CN109982717B - 用于修改昼夜节律钟的方法和化合物 - Google Patents

用于修改昼夜节律钟的方法和化合物 Download PDF

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CN109982717B
CN109982717B CN201880004492.8A CN201880004492A CN109982717B CN 109982717 B CN109982717 B CN 109982717B CN 201880004492 A CN201880004492 A CN 201880004492A CN 109982717 B CN109982717 B CN 109982717B
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dinaciclib
inhibitor
kinase
circadian
hper2
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余腾辉
饶毅
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Beijing Yuanji Huayi Biotechnology Co ltd
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Abstract

通过施用昼夜节律相位移动hPER2磷酸化激酶抑制剂来调控昼夜节律。

Description

用于修改昼夜节律钟的方法和化合物
本申请要求于2017年3月3日向中国知识产权局提交的题为“Method andCompound for Modifying Circadian Clock”的PCT专利申请PCT/CN2017/075570的优先权，其以其全部内容通过引用并入本文。
发明领域
本发明涉及调控昼夜节律的方法、化合物和组合物。
发明背景
由于每年有超过10亿的跨时区旅行者和20％的西方工人进行轮班工作(Roenneberg and Merrow,2016)，与昼夜节律的紊乱相关的健康问题是相当令人担忧的(Ohlander et al.,2015；Parsons et al.,2015；Reutrakul and Knutson,2015；Roenneberg et al.,2012；Schernhammer et al.,2003；Sigurdardottir et al.,2012；Tynes et al.,1996)。然而，用于治疗与昼夜节律相关的睡眠问题和时差的方法如黑视蛋白和GABA类似物靶向其他分子而非昼夜节律的核心钟上。因此，期望在昼夜节律的核心钟上起作用的化合物。昼夜节律钟(circadian clock)是内在的计时系统，其驱动多个系统的每天的节奏，所述系统包括睡眠/觉醒周期、免疫反应和新陈代谢(Asher and Sassone-Corsi,2015；Bass and Takahashi,2010；Chong et al.,2012；Curtis et al.,2014；Takahashi et al.,2008)。在哺乳动物中，主钟驻留在下丘脑视交叉上核(SCN)中，其同步全身细胞中存在的昼夜振荡(Dibner et al.,2010；Mohawk and Takahashi,2011；Welshet al.,2010)。多种生物体特别是果蝇和小鼠中的遗传研究已经建立了互锁的转录-翻译反馈环(TTFL)作为动物中昼夜节律钟的潜在分子机制(Allada et al.,2001；Andreani etal.,2015；Baker et al.,2012；Crane and Young,2014；Hall,2003；Hardin et al.,1990；Lowrey and Takahashi,2011；Nitabach and Taghert,2008；Panda et al.,2002；Reppertand Weaver,2001；Zheng and Sehgal,2012)。在哺乳动物中，第一环是通过Bmal1和Clock激活三种周期(period)基因(Per1、Per2、Per3)和两种隐花色素基因(Cry1、Cry2)，而Per/Cry形成异源二聚体以抑制其自身的表达(Bunger et al.,2000；Gekakis et al.,1998；Griffin et al.,1999；Hogenesch et al.,1998；Kume et al.,1999；Reppert andWeaver,2001；Shearman et al.,2000；Shearman et al.,1997；Shigeyoshi et al.,1997；Sun et al.,1997；Tei et al.,1997；van der Horst et al.,1999；Vitaterna et al.,1999；Zheng et al.,1999；Zylka et al.,1998)。第二环是通过Bmal1和Clock激活两个核受体基因(Rorα/β、Rev-erbα)，而Rorα/β和Rev-erbα反馈Bmal1表达(Preitner et al.,2002；Sato et al.,2004；Ueda et al.,2002)。
翻译后修饰，尤其是磷酸化，在生物钟调节中是重要的(Crane and Young,2014；Gallego and Virshup,2007；Mehra et al.,2009；Reischl and Kramer,2011)。在果蝇和哺乳动物两者中，PER蛋白(原型昼夜节律调节物)被磷酸化并且有缺陷的PER磷酸化导致昼夜节律破坏(Akashi et al.,2002；Blau,2008；Chiu et al.,2011；Chiu et al.,2008；Cyran et al.,2005；Edery et al.,1994；Eide et al.,2005；Etchegaray et al.,2009；Gallego et al.,2006；Garbe et al.,2013；Iitaka et al.,2005；Kaasik et al.,2013；Kim et al.,2007；Kivimae et al.,2008；Kloss et al.,1998；Kloss et al.,2001；Ko etal.,2010；Lee et al.,2001；Lee et al.,2004；Lee et al.,2011；Lin et al.,2005；Maywood et al.,2014；Meng et al.,2008；Miyazaki et al.,2003；Miyazaki et al.,2004；Nawathean and Rosbash,2004；Nawathean et al.,2007；Price et al.,1998；Reischl et al.,2007；Sathyanarayanan et al.,2004；Schmutz et al.,2011；Shanwareet al.,2011；Shirogane et al.,2005；Takano et al.,2004；Toh et al.,2001；Tsuchiyaet al.,2009b；Uchida et al.,2012；Vanselow et al.,2006；Vielhaber et al.,2000；Xuet al.,2005；Xu et al.,2007；Zhou et al.,2015)。在人家族中发现了点突变，其导致人PER2蛋白(hPER2)中第662位氨基酸残基处从丝氨酸转变为甘氨酸，与家族性超前睡眠期综合征(familial advanced sleep phase syndrome)(FASPS)相关，并证实在小鼠中构成原因(Toh et al.,2001；Xu et al.,2007)。hPER2S662是酪蛋白激酶1δ(CK1δ)的引发位点，发现其基因在另一个FASPS的家族中发生突变(Xu et al.,2005；Xu et al.,2007)。hPER2具有多个潜在的磷酸化位点，其中CK和糖原合成酶激酶(GSK)3与其磷酸化有关(Akashi etal.,2002；Eide et al.,2005；Hirota et al.,2010；Iitaka et al.,2005；Lowrey etal.,2000；Maier et al.,2009；Meng et al.,2008；Tsuchiya et al.,2009a；Vanselow etal.,2006；Xu et al.,2005；Xu et al.,2007)。CK和GSK3以外的激酶可能也参与调节hPER2和参与昼夜节律的其他蛋白质的功能。
已经通过对CK1、GSK3(Chen et al.,2013；Badura et al.,2007；Hirota et al.,2008；Isojima et al.,2009；Kennaway et al.,2015；Sprouse et al.,2010；Sprouse etal.,2009；Walton et al.,2009)、REV-ERB(Solt et al.,2012)或加压素受体(Yamaguchiet al.,2013)的抑制剂的研究揭示了昼夜节律的化学修饰剂。通过基于细胞的昼夜节律测定对化学文库的筛选也揭示了化学修饰剂(Chen et al.,2012；Hirota and Kay,2009；Hirota et al.,2010；Hirota et al.,2008；Isojima et al.,2009；Hirota et al.,2010；Hirota et al.,2012；Oshima et al.,2015)。
我们启动了涉及三个步骤的策略来识别昼夜节律的小分子修饰剂：1)我们筛选了能够磷酸化hPER2的激酶。鉴定新的hPER2激酶允许我们缩小抑制剂作为生物钟的潜在调节物的列表；2)然后我们使用培养的人U2OS细胞作为模型来测试hPER2激酶的抑制剂在体外修饰人生物钟的能力。我们发现了几种有效的修饰剂，其中我们在此详细例证dinaciclib。我们证明了dinaciclib对含有哺乳动物主钟的脑切片和最终在完整动物中的作用。Dinaciclib不仅在培养的U2OS细胞中而且在SCN切片中显著改变了生物钟的相位。向小鼠体内应用单剂量的dinaciclib有效缩短了在6小时(h)超前时差范例(paradigm)中调整所需的时间。因此，我们成功地使用合理的方法来获得用于改变昼夜节律钟的相位的化合物。
发明概述
本发明提供了用于调控，优选相位移动昼夜节律的方法和组合物。
在一方面，本发明提供了用于调控，优选相位移动昼夜节律的方法，其包括向有此需要的受试者施用hPER2磷酸化激酶抑制剂。
在一方面，本发明提供了化合物，用于有此需要的受试者中使用以治疗时差、轮班工作或年龄相关的睡眠障碍的用途或在制备用于治疗时差、轮班工作或年龄相关的睡眠障碍的药物中的用途，其中化合物是hPER2磷酸化激酶抑制剂。
在一方面，本发明提供了组合物，其包含(a)hPER2磷酸化激酶抑制剂，和(b)用于治疗时差(jet lag)、轮班工作(shift-work)或年龄相关的睡眠障碍(age-related sleepdisturbances)的不同的药物。
在实施方案中，
-激酶选自：CDK5、PRKACB、PRKG1、IKBKB、TSSK2和IKBKE；
-激酶和抑制剂选自：(a)CDK5和Dinaciclib、Cdk/crk抑制剂、Roscovitine或靛红-3’-单肟-5-磺酸、9-Cyanopaullone；(b)IKBKB和Ikk2抑制剂iv、Ikk2抑制剂v、Ikk2抑制剂vi或AS602868；和(c)IKBKE和Cay105765；
-抑制剂为单位剂型，优选肠的(例如口服)；
-抑制剂和药物共包装或共配制，优选以单位剂型，优选肠的；
-药物是咖啡因、褪黑激素、唑吡坦(zolpidem)、左旋佐匹克隆(eszopiclone)、扎来普隆(zaleplon)或三唑仑(triazolam)；
-受试者是暴露于或者确定有风险暴露于时差、轮班工作或年龄相关的睡眠障碍的人；和/或
-方法进一步包括确定受试者暴露于或有风险暴露于时差、轮班工作或年龄相关的睡眠障碍的先行步骤。
在一方面，本发明提供了检测哺乳动物细胞内生物钟的调控物的方法，其包括：(a)使激酶与化合物接触并检测由此导致的对激酶的抑制；和(b)使哺乳动物生物钟与化合物接触并检测由此导致的生物钟的调控。
在实施方案中，激酶测定法是体外或基于细胞的，并且生物钟测定法是基于细胞或动物的。
在一方面，本发明提供了调控昼夜节律的方法，其包括向有此需要的受试者施用有效量的dinaciclib。
在一方面，本发明提供了化合物dinaciclib，用于在有此需要的受试者中使用以治疗时差、轮班工作或年龄相关的睡眠障碍的用途或在制备用于治疗时差、轮班工作或年龄相关的睡眠障碍的药物中的用途。
在一方面，本发明提供了组合物，其包含dinaciclib，和用于治疗时差、轮班工作或年龄相关的睡眠障碍的不同的药物。
在实施方案中：
-dinaciclib处于：(a)用于癌症治疗的亚治疗剂量；(b)治疗或常规癌症剂量的小于50％、20％或10％的剂量；(c)小于1或2或5或10或20mg/m2的剂量；或(d)小于1、2、5、10或20mg的单位剂型；
-dinaciclib为单位剂型，优选肠的(例如口服)；
-dinaciclib和药物共包装或共配制，优选以单位剂型，优选肠的；
-药物是咖啡因、褪黑激素、唑吡坦、左旋佐匹克隆、扎来普隆或三唑仑；
-受试者是暴露于或者确定有风险暴露于时差、轮班工作或年龄相关的睡眠障碍的人。
-方法包括确定受试者暴露于或有风险暴露于时差、轮班工作或年龄相关的睡眠障碍的先行步骤。
本发明涵盖本文所述的具体实施方案的所有组合，如同每个单独地、艰涩地叙述。
附图简述
图1a、1b和1c：在激酶抑制剂修改U2OS细胞的昼夜节律的能力的化学筛选中发现的激酶抑制剂作用。(a)IKBKB抑制剂。所有抑制剂的浓度均为10μM，n＝5。(b)IKBKE抑制剂。抑制剂的浓度如下：Amelxanox，15μM，n＝4；Cay10576，15μM，n＝4；BX795，0.3μM，n＝4(c)CDK5抑制剂。抑制剂的浓度如下：dinaciclib，10nM，n＝4；Cdk/Crk抑制剂，10nM，n＝4；Cdk1/5抑制剂，10μM，n＝4。Dinaciclib在昼夜节律钟的相位中引起了最引人注目的移动。
发明详述
详细描述、具体实施方案和实施例是以说明而非限制的方式提供的。本领域技术人员将容易地认识到可以改变或修改各种非关键参数以产生基本相似的结果。本文引用的所有出版物、专利和专利申请，包括其中的引文，出于所有目的以其整体通过引用在此并入。
除非另有禁忌或另有说明，否则在这些描述和整个说明书中，术语“一”和“一个”意指一个或多个，术语“或”意指和/或以及多核苷酸序列理解为涵盖相反链以及本文所述的替代主链。此外，属叙述为对该属的所有成员的叙述的简写；例如，(C1-C3)烷基的叙述是对以下所有C1-C3烷基的叙述的简写：甲基、乙基和丙基，包括其异构体。
Dinaciclib配制和施用
Dinaciclib，配制和施用是容易凭经验确定的或以其他方式在本领域已知的，例如，US7,119,200；US20160193334；WO 2015130585：除非另有说明，否则以下对dinaciclib的提及还包括其药学上可接受的盐。为了制备dinaciclib药物组合物，惰性的药学上可接受的载体可以是固体或液体。固体形式的制剂包括粉末、片剂、可分散的颗粒、胶囊剂、扁囊剂和栓剂。粉末和片剂可以包含约5％至约95％的dinaciclib。合适的固体载体是本领域已知的，例如碳酸镁、硬脂酸镁、滑石、糖或乳糖。片剂、粉末、扁囊剂和胶囊剂可以用作适合于口服施用的固体剂型。药学上可接受的载体的实例和用于各种组合物的制备的方法可以在A.Gennaro(编),Remington's Pharmaceutical Sciences,第18版,(1990),MackPublishing Co.,Easton,Pennsylvania中找到。dinaciclib的液体形式制剂包括溶液、悬浮液和乳液。作为实例，可提及用于肠胃外注射的水或水-丙二醇溶液或用于口服溶液、悬浮液和乳液的甜味剂和遮光剂的添加。液体形式制剂还可以包括用于鼻内施用的溶液。适于吸入的dinaciclib的气雾剂制剂可以包括溶液和粉末形式的固体，其可以与药学上可接受的载体(如惰性压缩气体，例如氮气)组合。还包括dinaciclib的固体形式制剂，其意图在使用前不久转化为dinaciclib的液体形式制剂，用于口服或肠胃外施用。此类液体形式包括溶液、悬浮液和乳液。Dinaciclib也可以是可透皮递送的。透皮组合物可以采取乳膏剂、洗剂、气雾剂和/或乳液的形式，并且可以包括在基质或储库类型的透皮贴剂中，如本领域常规用于此目的。Dinaciclib也可以皮下递送。药物制剂可以是以单位剂型。在此类形式中，将制剂细分为合适大小的单位剂量，其含有适当量的dinaciclib，例如，达到所期望目的的有效量。根据具体应用，单位剂量的制剂中的dinaciclib的量可以从约1mg至约100mg，更具体地约1mg至约50mg，更具体地约1mg至约25mg变化或调整。所采用的实际剂量可以根据患者的需要和所治疗状况的严重程度而变化。确定用于具体情况的适当剂量方案在本领域技术范围内。为方便起见，可以根据需要将每日总剂量分开并在一天中按份施用。
实施例
期望调节昼夜节律的药物在患病或衰老人群中治疗时差、轮班工作相关问题和昼夜节律相关的睡眠病症。因为昼夜节律钟涉及蛋白质磷酸化，所以我们首先对能够磷酸化人Period 2(hPER2)蛋白的片段的人激酶进行筛选，导致发现以前在PER磷酸化上未知的6种激酶。然后，我们在培养的人细胞中进行了用于功能性调节昼夜节律的激酶抑制剂的筛选。发现几种抑制剂可以延长或缩短周期，而一种抑制剂dinaciclib则以剂量和相位依赖的方式显著移动了昼夜节律钟的相位：高达8.172±0.2194小时(h)(平均值±SEM，n＝4)。Dinaciclib可以移动视交叉上核(SCN)(小鼠大脑中的主钟)的相位。向小鼠单次注射dinaciclib显著改善了具有6h相位超前的时差范例中的调整。因此，偶联化学和生物筛选有效地揭示了生物钟移动中应用的小分子。
使hPER2磷酸化的激酶的筛选
为了找到hPER2的激酶，我们亚克隆并在HEK293T细胞中共转染了编码具有不同hPER2片段的激酶的cDNA。在我们的系统中成功测试了288种蛋白激酶。在筛选过程中通过phos-tag检测到hPER2的磷酸化，所述phos-tag可以结合蛋白质中的有机磷酸盐以延迟其在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)中的迁移(Kinoshita et al.,2006)。我们将分子量超过130千道尔顿(kD)的hPER2分成六个片段，每个片段的分子量在20-30kD之间。这旨在增加通过凝胶移位的磷酸化检测的灵敏度，并分离在不同位点上的磷酸化的干扰。因为片段771-1000和979-1240在HEK293T细胞中显示多个条带，所以我们的筛选聚焦于剩余的4个片段。
筛选揭示了使片段1-200磷酸化的0种激酶，使片段150-400磷酸化的一种激酶，使片段328-556磷酸化的三种激酶以及使片段556-771磷酸化的五种激酶。筛选的结果得到验证，并且通过磷酸酶处理证实凝胶移位是由于磷酸化所致。一种激酶使片段328-556和片段556-771两者磷酸化，而CSNK1D和CSNK1E先前已被鉴定(Akashi et al.,2002；Eide etal.,2005；Toh et al.,2001；Vanselow et al.,2006；Xu et al.,2005；Xu et al.,2007)。这给我们留下了6种新鉴定的hPER2磷酸化激酶。仅发现一种激酶CDK5使片段150-400磷酸化。PRKACB、PRKG1和IKBKB可以使片段328-556磷酸化。PRKACB、TSSK2、IKBKE、CSNK1D和CSNK1E使片段556-771磷酸化。
激酶抑制剂对培养的人细胞的昼夜节律的作用
我们测试了新发现的激酶的抑制剂在表达Per2-萤光素酶(其如实地报告了昼夜节律钟)的U2OS细胞中对昼夜节律的潜在调节(Zhang et al.,2009)。我们没有检测到PRKACB和PRKG1抑制剂对昼夜节律的任何作用。CDK5、IKBKB和IKBKE抑制剂的作用显示于图1和表1中。
表1
Figure BDA0002065644120000071
Figure BDA0002065644120000081
这里测试的所有IKBKB抑制剂均延长了周期。IKBKE的三种抑制剂显示出不同的作用：Amelxanox和BX795没有可检测的作用，而Cay10576将周期缩短0.3h。CDK5的三种抑制剂也显示出不同的作用。有趣的是，dinaciclib显著移动了U2OS细胞的昼夜节律的相位。
dinaciclib对人细胞的昼夜节律的作用
为了进一步测试dinaciclib的作用，我们使用表达Bmal1-萤光素酶的细胞系，其以不同于Per2-萤光素酶的相位振荡。在Bmal1-萤光素酶细胞中证实了dinaciclib的相位移动作用，支持了dinaciclib影响昼夜节律，而不仅仅是特异性影响一种报告基因的表达。当Per2-萤光素酶表达的第一个峰的时间用于计算相位移动的程度时，发现dinaciclib将峰延迟9.408h。当从测定的开始应用时，dinaciclib将振幅增加1.78倍并将周期缩短0.6h。
我们检查了dinaciclib对表达Per2-萤光素酶的细胞的剂量反应。dinaciclib对相位移动、振幅和周期长度的作用均为剂量依赖性。用于相位移动的dinaciclib的EC50(半数最大有效浓度)为8.363nM。
上述实验是从一开始就将药物加入到培养基进行的。为了研究药物治疗的时机是否影响反应，在不同的昼夜节律时间(CT)将dinaciclib添加到表达Per2-萤光素酶的细胞。Dinaciclib在CT0应用时延迟了相位4.092±0.9062h(p<0.01，n＝5)，在CT6延迟了相位7.543±0.6185h(p<0.001，n＝5)以及在CT12延迟了相位5.808±0.5455h(p<0.0001，n＝5)。然而，如果在CT18引入dinaciclib，则昼夜节律相位提前了1.7±0.81h。总之，昼夜节律的dinaciclib修改是剂量和相位依赖性的。
dinaciclib对SCN中昼夜节律的作用
U2OS细胞仅是培养的细胞，而SCN是哺乳动物中的主钟。我们从表达Per2::萤光素酶基因的小鼠中解剖出SCN，该基因如实地报告了昼夜节律(Yoo et al.,2004)。将SCN外植体切成切片，其不仅细胞自主地而且以细胞-细胞相互作用依赖性方式维持节律性。dinaciclib在CT12的应用使SCN外植体的相位提前2.488±0.7391h(p<0.01，n＝8)，而在CT0或CT18的dinaciclib应用延迟了相位。SCN切片和分离的组织培养细胞之间的差异可能是由同步所需的SCN细胞之间的相互作用引起的。
因此，dinaciclib可以调节含有哺乳动物主钟的SCN切片中的昼夜节律。当在CT12:00应用时，其显著提前了SCN相位。
dinaciclib对小鼠的时差范例的体内作用
dinaciclib可以修改培养的人细胞中的昼夜节律的相位，并且小鼠SCN外植体表明dinaciclib可以成为治疗完整动物的时差的强效药物的令人兴奋的可能性。
将成年小鼠置于12L:12D的明/暗(LD)循环中(在CT 3:00开灯，在CT15:00关灯)11天，在CT20:00应用dinaciclib(0.1mg/kg体重)。在CT21:00，将LD循环提前6h(在CT21:00开灯，在CT9:00关灯)，并将小鼠保持在新LD中14天。
与载体治疗的小鼠相比，用单剂量的dinaciclib治疗的小鼠在显著更短的时间中适应新的LD循环。dinaciclib治疗组的50％相位移动时间(PS50)为1.34天，而对照组为2.63天(p<0.01)。因此，dinaciclib在治疗时差中是有效的。
我们的用于使hPER2(生物钟组分)磷酸化的激酶的筛选促进了用于调节生物钟的小分子的筛选。通过进一步分析dinaciclib在培养的人细胞、大脑切片和完整小鼠中的作用，扩大了来自筛选的dinaciclib的发现。
Dinaciclib是抗癌药物，没有先前的证据表明其可以影响昼夜节律(Parry etal.,2010；Paruch et al.,2010)。它已经通过临床试验的I期和II期以及本文用于时差的剂量低于癌症治疗的剂量的事实支持了dinaciclib相对安全的可能性(Fabre et al.,2014；Flynn et al.,2015；Gojo et al.,2013；Gorlick et al.,2012；Hu et al.,2015；Kumar et al.,2015；Mita et al.,2014；Mitri et al.,2015；Nemunaitis et al.,2013a；Stephenson et al.,2014；Zhang et al.,2012)。
通过筛选288种蛋白激酶，我们已发现了6种能够磷酸化hPER2的新激酶。我们测试了典型的抑制剂在培养的人U2OS细胞中修改昼夜节律的能力。CDK5可以磷酸化hPER2片段150-400。CDK5是非典型的细胞周期蛋白依赖性激酶，其由P35或P39而不是细胞周期蛋白激活(Dhavan and Tsai,2001)。片段150-400含有一个PAS域，其用于PER蛋白的转录调节活性。然而，有报道称CDK5可以磷酸化CLOCK蛋白(Kwak et al.,2013)。
免疫系统受昼夜节律的控制(Curtis et al.,2014；Scheiermann et al.,2013)。在我们的激酶筛选中，已知在免疫系统中重要的两种激酶IKBKB和IKBKE(Chen,2012)可以磷酸化hPER2片段。在这里我们还发现了IKBKB抑制剂可以延长U2OS的周期。hPER2的降解需要区域328-771中的泛素化，其也是磷酸化依赖性的(Eide et al.,2005；Ohsaki et al.,2008)。例如，FASPS中发现的S662G突变在该区域中(Toh et al.,2001)。
Dinaciclib目前正在针对多种肿瘤的临床试验中(包括难治性慢性淋巴细胞白血病(III期))得以研究。其有效地和选择性地抑制CDK5、CDK9、CDK1和CDK2(Fabre et al.,2014；Flynn et al.,2015；Gojo et al.,2013；Gorlick et al.,2012；Guzi et al.,2011；Hu et al.,2015；Kumar et al.,2015；Mita et al.,2014；Mitri et al.,2015；Nemunaitis et al.,2013a；Nemunaitis et al.,2013b；Parry et al.,2010；Paruch etal.,2010；Stephenson et al.,2014；Zhang et al.,2012)。在评估的CDK抑制剂中，dinaciclib是最有效的并且具有独特的激酶选择性概况，尽管仍有待研究哪种CDK是dinaciclib用于其修改昼夜节律的内源性靶标。我们的工作表明激酶活性的调节物可以提供有效和安全的昼夜节律的修饰剂，不仅减轻与时差相关的问题，而且减轻与轮班工作和年龄相关的睡眠障碍相关的问题。
小鼠时差测定
将套管的尖端立体定向地插入到小鼠的侧脑室。手术后，将小鼠放回在LD循环(在CT3:00开灯，在CT15:00关灯)下的家笼中7天，然后开始视频记录小鼠活动。再过四天，在CT20:00，将溶于20％羟丙基-β-环糊精(hydroxypropyl-β-cyclodextran)的Dinaciclib(Parry et al.,2010)(浓度为5mg/ml，速度为0.5μl/min)或单独载体在Micro4控制器的帮助下，通过UltraMicroPumpIII泵入大脑中以达到0.1mg/kg体重的总量。在同一天的CT21:00，将LD循环移动为在CT21:00开灯，在CT9:00关灯，其是提前6小时。将小鼠保持在新LD中14天。处理数据并计算移动距离/30s。
激酶
CDK5(细胞周期蛋白依赖性激酶5)；PRKACB(蛋白激酶cAMP活化的催化亚基beta)；PRKG1(蛋白激酶cGMP依赖性，1型)；IKBKB(也是IKK2)-核因子kappa-B激酶亚基beta的抑制剂；IKBKE(核因子kappa-B激酶亚基epsilon的抑制剂)；TSSK2(睾丸特异性丝氨酸激酶2)。
激酶抑制剂
Dinaciclib，(2-[(2S)-1-[3-乙基-7-[(1-氧化吡啶-1-鎓-3-基)甲氨基]吡唑并[1,5-a]嘧啶-5-基]哌啶-2-基]乙醇)(2-[(2S)-1-[3-ethyl-7-[(1-oxidopyridin-1-ium-3-yl)methylamino]pyrazolo[1,5-a]pyrimidin-5-yl]piperidin-2-yl]ethanol)，Amelxanox：(2-氨基-5-氧代-7-丙-2-基色烯[2,3-b]吡啶-3-羧酸)(2-amino-5-oxo-7-propan-2-ylchromeno[2,3-b]pyridine-3-carboxylic acid)，Cdk1/5抑制剂(3-氨基-1H-吡唑并[3,4-b]喹喔啉)(3-Amino-1H-pyrazolo[3,4-b]quinoxaline)，Cdk/Crk抑制剂：(1-(2,6-二氯苯基)-6-[[4-(2-羟乙氧基)苯基]甲基]-3-丙-2-基2H-吡唑并[3,4-d]嘧啶-4-酮)(1-(2,6-dichlorophenyl)-6-[[4-(2-hydroxyethoxy)phenyl]methyl]-3-propan-2-yl-2H-pyrazolo[3,4-d]pyrimidin-4-one)，IKK-2抑制剂IV(TPCA-1或2-(氨基甲酰氨基)-5-(4-氟苯基)噻吩-3-甲酰胺(2-(carbamoylamino)-5-(4-fluorophenyl)thiophene-3-carboxamide))，IKK-2抑制剂V(IMD 0534或N-[3,5-双(三氟甲基)苯基]-5-氯代-2-羟基苯甲酰胺(N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide))，IKK-2抑制剂VI(2-[(氨羰基)氨基]-5-苯基-3-噻吩甲酰胺))(2-[(aminocarbonyl)amino]-5-phenyl-3-thiophenecarboxamide))，Cay10576(5-(5,6-二甲氧苯并咪唑-1-基)-3-[(2-甲磺酰苯基)甲氧基]噻吩-2-腈)(5-(5,6-dimethoxybenzimidazol-1-yl)-3-[(2-methylsulfonylphenyl)methoxy]thiophene-2-carbonitrile)，BX795(N-[3-[[5-碘代-4-[3-(噻吩-2-羰氨基)丙氨基]嘧啶-2-基]氨基]苯基]吡咯烷-1-甲酰胺)(N-[3-[[5-iodo-4-[3-(thiophene-2-carbonylamino)propylamino]pyrimidin-2-yl]amino]phenyl]pyrrolidine-1-carboxamide)，KT5720(己基6-羟基-5-甲基-13-氧代-5,6,7,8,14,15-六氢-13h-5,8-环氧-4b,8a,14-三氮杂二苯并[b,h]环辛[1,2,3,4-jkl]环戊[e]-不对称引达省-6-羧酸盐)(Hexyl6-hydroxy-5-methyl-13-oxo-5,6,7,8,14,15-hexahydro-13h-5,8-epoxy-4b,8a,14-triazadibenzo[b,h]cycloocta[1,2,3,4-jkl]cyclopenta[e]-as-indacene-6-carboxylate)，Rp-cAMPS((4aR,6R,7R,7aS)-6-(6-氨基嘌呤-9-基)-2-氧化-2-亚硫烷基-4a,6,7,7a-四氢-4H-呋喃并[3,2-d][1,3,2]二氧杂磷杂-7-醇；三乙基氨鎓)((4aR,6R,7R,7aS)-6-(6-aminopurin-9-yl)-2-oxido-2-sulfanylidene-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-7-ol；triethylazanium)，KT5823(9-甲氧基-9-甲氧羰基-8-甲基-2,3,9,10-四氢-8,11-环氧-1H,8H,11H-2,7b-11a-三氮杂二苯并(a,g)环辛(cde)-trinden-1-酮)(9-methoxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epxoy-1H,8H,11H-2,7b-11a-triazadibenzo(a,g)cycloocta(cde)-trinden-1-one)，Rp-8-Br-PET-cGMPS(钠；3-[(4aR,6R,7R,7aS)-7-羟基-2-氧化-2-亚硫烷基-4a,6,7,7a-四氢-4H-呋喃并[3,2-d][1,3,2]二氧杂磷杂-6-基]-2-溴代-6-苯基-5H-咪唑并[1,2-a]嘌呤-9-酮)(sodium；3-[(4aR,6R,7R,7aS)-7-hydroxy-2-oxido-2-sulfanylidene-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-6-yl]-2-bromo-6-phenyl-5H-imidazo[1,2-a]purin-9-one)。
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Claims (6)

1.调控昼夜节律的方法，其包括向有此需要的受试者施用hPER2磷酸化激酶抑制剂，所述方法是非治疗目的的方法，其中所述抑制剂是Dinaciclib。
2.hPER2磷酸化激酶抑制剂在制备用于调控受试者的昼夜节律的药物中的用途，其中所述抑制剂是Dinaciclib。
3.化合物在制备用于治疗时差、轮班工作或年龄相关的睡眠障碍的药物中的用途，其中所述化合物是Dinaciclib。
4.根据权利要求2所述的用途，其中所述受试者是暴露于或者确定有风险暴露于时差、轮班工作或年龄相关的睡眠障碍的人。
5.根据权利要求1所述的方法或根据权利要求2或3所述的用途，其中所述dinaciclib处于：
(a) 用于癌症治疗的亚治疗剂量；
(b) 治疗或常规癌症剂量的小于50%、20%或10%的剂量；
(c) 小于1或2或5或10或20 mg/m2的剂量；或
(d) 小于1、2、5、10或20 mg的单位剂型。
6.根据权利要求5所述的方法或用途，其中所述dinaciclib为单位剂型。
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