WO2024011657A1 - 细胞分裂调控重要着丝粒蛋白cenp-n靶向抑制剂cenpenlin及其应用 - Google Patents
细胞分裂调控重要着丝粒蛋白cenp-n靶向抑制剂cenpenlin及其应用 Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/415—1,2-Diazoles
- A61K31/4162—1,2-Diazoles condensed with heterocyclic ring systems
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the invention belongs to the field of medicine, and specifically relates to Cenpenlin, a targeted inhibitor of CENP-N, an important centromere protein that regulates cell division, and its application.
- the centromere-associated network protein CCAN is a sixteen-membered complex that is responsible for the connection between the CENP-A nucleosome and the centromere spindle.
- CENP-N plays an important role in the complex.
- the cryo-EM structure of CCAN shows the interaction between CENP-N and other components of the CCAN complex.
- CENP-N binds CNEP-A in a different mode than classical octamer nucleosomes. Kinetochores mediate the connection of chromosomes to spindle microtubules.
- CENP-A Centromeres are clustered in specialized chromatin regions, and CENP-A recruits the CCAN complex composed of 16 subunits.
- CENP-C and CENP-N specifically recognize CENP-A.
- CENP-C binds to CENP-A nucleosomes through the central region and CENP-C motif.
- CENP-N recognizes L1 of CENP-A, which is the RG loop.
- Knl1-Mis12-Ndc80 (KMN) complex Provides an anchoring region for the Knl1-Mis12-Ndc80 (KMN) complex, mediating the regulation of attachment and sliding of cell cycle microtubules.
- KN Knl1-Mis12-Ndc80
- the protein machine based on the CENP-A nucleosome and CCAN kinetochore complex has evolved from lower organisms to various eukaryotes such as humans. Its protein machine composition has changed, but it is conserved in metazoans.
- centromere In yeast, the centromere is built on a 125-base pair DNA segment restricted to the nucleosome of Cse4 (CENP-A) and is called a punctate centromere. Most of the subunits of CCAN are recognized in organisms and are collectively known as the Ctf19 complex. The recent cryo-electron microscopy analysis of the complex structure of Ctf19 and Cse4 nucleosomes in Saccharomyces cerevisiae revealed the interaction mode between subunits.
- the object of the present invention is to provide new pharmaceutical uses of the compound represented by formula I, its isomers and pharmaceutically acceptable salts thereof, wherein the compound represented by formula I is named Cenpenlin,
- Said 1) specifically affects the interaction between CENP-N and CENP-W and/or CENP-L, and targets the assembly of centromere protein CENP-N into CCAN complex;
- the tumor cells are osteosarcoma cells, cervical cancer cells, and gastric cancer cells;
- the cancer may specifically be osteosarcoma cancer, cervical cancer and gastric cancer.
- the present invention also provides a method for affecting the interaction between CENP-N and CENP-W, which involves adding the compound represented by formula I in claim 1, its isomers and their pharmaceutical properties into a system containing CENP-N and CENP-W. Acceptable salt, culture, that's it.
- the present invention also provides a method for affecting the interaction between CENP-N and CENP-L, which involves adding the compound represented by formula I in claim 1, its isomers and their pharmaceutical properties into a system containing CENP-N and CENP-L. Acceptable salt, culture, that's it.
- the spatiotemporal sequence of the assembly of the kinetochore protein CENP-N into the CCAN complex remains unresolved. If inhibitors of CENP-N can pause the assembly of CCAN complexes, we will be able to analyze the function of CENP-N assembly into CCAN complexes and the spatiotemporal regulation mechanism of its assembly in a short period of time.
- Figure 1 shows the synthetic route of Cenpenlin small molecule.
- Figure 2 shows the NMR spectrum of the synthesized Cenpenlin small molecule.
- Figure 3 shows the cryo-electron microscopy structure and analysis of the human centromere protein machine CCAN.
- A is the cryo-electron microscopy structure of the human centromere protein machine CCAN complex;
- B is the gray solid structure showing the resolved CENP-N structure; pink
- the stick structure shows the small molecule and action space of Cenpenlin.
- Figure 5 shows the mitotic block caused by the small molecule compound Cenpenlin. Judging from the movement of chromosomes, Cenpenlin treatment will cause some of the chromosomes of the cells to never line up correctly, and it will also cause an obvious delay in mitosis. The cells have not yet entered anaphase 120 minutes after the nuclear membrane ruptured. This conclusion also confirms that some previous studies believed that CENP-N has a certain function in the spindle assembly checkpoint.
- Figure 6 shows the effect of the small molecule compound Cenpenlin on the centromere proteins CENP-N and CENP-W.
- Cenpenlin was added at a final concentration of 300nM.
- Based on the display of GFP-CENP-N and GFP-CENP-W, it was found that small molecules have a certain impact on CENP-N and CENP-W, causing the production of lagging chromosomes. Scale bars 10 ⁇ m.
- Figure 7 shows the phenotypic analysis of Cenpenlin on cell growth and division.
- CENP-N When CENP-N is knocked out, the distance between the two poles of the spindle centriole is lengthened; when incubation with the small molecule Cenpenlin at a final concentration of 500 nM, the distance between the spindle poles of the CENP-N knockout cell line is not lengthened, and it is easier to form multiple Polar spindle.
- Scale bar 10 ⁇ m.
- Figure 8 is an analysis of the phenotypic effect of the small molecule compound Cenpenlin on the mitosis of CENP-A knockout cell lines.
- Figure 9 is a diagram showing the mitotic multipolar spindle phenotype caused by the biotin-labeled small molecule compound Bio-Cenpenlin.
- Figure 10 shows the inhibitory effect of Cenpenlin on the growth of gastric cancer organoid cells.
- the registration number of CENP-L used in the following examples in the NCBI database is: NP_001164653.1 (update: 30-JAN-2022).
- Example 2 The small molecule compound Cenpenlin causes chromosome alignment errors
- Example 3 The small molecule compound Cenpenlin causes mitotic arrest
- transfect plasmid DNA GFP-tubulin and mCherry-H2B (transfection plasmid cell density is 70%-80%);
- HeLa cells treated with Cenpenlin at a final concentration of 300 nM showed a large number of chromosomes that were not aligned to the equatorial plate. Judging from the chromosome movement, Cenpenlin treatment will cause some of the chromosomes of the cells to never be aligned correctly.
- counting the proportion of multipolar spindles formed (B) also caused an obvious delay in mitosis. The cells did not enter the anaphase 120 minutes after the nuclear membrane ruptured; Reversine with a final concentration of 250nM was added to alleviate the mitosis blockage of Cenpenlin. , making it enter the later stage smoothly.
- Cenpenlin was added at a final concentration of 300nM. Based on the display of GFP-CENP-N and GFP-CENP-W, it was found that small molecules have a certain impact on CENP-N and CENP-W, causing the production of lagging chromosomes.
- Example 5 Effect of the small molecule compound Cenpenlin on mitochore protein CENP-N and CENP-A knockout cell lines
- Biotin-labeled small molecule compound Bio-Cenpenlin causes mitotic multipolar spindle phenotype
- Example 7 Inhibitory effect of the small molecule compound Cenpenlin on the growth of gastric cancer organoid cells
- PI propidium iodide
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Abstract
公开了细胞分裂调控重要着丝粒蛋白CENP-N靶向抑制剂Cenpenlin及其应用。式I所示化合物在制备具有如下功能的产品中的应用：1)着丝粒蛋白CENP-N靶向抑制剂；2)影响染色体正常排列及延迟有丝分裂进程的试剂；3)抑制肿瘤细胞增殖的产品；4)预防和/或治疗癌症的产品。通过免疫荧光检测，证实小分子通过干扰CENP-N与CCAN复合物中其他组分的相互作用，从而发挥抑制效应。
Description
本发明属于医药领域，具体涉及细胞分裂调控重要着丝粒蛋白CENP-N靶向抑制剂Cenpenlin及其应用。
着丝粒区域的动粒复合物承载了染色体遗传信息。着丝粒相关网络蛋白CCAN为十六元复合物，负责CENP-A核小体和着丝粒纺锤体的连接，其中CENP-N在复合物中功能至关重要。CCAN的冷冻电镜结构，展示了CENP-N与CCAN复合物其他组分的互作关系。CENP-N以不同于经典八聚体核小体的模式结合CNEP-A。动粒介导染色体和纺锤体微管的连接。着丝粒聚集在特殊的染色质区域，CENP-A招募16个亚基组成的CCAN复合物。CENP-C和CENP-N特异识别CENP-A，CENP-C通过中央区域和CENP-C基序结合CENP-A核小体，CENP-N识别CENP-A的L1即RG loop。为Knl1-Mis12-Ndc80(KMN)复合物提供了锚定区域，介导细胞周期微管的附着和滑动调控。通过细胞有丝分裂遗传物质均等分到两个子细胞，补充CENP-A弥补其在DNA复制期间的减少。基于CENP-A核小体和CCAN动粒复合物组成的蛋白质机器，从低等生物进化到在人类等多种真核生物，其蛋白质机器组成发生了变化，但在后生动物中保守。
在酵母中，着丝粒建立在125个碱基对的DNA片段上，仅限于Cse4(CENP-A)的核小体上，被称为点着丝粒。大多数CCAN的亚基在生物体中被识别，统称为Ctf19复合体。近期冷冻电镜解析的酿酒酵母中Ctf19与Cse4核小体的复合物结构揭示了亚基之间的相互作用模式。
不同于酿酒酵母，大多数真核生物具有区域性着丝粒，通过负责的重复的DNA序列，例如人类着丝粒富含AT、171个碱基对的alpha卫星DNA重复序列。然而区域性着丝粒的组装和遗传可能与DNA序列相对独立。而CENP-A相关的CCAN蛋白质机器通过细胞周期调节CENP-A招募CCAN复合物组分。CCAN的保守性说明区域着丝粒的功能分区。
发明公开
本发明的目的是提供式I所示化合物、其异构体及其药学上可接受的盐的药物新用途，其中式I所示化合物命名为Cenpenlin，
本发明所提供的式I所示化合物、其异构体及其药学上可接受的盐的药物新用途，为式I所示化合物在制备具有如下功能的产品中的应用：
1)着丝粒蛋白CENP-N靶向抑制剂；
2)影响染色体正常排列及延迟有丝分裂进程的试剂；
3)抑制肿瘤细胞增殖的产品；
4)预防和/或治疗癌症的产品。
所述1)具体为影响CENP-N与CENP-W和/或CENP-L的相互作用，靶向抑制着丝粒蛋白CENP-N组装为CCAN复合物；
所述3)中，所述肿瘤细胞为骨肉瘤细胞、宫颈癌细胞、胃癌细胞；
所述4)中，所述癌症具体可为骨肉癌、宫颈癌和胃癌。
本发明还提供一种影响CENP-N与CENP-W相互作用的方法，为向含有CENP-N与CENP-W的体系中加入权利要求1中式I所示化合物、其异构体及其药学上可接受的盐，培养，即可。
本发明还提供一种影响CENP-N与CENP-L相互作用的方法，为向含有CENP-N与CENP-L的体系中加入权利要求1中式I所示化合物、其异构体及其药学上可接受的盐，培养，即可。
动粒蛋白CENP-N组装为CCAN复合物的时空顺序仍未解析。如果CENP-N的抑制剂能暂停CCAN复合物的组装，我们将能在短暂的时期内解析CENP-N组装为CCAN复合物的功能及其组装时空调控机制。
在已报道的CCAN复合物结构中，人类的CENP-N全长的结构仍未被解析。我们与合作团队基于冷冻电镜解析的人类包括CENP-N复合物的结构，并基于该结构设计干预CENP-N的抑制剂。我们基于分子对接从类药分子库中筛选出多个可能对CENP-N有影 响的化合物，并基于表型筛选，发现对有丝分裂进程有影响的表型。通过免疫荧光检测，证实小分子通过干扰CENP-N与CCAN复合物中其他组分的相互作用，从而发挥抑制效应。
图1为Cenpenlin小分子的合成路线。
图2为合成的Cenpenlin小分子的NMR谱图。
图3为人源着丝粒蛋白质机器CCAN的冷冻电镜结构与分析，其中，A为人源着丝粒蛋白质机器CCAN复合物冷冻电镜结构；B为灰色实体结构展示为已解析的CENP-N结构；粉色棍棒结构展示Cenpenlin的小分子及作用空间。
图4为小分子化合物Cenpenlin造成染色体排列错误图。加入终浓度为1μM的Cenpenlin小分子，对于人骨肉瘤细胞系U2OS(A)、人宫颈癌细胞系HeLa(B)有一定的抑制其增殖的作用；但是对正常的人视网膜色素上皮细胞系RPE1(C)没有明显的抑制增殖作用。Scale bars＝10μm。
图5为小分子化合物Cenpenlin造成有丝分裂阻滞图。从染色体运动的情况来看，Cenpenlin处理后会致使细胞的部分染色体始终无法正确队列，还造成了明显的有丝分裂延迟，细胞在核膜破裂120min后仍未进入后期。该结论同样证实了之前一些研究所认为的CENP-N对纺锤体组装检验点的具有一定功能。
图6为小分子化合物Cenpenlin对着丝粒蛋白CENP-N和CENP-W的影响图。以DMSO为对照，加入Cenpenlin的终浓度为300nM，基于GFP-CENP-N和GFP-CENP-W展示，发现小分子对CENP-N和CENP-W有一定的影响，造成后滞染色体的产生。Scale bars＝10μm。
图7为Cenpenlin对细胞生长与分裂的表型分析。当敲除CENP-N导致纺锤体中心粒两极之间的距离加长；而当加入终浓度500nM的Cenpenlin小分子孵育，诱导敲除CENP-N细胞系的纺锤体两极距离没有加长，而且更易形成多极纺锤体。Scale bar＝10μm。
图8为小分子化合物Cenpenlin对CENP-A敲除细胞系的有丝分裂的表型影响分析。当加入Cenpenlin小分子导致纺锤体中心粒两极之间的距离缩短；而当加入终浓度500nM的Cenpenlin小分子孵育，诱导敲除CENP-A细胞系的纺锤体微管稳定性受到影响更为剧烈，而且产生滞后染色体。Scale bar＝10μm。
图9为生物素标记的小分子化合物Bio-Cenpenlin导致有丝分裂多极纺锤体表型图。将Cenpenlin小分子加上生物素标记，在细胞中展现了导致细胞在有丝分裂时形成多极纺锤体。
图10为Cenpenlin对胃癌类器官细胞生长的抑制作用。A.胃癌类器官在基质胶中生长7天后，加入150nM的Cenpenlin。B.胃癌类器官细胞的生存率通过染PI来测定,Cenpenlin对胃癌类器官细胞生长的抑制作用达到90％。
实施发明的最佳方式
下述实施例中的实验方法，如无特别说明，均为常规方法
下面结合实施例对本发明作进一步的说明，但不以任何方式对本发明加以限制，基于本发明教导所做的任何变更或改进，均属于本发明的保护范围。
下述实施例中所用小分子化合物Cenpenlin按照图1所示的合成路线图合成。
具体操作步骤如下：
化合物2的合成：
将对甲基苯硫酚(7.6mmol)溶于50mL二氯甲烷，将4-氯乙酰乙酸乙酯(7.0mmol)溶于50mL二氯甲烷，将两个溶液分别在冰水浴中冷却到0度，然后混合并搅拌。将10.8mmol三乙胺逐滴加入上述混合液中，反应产生白色沉淀。将混合物在冰水浴中继续搅拌30分钟，反应完成。加入100mL水，分出有机相，用10*3mL乙酸乙酯萃取水相。合并有机相，依次用饱和碳酸氢钠溶液，0.25M的稀盐酸，饱和食盐水洗涤有机相，然后将有机相旋蒸浓缩，用乙酸乙酯：石油醚＝1:9作为洗脱剂过硅胶柱，制备得化合物1，为淡黄色油状物。然后，立即将化合物1溶于20mL乙醇，加入与化合物1等当量的水合肼(含NH
2NH
2为70％)，室温搅拌过夜，有白色沉淀产生。将沉淀过滤并用冷乙醇洗涤，干燥后得白色固体，为化合物2，两步反应的总产率为69％。化合物2的核磁氢谱数据为
1H NMR(500MHz,DMSO-d
6):11.55(br s,1H),9.47(br s,1H),7.09(d,2H),7.22(d,2H),5.31(s,1H),4.08(s,2H),2.32(s,3H)。
化合物3的合成：
将50mL丙二腈溶于25mL乙醇，将50mL 2,3-二甲氧基苯甲醛加入上述溶液中，室温搅拌4小时，反应完成。产物为沉淀，过滤并用冷乙醇洗涤，得固体粉末，产率92％。化合物3的核磁氢谱数据为1H NMR(500MHz,CDCl3):3.91(s,3H,CH
3O),3.95 (s,3H,CH
3O),7.15-7.21(m,2H,ArH),7.85(dd,J＝7.6Hz,J′＝2.8Hz,1H,ArH),8.26(s,1H,CH＝)。
化合物4的合成：
将化合物3(10mmol)和化合物2(11mmol)加入到20mL乙醇中，加入0.5mL三乙胺，加热回流4小时，趁热过滤除去不溶物，冷却至室温，产物逐渐结晶析出。过滤并用冷乙醇和冷石油醚洗涤，得白色固体产物，产率92％。化合物4的核磁氢谱数据为1H NMR(500MHz,DMSO-d
6),2.29(3H,s,CH
3)；3.59(1H,d,J＝14.4,SCH
2)；3.90(1H,d,J＝14.4,SCH
2)；4.60(1H,s,CH)；6.89-7.42(9H,m,NH
2+ArH)；12.42(1H,br.s,NH)。
化合物Cenpenlin的结构式如上所示，其600MHz
1HNMR谱图(图2)的归属情况如下：
1H NMR(600MHz,DMSO-d6)δ7.09(13-17,d,2CH),δ7.04(14-16,d,2CH),δ6.99(22,t,CH),δ6.92(21,d,CH),δ6.85(26,s,NH2),δ6.58(23,d,CH),δ6.54(4,s,NH),δ4.57(7,s,CH),δ3.79(10,s,CH2),δ2.55(27,s,CH3),δ2.28(29-31,s,2CH3),
下述实施例中所采用的CENP-N在NCBI数据库中的登录号：NP_001094094.2(update：09-JUN-2022)；
下述实施例中所采用的CENP-L在NCBI数据库中的登录号：NP_001164653.1 (update：30-JAN-2022)。
实施例1、小分子化合物Cenpenlin的分子对接模拟
1、实验步骤
1)采用DOCK软件，选取CENP-N的结构为靶标蛋白，选取最大的口袋，将候选小分子对接到结合口袋；
2)利用Pymol软件展示对接位置。基于分子对接模拟，展示Cenpenlin结合在CENP-L/CENP-N之间的界面之处。
2、结果如图3所示。
基于分子对接模拟分析，表明Cenpenlin结合在CENP-L/CENP-N之间的相互界面之处，推测通过变构作用破坏复合物之间相互作用的功能。
实施例2、小分子化合物Cenpenlin造成染色体排列错误
1、实验步骤
1)将稳定表达GFP-CENP-N的HeLa、U2OS、RPE1细胞接种到直径为12mm的圆盖玻片上；
2)24h后加入终浓度为2mM的Thymidine处理细胞16h；
3)用37℃预热的PBS清洗细胞3次将Thymidine洗脱，换上新鲜培养基继续培养8h；
4)实验组加入终浓度为1μM的Cenpenlin，对照组加入同等体积的DMSO，处理1h；
5)用含3.7％甲醛的PBS缓冲液固定细胞10min；
6)经过0.2％TritonX-100打孔和1％BSA封闭后，室温孵育ACA抗体1h；
7)DAPI染色3min后封片；
8)将玻片置于DV显微镜载物台上，在60×，NA＝1.42镜头下拍摄。
2、结果如图4所示。
加入终浓度为1μM的Cenpenlin小分子，对于人骨肉瘤细胞系U2OS(A)、人宫颈癌细胞系HeLa(B)有一定的抑制其增殖的作用；但是对正常的人视网膜色素上皮细胞系RPE1(C)没有明显的抑制增殖作用。Scale bars＝10μm。
实施例3、小分子化合物Cenpenlin造成有丝分裂阻滞
1、实验步骤
1)将HeLa细胞接种到35mm活细胞培养皿中；
2)24h后转染质粒DNA：GFP-tubulin和mCherry-H2B(转染质粒细胞密度为70％-80％)；
3)转染6h后换上新鲜培养基并加入终浓度为2mM的Thymidine处理细胞16h；
4)用37℃预热的PBS清洗细胞3次将细胞释放，随后换上新鲜培养基继续培养；
5)打开Applied Precision Personal DV显微镜及恒温装置使温度稳定在37℃；
6)释放后8h将细胞培养基换成CO2-independent Medium，加入终浓度为300nM的Cenpenlin或相同体积的DMSO；
7)将细胞置于DV显微镜37℃恒温载物台上，在60×，NA＝1.42镜头下实时拍摄，每隔3min拍摄一帧；
8)加入终浓度为250nM的Reversine，继续实时拍摄，每隔3min拍摄一帧。
2、结果如图5所示。
以DMSO为对照(A)，终浓度300nM的Cenpenlin处理后的HeLa细胞，可见大量未排列到赤道板的染色体，从染色体运动的情况来看，Cenpenlin处理后会致使细胞的部分染色体始终无法正确队列，统计形成的多极纺锤体的比例(B)，还造成了明显的有丝分裂延迟，细胞在核膜破裂120min后仍未进入后期；加入终浓度为250nM的Reversine，缓解Cenpenlin对细胞有丝分裂的阻滞，使其顺利进入后期。
实施例4、小分子化合物Cenpenlin对着丝粒蛋白CENP-N和CENP-W的影响
1、实验步骤
1)向24孔板中放入圆形盖玻片，在盖玻片上面涂抹一层Poly-Lysine溶液，增加粘附性；
2)加500μL PBS洗3次，吸去PBS，将24孔板打开置于超净工作台内，用UV照射15min；
3)分细胞到含有盖玻片的孔中，过夜生长至贴壁；
4)转染GFP-CENP-N和GFP-CENP-W质粒；
5)24h后加入终浓度为2mM的Thymidine处理细胞16h；
6)用37℃预热的PBS清洗细胞3次将Thymidine洗脱，换上新鲜培养基继续 培养8h；
7)分别加入终浓度为300nM的Cenpenlin，对照组加入等体积的DMSO；
8)多聚甲醛固定；固定和通透同步，吸去孔中培养基，加入500μL预热的含有3.7％甲醛的PTEM固定10min，加入PBST洗3次，每次5min；
9)用尖头镊子夹取出盖玻片刀铺平的封口膜上(有细胞的一面朝上)，滴加50μL含有1％BSA的PBST，室温孵育30min；
10)用吸水纸沿盖玻片边缘吸去液体，滴加50μL稀释的一抗溶液，室温孵育50min；
11)吸去一抗，滴加50μL PBST孵育5min，重复3次，洗去未结合的抗体；
12)滴加50μL稀释的荧光二抗溶液，室温孵育30min；滴加50μL PBST孵育5min，重复3次，洗去未结合的抗体；
13)吸去二抗，滴加50μL稀释的DAPI染料，室温孵育1min；
14)在洁净干燥的载玻片上滴1μL防淬灭剂，夹取盖玻片倒扣在载玻片上(有细胞的一面朝下)，沿盖玻片边缘涂指甲油封住，晾干后即可在显微镜上观察。
2、结果如图6所示。
以DMSO为对照，加入Cenpenlin的终浓度为300nM，基于GFP-CENP-N和GFP-CENP-W展示，发现小分子对CENP-N和CENP-W有一定的影响，造成后滞染色体的产生。
实施例5、小分子化合物Cenpenlin对着丝粒蛋白CENP-N、CENP-A敲除细胞系的影响
1、实验步骤
1)向24孔板中放入圆形盖玻片，在盖玻片上面涂抹一层Poly-Lysine溶液，增加粘附性；
2)加500μL PBS洗3次，吸去PBS，将24孔板打开置于超净工作台内，用UV照射15min；
3)分别将CENP-N、CENP-A敲除细胞系，分细胞到含有盖玻片的孔中，过夜生长至贴壁；
4)Doxycycline分别诱导敲除CENP-N、CENP-A；
5)24h后加入终浓度为2mM的Thymidine处理细胞16h；
6)用37℃预热的PBS清洗细胞3次将Thymidine洗脱，换上新鲜培养基继续 培养8h；
7)分别加入终浓度为500nM的Cenpenlin，对照组加入等体积的DMSO；
8)多聚甲醛固定；固定和通透同步，吸去孔中培养基，加入500μL预热的含有3.7％甲醛的PTEM固定10min，加入PBST洗3次，每次5min；
9)用尖头镊子夹取出盖玻片刀铺平的封口膜上(有细胞的一面朝上)，滴加50μL含有1％BSA的PBST，室温孵育30min；
10)用吸水纸沿盖玻片边缘吸去液体，滴加50μL稀释的一抗溶液，室温孵育50min；
11)吸去一抗，滴加50μL PBST孵育5min，重复3次，洗去未结合的抗体；
12)滴加50μL稀释的荧光二抗溶液，室温孵育30min；滴加50μL PBST孵育5min，重复3次，洗去未结合的抗体；
13)吸去二抗，滴加50μL稀释的DAPI染料，室温孵育1min；
14)在洁净干燥的载玻片上滴1μL防淬灭剂，夹取盖玻片倒扣在载玻片上(有细胞的一面朝下)，沿盖玻片边缘涂指甲油封住，晾干后即可在显微镜上观察。
2、结果如图7所示。
Cenpenlin对细胞生长与分裂的表型分析。当敲除CENP-N导致纺锤体中心粒两极之间的距离加长；而当加入终浓度500nM的Cenpenlin小分子孵育，诱导敲除CENP-N细胞系的纺锤体两极距离没有加长，而且更易形成多极纺锤体。
结果如图8所示。
小分子化合物Cenpenlin对CENP-A敲除细胞系的有丝分裂的表型影响分析。当加入Cenpenlin小分子导致纺锤体中心粒两极之间的距离缩短；而当加入终浓度500nM的Cenpenlin小分子孵育，诱导敲除CENP-A细胞系的纺锤体微管稳定性受到影响更为剧烈，而且产生滞后染色体。
实施例6、生物素标记的小分子化合物Bio-Cenpenlin导致有丝分裂多极纺锤体表型
1、实验步骤
1)向24孔板中放入圆形盖玻片，在盖玻片上面涂抹一层Poly-Lysine溶液，增加粘附性；
2)加500μL PBS洗3次，吸去PBS，将24孔板打开置于超净工作台内，用UV照射15min；
3)分细胞到含有盖玻片的孔中，过夜生长至贴壁；
4)24h后加入终浓度为2mM的Thymidine处理细胞16h；
5)用37℃预热的PBS清洗细胞3次将Thymidine洗脱，换上新鲜培养基继续培养8h；
6)分别加入终浓度为1μM的Biotin标记的Bio-Cenpenlin，对照组加入等体积的DMSO；
7)多聚甲醛固定；固定和通透同步，吸去孔中培养基，加入500μL预热的含有3.7％甲醛的PTEM固定10min，加入PBST洗3次，每次5min；
8)用尖头镊子夹取出盖玻片刀铺平的封口膜上(有细胞的一面朝上)，滴加50μL含有1％BSA的PBST，室温孵育30min；
10)用吸水纸沿盖玻片边缘吸去液体，滴加50μL稀释的一抗溶液，室温孵育50min；
11)吸去一抗，滴加50μL PBST孵育5min，重复3次，洗去未结合的抗体；
12)滴加50μL稀释的荧光二抗溶液，室温孵育30min；滴加50μL PBST孵育5min，重复3次，洗去未结合的抗体；
13)吸去二抗，滴加50μL稀释的DAPI染料，室温孵育1min；
14)在洁净干燥的载玻片上滴1μL防淬灭剂，夹取盖玻片倒扣在载玻片上(有细胞的一面朝下)，沿盖玻片边缘涂指甲油封住，晾干后即可在显微镜上观察。
2、结果如图9所示。
将Cenpenlin小分子加上生物素标记，在细胞中展现了导致细胞在有丝分裂时形成多极纺锤体。
实施例7、小分子化合物Cenpenlin对胃癌类器官细胞生长的抑制作用
1、实验步骤
1)向分离的胃癌组织细胞中加入适量的基质胶，然后分至10cm的培养皿中；
2)将培养皿放至37℃10-30min，等基质胶充分凝固后，向培养皿中加入完全培养液；
3)每3天更换一次培养液；
4)在基质胶中生长7天后，加入终浓度为150nM的Cenpenlin，对照组加入等体积的DMSO；
5)每隔24小时，通过染PI(荧光染料有碘化丙啶Propidium，简称PI)测定胃癌类器官细胞的生存率并统计。
2、结果如图10所示。
A.胃癌类器官在基质胶中生长7天后，加入150nM的Cenpenlin。B.胃癌类器官细胞的生存率通过染PI来测定,Cenpenlin对胃癌类器官细胞生长的抑制作用达到90％。
工业应用
基于分子对接从类药分子库中筛选出多个可能对CENP-N有影响的化合物，并基于表型筛选，发现对有丝分裂进程有影响的表型。通过免疫荧光检测，证实小分子通过干扰CENP-N与CCAN复合物中其他组分的相互作用，从而发挥抑制效应。
Claims (7)
- 式I所示化合物、其异构体及其药学上可接受的盐在制备具有如下功能的产品中的应用：
1)着丝粒蛋白CENP-N靶向抑制剂；2)影响染色体正常排列及延迟有丝分裂进程的试剂。 - 根据权利要求1所述的应用，其特征在于：1)式I所示化合物影响CENP-N与CENP-W和/或CENP-L的相互作用，靶向抑制着丝粒蛋白CENP-N组装为CCAN复合物。
- 一种影响CENP-N与CENP-W相互作用的方法，为向含有CENP-N与CENP-W的体系中加入权利要求1中式I所示化合物、其异构体及其药学上可接受的盐，培养，即可。
- 一种影响CENP-N与CENP-L相互作用的方法，为向含有CENP-N与CENP-L的体系中加入权利要求1中式I所示化合物、其异构体及其药学上可接受的盐，培养，即可。
- 式I所示化合物、其异构体及其药学上可接受的盐在制备具有如下功能的产品中的应用：
1)抑制肿瘤细胞增殖的产品；2)预防和/或治疗癌症的产品。 - 根据权利要求5所述的应用，其特征在于：所述1)中，所述肿瘤细胞为骨肉瘤细胞、宫颈癌细胞、胃癌细胞。
- 根据权利要求5所述的应用，其特征在于：所述2)中，所述癌症为骨肉癌、宫颈癌和胃癌。
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| WO2012078902A2 (en) * | 2010-12-08 | 2012-06-14 | Proteostasis Therapeutics, Inc. | Proteostasis regulators |
| WO2018183587A1 (en) * | 2017-03-29 | 2018-10-04 | Purdue Research Foundation | 6-amino-2,4-dihydropyrano [2,3-c] pyrazoles and methods of use |
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| WO2012078902A2 (en) * | 2010-12-08 | 2012-06-14 | Proteostasis Therapeutics, Inc. | Proteostasis regulators |
| WO2018183587A1 (en) * | 2017-03-29 | 2018-10-04 | Purdue Research Foundation | 6-amino-2,4-dihydropyrano [2,3-c] pyrazoles and methods of use |
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| Title |
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| ADIBI HADI, HOSSEINZADEH LEILA, FARHADI SEPIDEH, AHMADI FARAHNAZ: "Synthesis and cytotoxic evaluation of 6-amino-4-aryl-3-methyl-2,4-dihydropyrano[2,3-C]pyrazole-carbonitrile derivatives using borax with potential anticancer effects", JOURNAL OF REPORTS IN PHARMACEUTICAL SCIENCES, vol. 2, no. 2, 1 January 2013 (2013-01-01), pages 116, XP093129449, ISSN: 2322-1232, DOI: 10.4103/2322-1232.222528 * |
| DATABASE Registry 3 March 2003 (2003-03-03), ANONYMOUS: "Pyrano[2,3-c]pyrazole-5-carbonitrile, 6-amino-4-(2,3-dimethoxyphenyl)-2,4-dihydro-3-[[(4- methylphenyl)thio]methyl]- (CA INDEX NAME)", XP093129456, retrieved from STNext Database accession no. 496804-53-6 * |
| NIMBALKAR URJA, SEIJAS JULIO, VAZQUEZ-TATO MARIA, DAMALE MANOJ, SANGSHETTI JAIPRAKASH, NIKALJE ANNA: "Ionic Liquid-Catalyzed Green Protocol for Multi-Component Synthesis of Dihydropyrano[2,3-c]pyrazoles as Potential Anticancer Scaffolds", MOLECULES, vol. 22, no. 10, 28 September 2017 (2017-09-28), pages 1628, XP093129451, DOI: 10.3390/molecules22101628 * |
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