(mP)值减小。活性化合物在本筛选方法中表现出较低的mP值,非活性化合物则表现较高的mP值,以多功能酶标仪检测实验体系的mP值即可得知待筛选化合物的PARP2/PARP3抑制活性高低。The present invention combines the fluorescent probe and PARP2/PARP3 protein into a larger complex, so that it has a larger fluorescence polarization value, and then uses the compound to be screened to competitively replace the fluorescent probe to reduce the fluorescence polarization (mP) value. The active compound shows a lower mP value in this screening method, while the inactive compound shows a higher mP value. The PARP2/PARP3 inhibitory activity of the compound to be screened can be determined by detecting the mP value of the experimental system with a multifunctional microplate reader.

荧光偏振(FP)检测技术是一种以荧光标记的检测技术,用于测定小分子与蛋白质亲和力,通过检测样品孔中荧光偏振值的大小来确定小分子和蛋白质亲和力的大小。通过让两种化合物与蛋白相竞争结合,当反应液中待筛选的化合物没有抑制活性时,荧光探针与蛋白相结合形成复合物,由于复合物分子量大,受偏振光激发后具有较高的偏振值;当反应液中待筛选化合物有抑制活性时,因二者竞争蛋白的结合位点,结合到蛋白上的荧光探针减少,由于游离的荧光探针分子量较小,因此偏振值较小。待筛选的化合物的抑制活性越强,与荧光探针的竞争越激烈,对产生的激发光的性质影响越大,因此偏振值的改变直接与待筛选化合物的抑制活性相关,利用这一原理可以检测待筛选化合物的活性。Fluorescence polarization (FP) detection technology is a fluorescent-labeled detection technology used to determine the affinity between small molecules and proteins. The affinity between small molecules and proteins is determined by detecting the size of the fluorescence polarization value in the sample well. By allowing the two compounds to compete with the protein for binding, when the compound to be screened in the reaction solution has no inhibitory activity, the fluorescent probe combines with the protein to form a complex. Due to the large molecular weight of the complex, it has a higher polarization value after being excited by polarized light; when the compound to be screened in the reaction solution has inhibitory activity, the two compete for the binding site of the protein, and the fluorescent probe bound to the protein is reduced. Since the molecular weight of the free fluorescent probe is small, the polarization value is small. The stronger the inhibitory activity of the compound to be screened, the more intense the competition with the fluorescent probe, and the greater the impact on the properties of the generated excitation light. Therefore, the change in polarization value is directly related to the inhibitory activity of the compound to be screened. This principle can be used to detect the activity of the compound to be screened.

本发明一实施例的用于检测或筛选PARP2/PARP3抑制剂的试剂盒,包括上述荧光探针A4或A5,以及PARP2蛋白和PARP3蛋白中的至少一种。A kit for detecting or screening PARP2/PARP3 inhibitors according to an embodiment of the present invention comprises the fluorescent probe A4 or A5 , and at least one of PARP2 protein and PARP3 protein.

在一个具体示例中,试剂盒还可以包括DMSO和Tris缓冲液等其他常用的试剂。In a specific example, the kit may also include other commonly used reagents such as DMSO and Tris buffer.

本发明一实施例的检测或筛选PARP2/PARP3抑制剂的方法,包括以下步骤:A method for detecting or screening PARP2/PARP3 inhibitors according to an embodiment of the present invention comprises the following steps:

S1、将上述荧光探针和PARP2或PARP3蛋白在缓冲液中进行孵育得到缓冲溶液A,然后检测荧光偏振值,得到标准偏振值;S1, incubating the fluorescent probe and PARP2 or PARP3 protein in a buffer to obtain a buffer solution A, and then detecting the fluorescence polarization value to obtain a standard polarization value;

S2、在缓冲溶液A中加入待筛选化合物与荧光探针竞争PARP2或PARP3蛋白的结合位点,室温下避光孵育,然后检测荧光偏振值,得到目标偏振值;S2, adding the compound to be screened to the buffer solution A to compete with the fluorescent probe for the binding site of the PARP2 or PARP3 protein, incubating at room temperature in the dark, and then detecting the fluorescence polarization value to obtain the target polarization value;

S3、根据标准偏振值和目标偏振值的大小,判断待筛选化合物是否具有PARP2或PARP3抑制活性。S3. Determine whether the compound to be screened has PARP2 or PARP3 inhibitory activity based on the standard polarization value and the target polarization value.

优选的,荧光探针的工作浓度为5~50nM,PARP2或PARP3蛋白的工作浓度为10~1000nM,在此浓度范围内检测效果更好。Preferably, the working concentration of the fluorescent probe is 5 to 50 nM, and the working concentration of PARP2 or PARP3 protein is 10 to 1000 nM. The detection effect is better within this concentration range.

优选的,荧光探针和PARP2/PARP3蛋白在缓冲液中于室温下孵育1~5分钟,缓冲溶液A中加入待筛选化合物后室温下避光孵育5~30分钟。Preferably, the fluorescent probe and PARP2/PARP3 protein are incubated in a buffer at room temperature for 1 to 5 minutes, and the compound to be screened is added to the buffer solution A and incubated at room temperature in the dark for 5 to 30 minutes.

在一个具体示例中,方法包括以下步骤:配制不同浓度的待筛选化合物溶液与缓冲溶液A混合,室温下避光孵育,检测荧光偏振值,以检测的荧光偏振值为纵坐标,以待筛选化合物的浓度的对数值为横坐标,绘制待筛选化合物的抑制曲线,拟合得到待筛选化合物的Ki值(Ki=IC50/(1+[S]/Km),Ki是抑制剂的结合亲和力,IC50是抑制剂的功能强度;[S]为固定的底物浓度,Km为酶促反应速度达到最大反应速度一半时所对应的底物浓度,是酶的特征常数。如果底物浓度远小于Km值,IC50等于Ki),从而量化待筛选化合物的PARP2/PARP3抑制活性。绘制待筛选化合物的抑制曲线的步骤中,其抑制率的计算公式如下:In a specific example, the method includes the following steps: preparing solutions of the compound to be screened of different concentrations and mixing them with buffer solution A, incubating at room temperature in the dark, detecting the fluorescence polarization value, plotting the inhibition curve of the compound to be screened with the detected fluorescence polarization value as the ordinate and the logarithmic value of the concentration of the compound to be screened as the abscissa, and fitting to obtain the K i value of the compound to be screened (K i =IC50/(1+[S]/K m ), K i is the binding affinity of the inhibitor, IC 50 is the functional strength of the inhibitor; [S] is a fixed substrate concentration, K m is the substrate concentration corresponding to the enzyme reaction rate reaching half of the maximum reaction rate, which is the characteristic constant of the enzyme. If the substrate concentration is much smaller than the K m value, IC 50 is equal to K i ), thereby quantifying the PARP2/PARP3 inhibitory activity of the compound to be screened. In the step of plotting the inhibition curve of the compound to be screened, the calculation formula of the inhibition rate is as follows:

在一个具体示例中,待筛选化合物溶液的最大浓度为10μM,以3倍浓度梯度稀释从而得到不同浓度的待筛选化合物溶液。In a specific example, the maximum concentration of the solution of the compound to be screened is 10 μM, and the solution is diluted in a 3-fold concentration gradient to obtain solutions of the compound to be screened with different concentrations.

可选的,PARP2/PARP3蛋白通过大肠杆菌原核表达的方法制备得到,但不限于此,也可以直接通过商业渠道购买得到。Optionally, the PARP2/PARP3 protein is prepared by prokaryotic expression in Escherichia coli, but is not limited thereto and can also be purchased directly through commercial channels.

在一个具体示例中,大肠杆菌原核表达制备PARP2/PARP3蛋白的方法包括以下步骤:将表达质粒从DH5α菌株中提取出来,导入表达菌株BL21 DE3感受态细胞中进行表达,以Ni-NTA亲和层析柱分离纯化PARP2/PARP3蛋白。In a specific example, the method for preparing PARP2/PARP3 protein by prokaryotic expression in Escherichia coli includes the following steps: extracting the expression plasmid from the DH5α strain, introducing it into the expression strain BL21 DE3 competent cells for expression, and separating and purifying the PARP2/PARP3 protein with a Ni-NTA affinity chromatography column.

以下为具体实施例。The following are specific embodiments.

实施例1Example 1

1、用于分析检测和筛选PARP2/PARP3抑制剂的荧光探针A4或A5可经过下述合成路线制备获得:1. The fluorescent probe A4 or A5 for analyzing, detecting and screening PARP2/PARP3 inhibitors can be prepared by the following synthetic route:

准备溶液A:将0.043mmol的5-FAM或者3-氟硼吡咯-丙酸(BDP FLacid),0.129mmol的N,N-二异丙基乙胺(DIPEA)和0.072mmol的六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(PyBOP)溶解在2.5ml N,N-二甲基甲酰胺(DMF)中,室温下搅拌1小时。准备溶液B:将0.052mmol的化合物4-(4-氟-3-(哌嗪-1-羰基)苄基)酞嗪-1(2H)-酮即A3溶解在2.5ml的N,N-二甲基甲酰胺中。然后,将溶液A缓慢滴入溶液B中,并在室温下搅拌过夜,用TLC监测反应。反应完成后,先用石油醚洗涤3次,后加入去离子水并抽滤得到沉淀,再用甲醇洗涤样品并离心得到粗产物A4或者A5,粗产物经硅胶柱分离得纯品A4或者A5Prepare solution A: Dissolve 0.043 mmol of 5-FAM or 3-fluoroboropyrrole-propionic acid (BDP FLacid), 0.129 mmol of N,N-diisopropylethylamine (DIPEA) and 0.072 mmol of benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBOP) in 2.5 ml of N,N-dimethylformamide (DMF) and stir at room temperature for 1 hour. Prepare solution B: Dissolve 0.052 mmol of compound 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, i.e. A3, in 2.5 ml of N,N-dimethylformamide. Then, slowly drop solution A into solution B and stir at room temperature overnight, and monitor the reaction by TLC. After the reaction is completed, the sample is first washed with petroleum ether for three times, then deionized water is added and filtered to obtain a precipitate, and then the sample is washed with methanol and centrifuged to obtain a crude product A4 or A5 . The crude product is separated by a silica gel column to obtain a pure product A4 or A5 .

上述制备方法中的荧光素(5-FAM或3-氟硼吡咯-丙酸)、4-(4-氟-3-(哌嗪-1-羰基)苄基)酞嗪-1(2H)-酮、六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(PyBOP)、有机碱N,N-二异丙基乙胺(DIPEA)、溶剂N,N-二甲基甲酰胺(DMF)均可通过商业途径购买获得。Fluorescein (5-FAM or 3-fluoroboropyrrole-propionic acid), 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazine-1(2H)-one, benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBOP), organic base N,N-diisopropylethylamine (DIPEA), and solvent N,N-dimethylformamide (DMF) in the above preparation method can all be purchased through commercial channels.

2、PARP2的原核表达与分离纯化2. Prokaryotic expression and purification of PARP2

如图1所示为原核表达载体的图谱,利用大肠杆菌原核表达技术,将表达质粒从DH5α菌株中提取出来,导入到蛋白表达菌株BL21 DE3感受态细胞中,以卡那霉素抗性法筛选重组子。将重组子接种到1升LB液体培养基(含50μg/mL卡那霉素)中,37℃培养4h,加入0.1mM IPTG,23℃过夜引发。菌体以超声波法破碎后,裂解上清液,再以Ni-NTA亲和层析柱分离纯化,纯化的PARP2表观分子量为40kDa,如图2所示为SDS-PAGE检测结果。As shown in Figure 1, the map of the prokaryotic expression vector is shown. The expression plasmid is extracted from the DH5α strain using the E. coli prokaryotic expression technology and introduced into the protein expression strain BL21 DE3 competent cells. The recombinants are screened by the kanamycin resistance method. The recombinants are inoculated into 1 liter of LB liquid culture medium (containing 50 μg/mL kanamycin), cultured at 37°C for 4 hours, and 0.1 mM IPTG is added to initiate overnight at 23°C. After the bacteria are broken by ultrasonication, the supernatant is lysed and then separated and purified by Ni-NTA affinity chromatography column. The apparent molecular weight of the purified PARP2 is 40 kDa. The results of SDS-PAGE detection are shown in Figure 2.

3、确定荧光探针在体系中使用浓度及缓冲溶液的选择:3. Determine the concentration of fluorescent probe used in the system and the choice of buffer solution:

荧光探针在使用前需要冷冻干燥过夜,后配制成10mM的溶液备用。将荧光探针A5进行3倍梯度稀释:96孔板一排12个孔,第一个孔加入5μL 100μM A5,在第2~12个孔加入100μL的DMSO,后取50μL的100μM的探针A5加入到第二个孔,用移液枪上下吹打10次使其混匀,从第二个孔中取出50μL加入到第三个孔,上下吹打10次使其混匀,并从第三个孔取出50μL重复上述操作。操作到第十一个孔时,上下吹打10次后取出50μl液体丢弃,使得第12个孔里是纯DMSO,这样就可得到3倍梯度稀释的探针A5。将梯度稀释后的探针A5稀释100倍:从96孔板的12个孔中分别取出1μL液体加入到384孔板中的横排奇数孔中(1、3、5、7、9、11、13、15、17、19、21、23),后加入99μL缓冲液,使探针A5浓度稀释100倍成为1μM。(取1μL液体加入384孔板时,枪头贴住小孔底部一端,加入后小孔会从正方形变成圆形)。在多功能酶标仪上选择Fluorescene Intensity,测量其荧光强度数值。再在酶标仪上选择Fluorescencepolarization,测量其mP(荧光偏振)值。(plate选择384flat black,发射波长选择485nm,吸收选择535nm,Fluorophore选择DAPI,Z-position选择Calcaμlate from well(DMSO空白对照)。根据图3的数据,我们优先选择荧光探针使用浓度5~50nM。The fluorescent probe needs to be freeze-dried overnight before use, and then prepared into a 10mM solution for standby use. Perform a 3-fold gradient dilution of the fluorescent probe A 5 : There are 12 wells in a row of a 96-well plate. Add 5μL of 100μM A 5 to the first well, add 100μL of DMSO to the 2nd to 12th wells, then take 50μL of 100μM probe A 5 and add it to the second well. Use a pipette to blow up and down 10 times to mix it. Take out 50μL from the second well and add it to the third well. Blow up and down 10 times to mix it, and take out 50μL from the third well and repeat the above operation. When operating to the eleventh well, blow up and down 10 times and take out 50μl of liquid and discard it, so that the 12th well contains pure DMSO, so that a 3-fold gradient dilution of probe A 5 can be obtained. Dilute the gradient diluted probe A 5 100 times: Take 1 μL of liquid from each of the 12 wells of the 96-well plate and add it to the odd-numbered wells in the horizontal row of the 384-well plate (1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23), and then add 99 μL of buffer to dilute the probe A 5 concentration 100 times to 1 μM. (When taking 1 μL of liquid and adding it to the 384-well plate, the tip of the gun should be placed against one end of the bottom of the well. After adding, the well will change from a square to a circle). Select Fluorescene Intensity on the multifunctional microplate reader and measure its fluorescence intensity value. Then select Fluorescencepolarization on the microplate reader and measure its mP (fluorescence polarization) value. (Plate selects 384 flat black, emission wavelength selects 485 nm, absorption selects 535 nm, Fluorophore selects DAPI, and Z-position selects Calcaμlate from well (DMSO blank control). Based on the data in Figure 3, we prefer to use fluorescent probes at a concentration of 5 to 50 nM.

4、确定PARP2蛋白最佳使用浓度:4. Determine the optimal concentration of PARP2 protein:

与前述操作相同,在96孔板中稀释蛋白浓度,先在96孔板的2~12孔加入100μL的缓冲液,后取50μL的蛋白加入2号孔,与前述稀释操作类似,以3倍浓度梯度稀释蛋白溶液。后取荧光探针A5加入384孔板(1、3、5、7、9、11、13、15、17、19、21、23)孔中,再从对应的96孔板取出99μL的蛋白溶液加入,使得荧光探针A5的最终浓度为5~50nM。在酶标仪中测量其偏振值,从而计算其Kd值(在酶标仪上选择Fluorescence polarisation)。避光5~30分钟后测量其mP值(荧光偏振值),后计算其Kd值(图4),确定选择PARP2蛋白的使用浓度为10~1000nM。As in the previous operation, dilute the protein concentration in a 96-well plate. First, add 100 μL of buffer to wells 2 to 12 of the 96-well plate, then take 50 μL of protein and add it to well 2. Similar to the previous dilution operation, dilute the protein solution in a 3-fold concentration gradient. Then take the fluorescent probe A 5 and add it to the wells (1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23) of the 384-well plate, and then take 99 μL of protein solution from the corresponding 96-well plate and add it, so that the final concentration of the fluorescent probe A 5 is 5 to 50 nM. Measure its polarization value in an ELISA reader to calculate its K d value (select Fluorescence polarisation on the ELISA reader). After 5 to 30 minutes of light protection, measure its mP value (fluorescence polarization value), and then calculate its K d value (Figure 4), and determine that the concentration of the selected PARP2 protein is 10 to 1000 nM.

5、金标准试验(对阳性药物奥拉帕尼进行活性测试):5. Gold standard test (activity test of positive drug Olaparib):

配置浓度为5~50nM荧光探针A5、浓度为10~1000nM蛋白质PARP2的缓冲溶液,将其定义为缓冲溶液A。3倍浓度梯度稀释奥拉帕尼(初始浓度为10mM):96孔板的一排12个孔,在第2~12个孔加入10μL的DMSO,后取5μL的10mM的奥拉帕尼加入到第二个孔,用移液枪上下吹打10次使其混匀,从第二个孔中取出5μL加入到第三个孔,上下吹打10次使其混匀,并从第三个孔取出5μL重复上述操作。操作到第十一个孔时,上下吹打10次后取出5μL液体丢弃,使得第12个孔里是纯DMSO。从96孔板的12个孔中分别取出1μL液体加入到384孔板中的横排奇数孔中(1、3、5、7、9、11、13、15、17、19、21、23),后加入99μL缓冲溶液A,使奥拉帕尼浓度稀释100倍,避光5~30分钟后测量其mP值(荧光偏振值),得到mP数据计算其Ki值,奥拉帕尼活性测试具体结果如图5所示,奥拉帕尼是一个FDA批准上市的PARP抑制剂,其对PARP2的抑制值Ki也是几个nM,这也侧面证实了建立的荧光偏振筛选方法具有良好的实用性和推广性。Prepare a buffer solution with a concentration of 5-50nM fluorescent probe A 5 and a concentration of 10-1000nM protein PARP2, which is defined as buffer solution A. 3-fold concentration gradient dilution of olaparib (initial concentration is 10mM): 12 wells in a row of 96-well plates, add 10μL of DMSO to the 2nd to 12th wells, then take 5μL of 10mM olaparib and add it to the second well, pipette up and down 10 times to mix it, take 5μL from the second well and add it to the third well, pipette up and down 10 times to mix it, and take 5μL from the third well and repeat the above operation. When operating to the eleventh well, pipette up and down 10 times and take out 5μL of liquid and discard it, so that the 12th well contains pure DMSO. 1 μL of liquid was taken out from 12 wells of the 96-well plate and added to the odd-numbered wells in the horizontal row of the 384-well plate (1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23), and then 99 μL of buffer solution A was added to dilute the concentration of olaparib 100 times. After protecting from light for 5 to 30 minutes, the mP value (fluorescence polarization value) was measured, and the K i value was calculated by obtaining the mP data. The specific results of the olaparib activity test are shown in Figure 5. Olaparib is a PARP inhibitor approved by the FDA, and its inhibition value K i for PARP2 is also several nM, which also indirectly confirms that the established fluorescence polarization screening method has good practicality and promotion.

6、PARP2抑制剂的荧光偏振高通量筛选的方法6. Fluorescence polarization high-throughput screening method for PARP2 inhibitors

待筛选化合物的原始浓度为10μM,取待筛选化合物1μL与缓冲溶液A(99μL/孔)加入到384孔板中,避光5~30分钟后测量其mP值。再取1μL的DMSO与缓冲溶液A(99μL/孔)加入到384孔板中,避光5~30分钟后测量其mP值,标记为标准mP值。当待筛选化合物的mP值小于标准mP值的一半时,我们认为该待筛选化合物具有一定的PARP2抑制活性。The original concentration of the compound to be screened is 10 μM. 1 μL of the compound to be screened and buffer solution A (99 μL/well) are added to a 384-well plate, and the mP value is measured after 5 to 30 minutes away from light. Then 1 μL of DMSO and buffer solution A (99 μL/well) are added to a 384-well plate, and the mP value is measured after 5 to 30 minutes away from light, and marked as the standard mP value. When the mP value of the compound to be screened is less than half of the standard mP value, we believe that the compound to be screened has a certain PARP2 inhibitory activity.

7、PARP2抑制剂活性评估的方法7. Methods for evaluating the activity of PARP2 inhibitors

化合物库待筛选化合物的原始浓度为10μM,将待筛选化合物进行3倍梯度稀释得到一组浓度梯度下降的待筛选化合物,取稀释后的一组待筛选化合物1μL与缓冲溶液A(99μL/孔)加入到384孔板中,避光5~30分钟后测量其mP值(重复3次),得到数据,计算其Ki值,可以量化该待筛选化合物对PARP2抑制活性大小。The original concentration of the compounds to be screened in the compound library is 10 μM. The compounds to be screened are diluted 3-fold to obtain a group of compounds to be screened with decreasing concentration gradients. 1 μL of the diluted group of compounds to be screened and buffer solution A (99 μL/well) are added to a 384-well plate, and the mP value is measured after 5 to 30 minutes in a dark place (repeated 3 times). The data is obtained and the Ki value is calculated, which can quantify the inhibitory activity of the compounds to be screened on PARP2.

实施例2Example 2

1、PARP3的原核表达与分离纯化1. Prokaryotic expression and purification of PARP3

如图6所示为原核表达载体的图谱,利用大肠杆菌原核表达技术,将表达质粒从DH5α菌株中提取出来,导入到蛋白表达菌株BL21 DE3感受态细胞中,以卡那霉素抗性法筛选重组子。将重组子接种到1升LB液体培养基(含50μg/mL卡那霉素)中,37℃培养4h,加入0.6mM IPTG,25℃过夜引发。菌体以超声波法破碎后,裂解上清液,再以Ni-NTA亲和层析柱分离纯化,纯化的PARP3表观分子量为67Kda,如图7所示为SDS-PAGE检测结果。As shown in Figure 6, the map of the prokaryotic expression vector is shown. The expression plasmid is extracted from the DH5α strain using the E. coli prokaryotic expression technology and introduced into the protein expression strain BL21 DE3 competent cells. The recombinants are screened by the kanamycin resistance method. The recombinants are inoculated into 1 liter of LB liquid culture medium (containing 50 μg/mL kanamycin), cultured at 37°C for 4 hours, and 0.6 mM IPTG is added to initiate at 25°C overnight. After the bacteria are broken by ultrasonication, the supernatant is lysed and then separated and purified by Ni-NTA affinity chromatography column. The apparent molecular weight of the purified PARP3 is 67Kda. The results of SDS-PAGE detection are shown in Figure 7.

2、确定PARP3蛋白最佳使用浓度2. Determine the optimal concentration of PARP3 protein

与前述操作相同,在96孔板中稀释蛋白浓度,先在96孔板的2~12孔加入100μL的缓冲液,后取50μL的蛋白加入2号孔,与前述稀释操作类似,以3倍浓度梯度稀释蛋白溶液。后取荧光探针A5加入384孔板(1、3、5、7、9、11、13、15、17、19、21、23)孔中,再从对应的96孔板取出99μL的蛋白溶液加入,使得荧光探针A5的最终浓度为5~50nM。在酶标仪中测量其偏振值,从而计算其Kd值(在酶标仪上选择Fluorescence polarisation)。避光5~30分钟后测量其mP值(荧光偏振值),后计算其Kd值(图8),确定选择PARP3蛋白的使用浓度为10~1000nM。As in the previous operation, dilute the protein concentration in a 96-well plate. First, add 100 μL of buffer to wells 2 to 12 of the 96-well plate, then take 50 μL of protein and add it to well 2. Similar to the previous dilution operation, dilute the protein solution in a 3-fold concentration gradient. Then take the fluorescent probe A 5 and add it to the wells (1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23) of the 384-well plate, and then take 99 μL of protein solution from the corresponding 96-well plate and add it, so that the final concentration of the fluorescent probe A 5 is 5 to 50 nM. Measure its polarization value in the microplate reader to calculate its K d value (select Fluorescence polarisation on the microplate reader). After 5 to 30 minutes of light protection, measure its mP value (fluorescence polarization value), and then calculate its K d value (Figure 8), and determine that the concentration of the selected PARP3 protein is 10 to 1000 nM.

3、金标准试验(对阳性药物奥拉帕尼进行活性测试)3. Gold standard test (activity test of positive drug Olaparib)

配置浓度为5~50nM荧光探针A5、浓度为10~1000nM蛋白质PARP3的缓冲溶液,将其定义为缓冲溶液A。3倍浓度梯度稀释奥拉帕尼(初始浓度为10mM):96孔板的一排12个孔,在第2~12个孔加入10μL的DMSO,后取5μL的10μM的奥拉帕尼加入到第二个孔,用移液枪上下吹打10次使其混匀,从第二个孔中取出5μL加入到第三个孔,上下吹打10次使其混匀,并从第三个孔取出5μL重复上述操作。操作到第十一个孔时,上下吹打10次后取出5μL液体丢弃,使得第12个孔里是纯DMSO。从96孔板的12个孔中分别取出1μL液体加入到384孔板中的横排奇数孔中(1、3、5、7、9、11、13、15、17、19、21、23),后加入99μL缓冲溶液A,使奥拉帕尼浓度稀释100倍,避光5~30分钟后测量其mP值(荧光偏振值),得到mP数据计算其Ki值,奥拉帕尼活性测试具体结果如图9所示,奥拉帕尼是一个FDA批准上市的PARP抑制剂,其对PARP3的抑制值Ki也是几十个nM,这也侧面证实了建立的荧光偏振筛选方法具有良好的实用性和推广性。Prepare a buffer solution with a concentration of 5-50nM fluorescent probe A 5 and a concentration of 10-1000nM protein PARP3, which is defined as buffer solution A. 3-fold concentration gradient dilution of olaparib (initial concentration is 10mM): 12 wells in a row of 96-well plates, add 10μL of DMSO to the 2nd to 12th wells, then take 5μL of 10μM olaparib and add it to the second well, pipette up and down 10 times to mix it, take 5μL from the second well and add it to the third well, pipette up and down 10 times to mix it, and take 5μL from the third well and repeat the above operation. When operating to the eleventh well, pipette up and down 10 times and take out 5μL of liquid and discard it, so that the 12th well contains pure DMSO. 1 μL of liquid was taken out from 12 wells of the 96-well plate and added to the odd-numbered wells in the horizontal row of the 384-well plate (1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23), and then 99 μL of buffer solution A was added to dilute the concentration of olaparib 100 times. After protecting from light for 5 to 30 minutes, the mP value (fluorescence polarization value) was measured, and the K i value was calculated by obtaining the mP data. The specific results of the olaparib activity test are shown in Figure 9. Olaparib is a PARP inhibitor approved by the FDA, and its inhibition value K i for PARP3 is also tens of nM, which also indirectly confirms that the established fluorescence polarization screening method has good practicality and promotion.

4、PARP3抑制剂的荧光偏振高通量筛选的方法4. Fluorescence polarization high-throughput screening method for PARP3 inhibitors

待筛选化合物的原始浓度为10μM,取待筛选化合物1μL与缓冲溶液A(99μL/孔)加入到384孔板中,避光5~30分钟后测量其mP值。再取1μL的DMSO与缓冲溶液A(99μL/孔)加入到384孔板中,避光5~30分钟后测量其mP值,标记为标准mP值。当待筛选化合物的mP值小于标准mP值的一半时,我们认为该待筛选化合物具有一定的活性。The original concentration of the compound to be screened is 10 μM. 1 μL of the compound to be screened and buffer solution A (99 μL/well) are added to a 384-well plate, and the mP value is measured after 5 to 30 minutes away from light. Then 1 μL of DMSO and buffer solution A (99 μL/well) are added to a 384-well plate, and the mP value is measured after 5 to 30 minutes away from light, and marked as the standard mP value. When the mP value of the compound to be screened is less than half of the standard mP value, we believe that the compound to be screened has a certain activity.

5、PARP3抑制剂活性评估的方法5. Methods for evaluating the activity of PARP3 inhibitors

化合物库待筛选化合物的原始浓度为10μM,将待筛选化合物进行3倍梯度稀释得到一组浓度梯度下降的待筛选化合物,取稀释后的一组待筛选化合物1μL与缓冲溶液A(99μL/孔)加入到384孔板中,避光5~30分钟后测量其mP值(重复3次),得到数据,计算其Ki值,可以量化该待筛选化合物对PARP3抑制活性大小。The original concentration of the compounds to be screened in the compound library is 10 μM. The compounds to be screened are diluted 3-fold to obtain a group of compounds to be screened with decreasing concentration gradients. 1 μL of the diluted group of compounds to be screened and buffer solution A (99 μL/well) are added to a 384-well plate, and the mP value is measured after 5 to 30 minutes in a dark place (repeated 3 times). The data is obtained and the Ki value is calculated, so as to quantify the inhibitory activity of the compounds to be screened on PARP3.

以上所述是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明所述原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (10)

1. Use of a fluorescent probe for the preparation of a product for detecting or screening PARP2/PARP3 inhibitors, wherein the fluorescent probe has the structural formula shown in a 4 or a 5:
2. The use according to claim 1, wherein the product is a kit, a test chip or a test strip.
3. A kit for detecting or screening for PARP2/PARP3 inhibitors, comprising the fluorescent probe of claim 1 and at least one of PARP2 protein and PARP3 protein.
4. The kit of claim 3, further comprising DMSO and Tris buffer.
5. A method of detecting or screening for PARP2/PARP3 inhibitors comprising the steps of:
Incubating the fluorescent probe according to claim 1 and PARP2 or PARP3 protein in a buffer solution to obtain a buffer solution a, and then detecting a fluorescence polarization value to obtain a standard polarization value;
Adding a compound to be screened and the fluorescent probe to compete for the binding site of PARP2 or PARP3 protein into the buffer solution A, incubating at room temperature in a dark place, and then detecting a fluorescence polarization value to obtain a target polarization value;
judging whether the compound to be screened has PARP2 or PARP3 inhibition activity according to the standard polarization value and the target polarization value.
6. The method of claim 5, wherein the working concentration of the fluorescent probe is 5-50 nM and the working concentration of the PARP2 or PARP3 protein is 10-1000 nM.
7. The method according to claim 5, comprising the steps of: preparing solutions of compounds to be screened with different concentrations, mixing the solutions with the buffer solution A, incubating the solutions at room temperature in a dark place, detecting fluorescence polarization values, taking the detected fluorescence polarization values as ordinate, taking the logarithmic value of the concentration of the compounds to be screened as abscissa, drawing an inhibition curve of the compounds to be screened, and calculating to obtain the K i value of the compounds to be screened, thereby quantifying the PARP2 or PARP3 inhibition activity of the compounds to be screened.
8. The method according to claim 7, wherein the maximum concentration of the compound solution to be screened is 10. Mu.M, and the compound solution to be screened is diluted in a concentration gradient of 3 times to obtain different concentrations of the compound solution to be screened.
9. The method of claim 5, wherein the buffer comprises 50mM Tris HCl, 150mM NaCl, pH 8.
10. The method of claim 5, wherein the PARP2 or PARP3 protein is prepared by a method of prokaryotic expression of escherichia coli.
CN202410948763.2A 2024-07-16 2024-07-16 A kit and method for detecting or screening PARP2/PARP3 inhibitors Pending CN118895121A (en)

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