CN100340288C - 钙调磷酸酶的持久的活化抑制剂 - Google Patents
钙调磷酸酶的持久的活化抑制剂 Download PDFInfo
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Abstract
本发明目的在于提供抑制持久的钙调磷酸酶活化、副作用少、对各种各样疾病有效的神经细胞死亡抑制剂。详细来说钙调磷酸酶的持久的活化抑制剂是抑制钙激活蛋白酶切断钙调磷酸酶A亚基(CaNA)的药物。例如,具有FDGATAAARKEVIRNK(序列1)和REESESVLTLKGLTPTG(序列2)的氨基酸序列的肽。
Description
技术领域
本发明涉及到钙调磷酸酶的持久的活化抑制剂。详细讲本发明涉及到抑制钙调磷酸酶A亚基(CaNA)被钙激活蛋白酶切断的药物。
背景技术
钙调磷酸酶(calcineurin)是依赖于钙和钙调蛋白的被活化的脱磷酸化酶,是由具有活性中心的钙调磷酸酶A亚基(CaNA)和作为调节因子的钙调磷酸酶B亚基(CaNB)构成的复合体。钙调磷酸酶由于通过自身抑制结构域抑制活性中心与底物的结合,所以通常在细胞内是非活性型。如果细胞内的钙浓度上升,钙调磷酸酶的结构变化,自身抑制结构域打开(活性型),底物就可以与活性中心结合。另外,如果钙浓度减少,钙调磷酸酶再恢复到非活性型。如上所述,可知钙调磷酸酶被可逆活化依赖于钙浓度。
1999年报道了钙调磷酸酶对神经细胞死亡具有重要的作用(参照Asai Akio等、J.Biological Chemistry、第274卷、P.34450、1999年)。另外还报道了钙调磷酸酶的特异抑制剂(免疫抑制剂FK506和环孢子菌素A)也抑制神经细胞死亡(参照Morioka Motohiro等、Progressin Neurobiology、第58卷、P.1、1999年和Springer Joe E等、TheJournal of Neuroscience、第20卷、P.7246、2000年)。根据包括这些文献在内的很多报告,关于在脑缺血和脊椎损伤等中所看到的神经细胞死亡,现在提出了由于神经细胞的兴奋,作为谷氨酸受体之一的N-甲基-D-天冬氨酸受体(NMDA受体)异常活化,大量的钙经钙受体流入细胞内导致钙调磷酸酶被活化,诱发细胞死亡的机制。然而,该钙的流入是瞬时的,钙调磷酸酶的活化状态不能持续很长时间。因此,想象了存在着另外的不是一过性细胞内钙浓度上升引起的钙调磷酸酶被长期活化的机制,但有关这样的机制还没有报道。
如上所述,对于神经细胞死亡的抑制、具体来说对于缺血性和兴奋性神经细胞死亡的抑制,已知免疫抑制剂FK506和环孢子菌素A有效。然而,已知这些药物由于抑制钙调磷酸酶参与的所有信息传递,所以副作用大(例如肾毒性和糖尿病等)。因此强烈盼望抑制长时间的钙调磷酸酶活化、而且与以往药物相比副作用更小的神经细胞死亡的抑制剂。
发明内容
本发明目的在于解决上述那样问题,提供副作用小、对各种各样疾病有效的神经细胞死亡抑制剂。
为了解决上述课题,进行不断研究,结果获得了以下见解。
1)通过给予谷氨酸诱导神经细胞死亡,确认钙调磷酸酶A亚基(CaNA)的断裂。
2)CaNA的切断部位在氨基酸序列中的第392位(精氨酸)和393位(赖氨酸)之间以及第421位和425位之间。
3)如果钙调磷酸酶在上述部位被切断,表明钙调磷酸酶具有钙和钙调蛋白非依赖性活性。
4)上述断裂是由认为属于与钙调磷酸酶不同的信息传递途经的钙激活蛋白酶引起的,可被钙激活蛋白酶抑制剂抑制。
5)如果将含有CaNA的392~393氨基酸残基的肽或含有CaNA的421~425氨基酸残基的肽导入神经细胞内,就会抑制给予谷氨酸引起的神经细胞死亡。
根据以上见解,开发以不是由于瞬时的细胞内钙浓度上升引起的钙调磷酸酶活化、而是以长期的钙调磷酸酶活化的机理作为目标的细胞死亡抑制剂,完成了本发明。即本发明涉及到:
(1)一种含有序列1的肽、序列2的肽和/或他们的类似物的抑制CaNA被钙激活蛋白酶切断的抑制剂,
(2)一种以上述(1)所述CaNA的切断抑制剂为有效成分的神经细胞死亡抑制剂,
(3)一种以上述(1)所述CaNA的切断抑制剂为有效成分的痴呆性疾病进展的抑制剂,以及
(4)一种含有上述(1)所述CaNA的切断抑制剂的细胞和脑切片培养基的添加剂。
附图的简单说明
图1是表示通过添加谷氨酸诱导神经细胞死亡时的CaNA的切断、以及通过本发明的CaNA的抑制剂产生的抑制CaNA切断的抑制效果的照片。图1(a)表示添加谷氨酸之后温育3小时后的样品的结果,图1(b)表示添加谷氨酸之后温育24小时后的样品的结果。在图1(a)和(b)中,带1表示对照、带2表示只添加谷氨酸的样品、带3表示只添加切断抑制肽的样品、带4表示添加谷氨酸和切断抑制肽的样品。
图2表示由于添加谷氨酸诱导神经细胞死亡的神经细胞的数目。
具体实施方式
本发明的抑制钙调磷酸酶A亚基(CaNA:序列3)被钙激活蛋白酶切断的抑制剂,是含有序列1的肽、序列2的肽和/或他们的类似物的化合物,指的是抑制钙激活蛋白酶切断CaNA的392~393氨基酸残基间以及421~425氨基酸残基之间部位,抑制钙调磷酸酶持久活化的物质。
作为上述序列1的肽的类似物,例如由在序列1肽的氨基酸序列中的一部分被缺失、置换和/或插入其他氨基酸序列,或在肽末端附加其它氨基酸序列的氨基酸序列构成,而且包含抑制钙激活蛋白酶切断CaNA的肽。本发明的序列1的肽的类似物中包括例如,序列1的肽序列中的第9位氨基酸残基Arg被置换为Lys后的肽和/或第10位的氨基酸残基Lys被置换为Arg的肽。
作为上述序列2的肽的类似物,例如由在序列2肽的氨基酸序列中的一部分被缺失、置换和/或插入其他氨基酸序列,或在肽末端附加其它氨基酸序列的氨基酸序列构成,而且包含抑制钙激活蛋白酶切断CaNA的肽。在本发明的序列2的肽的类似物中包括例如,序列2的肽序列中的第11位氨基酸残基Lys被置换为Arg后的肽。
序列1或序列2的肽类似物(类似肽)的抑制CaNA切断的抑制活性可以象以下那样进行评价。即,在制备试验溶液(含有0.1μM或10μM类似肽、5μM纯化钙激活蛋白酶,20mM Tris-HCl(pH7.4)和1mMCaCl2)后,添加纯化钙调磷酸酶(终浓度1μM),于30℃下进行温育1小时。然后通过10%SDS-PAGE对反应液在凝胶上进行电泳,凝胶用考马斯亮篮染色。通过对染色结果看到的分子量45kDa和48kDa的钙激活蛋白酶依赖性片段化钙调磷酸酶进行定量,可以进行评价。
上述肽可以使用Boc法或Fmoc法的固相或液相合成等众所周知的方法进行制造。另外,这样制造的肽也可以用醚沉淀-过滤法、高效液相色谱(HPLC)、Perfusion Chromatography等众所周知的方法进行纯化。
本发明的抑制钙激活蛋白酶切断CaNA的抑制剂只要是含有序列1的肽、序列2的肽和/或他们的类似物,也可以含有其他的化合物。其他的化合物,例如聚精氨酸肽(例如,由5个精氨酸构成的聚精氨酸肽)、由HIV病毒的TAT蛋白质中含有的11个氨基酸残基构成的蛋白质导入结构域(PTD;序列4)这样的含有50%以上的精氨酸或赖氨酸的7~30个构成的细胞内导入信号肽,或作为阳离子性水溶性聚合物的直链聚乙烯亚胺(PEI)等。这些化合物利用(i)通过通常的肽合成连接到序列1或序列2的肽和/或他们的类似物的合成的方法,或(ii)使其中的一个肽连接2价性交联剂,另一个肽的末端连接半胱氨酸残基,通过使两个肽反应的方法等,可以与序列1或序列2的肽和/或他们的类似物结合(融合)。这样得到的CaNA的切断抑制剂可以使用上述的纯化方法进行纯化。
本发明的神经细胞死亡抑制剂作为有效成分含有上述抑制钙激活蛋白酶切断CaNA的抑制剂,指的是抑制神经细胞中长时间的神经细胞死亡诱导的药物。神经细胞死亡抑制剂也可以作为与神经细胞死亡有关的疾病的预防或治疗剂使用。本发明的神经细胞死亡抑制剂优选是含有由序列2的肽和细胞内导入信号肽构成的CaNA的切断抑制剂,更优选含有由序列1的肽、序列2的肽和细胞内导入信号肽构成的CaNA的切断抑制剂。与神经细胞死亡有关的疾病的预防或治疗剂指的是含有用于与神经细胞死亡有关的疾病的预防或治疗的有效量的神经细胞死亡抑制剂的药物。作为与神经细胞死亡有关的疾病,如阿耳茨海默病、痴呆性疾病、脑缺血性疾病、蛛网膜下出血等脑内出血、脊髓损伤、帕金森病以及癫痫等。
作为本发明的神经细胞死亡抑制剂的给药途经,可举出如经口给药、经静脉给药以及脑内直接给药等,从对患者的负担、副作用观点考虑经口给药更好。
本发明的神经细胞死亡抑制剂的剂型,可以根据给药方法适当设定。具体来说,如水溶液、乳剂、悬浊液等液剂、片剂以及胶囊等。例如,经口给药时,优选片剂或胶囊,经静脉给药或脑内直接给药时优选液体剂。本发明的神经细胞死亡抑制剂的制剂化中,可以使用与该剂型相匹配的通常本领域技术人员都使用的各种各样的添加物。例如防止氧化剂、pH调整剂、防腐剂等。
上述神经细胞死亡抑制剂的给药量可以根据给药方法、适用的患者的年龄、体重和病状等适当设定。例如,换算为本发明的抑制钙激活蛋白酶切断CaNA的抑制剂,优选每天0.1mg/kg以上,更优选1mg/kg以上。给药量比0.1mg/kg少时,存在着CaNA切断抑制效果减半的倾向。另外,换算为本发明的抑制钙激活蛋白酶切断CaNA的抑制剂,优选每天100mg/kg以下,更优选20mg/kg以下。给药量超过100mg/kg时,存在表现出细胞毒性的倾向。本发明的神经细胞死亡抑制剂的给药可以一次或多次进行。
本发明的细胞培养基或脑切片培养基的添加剂指的是至少含有CaNA的切断抑制剂的培养基的添加剂。本发明的细胞培养基的添加剂优选含有由序列2的肽和细胞内导入信号肽构成的CaNA的切断抑制剂,更优选含有由序列1的肽、序列2的肽和细胞内导入信号肽构成的CaNA的切断抑制剂。
应用于培养细胞时,作为本发明的抑制钙激活蛋白酶切断CaNA的切断抑制剂的添加量,在细胞浓度1×105细胞/ml的培养液优选0.01~100nmol/ml,更优选0.1~10nmol/ml。添加量少于0.01nmol/ml时,CaNA的切断抑制剂的效果有减半的倾向,超过100nmol/ml存在表现出细胞毒性的倾向。
以下按照实施例对本发明进行更详细说明,但本发明并不受这些实施例的限制。
实施例1
作为本发明的CaNA的切断抑制剂,为了将FDGATAAARKEVIRNK(序列1)和REESESVLTLKGLTPTG(序列2)导入到培养细胞内,制造如下所示在N末端附加了细胞内导入信号肽(10个精氨酸)的寡肽(肽研究所公司生产)。
RRRRRRRRRRFDGATAAARKEVIRNK(序列5)
RRRRRRRRRRREESESVLTLKGLTPTG(序列6)
(神经细胞的制备)
将Wister大鼠胎仔第18天的脑海马摘出后,用含有0.05%胰蛋白酶的PBS于37℃处理15分钟。使用玻璃移液管使神经细胞分散后,在预先用聚-D-赖氨酸包被的直径3.5cm培养皿中培养1×106个细胞。培养基使用添加了B27supplement(0.03ml;Invitrogen,Inc.公司生产)、盘尼西林(终浓度100单位/ml;Invitrogen公司生产)以及链霉素(终浓度100μg/ml;Invitrogen公司生产)的3ml Neuro Basa1培养基(Invitrogen公司生产),于二氧化碳培养箱(5%CO2、37℃)中进行培养。
(肽的添加)
在培养开始10天后,向培养液中添加终浓度为1μM的上述序列5和6的肽,于二氧化碳培养箱(5%CO2、37℃)中进行培养。添加3小时后,添加终浓度500μM的谷氨酸,培养15分钟。然后交换培养液,再进行培养。在添加谷氨酸后3小时和24小时后,回收细胞,于1%SDS溶液中对细胞进行超声粉碎,加SDS-PAGE缓冲液。相同样品经SDS-PAGE凝胶电泳后,用识别CaNA的抗体(兔血清、Santa CruzBiotechnology,Inc公司生产)进行免疫印迹(Western blotting)。另外,在本实施例中,作为对照使用不添加谷氨酸和切断抑制肽的细胞样品。另外作为平行,也制作只添加谷氨酸的细胞样品和只添加切断抑制肽的细胞样品。
结果如图1所示。在添加谷氨酸组中,确认钙调磷酸酶的片段化。而在导入切断抑制肽的神经细胞中,由于添加谷氨酸,通常可发生的钙调磷酸酶的切断被抑制。
实施例2
与实施例1同样,对神经细胞进行培养,然后与实施例1同样向神经细胞中添加上述那样的序列5和6的肽使终浓度为1μM。作为对照,加钙调磷酸酶抑制剂FK506(フジサワ制药株式会社;终浓度1μM)或钙激活蛋白酶抑制剂ALLM(Merk公司生产;终浓度25μM)。将各个药物温育2小时后,添加终浓度500μM的谷氨酸,温育15分钟。然后,交换培养液,再进行培养。添加后3小时、6小时、12小时或24小时后,各个神经细胞用4%多聚甲醛固定。然后,为了鉴定发生细胞死亡的神经细胞,进行TUNEL染色(Roche Diagnostics,Inc生产)。对TUNEL阳性细胞进行计数。
结果如图2所示。在给予谷氨酸组中,随着给药后时间推移,发生细胞死亡的神经细胞数上升。判明切断抑制肽具有抑制因谷氨酸诱导的神经细胞死亡的效果。另外,其效果具有与FK506、ALLM同等程度的效果。
参考例1
将从牛脑中纯化的钙调磷酸酶1μM在1μM钙调蛋白(Merck,Inc生产)和/或1μM m-钙激活蛋白酶(Merck,Inc生产)反应液中(含有20mM Tris-HCl、pH7.4、1mM CaCl2、1mM MgCl2)于30℃分别反应1小时,然后进行12%SDS-PAGE凝胶电泳后,用考马斯亮篮对该凝胶进行染色。
结果没有添加钙激活蛋白酶时,看到纯化CaNA在电泳凝胶上60Kda的大小。这与到目前为止报道的CaNA的大小一致。而添加钙调蛋白和钙激活蛋白酶时,在60Kda没有看到带,在48和45Kda看到了带。如果只与钙激活蛋白酶反应,确认被切成45Kda大小的CaNA。
上述结果表明钙调磷酸酶被钙激活蛋白酶切断了。
另外确定钙激活蛋白酶切断CaNA的切断部位的结果表明,45Kda切断蛋白由到氨基酸序列第392位之前的氨基酸构成,48Kda的切断蛋白由到421位之前、422之前、423之前、以及424之前的氨基酸构成。
本发明提供CaNA的切断抑制剂。本发明的CaNA切断抑制剂由于可以抑制钙调磷酸酶的不可逆活化,所有可以抑制由该活化诱发的神经细胞死亡。另外,含有CaNA的切断抑制剂的本发明的神经细胞死亡抑制剂由于可以作为与痴呆性疾病等神经细胞死亡有关的疾病的预防或治疗药使用,所有非常有用。另外,如果使用含有CaNA的切断抑制剂的本发明的培养细胞培养基的添加剂,也可以更好地使培养细胞增殖。另外,本发明的CaNA的切断抑制剂也可以作为与神经细胞死亡有关的研究用试剂使用。
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Claims (4)
1.一种抑制钙激活蛋白酶切断钙调磷酸酶A亚基的切断抑制剂,其含有序列1的肽和/或序列2所示的肽。
2.一种神经细胞死亡抑制剂,以权利要求1所述的钙调磷酸酶A亚基的切断抑制剂为有效成分。
3.一种痴呆性疾病的进行抑制剂,以权利要求1所述的钙调磷酸酶A亚基的切断抑制剂为有效成分。
4.一种细胞和脑切片培养基的添加剂,含有权利要求1所述的钙调磷酸酶A亚基的切断抑制剂。
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- 2003-10-07 WO PCT/JP2003/012816 patent/WO2004032955A1/ja active Application Filing
- 2003-10-07 KR KR1020057005991A patent/KR100833728B1/ko not_active IP Right Cessation
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Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2002000872A2 (fr) * | 2000-06-29 | 2002-01-03 | Exonhit Therapeutics Sa | Compositions et methodes pour le traitement ou la detection de pathologies neurodegeneratives |
Non-Patent Citations (2)
Title |
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activation of a calmodulin-depentdetn phosphatase by aca2+dependetn protease TALLTANT EA et al,Biochemistry,Vol.27 No.6 1988 * |
Calpain-dependent cleavage of cain/cabin1 activatescalcineurin to mediate calcium-triggered cell death. KIM,M.J. et al,Proc.Natl.Acad.Sci.,Vol.99 No.15 2002 * |
Also Published As
Publication number | Publication date |
---|---|
JPWO2004032955A1 (ja) | 2006-02-09 |
EP1552847A1 (en) | 2005-07-13 |
KR20050065581A (ko) | 2005-06-29 |
WO2004032955A1 (ja) | 2004-04-22 |
KR100833728B1 (ko) | 2008-05-29 |
TW200407160A (en) | 2004-05-16 |
US7183375B2 (en) | 2007-02-27 |
EP1552847A4 (en) | 2009-10-28 |
JP4642469B2 (ja) | 2011-03-02 |
TWI341208B (zh) | 2011-05-01 |
US20060177431A1 (en) | 2006-08-10 |
CN1691960A (zh) | 2005-11-02 |
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