CN105481949B - 一种治疗癌症的肽变体及其应用 - Google Patents
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Abstract
本发明涉及一种治疗癌症的肽变体及其应用，这些肽变体的氨基酸序列分别为SEQ ID No:2‑15。这些肽变体相对于野生型具有更强的杀伤肿瘤细胞的效果，有着广阔的制药前景。
Description
技术领域
本发明属于生物技术领域，具体地说，本发明涉及治疗癌症的肽变体。
背景技术
微管是细胞骨架的三个丝状网络之一的组成成分，是复杂的、不稳定的聚合物，微管的基本单元是α-和β-微管蛋白二聚体。例如，它们负责多种细胞运动、轴突运输、或运动性，特别是，它们提供用于形成有丝分裂纺锤体的基本材料。微管不断地重新形成不同长度的中空管。因此，通过破坏癌细胞的微管动力学，本发明的肽能起到阻止癌细胞增殖的抗有丝分裂剂的作用（减慢或阻止癌细胞的有丝分裂）。
本发明的活性肽还显现出特异性地改变癌细胞的质膜，并且显现出在宿主生物体的健康细胞中不引起这些相同的改变。所述活性肽具有聚阳离子的性质，因此它们会作用在富含负电荷的癌细胞的质膜上。
在现有技术中，为了提高蛋白或肽的活性，针对蛋白或肽的活性位点进行特异性的突变和取代，从而寻找活性更高的变体是常规的做法。
发明内容
序列SEQ ID No:1所示的肽是现有技术中已知的可以被被用于治疗癌症。但是其活性并不是非常令人满意。为了进一步提高该肽的生物活性，申请人通过大量的实验，针对SEQ ID No:1所示的肽中特定的位点进行了点突变以及两端分别进行特异性的修饰，从而获得了比SEQ ID No:1所示的肽本身具有更高抑癌活性的变体。
具体地，被用作治疗癌症的活性成分的肽的序列如下所示（表I）：
| SEQ序列号（XA代号） | 序列 | 氨基酸数目 |
| 1（XA1） | INWLKIAKKVAGML-NH<sub>2</sub> | 14 |
| 2（XA2） | SINWLKIAKKVAGML-NH<sub>2</sub> | 15 |
| 3（XA3） | SINWLAIAKKVAGML-NH<sub>2</sub> | 15 |
| 4（XA4） | SINWLAIAAKVAGML-NH<sub>2</sub> | 15 |
| 5（XA5） | SINWLAIAAKVGGML-NH<sub>2</sub> | 15 |
| 6（XA6） | SINWLKIAKKVAGMLL-NH<sub>2</sub> | 16 |
| 7（XA7） | SINWLKIAKKVAGMLL-NH<sub>2</sub> | 16 |
| 8（XA8） | SINWLAIAKKVAGMLL-NH<sub>2</sub> | 16 |
| 9（XA9） | SINWLAIAAKVAGMLL-NH<sub>2</sub> | 16 |
| 10（XA10） | SINWLAIAAKVGGMLL-NH<sub>2</sub> | 16 |
| 11（XA11） | INWLINWLKIAKKVAGML-NH<sub>2</sub> | 18 |
| 12（XA12） | INWLSINWLKIAKKVAGML-NH<sub>2</sub> | 19 |
| 13（XA13） | INWLSINWLAIAKKVAGML-NH<sub>2</sub> | 19 |
| 14（XA14） | INWLSINWLAIAAKVGGMLL-NH<sub>2</sub> | 20 |
| 15（XA15） | INWLSINWLKIAKKVAGMLL-NH<sub>2</sub> | 20 |
| 16（XA16） | INWL INWLAIAKKVAGML-NH<sub>2</sub> | 18 |
| 17（XA17） | INWLSINWLAIAKKVAGMLL-NH<sub>2</sub> | 20 |
申请人对SEQ ID No:2至SEQ ID No:15这些肽的研究有证实，这些肽相对于SEQID No:1的肽对癌细胞具有更高的细胞毒活性，同时对待治疗的宿主的细胞具有非常弱的毒性或者无毒性。本发明的活性肽特异性地改变癌细胞的微管。
表I中所引用的肽被用作治疗癌症的药物，具体地，可有效地治疗白血病、肝癌、胶质母细胞瘤等。
根据本发明的另一个特点，通过化学合成产生序列SEQ ID No:1至SEQ ID No:15的肽。
发明的另一个目的涉及包含上文所定义的肽之一的药物组合物。
有利地，所述组合物是水性悬浮液或溶液形式，或者是干燥状态的未包衣或包衣片剂形式，例如丸剂、凝胶胶囊、胶囊或者粉末。
如果所述药物组合物处于干燥状态，则它可以与一种或多种惰性稀释剂例如淀粉、纤维素、蔗糖、乳糖或二氧化硅等混合。它在干燥状态下还可以包含其他物质，例如一种或多种润滑剂如硬脂酸镁或滑石粉、着色剂、包衣（糖包衣片剂）或清漆。
如果本发明的药物组合物是液体形式，则它可以包括含有惰性稀释剂例如水、乙醇、甘油、植物油或液体石蜡的可药用的溶液、悬浮液、乳剂、糖浆剂和酏剂。所述药物组合物还可以包含除稀释剂以外的其他液体物质，例如润湿剂、甜味剂、增稠剂、调味剂或稳定剂产品。
所述组合物任选与可药用的无毒惰性赋形剂或介质组合，其特征在于，可以以皮肤、透皮或经皮；口服；胃肠外；鼻或支气管应用的形式局部施用所述组合物。
用于胃肠外施用的药物组合物可以是水性或非水性溶液、悬浮液或乳剂。这些制剂由作为溶剂或介质的水、丙二醇、植物油特别是橄榄油和芝麻油、可注射的有机酯例如油酸乙酯、或其他合适的有机溶剂制成。所述药物组合物还可以含有佐剂，特别是润湿剂、等渗剂、乳化剂、分散剂和稳定剂。
对于局部施用来说，有利地，以软膏、乳膏、凝胶或斑贴的形式施用所述药物。
另外，所述药物组合物可被单独使用或在给定的治疗中与其他药物联合使用，或者与放射疗法或手术联合。
具体实施方式
A)肽的合成
肽SEQ ID No:1至SEQ ID No:17的合成与纯化
通过使用N-9-氟代苯基甲氧基羰基和Novasyn TGS树脂手动分离固相合成来制备所述肽。侧链保护的基团包括丝氨酸的叔丁基和赖氨酸的叔丁氧基羰基。为了制备具有乙酰化的Ｎ末端残基的肽，在连接了最后的氨基酸残基后，将乙酸酐加入到树脂-肽复合物中，并保持搅拌1小时以促进乙酰化。
通过用浓度为10mL.g-1的三氟乙酸/1,2-乙二硫醇/苯甲醚/苯酚/水（体积比82.5:2.5:5:5:5）在室温下处理2小时来剪切所述树脂-肽复合物。随后进行过滤以去除树脂。然后加入4°C的乙醚，使粗肽沉淀，随后在室温下以1000g离心15分钟后收集粗肽。随后将粗肽溶解在水中，并使用Shiseido C-18半制备柱（250mm x 10.0mm；5μm）在用含有0.1%（v/v）TFA的40%（v/v）乙腈以2mL/min的速率恒溶剂洗脱下通过RP-HPLC进行层析分离。用UV-DAD检测器（紫外线二极管阵列检测器）（Shimadzu，型号SPD-M10A）在215nm下调节洗脱，并且每个洗脱的馏分都被手动收集在2mL的玻璃小瓶中。通过HPLC和ESI-MS分析来测定合成的肽（乙酰化和非乙酰化两种肽）的序列。 在肽被合成、纯化并检验后，确认得到了表I所示的肽序列。
B)肽SEQ ID No:1-17对健康细胞的活性
使两种非致瘤的成纤维细胞FIBRO1和FIBRO2与肽SEQ ID No:1-17接触，以观察健康细胞的微管网络是否也被该肽破坏。在使用肽浓度为15μM的情况下，代表了多种正常细胞的人成纤维细胞对肽SEQ ID No:1-15根本没有响应（聚合的微管蛋白密度没有改变）。而SEQ ID No:16-17处理的成纤维细胞对微管蛋白密度的影响较大，其厚度变薄了大约15%。这说明，肽SEQ ID No:1-15对健康细胞无活性。当肽SEQ ID No:1-15被用作哺乳动物、尤其是人的治疗剂时，事实上非常有可能发生很少的副作用。
C)细胞增殖和细胞存活率的测定
分析以下细胞系（表II），以确定并定量肽SEQ ID No:1-17的活性。
表II
| 组织类型 | 细胞系 |
| 急性早幼粒细胞白血病细胞系 | NB4 |
| 肝癌 | HepG2 |
| 胶质母细胞瘤 | U87M6 |
以每孔0.2mL的体积将约10000个细胞接种到96孔微板中，24小时后，用肽SEQ IDNo:1-17进行处理。在存在肽SEQ ID No:1-17的条件下将细胞在CO2培养箱中在37°C下孵育48小时。在孵育后，将MTT试剂或(3-(4,5-二甲基-2-基)-2,5-二苯基溴化四唑（0.5mg/ml，在0.1ml的体积中）加入到每个孔中并在37°C下孵育2小时。 然后，MTT所含有的四唑环被活细胞的琥珀酸脱氢酶还原。然后，去除上清，并加入0.1ml裂解缓冲液（含有10%tritonX100和10%1N HCl的异丙醇）以溶解所形成的蓝紫色甲臜沉淀。几分钟后，在室温下用酶标仪在562nm的检测波长下测定吸光度。使用不含肽的孔来证实对照细胞的存活率。通过处理后细胞的吸光度与对照细胞的吸光度的比率来评估每一种细胞的IC50（肽SEQ ID No:1-17在这些细胞系上引起50%细胞毒性时的浓度）值，μM。
表III
| SEQ序列号（XA代号） | NB4（IC50） | HepG2（IC50） | U87M6（IC50） |
| 1（XA1） | 52.2 | 35.8 | 15.41 |
| 2（XA2） | 37.3 | 29.3 | 10.9 |
| 3（XA3） | 32.5 | 27.4 | 9.8 |
| 4（XA4） | 34.6 | 25.4 | 10.3 |
| 5（XA5） | 31.9 | 26.9 | 10.1 |
| 6（XA6） | 40.1 | 24.8 | 9.7 |
| 7（XA7） | 34.9 | 28.7 | 9.6 |
| 8（XA8） | 35.2 | 27.3 | 10.0 |
| 9（XA9） | 37.8 | 28.1 | 10.2 |
| 10（XA10） | 36.3 | 25.1 | 10.4 |
| 11（XA11） | 30.9 | 20.9 | 9.9 |
| 12（XA12） | 29.8 | 19.9 | 9.3 |
| 13（XA13） | 31.7 | 21.7 | 9.7 |
| 14（XA14） | 39.5 | 22.5 | 9.7 |
| 15（XA15） | 28.7 | 20.1 | 10.1 |
| 16（XA16） | 60.9 | 45.9 | 30.5 |
| 17（XA17） | 65.3 | 50.5 | 35.7 |
如表III所示，肽SEQ ID No:2-15显著提高了以上三种肿瘤细胞的增殖。比肽SEQID No:1对癌细胞有毒性的剂量得到显著的降低，而SEQ ID No:16和17反而提高了IC50值，说明本申请的肽SEQ ID No:2-15相对于现有技术有着对肿瘤细胞更好的抑制效果，可进一步用于肿瘤的相关治疗。
癌细胞对肽的耐药性
将SEQ ID No:1-15分别对癌细胞进行耐药性实验，实验方法为本领域常规的方法，结果显示该肽具有不诱导快速耐药过程。
序列表
<110>朱凌玮
〈120〉一种治疗癌症的肽变体XA10及其应用
〈160〉17
〈210〉1
〈211〉14
〈212〉PRT
〈213〉artificial sequence
〈400〉1
INWLKIAKKVAGML
〈210〉2
〈211〉14
〈212〉PRT
〈213〉artificial sequence
〈400〉2
SINWLKIAKKVAGML
〈210〉3
〈211〉15
〈212〉PRT
〈213〉artificial sequence
〈400〉3
SINWLAIAKKVAGML
〈210〉4
〈211〉15
〈212〉PRT
〈213〉artificial sequence
〈400〉4
SINWLAIAAKVAGML
〈210〉5
〈211〉15
〈212〉PRT
〈213〉artificial sequence
〈400〉5
SINWLAIAAKVGGML
〈210〉6
〈211〉16
〈212〉PRT
〈213〉artificial sequence
〈400〉6
SINWLKIAKKVAGMLL
〈210〉7
〈211〉16
〈212〉PRT
〈213〉artificial sequence
〈400〉7
SINWLKIAKKVAGMLL
〈210〉8
〈211〉16
〈212〉PRT
〈213〉artificial sequence
〈400〉8
SINWLAIAKKVAGMLL
〈210〉9
〈211〉16
〈212〉PRT
〈213〉artificial sequence
〈400〉9
SINWLAIAAKVAGMLL
〈210〉10
〈211〉16
〈212〉PRT
〈213〉artificial sequence
〈400〉10
SINWLAIAAKVGGMLL
〈210〉11
〈211〉18
〈212〉PRT
〈213〉artificial sequence
〈400〉12
INWLINWLKIAKKVAGML
〈210〉12
〈211〉19
〈212〉PRT
〈213〉artificial sequence
〈400〉12
INWLSINWLKIAKKVAGML
〈210〉13
〈211〉19
〈212〉PRT
〈213〉artificial sequence
〈400〉13
INWLSINWLAIAKKVAGML
〈210〉14
〈211〉20
〈212〉PRT
〈213〉artificial sequence
〈400〉14
INWLSINWLAIAAKVGGMLL
〈210〉15
〈211〉20
〈212〉PRT
〈213〉artificial sequence
〈400〉15
INWLSINWLKIAKKVAGMLL
〈210〉16
〈211〉18
〈212〉PRT
〈213〉artificial sequence
〈400〉16
INWL INWLAIAKKVAGML
〈210〉17
〈211〉20
〈212〉PRT
〈213〉artificial sequence
〈400〉17
INWLSINWLAIAKKVAGMLL
Claims (2)
1.一种用于治疗癌症的肽，其特征在于：为XA10，其氨基酸序列为SEQ ID No:10。
2.权利要求1所述的肽用于制备药物的用途，所述药物用于治疗白血病或肝癌。
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