CN113490739A - 通过磷酸二酯酶4(pde4)抑制治疗癫痫的方法 - Google Patents

通过磷酸二酯酶4(pde4)抑制治疗癫痫的方法 Download PDF

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CN113490739A
CN113490739A CN202080013929.1A CN202080013929A CN113490739A CN 113490739 A CN113490739 A CN 113490739A CN 202080013929 A CN202080013929 A CN 202080013929A CN 113490739 A CN113490739 A CN 113490739A
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黛博拉·M·库拉施
金斯利·伊布哈泽赫博
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Abstract

提供治疗癫痫的方法。该方法包括向患有癫痫症的个体施用治疗有效量的磷酸二酯酶4(PDE4)抑制剂。还提供了鉴定抗癫痫剂的方法。此类方法包括在PDE4活性测定中将PDE4多肽与候选试剂接触，其中候选试剂对PDE4多肽活性的抑制将候选试剂识别为抗癫痫剂。

Description

通过磷酸二酯酶4(PDE4)抑制治疗癫痫的方法
相关申请的交叉引用
本申请要求2019年1月23日提交的美国临时专利申请62/796,002的权益，所述申请通 过引用以其全部并入本文。
背景技术
数以千万计的人患有癫痫。尽管经过近八十年的研究和许多新药的问世，缓解癫痫发作 的有效率并没有显着变化。药物是癫痫的主要治疗方法，但顽固的30％～40％的癫痫患者对 目前的药物无效，并且会出现持续反复的发作和随之而来的终生健康问题。举例来说，德拉 韦综合征(DS)是一种儿童癫痫，通常在健康婴儿出生后的第一年出现，表现为持续超过五分 钟(通常超过30分钟)的热性惊厥。大多数DS病例是由于编码脑部电压门控钠通道I型 NaV1.1的Scn1a基因的功能丧失突变所致。尽管了解了其遗传学，但DS仍然具有高度的抗 药性，成千上万的儿童挣扎于无数无效的治疗。
在诊断出DS后，随着临床医生试图了解疾病并评估治疗策略，家庭会被安排进出诊所 和急诊室，孩子们反复进行脑电图、CT、MRI和脊椎穿刺。这意味着年幼的孩子(及其家人)面临着单独或联合地尝试各种主要的成人抗癫痫疗法(n＝8药物和/或手术和/或生酮饮 食)的艰巨任务，希望找到一种有效的策略。鉴于测试一种策略可能需要8-10周时间，一年 内可能只测试5-6种组合，而确定治疗策略通常需要多年时间——而与此同时，儿童仍会继 续出现不受控制的癫痫发作和到急诊室就诊。
2018年六月，FDA批准大麻二酚(CBD；Epidiolex)作为第一种用于DS的抗癫痫药物。 虽然向前迈出了重要的一步，但CBD仅降低了43％的患者的癫痫发作频率，并且只有不到 5％的患者实现了完全无癫痫发作(最终目标)。因此，对于有效的抗癫痫剂仍然存在显着 未满足的需求。
发明内容
提供了治疗癫痫的方法。该方法包括向患有癫痫症的个体施用治疗有效量的磷酸二酯酶 4(PDE4)抑制剂。还提供了鉴定抗癫痫剂的方法。此类方法包括在PDE4活性测定中将PDE4 多肽与候选药剂接触，其中候选药剂对PDE4多肽的活性的抑制将候选药剂鉴定为抗癫痫剂。
附图说明
图1：代表性PDE4基因结构概述。
图2：根据本公开的实施例可以被抑制的人PDE4A多肽的氨基酸序列比对。
图3：根据本公开的实施例可以被抑制的人PDE4B多肽的氨基酸序列比对。
图4：根据本公开的实施例可以被抑制的人PDE4C多肽的氨基酸序列比对。
图5：根据本公开的实施例可以被抑制的人PDE4D多肽的氨基酸序列比对。
图6：A组：数据表明实例PDE4抑制剂(AN2728)剂量依赖性地将scn1lab突变斑马鱼的 生物能(bioenergetics)恢复到基线水平。B组：数据表明多种PDE4抑制剂(在本例中为咯利 普兰、西洛司特、罗氟司特、异丁司特、茶碱、屈他维林和伊索格拉定)可有效将scn1lab 突变斑马鱼的生物能恢复到基线水平。
图7：数据表明PDE4B-、4C-和4D-吗啉(morpholinos)可有效将scn1lab突变斑马鱼的生 物能恢复到基线水平。
图8：数据表明PDE4抑制剂的两个实例(罗氟司特和茶碱)在全身性癫痫的kcna1模型中恢复生物能。
图9：A组和B组：数据表明PDE4抑制剂实例(AN2728)可将scn1lab斑马鱼的癫痫样过 度兴奋阻断至基线水平，包括癫痫发作频率和峰值振幅的降低。
图10：A组和B组：数据表明PDE4B吗啉可有效将scn1lab斑马鱼的癫痫样过度兴奋阻 断至基线水平。
图11：数据表明PDE4抑制剂实例(AN2728)降低了小鼠Scn1a突变脑离体切片的过度兴 奋。
图12：数据表明PDE4抑制剂实例(AN2728)在6Hz诱导小鼠模型中防止癫痫发作。
图13：数据表明PDE4抑制剂实例(AN2728)可防止Scn1a突变小鼠中的高温诱导的癫痫 发作。
图14：数据表明双重选择性PDE4B抑制剂(咯利普兰)可有效防止Scn1a突变小鼠中的高温诱导的癫痫发作。
图15：数据证明PDE4抑制剂的三个实例(AN2728、咯利普兰、罗氟司特)部分或完全阻断Scn1a突变小鼠的癫痫发作。
具体实施方式
提供了治疗癫痫的方法。该方法包括向患有癫痫症的个体施用治疗有效量的磷酸二酯酶 4(PDE4)抑制剂。还提供了鉴定抗癫痫剂的方法。此类方法包括在PDE4活性测定中将PDE4 多肽与候选药剂接触，其中候选药剂对PDE4多肽的活性的抑制将候选药剂鉴定为抗癫痫剂。
在更详细地描述本公开的方法之前，应当理解，这些方法不限于所描述的特定实施例， 因此当然可以改变。还应理解，本文所用的术语仅用于描述特定实施例的目的，而不是旨在 限制，因为所述方法的范围将仅由所附权利要求书限制。
在提供了值范围的情况下，应当理解的是，介于该范围的上限与下限之间的每个中间值 (到下限的单位的十分之一，除非另外明确说明)以及所述范围中的任何其它所陈述或中间 值均涵盖于本方法内。这些较小范围的上限和下限可以独立地包含在更小的范围中，并且也 涵盖在本方法内，这受制于所陈述的范围中的任何具体排除的极限。当规定范围包括一个或 两个限值时，排除了那些所包括的限值中的任一个或两个的范围也包括在本方法中。
本文给出的某些范围的数值前面有术语“约”。术语“约”在本文中用于提供对其之前的确 切数字以及接近或近似该术语之前的数字的文字支持。在确定一个数字是否接近或近似一个 具体列举的数字时，接近或近似的未列举的数字可以是在其出现的上下文中提供该具体列举 的数字的实质等同物的数字。
除非另有定义，否则本文中使用的所有技术和科学术语具有与方法所属领域的普通技术 人员通常理解的相同含义。尽管与本文所述方法类似或等效的任何方法也可用于方法的实践 或测试中，但现在描述了代表性说明性方法。
在本说明书中引用的所有公开和专利通过引用并入本文，如同每个单独的公开或专利被 具体和单独地指示通过引用并入并且通过引用并入本文以公开和描述与所引用的公开有关的 材料和/或方法。引用任何出版物是为了在提交日期之前公开，不应被解释为承认目前的方 法无权提前发布，因为提供的发布日期可能不同于实际发布日期，可能需要独立确认。
注意，如本文和所附权利要求书中所使用的，除非上下文另有明确指示，否则单数形式 “一(a)”、“一种(an)”和“所述(the)”包括复数指示物。还应当注意，权利要求书可以被起草为排除任何任选的元素。因此，所述陈述旨在用作前置基础，用于结合权利要求要素的叙述使用诸如“单独地”、“仅”等排他性术语或使用“负面”限制。
应理解，为清楚起见，在单独实施例的上下文中描述的方法的某些特征也可以组合在单 个实施例中提供。相反，为简洁起见，在单个实施例的上下文中描述的方法的各种特征也可 以单独提供或以任何合适的子组合提供。本发明具体包含实施例的所有组合，并在本文中公 开，如同单独且明确地公开了每个组合一样，只要这些组合包含可操作的过程和/或组合物。 此外，在描述这些变量的实施例中列出的所有子组合也被本方法具体地包含，并且在本文中 被公开，就如同每个这样的子组合在本文中被单独和明确地公开一样。
正如本领域技术人员在阅读本公开内容后将显而易见的那样，本文描述和图示的每个单 独实施例具有离散的组件和特征，这些组件和特征可以容易地与其他几个实施例中的任何一 个的特征分离或组合，而不脱离本方法的范围或精神。任何所述的方法可以以所述的事件的 顺序或以逻辑上可能的任何其它顺序执行。
治疗方法
如上所述，本公开提供了治疗癫痫的方法。治疗癫痫的方法包括向患有癫痫的个体施用 治疗有效量的磷酸二酯酶4(PDE4)抑制剂。该方法部分基于本文所述的意外发现，即PDE4 抑制：在癫痫的斑马鱼和小鼠模型中阻断生物能和过度兴奋的神经元表型；在癫痫小鼠模型 中防止高温诱导的癫痫发作；并使用6Hz测试阻止癫痫发作--NIH支持的癫痫治疗筛查计划 的入口点模型。
环核苷酸磷酸二酯酶(PDE)催化环核苷酸第二信使——环磷酸腺苷(cAMP)和环磷酸鸟 苷(cGMP)的水解。PDE4家族是三个cAMP特定的PDE家族之一。PDE4已被证明可调节多种细胞生理过程，包括通过cAMP依赖性蛋白激酶A(PKA)进行的蛋白质磷酸化、通过cAMP 应答元件进行的基因转录和环核苷酸门控离子通道。这些过程与认知功能、抑郁症、精神分裂症、高血压和心肌细胞收缩力有关。
PDE4基因家族由四种基因亚型组成：PDE4A、PDE4B、PDE4C和PDE4D。这些亚型是 在海绵动物和真寄生虫分离之前，通过一个共同的真核生物祖先中的基因复制事件产生的。已在哺乳动物物种中检测到所有四种PDE4基因亚型的转录本。图1中提供了代表性PDE4 基因结构的概述。PDE4基因由通过虚线(兼性外显子)或实线(组成性外显子)连接的多 个外显子组成。PDE4长型氨基末端指定外显子(1)位于基因的上游区域，每个基因受不同的 启动子控制。上游保守区-1(UCR1)由三个外显子(UCR1a、UCR1b和UCR1c)组成。PDE4 短型氨基末端指定外显子(1a)位于接头区域1(LR1)外显子的下游。上游保守区2(UCR2)由 三个外显子(UCR2a、UCR2b和UCR2c)组成，它们被指定外显子(1b)的超短型氨基末端中断。 截短的超短PDE4剪接变体的氨基末端位于UCR2b外显子(1c)内。PDE4的酶促核心由位于 基因最远下游区域的几个组成型外显子(在所有亚型中发现)编码。关于PDE4亚型的基因 结构和剪接变体的更多细节见于例如Johnson et al.(2010)BMC Evol Biol.10:247，其公开内 容出于所有目的通过引用整体并入本文。在一些实施例中，根据本公开方法抑制的PDE4多 肽是Johnson等人(2010)BMC Evol Biol.10:247的图S1中提供的PDE4多肽。
表1中提供了根据本发明方法可被抑制(单独或以任何组合)的PDE4多肽的非限制性 实例。
表1–人类PDE4目标多肽实例
Figure BDA0003208087430000051
Figure BDA0003208087430000061
Figure BDA0003208087430000071
Figure BDA0003208087430000081
Figure BDA0003208087430000091
Figure BDA0003208087430000101
Figure BDA0003208087430000111
如本文所用，“PDE4抑制剂”或“PDE4的抑制剂”是与一种或多种PDE4亚型在不存在该 试剂的情况下的磷酸二酯酶活性相比，抑制(例如，减少或消除)一种或多种PDE4亚型的磷酸二酯酶活性。在某些实施例中，与在不存在抑制剂的情况下一种或多种PDE4亚型中的至少一种的磷酸二酯酶活性相比，抑制剂导致一种或多种PDE4亚型中的至少一种的磷酸二酯酶活性为80％或更低、75％或更低、70％或更低、65％或更低、60％或更低、55％或更低、50％或更低、45％或更低、40％或更低、35％或更低、30％或更低、25％或更低、20％或更低、15％或更低、10％或更低、5％为或更低、4％或更低、3％或更低、2％或更低、或1％或更低。
PDE4A在中枢神经系统中表达，包括在大脑皮层、海马体和小脑中。PDE4B在中枢神经系统中广泛表达，包括纹状体、杏仁核、丘脑和下丘脑。PDE4C在中枢神经系统中并未 广泛表达，尽管已在嗅球中观察到表达，但在大脑皮层中表达有限。PDE4D在中枢神经系 统中表达，包括在海马体和其他地方。
在一些实施例中，所述方法包括施用抑制PDE4A、PDE4B、PDE4C和PDE4D中的一种或多种的PDE4抑制剂。在某些实施例中，PDE4抑制剂抑制PDE4A、PDE4B、PDE4C和 PDE4D中的两种、三种或每一种。该方法可以包括施用在PDE4A、PDE4B、PDE4C和 PDE4D中表现出选择性的PDE4抑制剂。“选择性”是指PDE4抑制剂：专门抑制PDE4A、 PDE4B、PDE4C或PDE4D；或抑制PDE4A、PDE4B、PDE4C和PDE4D中的两种或更多种， 其中两种或更多种PDE4亚型中至少一种的抑制大于被抑制剂抑制的两种或更多种PDE4亚 型中不同亚型的抑制。在一些实施例中，PDE4抑制剂对PDE4亚型具有选择性。如本文所 用，PDE4抑制剂在以下任一情况下对PDE4亚型是“选择性的”：仅抑制PDE4亚型(PDE4A、 PDE4B、PDE4C或PDE4D)；或比至少一种其他PDE4亚型在更大程度上抑制PDE4亚型。 例如，在某些实施例中，PDE4抑制剂对PDE4B具有选择性，使得对PDE4B的抑制大于对 第二种PDE4亚型的抑制，例如大于对PDE4D的抑制。
在一些实施例中，PDE4抑制剂不抑制(或基本上不抑制)PDE4A、PDE4B、PDE4C和PDE4D中的一种、两种或三种。如本文所用，与不存在抑制剂时亚型的活性相比，当使 PDE4亚型与抑制剂接触时，抑制剂不会“基本上抑制”PDE4亚型的活性，不会抑制亚型的活 性超过20％、15％、10％、5％、4％、3％、2％或1％。在某些实施例中，该方法包括施用不 抑制(或基本上不抑制)PDE4D的PDE4B抑制剂。
在一些实施例中，PDE4抑制剂通过与PDE4的催化结构域(例如，活性位点)结合而起作用。在其他实施例中，PDE4抑制剂通过变构地抑制PDE4活性起作用。所有十一个PDE 超家族成员(PDE1-11)在整个催化结构域中都表现出高度的序列保守性，从而使PDE家族以编码唯一调节结构域(unique regulatory domain)的基序来区分。针对PDE的传统策略主要集中 在结合催化结构域的配体上，从而导致完全抑制cAMP水解和相关毒性，以及由于非选择性 结合而导致的狭窄治疗范围。PDE4A、PDE4B、PDE4C和PDE4D均包含三个特征调节域(signature regulatory domains)：上游保守区域1(UCR1)和2(UCR2)形成一个调节模块，以及位 于C-末端的控制区(CR3)结构域。PDE4B和PDE4D晶体结构表明磷酸二酯酶活性受催化活 性位点的UCR2变构控制来调节。因此，PDE4的变构调节是一种被低估的选择性干扰PDE4 活性的方法。事实上，小分子PDE4D变构调节剂在细胞和体内测定中是有效的。此外，PDE4B的UCR2已显示与催化结构域中的活性位点发生反式紧密相互作用。在一些实施例中，所述方法包括施用选择性抑制PDE4B(例如，相对于PDE4D)的抑制剂，例如通过以 UCR2闭合活性位点的方式结合PDE4B，从而防止cAMP进入。
在一些实施例中，PDE4抑制剂是变构调节剂，其通过在闭合cAMP进入的构象中跨越 活性位点捕获C-末端调节螺旋(CR3)而选择性抑制PDE4A、PDE4B和/或PDE4C(例如，相对于PDE4D)。例如，小分子可以与沿CR3螺旋的不同残基相互作用，导致多个“闭合”构象。Fox et al.(2014)Cell Signal.26(3):657-63.CR3螺旋可以在整个活性位点采用略微不同的方向， 每个方向都有独特的螺旋登记(unique helical registries)。对PDE4B相对于PDE4D表现出 选择性并且可以根据本公开内容的方法施用的变构抑制剂的实例是已知的并且包括2-芳基嘧 啶衍生物化合物A-33(如Hagan等人的(2014)Bioorg Med ChemLett.24(16):4031-4and Fox et al.(2014)Cell Signal.26(3):657-63所述)和Hagan等人的(2014)Bioorg Med Chem Lett.24(16): 4031-4中所述的三嗪类化合物。
当实践本公开的方法时，可以使用任何合适类型的PDE4抑制剂。在一些实施例中，PDE4抑制剂是小分子。“小分子”是指分子量为1000原子质量单位(amu)或更小的化合物。在一些实施例中，小分子为750amu或更低、500amu或更低、400amu或更低、300amu或 更低、或者200amu或更低。在某些实施例，小分子不是由重复的分子单元(诸如存在于聚 合物中)制成的。可以根据本公开内容的方法施用的PDE4活性的小分子变构调节剂是已知 的并且包括在Gurney等人的(2011)Handb Exp Pharmacol。204:167-92；Burgin等人的(2010) NatBiotechnol。28(1)：63-70；Fox等人的(同上)；哈根等人的(同上)；和其他地方描述的那些。在一些实施例中，当PDE4抑制剂是小分子时，PDE4抑制剂选自AN2728(5-(4-氰基 苯氧基)-2,3-二氢-1-羟基-2,1-苯并恶唑)、屈他维林、异丁司特、伊索格列定、匹拉米司特、 罗氟司特、咯利普兰、茶碱、阿普司特、化合物A-33(如Hagan等人的(2014)Bioorg Med ChemLett.24(16):4031-4和Fox等人的(2014)Cell Signal.26(3):657-63)所述，如Hagan等人的(2014)Bioorg Med Chem Lett。24(16):4031-4所述的三嗪化合物。及其任意组合。在某些 实施例中，当PDE4抑制剂是小分子时，PDE4抑制剂是AN2728。根据一些实施例，PDE4 抑制剂不是咯利普兰。在某些实施例中，PDE4抑制剂不是咯利普兰并且在PDE4A、PDE4B、PDE4C和PDE4D中表现出与咯利普兰的选择性不同的选择性。在一些实施例中，该方法使 用表现出比咯利普兰更小的PDE4D抑制的PDE4抑制剂。
根据一些实施例，PDE4抑制剂抑制PDE4的表达。例如，PDE4抑制剂可以抑制PDE4A、PDE4B、PDE4C和PDE4D中的一种、两种、三种或每一种的表达。在某些实施例中，当 PDE4抑制剂抑制PDE4的表达时，PDE4抑制剂是基于核酸的抑制剂。“基于核酸的抑制剂” 是指两个或多个连接的核苷酸的聚合物，其中聚合物可以包括天然存在的核苷酸、非天然存 在的核苷酸(例如，核苷酸类似物，例如LNA、FANA、2'-O-Me RNA、2'-氟RNA和/或类 似物)或其组合。
在一些实施例中，基于核酸的PDE4抑制剂包括与编码PDE4A的信使RNA(mRNA)、编码PDE4B的mRNA、编码PDE4C的mRNA、编码PDE4D的mRNA或任何组合的一部分互 补的区域。如本文所用，术语“互补”是指通过非共价键与靶核酸的全部或区域碱基配对的核 苷酸序列。在经典的Watson-Crick碱基配对中，腺嘌呤(A)与胸腺嘧啶(T)形成碱基对， 和鸟嘌呤(G)在DNA中与胞嘧啶(C)形成碱基对一样。在RNA中，胸腺嘧啶被尿嘧啶 (U)替代。因此，A与T互补，并且G与C互补。在RNA中，A与U互补，并且反之亦 然。通常，“互补的”是指至少部分互补的核苷酸序列。这些术语还可以涵盖完全互补的双链 体，使得一条链中的每个核苷酸与对应位置中另一条链中的每个核苷酸互补。在某些情况下， 核苷酸序列可以与靶标部分互补，其中并非所有核苷酸在所有对应位置都与靶核酸中的每个 核苷酸互补。例如，引物可以与靶核酸完全(即，100％)互补，或者引物和靶核酸可能具 有某种程度的互补，但这种互补不够完美(例如，70％、75％、85％、90％、95％、99％)。 可以通过出于最佳比较的目的比对序列来确定两个核苷酸序列的同一性百分比(例如，可以 在第一序列的序列中引入空位以进行最佳比对)。然后比较对应位置处的核苷酸，并且两个 序列之间的同一性百分比是序列共有的相同位置的数量的函数(即，％同一性＝相同位置的#/位置的总#×100)。当一个序列中的位置被与另一个序列中的对应位置相同的核苷酸占据 时，则所述位置处的分子是相同的。此数学算法的非限制性实例描述于Karlin等人,《美国 国家科学院院刊》90:5873-5877(1993)中。此算法并入到NBLAST和XBLAST程序(版本 2.0)中，如Altschul等人,《核酸研究(Nucleic Acids Res.)》25:389-3402(1997)中所描述 的。当利用BLAST和Gapped BLAST程序时，可以使用相应程序(例如，NBLAST)的默 认参数。在一个方面，用于序列比较的参数可以被设置为评分＝100、字长＝12，或者可以 改变(例如，字长＝5或字长＝20)。
根据一些实施例，选择/设计基于核酸的PDE4抑制剂的核苷酸序列，使得基于核酸的 PDE4抑制剂对PDE4亚型具有选择性。例如，可以选择/设计基于核酸的PDE4抑制剂的核苷酸序列，使得抑制剂选择性抑制PDE4A、PDE4B和/或PDE4C的表达(例如，相对于 PDE4D)。在一些实施例中，选择/设计核苷酸序列使得抑制剂选择性地抑制PDE4B表达， 例如，相对于PDE4D表达，选择性地抑制PDE4B表达。
核酸抑制剂的非限制性实例当实践本公开的方法时可以采用的包括短干扰RNA(siRNA)、 微小RNA(miRNA)、吗啉等。基于PDE4A(NCBI基因ID：5141，对于人PDE4A)，PDE4B(NCBI基因ID：5142，对于人PDE4B)，PDE4C(NCBI基因ID：5143，对于人PDE4C)和 PDE4D(NCBI基因ID：5144，对于人PDE4C)的可获得的序列信息，和相应的转录物，基于 核酸的抑制剂如siRNA，miRNA，吗啉等，可以使用可获得的工具，例如Invivogen的 siRNA Wizard，Dharmacon的siDESIDN中心，Invitrogen的BLOCK-iTTM RNAi设计器，在microrna.osumc.edu/mir-synth上获得的microSynth，WMD3-Web MicroRNA基因工具提供的 吗啉设计工具等来设计。设计和递送siRNA、miRNA、吗啉等以靶向特定mRNA的方法是 已知的，并且描述于例如Chakraborty等(2017)Mol Ther Nucleic Acids 8:132-143；Ahmadzada 等(2018)Biophys Rev。10(1)：69-86；郑等(2018)Trends Biotechnol。36(5)：562-575； Mohanty等人(2015)Curr Pharm Des。21(31)：4606-13；Gomes等(2015)Delisting Res Rev。 21:43-54；Gusticich等(2017)Prog Neurobiol。155:194-211；Monsoori等(2014)Adv Pharm Bull。4(4)：313-321；和Xin等(2017)Mol Cancer 16:134。
如上所述，本公开的方面包括通过向患有癫痫症的个体(例如，诊断为患有癫痫症的个 体)施用治疗有效量的PDE4抑制剂来治疗癫痫(有时称为“癫痫发作症”)。在一些实施例 中，PDE4抑制剂是本文别处描述的任何PDE4抑制剂，包括使用本公开内容的鉴定抗癫痫剂的方法鉴定的任何PDE4抑制剂。较老的抗癫痫药物(AED)苯妥英、卡马西平、氯硝西泮、乙琥胺、丙戊酸和巴比妥类药物被广泛使用，但也有一系列副作用。此外，有相当一部分患者(30-40％)对当前可用的治疗剂具有抗药性。最近推出了几种药物，包括非氨酯、加巴喷丁、 拉莫三嗪、奥卡西平、噻加宾、托吡酯、氨己烯酸、唑尼沙胺和左乙拉西坦。虽然一些此类 较新的药物显示出改善的功效和副作用，但仍有约30％的癫痫患者未得到治疗。
鉴于本文所述的意外发现，PDE4抑制在斑马鱼和癫痫小鼠模型中阻断生物能和过度兴 奋的神经元表型，在癫痫小鼠模型中防止高温诱导的癫痫发作，并使用6Hz测试阻止癫痫发 作，它将应当理解，该方法可用于治疗多种癫痫。在某些实施例中，个体患有癫痫，其选自 良性罗兰癫痫、额叶癫痫、婴儿痉挛、青少年肌阵挛癫痫(JME)、青少年失神癫痫、儿童失 神癫痫、密集性癫痫、热性惊厥、进行性肌阵挛性癫痫、伦诺克斯-加斯托综合征、兰道-克 勒夫纳综合征、德拉韦综合征(DS)、全身性癫痫伴热性惊厥(GEFS+)、婴儿严重肌阵挛性癫 痫(SMEI)、良性家族性新生儿惊厥(BFNC)、韦斯特综合征、大田原综合征、早期肌阵挛脑 病、迁移性部分性癫痫、婴儿癫痫性脑病、结节性硬化症(TSC)、局灶性皮质发育不良、I 型无脑回畸形、Miller-Dieker综合征、快乐木偶综合征、脆性X综合征、自闭症谱系障碍中 的癫痫、皮质下带异位症、Walker-Warburg综合征、阿尔茨海默病癫痫、创伤后癫痫、进行 性肌阵挛癫痫、反射性癫痫、拉斯穆森病综合征、颞叶癫痫、边缘癫痫、癫痫持续状态、腹部癫痫、双侧大量肌阵挛、月经性癫痫、杰克逊癫痫症、Unverricht-Lundborg病和光敏性癫痫。根据一些实施例，个体患有德拉韦综合征(DS)。在某些实施例中，个体患有由基因突变引起的癫痫症。在一些实施例中，个体患有具有非遗传病因的癫痫，其非限制性实例包括由脑震荡、脑损伤等引起的癫痫。根据一些实施例，基于个体具有两次或更多次无端癫痫发作， 个体已被诊断为患有癫痫症。个体的癫痫可能包括全身性癫痫发作或部分(例如，局灶性) 癫痫发作。
根据受试者的治疗方法，可以治疗各种的个体。通常这种个体是“哺乳动物”或“哺乳类 动物”，其中这些术语广泛用于描述哺乳纲内的生物体，包括食肉目(例如狗和猫)，啮齿 目(例如小鼠、豚鼠和大鼠)和灵长目(例如人、黑猩猩和猴)。在一些实施例中，个体是人。
“治疗(treat)”或“治疗(treatment)”是指至少改善与折磨个人的病理状况有关的症状， 其中广义上使用改善是指至少减少与所治疗的病理状况有关的参数，例如症状的轻重，例如 与PDE4活性相关的疾病或病症(例如癫痫)，其中抑制个体中的PDE4活性是有益的。因 此，治疗还包括以下情况：病理状况(例如癫痫)或至少与之相关的症状被完全抑制(例如 防止发生)或停止(例如终止)使得个体不再患有病理状况，或至少表征病理状况的症状。 在一些实施例中，用本发明方法治疗患有癫痫的个体，与没有一次或多次施用PDE4抑制剂 时个体经历的癫痫发作的频率和/或严重程度相比，当一次或多次施用PDE4抑制剂导致个体 经历的癫痫发作的频率和/或严重性为90％或更低、80％或更低、70％或更低、60％或更低、 50％或更低、40％或更低、30％或更低、20％或更低、10％或更低、为5％或更低、或1％或更 低。
剂量取决于待治疗疾病状态的严重程度和反应性。最佳给药方案可以通过测量PDE4抑 制剂在个体体内的积累来计算。给药医师可以确定最佳剂量、给药方法和重复率。最佳剂量 可以根据PDE4抑制剂的相对效力而变化，并且通常可以基于发现在体外和体内动物模型等 中有效的EC50来估计。通常，剂量为每千克体重0.01μg至100g，并且可以每天、每周、每 月或每年给予一次或多次。治疗医师可以基于测量体液或组织中PDE4抑制剂的的停留时间 和浓度来估计给药的重复率。成功治疗后，可能需要使受试者接受维持治疗以防止疾病状态 的复发，其中PDE4抑制剂以维持剂量每天一次或多次施用至每几个月一次，每六个月一次， 每年一次，或以任何其他合适的频率。
本公开的治疗方法可以包括向个体施用单一类型的PDE4抑制剂，或者可以包括向个体 施用两种或更多种类型的PDE4抑制剂，例如不同PDE4抑制剂的混合物。
在某些方面，作为组合疗法的一部分，将PDE4抑制剂与第二治疗剂组合施用于个体。 例如，本公开的方法可以包括向患有癫痫症的个体与第二抗癫痫药的组合施用治疗有效量的 PDE4抑制剂。可与PDE4抑制剂联合使用的第二种抗癫痫药的非限制性实例包括乙酰唑胺、 地西泮、苯二氮卓、大麻二酚、卡马西平、氯巴占、氯硝西泮、醋酸艾司利卡西平、乙琥胺、 乙妥英、非尔氨酯、芬氟拉明、磷苯妥因、加巴喷丁、加那索酮、石杉碱甲、拉考沙胺、拉莫三嗪、左乙拉西坦、劳拉西泮、硝西泮、奥卡西平、哌仑帕尼、吡拉西坦、苯巴比妥、 苯妥英、溴化钾、普瑞巴林、普里米酮、瑞替加滨、鲁非那胺、丙戊酸钠、司他丁、硫加宾、 托吡酯、丙戊酸、维加巴林或唑尼沙胺。根据其中组合施用PDE4抑制剂和第二治疗剂的实 施例，PDE4抑制剂和第二治疗剂可以同时或顺序施用。
可使用任何适用于药物递送的可用方法和途径(包括体内和体外方法)向个体施用 PDE4抑制剂，以及全身和局部施用途径。常规和药学上可接受的给药路径包括鼻内、肌内、 气管内、皮下、皮内、局部应用、眼内、静脉内、动脉内、鼻、口服和其它肠内和肠胃外给药路径。如果需要，给药途径可以为组合使用，或根据PDE4抑制剂和/或预期效果进行调整。PDE4抑制剂可以单剂量或多剂量施用。在一些实施例中，PDE4抑制剂通过口服、肠胃外、 鼻内、鞘内、颅内或经皮施用来施用。在一些实施例中，PDE4抑制剂口服给药。在一些实 施例中，局部施用PDE4抑制剂。在一些实施例中，PDE4抑制剂经眼部施用。在一些实施 例中，颅内施用PDE4抑制剂。在一些实施例中，静脉内施用PDE4抑制剂。在一些实施例 中，通过注射例如用于全身递送(例如，静脉内输注)或注射至局部部位来施用PDE4抑制 剂。在一些实施例中，通过吸入途径施用PDE4抑制剂。在一些实施例中，PDE4抑制剂通 过鼻内施用。在一些实施例中，PDE4抑制剂不容易穿过血脑屏障(BBB)，并且鼻内施用 PDE4抑制剂以绕过BBB。关于通过鼻内给药绕过BBB的更多细节可以在例如Mohanty等人 (2015)Curr Pharm Des21(31):4606-13和其他地方中找到。
鉴定抗癫痫剂的方法
如上所概述，本公开的方面包括鉴定抗癫痫剂的方法。鉴定抗癫痫剂的方法包括在 PDE4活性测定中将PDE4多肽与候选药物接触，其中候选药物对PDE4多肽的活性的抑制将 候选药物鉴定为抗癫痫药。
与候选试剂接触的PDE4多肽可以是任何感兴趣的PDE4多肽。在一些实施例中，PDE4 多肽是PDE4A、PDE4B、PDE4C或PDE4D，包括图2-5或表1中提供的任何PDE4多肽， 或其具有磷酸二酯酶活性的功能变体(functional variant)。“功能变体”是指具有磷酸二酯酶活 性的PDE4多肽，相对于相应的野生型PDE4多肽，其包括一个或多个氨基酸取代、缺失、插入或其组合。例如，PDE4B的功能变体可以是相对于野生型PDE4B包括一个或多个氨基 酸取代、缺失、插入或其组合的PDE4B多肽。功能性变体可包括相应野生型PDE4多肽长度 的70％或更高、75％或更多、80％或更多、85％或更多、90％或更多、95％或更多、99％或更 多氨基酸序列同源性或同一性跨越40％或更高、50％或更多、60％或更多、70％或更多、80％或更多、90％或更多、95％或更多。PDE4多肽或其功能变体可以与异源结构域融合。异源 结构域的非限制性实例包括接头结构域(例如，甘氨酸-丝氨酸接头或其他合适的接头结构域)、可用于检测PDE4多肽的结构域(例如，荧光素酶结构域或其他合适的可检测结构 域)、结构域可用于纯化PDE4多肽(例如，HIS标签，或其他合适的纯化标签)，和/或诸 如此类的。
PDE4活性测定可以使用任何合适的试剂(例如，PDE4底物等)和用于测定磷酸二酯酶 活性的形式进行。在一些实施例中，PDE4活性测定包括检测PDE4多肽对cAMP或cGMP 的切割。PDE4磷酸二酯酶活性的检测可以是基于色度的、基于发光的、基于荧光的、基于 放射性的和/或类似的。在一些实施例中，测定在单管、单孔、多管、多孔(例如，24孔、 48孔、96孔、384孔或其他孔格式)或其他合适的形式中进行。PDE4活性测定适合高通量 形式，使得大量候选试剂(例如小分子文库的小分子)可以容易地与并行的PDE4多肽接触， 例如单独的孔。
在一些实施例中，在鉴定抗癫痫剂的方法中，接触包括在无细胞PDE4活性测定中组合 PDE4多肽和候选药剂。用于评估磷酸二酯酶活性及其抑制的无细胞测定试剂和试剂盒在实 践本公开内容的方法时可以使用是已知的并且包括可从Abcam获得的PDE活性测定试剂盒 (96孔比色法)、PDE-GloTM Promega公司提供的磷酸二酯酶检测试剂盒(1管至1536孔发 光)、FabGennix公司提供的PDEase试剂盒、Mediomics公司提供的
Figure BDA0003208087430000181
cAMP-磷酸 二酯酶检测试剂盒等。在无细胞形式中评估磷酸二酯酶活性及其抑制的其他方法在例如 Rybalkin等人的(2013)Methods Mol Biol.1020:51-62.中有所描述。
在一些实施例中，在鉴定抗癫痫剂的方法中，接触包括在基于细胞的PDE4活性测定中 组合PDE4多肽和候选药剂。“基于细胞的”是指PDE4多肽和候选药剂在细胞内接触。在一 些实施例中，PDE4多肽由发生接触的细胞表达。用于评估磷酸二酯酶活性及其抑制的基于 细胞的测定试剂和试剂盒在实践本公开的方法时可以使用是已知的，并且包括可从eEnzyme 获得的ACTOne PDE测定试剂盒、可从SB Drug Discovery获得的基于细胞的PDE测定试剂 盒、可从Biovision获得的K927-总磷酸二酯酶活性测定试剂盒等。在无细胞形式中评估磷酸 二酯酶活性及其抑制的其他方法在例如Titus等人的(2008)J Biomol Screen13(7):609-618中 有所描述。(描述了用于高通量筛选的1536孔板格式的基于细胞的PDE4测定，涉及组成 型活性GPCR作为cAMP生产的驱动力和环核苷酸门控(CNG)阳离子通道作为1536孔板中 的生物传感器)；Allen et al.(1999)Biochem Pharmacol.57(12):1375-82；和Qiu et al.(2003) Eur J Pharmacol.472(1-2):73-80。
在一些实施例中，PDE4活性测定进一步包括将PDE4多肽与已知抑制PDE4活性的阳性 对照剂接触。可采用的阳性对照剂的非限制性实例包括AN2728、屈他维林、异丁司特、伊索格列定、匹拉米司特、罗氟司特、咯利普兰、茶碱、阿普司特及其任何组合。
候选药剂可以是任何类型的感兴趣的候选药剂。在一些实施例中，候选药剂是小分子。 在一些实施例中，基于PDE4抑制剂的计算机筛选，选择小分子作为候选药剂。计算机筛选 可能是对PDE4抑制剂的筛选，该抑制剂在PDE4A、PDE4B、PDE4C和PDE4D中具有选择性。例如，计算机筛选可以是选择性抑制PDE4A、PDE4B和/或PDE4C(例如，相对于 PDE4D)的PDE4抑制剂的计算机筛选。在一些实施例中，基于对PDE4B相对于PDE4D具 有选择性的抑制剂的计算机筛选，选择小分子作为候选药剂。因此，在一些实施例中，该方 法还可包括——当确定候选药剂抑制PED4多肽的活性时——确定候选药剂是否表现出对 PDE4A、PDE4B、PDE4C和PDE4D的选择性抑制。
在一些实施例中，当候选药剂是小分子时，小分子是小分子候选药剂文库的一部分。在 一些实施例中，该方法包括将小分子候选试剂文库以高通量形式与PDE4多肽接触，例如96 孔、384孔、1536孔或其他高通量形式。
组合物
本公开的方面包括组合物。在一些实施例中，该组合物可用于例如实践本公开的方法。
在一些实施例中，本发明的组合物包括本文别处所述的任何PDE4抑制剂，包括通过本 发明的抗癫痫剂识别方法识别的任何抗癫痫剂。
在某些方面，本公开的组合物包含存在于液体介质中的PDE4抑制剂(和任选地，第二 抗癫痫剂)。液体介质可以是水性液体介质，例如水、缓冲溶液等。一种或多种添加剂，如盐(例如NaCl、MgCl2、KCl、MgSO4)、缓冲剂(Tris缓冲剂、N-(2-羟乙基)哌嗪-N'-(2-乙 磺酸)(HEPES)、2-(N-吗啉)乙磺酸(MES)、2-(N-吗啉)乙磺酸钠盐(MES)、3-(N-吗啉) 丙磺酸(MOPS)、N-三[羟基甲基]甲基-3-氨基丙磺酸(TAPS)等)、增溶剂、洗涤剂(例 如，非离子型洗涤剂，如吐温(Tween)-20等)、核酸酶抑制剂、蛋白酶抑制剂、甘油、螯 合剂等可以存在于此类组合物中。
还提供了药物组合物。本公开的药物组合物包括PDE4抑制剂和药学上可接受的载体。 本公开的任何药物组合物可以包括——除了PDE4抑制剂之外——第二抗癫痫剂。例如，提 供了包含PDE4抑制剂和第二抗癫痫剂的药物组合物。可以在本公开的药物组合物中提供的 第二抗癫痫剂的非限制性实例包括乙酰唑胺、地西泮、苯二氮卓、大麻二酚、卡马西平、氯 巴占、氯硝西泮、醋酸艾司利卡西平、乙琥胺、乙妥英、非尔氨酯、芬氟拉明、磷苯妥因、 加巴喷丁、加那索酮、石杉碱甲、拉考沙胺、拉莫三嗪、左乙拉西坦、劳拉西泮、硝西泮、奥卡西平、哌仑帕尼、吡拉西坦、苯巴比妥、苯妥英、溴化钾、普瑞巴林、普里米酮、瑞替 加滨、鲁非那胺、丙戊酸钠、司他丁、硫加宾、托吡酯、丙戊酸、维加巴林或唑尼沙胺。
PDE4抑制剂(和任选的第二抗癫痫剂)可以掺入多种制剂中以施用于个体。更具体地 说，PDE4抑制剂可以通过与适当的药学上可接受的赋形剂或稀释剂组合配制成药物组合物， 并且可以配制成固体、半固体、液体或气体形式的制剂，如片剂、胶囊、粉末、颗粒、软膏、 溶液、注射剂、吸入剂和气雾剂。
适用于个体给药(例如，适用于人类给药)的PDE4抑制剂的制剂通常是无菌的，并且 根据所选给药途径，还不含可检测热原或禁忌给个体给药的其他污染物。
在药物剂型中，PDE4抑制剂可以单独或与药物活性化合物(例如第二抗癫痫剂)以适 当的联合以及组合施用。以下方法和载体/赋形剂仅仅是实例，而决不是限制性的。
对于口服制剂，PDE4抑制剂可以单独使用或与适当的添加剂组合使用以制备片剂、粉 剂、颗粒剂或胶囊剂，例如，使用常规添加剂，诸如乳糖、甘露醇、玉米淀粉或马铃薯淀粉； 使用粘合剂，诸如结晶纤维素、纤维素衍生物、阿拉伯树胶、玉米淀粉或明胶；使用崩解剂， 诸如玉米淀粉、马铃薯淀粉或羧甲基纤维素钠；使用润滑剂，诸如滑石粉或硬脂酸镁；并且 如果需要，使用稀释剂、缓冲剂、润湿剂、防腐剂和调味剂。
PDE4抑制剂可配制用于肠外(例如，静脉、动脉内、骨内、肌肉内、脑内、脑室内、 颅内、鞘内、皮下等)给药。在一些实施例中，PDE4抑制剂被配制用于口服、肠胃外、鼻 内、鞘内、颅内、脑内、脑室内或透皮给药。在一些实施例中，通过将PDE4抑制剂溶解、 悬浮或乳化在水性或非水性溶剂中，例如植物油或其他类似油、合成脂肪酸甘油酯、高级脂 肪酸酯或丙二醇，来配制PDE4抑制剂用于注射；如果需要的话，可以使用常规添加剂，如 增溶剂、等渗剂、悬浮剂、乳化剂、稳定剂和防腐剂。
包括PDE4抑制剂的药物组合物可通过将具有所需纯度的PDE4抑制剂与可选生理上可 接受的载体、赋形剂、稳定剂、表面活性剂、缓冲剂和/或强化剂混合来制备。可接受的载 体、赋形剂和/或稳定剂在所用的剂量和浓度下对接受者无毒，并且包含：缓冲剂，如磷酸 盐、柠檬酸盐和其它有机酸；抗氧化剂，包含抗坏血酸、谷胱甘肽、半胱氨酸、蛋氨酸和柠檬酸；防腐剂(如乙醇、苯甲醇、苯酚、间甲酚、对氯间甲酚、对羟基苯甲酸甲酯或对羟基 苯甲酸丙酯、苯扎氯铵或其组合)；氨基酸，如精氨酸、甘氨酸、鸟氨酸、赖氨酸、组氨酸、 谷氨酸、天冬氨酸、异亮氨酸、亮氨酸、丙氨酸、苯丙氨酸、酪氨酸、色氨酸、蛋氨酸、丝 氨酸、脯氨酸及其组合；单糖、二糖和其它碳水化合物；低分子量(少于约10个残基)多 肽；蛋白质，如明胶或血清白蛋白；螯合剂，如EDTA；糖，如海藻糖、蔗糖、乳糖、葡萄 糖、甘露糖、麦芽糖、半乳糖、果糖、山梨糖、棉子糖、葡糖胺、N-甲基葡糖胺、半乳糖胺 和神经氨酸；和/或非离子表面活性剂，如Tween、Brij Pluronics、Triton-X或聚乙二醇 (PEG)。
药物组合物可以是液体形式、冻干形式或从冻干形式重构的液体形式，其中在施用之前 用无菌溶液重构冻干制剂。用于重构冻干组合物的标准方法是加回一定体积的纯水(通常等 于冻干期间去除的体积)；然而，包括抗菌剂的溶液可用于生产用于肠胃外给药的药物组合 物。
可以在pH缓冲溶液中制备PDE4抑制剂的含水制剂，例如，在约4.0到约7.0、或约5.0 到约6.0、或者替代性地约5.5的pH范围下。适于此范围内的pH的缓冲剂的实例包括磷酸 盐缓冲剂、组氨酸缓冲剂、柠檬酸盐缓冲剂、琥珀酸盐缓冲剂、乙酸盐缓冲剂和其它有机酸 缓冲剂。缓冲剂浓度可为约1mM至约100mM，或约5mM至约50mM，这取决于例如缓 冲剂和制剂的所需张力。
制剂中可包含张力剂以调节制剂的张力。张力剂实例包括氯化钠、氯化钾、甘油和来自 氨基酸、糖以及其组合的群组的任何组分。在一些实施例中，水性制剂是等渗的，尽管高渗 或低渗溶液可能是合适的。术语“等渗”表示具有与其比较的一些其它溶液相同的张力的溶液， 如生理盐溶液或血清。可以以约5mM到约350mM的量使用张力剂，例如以100mM到350 mM的量使用。
还可以将表面活性剂添加到制剂中以减少聚集和/或使所述制剂中颗粒的形成最小化和/ 或减少吸附。表面活性剂实例包含聚氧乙烯山梨糖醇酐脂肪酸酯(吐温)、聚氧乙烯烷基醚 (Brij)、烷基苯基聚氧乙烯醚(Triton-X)、聚氧乙烯-聚氧丙烯共聚物(泊洛沙姆， Pluronic)和十二烷基硫酸钠(SDS)。合适的聚氧乙烯山梨糖醇酐-脂肪酸酯的实例是聚山 梨醇酯20(以商标Tween 20TM销售)和聚山梨醇酯80(以商标Tween 80TM销售)。合适的聚乙烯-聚丙烯共聚物的实例是以名称
Figure BDA0003208087430000211
F68或Poloxamer 188TM销售的聚乙烯-聚丙 烯共聚物。合适的聚氧乙烯烷基醚的实例是以商标BrijTM销售的聚氧乙烯烷基醚。表面活性 剂的实例浓度范围可以为约0.001％到约1％w/v。
还可以加入冻干保护剂，以在冻干过程期间保护PDE4抑制剂免于去稳定化条件。例如， 已知的冻干保护剂包含糖(包含葡萄糖和蔗糖)；多元醇(包含甘露醇、山梨醇和甘油)； 和氨基酸(包含丙氨酸、甘氨酸和谷氨酸)。可以包含约10mM到500nM的量的冻干保护剂。
在一些实施例中，药物组合物包含PDE4抑制剂以及上述鉴定剂(例如，表面活性剂、 缓冲剂、稳定剂、张力剂)中的一种或多种，并且基本上不含一种或多种防腐剂，如乙醇、 苯甲醇、苯酚、间甲酚、对氯间甲酚、对羟基苯甲酸甲酯或丙酯、苯扎氯铵及其组合。在其 它实施例中，制剂中包含防腐剂，例如，以约0.001到约2％(w/v)的浓度范围。
试剂盒
本公开还提供试剂盒。在一些实施例中，试剂盒可用于例如实践本公开的方法。
在一些实施例中，本公开的试剂盒包括本文别处描述的任何PDE4抑制剂，包括通过本 公开的鉴定抗癫痫剂的方法鉴定的任何抗癫痫剂。
在一些实施例中，本公开的试剂盒包括包含PDE4抑制剂的药物组合物和药学上可接受 的载体。例如，提供的试剂盒包括本发明的任何药物组合物，包括上文组合物部分所述的任 何药物组合物。在一些实施例中，本公开的试剂盒包括药物组合物，其–除了PDE4抑制剂 之外–还包括第二种抗癫痫剂。例如，可用于治疗癫痫的方法中的试剂盒可包括包含PDE4 抑制剂和第二抗癫痫剂的药物组合物。在一些实施例中，本公开的试剂盒进一步包括在与包 含PDE4抑制剂的药物组合物分开的药物组合物中提供的第二抗癫痫剂。可以在本公开的试 剂盒中提供的第二抗癫痫剂的非限制性实例(在与PDE4抑制剂相同或不同的药物组合物中) 包括乙酰唑胺、地西泮、苯二氮卓、大麻二酚、卡马西平、氯巴占、氯硝西泮、醋酸艾司利 卡西平、乙琥胺、乙妥英、非尔氨酯、芬氟拉明、磷苯妥因、加巴喷丁、加那索酮、石杉 碱甲、拉考沙胺、拉莫三嗪、左乙拉西坦、劳拉西泮、硝西泮、奥卡西平、哌仑帕尼、吡拉西坦、苯巴比妥、苯妥英、溴化钾、普瑞巴林、普里米酮、瑞替加滨、鲁非那胺、丙戊酸钠、 司他丁、硫加宾、托吡酯、丙戊酸、维加巴林或唑尼沙胺。
用于实践主题方法的试剂盒可以包括一定量的PDE4抑制剂(和任选的第二抗癫痫剂)， 以单位剂量(例如安瓿)或多剂量形式存在。因此，在某些实施例中，试剂盒可以包括一种 或多种(例如，两种或更多种)单位剂量(例如，安瓿)的药物组合物，该药物组合物包括 PDE4抑制剂(和任选的第二种抗癫痫剂)和/或一种或多种(例如，两种或多种)单位剂量(例如，安瓿)的药物组合物，其包括第二种抗癫痫剂。
如本文中所使用的，术语“单位剂量”是指适合作为用于人和动物受试者的单位剂量的物 理上离散的单位，每个单位包含按足以产生期望效果的量计算的预定量的组合物。单位剂量 的量取决于各种因素，例如在个体中使用的特定PDE4抑制剂、要达到的效果以及与PDE4 抑制剂相关的药效学。在其他实施例中，试剂盒可包括单一多剂量的组合物，所述组合物包 括PDE4抑制剂(以及可选的第二种抗癫痫剂)。
试剂盒的组分可以存在于分开的容器中，或多种组分可以存在于单一容器中。例如，在 包含PDE4抑制剂和第二种抗癫痫剂的试剂盒中，PDE4抑制剂和第二种抗癫痫剂可以以相 同的组合物(例如，在一个或多个容器中)提供或可以以分开的组合物在分开的容器中提供。 合适的容器包括单独的管(例如，小瓶)、安瓿等。
本公开的试剂盒还可包括说明书。例如，包括PDE4抑制剂的试剂盒可以包括向有需要 的个体施用PDE4抑制剂的说明。在一些实施例中，说明书包括用于向患有癫痫的个体施用 PDE4抑制剂的说明书，所述癫痫包括本文别处描述的任何类型的癫痫中的一种或多种。
可以将说明书记录在合适的记录媒体上。例如，说明书可以印刷在基材上，如纸或塑料 等。因此，说明书可以作为包装说明书存在于试剂盒中、在试剂盒的容器或其组分的标签中 (即与包装或次包装有关联)等。在其它实施例中，说明书作为存在于合适的计算机可读存 储介质上的电子存储数据文件存在，例如便携式闪存驱动器、DVD、CD-ROM，软盘等。在又其它实施例中，实际说明书不存在于试剂盒中，但是提供了用于从远程源，例如通过因特网获得说明书的手段。该实施例的一个实例是包括网址的试剂盒，可以在所述网址中查看 说明书和/或可以从所述网址下载说明书。与说明书一样，用于获得说明书的方式被记录在 合适的基材上。
尽管有所附权利要求，但本公开还由以下实施例定义:
1.一种治疗癫痫的方法，包括向患有癫痫的个体施用治疗有效量的磷酸二酯酶4(PDE4)抑制 剂。
2.根据实施例1所述的方法，其中PDE4抑制剂是小分子。
3.根据实施例2所述的方法，其中PDE4抑制剂选自以下所组成的组：AN2728、屈他维林、 异丁司特、伊索格列定、匹拉米司特、罗氟司特、咯利普兰、茶碱、阿普司特及其任意组合。
4.根据实施例2所述的方法，其中PDE4抑制剂是AN2728。
5.根据实施例1所述的方法，其中PDE4抑制剂抑制PDE4A、PDE4B、PDE4C或PDE4D中的一种或多种。
6.根据实施例1所述的方法，其中PDE4抑制剂在PDE4A、PDE4B、PDE4C和PDE4D中表现出选择性。
7.根据实施例6所述的方法，其中PDE4抑制剂对PDE4B具有选择性。
8.根据实施例1-7任一项所述的方法，其中给药是通过口服、肠胃外、鼻内、鞘内、颅内或 经皮给药。
9.根据实施例1所述的方法，其中PDE4抑制剂抑制PDE4的表达。
10.根据实施例9所述的方法，其中PDE4抑制剂是基于核酸的抑制剂，其包括与编码PDE4A 的信使RNA(mRNA)、编码PDE4B的mRNA、编码PDE4C的mRNA、编码PDE4D的mRNA 或任何组合的一部分互补的区域。
11.根据实施例10所述的方法，其中基于核酸的抑制剂与编码PDE4A、PDE4B、PDE4C或 PDE4D的mRNA选择性杂交。
12.根据实施例11所述的方法，其中基于核酸的抑制剂与编码PDE4B的mRNA选择性杂交。
13.根据实施例10-12任一项所述的方法，其中基于核酸的抑制剂是吗啉、短干扰RNA(siRNA)或微RNA(miRNA)。
14.根据实施例1-13任一项所述的方法，其中个体患有的癫痫，其选自以下的癫痫症组成的 组：良性罗兰癫痫、额叶癫痫、婴儿痉挛、青少年肌阵挛癫痫(JME)、青少年失神癫痫、儿 童失神癫痫、密集性癫痫、热性惊厥、进行性肌阵挛性癫痫、伦诺克斯-加斯托综合征、兰 道-克勒夫纳综合征、德拉韦综合征(DS)、全身性癫痫伴热性惊厥(GEFS+)、婴儿严重肌阵 挛性癫痫(SMEI)、良性家族性新生儿惊厥(BFNC)、韦斯特综合征、大田原综合征、早期肌 阵挛脑病、迁移性部分性癫痫、婴儿癫痫性脑病、结节性硬化症(TSC)、局灶性皮质发育不 良、I型无脑回畸形、Miller-Dieker综合征、快乐木偶综合征、脆性X综合征、自闭症谱系障碍中的癫痫、皮质下带异位症、Walker-Warburg综合征、阿尔茨海默病癫痫、创伤后癫痫、进行性肌阵挛癫痫、反射性癫痫、拉斯穆森病综合征、颞叶癫痫、边缘癫痫、癫痫持续状态、腹部癫痫、双侧大量肌阵挛、月经性癫痫、杰克逊癫痫症、Unverricht-Lundborg病和光敏性癫痫。
15.根据实施例1-13任一项所述的方法，其中个体患有由基因突变引起的癫痫。
16.一种鉴定抗癫痫剂的方法，包括在PDE4活性测定中使磷酸二酯酶4(PDE4)多肽与候选药 剂接触，其中候选药物对PDE4多肽的活性的抑制将候选药物鉴定为抗癫痫剂。
17.根据实施例16所述的方法，其中接触包括在无细胞PDE4活性测定中组合PDE4多肽和候 选药剂。
18.根据实施例16所述的方法，其中接触包括在基于细胞的PDE4活性测定中组合PDE4多肽 和候选药剂。
19.根据实施例16-18任一项所述的方法，其中PDE4活性测定进一步包括将PDE4多肽与已 知抑制PDE4活性的阳性对照剂接触。
20.根据实施例19所述的方法，其中阳性对照剂选自以下所组成的组：AN2728、屈他维林、 异丁司特、伊索格列定、匹拉米司特、罗氟司特、咯利普兰、茶碱、阿普司特及其任意组合。
21.根据实施例16-20中任一项所述的方法，其中所述候选药剂是小分子。
22.根据实施例16-21中任一项所述的方法，其中PDE4多肽是PDE4A、PDE4B、PDE4C或 PDE4D。
23.根据实施例22的方法，其中PDE4多肽是PDE4B。
24.根据实施例16-23中任一项所述的方法，进一步包括，当确定候选药剂抑制PED4多肽的 活性时，确定候选药剂是否表现出对PDE4A、PDE4B、PDE4C和PDE4D的选择性抑制。
25.一种药物组合物，其包括通过根据实施例16-24中任一项所述的方法鉴定的抗癫痫剂。
26.一种方法，包括向患有癫痫的个体施用治疗有效量的抗癫痫剂，该癫痫剂通过根据实施 例16-24中任一项所述的方法鉴定。
提供以下实例是为了说明而非限制。
实验
实例1–PDE4抑制将scn1lab和kcna1突变体斑马鱼中的生物能恢复到基线水平
如图6(A组)所示是证明PDE4抑制剂(AN2728)实例在scn1lab突变体斑马鱼中剂量依赖 性地将生物能恢复至基线水平的数据。(在图6-8中，“Scn1a”突变斑马鱼是scn1lab突变斑 马鱼)。本实验部分使用的scn1lab突变斑马鱼是纯合的、功能缺失突变体。为了全面测量 生物能，将活斑马鱼(其详细信息可在Ibhazehiebo等人(2018)Brain 141(3)：744-761 中找到)，斑马鱼幼体(受精后5天，dpf)接种到24-孔胰岛捕获微孔板(安捷伦)并在测定开始前预先暴露于载体或药物20分钟。使用海马生物分析仪(安捷伦)测量耗氧率(OCR)。在这个例子中，scn1lab突变体斑马鱼幼体相对于野生型(WT)对照显示出较低的OCR，表明线粒体呼吸在scn1lab突变体背景中降低。在AN2728存在下，观察到生物能的 剂量依赖性恢复，1μM和10μM将OCR应答完全恢复到WT水平(图6，A组)。1nM- 100nM剂量无效，100μM剂量有毒，如OCR反应低于突变体水平的结果所证实。
如图6(B组)所示的是证明多种PDE4抑制剂(在本例中为咯利普兰、西洛司特、罗氟司 特、异布司特、茶碱、屈他维林和伊索格莱定)可有效将scn1lab突变体斑马鱼的生物能恢 复到基线水平的数据。在与上述类似的测定中，scn1lab突变体暴露于40μM剂量的一系列 PDE4抑制剂。咯利普兰、西洛司特、罗氟司特、异丁司特都将OCR应答恢复到在WT幼体 中观察到的基线水平，表明这些药物对PDE4的抑制逆转了scn1lab突变斑马鱼暴露于载体时生物能的降低。
如图7所示的是证明PDE4B-、4C-和4D-吗啉可有效将scn1lab突变体斑马鱼中的生物 能恢复到基线水平的数据。scn1lab突变斑马鱼胚胎在一个细胞阶段注射ATG和/或剪接阻断 吗啉(Gene-Tools,Philomath,OR)，设计为敲除癫痫斑马鱼遗传背景中的特定PDE4亚型。由 于斑马鱼基因组中的基因重复，许多在人类中具有单一直系同源物的基因在斑马鱼中具有两 种亚型。吗啉旨在单独阻止每个斑马鱼亚型。Morpholinos注射的scn1lab突变斑马鱼在24hpf 进行分类，并将存活的胚胎接种到胰岛微孔板中，用于在5dpf的生物能测定(海马生物分析 仪)。靶向PDE4Ba、PDE4Bb、PDE4Cb和PDE4D的吗啉在癫痫背景下恢复了对WT基线 水平的OCR应答，表明PDE4信号转导的敲除减轻了在scn1lab突变体中观察到的线粒体功 能的降低。
如图8所示的是证明两个PDE4抑制剂实例(罗氟司特和茶碱)将kcna1突变斑马鱼中 的生物能恢复到在野生型鱼中观察到的基线水平的数据。kcna1突变体为全身性癫痫模型， 也在癫痫(SUDEP)中突然意外死亡，这表明抑制PDE4很可能在更广泛的癫痫条件下激发 有效的抗癫痫特性。
实例2–PDE4抑制在scn1lab斑马鱼中阻止癫痫样过度兴奋达到基线水平
如图9所示的是表明一个PDE4抑制剂实例(AN2728)在scn1lab斑马鱼中阻止癫痫样过 度兴奋达到基线水平。简而言之，6dpf斑马鱼(WT和scn1lab突变体)麻痹后(α-银环蛇毒素，1mg/ml，Tocris)并植入在琼脂糖中。将斑马鱼的背侧暴露于琼脂糖凝胶表面，将玻璃微电极放入斑马鱼的顶盖中并记录电生理测量值。基线记录20分钟后，药物(终浓度 20μM)被直接添加到胚胎培养基中，在接下来的20分钟内，对同一条斑马鱼进行连续记录。 注意不要干扰移液管或以任何方式移动鱼。癫痫样活动由高频、大振幅尖峰定义，如其他地方所定义(Baraban等人，2013)。在放大EEG轨迹的一部分(数据未显示)时观察到itcal (持续时间>1000ms)和发作间期(持续时间<300ms)活动，与癫痫样事件一致。暴露于 PDE抑制剂(AN2728,40μM)将这种癫痫样活动阻止到基线水平。对多条scn1lab突变斑马鱼 (n＝6-10)的这些过度兴奋性事件进行量化(图9，B组)表明，AN2728治疗后，突变体 的癫痫发作频率和峰值振幅降低。
如图10所示的是证明PDE4B吗啉能有效阻止scn1lab斑马鱼的癫痫样兴奋性达到基线 水平的数据。如上所述，注射靶向PDE4Ba和PDE4Bb的PDE4亚型特异性吗啉可阻断在scn1lab突变体中观察到的过度兴奋表型。PDE4Ca-、PDE4Cb-和PDE4D-吗啉都部分阻断了这种癫痫样活动，而PDE4A-morpholino没有效果，甚至可能加剧过度兴奋的表型。与图9， B组中所示的scn1lab水平相比，注射了PDE4亚型靶向的吗啉的scn1lab突变斑马鱼(n＝6-10)中的这些过度兴奋性事件的量化显示，PDE4B-、PDE4C-和PDE4D-morpolinos的发作频率和幅度降低。
实例3–PDE4抑制降低小鼠Scn1a突变脑离体切片的过度兴奋
图11提供了EEG数据，证明PDE4抑制剂(AN2728)实例降低了小鼠Scn1a突变体脑离体切片的过度兴奋性。本实验部分采用的小鼠Scn1a突变体是杂合突变体(Scn1a-/-小鼠在出生后过早死亡)。如图所示，离体Scn1a突变小鼠的脑电图记录显示AN2728降低了过度 兴奋表型(n＝3)。在该测定中，成人Scn1a突变体大脑被迅速取出并切片用于电生理测定。 在此，在添加PDE4抑制剂(AN2728,40μM)之前，进行细胞外场记录以测量基线电生理活动。 在AN2728存在下测量到过度兴奋的振幅和频率的降低。
实例4–PDE4抑制可防止在6Hz诱导小鼠模型中诱发癫痫发作
图12提供的是证明PDE4抑制剂(AN2728)实例在6Hz诱导小鼠模型中防止癫痫诱导的 数据。为了检查PDE4抑制对癫痫发作的急性影响，采用了6Hz精神运动模型，代表了治疗抵抗性边缘系统癫痫发作(White et al.,1995；Barton et al.,2001)。简单地说，通过角膜电极传 递低频(6Hz)、长时间(3秒)刺激，筛选化合物是否能够阻止精神运动性癫痫发作。据 信这些癫痫发作模拟了在人类中观察到的部分癫痫发作。当在6Hz测试前30分钟给动物给 药时，只有1只动物在最高剂量(300mg/kg)下有反应。然而，当在6Hz试验前两小时用 AN2728治疗时，四分之三的动物有反应。
实例5–PDE4抑制在Scn1a突变小鼠中防止高温诱导的癫痫发作
如图13所示(A组和B组)，一个PDE4抑制剂(AN2728)实例在Scn1a突变小鼠中防止高温诱导的癫痫发作。在该模型中，Scn1a突变体动物的核心温度因使用外部热源而升高，因此，大脑温度升高，这导致高热癫痫发作。这些高热癫痫发作被认为代表热性惊厥。在Scn1a突变体中，癫痫发作开始于41℃，而在WT中，即使在高达43℃的温度下也很少检 测到癫痫发作。用PDE4抑制剂(AN2728)治疗后，Scn1a突变体可免受高温诱导的癫痫发作， 而一些动物从未经历过癫痫发作(n＝3)，而其余动物仅在较高温度下经历过癫痫发作(例如，42℃)。图12B是以不同方式绘制的相同数据。
如图14所示的是证明双重选择性PDE4B抑制剂(咯利普兰)在Scn1a突变小鼠中有效防 止高温诱导的癫痫发作的数据。使用上述高温诱导测定，用不同的PDE4B抑制剂(咯利普 兰，3mg/kg)对Scn1a突变小鼠进行治疗也对高温诱导的癫痫发作具有神经保护作用，不仅 提高了诱发癫痫发作所需的温度(A组)还提高了癫痫发作的严重程度，使用Racine量表测 量(B组)，其严重程度为从大多数4-5级癫痫发作到3级。
如图15所示是使用上述小鼠Scn1a高温模型比较三种示例PDE4抑制剂(AN2728、咯利普兰、罗氟司特)在阻断癫痫发作中的作用的数据。当诱导发作所需的温度与野生型统计学不同时，注意到发作中的部分阻滞，而当用PDE4抑制剂预处理后未引起发作时，发生发作中的完全阻滞。
因此，以上仅说明本公开的原理。应当理解的是，本领域技术人员能够设计各种布置， 尽管未在本文明确描述或示出，但所述各种布置体现了本发明的原理并且包括在本发明的精 神和范围内。此外，本文列举的所有实例和条件性语言主要旨在帮助读者理解本发明的原理 和发明人为促进本领域的发展而贡献的概念，并且被解释为不限于这些具体列举的实例和条 件。此外，本文叙述本发明的原理、方面和实施例以及其具体实例的所有陈述旨在涵盖其结 构和功能等同物。另外，预期此类等同物包括当前已知的等同物以及未来开发的等同物，即， 开发的不论结构如何执行相同功能的任何要素。因此，本发明的范围并不旨在限于本文示出 和描述的示范性实施例。
序列表
<110> 通路治疗公司
　黛博拉·库拉施
　金斯利·伊布哈泽赫博
<120> 通过磷酸二酯酶4(PDE4)抑制治疗癫痫的方法
<130> PATH-001WO
<150> US 62/796,002
<151> 2019-01-23
<160> 26
<170> PatentIn version 3.5
<210> 1
<211> 886
<212> PRT
<213> 智人
<400> 1
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His Asp Val Asp His Pro Gly Val Ser Asn Gln Phe Leu Ile Asn Thr
275 280 285
Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Glu Ser Val Leu Glu Asn
290 295 300
His His Leu Ala Val Gly Phe Lys Leu Leu Gln Glu Asp Asn Cys Asp
305 310 315 320
Ile Phe Gln Asn Leu Ser Lys Arg Gln Arg Gln Ser Leu Arg Lys Met
325 330 335
Val Ile Asp Met Val Leu Ala Thr Asp Met Ser Lys His Met Thr Leu
340 345 350
Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr Ser Ser
355 360 365
Gly Val Leu Leu Leu Asp Asn Tyr Ser Asp Arg Ile Gln Val Leu Arg
370 375 380
Asn Met Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys Pro Leu Glu
385 390 395 400
Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Ala Glu Phe Phe Gln Gln
405 410 415
Gly Asp Arg Glu Arg Glu Arg Gly Met Glu Ile Ser Pro Met Cys Asp
420 425 430
Lys His Thr Ala Ser Val Glu Lys Ser Gln Val Gly Phe Ile Asp Tyr
435 440 445
Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp Leu Val His Pro Asp
450 455 460
Ala Gln Glu Ile Leu Asp Thr Leu Glu Asp Asn Arg Asp Trp Tyr Tyr
465 470 475 480
Ser Ala Ile Arg Gln Ser Pro Ser Pro Pro Pro Glu Glu Glu Ser Arg
485 490 495
Gly Pro Gly His Pro Pro Leu Pro Asp Lys Phe Gln Phe Glu Leu Thr
500 505 510
Leu Glu Glu Glu Glu Glu Glu Glu Ile Ser Met Ala Gln Ile Pro Cys
515 520 525
Thr Ala Gln Glu Ala Leu Thr Ala Gln Gly Leu Ser Gly Val Glu Glu
530 535 540
Ala Leu Asp Ala Thr Ile Ala Trp Glu Ala Ser Pro Ala Gln Glu Ser
545 550 555 560
Leu Glu Val Met Ala Gln Glu Ala Ser Leu Glu Ala Glu Leu Glu Ala
565 570 575
Val Tyr Leu Thr Gln Gln Ala Gln Ser Thr Gly Ser Ala Pro Val Ala
580 585 590
Pro Asp Glu Phe Ser Ser Arg Glu Glu Phe Val Val Ala Val Ser His
595 600 605
Ser Ser Pro Ser Ala Leu Ala Leu Gln Ser Pro Leu Leu Pro Ala Trp
610 615 620
Arg Thr Leu Ser Val Ser Glu His Ala Pro Gly Leu Pro Gly Leu Pro
625 630 635 640
Ser Thr Ala Ala Glu Val Glu Ala Gln Arg Glu His Gln Ala Ala Lys
645 650 655
Arg Ala Cys Ser Ala Cys Ala Gly Thr Phe Gly Glu Asp Thr Ser Ala
660 665 670
Leu Pro Ala Pro Gly Gly Gly Gly Ser Gly Gly Asp Pro Thr
675 680 685
<210> 4
<211> 647
<212> PRT
<213> 智人
<400> 4
Met Pro Leu Val Asp Phe Phe Cys Glu Thr Cys Ser Lys Pro Trp Leu
1 5 10 15
Val Gly Trp Trp Asp Gln Phe Lys Arg Met Leu Asn Arg Glu Leu Thr
20 25 30
His Leu Ser Glu Met Ser Arg Ser Gly Asn Gln Val Ser Glu Tyr Ile
35 40 45
Ser Thr Thr Phe Leu Asp Lys Gln Asn Glu Val Glu Ile Pro Ser Pro
50 55 60
Thr Met Lys Glu Arg Glu Lys Gln Gln Ala Pro Arg Pro Arg Pro Ser
65 70 75 80
Gln Pro Pro Pro Pro Pro Val Pro His Leu Gln Pro Met Ser Gln Ile
85 90 95
Thr Gly Leu Lys Lys Leu Met His Ser Asn Ser Leu Asn Asn Ser Asn
100 105 110
Ile Pro Arg Phe Gly Val Lys Thr Asp Gln Glu Glu Leu Leu Ala Gln
115 120 125
Glu Leu Glu Asn Leu Asn Lys Trp Gly Leu Asn Ile Phe Cys Val Ser
130 135 140
Asp Tyr Ala Gly Gly Arg Ser Leu Thr Cys Ile Met Tyr Met Ile Phe
145 150 155 160
Gln Glu Arg Asp Leu Leu Lys Lys Phe Arg Ile Pro Val Asp Thr Met
165 170 175
Val Thr Tyr Met Leu Thr Leu Glu Asp His Tyr His Ala Asp Val Ala
180 185 190
Tyr His Asn Ser Leu His Ala Ala Asp Val Leu Gln Ser Thr His Val
195 200 205
Leu Leu Ala Thr Pro Ala Leu Asp Ala Val Phe Thr Asp Leu Glu Ile
210 215 220
Leu Ala Ala Leu Phe Ala Ala Ala Ile His Asp Val Asp His Pro Gly
225 230 235 240
Val Ser Asn Gln Phe Leu Ile Asn Thr Asn Ser Glu Leu Ala Leu Met
245 250 255
Tyr Asn Asp Glu Ser Val Leu Glu Asn His His Leu Ala Val Gly Phe
260 265 270
Lys Leu Leu Gln Glu Asp Asn Cys Asp Ile Phe Gln Asn Leu Ser Lys
275 280 285
Arg Gln Arg Gln Ser Leu Arg Lys Met Val Ile Asp Met Val Leu Ala
290 295 300
Thr Asp Met Ser Lys His Met Thr Leu Leu Ala Asp Leu Lys Thr Met
305 310 315 320
Val Glu Thr Lys Lys Val Thr Ser Ser Gly Val Leu Leu Leu Asp Asn
325 330 335
Tyr Ser Asp Arg Ile Gln Val Leu Arg Asn Met Val His Cys Ala Asp
340 345 350
Leu Ser Asn Pro Thr Lys Pro Leu Glu Leu Tyr Arg Gln Trp Thr Asp
355 360 365
Arg Ile Met Ala Glu Phe Phe Gln Gln Gly Asp Arg Glu Arg Glu Arg
370 375 380
Gly Met Glu Ile Ser Pro Met Cys Asp Lys His Thr Ala Ser Val Glu
385 390 395 400
Lys Ser Gln Val Gly Phe Ile Asp Tyr Ile Val His Pro Leu Trp Glu
405 410 415
Thr Trp Ala Asp Leu Val His Pro Asp Ala Gln Glu Ile Leu Asp Thr
420 425 430
Leu Glu Asp Asn Arg Asp Trp Tyr Tyr Ser Ala Ile Arg Gln Ser Pro
435 440 445
Ser Pro Pro Pro Glu Glu Glu Ser Arg Gly Pro Gly His Pro Pro Leu
450 455 460
Pro Asp Lys Phe Gln Phe Glu Leu Thr Leu Glu Glu Glu Glu Glu Glu
465 470 475 480
Glu Ile Ser Met Ala Gln Ile Pro Cys Thr Ala Gln Glu Ala Leu Thr
485 490 495
Ala Gln Gly Leu Ser Gly Val Glu Glu Ala Leu Asp Ala Thr Ile Ala
500 505 510
Trp Glu Ala Ser Pro Ala Gln Glu Ser Leu Glu Val Met Ala Gln Glu
515 520 525
Ala Ser Leu Glu Ala Glu Leu Glu Ala Val Tyr Leu Thr Gln Gln Ala
530 535 540
Gln Ser Thr Gly Ser Ala Pro Val Ala Pro Asp Glu Phe Ser Ser Arg
545 550 555 560
Glu Glu Phe Val Val Ala Val Ser His Ser Ser Pro Ser Ala Leu Ala
565 570 575
Leu Gln Ser Pro Leu Leu Pro Ala Trp Arg Thr Leu Ser Val Ser Glu
580 585 590
His Ala Pro Gly Leu Pro Gly Leu Pro Ser Thr Ala Ala Glu Val Glu
595 600 605
Ala Gln Arg Glu His Gln Ala Ala Lys Arg Ala Cys Ser Ala Cys Ala
610 615 620
Gly Thr Phe Gly Glu Asp Thr Ser Ala Leu Pro Ala Pro Gly Gly Gly
625 630 635 640
Gly Ser Gly Gly Asp Pro Thr
645
<210> 5
<211> 323
<212> PRT
<213> 智人
<400> 5
Met Val Leu Pro Ser Asp Gln Gly Phe Lys Leu Leu Gly Asn Val Leu
1 5 10 15
Gln Gly Pro Glu Pro Tyr Arg Leu Leu Thr Ser Gly Leu Arg Leu His
20 25 30
Gln Glu Leu Glu Asn Leu Asn Lys Trp Gly Leu Asn Ile Phe Cys Val
35 40 45
Ser Asp Tyr Ala Gly Gly Arg Ser Leu Thr Cys Ile Met Tyr Met Ile
50 55 60
Phe Gln Glu Arg Asp Leu Leu Lys Lys Phe Arg Ile Pro Val Asp Thr
65 70 75 80
Met Val Thr Tyr Met Leu Thr Leu Glu Asp His Tyr His Ala Asp Val
85 90 95
Ala Tyr His Asn Ser Leu His Ala Ala Asp Val Leu Gln Ser Thr His
100 105 110
Val Leu Leu Ala Thr Pro Ala Leu Asp Ala Val Phe Thr Asp Leu Glu
115 120 125
Ile Leu Ala Ala Leu Phe Ala Ala Ala Ile His Asp Val Asp His Pro
130 135 140
Gly Val Ser Asn Gln Phe Leu Ile Asn Thr Asn Ser Glu Leu Ala Leu
145 150 155 160
Met Tyr Asn Asp Glu Ser Val Leu Glu Asn His His Leu Ala Val Gly
165 170 175
Phe Lys Leu Leu Gln Glu Asp Asn Cys Asp Ile Phe Gln Asn Leu Ser
180 185 190
Lys Arg Gln Arg Gln Ser Leu Arg Lys Met Val Ile Asp Met Val Leu
195 200 205
Ala Thr Asp Met Ser Lys His Met Thr Leu Leu Ala Asp Leu Lys Thr
210 215 220
Met Val Glu Thr Lys Lys Val Thr Ser Ser Gly Val Leu Leu Leu Asp
225 230 235 240
Asn Tyr Ser Asp Arg Ile Gln Val Leu Arg Asn Met Val His Cys Ala
245 250 255
Asp Leu Ser Asn Pro Thr Lys Pro Leu Glu Leu Tyr Arg Gln Trp Thr
260 265 270
Asp Arg Ile Met Ala Glu Phe Phe Gln Gln Gly Asp Arg Glu Arg Glu
275 280 285
Arg Gly Met Glu Ile Ser Pro Met Cys Asp Lys His Thr Ala Ser Val
290 295 300
Glu Lys Ser Gln Val Gln Ala Arg Gly Ile Asp Gly Arg Ala Gln Gly
305 310 315 320
Gly Phe Tyr
<210> 6
<211> 825
<212> PRT
<213> 智人
<400> 6
Met Arg Ser Gly Ala Ala Pro Arg Ala Arg Pro Arg Pro Pro Ala Leu
1 5 10 15
Ala Leu Pro Pro Thr Gly Pro Glu Ser Leu Thr His Phe Pro Phe Ser
20 25 30
Asp Glu Asp Thr Arg Arg His Pro Pro Gly Arg Ser Val Ser Phe Glu
35 40 45
Ala Glu Asn Gly Pro Thr Pro Ser Pro Gly Arg Ser Pro Leu Asp Ser
50 55 60
Gln Ala Ser Pro Gly Leu Val Leu His Ala Gly Ala Ala Thr Ser Gln
65 70 75 80
Arg Arg Glu Ser Phe Leu Tyr Arg Ser Asp Ser Asp Tyr Asp Met Ser
85 90 95
Pro Lys Thr Met Ser Arg Asn Ser Ser Val Thr Ser Glu Ala His Ala
100 105 110
Glu Asp Leu Ile Val Thr Pro Phe Ala Gln Val Leu Ala Ser Leu Arg
115 120 125
Ser Val Arg Ser Asn Phe Ser Leu Leu Thr Asn Val Pro Val Pro Ser
130 135 140
Asn Lys Arg Ser Pro Leu Gly Gly Pro Thr Pro Val Cys Lys Ala Thr
145 150 155 160
Leu Ser Glu Glu Thr Cys Gln Gln Leu Ala Arg Glu Thr Leu Glu Glu
165 170 175
Leu Asp Trp Cys Leu Glu Gln Leu Glu Thr Met Gln Thr Tyr Arg Ser
180 185 190
Val Ser Glu Met Ala Ser His Lys Phe Lys Arg Met Leu Asn Arg Glu
195 200 205
Leu Thr His Leu Ser Glu Met Ser Arg Ser Gly Asn Gln Val Ser Glu
210 215 220
Tyr Ile Ser Thr Thr Phe Leu Asp Lys Gln Asn Glu Val Glu Ile Pro
225 230 235 240
Ser Pro Thr Met Lys Glu Arg Glu Lys Gln Gln Ala Pro Arg Pro Arg
245 250 255
Pro Ser Gln Pro Pro Pro Pro Pro Val Pro His Leu Gln Pro Met Ser
260 265 270
Gln Ile Thr Gly Leu Lys Lys Leu Met His Ser Asn Ser Leu Asn Asn
275 280 285
Ser Asn Ile Pro Arg Phe Gly Val Lys Thr Asp Gln Glu Glu Leu Leu
290 295 300
Ala Gln Glu Leu Glu Asn Leu Asn Lys Trp Gly Leu Asn Ile Phe Cys
305 310 315 320
Val Ser Asp Tyr Ala Gly Gly Arg Ser Leu Thr Cys Ile Met Tyr Met
325 330 335
Ile Phe Gln Glu Arg Asp Leu Leu Lys Lys Phe Arg Ile Pro Val Asp
340 345 350
Thr Met Val Thr Tyr Met Leu Thr Leu Glu Asp His Tyr His Ala Asp
355 360 365
Val Ala Tyr His Asn Ser Leu His Ala Ala Asp Val Leu Gln Ser Thr
370 375 380
His Val Leu Leu Ala Thr Pro Ala Leu Asp Ala Val Phe Thr Asp Leu
385 390 395 400
Glu Ile Leu Ala Ala Leu Phe Ala Ala Ala Ile His Asp Val Asp His
405 410 415
Pro Gly Val Ser Asn Gln Phe Leu Ile Asn Thr Asn Ser Glu Leu Ala
420 425 430
Leu Met Tyr Asn Asp Glu Ser Val Leu Glu Asn His His Leu Ala Val
435 440 445
Gly Phe Lys Leu Leu Gln Glu Asp Asn Cys Asp Ile Phe Gln Asn Leu
450 455 460
Ser Lys Arg Gln Arg Gln Ser Leu Arg Lys Met Val Ile Asp Met Val
465 470 475 480
Leu Ala Thr Asp Met Ser Lys His Met Thr Leu Leu Ala Asp Leu Lys
485 490 495
Thr Met Val Glu Thr Lys Lys Val Thr Ser Ser Gly Val Leu Leu Leu
500 505 510
Asp Asn Tyr Ser Asp Arg Ile Gln Val Leu Arg Asn Met Val His Cys
515 520 525
Ala Asp Leu Ser Asn Pro Thr Lys Pro Leu Glu Leu Tyr Arg Gln Trp
530 535 540
Thr Asp Arg Ile Met Ala Glu Phe Phe Gln Gln Gly Asp Arg Glu Arg
545 550 555 560
Glu Arg Gly Met Glu Ile Ser Pro Met Cys Asp Lys His Thr Ala Ser
565 570 575
Val Glu Lys Ser Gln Val Gly Phe Ile Asp Tyr Ile Val His Pro Leu
580 585 590
Trp Glu Thr Trp Ala Asp Leu Val His Pro Asp Ala Gln Glu Ile Leu
595 600 605
Asp Thr Leu Glu Asp Asn Arg Asp Trp Tyr Tyr Ser Ala Ile Arg Gln
610 615 620
Ser Pro Ser Pro Pro Pro Glu Glu Glu Ser Arg Gly Pro Gly His Pro
625 630 635 640
Pro Leu Pro Asp Lys Phe Gln Phe Glu Leu Thr Leu Glu Glu Glu Glu
645 650 655
Glu Glu Glu Ile Ser Met Ala Gln Ile Pro Cys Thr Ala Gln Glu Ala
660 665 670
Leu Thr Ala Gln Gly Leu Ser Gly Val Glu Glu Ala Leu Asp Ala Thr
675 680 685
Ile Ala Trp Glu Ala Ser Pro Ala Gln Glu Ser Leu Glu Val Met Ala
690 695 700
Gln Glu Ala Ser Leu Glu Ala Glu Leu Glu Ala Val Tyr Leu Thr Gln
705 710 715 720
Gln Ala Gln Ser Thr Gly Ser Ala Pro Val Ala Pro Asp Glu Phe Ser
725 730 735
Ser Arg Glu Glu Phe Val Val Ala Val Ser His Ser Ser Pro Ser Ala
740 745 750
Leu Ala Leu Gln Ser Pro Leu Leu Pro Ala Trp Arg Thr Leu Ser Val
755 760 765
Ser Glu His Ala Pro Gly Leu Pro Gly Leu Pro Ser Thr Ala Ala Glu
770 775 780
Val Glu Ala Gln Arg Glu His Gln Ala Ala Lys Arg Ala Cys Ser Ala
785 790 795 800
Cys Ala Gly Thr Phe Gly Glu Asp Thr Ser Ala Leu Pro Ala Pro Gly
805 810 815
Gly Gly Gly Ser Gly Gly Asp Pro Thr
820 825
<210> 7
<211> 864
<212> PRT
<213> 智人
<400> 7
Met Lys Arg Ser Arg Ser Ala Leu Ser Val Ala Gly Thr Gly Asp Glu
1 5 10 15
Arg Ser Arg Glu Thr Pro Glu Ser Asp Arg Ala Asn Met Leu Gly Ala
20 25 30
Asp Leu Arg Arg Pro Arg Arg Arg Leu Ser Ser Gly Pro Gly Leu Gly
35 40 45
Trp Ala Gln Pro Glu Pro Ser Asp Pro Gly Val Pro Leu Pro Pro Arg
50 55 60
Pro Thr Thr Leu Pro Leu Leu Ile Pro Pro Arg Ile Ser Ile Thr Arg
65 70 75 80
Ala Glu Asn Asp Ser Phe Glu Ala Glu Asn Gly Pro Thr Pro Ser Pro
85 90 95
Gly Arg Ser Pro Leu Asp Ser Gln Ala Ser Pro Gly Leu Val Leu His
100 105 110
Ala Gly Ala Ala Thr Ser Gln Arg Arg Glu Ser Phe Leu Tyr Arg Ser
115 120 125
Asp Ser Asp Tyr Asp Met Ser Pro Lys Thr Met Ser Arg Asn Ser Ser
130 135 140
Val Thr Ser Glu Ala His Ala Glu Asp Leu Ile Val Thr Pro Phe Ala
145 150 155 160
Gln Val Leu Ala Ser Leu Arg Ser Val Arg Ser Asn Phe Ser Leu Leu
165 170 175
Thr Asn Val Pro Val Pro Ser Asn Lys Arg Ser Pro Leu Gly Gly Pro
180 185 190
Thr Pro Val Cys Lys Ala Thr Leu Ser Glu Glu Thr Cys Gln Gln Leu
195 200 205
Ala Arg Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu Glu Gln Leu Glu
210 215 220
Thr Met Gln Thr Tyr Arg Ser Val Ser Glu Met Ala Ser His Lys Phe
225 230 235 240
Lys Arg Met Leu Asn Arg Glu Leu Thr His Leu Ser Glu Met Ser Arg
245 250 255
Ser Gly Asn Gln Val Ser Glu Tyr Ile Ser Thr Thr Phe Leu Asp Lys
260 265 270
Gln Asn Glu Val Glu Ile Pro Ser Pro Thr Met Lys Glu Arg Glu Lys
275 280 285
Gln Gln Ala Pro Arg Pro Arg Pro Ser Gln Pro Pro Pro Pro Pro Val
290 295 300
Pro His Leu Gln Pro Met Ser Gln Ile Thr Gly Leu Lys Lys Leu Met
305 310 315 320
His Ser Asn Ser Leu Asn Asn Ser Asn Ile Pro Arg Phe Gly Val Lys
325 330 335
Thr Asp Gln Glu Glu Leu Leu Ala Gln Glu Leu Glu Asn Leu Asn Lys
340 345 350
Trp Gly Leu Asn Ile Phe Cys Val Ser Asp Tyr Ala Gly Gly Arg Ser
355 360 365
Leu Thr Cys Ile Met Tyr Met Ile Phe Gln Glu Arg Asp Leu Leu Lys
370 375 380
Lys Phe Arg Ile Pro Val Asp Thr Met Val Thr Tyr Met Leu Thr Leu
385 390 395 400
Glu Asp His Tyr His Ala Asp Val Ala Tyr His Asn Ser Leu His Ala
405 410 415
Ala Asp Val Leu Gln Ser Thr His Val Leu Leu Ala Thr Pro Ala Leu
420 425 430
Asp Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Leu Phe Ala Ala
435 440 445
Ala Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln Phe Leu Ile
450 455 460
Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Glu Ser Val Leu
465 470 475 480
Glu Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln Glu Asp Asn
485 490 495
Cys Asp Ile Phe Gln Asn Leu Ser Lys Arg Gln Arg Gln Ser Leu Arg
500 505 510
Lys Met Val Ile Asp Met Val Leu Ala Thr Asp Met Ser Lys His Met
515 520 525
Thr Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr
530 535 540
Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr Ser Asp Arg Ile Gln Val
545 550 555 560
Leu Arg Asn Met Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys Pro
565 570 575
Leu Glu Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Ala Glu Phe Phe
580 585 590
Gln Gln Gly Asp Arg Glu Arg Glu Arg Gly Met Glu Ile Ser Pro Met
595 600 605
Cys Asp Lys His Thr Ala Ser Val Glu Lys Ser Gln Val Gly Phe Ile
610 615 620
Asp Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp Leu Val His
625 630 635 640
Pro Asp Ala Gln Glu Ile Leu Asp Thr Leu Glu Asp Asn Arg Asp Trp
645 650 655
Tyr Tyr Ser Ala Ile Arg Gln Ser Pro Ser Pro Pro Pro Glu Glu Glu
660 665 670
Ser Arg Gly Pro Gly His Pro Pro Leu Pro Asp Lys Phe Gln Phe Glu
675 680 685
Leu Thr Leu Glu Glu Glu Glu Glu Glu Glu Ile Ser Met Ala Gln Ile
690 695 700
Pro Cys Thr Ala Gln Glu Ala Leu Thr Ala Gln Gly Leu Ser Gly Val
705 710 715 720
Glu Glu Ala Leu Asp Ala Thr Ile Ala Trp Glu Ala Ser Pro Ala Gln
725 730 735
Glu Ser Leu Glu Val Met Ala Gln Glu Ala Ser Leu Glu Ala Glu Leu
740 745 750
Glu Ala Val Tyr Leu Thr Gln Gln Ala Gln Ser Thr Gly Ser Ala Pro
755 760 765
Val Ala Pro Asp Glu Phe Ser Ser Arg Glu Glu Phe Val Val Ala Val
770 775 780
Ser His Ser Ser Pro Ser Ala Leu Ala Leu Gln Ser Pro Leu Leu Pro
785 790 795 800
Ala Trp Arg Thr Leu Ser Val Ser Glu His Ala Pro Gly Leu Pro Gly
805 810 815
Leu Pro Ser Thr Ala Ala Glu Val Glu Ala Gln Arg Glu His Gln Ala
820 825 830
Ala Lys Arg Ala Cys Ser Ala Cys Ala Gly Thr Phe Gly Glu Asp Thr
835 840 845
Ser Ala Leu Pro Ala Pro Gly Gly Gly Gly Ser Gly Gly Asp Pro Thr
850 855 860
<210> 8
<211> 736
<212> PRT
<213> 智人
<400> 8
Met Lys Lys Ser Arg Ser Val Met Thr Val Met Ala Asp Asp Asn Val
1 5 10 15
Lys Asp Tyr Phe Glu Cys Ser Leu Ser Lys Ser Tyr Ser Ser Ser Ser
20 25 30
Asn Thr Leu Gly Ile Asp Leu Trp Arg Gly Arg Arg Cys Cys Ser Gly
35 40 45
Asn Leu Gln Leu Pro Pro Leu Ser Gln Arg Gln Ser Glu Arg Ala Arg
50 55 60
Thr Pro Glu Gly Asp Gly Ile Ser Arg Pro Thr Thr Leu Pro Leu Thr
65 70 75 80
Thr Leu Pro Ser Ile Ala Ile Thr Thr Val Ser Gln Glu Cys Phe Asp
85 90 95
Val Glu Asn Gly Pro Ser Pro Gly Arg Ser Pro Leu Asp Pro Gln Ala
100 105 110
Ser Ser Ser Ala Gly Leu Val Leu His Ala Thr Phe Pro Gly His Ser
115 120 125
Gln Arg Arg Glu Ser Phe Leu Tyr Arg Ser Asp Ser Asp Tyr Asp Leu
130 135 140
Ser Pro Lys Ala Met Ser Arg Asn Ser Ser Leu Pro Ser Glu Gln His
145 150 155 160
Gly Asp Asp Leu Ile Val Thr Pro Phe Ala Gln Val Leu Ala Ser Leu
165 170 175
Arg Ser Val Arg Asn Asn Phe Thr Ile Leu Thr Asn Leu His Gly Thr
180 185 190
Ser Asn Lys Arg Ser Pro Ala Ala Ser Gln Pro Pro Val Ser Arg Val
195 200 205
Asn Pro Gln Glu Glu Ser Tyr Gln Lys Leu Ala Met Glu Thr Leu Glu
210 215 220
Glu Leu Asp Trp Cys Leu Asp Gln Leu Glu Thr Ile Gln Thr Tyr Arg
225 230 235 240
Ser Val Ser Glu Met Ala Ser Asn Lys Phe Lys Arg Met Leu Asn Arg
245 250 255
Glu Leu Thr His Leu Ser Glu Met Ser Arg Ser Gly Asn Gln Val Ser
260 265 270
Glu Tyr Ile Ser Asn Thr Phe Leu Asp Lys Gln Asn Asp Val Glu Ile
275 280 285
Pro Ser Pro Thr Gln Lys Asp Arg Glu Lys Lys Lys Lys Gln Gln Leu
290 295 300
Met Thr Gln Ile Ser Gly Val Lys Lys Leu Met His Ser Ser Ser Leu
305 310 315 320
Asn Asn Thr Ser Ile Ser Arg Phe Gly Val Asn Thr Glu Asn Glu Asp
325 330 335
His Leu Ala Lys Glu Leu Glu Asp Leu Asn Lys Trp Gly Leu Asn Ile
340 345 350
Phe Asn Val Ala Gly Tyr Ser His Asn Arg Pro Leu Thr Cys Ile Met
355 360 365
Tyr Ala Ile Phe Gln Glu Arg Asp Leu Leu Lys Thr Phe Arg Ile Ser
370 375 380
Ser Asp Thr Phe Ile Thr Tyr Met Met Thr Leu Glu Asp His Tyr His
385 390 395 400
Ser Asp Val Ala Tyr His Asn Ser Leu His Ala Ala Asp Val Ala Gln
405 410 415
Ser Thr His Val Leu Leu Ser Thr Pro Ala Leu Asp Ala Val Phe Thr
420 425 430
Asp Leu Glu Ile Leu Ala Ala Ile Phe Ala Ala Ala Ile His Asp Val
435 440 445
Asp His Pro Gly Val Ser Asn Gln Phe Leu Ile Asn Thr Asn Ser Glu
450 455 460
Leu Ala Leu Met Tyr Asn Asp Glu Ser Val Leu Glu Asn His His Leu
465 470 475 480
Ala Val Gly Phe Lys Leu Leu Gln Glu Glu His Cys Asp Ile Phe Met
485 490 495
Asn Leu Thr Lys Lys Gln Arg Gln Thr Leu Arg Lys Met Val Ile Asp
500 505 510
Met Val Leu Ala Thr Asp Met Ser Lys His Met Ser Leu Leu Ala Asp
515 520 525
Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr Ser Ser Gly Val Leu
530 535 540
Leu Leu Asp Asn Tyr Thr Asp Arg Ile Gln Val Leu Arg Asn Met Val
545 550 555 560
His Cys Ala Asp Leu Ser Asn Pro Thr Lys Ser Leu Glu Leu Tyr Arg
565 570 575
Gln Trp Thr Asp Arg Ile Met Glu Glu Phe Phe Gln Gln Gly Asp Lys
580 585 590
Glu Arg Glu Arg Gly Met Glu Ile Ser Pro Met Cys Asp Lys His Thr
595 600 605
Ala Ser Val Glu Lys Ser Gln Val Gly Phe Ile Asp Tyr Ile Val His
610 615 620
Pro Leu Trp Glu Thr Trp Ala Asp Leu Val Gln Pro Asp Ala Gln Asp
625 630 635 640
Ile Leu Asp Thr Leu Glu Asp Asn Arg Asn Trp Tyr Gln Ser Met Ile
645 650 655
Pro Gln Ser Pro Ser Pro Pro Leu Asp Glu Gln Asn Arg Asp Cys Gln
660 665 670
Gly Leu Met Glu Lys Phe Gln Phe Glu Leu Thr Leu Asp Glu Glu Asp
675 680 685
Ser Glu Gly Pro Glu Lys Glu Gly Glu Gly His Ser Tyr Phe Ser Ser
690 695 700
Thr Lys Thr Leu Cys Val Ile Asp Pro Glu Asn Arg Asp Ser Leu Gly
705 710 715 720
Glu Thr Asp Ile Asp Ile Ala Thr Glu Asp Lys Ser Pro Val Asp Thr
725 730 735
<210> 9
<211> 564
<212> PRT
<213> 智人
<400> 9
Met Lys Glu His Gly Gly Thr Phe Ser Ser Thr Gly Ile Ser Gly Gly
1 5 10 15
Ser Gly Asp Ser Ala Met Asp Ser Leu Gln Pro Leu Gln Pro Asn Tyr
20 25 30
Met Pro Val Cys Leu Phe Ala Glu Glu Ser Tyr Gln Lys Leu Ala Met
35 40 45
Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu Asp Gln Leu Glu Thr Ile
50 55 60
Gln Thr Tyr Arg Ser Val Ser Glu Met Ala Ser Asn Lys Phe Lys Arg
65 70 75 80
Met Leu Asn Arg Glu Leu Thr His Leu Ser Glu Met Ser Arg Ser Gly
85 90 95
Asn Gln Val Ser Glu Tyr Ile Ser Asn Thr Phe Leu Asp Lys Gln Asn
100 105 110
Asp Val Glu Ile Pro Ser Pro Thr Gln Lys Asp Arg Glu Lys Lys Lys
115 120 125
Lys Gln Gln Leu Met Thr Gln Ile Ser Gly Val Lys Lys Leu Met His
130 135 140
Ser Ser Ser Leu Asn Asn Thr Ser Ile Ser Arg Phe Gly Val Asn Thr
145 150 155 160
Glu Asn Glu Asp His Leu Ala Lys Glu Leu Glu Asp Leu Asn Lys Trp
165 170 175
Gly Leu Asn Ile Phe Asn Val Ala Gly Tyr Ser His Asn Arg Pro Leu
180 185 190
Thr Cys Ile Met Tyr Ala Ile Phe Gln Glu Arg Asp Leu Leu Lys Thr
195 200 205
Phe Arg Ile Ser Ser Asp Thr Phe Ile Thr Tyr Met Met Thr Leu Glu
210 215 220
Asp His Tyr His Ser Asp Val Ala Tyr His Asn Ser Leu His Ala Ala
225 230 235 240
Asp Val Ala Gln Ser Thr His Val Leu Leu Ser Thr Pro Ala Leu Asp
245 250 255
Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Ile Phe Ala Ala Ala
260 265 270
Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln Phe Leu Ile Asn
275 280 285
Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Glu Ser Val Leu Glu
290 295 300
Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln Glu Glu His Cys
305 310 315 320
Asp Ile Phe Met Asn Leu Thr Lys Lys Gln Arg Gln Thr Leu Arg Lys
325 330 335
Met Val Ile Asp Met Val Leu Ala Thr Asp Met Ser Lys His Met Ser
340 345 350
Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr Ser
355 360 365
Ser Gly Val Leu Leu Leu Asp Asn Tyr Thr Asp Arg Ile Gln Val Leu
370 375 380
Arg Asn Met Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys Ser Leu
385 390 395 400
Glu Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Glu Glu Phe Phe Gln
405 410 415
Gln Gly Asp Lys Glu Arg Glu Arg Gly Met Glu Ile Ser Pro Met Cys
420 425 430
Asp Lys His Thr Ala Ser Val Glu Lys Ser Gln Val Gly Phe Ile Asp
435 440 445
Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp Leu Val Gln Pro
450 455 460
Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp Asn Arg Asn Trp Tyr
465 470 475 480
Gln Ser Met Ile Pro Gln Ser Pro Ser Pro Pro Leu Asp Glu Gln Asn
485 490 495
Arg Asp Cys Gln Gly Leu Met Glu Lys Phe Gln Phe Glu Leu Thr Leu
500 505 510
Asp Glu Glu Asp Ser Glu Gly Pro Glu Lys Glu Gly Glu Gly His Ser
515 520 525
Tyr Phe Ser Ser Thr Lys Thr Leu Cys Val Ile Asp Pro Glu Asn Arg
530 535 540
Asp Ser Leu Gly Glu Thr Asp Ile Asp Ile Ala Thr Glu Asp Lys Ser
545 550 555 560
Pro Val Asp Thr
<210> 10
<211> 721
<212> PRT
<213> 智人
<400> 10
Met Thr Ala Lys Asp Ser Ser Lys Glu Leu Thr Ala Ser Glu Pro Glu
1 5 10 15
Val Cys Ile Lys Thr Phe Lys Glu Gln Met His Leu Glu Leu Glu Leu
20 25 30
Pro Arg Leu Pro Gly Asn Arg Pro Thr Ser Pro Lys Ile Ser Pro Arg
35 40 45
Ser Ser Pro Arg Asn Ser Pro Cys Phe Phe Arg Lys Leu Leu Val Asn
50 55 60
Lys Ser Ile Arg Gln Arg Arg Arg Phe Thr Val Ala His Thr Cys Phe
65 70 75 80
Asp Val Glu Asn Gly Pro Ser Pro Gly Arg Ser Pro Leu Asp Pro Gln
85 90 95
Ala Ser Ser Ser Ala Gly Leu Val Leu His Ala Thr Phe Pro Gly His
100 105 110
Ser Gln Arg Arg Glu Ser Phe Leu Tyr Arg Ser Asp Ser Asp Tyr Asp
115 120 125
Leu Ser Pro Lys Ala Met Ser Arg Asn Ser Ser Leu Pro Ser Glu Gln
130 135 140
His Gly Asp Asp Leu Ile Val Thr Pro Phe Ala Gln Val Leu Ala Ser
145 150 155 160
Leu Arg Ser Val Arg Asn Asn Phe Thr Ile Leu Thr Asn Leu His Gly
165 170 175
Thr Ser Asn Lys Arg Ser Pro Ala Ala Ser Gln Pro Pro Val Ser Arg
180 185 190
Val Asn Pro Gln Glu Glu Ser Tyr Gln Lys Leu Ala Met Glu Thr Leu
195 200 205
Glu Glu Leu Asp Trp Cys Leu Asp Gln Leu Glu Thr Ile Gln Thr Tyr
210 215 220
Arg Ser Val Ser Glu Met Ala Ser Asn Lys Phe Lys Arg Met Leu Asn
225 230 235 240
Arg Glu Leu Thr His Leu Ser Glu Met Ser Arg Ser Gly Asn Gln Val
245 250 255
Ser Glu Tyr Ile Ser Asn Thr Phe Leu Asp Lys Gln Asn Asp Val Glu
260 265 270
Ile Pro Ser Pro Thr Gln Lys Asp Arg Glu Lys Lys Lys Lys Gln Gln
275 280 285
Leu Met Thr Gln Ile Ser Gly Val Lys Lys Leu Met His Ser Ser Ser
290 295 300
Leu Asn Asn Thr Ser Ile Ser Arg Phe Gly Val Asn Thr Glu Asn Glu
305 310 315 320
Asp His Leu Ala Lys Glu Leu Glu Asp Leu Asn Lys Trp Gly Leu Asn
325 330 335
Ile Phe Asn Val Ala Gly Tyr Ser His Asn Arg Pro Leu Thr Cys Ile
340 345 350
Met Tyr Ala Ile Phe Gln Glu Arg Asp Leu Leu Lys Thr Phe Arg Ile
355 360 365
Ser Ser Asp Thr Phe Ile Thr Tyr Met Met Thr Leu Glu Asp His Tyr
370 375 380
His Ser Asp Val Ala Tyr His Asn Ser Leu His Ala Ala Asp Val Ala
385 390 395 400
Gln Ser Thr His Val Leu Leu Ser Thr Pro Ala Leu Asp Ala Val Phe
405 410 415
Thr Asp Leu Glu Ile Leu Ala Ala Ile Phe Ala Ala Ala Ile His Asp
420 425 430
Val Asp His Pro Gly Val Ser Asn Gln Phe Leu Ile Asn Thr Asn Ser
435 440 445
Glu Leu Ala Leu Met Tyr Asn Asp Glu Ser Val Leu Glu Asn His His
450 455 460
Leu Ala Val Gly Phe Lys Leu Leu Gln Glu Glu His Cys Asp Ile Phe
465 470 475 480
Met Asn Leu Thr Lys Lys Gln Arg Gln Thr Leu Arg Lys Met Val Ile
485 490 495
Asp Met Val Leu Ala Thr Asp Met Ser Lys His Met Ser Leu Leu Ala
500 505 510
Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr Ser Ser Gly Val
515 520 525
Leu Leu Leu Asp Asn Tyr Thr Asp Arg Ile Gln Val Leu Arg Asn Met
530 535 540
Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys Ser Leu Glu Leu Tyr
545 550 555 560
Arg Gln Trp Thr Asp Arg Ile Met Glu Glu Phe Phe Gln Gln Gly Asp
565 570 575
Lys Glu Arg Glu Arg Gly Met Glu Ile Ser Pro Met Cys Asp Lys His
580 585 590
Thr Ala Ser Val Glu Lys Ser Gln Val Gly Phe Ile Asp Tyr Ile Val
595 600 605
His Pro Leu Trp Glu Thr Trp Ala Asp Leu Val Gln Pro Asp Ala Gln
610 615 620
Asp Ile Leu Asp Thr Leu Glu Asp Asn Arg Asn Trp Tyr Gln Ser Met
625 630 635 640
Ile Pro Gln Ser Pro Ser Pro Pro Leu Asp Glu Gln Asn Arg Asp Cys
645 650 655
Gln Gly Leu Met Glu Lys Phe Gln Phe Glu Leu Thr Leu Asp Glu Glu
660 665 670
Asp Ser Glu Gly Pro Glu Lys Glu Gly Glu Gly His Ser Tyr Phe Ser
675 680 685
Ser Thr Lys Thr Leu Cys Val Ile Asp Pro Glu Asn Arg Asp Ser Leu
690 695 700
Gly Glu Thr Asp Ile Asp Ile Ala Thr Glu Asp Lys Ser Pro Val Asp
705 710 715 720
Thr
<210> 11
<211> 503
<212> PRT
<213> 智人
<400> 11
Met Pro Glu Ala Asn Tyr Leu Leu Ser Val Ser Trp Gly Tyr Ile Lys
1 5 10 15
Phe Lys Arg Met Leu Asn Arg Glu Leu Thr His Leu Ser Glu Met Ser
20 25 30
Arg Ser Gly Asn Gln Val Ser Glu Tyr Ile Ser Asn Thr Phe Leu Asp
35 40 45
Lys Gln Asn Asp Val Glu Ile Pro Ser Pro Thr Gln Lys Asp Arg Glu
50 55 60
Lys Lys Lys Lys Gln Gln Leu Met Thr Gln Ile Ser Gly Val Lys Lys
65 70 75 80
Leu Met His Ser Ser Ser Leu Asn Asn Thr Ser Ile Ser Arg Phe Gly
85 90 95
Val Asn Thr Glu Asn Glu Asp His Leu Ala Lys Glu Leu Glu Asp Leu
100 105 110
Asn Lys Trp Gly Leu Asn Ile Phe Asn Val Ala Gly Tyr Ser His Asn
115 120 125
Arg Pro Leu Thr Cys Ile Met Tyr Ala Ile Phe Gln Glu Arg Asp Leu
130 135 140
Leu Lys Thr Phe Arg Ile Ser Ser Asp Thr Phe Ile Thr Tyr Met Met
145 150 155 160
Thr Leu Glu Asp His Tyr His Ser Asp Val Ala Tyr His Asn Ser Leu
165 170 175
His Ala Ala Asp Val Ala Gln Ser Thr His Val Leu Leu Ser Thr Pro
180 185 190
Ala Leu Asp Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Ile Phe
195 200 205
Ala Ala Ala Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln Phe
210 215 220
Leu Ile Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Glu Ser
225 230 235 240
Val Leu Glu Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln Glu
245 250 255
Glu His Cys Asp Ile Phe Met Asn Leu Thr Lys Lys Gln Arg Gln Thr
260 265 270
Leu Arg Lys Met Val Ile Asp Met Val Leu Ala Thr Asp Met Ser Lys
275 280 285
His Met Ser Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys
290 295 300
Val Thr Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr Thr Asp Arg Ile
305 310 315 320
Gln Val Leu Arg Asn Met Val His Cys Ala Asp Leu Ser Asn Pro Thr
325 330 335
Lys Ser Leu Glu Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Glu Glu
340 345 350
Phe Phe Gln Gln Gly Asp Lys Glu Arg Glu Arg Gly Met Glu Ile Ser
355 360 365
Pro Met Cys Asp Lys His Thr Ala Ser Val Glu Lys Ser Gln Val Gly
370 375 380
Phe Ile Asp Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp Leu
385 390 395 400
Val Gln Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp Asn Arg
405 410 415
Asn Trp Tyr Gln Ser Met Ile Pro Gln Ser Pro Ser Pro Pro Leu Asp
420 425 430
Glu Gln Asn Arg Asp Cys Gln Gly Leu Met Glu Lys Phe Gln Phe Glu
435 440 445
Leu Thr Leu Asp Glu Glu Asp Ser Glu Gly Pro Glu Lys Glu Gly Glu
450 455 460
Gly His Ser Tyr Phe Ser Ser Thr Lys Thr Leu Cys Val Ile Asp Pro
465 470 475 480
Glu Asn Arg Asp Ser Leu Gly Glu Thr Asp Ile Asp Ile Ala Thr Glu
485 490 495
Asp Lys Ser Pro Val Asp Thr
500
<210> 12
<211> 712
<212> PRT
<213> 智人
<400> 12
Met Glu Asn Leu Gly Val Gly Glu Gly Ala Glu Ala Cys Ser Arg Leu
1 5 10 15
Ser Arg Ser Arg Gly Arg His Ser Met Thr Arg Ala Pro Lys His Leu
20 25 30
Trp Arg Gln Pro Arg Arg Pro Ile Arg Ile Gln Gln Arg Phe Tyr Ser
35 40 45
Asp Pro Asp Lys Ser Ala Gly Cys Arg Glu Arg Asp Leu Ser Pro Arg
50 55 60
Pro Glu Leu Arg Lys Ser Arg Leu Ser Trp Pro Val Ser Ser Cys Arg
65 70 75 80
Arg Phe Asp Leu Glu Asn Gly Leu Ser Cys Gly Arg Arg Ala Leu Asp
85 90 95
Pro Gln Ser Ser Pro Gly Leu Gly Arg Ile Met Gln Ala Pro Val Pro
100 105 110
His Ser Gln Arg Arg Glu Ser Phe Leu Tyr Arg Ser Asp Ser Asp Tyr
115 120 125
Glu Leu Ser Pro Lys Ala Met Ser Arg Asn Ser Ser Val Ala Ser Asp
130 135 140
Leu His Gly Glu Asp Met Ile Val Thr Pro Phe Ala Gln Val Leu Ala
145 150 155 160
Ser Leu Arg Thr Val Arg Ser Asn Val Ala Ala Leu Ala Arg Gln Gln
165 170 175
Cys Leu Gly Ala Ala Lys Gln Gly Pro Val Gly Asn Pro Ser Ser Ser
180 185 190
Asn Gln Leu Pro Pro Ala Glu Asp Thr Gly Gln Lys Leu Ala Leu Glu
195 200 205
Thr Leu Asp Glu Leu Asp Trp Cys Leu Asp Gln Leu Glu Thr Leu Gln
210 215 220
Thr Arg His Ser Val Gly Glu Met Ala Ser Asn Lys Phe Lys Arg Ile
225 230 235 240
Leu Asn Arg Glu Leu Thr His Leu Ser Glu Thr Ser Arg Ser Gly Asn
245 250 255
Gln Val Ser Glu Tyr Ile Ser Arg Thr Phe Leu Asp Gln Gln Thr Glu
260 265 270
Val Glu Leu Pro Lys Val Thr Ala Glu Glu Ala Pro Gln Pro Met Ser
275 280 285
Arg Ile Ser Gly Leu His Gly Leu Cys His Ser Ala Ser Leu Ser Ser
290 295 300
Ala Thr Val Pro Arg Phe Gly Val Gln Thr Asp Gln Glu Glu Gln Leu
305 310 315 320
Ala Lys Glu Leu Glu Asp Thr Asn Lys Trp Gly Leu Asp Val Phe Lys
325 330 335
Val Ala Glu Leu Ser Gly Asn Arg Pro Leu Thr Ala Ile Ile Phe Ser
340 345 350
Ile Phe Gln Glu Arg Asp Leu Leu Lys Thr Phe Gln Ile Pro Ala Asp
355 360 365
Thr Leu Ala Thr Tyr Leu Leu Met Leu Glu Gly His Tyr His Ala Asn
370 375 380
Val Ala Tyr His Asn Ser Leu His Ala Ala Asp Val Ala Gln Ser Thr
385 390 395 400
His Val Leu Leu Ala Thr Pro Ala Leu Glu Ala Val Phe Thr Asp Leu
405 410 415
Glu Ile Leu Ala Ala Leu Phe Ala Ser Ala Ile His Asp Val Asp His
420 425 430
Pro Gly Val Ser Asn Gln Phe Leu Ile Asn Thr Asn Ser Glu Leu Ala
435 440 445
Leu Met Tyr Asn Asp Ala Ser Val Leu Glu Asn His His Leu Ala Val
450 455 460
Gly Phe Lys Leu Leu Gln Ala Glu Asn Cys Asp Ile Phe Gln Asn Leu
465 470 475 480
Ser Ala Lys Gln Arg Leu Ser Leu Arg Arg Met Val Ile Asp Met Val
485 490 495
Leu Ala Thr Asp Met Ser Lys His Met Asn Leu Leu Ala Asp Leu Lys
500 505 510
Thr Met Val Glu Thr Lys Lys Val Thr Ser Leu Gly Val Leu Leu Leu
515 520 525
Asp Asn Tyr Ser Asp Arg Ile Gln Val Leu Gln Asn Leu Val His Cys
530 535 540
Ala Asp Leu Ser Asn Pro Thr Lys Pro Leu Pro Leu Tyr Arg Gln Trp
545 550 555 560
Thr Asp Arg Ile Met Ala Glu Phe Phe Gln Gln Gly Asp Arg Glu Arg
565 570 575
Glu Ser Gly Leu Asp Ile Ser Pro Met Cys Asp Lys His Thr Ala Ser
580 585 590
Val Glu Lys Ser Gln Val Gly Phe Ile Asp Tyr Ile Ala His Pro Leu
595 600 605
Trp Glu Thr Trp Ala Asp Leu Val His Pro Asp Ala Gln Asp Leu Leu
610 615 620
Asp Thr Leu Glu Asp Asn Arg Glu Trp Tyr Gln Ser Lys Ile Pro Arg
625 630 635 640
Ser Pro Ser Asp Leu Thr Asn Pro Glu Arg Asp Gly Pro Asp Arg Phe
645 650 655
Gln Phe Glu Leu Thr Leu Glu Glu Ala Glu Glu Glu Asp Glu Glu Glu
660 665 670
Glu Glu Glu Gly Glu Glu Thr Ala Leu Ala Lys Glu Ala Leu Glu Leu
675 680 685
Pro Asp Thr Glu Leu Leu Ser Pro Glu Ala Gly Pro Asp Pro Gly Asp
690 695 700
Leu Pro Leu Asp Asn Gln Arg Thr
705 710
<210> 13
<211> 606
<212> PRT
<213> 智人
<400> 13
Met Gln Ala Pro Val Pro His Ser Gln Arg Arg Glu Ser Phe Leu Tyr
1 5 10 15
Arg Ser Asp Ser Asp Tyr Glu Leu Ser Pro Lys Ala Met Ser Arg Asn
20 25 30
Ser Ser Val Ala Ser Asp Leu His Gly Glu Asp Met Ile Val Thr Pro
35 40 45
Phe Ala Gln Val Leu Ala Ser Leu Arg Thr Val Arg Ser Asn Val Ala
50 55 60
Ala Leu Ala Arg Gln Gln Cys Leu Gly Ala Ala Lys Gln Gly Pro Val
65 70 75 80
Gly Asn Pro Ser Ser Ser Asn Gln Leu Pro Pro Ala Glu Asp Thr Gly
85 90 95
Gln Lys Leu Ala Leu Glu Thr Leu Asp Glu Leu Asp Trp Cys Leu Asp
100 105 110
Gln Leu Glu Thr Leu Gln Thr Arg His Ser Val Gly Glu Met Ala Ser
115 120 125
Asn Lys Phe Lys Arg Ile Leu Asn Arg Glu Leu Thr His Leu Ser Glu
130 135 140
Thr Ser Arg Ser Gly Asn Gln Val Ser Glu Tyr Ile Ser Arg Thr Phe
145 150 155 160
Leu Asp Gln Gln Thr Glu Val Glu Leu Pro Lys Val Thr Ala Glu Glu
165 170 175
Ala Pro Gln Pro Met Ser Arg Ile Ser Gly Leu His Gly Leu Cys His
180 185 190
Ser Ala Ser Leu Ser Ser Ala Thr Val Pro Arg Phe Gly Val Gln Thr
195 200 205
Asp Gln Glu Glu Gln Leu Ala Lys Glu Leu Glu Asp Thr Asn Lys Trp
210 215 220
Gly Leu Asp Val Phe Lys Val Ala Glu Leu Ser Gly Asn Arg Pro Leu
225 230 235 240
Thr Ala Ile Ile Phe Ser Ile Phe Gln Glu Arg Asp Leu Leu Lys Thr
245 250 255
Phe Gln Ile Pro Ala Asp Thr Leu Ala Thr Tyr Leu Leu Met Leu Glu
260 265 270
Gly His Tyr His Ala Asn Val Ala Tyr His Asn Ser Leu His Ala Ala
275 280 285
Asp Val Ala Gln Ser Thr His Val Leu Leu Ala Thr Pro Ala Leu Glu
290 295 300
Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Leu Phe Ala Ser Ala
305 310 315 320
Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln Phe Leu Ile Asn
325 330 335
Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Ala Ser Val Leu Glu
340 345 350
Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln Ala Glu Asn Cys
355 360 365
Asp Ile Phe Gln Asn Leu Ser Ala Lys Gln Arg Leu Ser Leu Arg Arg
370 375 380
Met Val Ile Asp Met Val Leu Ala Thr Asp Met Ser Lys His Met Asn
385 390 395 400
Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr Ser
405 410 415
Leu Gly Val Leu Leu Leu Asp Asn Tyr Ser Asp Arg Ile Gln Val Leu
420 425 430
Gln Asn Leu Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys Pro Leu
435 440 445
Pro Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Ala Glu Phe Phe Gln
450 455 460
Gln Gly Asp Arg Glu Arg Glu Ser Gly Leu Asp Ile Ser Pro Met Cys
465 470 475 480
Asp Lys His Thr Ala Ser Val Glu Lys Ser Gln Val Gly Phe Ile Asp
485 490 495
Tyr Ile Ala His Pro Leu Trp Glu Thr Trp Ala Asp Leu Val His Pro
500 505 510
Asp Ala Gln Asp Leu Leu Asp Thr Leu Glu Asp Asn Arg Glu Trp Tyr
515 520 525
Gln Ser Lys Ile Pro Arg Ser Pro Ser Asp Leu Thr Asn Pro Glu Arg
530 535 540
Asp Gly Pro Asp Arg Phe Gln Phe Glu Leu Thr Leu Glu Glu Ala Glu
545 550 555 560
Glu Glu Asp Glu Glu Glu Glu Glu Glu Gly Glu Glu Thr Ala Leu Ala
565 570 575
Lys Glu Ala Leu Glu Leu Pro Asp Thr Glu Leu Leu Ser Pro Glu Ala
580 585 590
Gly Pro Asp Pro Gly Asp Leu Pro Leu Asp Asn Gln Arg Thr
595 600 605
<210> 14
<211> 680
<212> PRT
<213> 智人
<400> 14
Met Gln Gly Pro Pro Ala Pro Ala Pro Val Pro Gly Pro Gly Ser Pro
1 5 10 15
Arg Gly Ser Pro Arg Gly Ser Pro Gly Leu Phe Arg Lys Leu Leu Val
20 25 30
Asn Gln Ser Ile Arg Leu Gln Arg Arg Phe Thr Val Ala His Pro Leu
35 40 45
Cys Phe Asp Leu Glu Asn Gly Leu Ser Cys Gly Arg Arg Ala Leu Asp
50 55 60
Pro Gln Ser Ser Pro Gly Leu Gly Arg Ile Met Gln Ala Pro Val Pro
65 70 75 80
His Ser Gln Arg Arg Glu Ser Phe Leu Tyr Arg Ser Asp Ser Asp Tyr
85 90 95
Glu Leu Ser Pro Lys Ala Met Ser Arg Asn Ser Ser Val Ala Ser Asp
100 105 110
Leu His Gly Glu Asp Met Ile Val Thr Pro Phe Ala Gln Val Leu Ala
115 120 125
Ser Leu Arg Thr Val Arg Ser Asn Val Ala Ala Leu Ala Arg Gln Gln
130 135 140
Cys Leu Gly Ala Ala Lys Gln Gly Pro Val Gly Asn Pro Ser Ser Ser
145 150 155 160
Asn Gln Leu Pro Pro Ala Glu Asp Thr Gly Gln Lys Leu Ala Leu Glu
165 170 175
Thr Leu Asp Glu Leu Asp Trp Cys Leu Asp Gln Leu Glu Thr Leu Gln
180 185 190
Thr Arg His Ser Val Gly Glu Met Ala Ser Asn Lys Phe Lys Arg Ile
195 200 205
Leu Asn Arg Glu Leu Thr His Leu Ser Glu Thr Ser Arg Ser Gly Asn
210 215 220
Gln Val Ser Glu Tyr Ile Ser Arg Thr Phe Leu Asp Gln Gln Thr Glu
225 230 235 240
Val Glu Leu Pro Lys Val Thr Ala Glu Glu Ala Pro Gln Pro Met Ser
245 250 255
Arg Ile Ser Gly Leu His Gly Leu Cys His Ser Ala Ser Leu Ser Ser
260 265 270
Ala Thr Val Pro Arg Phe Gly Val Gln Thr Asp Gln Glu Glu Gln Leu
275 280 285
Ala Lys Glu Leu Glu Asp Thr Asn Lys Trp Gly Leu Asp Val Phe Lys
290 295 300
Val Ala Glu Leu Ser Gly Asn Arg Pro Leu Thr Ala Ile Ile Phe Ser
305 310 315 320
Ile Phe Gln Glu Arg Asp Leu Leu Lys Thr Phe Gln Ile Pro Ala Asp
325 330 335
Thr Leu Ala Thr Tyr Leu Leu Met Leu Glu Gly His Tyr His Ala Asn
340 345 350
Val Ala Tyr His Asn Ser Leu His Ala Ala Asp Val Ala Gln Ser Thr
355 360 365
His Val Leu Leu Ala Thr Pro Ala Leu Glu Ala Val Phe Thr Asp Leu
370 375 380
Glu Ile Leu Ala Ala Leu Phe Ala Ser Ala Ile His Asp Val Asp His
385 390 395 400
Pro Gly Val Ser Asn Gln Phe Leu Ile Asn Thr Asn Ser Glu Leu Ala
405 410 415
Leu Met Tyr Asn Asp Ala Ser Val Leu Glu Asn His His Leu Ala Val
420 425 430
Gly Phe Lys Leu Leu Gln Ala Glu Asn Cys Asp Ile Phe Gln Asn Leu
435 440 445
Ser Ala Lys Gln Arg Leu Ser Leu Arg Arg Met Val Ile Asp Met Val
450 455 460
Leu Ala Thr Asp Met Ser Lys His Met Asn Leu Leu Ala Asp Leu Lys
465 470 475 480
Thr Met Val Glu Thr Lys Lys Val Thr Ser Leu Gly Val Leu Leu Leu
485 490 495
Asp Asn Tyr Ser Asp Arg Ile Gln Val Leu Gln Asn Leu Val His Cys
500 505 510
Ala Asp Leu Ser Asn Pro Thr Lys Pro Leu Pro Leu Tyr Arg Gln Trp
515 520 525
Thr Asp Arg Ile Met Ala Glu Phe Phe Gln Gln Gly Asp Arg Glu Arg
530 535 540
Glu Ser Gly Leu Asp Ile Ser Pro Met Cys Asp Lys His Thr Ala Ser
545 550 555 560
Val Glu Lys Ser Gln Val Gly Phe Ile Asp Tyr Ile Ala His Pro Leu
565 570 575
Trp Glu Thr Trp Ala Asp Leu Val His Pro Asp Ala Gln Asp Leu Leu
580 585 590
Asp Thr Leu Glu Asp Asn Arg Glu Trp Tyr Gln Ser Lys Ile Pro Arg
595 600 605
Ser Pro Ser Asp Leu Thr Asn Pro Glu Arg Asp Gly Pro Asp Arg Phe
610 615 620
Gln Phe Glu Leu Thr Leu Glu Glu Ala Glu Glu Glu Asp Glu Glu Glu
625 630 635 640
Glu Glu Glu Gly Glu Glu Thr Ala Leu Ala Lys Glu Ala Leu Glu Leu
645 650 655
Pro Asp Thr Glu Leu Leu Ser Pro Glu Ala Gly Pro Asp Pro Gly Asp
660 665 670
Leu Pro Leu Asp Asn Gln Arg Thr
675 680
<210> 15
<211> 809
<212> PRT
<213> 智人
<400> 15
Met Glu Ala Glu Gly Ser Ser Ala Pro Ala Arg Ala Gly Ser Gly Glu
1 5 10 15
Gly Ser Asp Ser Ala Gly Gly Ala Thr Leu Lys Ala Pro Lys His Leu
20 25 30
Trp Arg His Glu Gln His His Gln Tyr Pro Leu Arg Gln Pro Gln Phe
35 40 45
Arg Leu Leu His Pro His His His Leu Pro Pro Pro Pro Pro Pro Ser
50 55 60
Pro Gln Pro Gln Pro Gln Cys Pro Leu Gln Pro Pro Pro Pro Pro Pro
65 70 75 80
Leu Pro Pro Pro Pro Pro Pro Pro Gly Ala Ala Arg Gly Arg Tyr Ala
85 90 95
Ser Ser Gly Ala Thr Gly Arg Val Arg His Arg Gly Tyr Ser Asp Thr
100 105 110
Glu Arg Tyr Leu Tyr Cys Arg Ala Met Asp Arg Thr Ser Tyr Ala Val
115 120 125
Glu Thr Gly His Arg Pro Gly Leu Lys Lys Ser Arg Met Ser Trp Pro
130 135 140
Ser Ser Phe Gln Gly Leu Arg Arg Phe Asp Val Asp Asn Gly Thr Ser
145 150 155 160
Ala Gly Arg Ser Pro Leu Asp Pro Met Thr Ser Pro Gly Ser Gly Leu
165 170 175
Ile Leu Gln Ala Asn Phe Val His Ser Gln Arg Arg Glu Ser Phe Leu
180 185 190
Tyr Arg Ser Asp Ser Asp Tyr Asp Leu Ser Pro Lys Ser Met Ser Arg
195 200 205
Asn Ser Ser Ile Ala Ser Asp Ile His Gly Asp Asp Leu Ile Val Thr
210 215 220
Pro Phe Ala Gln Val Leu Ala Ser Leu Arg Thr Val Arg Asn Asn Phe
225 230 235 240
Ala Ala Leu Thr Asn Leu Gln Asp Arg Ala Pro Ser Lys Arg Ser Pro
245 250 255
Met Cys Asn Gln Pro Ser Ile Asn Lys Ala Thr Ile Thr Glu Glu Ala
260 265 270
Tyr Gln Lys Leu Ala Ser Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu
275 280 285
Asp Gln Leu Glu Thr Leu Gln Thr Arg His Ser Val Ser Glu Met Ala
290 295 300
Ser Asn Lys Phe Lys Arg Met Leu Asn Arg Glu Leu Thr His Leu Ser
305 310 315 320
Glu Met Ser Arg Ser Gly Asn Gln Val Ser Glu Phe Ile Ser Asn Thr
325 330 335
Phe Leu Asp Lys Gln His Glu Val Glu Ile Pro Ser Pro Thr Gln Lys
340 345 350
Glu Lys Glu Lys Lys Lys Arg Pro Met Ser Gln Ile Ser Gly Val Lys
355 360 365
Lys Leu Met His Ser Ser Ser Leu Thr Asn Ser Ser Ile Pro Arg Phe
370 375 380
Gly Val Lys Thr Glu Gln Glu Asp Val Leu Ala Lys Glu Leu Glu Asp
385 390 395 400
Val Asn Lys Trp Gly Leu His Val Phe Arg Ile Ala Glu Leu Ser Gly
405 410 415
Asn Arg Pro Leu Thr Val Ile Met His Thr Ile Phe Gln Glu Arg Asp
420 425 430
Leu Leu Lys Thr Phe Lys Ile Pro Val Asp Thr Leu Ile Thr Tyr Leu
435 440 445
Met Thr Leu Glu Asp His Tyr His Ala Asp Val Ala Tyr His Asn Asn
450 455 460
Ile His Ala Ala Asp Val Val Gln Ser Thr His Val Leu Leu Ser Thr
465 470 475 480
Pro Ala Leu Glu Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Ile
485 490 495
Phe Ala Ser Ala Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln
500 505 510
Phe Leu Ile Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Ser
515 520 525
Ser Val Leu Glu Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln
530 535 540
Glu Glu Asn Cys Asp Ile Phe Gln Asn Leu Thr Lys Lys Gln Arg Gln
545 550 555 560
Ser Leu Arg Lys Met Val Ile Asp Ile Val Leu Ala Thr Asp Met Ser
565 570 575
Lys His Met Asn Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys
580 585 590
Lys Val Thr Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr Ser Asp Arg
595 600 605
Ile Gln Val Leu Gln Asn Met Val His Cys Ala Asp Leu Ser Asn Pro
610 615 620
Thr Lys Pro Leu Gln Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Glu
625 630 635 640
Glu Phe Phe Arg Gln Gly Asp Arg Glu Arg Glu Arg Gly Met Glu Ile
645 650 655
Ser Pro Met Cys Asp Lys His Asn Ala Ser Val Glu Lys Ser Gln Val
660 665 670
Gly Phe Ile Asp Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp
675 680 685
Leu Val His Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp Asn
690 695 700
Arg Glu Trp Tyr Gln Ser Thr Ile Pro Gln Ser Pro Ser Pro Ala Pro
705 710 715 720
Asp Asp Pro Glu Glu Gly Arg Gln Gly Gln Thr Glu Lys Phe Gln Phe
725 730 735
Glu Leu Thr Leu Glu Glu Asp Gly Glu Ser Asp Thr Glu Lys Asp Ser
740 745 750
Gly Ser Gln Val Glu Glu Asp Thr Ser Cys Ser Asp Ser Lys Thr Leu
755 760 765
Cys Thr Gln Asp Ser Glu Ser Thr Glu Ile Pro Leu Asp Glu Gln Val
770 775 780
Glu Glu Glu Ala Val Gly Glu Glu Glu Glu Ser Gln Pro Glu Ala Cys
785 790 795 800
Val Ile Asp Asp Arg Ser Pro Asp Thr
805
<210> 16
<211> 673
<212> PRT
<213> 智人
<400> 16
Met Met His Val Asn Asn Phe Pro Phe Arg Arg His Ser Trp Ile Cys
1 5 10 15
Phe Asp Val Asp Asn Gly Thr Ser Ala Gly Arg Ser Pro Leu Asp Pro
20 25 30
Met Thr Ser Pro Gly Ser Gly Leu Ile Leu Gln Ala Asn Phe Val His
35 40 45
Ser Gln Arg Arg Glu Ser Phe Leu Tyr Arg Ser Asp Ser Asp Tyr Asp
50 55 60
Leu Ser Pro Lys Ser Met Ser Arg Asn Ser Ser Ile Ala Ser Asp Ile
65 70 75 80
His Gly Asp Asp Leu Ile Val Thr Pro Phe Ala Gln Val Leu Ala Ser
85 90 95
Leu Arg Thr Val Arg Asn Asn Phe Ala Ala Leu Thr Asn Leu Gln Asp
100 105 110
Arg Ala Pro Ser Lys Arg Ser Pro Met Cys Asn Gln Pro Ser Ile Asn
115 120 125
Lys Ala Thr Ile Thr Glu Glu Ala Tyr Gln Lys Leu Ala Ser Glu Thr
130 135 140
Leu Glu Glu Leu Asp Trp Cys Leu Asp Gln Leu Glu Thr Leu Gln Thr
145 150 155 160
Arg His Ser Val Ser Glu Met Ala Ser Asn Lys Phe Lys Arg Met Leu
165 170 175
Asn Arg Glu Leu Thr His Leu Ser Glu Met Ser Arg Ser Gly Asn Gln
180 185 190
Val Ser Glu Phe Ile Ser Asn Thr Phe Leu Asp Lys Gln His Glu Val
195 200 205
Glu Ile Pro Ser Pro Thr Gln Lys Glu Lys Glu Lys Lys Lys Arg Pro
210 215 220
Met Ser Gln Ile Ser Gly Val Lys Lys Leu Met His Ser Ser Ser Leu
225 230 235 240
Thr Asn Ser Ser Ile Pro Arg Phe Gly Val Lys Thr Glu Gln Glu Asp
245 250 255
Val Leu Ala Lys Glu Leu Glu Asp Val Asn Lys Trp Gly Leu His Val
260 265 270
Phe Arg Ile Ala Glu Leu Ser Gly Asn Arg Pro Leu Thr Val Ile Met
275 280 285
His Thr Ile Phe Gln Glu Arg Asp Leu Leu Lys Thr Phe Lys Ile Pro
290 295 300
Val Asp Thr Leu Ile Thr Tyr Leu Met Thr Leu Glu Asp His Tyr His
305 310 315 320
Ala Asp Val Ala Tyr His Asn Asn Ile His Ala Ala Asp Val Val Gln
325 330 335
Ser Thr His Val Leu Leu Ser Thr Pro Ala Leu Glu Ala Val Phe Thr
340 345 350
Asp Leu Glu Ile Leu Ala Ala Ile Phe Ala Ser Ala Ile His Asp Val
355 360 365
Asp His Pro Gly Val Ser Asn Gln Phe Leu Ile Asn Thr Asn Ser Glu
370 375 380
Leu Ala Leu Met Tyr Asn Asp Ser Ser Val Leu Glu Asn His His Leu
385 390 395 400
Ala Val Gly Phe Lys Leu Leu Gln Glu Glu Asn Cys Asp Ile Phe Gln
405 410 415
Asn Leu Thr Lys Lys Gln Arg Gln Ser Leu Arg Lys Met Val Ile Asp
420 425 430
Ile Val Leu Ala Thr Asp Met Ser Lys His Met Asn Leu Leu Ala Asp
435 440 445
Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr Ser Ser Gly Val Leu
450 455 460
Leu Leu Asp Asn Tyr Ser Asp Arg Ile Gln Val Leu Gln Asn Met Val
465 470 475 480
His Cys Ala Asp Leu Ser Asn Pro Thr Lys Pro Leu Gln Leu Tyr Arg
485 490 495
Gln Trp Thr Asp Arg Ile Met Glu Glu Phe Phe Arg Gln Gly Asp Arg
500 505 510
Glu Arg Glu Arg Gly Met Glu Ile Ser Pro Met Cys Asp Lys His Asn
515 520 525
Ala Ser Val Glu Lys Ser Gln Val Gly Phe Ile Asp Tyr Ile Val His
530 535 540
Pro Leu Trp Glu Thr Trp Ala Asp Leu Val His Pro Asp Ala Gln Asp
545 550 555 560
Ile Leu Asp Thr Leu Glu Asp Asn Arg Glu Trp Tyr Gln Ser Thr Ile
565 570 575
Pro Gln Ser Pro Ser Pro Ala Pro Asp Asp Pro Glu Glu Gly Arg Gln
580 585 590
Gly Gln Thr Glu Lys Phe Gln Phe Glu Leu Thr Leu Glu Glu Asp Gly
595 600 605
Glu Ser Asp Thr Glu Lys Asp Ser Gly Ser Gln Val Glu Glu Asp Thr
610 615 620
Ser Cys Ser Asp Ser Lys Thr Leu Cys Thr Gln Asp Ser Glu Ser Thr
625 630 635 640
Glu Ile Pro Leu Asp Glu Gln Val Glu Glu Glu Ala Val Gly Glu Glu
645 650 655
Glu Glu Ser Gln Pro Glu Ala Cys Val Ile Asp Asp Arg Ser Pro Asp
660 665 670
Thr
<210> 17
<211> 604
<212> PRT
<213> 智人
<400> 17
Met Ser Arg Asn Ser Ser Ile Ala Ser Asp Ile His Gly Asp Asp Leu
1 5 10 15
Ile Val Thr Pro Phe Ala Gln Val Leu Ala Ser Leu Arg Thr Val Arg
20 25 30
Asn Asn Phe Ala Ala Leu Thr Asn Leu Gln Asp Arg Ala Pro Ser Lys
35 40 45
Arg Ser Pro Met Cys Asn Gln Pro Ser Ile Asn Lys Ala Thr Ile Thr
50 55 60
Glu Glu Ala Tyr Gln Lys Leu Ala Ser Glu Thr Leu Glu Glu Leu Asp
65 70 75 80
Trp Cys Leu Asp Gln Leu Glu Thr Leu Gln Thr Arg His Ser Val Ser
85 90 95
Glu Met Ala Ser Asn Lys Phe Lys Arg Met Leu Asn Arg Glu Leu Thr
100 105 110
His Leu Ser Glu Met Ser Arg Ser Gly Asn Gln Val Ser Glu Phe Ile
115 120 125
Ser Asn Thr Phe Leu Asp Lys Gln His Glu Val Glu Ile Pro Ser Pro
130 135 140
Thr Gln Lys Glu Lys Glu Lys Lys Lys Arg Pro Met Ser Gln Ile Ser
145 150 155 160
Gly Val Lys Lys Leu Met His Ser Ser Ser Leu Thr Asn Ser Ser Ile
165 170 175
Pro Arg Phe Gly Val Lys Thr Glu Gln Glu Asp Val Leu Ala Lys Glu
180 185 190
Leu Glu Asp Val Asn Lys Trp Gly Leu His Val Phe Arg Ile Ala Glu
195 200 205
Leu Ser Gly Asn Arg Pro Leu Thr Val Ile Met His Thr Ile Phe Gln
210 215 220
Glu Arg Asp Leu Leu Lys Thr Phe Lys Ile Pro Val Asp Thr Leu Ile
225 230 235 240
Thr Tyr Leu Met Thr Leu Glu Asp His Tyr His Ala Asp Val Ala Tyr
245 250 255
His Asn Asn Ile His Ala Ala Asp Val Val Gln Ser Thr His Val Leu
260 265 270
Leu Ser Thr Pro Ala Leu Glu Ala Val Phe Thr Asp Leu Glu Ile Leu
275 280 285
Ala Ala Ile Phe Ala Ser Ala Ile His Asp Val Asp His Pro Gly Val
290 295 300
Ser Asn Gln Phe Leu Ile Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr
305 310 315 320
Asn Asp Ser Ser Val Leu Glu Asn His His Leu Ala Val Gly Phe Lys
325 330 335
Leu Leu Gln Glu Glu Asn Cys Asp Ile Phe Gln Asn Leu Thr Lys Lys
340 345 350
Gln Arg Gln Ser Leu Arg Lys Met Val Ile Asp Ile Val Leu Ala Thr
355 360 365
Asp Met Ser Lys His Met Asn Leu Leu Ala Asp Leu Lys Thr Met Val
370 375 380
Glu Thr Lys Lys Val Thr Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr
385 390 395 400
Ser Asp Arg Ile Gln Val Leu Gln Asn Met Val His Cys Ala Asp Leu
405 410 415
Ser Asn Pro Thr Lys Pro Leu Gln Leu Tyr Arg Gln Trp Thr Asp Arg
420 425 430
Ile Met Glu Glu Phe Phe Arg Gln Gly Asp Arg Glu Arg Glu Arg Gly
435 440 445
Met Glu Ile Ser Pro Met Cys Asp Lys His Asn Ala Ser Val Glu Lys
450 455 460
Ser Gln Val Gly Phe Ile Asp Tyr Ile Val His Pro Leu Trp Glu Thr
465 470 475 480
Trp Ala Asp Leu Val His Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu
485 490 495
Glu Asp Asn Arg Glu Trp Tyr Gln Ser Thr Ile Pro Gln Ser Pro Ser
500 505 510
Pro Ala Pro Asp Asp Pro Glu Glu Gly Arg Gln Gly Gln Thr Glu Lys
515 520 525
Phe Gln Phe Glu Leu Thr Leu Glu Glu Asp Gly Glu Ser Asp Thr Glu
530 535 540
Lys Asp Ser Gly Ser Gln Val Glu Glu Asp Thr Ser Cys Ser Asp Ser
545 550 555 560
Lys Thr Leu Cys Thr Gln Asp Ser Glu Ser Thr Glu Ile Pro Leu Asp
565 570 575
Glu Gln Val Glu Glu Glu Ala Val Gly Glu Glu Glu Glu Ser Gln Pro
580 585 590
Glu Ala Cys Val Ile Asp Asp Arg Ser Pro Asp Thr
595 600
<210> 18
<211> 584
<212> PRT
<213> 智人
<400> 18
Met Lys Glu Gln Pro Ser Cys Ala Gly Thr Gly His Pro Met Ala Gly
1 5 10 15
Tyr Gly Arg Met Ala Pro Phe Glu Leu Ala Ser Gly Pro Val Lys Arg
20 25 30
Leu Arg Thr Glu Ser Pro Phe Pro Cys Leu Phe Ala Glu Glu Ala Tyr
35 40 45
Gln Lys Leu Ala Ser Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu Asp
50 55 60
Gln Leu Glu Thr Leu Gln Thr Arg His Ser Val Ser Glu Met Ala Ser
65 70 75 80
Asn Lys Phe Lys Arg Met Leu Asn Arg Glu Leu Thr His Leu Ser Glu
85 90 95
Met Ser Arg Ser Gly Asn Gln Val Ser Glu Phe Ile Ser Asn Thr Phe
100 105 110
Leu Asp Lys Gln His Glu Val Glu Ile Pro Ser Pro Thr Gln Lys Glu
115 120 125
Lys Glu Lys Lys Lys Arg Pro Met Ser Gln Ile Ser Gly Val Lys Lys
130 135 140
Leu Met His Ser Ser Ser Leu Thr Asn Ser Ser Ile Pro Arg Phe Gly
145 150 155 160
Val Lys Thr Glu Gln Glu Asp Val Leu Ala Lys Glu Leu Glu Asp Val
165 170 175
Asn Lys Trp Gly Leu His Val Phe Arg Ile Ala Glu Leu Ser Gly Asn
180 185 190
Arg Pro Leu Thr Val Ile Met His Thr Ile Phe Gln Glu Arg Asp Leu
195 200 205
Leu Lys Thr Phe Lys Ile Pro Val Asp Thr Leu Ile Thr Tyr Leu Met
210 215 220
Thr Leu Glu Asp His Tyr His Ala Asp Val Ala Tyr His Asn Asn Ile
225 230 235 240
His Ala Ala Asp Val Val Gln Ser Thr His Val Leu Leu Ser Thr Pro
245 250 255
Ala Leu Glu Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Ile Phe
260 265 270
Ala Ser Ala Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln Phe
275 280 285
Leu Ile Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Ser Ser
290 295 300
Val Leu Glu Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln Glu
305 310 315 320
Glu Asn Cys Asp Ile Phe Gln Asn Leu Thr Lys Lys Gln Arg Gln Ser
325 330 335
Leu Arg Lys Met Val Ile Asp Ile Val Leu Ala Thr Asp Met Ser Lys
340 345 350
His Met Asn Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys
355 360 365
Val Thr Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr Ser Asp Arg Ile
370 375 380
Gln Val Leu Gln Asn Met Val His Cys Ala Asp Leu Ser Asn Pro Thr
385 390 395 400
Lys Pro Leu Gln Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Glu Glu
405 410 415
Phe Phe Arg Gln Gly Asp Arg Glu Arg Glu Arg Gly Met Glu Ile Ser
420 425 430
Pro Met Cys Asp Lys His Asn Ala Ser Val Glu Lys Ser Gln Val Gly
435 440 445
Phe Ile Asp Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp Leu
450 455 460
Val His Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp Asn Arg
465 470 475 480
Glu Trp Tyr Gln Ser Thr Ile Pro Gln Ser Pro Ser Pro Ala Pro Asp
485 490 495
Asp Pro Glu Glu Gly Arg Gln Gly Gln Thr Glu Lys Phe Gln Phe Glu
500 505 510
Leu Thr Leu Glu Glu Asp Gly Glu Ser Asp Thr Glu Lys Asp Ser Gly
515 520 525
Ser Gln Val Glu Glu Asp Thr Ser Cys Ser Asp Ser Lys Thr Leu Cys
530 535 540
Thr Gln Asp Ser Glu Ser Thr Glu Ile Pro Leu Asp Glu Gln Val Glu
545 550 555 560
Glu Glu Ala Val Gly Glu Glu Glu Glu Ser Gln Pro Glu Ala Cys Val
565 570 575
Ile Asp Asp Arg Ser Pro Asp Thr
580
<210> 19
<211> 507
<212> PRT
<213> 智人
<400> 19
Met Ala Ser Asn Lys Phe Lys Arg Met Leu Asn Arg Glu Leu Thr His
1 5 10 15
Leu Ser Glu Met Ser Arg Ser Gly Asn Gln Val Ser Glu Phe Ile Ser
20 25 30
Asn Thr Phe Leu Asp Lys Gln His Glu Val Glu Ile Pro Ser Pro Thr
35 40 45
Gln Lys Glu Lys Glu Lys Lys Lys Arg Pro Met Ser Gln Ile Ser Gly
50 55 60
Val Lys Lys Leu Met His Ser Ser Ser Leu Thr Asn Ser Ser Ile Pro
65 70 75 80
Arg Phe Gly Val Lys Thr Glu Gln Glu Asp Val Leu Ala Lys Glu Leu
85 90 95
Glu Asp Val Asn Lys Trp Gly Leu His Val Phe Arg Ile Ala Glu Leu
100 105 110
Ser Gly Asn Arg Pro Leu Thr Val Ile Met His Thr Ile Phe Gln Glu
115 120 125
Arg Asp Leu Leu Lys Thr Phe Lys Ile Pro Val Asp Thr Leu Ile Thr
130 135 140
Tyr Leu Met Thr Leu Glu Asp His Tyr His Ala Asp Val Ala Tyr His
145 150 155 160
Asn Asn Ile His Ala Ala Asp Val Val Gln Ser Thr His Val Leu Leu
165 170 175
Ser Thr Pro Ala Leu Glu Ala Val Phe Thr Asp Leu Glu Ile Leu Ala
180 185 190
Ala Ile Phe Ala Ser Ala Ile His Asp Val Asp His Pro Gly Val Ser
195 200 205
Asn Gln Phe Leu Ile Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn
210 215 220
Asp Ser Ser Val Leu Glu Asn His His Leu Ala Val Gly Phe Lys Leu
225 230 235 240
Leu Gln Glu Glu Asn Cys Asp Ile Phe Gln Asn Leu Thr Lys Lys Gln
245 250 255
Arg Gln Ser Leu Arg Lys Met Val Ile Asp Ile Val Leu Ala Thr Asp
260 265 270
Met Ser Lys His Met Asn Leu Leu Ala Asp Leu Lys Thr Met Val Glu
275 280 285
Thr Lys Lys Val Thr Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr Ser
290 295 300
Asp Arg Ile Gln Val Leu Gln Asn Met Val His Cys Ala Asp Leu Ser
305 310 315 320
Asn Pro Thr Lys Pro Leu Gln Leu Tyr Arg Gln Trp Thr Asp Arg Ile
325 330 335
Met Glu Glu Phe Phe Arg Gln Gly Asp Arg Glu Arg Glu Arg Gly Met
340 345 350
Glu Ile Ser Pro Met Cys Asp Lys His Asn Ala Ser Val Glu Lys Ser
355 360 365
Gln Val Gly Phe Ile Asp Tyr Ile Val His Pro Leu Trp Glu Thr Trp
370 375 380
Ala Asp Leu Val His Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu
385 390 395 400
Asp Asn Arg Glu Trp Tyr Gln Ser Thr Ile Pro Gln Ser Pro Ser Pro
405 410 415
Ala Pro Asp Asp Pro Glu Glu Gly Arg Gln Gly Gln Thr Glu Lys Phe
420 425 430
Gln Phe Glu Leu Thr Leu Glu Glu Asp Gly Glu Ser Asp Thr Glu Lys
435 440 445
Asp Ser Gly Ser Gln Val Glu Glu Asp Thr Ser Cys Ser Asp Ser Lys
450 455 460
Thr Leu Cys Thr Gln Asp Ser Glu Ser Thr Glu Ile Pro Leu Asp Glu
465 470 475 480
Gln Val Glu Glu Glu Ala Val Gly Glu Glu Glu Glu Ser Gln Pro Glu
485 490 495
Ala Cys Val Ile Asp Asp Arg Ser Pro Asp Thr
500 505
<210> 20
<211> 745
<212> PRT
<213> 智人
<400> 20
Met Ala Gln Gln Thr Ser Pro Asp Thr Leu Thr Val Pro Glu Val Asp
1 5 10 15
Asn Pro His Cys Pro Asn Pro Trp Leu Asn Glu Asp Leu Val Lys Ser
20 25 30
Leu Arg Glu Asn Leu Leu Gln His Glu Lys Ser Lys Thr Ala Arg Lys
35 40 45
Ser Val Ser Pro Lys Leu Ser Pro Val Ile Ser Pro Arg Asn Ser Pro
50 55 60
Arg Leu Leu Arg Arg Met Leu Leu Ser Ser Asn Ile Pro Lys Gln Arg
65 70 75 80
Arg Phe Thr Val Ala His Thr Cys Phe Asp Val Asp Asn Gly Thr Ser
85 90 95
Ala Gly Arg Ser Pro Leu Asp Pro Met Thr Ser Pro Gly Ser Gly Leu
100 105 110
Ile Leu Gln Ala Asn Phe Val His Ser Gln Arg Arg Glu Ser Phe Leu
115 120 125
Tyr Arg Ser Asp Ser Asp Tyr Asp Leu Ser Pro Lys Ser Met Ser Arg
130 135 140
Asn Ser Ser Ile Ala Ser Asp Ile His Gly Asp Asp Leu Ile Val Thr
145 150 155 160
Pro Phe Ala Gln Val Leu Ala Ser Leu Arg Thr Val Arg Asn Asn Phe
165 170 175
Ala Ala Leu Thr Asn Leu Gln Asp Arg Ala Pro Ser Lys Arg Ser Pro
180 185 190
Met Cys Asn Gln Pro Ser Ile Asn Lys Ala Thr Ile Thr Glu Glu Ala
195 200 205
Tyr Gln Lys Leu Ala Ser Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu
210 215 220
Asp Gln Leu Glu Thr Leu Gln Thr Arg His Ser Val Ser Glu Met Ala
225 230 235 240
Ser Asn Lys Phe Lys Arg Met Leu Asn Arg Glu Leu Thr His Leu Ser
245 250 255
Glu Met Ser Arg Ser Gly Asn Gln Val Ser Glu Phe Ile Ser Asn Thr
260 265 270
Phe Leu Asp Lys Gln His Glu Val Glu Ile Pro Ser Pro Thr Gln Lys
275 280 285
Glu Lys Glu Lys Lys Lys Arg Pro Met Ser Gln Ile Ser Gly Val Lys
290 295 300
Lys Leu Met His Ser Ser Ser Leu Thr Asn Ser Ser Ile Pro Arg Phe
305 310 315 320
Gly Val Lys Thr Glu Gln Glu Asp Val Leu Ala Lys Glu Leu Glu Asp
325 330 335
Val Asn Lys Trp Gly Leu His Val Phe Arg Ile Ala Glu Leu Ser Gly
340 345 350
Asn Arg Pro Leu Thr Val Ile Met His Thr Ile Phe Gln Glu Arg Asp
355 360 365
Leu Leu Lys Thr Phe Lys Ile Pro Val Asp Thr Leu Ile Thr Tyr Leu
370 375 380
Met Thr Leu Glu Asp His Tyr His Ala Asp Val Ala Tyr His Asn Asn
385 390 395 400
Ile His Ala Ala Asp Val Val Gln Ser Thr His Val Leu Leu Ser Thr
405 410 415
Pro Ala Leu Glu Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Ile
420 425 430
Phe Ala Ser Ala Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln
435 440 445
Phe Leu Ile Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Ser
450 455 460
Ser Val Leu Glu Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln
465 470 475 480
Glu Glu Asn Cys Asp Ile Phe Gln Asn Leu Thr Lys Lys Gln Arg Gln
485 490 495
Ser Leu Arg Lys Met Val Ile Asp Ile Val Leu Ala Thr Asp Met Ser
500 505 510
Lys His Met Asn Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys
515 520 525
Lys Val Thr Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr Ser Asp Arg
530 535 540
Ile Gln Val Leu Gln Asn Met Val His Cys Ala Asp Leu Ser Asn Pro
545 550 555 560
Thr Lys Pro Leu Gln Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Glu
565 570 575
Glu Phe Phe Arg Gln Gly Asp Arg Glu Arg Glu Arg Gly Met Glu Ile
580 585 590
Ser Pro Met Cys Asp Lys His Asn Ala Ser Val Glu Lys Ser Gln Val
595 600 605
Gly Phe Ile Asp Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp
610 615 620
Leu Val His Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp Asn
625 630 635 640
Arg Glu Trp Tyr Gln Ser Thr Ile Pro Gln Ser Pro Ser Pro Ala Pro
645 650 655
Asp Asp Pro Glu Glu Gly Arg Gln Gly Gln Thr Glu Lys Phe Gln Phe
660 665 670
Glu Leu Thr Leu Glu Glu Asp Gly Glu Ser Asp Thr Glu Lys Asp Ser
675 680 685
Gly Ser Gln Val Glu Glu Asp Thr Ser Cys Ser Asp Ser Lys Thr Leu
690 695 700
Cys Thr Gln Asp Ser Glu Ser Thr Glu Ile Pro Leu Asp Glu Gln Val
705 710 715 720
Glu Glu Glu Ala Val Gly Glu Glu Glu Glu Ser Gln Pro Glu Ala Cys
725 730 735
Val Ile Asp Asp Arg Ser Pro Asp Thr
740 745
<210> 21
<211> 215
<212> PRT
<213> 智人
<400> 21
Met Ala Gln Gln Thr Ser Pro Asp Thr Leu Thr Val Pro Glu Val Asp
1 5 10 15
Asn Pro His Cys Pro Asn Pro Trp Leu Asn Glu Asp Leu Val Lys Ser
20 25 30
Leu Arg Glu Asn Leu Leu Gln His Glu Lys Ser Lys Thr Ala Arg Lys
35 40 45
Ser Val Ser Pro Lys Leu Ser Pro Val Ile Ser Pro Arg Asn Ser Pro
50 55 60
Arg Leu Leu Arg Arg Met Leu Leu Ser Ser Asn Ile Pro Lys Gln Arg
65 70 75 80
Arg Phe Thr Val Ala His Thr Cys Phe Asp Val Asp Asn Gly Thr Ser
85 90 95
Ala Gly Arg Ser Pro Leu Asp Pro Met Thr Ser Pro Gly Ser Gly Leu
100 105 110
Ile Leu Gln Ala Asn Phe Val His Ser Gln Arg Arg Glu Ser Phe Leu
115 120 125
Tyr Arg Ser Asp Ser Asp Tyr Asp Leu Ser Pro Lys Ser Met Ser Arg
130 135 140
Asn Ser Ser Ile Ala Ser Asp Ile His Gly Asp Asp Leu Ile Val Thr
145 150 155 160
Pro Phe Ala Gln Val Leu Ala Ser Leu Arg Thr Val Arg Asn Asn Phe
165 170 175
Ala Ala Leu Thr Asn Leu Gln Asp Arg Ala Pro Ser Lys Arg Ser Pro
180 185 190
Met Cys Asn Gln Pro Ser Ile Asn Lys Ala Thr Ile Thr Gly Leu Tyr
195 200 205
Asn Gly Ile Ile Ala Phe Leu
210 215
<210> 22
<211> 518
<212> PRT
<213> 智人
<400> 22
Met Pro Glu Ala Asn Tyr Leu Leu Ser Val Ser Trp Gly Tyr Ile Lys
1 5 10 15
Phe Lys Arg Met Leu Asn Arg Glu Leu Thr His Leu Ser Glu Met Ser
20 25 30
Arg Ser Gly Asn Gln Val Ser Glu Phe Ile Ser Asn Thr Phe Leu Asp
35 40 45
Lys Gln His Glu Val Glu Ile Pro Ser Pro Thr Gln Lys Glu Lys Glu
50 55 60
Lys Lys Lys Arg Pro Met Ser Gln Ile Ser Gly Val Lys Lys Leu Met
65 70 75 80
His Ser Ser Ser Leu Thr Asn Ser Ser Ile Pro Arg Phe Gly Val Lys
85 90 95
Thr Glu Gln Glu Asp Val Leu Ala Lys Glu Leu Glu Asp Val Asn Lys
100 105 110
Trp Gly Leu His Val Phe Arg Ile Ala Glu Leu Ser Gly Asn Arg Pro
115 120 125
Leu Thr Val Ile Met His Thr Ile Phe Gln Glu Arg Asp Leu Leu Lys
130 135 140
Thr Phe Lys Ile Pro Val Asp Thr Leu Ile Thr Tyr Leu Met Thr Leu
145 150 155 160
Glu Asp His Tyr His Ala Asp Val Ala Tyr His Asn Asn Ile His Ala
165 170 175
Ala Asp Val Val Gln Ser Thr His Val Leu Leu Ser Thr Pro Ala Leu
180 185 190
Glu Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Ile Phe Ala Ser
195 200 205
Ala Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln Phe Leu Ile
210 215 220
Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Ser Ser Val Leu
225 230 235 240
Glu Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln Glu Glu Asn
245 250 255
Cys Asp Ile Phe Gln Asn Leu Thr Lys Lys Gln Arg Gln Ser Leu Arg
260 265 270
Lys Met Val Ile Asp Ile Val Leu Ala Thr Asp Met Ser Lys His Met
275 280 285
Asn Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr
290 295 300
Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr Ser Asp Arg Ile Gln Val
305 310 315 320
Leu Gln Asn Met Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys Pro
325 330 335
Leu Gln Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Glu Glu Phe Phe
340 345 350
Arg Gln Gly Asp Arg Glu Arg Glu Arg Gly Met Glu Ile Ser Pro Met
355 360 365
Cys Asp Lys His Asn Ala Ser Val Glu Lys Ser Gln Val Gly Phe Ile
370 375 380
Asp Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp Leu Val His
385 390 395 400
Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp Asn Arg Glu Trp
405 410 415
Tyr Gln Ser Thr Ile Pro Gln Ser Pro Ser Pro Ala Pro Asp Asp Pro
420 425 430
Glu Glu Gly Arg Gln Gly Gln Thr Glu Lys Phe Gln Phe Glu Leu Thr
435 440 445
Leu Glu Glu Asp Gly Glu Ser Asp Thr Glu Lys Asp Ser Gly Ser Gln
450 455 460
Val Glu Glu Asp Thr Ser Cys Ser Asp Ser Lys Thr Leu Cys Thr Gln
465 470 475 480
Asp Ser Glu Ser Thr Glu Ile Pro Leu Asp Glu Gln Val Glu Glu Glu
485 490 495
Ala Val Gly Glu Glu Glu Glu Ser Gln Pro Glu Ala Cys Val Ile Asp
500 505 510
Asp Arg Ser Pro Asp Thr
515
<210> 23
<211> 687
<212> PRT
<213> 智人
<400> 23
Met Ala Phe Val Trp Asp Pro Leu Gly Ala Thr Val Pro Gly Pro Ser
1 5 10 15
Thr Arg Ala Lys Ser Arg Leu Arg Phe Ser Lys Ser Tyr Ser Phe Asp
20 25 30
Val Asp Asn Gly Thr Ser Ala Gly Arg Ser Pro Leu Asp Pro Met Thr
35 40 45
Ser Pro Gly Ser Gly Leu Ile Leu Gln Ala Asn Phe Val His Ser Gln
50 55 60
Arg Arg Glu Ser Phe Leu Tyr Arg Ser Asp Ser Asp Tyr Asp Leu Ser
65 70 75 80
Pro Lys Ser Met Ser Arg Asn Ser Ser Ile Ala Ser Asp Ile His Gly
85 90 95
Asp Asp Leu Ile Val Thr Pro Phe Ala Gln Val Leu Ala Ser Leu Arg
100 105 110
Thr Val Arg Asn Asn Phe Ala Ala Leu Thr Asn Leu Gln Asp Arg Ala
115 120 125
Pro Ser Lys Arg Ser Pro Met Cys Asn Gln Pro Ser Ile Asn Lys Ala
130 135 140
Thr Ile Thr Glu Glu Ala Tyr Gln Lys Leu Ala Ser Glu Thr Leu Glu
145 150 155 160
Glu Leu Asp Trp Cys Leu Asp Gln Leu Glu Thr Leu Gln Thr Arg His
165 170 175
Ser Val Ser Glu Met Ala Ser Asn Lys Phe Lys Arg Met Leu Asn Arg
180 185 190
Glu Leu Thr His Leu Ser Glu Met Ser Arg Ser Gly Asn Gln Val Ser
195 200 205
Glu Phe Ile Ser Asn Thr Phe Leu Asp Lys Gln His Glu Val Glu Ile
210 215 220
Pro Ser Pro Thr Gln Lys Glu Lys Glu Lys Lys Lys Arg Pro Met Ser
225 230 235 240
Gln Ile Ser Gly Val Lys Lys Leu Met His Ser Ser Ser Leu Thr Asn
245 250 255
Ser Ser Ile Pro Arg Phe Gly Val Lys Thr Glu Gln Glu Asp Val Leu
260 265 270
Ala Lys Glu Leu Glu Asp Val Asn Lys Trp Gly Leu His Val Phe Arg
275 280 285
Ile Ala Glu Leu Ser Gly Asn Arg Pro Leu Thr Val Ile Met His Thr
290 295 300
Ile Phe Gln Glu Arg Asp Leu Leu Lys Thr Phe Lys Ile Pro Val Asp
305 310 315 320
Thr Leu Ile Thr Tyr Leu Met Thr Leu Glu Asp His Tyr His Ala Asp
325 330 335
Val Ala Tyr His Asn Asn Ile His Ala Ala Asp Val Val Gln Ser Thr
340 345 350
His Val Leu Leu Ser Thr Pro Ala Leu Glu Ala Val Phe Thr Asp Leu
355 360 365
Glu Ile Leu Ala Ala Ile Phe Ala Ser Ala Ile His Asp Val Asp His
370 375 380
Pro Gly Val Ser Asn Gln Phe Leu Ile Asn Thr Asn Ser Glu Leu Ala
385 390 395 400
Leu Met Tyr Asn Asp Ser Ser Val Leu Glu Asn His His Leu Ala Val
405 410 415
Gly Phe Lys Leu Leu Gln Glu Glu Asn Cys Asp Ile Phe Gln Asn Leu
420 425 430
Thr Lys Lys Gln Arg Gln Ser Leu Arg Lys Met Val Ile Asp Ile Val
435 440 445
Leu Ala Thr Asp Met Ser Lys His Met Asn Leu Leu Ala Asp Leu Lys
450 455 460
Thr Met Val Glu Thr Lys Lys Val Thr Ser Ser Gly Val Leu Leu Leu
465 470 475 480
Asp Asn Tyr Ser Asp Arg Ile Gln Val Leu Gln Asn Met Val His Cys
485 490 495
Ala Asp Leu Ser Asn Pro Thr Lys Pro Leu Gln Leu Tyr Arg Gln Trp
500 505 510
Thr Asp Arg Ile Met Glu Glu Phe Phe Arg Gln Gly Asp Arg Glu Arg
515 520 525
Glu Arg Gly Met Glu Ile Ser Pro Met Cys Asp Lys His Asn Ala Ser
530 535 540
Val Glu Lys Ser Gln Val Gly Phe Ile Asp Tyr Ile Val His Pro Leu
545 550 555 560
Trp Glu Thr Trp Ala Asp Leu Val His Pro Asp Ala Gln Asp Ile Leu
565 570 575
Asp Thr Leu Glu Asp Asn Arg Glu Trp Tyr Gln Ser Thr Ile Pro Gln
580 585 590
Ser Pro Ser Pro Ala Pro Asp Asp Pro Glu Glu Gly Arg Gln Gly Gln
595 600 605
Thr Glu Lys Phe Gln Phe Glu Leu Thr Leu Glu Glu Asp Gly Glu Ser
610 615 620
Asp Thr Glu Lys Asp Ser Gly Ser Gln Val Glu Glu Asp Thr Ser Cys
625 630 635 640
Ser Asp Ser Lys Thr Leu Cys Thr Gln Asp Ser Glu Ser Thr Glu Ile
645 650 655
Pro Leu Asp Glu Gln Val Glu Glu Glu Ala Val Gly Glu Glu Glu Glu
660 665 670
Ser Gln Pro Glu Ala Cys Val Ile Asp Asp Arg Ser Pro Asp Thr
675 680 685
<210> 24
<211> 679
<212> PRT
<213> 智人
<400> 24
Met Ser Ile Ile Met Lys Pro Arg Ser Arg Ser Thr Ser Ser Leu Arg
1 5 10 15
Thr Ala Glu Ala Val Cys Glu Asp Val Asp Asn Gly Thr Ser Ala Gly
20 25 30
Arg Ser Pro Leu Asp Pro Met Thr Ser Pro Gly Ser Gly Leu Ile Leu
35 40 45
Gln Ala Asn Phe Val His Ser Gln Arg Arg Glu Ser Phe Leu Tyr Arg
50 55 60
Ser Asp Ser Asp Tyr Asp Leu Ser Pro Lys Ser Met Ser Arg Asn Ser
65 70 75 80
Ser Ile Ala Ser Asp Ile His Gly Asp Asp Leu Ile Val Thr Pro Phe
85 90 95
Ala Gln Val Leu Ala Ser Leu Arg Thr Val Arg Asn Asn Phe Ala Ala
100 105 110
Leu Thr Asn Leu Gln Asp Arg Ala Pro Ser Lys Arg Ser Pro Met Cys
115 120 125
Asn Gln Pro Ser Ile Asn Lys Ala Thr Ile Thr Glu Glu Ala Tyr Gln
130 135 140
Lys Leu Ala Ser Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu Asp Gln
145 150 155 160
Leu Glu Thr Leu Gln Thr Arg His Ser Val Ser Glu Met Ala Ser Asn
165 170 175
Lys Phe Lys Arg Met Leu Asn Arg Glu Leu Thr His Leu Ser Glu Met
180 185 190
Ser Arg Ser Gly Asn Gln Val Ser Glu Phe Ile Ser Asn Thr Phe Leu
195 200 205
Asp Lys Gln His Glu Val Glu Ile Pro Ser Pro Thr Gln Lys Glu Lys
210 215 220
Glu Lys Lys Lys Arg Pro Met Ser Gln Ile Ser Gly Val Lys Lys Leu
225 230 235 240
Met His Ser Ser Ser Leu Thr Asn Ser Ser Ile Pro Arg Phe Gly Val
245 250 255
Lys Thr Glu Gln Glu Asp Val Leu Ala Lys Glu Leu Glu Asp Val Asn
260 265 270
Lys Trp Gly Leu His Val Phe Arg Ile Ala Glu Leu Ser Gly Asn Arg
275 280 285
Pro Leu Thr Val Ile Met His Thr Ile Phe Gln Glu Arg Asp Leu Leu
290 295 300
Lys Thr Phe Lys Ile Pro Val Asp Thr Leu Ile Thr Tyr Leu Met Thr
305 310 315 320
Leu Glu Asp His Tyr His Ala Asp Val Ala Tyr His Asn Asn Ile His
325 330 335
Ala Ala Asp Val Val Gln Ser Thr His Val Leu Leu Ser Thr Pro Ala
340 345 350
Leu Glu Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Ile Phe Ala
355 360 365
Ser Ala Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln Phe Leu
370 375 380
Ile Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Ser Ser Val
385 390 395 400
Leu Glu Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln Glu Glu
405 410 415
Asn Cys Asp Ile Phe Gln Asn Leu Thr Lys Lys Gln Arg Gln Ser Leu
420 425 430
Arg Lys Met Val Ile Asp Ile Val Leu Ala Thr Asp Met Ser Lys His
435 440 445
Met Asn Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val
450 455 460
Thr Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr Ser Asp Arg Ile Gln
465 470 475 480
Val Leu Gln Asn Met Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys
485 490 495
Pro Leu Gln Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Glu Glu Phe
500 505 510
Phe Arg Gln Gly Asp Arg Glu Arg Glu Arg Gly Met Glu Ile Ser Pro
515 520 525
Met Cys Asp Lys His Asn Ala Ser Val Glu Lys Ser Gln Val Gly Phe
530 535 540
Ile Asp Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp Leu Val
545 550 555 560
His Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp Asn Arg Glu
565 570 575
Trp Tyr Gln Ser Thr Ile Pro Gln Ser Pro Ser Pro Ala Pro Asp Asp
580 585 590
Pro Glu Glu Gly Arg Gln Gly Gln Thr Glu Lys Phe Gln Phe Glu Leu
595 600 605
Thr Leu Glu Glu Asp Gly Glu Ser Asp Thr Glu Lys Asp Ser Gly Ser
610 615 620
Gln Val Glu Glu Asp Thr Ser Cys Ser Asp Ser Lys Thr Leu Cys Thr
625 630 635 640
Gln Asp Ser Glu Ser Thr Glu Ile Pro Leu Asp Glu Gln Val Glu Glu
645 650 655
Glu Ala Val Gly Glu Glu Glu Glu Ser Gln Pro Glu Ala Cys Val Ile
660 665 670
Asp Asp Arg Ser Pro Asp Thr
675
<210> 25
<211> 748
<212> PRT
<213> 智人
<400> 25
Met Lys Arg Asn Thr Cys Asp Leu Leu Ser Arg Ser Lys Ser Ala Ser
1 5 10 15
Glu Glu Thr Leu His Ser Ser Asn Glu Glu Glu Asp Pro Phe Arg Gly
20 25 30
Met Glu Pro Tyr Leu Val Arg Arg Leu Ser Cys Arg Asn Ile Gln Leu
35 40 45
Pro Pro Leu Ala Phe Arg Gln Leu Glu Gln Ala Asp Leu Lys Ser Glu
50 55 60
Ser Glu Asn Ile Gln Arg Pro Thr Ser Leu Pro Leu Lys Ile Leu Pro
65 70 75 80
Leu Ile Ala Ile Thr Ser Ala Glu Ser Ser Gly Phe Asp Val Asp Asn
85 90 95
Gly Thr Ser Ala Gly Arg Ser Pro Leu Asp Pro Met Thr Ser Pro Gly
100 105 110
Ser Gly Leu Ile Leu Gln Ala Asn Phe Val His Ser Gln Arg Arg Glu
115 120 125
Ser Phe Leu Tyr Arg Ser Asp Ser Asp Tyr Asp Leu Ser Pro Lys Ser
130 135 140
Met Ser Arg Asn Ser Ser Ile Ala Ser Asp Ile His Gly Asp Asp Leu
145 150 155 160
Ile Val Thr Pro Phe Ala Gln Val Leu Ala Ser Leu Arg Thr Val Arg
165 170 175
Asn Asn Phe Ala Ala Leu Thr Asn Leu Gln Asp Arg Ala Pro Ser Lys
180 185 190
Arg Ser Pro Met Cys Asn Gln Pro Ser Ile Asn Lys Ala Thr Ile Thr
195 200 205
Glu Glu Ala Tyr Gln Lys Leu Ala Ser Glu Thr Leu Glu Glu Leu Asp
210 215 220
Trp Cys Leu Asp Gln Leu Glu Thr Leu Gln Thr Arg His Ser Val Ser
225 230 235 240
Glu Met Ala Ser Asn Lys Phe Lys Arg Met Leu Asn Arg Glu Leu Thr
245 250 255
His Leu Ser Glu Met Ser Arg Ser Gly Asn Gln Val Ser Glu Phe Ile
260 265 270
Ser Asn Thr Phe Leu Asp Lys Gln His Glu Val Glu Ile Pro Ser Pro
275 280 285
Thr Gln Lys Glu Lys Glu Lys Lys Lys Arg Pro Met Ser Gln Ile Ser
290 295 300
Gly Val Lys Lys Leu Met His Ser Ser Ser Leu Thr Asn Ser Ser Ile
305 310 315 320
Pro Arg Phe Gly Val Lys Thr Glu Gln Glu Asp Val Leu Ala Lys Glu
325 330 335
Leu Glu Asp Val Asn Lys Trp Gly Leu His Val Phe Arg Ile Ala Glu
340 345 350
Leu Ser Gly Asn Arg Pro Leu Thr Val Ile Met His Thr Ile Phe Gln
355 360 365
Glu Arg Asp Leu Leu Lys Thr Phe Lys Ile Pro Val Asp Thr Leu Ile
370 375 380
Thr Tyr Leu Met Thr Leu Glu Asp His Tyr His Ala Asp Val Ala Tyr
385 390 395 400
His Asn Asn Ile His Ala Ala Asp Val Val Gln Ser Thr His Val Leu
405 410 415
Leu Ser Thr Pro Ala Leu Glu Ala Val Phe Thr Asp Leu Glu Ile Leu
420 425 430
Ala Ala Ile Phe Ala Ser Ala Ile His Asp Val Asp His Pro Gly Val
435 440 445
Ser Asn Gln Phe Leu Ile Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr
450 455 460
Asn Asp Ser Ser Val Leu Glu Asn His His Leu Ala Val Gly Phe Lys
465 470 475 480
Leu Leu Gln Glu Glu Asn Cys Asp Ile Phe Gln Asn Leu Thr Lys Lys
485 490 495
Gln Arg Gln Ser Leu Arg Lys Met Val Ile Asp Ile Val Leu Ala Thr
500 505 510
Asp Met Ser Lys His Met Asn Leu Leu Ala Asp Leu Lys Thr Met Val
515 520 525
Glu Thr Lys Lys Val Thr Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr
530 535 540
Ser Asp Arg Ile Gln Val Leu Gln Asn Met Val His Cys Ala Asp Leu
545 550 555 560
Ser Asn Pro Thr Lys Pro Leu Gln Leu Tyr Arg Gln Trp Thr Asp Arg
565 570 575
Ile Met Glu Glu Phe Phe Arg Gln Gly Asp Arg Glu Arg Glu Arg Gly
580 585 590
Met Glu Ile Ser Pro Met Cys Asp Lys His Asn Ala Ser Val Glu Lys
595 600 605
Ser Gln Val Gly Phe Ile Asp Tyr Ile Val His Pro Leu Trp Glu Thr
610 615 620
Trp Ala Asp Leu Val His Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu
625 630 635 640
Glu Asp Asn Arg Glu Trp Tyr Gln Ser Thr Ile Pro Gln Ser Pro Ser
645 650 655
Pro Ala Pro Asp Asp Pro Glu Glu Gly Arg Gln Gly Gln Thr Glu Lys
660 665 670
Phe Gln Phe Glu Leu Thr Leu Glu Glu Asp Gly Glu Ser Asp Thr Glu
675 680 685
Lys Asp Ser Gly Ser Gln Val Glu Glu Asp Thr Ser Cys Ser Asp Ser
690 695 700
Lys Thr Leu Cys Thr Gln Asp Ser Glu Ser Thr Glu Ile Pro Leu Asp
705 710 715 720
Glu Gln Val Glu Glu Glu Ala Val Gly Glu Glu Glu Glu Ser Gln Pro
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Glu Ala Cys Val Ile Asp Asp Arg Ser Pro Asp Thr
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<211> 219
<212> PRT
<213> 智人
<400> 26
Met Ala Gln Gln Thr Ser Pro Asp Thr Leu Thr Val Pro Glu Val Asp
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Asn Pro His Cys Pro Asn Pro Trp Leu Asn Glu Asp Leu Val Lys Ser
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Ser Val Ser Pro Lys Leu Ser Pro Val Ile Ser Pro Arg Asn Ser Pro
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Arg Leu Leu Arg Arg Met Leu Leu Ser Ser Asn Ile Pro Lys Gln Arg
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Arg Phe Thr Val Ala His Thr Cys Phe Asp Val Asp Asn Gly Thr Ser
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Ala Gly Arg Ser Pro Leu Asp Pro Met Thr Ser Pro Gly Ser Gly Leu
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Ile Leu Gln Ala Asn Phe Val His Ser Gln Arg Arg Glu Ser Phe Leu
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Tyr Arg Ser Asp Ser Asp Tyr Asp Leu Ser Pro Lys Ser Met Ser Arg
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Asn Ser Ser Ile Ala Ser Asp Ile His Gly Asp Asp Leu Ile Val Thr
145 150 155 160
Pro Phe Ala Gln Val Leu Ala Ser Leu Arg Thr Val Arg Asn Asn Phe
165 170 175
Ala Ala Leu Thr Asn Leu Gln Asp Arg Ala Pro Ser Lys Arg Ser Pro
180 185 190
Met Cys Asn Gln Pro Ser Ile Asn Lys Ala Thr Ile Thr Gly Ser Trp
195 200 205
Met Glu Leu Asn Pro Tyr Thr Leu Leu Asp Met
210 215

Claims (26)

1.一种治疗癫痫的方法，其特征在于：包括向患有癫痫的个体施用治疗有效量的磷酸二酯酶4(PDE4)抑制剂。
2.根据权利要求1所述的方法，其特征在于：PDE4抑制剂是小分子。
3.根据权利要求2所述的方法，其特征在于：其中PDE4抑制剂选自以下所组成的组：AN2728、屈他维林、异丁司特、伊索格列定、匹拉米司特、罗氟司特、咯利普兰、茶碱、阿普司特及其任意组合。
4.根据权利要求2所述的方法，其特征在于：PDE4抑制剂是AN2728。
5.根据权利要求1所述的方法，其特征在于：PDE4抑制剂抑制PDE4A、PDE4B、PDE4C或PDE4D中的一种或多种。
6.根据权利要求1所述的方法，其特征在于：PDE4抑制剂在PDE4A、PDE4B、PDE4C和PDE4D中表现出选择性。
7.根据权利要求6所述的方法，其特征在于：PDE4抑制剂对PDE4B具有选择性。
8.根据权利要求1-7中任一项所述的方法，其特征在于：给药是通过口服、肠胃外、鼻内、鞘内、颅内或经皮给药。
9.根据权利要求1所述的方法，其特征在于：PDE4抑制剂抑制PDE4的表达。
10.根据权利要求9所述的方法，其特征在于：PDE4抑制剂是基于核酸的抑制剂，PDE4抑制剂是基于核酸的抑制剂，其包括与编码PDE4A的信使RNA(mRNA)、编码PDE4B的mRNA、编码PDE4C的mRNA、编码PDE4D的mRNA或任何组合的一部分互补的区域。
11.根据权利要求10所述的方法，其特征在于：基于核酸的抑制剂与编码PDE4A、PDE4B、PDE4C或PDE4D的mRNA选择性杂交。
12.根据权利要求11所述的方法，其特征在于：基于核酸的抑制剂与编码PDE4B的mRNA选择性杂交。
13.根据权利要求10-12中任一项所述的方法，其特征在于：基于核酸的抑制剂是吗啉、短干扰RNA(siRNA)或微RNA(miRNA)。
14.根据权利要求1-13中任一项所述的方法，其特征在于：个体患有的癫痫，其选自以下的癫痫症组成的组：良性罗兰癫痫、额叶癫痫、婴儿痉挛、青少年肌阵挛癫痫(JME)、青少年失神癫痫、儿童失神癫痫、密集性癫痫、热性惊厥、进行性肌阵挛性癫痫、伦诺克斯-加斯托综合征、兰道-克勒夫纳综合征、德拉韦综合征(DS)、全身性癫痫伴热性惊厥(GEFS+)、婴儿严重肌阵挛性癫痫(SMEI)、良性家族性新生儿惊厥(BFNC)、韦斯特综合征、大田原综合征、早期肌阵挛脑病、迁移性部分性癫痫、婴儿癫痫性脑病、结节性硬化症(TSC)、局灶性皮质发育不良、I型无脑回畸形、Miller-Dieker综合征、快乐木偶综合征、脆性X综合征、自闭症谱系障碍中的癫痫、皮质下带异位症、Walker-Warburg综合征、阿尔茨海默病癫痫、创伤后癫痫、进行性肌阵挛癫痫、反射性癫痫、拉斯穆森病综合征、颞叶癫痫、边缘癫痫、癫痫持续状态、腹部癫痫、双侧大量肌阵挛、月经性癫痫、杰克逊癫痫症、Unverricht-Lundborg病和光敏性癫痫。
15.根据权利要求1-13中任一项所述的方法，其特征在于：个体患有由基因突变引起的癫痫。
16.一种鉴定抗癫痫药的方法，其特征在于：包括在PDE4活性测定中使磷酸二酯酶4(PDE4)多肽与候选药剂接触，其中候选药物对PDE4多肽的活性的抑制将候选药物鉴定为抗癫痫剂。
17.根据权利要求16所述的方法，其特征在于：接触包括在无细胞PDE4活性测定中组合PDE4多肽和候选药剂。
18.根据权利要求16所述的方法，其特征在于：接触包括在基于细胞的PDE4活性测定中组合PDE4多肽和候选药剂。
19.根据权利要求16-18中任一项所述的方法，其特征在于：PDE4活性测定进一步包括将PDE4多肽与已知抑制PDE4活性的阳性对照剂接触。
20.根据权利要求19所述的方法，其特征在于：阳性对照剂选自以下所组成的组：AN2728、屈他维林、异丁司特、伊索格列定、匹拉米司特、罗氟司特、咯利普兰、茶碱、阿普司特及其任意组合。
21.根据权利要求16-20中任一项所述的方法，其特征在于：所述候选药剂是小分子。
22.根据权利要求16-21中任一项所述的方法，其特征在于：PDE4多肽是PDE4A、PDE4B、PDE4C或PDE4D。
23.根据权利要求22所述的方法，其特征在于：PDE4多肽是PDE4B。
24.根据权利要求16-23中任一项所述的方法，其特征在于，进一步包括，当确定候选药剂抑制PED4多肽的活性时，确定候选药剂是否表现出对PDE4A、PDE4B、PDE4C和PDE4D的选择性抑制。
25.一种药物组合物，其特征在于：包括通过根据权利要求16-24中任一项所述的方法鉴定的抗癫痫剂。
26.一种方法，其特征在于：包括向患有癫痫的个体施用治疗有效量的抗癫痫剂，该癫痫剂通过根据权力要求16-24中任一项所述的方法鉴定。
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