CN102796050A - 一种Cdc42抑制剂及其应用 - Google Patents
一种Cdc42抑制剂及其应用 Download PDFInfo
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- CN102796050A CN102796050A CN2012101582763A CN201210158276A CN102796050A CN 102796050 A CN102796050 A CN 102796050A CN 2012101582763 A CN2012101582763 A CN 2012101582763A CN 201210158276 A CN201210158276 A CN 201210158276A CN 102796050 A CN102796050 A CN 102796050A
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Abstract
本发明涉及一种具有式I结构的化合物,该化合物能够用于制备Cdc42抑制剂。丝状伪足的形态学分析,Western blot Cdc42磷酸化及下游效应蛋白WASP的分析,以及细胞伤口愈合试验和生长锥的形成试验,均表明本发明提供的化合物能够抑制Cdc42参与的所有过程,有效抑制Cdc42的作用。有效抑制actin参与的细胞功能,如高尔基体组织和细胞运动。
Description
技术领域
本发明涉及一种化合物,具体地涉及一种Cdc42抑制剂及其应用。
背景技术
细胞分裂周期蛋白Cdc42是小G蛋白的Rho GTP酶家族的一个亚类,是许多细胞生物学功能的重要调控蛋白。首次在Saccharomyces cerevisiae中发现,参与细胞极化,随后认识到其在细胞骨架重组、细胞的胞吞运输途径、细胞周期调控和细胞转录中都发挥着重要作用。与多数GTP酶一样,通过GDP交换为GTP结合实现信号转导,激活Cdc42。Rho GTP酶家族的这种核苷酸限制型构象之间的循环由3类重要蛋白调节:鸟嘌呤交换因子(GEF),催化GDP的释放和GTP的结合;GTP酶激活蛋白(GAP),作为负向调节因子加速Rho GTP酶的水解,使Rho GTP酶由活性状态变为无活性状态;GDP解离抑制因子(GDI),阻止GDP从Rho GTP酶上分离,抑制Rho GTP酶活性。
近年的研究揭示Cdc42的异常活性广泛参与了包括肿瘤和神经退行性疾病等人类疾病的病理生理。有趣的是在人类肿瘤中没有发现Cdc42的突变基因,它的异常形式主要表现为在组织和依赖的微环境中失调或过表达,与肿瘤细胞的转化及转移密切相关。作为关键的神经元形态形成的调控器,Cdc42掌控着正常大脑发育的命运,Cdc42敲除的小鼠不能活至出生并表现明显的大脑畸形。既往不同的研究显示Cdc42的活化对上皮细胞间充质细胞转变(EMT)和因此发生的胞内物质运输十分重要,这对肿瘤细胞的侵袭是非常必要的。
但是,在三大典型的Rho超家族亚类中,对于Cdc42的研究远滞后于RhoA和Rac1。这部分是由于Cdc42的活化/失活形式的转换非常快速,也缺乏选择性的小分子研究工具来帮助直接了解这个过程。
Rho GTP酶家族参与的信号通路调控细胞多种生化功能,诸如细胞膜的物质运输、细胞周期调控和细胞骨架的组织,这关乎细胞形态、细胞运动以及细胞命运。近年的研究显示很多疾病的病理发生和进程与Rho GTP酶家族功能蛋白的失常或失控有关,因此成为药物开发的重要靶点。
同时,Rho GTP酶家族的小分子调节剂的应用也促进了对其中功能蛋白的研究。例如,新型脑、心血管活性药——法舒地尔和小分子化合物Y27632是公认的RhoA下游效应信号分子Rho/p160ROCK强效抑制剂。作为Rac1蛋白的选择性抑制剂,近来对靶向Rac1-GEF连接的小分子化合物NSC23766的研究也大大促进了对Rac1蛋白功能的认识。但是,却几乎没有有效的Cdc42选择性抑制剂。Secramine,天然产物加兰他敏的类似物,近来认为它通过RhoGDI1可抑制依赖于Cdc42的高尔基体-细胞膜间物质转运。不同于广泛应用的Y27632(1903篇相关文献)和NSC23766(115篇相关文献),Secramine的获得十分有限,研究甚少(仅有9篇相关文献)。Cdc42的失调在很多方面与肿瘤发生相关,包括肿瘤的转化和转移;另外神经元的发展与维持也严重依赖于正常的Cdc42活性。
NSC23766是一个基于计算机模拟的结构筛选的化合物,与Rac1分子的表面结构契合,而已知Rac1对GEF的结合至关重要(Gao,Y.,J.B.Dickerson,et al.(2004).Rational design and characterization of a RacGTPase-specific small molecule inhibitor.Proc Natl Acad Sci USA.101(20):7618-7623)。NSC23766可以抑制血清中或生长因子诱导的Rac1活化和Rac1片状伪的形成。NSC23766作用于人类前列腺癌细胞株,能抑制细胞增殖,抑制非停泊性生长,并且降低细胞的侵袭表型,而肿瘤细胞的这些表型均依赖于内源性Rac1的活性。此外,新的研究表明,NSC23766可以改善由Rac1所介导的脊髓损伤(SCI)诱发的神经性疼痛(Tan,A.M.,S.Stamboulian,et al.(2008).Neuropathic pain memory is maintained byRac1-regulated dendritic spine remodeling after spinal cord injury.J Neurosci.28(49):13173-13183)。
发明内容
本发明的目的在于提供一种化合物,其具有下式I的结构:
式I,
其名称为:
4-(3-(2-(4-Bromo-2-chlorophenoxy)acetyl)thioureido)-N-(4,6-dimethylpyrimidin-2-yl)benzenesulfonamide,即4-(3-(2-(4-溴-2-氯-苯氧基)-乙酰基)-硫脲基)-氮-(4,6-二甲基嘧啶-2-基)苯磺酰胺。为了简便起见,本申请文件中将其命名为ZCL278。
本发明提供的ZCL278,能够抑制Cdc42参与的所有过程,有效抑制Cdc42的作用。
因为Cdc42在细胞周期、运动、黏附、细胞凋亡和细胞内运输等中都具有重要的作用。同时在癌症的发展和侵袭,心血管系统和呼吸系统疾病,神经系统疾病,以及其它许多疾病中均有重要的作用。所以本发明提供的ZCL278可以制备用于治疗恶性肿瘤的药物,具体说,可以制备用于防治恶性肿瘤的发展和侵袭的药物。
上述的化合物还可以制备用于治疗心血管系统疾病的药物。
上述的化合物还可以制备用于治疗呼吸系统疾病的药物。
上述的化合物还可以制备用于治疗神经系统疾病的药物。
其中,上述的化合物可以通过抑制Cdc42的功能发挥上述作用。
本发明的另一目的是提供一种Cdc42抑制剂,其包含上述式I所示的化合物。
本发明提供的化合物ZCL278能够有效抑制GTP结合Cdc42的活性。在小鼠成纤维细胞Swiss 3T3中,化合物ZCL278影响Cdc42调控亚细胞结构的两个最主要的效应表现:消除细胞微棘的形成,破坏GM130对接高尔基体的结构。相比Rac的选择性抑制剂NSC23766,ZCL278可减少细胞核外缘活性Cdc42的积聚。ZCL278抑制Cdc42介导的神经元分支及其生长锥动力学,同时发现其在不破坏细胞活率的条件下即可抑制转移性前列腺癌细胞PC-3的actin为基础的细胞运动及迁移。所以,ZCL278能够有效抑制Cdc42对细胞形态及行为学的调控,在癌症的发展和侵袭,心血管系统和呼吸系统疾病,以及神经系统等疾病中均能发挥重要的作用。
为让本发明之上述和其它目的、特征和优点能更明显易懂,下文特举较佳实施例,并配合附图,作详细说明如下。
附图说明
图1A-图1C:ZCL靶向Cdc42-ITSN(交叉蛋白)连接的结构鉴定。其中,
图1A为计算机模拟ZCL278结合Cdc42分子(实际试验中生成的为彩色图):灰色表面(如图中的箭头1)所示为蛋白质,绿色棒(如图中的箭头2)所示为配基。图1B为ZCL278与Cdc42氨基酸残基的结合:绿色棒为ZCL278分子(如图中的箭头3),灰色(如图中的箭头4)示意Cdc42结构,橙色线条(如图中的箭头5)示意两分子间形成氢键。图1C表示将Cdc42-ZCL278复合物与GMP-PCP蛋白质分子(蛋白质数据库:2QRZ)的结构进行叠合:绿色棒为ZCL278分子,灰色示意Cdc42结构,青色棒示意GMP-PCP结构(GMP-PCP是GTP类似物,β,γ-亚甲基二磷酸鸟苷酸)。
图2A-图2C表示ZCL278的活性特性。其中:图2A表示ZCL278抑制Cdc42介导的细胞微棘形成。图2B表示ZCL278抑制内生型Rac/Cdc42的活化。图2C表示ZCL278抑制刺激型Cdc42的活化。
图3A-图3C表示活化Cdc42/磷酸化RhoA的免疫荧光染色。
图4表示ZCL278破坏细胞内GM130蛋白对接高尔基体的组织。
图5A-图5C表示ZCL278阻碍细胞迁移,但不影响细胞活率。
图6A-图6C表示ZCL278抑制神经元分支和生长锥动力学。
具体实施方式
实施例1:Cdc42抑制剂的虚拟筛选
分析Cdc42-ITSN复合物的三维结构可发现介于两分子之间一个主要的结合域,ITSN分子中Gln1380残基和Arg1384残基之间以及Cdc42上Asn39和Phe37之间的氢键,ITSN分子中Leu1376、Met1379残基和Thr1383残基之间以及Cdc42上Phe56、Tyr64、Leu67和Leu70之间的两个疏水簇。为筛选Cdc42抑制剂,推定Cdc42分子上结合口袋为ITSN蛋白表面与Cdc42结合的半径距离在7A内的氨基酸残基形成的结构。这其中含有Cdc42蛋白分子中Thr35,Val36,Asn39,Phe56和Asp57等16个氨基酸残基,如图1A-图1C所示。
用Glide程序筛选SPECS数据库中能破坏Cdc42与ITSN连接的小分子化合物。Cdc42-ITSN复合物的晶体结构来源于蛋白质数据库,ID号:1KI1.占据Cdc42结合位点的ISTN中氨基酸残基为Leu376,Met379,Gln1380,Thr1383,Arg1384.距离这五个氨基酸中心在7A内的Cdc42氨基酸残基形成Cdc42的结合口袋。运用蛋白准备模块Protein Preparation Wizard处理模型的初始结构,使用Glide/Receptor Grid Generation模块分子对接,使用配体准备模块Ligprep将对Specs数据库中197000个化合物进行进行高通量虚拟筛选,按照以下标准筛选出来的排名前100位的化合物将入选:ITSN样的结合态,可占据ITSN上Leu1376,Gln1380,Arg1384,Met1379和Thr1383残基的空间结构;至少形成3个氢键;与Cdc42分子中保守的Asn39或Phe37残基能形成氢键;分子骨架的多样性。排名前50000个分子再进行标准精度计算排出前100个分子,最后选出30个化合物测试它们对Cdc42的活性和/或功能的影响。
Cdc42分子上ZCL278键合的电脑模拟模式:如图1A所示,ZCL278嵌合Cdc42蛋白中Thr35,Val36,Asp38,Asn39,Phe56,Tyr64,Leu67和Leu70残基顺序形成的口袋结构。如图1B所示,两分子之间还发现外延的良性相互作用。与涉及Thr35,Asn39和Asp57残基在内的氨基酸残基形成5个氢键,同时与Val36和Phe56残基形成有疏水作用的结构。如图1C所示,分子中的溴苯环插入到GDP/GTP的结合口袋里。电脑模拟的ZCL278与Cdc42的结合可破坏Cdc42-ISTN的GDP/GTP转换态结合。
实施例2:化合物ZCL278的合成:
ZCL278可以通过下述合成方法合成,以下合成方法仅用于示例,而非对本发明的限制,本领域的技术人员可以理解并想到可采用其它合成方法来合成ZCL278,其也属于本发明的保护范围。
反应试剂和条件:(a)K2CO3,DMF(N,N-二甲基甲酰胺),70℃;(b)NaOH,dioxane(二氧杂环乙烷)/H2O;(c)SOCl2(二氯亚砜),DMF,reflux(回流);(d)NaSCN(硫氰酸钠),acetone(丙酮),0℃-rt;(e)4-amino-N-(4,6-dimethylpyrimidin-2-yl)benzene-sulfonamide(4-氨基-N-(4,6-二甲基-2-嘧啶基)苯磺酰胺),0℃-r.t.
反应式如下:
化合物1(4-溴-2-氯酚)与2-溴乙酸乙酯在K2CO3存在下发生亲核取代反应可得到化合物2(2-(4-溴-2-氯苯氧基)乙酸乙酯)。化合物2在碱性条件下水解得到化合物3(2-(4-溴-2-氯苯氧基)乙酸)。化合物3在N,N-二甲基甲酰胺催化下在二氯亚砜中回流可制备得到化合物4。化合物4(2-(4-溴-2-氯苯氧基)乙酰氯)与硫氰酸钠反应后形成相应的硫氰酸酯中间体,进一步在反应体系中加入4-氨基-N-(4,6-二甲基-2-嘧啶基)苯磺酰胺可得到化合物5(即本发明的ZCL278)。
仪器与试剂:Bruker Avance III 400MHz核磁共振仪;SGWX-4熔点仪;Agilent 1200型高效液相色谱;ZORBAX Eclipse XDB-C18色谱柱(4.6mm×150mm,5μM);所有试剂均为分析纯或化学纯。
步骤1:2-(4-溴-2-氯苯氧基)乙酸乙酯(即化合物2)的合成
在反应瓶中依次加入化合物1(4-溴-2-氯酚)(5.2g,25.0mmol),50mLN,N-二甲基甲酰胺(DMF),2-溴乙酸乙酯(4.3,25.7mmol)以及K2CO3(3.45g,25.0mmol)。在70℃搅拌过夜后将反应混合物倒入150mL水中,用乙酸乙酯萃取(70mL×4)。合并有机相,用饱和食盐水洗涤(100mL×3)后用无水硫酸钠干燥,过滤后减压除去溶剂,柱层析纯化后得到浅色油状物2(6.18g)。产率84.1%。1H NMR(400MHz,CDCl3):7.53(s,1H),7.30(d,1H,J=8.4Hz),6.72(d,1H,J=8.4Hz),4.68(s,2H),4.26(q,2H,J=7.2Hz)and 1.29(t,3H,J=7.2Hz)。
步骤2:2-(4-溴-2-氯苯氧基)乙酸(即化合物3)的合成
在反应瓶重加入化合物2(5.0g,17.0mmol),50mL二氧六环和1M NaOH (50mL),室温下搅拌过夜后,将反应混合物用1M HCl酸化至pH=3。将酸化后的反应液用乙酸乙酯(50mL×4)萃取,有机相合并后用饱和食盐水洗涤(50mL),无水硫酸钠干燥。过滤除去干燥剂后,减压浓缩可得白色固体3(4.69g)。产率94.3%。1H NMR(400MHz,DMSO-d6):7.66(s,1H),7.44(d,1H,J=8.8Hz),6.97(d,1H,J=8.8Hz),4.72(s,2H)。
步骤3:4-(3-(2-(4-溴-2-氯苯氧基)乙酰基)硫脲基)-N-(4,6-二甲基-2-嘧啶基)苯磺酰胺(即化合物5)的合成
在反应瓶中依次加入化合物3,25mL二氯亚砜以及1滴DMF,将反应体系加热至回流。回流3小时后,常压蒸馏除去二氯亚砜,将剩余的液体用油泵减压干燥5分钟后即得化合物4(2-(4-溴-2-氯苯氧基)乙酰氯)。在另一反应瓶中将硫氰酸钠(326.8mg,4.0mmol)溶于10mL丙酮冰浴冷却至0℃,将化合物4用10mL丙酮稀释后逐滴加入到上述反应液中,滴加完毕后撤去冰浴,用油浴加热反应体系至30℃反应。在30℃反应2小时后,再次将反应液冷却至0℃,加入4-氨基-N-(4,6-二甲基-2-嘧啶基)-苯磺酰胺(556mg,2.0mmol)后升至室温搅拌过夜。反应完毕后,过滤反应液得到的固体用水和丙酮洗涤后,可得黄色粉末5(276mg)。产率23.6%。1H NMR(400MHz,DMSO-d6):12.19(s,1H),11.68(s,1H),11.52(br s,1H),7.99(d,2H,J=8.4Hz),7.86(d,2H,J=8.4Hz),7.70(d,1H,J=1.6Hz),7.49(d,1H,J=8.8Hz),7.10(d,1H,J1=8.8Hz,J2=1.6Hz),6.75(s,1H),5.02(s,2H),2.25(s,6H).HPLC纯度95.9%(254nm)。
实施例3:ZCL278活性特性
1、ZCL278抑制Cdc42介导的微棘形成
以去血清培养的小鼠成纤维细胞Swiss 3T3测试候选30个化合物对Cdc42介导的细胞微棘/丝状伪足形成的作用。成纤维细胞actin组成的微棘/丝状伪足是Cdc42活性的特征。如图2A所示,对照组细胞边缘可见少数微棘(小箭头标识)和特征性的RhoA介导形成的应力纤维(星号标识)。用1单位/mL的Cdc42激动剂(cytoskeleton公司产品)短暂地刺激细胞后,可见明显的微棘数量增多和应力纤维的减少。加入以上激动剂作用2分钟,然后以50uM的ZCL278处理细胞1小时,相比单独的激动剂作用ZCL278明显抑制微棘的形成。这结果显示该化合物可作为Cdc42抑制剂的候选进行后续研究。在30个化合物的测试中ZCL278显示最强的抑制作用。
具体如图2A所示。图2A表示ZCL278抑制Cdc42介导的细胞微棘形成。计算机虚拟筛选出的小分子配体分别作用于无血清培养的Swiss3T3细胞。DMSO作为阴性对照,1单位/mL的Cdc42激动剂(cytoskeleton公司产品)短暂地刺激细胞(1分钟)。激动剂作用1分钟,然后以50uMZCL278处理细胞1小时。同样条件用10uM的NSC23766作用细胞作为参比。细胞固定后用rhodamine-Phalloidin荧光抗体杂交标识F-actin。细胞边缘可见极少的微棘(箭头标识),星号指示正常的应力纤维分布。Bar:5um。
2、ZCL278抑制Cdc42活性
Cdc42活性相关的形态学测试中ZCL278表现最好,接下来则在生化水平上测试它的活性。首先,分别以Cdc42激动剂和ZCL278(50uM)作用人转移性前列腺癌细胞PC3 5、10、15分钟,细胞裂解液提取蛋白样品进行Westernblot,检测其中磷酸化Rac/Cdc42(上层)、磷酸化WASP蛋白的水平(中层),GAPDH蛋白作参比(下层)。71位丝氨酸残基的磷酸化是RAC/Cdc42蛋白活性的负调控机制,所以磷酸化Rac/Cdc42蛋白表达的增加指示活性Rac/Cdc42(GTP结合型)的减低。如图2B所示,激动剂作用下磷酸化Rac/Cdc42蛋白的表达持续降低,而50uM的ZCL278则时间依赖性地提高了该蛋白的表达水平。
WASP蛋白是Cdc42活化的下游效应器,通过Arp2/3复合物作用诱导细胞骨架actin重组和微棘、丝状伪足的形成,其酪氨酸磷酸化与Cdc42活化后快速降解相关。如图2B所示,激动剂作用15分钟后减少磷酸化WASP蛋白的表达,而相应时间内ZCL278并没抑制该蛋白的表达。此数据显示ZCL278能够抑制磷酸化Rac/Cdc42蛋白的内生水平,呈时间依赖性,并可维持酪氨酸磷酸化WASP蛋白的水平。
Rac和Cdc42蛋白都可以发生71位丝氨酸残基的磷酸化。为直接检测Cdc42的活化与失活,接下来用G-LISA试剂盒定量检测GTP结合型Cdc42的水平。用1单位/mL的Cdc42激动剂刺激细胞1分钟,然后分别用50uM的ZCL278或10uM的Rac激动剂NSC23766处理swiss 3T3细胞1小时,提取细胞蛋白样品,用G-LISA试剂盒定量检测GTP结合型Cdc42的水平。未加任何处理的细胞作为阴性对照,仅激动剂处理的细胞作为阳性对照,另外缓冲液和活化Cdc42蛋白也分别作为阴性对照和阳性对照。如图2C所示(图中数据为三次独立实验的平均结果,±标示平均差,**:p<0.01,*:p<0.05),相比阴性对照,激动剂作用使活化的Cdc42蛋白水平明显增高(70%);对比阳性对照,ZCL278则使活化的Cdc42蛋白水平明显降低(80%)。NSC23766如预期地对Cdc42没有影响。这些数据说明在两种类型的细胞上,ZCL278既可抑制刺激型也可抑制内生型的Cdc42活性。
实施例4:活化Cdc42/磷酸化RhoA的免疫荧光染色
1、ZCL278,而非NSC23766,破坏活化Cdc42在细胞核外周的分布
免疫荧光染色:Swiss3T3细胞在盖片上生长密度大约30%.加药时去血清培养,加入1单位/mL的Cdc42激动剂(cytoskeleton公司产品)作用1分钟,然后以50uM的ZCL278或10uM的NSC23766分别处理细胞1小时。单独加激动剂作为阳性对照,DMSO作为阴性对照。4%多聚甲醛固定细胞盖片15分钟,0.2%Triton X-100透化15分钟,10%BSA 37度封闭30分钟。
抗体孵育:一抗:活化Cdc42(鼠抗,Neweast Biosciences公司);磷酸化RhoA抗体(兔抗,Santa Cruze Biotechnology公司);GM130抗体(鼠抗,BD Biosciences公司),1∶100稀释使用。相应二抗杂交后,rhodaminePhalloidin室温孵育1小时用以观察actin。Zeiss Axiovert电子显微镜观察细胞染色。用图像处理软件MetaMorph随机选取5个细胞随机选取5个点的pixel进行均数计算即得各组平均像素强度值。
Westernblot:PC3细胞培养至约70%的密度,撤除血清继续培养16小时,分别加药物:1单位/mL的Cdc42激动剂(cytoskeleton公司产品),5uM、50uM的ZCL278处置细胞5、10、15分钟。细胞裂解缓冲液(配方:50mMTris缓冲液PH7.5,10mM氯化镁,0.5M氯化钠,1%Triton X-100,蛋白酶抑制剂)裂解细胞,14000转4度离心提取细胞蛋白样品。等浓度蛋白样品进行Westernblot分析,抗体:磷酸化Rac1/Cdc42(Milipore公司)、磷酸化WASP(Assay Biotech公司),均以1∶1000比例稀释杂交,GAPDH抗体(Calbiochem公司)1∶2000稀释杂交。PVDF膜用化学发光法显影。
G-LISA试剂盒分析:Swiss3T3细胞培养至40%的生长密度,撤除血清培养48小时,加Cdc42激动剂作用1分钟,再分别以50uM的ZCL278和10uM的NSC23766处理。按照试剂盒说明书裂解细胞提取蛋白、定量总蛋白浓度0.15mg/ml进行分析。未加任何处理的细胞和缓冲液作为阴性对照,仅激动剂处理的细胞和活化的Cdc42蛋白作为阳性对照。酶标法测定各样品光波490nm时的吸光度值。
细胞水平上检测ZCL278对Cdc42活化的选择性抑制:去血清的Swiss3T3细胞加1单位/ml Cdc42激动剂作用2分钟,再分别以50uM的ZCL278或10uM的NSC23766处理,DMSO做阴性对照。为检测ZCL278的作用是选择性抑制Cdc42活性而非对RhoA的作用,细胞分别用活化Cdc42(图3A、图3B)和磷酸化RhoA(图3C)抗体免疫杂交。箭头:细胞核外缘高尔基体-内质网网络;Hoechest染色剂标识细胞核。Bar:15um。如图3A所示,细胞化学免疫荧光染色:小鼠单抗GTP结合型Cdc42抗体和Hoechest(赫斯特)染色剂(标识细胞核)。对照组细胞呈现活化的Cdc42在细胞核外周有组织的分布;激动剂作用使其分布明显增加,并在核内也有分布,这与Cdc42参与高尔基体蛋白运输的作用相一致;ZCL278明显破坏了这种组织性的分布,并降低与抗活化Cdc42抗体的免疫反应性;而NSC23766对此没有影响。图3B显示各组高尔基体样分布的细胞数:活化Cdc42抗体识别的细胞核外周高尔基体-内质网网络有组织分布的量化:随机选取细胞的6个各自独立的区间计数高尔基体-内质网网络(*:p<0.05)。图3C:磷酸化RhoA信号的平均像素强度值定量,数据来自5个独立细胞随机5个信号的像素强度值取平均值。±标示平均差,*:p<0.03:激动剂、ZCL278、NSC23766均未显示对磷酸化RhoA的影响。这结果表明ZCL278选择性抑制Cdc42活性。
2、ZCL278,而非NSC23766,破坏细胞内GM130蛋白对接高尔基体的组织
为了解ZCL278破坏细胞核外周的活化Cdc42的分布是否反映其对高尔基体的组织也有作用,设计实验检测GM130蛋白-一种细胞质外缘的蛋白质,紧密连接高尔基体膜,以维持高尔基体的顺式结构。
Swiss3T3细胞去血清培养,分别加入Cdc42激动剂、ZCL278、NSC23766,DMSO作为阴性对照。细胞用Rhodamine-phalloidin(红色)、抗GM130抗体(绿色)、Hoechest(蓝色)免疫荧光染色。如图4所示,其中箭头指示细胞微棘(实际试验中为红色荧光),星号指示GM130抗体标记的高尔基体结构(实际试验中为绿色荧光)。去血清的Swiss3T3细胞对照组显示特征性应力纤维(如图中的箭头所示,实际试验中为红色荧光),对应细胞核单侧边的GM130抗体分布(如图中的星号所示,实际试验中为绿色荧光)。Cdc42激动剂处理的细胞可见微棘的增多,GM130在细胞核外缘分布增多。ZCL278处理的细胞表现明显的微棘减少和GM130的减少(ZCL278:实际试验中为红色荧光),并且向细胞核两侧驱散(如图中的星号所示,实际试验中为绿色荧光)。NSC23766对GM130的表达以及分布没有明显影响。Bar:10um。这结果不仅更加说明ZCL278对Cdc42的选择性抑制作用,还证明了Cdc42在高尔基体的组织和物质运输中发挥重要作用。
实施例5:细胞伤口愈合实验:ZCL278阻碍细胞伤口愈合,但不影响细胞活率
丝状伪足是细胞的运动结构,是细胞迁移导向和生长路径导引,主要由Cdc42活性调控。PC3细胞单层长满6孔板,去血清培养,用1ml枪头每孔分别做三个愈伤区域,无血清培养基洗去剥落下的细胞。分别以1单位/mlCdc42激动剂、50uM、5uM的ZCL278和10uM的NSC23766处置细胞24小时。Cdc42激动剂作为阳性对照,未加任何处置的作为阴性对照。药物作用0时和24小时分别拍下细胞影像,用MetaMorph图形软件分析细胞迁移的距离。黑色线条指示愈伤区域的界限。每次实验结果均进行假设检验p值分析(p>0.05)。
细胞活率检测:以75000/ml细胞密度种下培养PC3细胞48小时,撤除血清与50uM的ZCL278或10uM的NSC23766继续培养24小时,胎盘蓝染色计算细胞活率。
如图5A和图5B(取愈伤区域边界间最短距离定量区域宽度。柱状图表示对比初始愈伤区域的百分比,三次独立实验取平均值,±标示平均差,**:p<0.01,*:p<0.05)所示,对比阴性对照组(41%愈合率),激动剂显著增强细胞伤口的愈合能力(59%);ZCL278两个浓度条件下均能抑制细胞的迁移,高浓度时抑制效应更强(50uM-8%;5uM-30%);NSC23766也显示了明显的抑制细胞迁移效应(因为Rac蛋白调控细胞片状伪足--同样是细胞运动的结构的形成)。这一结果与生化检测数据结果一致,提示ZCL278不仅是Cdc42的抑制剂,还可有效抑制依赖于Cdc42的细胞运动。
为说明ZCL278抑制细胞迁移是源于抑制Cdc42活性的作用(NSC23766抑制Rac活性作用),而非因为导致细胞死亡,以胎盘蓝染色法测试细胞活率。PC3细胞阻滞于G0期,分别用50uM的ZCL278和10uM的NSC23766处置细胞24小时,如图5C所示(PC3细胞以75000/ml细胞密度种下,培养48小时,撤除血清与50uM的ZCL278或10uM的NSC23766继续培养24小时,胎盘蓝染色计算细胞活率),对比阴性对照组,药物处理的细胞活率没有差别。因此可以认为药物未影响细胞生长而是抑制Cdc42或Rac蛋白活性导致的抑制细胞迁移作用。
实施例6:ZCL278抑制神经元分支和生长锥动力学
Cdc42调控神经突的分支与生长。Garvalor等人通过Cdc42基因敲除技术证实了Cdc42对于神经元形态生成的关键性作用,决定着神经元命运,采集的神经元中Cdc42的缺失导致明显的神经突数量减少,丝状伪足功能的严重破坏。所以将检测ZCL278抑制神经元分支的作用。
原代培养一日龄出生小鼠取其大脑组织,放置于含0.25%胰酶的HBSS缓冲液中37度培养15分钟,小心吹打分离神经元铺种细胞于Poly-L-lysine包被的细胞盖片上,用含胎牛血清的DMEM培养液培养16小时,然后换Neurobasal培养液(Invitrogen公司)继续培养。至培养第五天分别用DMSO或50uM的ZCL278作用细胞5、10分钟,随后即以4%多聚甲醛固定细胞15分钟,荧光抗体Fluorescein-phalloidin杂交标记F-actin结构,Zeiss显微镜下观察神经元形态。
为观察ZCL278对神经元生长锥动力学的作用采用了显微镜下的缩时摄影技术,用Hamamatsu Orca数字照相机记录十分钟内放大63倍的细胞影像,300毫秒曝光拍摄以最大程度减低对细胞的光毒性,用MetaMorph图形软件分析影像并统计分析。
原代培养新生中枢的神经元,如图6A所示,培养的第五天,可见神经元长出一定的分支,用50uM的ZCL278分别处置神经元5、10分钟,DMSO作为阴性对照。随时间推移,ZCL278抑制神经元的分支,定量分析显示药物处置后神经元分支数量明显减少(图6B:定量ZCL278处置后神经元分支数量:3次独立实验取平均值。±标示平均差,*:p<0.01)。
Cdc42被公认可调控生长锥导向端微棘突和丝状伪足的形成。缩时摄影图像(图6C)展示对照组神经元细胞从生长锥处外生出不少微棘突或丝状伪足;但是,ZCL278在四分钟内即引起丝状伪足快速的收回。Bar:1um。所以,这进一步说明ZCL278是一个有效调控Cdc42介导神经元分支和生长锥动力学的小分子抑制剂。
本发明应用高通量计算模拟方法筛选嵌合Cdc42分子结合GEFs的关键结构的化合物。基于Cdc42结合其特异性GEF分子intersectin(ITSN)的结构特征,化合物的三维结构恰好可填充intersectin分子连接的口袋的被继续进行相关研究。由此成功筛选出一个化合物ZCL278,具有细胞透性的Cdc42特异性抑制剂。
本发明证实ZCL278的活性特性,作为第一个小分子Cdc42抑制剂,选择性地直接靶向作用于Cdc42与其GEF的连接。利用细胞伤口愈合实验,可见Cdc42激活后促进愈伤区域的合拢,说明Cdc42活化后促进肿瘤细胞的转移。ZCL278则显著抑制PC3细胞的迁移,具浓度依赖性。而且ZCL278不是细胞毒类物质,并非引起肿瘤细胞的死亡而导致的抑制细胞迁移效应。
本发明中应用新生中枢神经元的实验也证明了Cdc42在神经元发展中所起的重要作用。Garalov等的实验显示Cdc42缺陷型小鼠的大脑和神经元发展被严重破坏,这些小鼠表现一系列的大脑畸形,包括轴突束的减少等,以及神经元丝状伪足动力学降低,生长锥变大,轴突生成抑制。其实轴突和树突的运动主要是以actin为基础,这一进程是由Cdc42调控的。ZCL278能够减少新生中枢神经元的分支数,并抑制生长锥的动力学。综上所述,ZCL278是第一个靶向Cdc42-ITSN连接的小分子抑制剂,能够有效用于肿瘤和神经病变中Cdc42分子功能研究。
虽然本发明已以较佳实施例披露如上,然其并非用以限定本发明,任何所属技术领域的技术人员,在不脱离本发明之精神和范围内,当可作些许之更动与改进,因此本发明之保护范围当视权利要求所界定者为准。
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US11439608B2 (en) | 2017-09-25 | 2022-09-13 | Qun Lu | Roles of modulators of intersectin-CDC42 signaling in Alzheimer's disease |
CN115068480B (zh) * | 2022-08-09 | 2023-10-20 | 郑州大学第一附属医院 | 细胞分裂周期蛋白42小分子抑制剂在制备治疗慢性肾脏病药物中的应用 |
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EP3999053A4 (en) * | 2019-07-17 | 2023-07-19 | Children's Hospital Medical Center | METHOD OF TREATING NEOPLASTIC DISEASES USING A SPECIFIC CDC42 INHIBITOR |
CA3151110A1 (en) * | 2019-08-16 | 2021-02-25 | Children's Hospital Medical Center | Methods of treating a subject with a cdc42-specific inhibitor |
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