CN115624554A - 化合物arv-825在治疗nut癌中的应用 - Google Patents
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Abstract
本发明属于生物医药领域，涉及化合物ARV‑825新的医药用途，具体涉及化合物ARV‑825通过靶向BRD4显示抗BRD4‑NUT融合蛋白的抗肿瘤活性，在制备治疗NUT癌药物中的用途。实验结果表明ARV‑825在体外和体内均能快速有效地诱导BRD4‑NUT蛋白降解，并抑制3T3‑BRD4‑NUT细胞的生长。本发明对细胞系异位表达系统的研究极大地提高了对NUTM1融合蛋白产生的分子改变的理解，为新的靶向药物的创建提供了新的视角，为个性化治疗提供了理论支持，并指出了有前途的靶向治疗途径，促进了NUT癌的治疗进展。这些结果表明，ARV‑825是治疗BRD4‑NUT癌的有效方法。
Description
技术领域
本发明属于生物医药领域，涉及化合物ARV-825新的医药用途，具体涉及化合物ARV-825通过靶向BRD4显示抗BRD4-NUT融合蛋白的抗肿瘤活性，在制备治疗NUT癌药物中的用途。
背景技术
目前BET蛋白家族成员中研究最多的时BRD4蛋白。BRD4 是一种转录和表观遗传调节因子，在胚胎发生和癌症发展过程中起着关键作用。作为溴结构域和末端外结构域(BET) 家族的成员，BRD4 的特征在于具有两个串联溴结构域（BD1、BD2）。BDs 结合靶蛋白（包括组蛋白）上的乙酰化赖氨酸残基，因而对具有多个乙酰化残基的蛋白质的亲和力更高。BRD4与染色质上的超乙酰化组蛋白区域相互作用，在转录活性调控元件上积累并在起始和延伸步骤中促进基因转录。BRD4功能障碍与多种癌症的发生和进展相关，包括急性髓系白血病、结肠癌、Burkitt氏淋巴瘤、乳腺癌、弥漫性大b细胞淋巴瘤、多发性骨髓瘤和卵巢癌。此外，它是染色体15和19之间的染色体易位的位置，这是侵袭性NUT癌的原因，通常表现为单个t易位，并产生新的融合癌基因BRD4-NUT。
NUTM1（也称NUT）是NUT中线癌家族成员1，是仅在睾丸表达的核蛋白，其功能未知。NUTM1与BRD4融合通常在鳞状细胞癌的NUT中线亚群中发现，并在软组织肿瘤中有报道，许多其他的NUTM1融合在各种其他类型的肿瘤中也有报道。BRD4-NUT由BRD4和NUTM1融合产生，导致细胞分化抑制。BRD4-NUTM1融合与NUT中线癌相关。NUT基因重排可能导致NUT癌(NC)，这是一种鳞状细胞癌的侵袭性亚型。NC主要作用于身体中线器官。它表现为一种单胚性低分化鳞状细胞癌。BRD4-NUT融合引起的染色体易位是遗传性疾病NC最常见的原因。这种癌症的发病年龄从不等。NC几乎是致命的，而且对目前已知的治疗方法几乎完全耐药，即使是积极的化疗，诊断为NC后的典型生存期不到一年(9.5个月)。
PROTAC（proteolysis-targetingchimeras, 蛋白降解靶向联合体）是一种利用泛素-蛋白酶系（Ubiquitin-ProteasomeSystem,UPS）对靶蛋白进行降解的药物开发技术。PROTAC是一个两头含有不同配体的化学分子，一头是结合E3连接酶的配体，另一头是结合细胞内蛋白质的配体，这两个配体再由一段linker连接起来。这样的化学分子既可以结合E3泛素连接酶，又可以结合胞内蛋白质，通过把靶向的蛋白质招募到E3泛素连接酶附近来实现靶向蛋白质的多泛素化，最后被蛋白酶体降解。PROTAC可以循环使用，不被蛋白酶体降解。在患者体内，PROTAC的靶蛋白配体和靶蛋白结合，E3泛素连接酶配体和细胞内的E3泛素连接酶的底物结合区结合，从而通过Linker把靶蛋白拉近到E3泛素连接酶旁边，实现UPS系统将靶蛋白降解。已有研究表明，使用PROTAC技术将BRD4抑制剂与配体结合的ARV-825能更有效地分解BRD4，有效地抑制肿瘤生长，并能持续地抑制。研究表明ARV-825在胰腺癌、vemurafenib耐药黑色素瘤、胆管癌、甲状腺癌、急性髓系白血病、T细胞急性淋巴细胞白血病和神经母细胞瘤中的抗肿瘤作用。然而在ARV-825化合物对NUT癌的作用机制和抗肿瘤活性鲜有报道。
发明内容
鉴于现有技术存在的问题，本发明的目的在于提供化合物ARV-825新的医药用途，化合物ARV-825通过靶向BRD4显示抗BRD4-NUT融合蛋白的抗肿瘤活性，在制备治疗NUT癌药物中的用途。本发明利用ARV-825靶向BRD4-NUT，并在3T3细胞中使用BRD4-NUT融合结构检测其体内外抗肿瘤活性，为NUT癌治疗提供了潜在的治疗药物。
为实现上述目的，本发明采用以下技术方案。
化合物ARV-825在制备治疗NUT癌药物中的应用，所述化合物ARV-825的结构式如下式所示：
。
进一步地，所述化合物ARV-825通过靶向BRD4，实现治疗或预防NUT癌。
进一步地，所述化合物ARV-825通过抑制BRD4-NUT融合蛋白表达，实现治疗或预防NUT癌。
进一步地，所述的化合物ARV-825加入药学上可接受的载体和/或辅料，制成片剂、喷雾剂、颗粒剂、胶囊剂、口服液、针剂的任一种剂型。
与现有技术比，本发明的有益效果如下。
在本发明研究中，发现ARV-825在体外和体内均能快速有效地诱导BRD4-NUT蛋白降解，并抑制3T3-BRD4-NUT细胞的生长。对细胞系异位表达系统的研究极大地提高了对NUTM1融合蛋白产生的分子改变的理解，为新的靶向药物的创建提供了新的视角，为个性化治疗提供了理论支持，并指出了有前途的靶向治疗途径，促进了NUT癌的治疗进展。这些结果表明，ARV-825是治疗BRD4-NUT癌的有效方法。
附图说明
图1是过表达BRD4-NUT促进细胞活力、增殖和迁移。其中A是CMV-BRD4-NUT 载体构建方案。B是BRD4-NUT过表达和对照转染48小时WB检测。C是3T3细胞和3T3- BRD4-NUT细胞显微镜明场下观察，比例尺为200 μm，细胞进一步在完全培养基中并在指定的时间段进行培养；D实细胞迁移(划痕试验)检测；E是细胞活力(CCK8 OD)检测，数据以均数±标准差(SD, n=3)表示(所有数据相同)。实验重复了三次，得到了相似的结果。
图2A-2G是ARV-825抑制BRD4-NUT 3T3细胞的活性。其中2A是过表达BRD4-NUT的细胞会降低细胞活力；2B是不同浓度ARV-825处理3T3细胞后细胞增殖情况；2C是不同浓度ARV-825处理后，3T3细胞在特定时间的迁移变化，比例尺为200 μm；2D是不同浓度ARV-825处理后，3T3- BRD4-NUT细胞在特定时间的迁移变化，比例尺为200 μm；2E是ARV-825处理后过表达BRD4- NUT的细胞减少了的3T3细胞的迁移；2F是ARV-825给药后伤口愈合情况；2G是WB检测分析不同浓度ARV-825处理后3T3-BRD4-NUT细胞中BRD4蛋白表达水平的。
图3A-3E是显示ARV-825治疗后的动态变化的转录组差异。其中3A是PCA分析了四组数据,输入数据集在前两个主成分上的投影。 BN, 3T3-BRD4-NUT细胞，BN+0.003, 3T3-BRD4-NUT细胞用0.003 μM ARV-825处理，BN+0.03,3T3-BRD4-NUT细胞用0.03μ m ARV-825处理；3B是RNA-seq分析BRD4-NUT3T3和对照细胞基因表达变化的火山图，蓝色标记的基因是向上变化的基因，蓝绿色表示下调的基因，黑色表示未改变的基因；3C是3T3-BRD4-NUT细胞与3T3细胞比较差异表达基因的热图，红色表示上调，蓝色表示下调；3D是关键靶点的KEGG途径富集分析(前10位)，横坐标标记为途径富集倍数；3E是将ARV-825给药前后差异表达最高的基因进行细胞功能富集分析(p < 0.05, |log2FC|>1)；每一列代表一个不同的样本，每一行代表一个基因，一行中的颜色变化表示相对于同一种群的平均值的表达水平，红色表示上调，蓝色表示下调，白色表示基本表达水平。
图4-1至4-6是显示ARV-825给药后RNA-Seq中基因集的基因富集的GSEA图。
图5是ARV-825在BRD4-NUT3T3异种移植瘤模型中显示抗肿瘤作用。其中A是异种移植流程图，携带异种移植瘤的裸鼠分别以10 mg/kg ARV-825或对照剂腹腔注射，持续21天。数据为平均值±SEM (n = 6)；B是每3天称一次小鼠体重。C是每3天记录一次肿瘤体积，计算公式为width × width × length×0.52；D是ARV-825或安慰剂处理的小鼠的异种移植瘤照片。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。细胞系3T3和293T细胞株由沈阳化工大学应用生物学实验室保存。细胞在37°C条件下提5%的二氧化碳，DMEM培养基 (Dulbecco’sModified Eagle’s)，培养基(Thermo Fisher Scientific)，含有100 U/ml青霉素-链霉素的培养基(Millipore Sigma)和10%胎牛血清(FBS) (ExCell Bio)中培养。
实施例1 质粒构建。
从VectorBuilder中合成pLV-EGFP T2A Puro-EF1A hNUTM1质粒;pcDNA3.1-CMV-BRD4 -Flag质粒购自YouBio。构建pcDNA3.1-CMV-BRD4 NUT质粒，采用PCR扩增NUT靶基因，采用正向5 ' -aaacaggtcctgccctcgagGTTACTCTGGGTCCTGGACCTG和反向5 ' -gggccctctagactcgagCTGGCTACGACGTCGTTTCTTC引物。PCR条件：95°C孵育2分钟，然后95°C孵育30秒，50°C孵育30秒，72°C孵育4分钟35个循环，72°C孵育7分钟。pcDNA3.1-CMV-BRD4-FLAG质粒用XhoI酶切。然后，从转化的DH5α细胞和单个菌落中分离质粒DNA，连接到消化产物(EasyGeno试剂盒)中，用上述引物进行测序。
实施例2 细胞转染。
3T3细胞用pcDNA3.1-CMV-BRD4 NUT (3 μg)电穿孔。转染后将细胞轻轻地重悬在1.5 mL预热的DMEM培养基中。所有细胞在37℃、5% CO2培养箱中培养。第二天，培养基改为完全DMEM培养基，细胞保持原样。48小时后，应用基因素(Sigma-Aldrich)来选择稳定的细胞系。
实施例3 WB检测。
6-10% SDS-PAGE凝胶用于分离整个细胞裂解物(每个通道每次处理10-20 g蛋白质)，然后转移到PVDF印迹上。阻断后，应用的一、二抗体与印迹孵育，用ECL试剂盒鉴定抗体-抗原结合。
实施例4 细胞活性分析。
每孔2000个细胞被三倍地接种到96孔板上，细胞生长一夜，然后在规定的时间内使用药物。按照制造商的说明，CCK-8测试被用来测量细胞活性。
实施例5 划痕试验。
6孔板培养24-48小时。当细胞达到100%融合时，使用1毫升的微移液管尖端制造伤口。去除培养基并用1ml PBS洗涤细胞后，每个孔获得2ml含有化合物的完整DMEM培养基。每12小时拍摄一次照片。导出照片后使用ImageJ测量伤口大小。
实施例6 RNA-Sequencing和分析。
利用promegene提供的程序(深圳)进行RNA-测序(RNA-seq)。使用TRIzol®试剂(Invitrogen)从细胞中提取总RNA。为了建立和测序文库，RNA首先被反向转录成cDNA。使用Bioconductor limma分析，差异表达基因(|log2fold change| >1 and p < 0.05)被发现。(http://www.bioinformatics.com.cn/)利用基因集富集分析(GSEA)选择了多条细胞途径进行基因富集。
实施例7体内异种移植。
从中国沈阳Lanpuda, Ltd.购买了裸鼠。800万个3T3-BRD4-NUT细胞皮下植入4周龄雄性裸鼠背部(每组6个)，当移植肿瘤大小达到200 mm3左右时，每天腹腔给予ARV-82510 mg/kg或对照品。当对照组肿瘤大小达到1000 mm3时，处死小鼠。每三天，用卡尺测量皮下肿瘤的大小。肿瘤体积由公式(宽×宽×长× 0.52)确定。
结果。
过表达BRD4-NUT促进3T3细胞活性。
考虑到BRD4-NUT融合蛋白是NUT癌的癌蛋白，构建了过表达BRD4-NUT的CMV-BRD4-NUT质粒来模拟NUT癌的致病基因，如图1A所示。BRD4的外显子2-11和hNUT的2-8无缝连接，模拟NC患者的BRD4-NUT融合。Western blotting结果显示，3T3细胞中BRD4-NUT过表达，如图1B所示，但3T3细胞与3T3细胞在形态上无明显差异，如图1C所示。CCK8试验显示，过表达BRD4-NUT可显著促进3T3-BRD4-NUT细胞增殖，如图1D所示。综上所述，这些结果表明本发明成功构建了过表达BRD4-NUT融合蛋白的稳定细胞系，并发现过表达BRD4-NUT可以促进3T3细胞的增殖和活性。
ARV-825抑制3T3-BRD4-NUT细胞的增殖和迁移。
用不同剂量的ARV-825 (0.001 ~ 0.1 M)处理过表达的BRD4-NUT细胞系48小时，以评估药物对细胞的作用。接受ARV-825治疗后，3T3-BRD4-NUT细胞增殖呈剂量依赖性下降，如图2A所示。在ARV-825处理后，细胞活性大幅下降，如图2B所示。即使在测试的最低浓度(0.001 μM)下，ARV-825也显示出抑制作用，如图2A所示。此外，BRD4 PROTAC药物以时间依赖性的方式抑制了3T3-BRD4-NUT细胞活性，如图2A所示。ARV-825 (0.001 ~ 0.1 μM)处理24小时后，活性开始下降，如图2B所示，表明增殖率下降。划痕试验结果显示，ARV-825(0.001-0.03 μM, 24h)显著减少迁移的3T3-BRD4-NUT细胞数量，如图2D所示。0.1 μM ARV-825能显著抑制3T3-BRD4-NUT细胞的迁移，呈现剂量依赖性，如图2D所示。ARV-825 (0.001~ 0.03 μM, 24h)对3T3细胞的迁移和增殖没有抑制作用，而0.1μM ARV-825对3T3细胞的迁移和增殖有抑制作用，说明0.1μM ARV-825对3T3细胞具有毒性，如图2C、2D和2E所示。这些结果表明，ARV-825有效地降低了3T3-BRD4-NUT细胞的活性、增殖和迁移。Westernblotting分析结果显示，ARV-825剂量依赖性地抑制了3T3-BRD4-NUT细胞中的BRD4-NUT蛋白水平，如图2G所示。
基因转录丰度的比较分析。
通过RNA-seq来研究BRD4-NUT在转录水平上的潜在机制。测试了3T3细胞、3T3-BRD4-NUT细胞、0.003 μM和0.03μMARV-825处理后的4组数据。PCA分析将四组患者分离开来。0.003 μMARV-825的低浓度处理与0.03μMARV-825聚集在一起，而不是BRD4-NUT，说明药物处理敏感，如图3A所示。由图3B可以看出，在|log2fold change| > 1 and an 调整后的p< 0.01的条件下，对两个过度表达BRD4-NUT的成对3T3细胞的转录组数据进行比较，103个基因上调，159个基因下调。与3T3细胞相比，这些基因在3T3- BRD4-NUT细胞中的表达水平有显著差异。对异常基因进行功能富集分析，如图3C所示。发现Rap1信号通路、小细胞肺癌、非小细胞肺癌、膀胱癌相关基因富集，提示这些通路可能与BRD4-NUT过表达相关，如图3C所示。而在加入ARV-825后，BRD4过表达引起的异常上下调基因发生了变化，在转录水平呈现出拯救趋势，如图3E所示。同样，在GSEA富集通路中，加入ARV-825后，通路变化也呈现拯救趋势，如图4-1至4-6所示。
ARV-825在异种移植瘤模型中抑制肿瘤生长 。
利用3T3-BRD4-NUT细胞，建立NUT癌移植瘤模型，观察ARV-825在体内的抗癌作用，如图5A所示。当皮下肿瘤体积达到约200 mm3时，裸鼠每天腹腔注射ARV-825，剂量为10 mg/kg。与对照组相比，ARV-825治疗组的肿瘤体积明显降低，如图5B、5C和5D所示，但体重与治疗组和对照组无明显差异，如图5B所示。这些结果表明，ARV-825可能显著减缓NUT癌肿瘤的生长。
ARV-825治疗导致了3T3-BRD4-NUT细胞中BRD4水平更显著和更持久的下降。 RNA-seq和Western blotting分析证实了ARV-825对3T3-BRD4-NUT细胞基因表达的影响。结果表明，ARV-825抑制BRD4可导致3T3-BRD4-NUT细胞中E2F、TRAFs、Wnt、Gadd 45 g、Sox 6 mRNA的改变。对NUT癌RNA-seq数据的进一步研究可能会发现新的治疗靶点和重要的信号机制。ARV-825在3T3细胞移植模型中抑制BRD4-NUT肿瘤的过表达。根据体外实验结果，ARV-825可以降低体内BRD4蛋白水平。这进一步证明了ARV-825可能有效调控BRD4-NUT关键基因调控网络。此外，研究还表明，接受ARV-825治疗的小鼠与对照组之间的体重没有统计学上的显著差异。接受ARV-825治疗的小鼠的器官没有显示出任何其他明显的负面影响。使用JQ1治疗的小鼠体重下降，脂肪生成受损，但在ARV-825治疗的器官中，除了体重外，未观察到明显的毒性作用。这些发现表明，ARV-825既安全又有效。本发明试验研究结果表明，ARV-825治疗NUT癌是一种新的治疗策略。总之，在本发明研究中，发现ARV-825在体外和体内均能快速有效地诱导BRD4-NUT蛋白降解，并抑制3T3-BRD4-NUT细胞的生长。对细胞系异位表达系统的研究极大地提高了对NUTM1融合蛋白产生的分子改变的理解，为新的靶向药物的创建提供了新的视角，为个性化治疗提供了理论支持，并指出了有前途的靶向治疗途径，促进了NUT癌的治疗进展。这些结果表明，ARV-825是治疗BRD4-NUT癌的有效方法。
Claims (5)
1.化合物ARV-825在制备治疗NUT癌药物中的应用，其特征在于，所述化合物ARV-825的结构式如下式所示：
。
2.如权利要求1所述的应用，其特征在于，所述化合物ARV-825通过靶向BRD4，实现治疗或预防NUT癌。
3.如权利要求1所述的应用，其特征在于，所述化合物ARV-825通过抑制BRD4-NUT融合蛋白表达，实现治疗或预防NUT癌。
4.如权利要求1所述的应用，其特征在于，所述的化合物ARV-825加入药学上可接受的载体和/或辅料，制成片剂、喷雾剂、颗粒剂、胶囊剂、口服液、针剂的任一种剂型。
5.一种治疗NUT癌药物，其特征在于，含有化合物ARV-825，化合物ARV-825的结构式如下式所示： 
。
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