CN113549129B - 一种d-构型抗肿瘤肽及其制备方法和应用 - Google Patents
一种d-构型抗肿瘤肽及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供一种D‑构型抗肿瘤肽及其制备方法和应用,属于多肽制备和生物医药技术领域。本发明针对L‑型肽易被蛋白酶降解等缺点,合成由D‑型氨基酸构成的D‑型抗肿瘤肽。D‑型多肽对蛋白酶降解有着天然的抗性,这极大地增加了多肽的稳定性。时效曲线实验表明,D‑型多肽的抗肿瘤效果的稳定性是L‑型多肽的5倍以上,因此具有良好的实际应用之价值。
Description
技术领域
本发明属于多肽制备和生物医药技术领域,具体涉及一种D-构型抗肿瘤肽及其制备方法和应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
肿瘤是严重威胁人类生命和健康的重大疾病,其发病率和死亡率在近几年呈持续上升的趋势,我国的癌症防控形势依然严峻。2015年,中国新发约400万恶性肿瘤病例,有超过200万肿瘤症患者因治疗无效而死亡。尽管近年来免疫治疗取得了突破性进展,手术、放疗和化疗仍然是临床上普遍采用的肿瘤治疗方法,寻找安全、合理、有效的肿瘤治疗方法是目前肿瘤治疗领域亟待解决的问题。
抗菌肽是一类天然产生的重要先天免疫防御物质,抗菌肽功能多样,其中将具有抗肿瘤活性的抗菌肽叫做抗肿瘤肽(溶瘤肽)。抗肿瘤肽具有很好的研究价值和应用前景,其长度和序列多样,但大部分抗肿瘤肽都有两个共同特征:阳离子性和两亲性,其通常由5~40个氨基酸组成,其中精氨酸、赖氨酸和组氨酸的存在使其表现出较强的阳离子特性,表面净电荷范围为+2~+9,并且绝大多数的抗肿瘤肽含有α-螺旋或β-折叠结构,这种两亲性侧链在具有α-螺旋结构的抗肿瘤肽链上分别排布在螺旋的两侧,或者集中于两端,因此可形成亲水面和疏水面或者明显的亲水端和疏水端。当抗肿瘤肽与肿瘤细胞膜相互作用时,疏水区域与胞膜脂质结合,带正电荷的亲水区域与带有负电荷的肿瘤细胞膜表面通过静电吸附而有效结合,这为抗肿瘤肽能够选择性作用于肿瘤细胞和在膜上打孔发挥抗肿瘤作用奠定了基础。
牛乳铁蛋白肽(LfcinB)是一种经典的抗菌肽,由25个氨基酸构成,具有两个两亲性结构,实验证明其对多种肿瘤具有杀伤作用。根据LfcinB的活性结构设计的新型阳离子抗肿瘤肽LTX-315对普通肿瘤细胞系和耐药肿瘤细胞系均显示出明显的抗肿瘤活性,并且对正常细胞的毒性较低。LTX-315可通过双重作用诱导细胞死亡,即细胞溶解和诱导免疫反应。实验证明,细胞溶解后释放到细胞间质的抗原分子,会激活未成熟的树突状细胞,随后产生特异性抗肿瘤细胞的细胞毒性T淋巴细胞,进而消灭肿瘤细胞。
LTX-315可以有效抑制小鼠移植瘤的生长,并保护小鼠免受第二次同肿瘤的侵袭。此后,LTX-315已经在人类临床研究的第一阶段进行了评估,并被证明其在肿瘤微环境中能引起T细胞增多,免疫抑制细胞减少,并且最终能诱导肿瘤坏死。由于这些特点,LTX-315被认为是一种具有选择性的强效抗肿瘤药物。但是,发明人发现,LTX-315由L-型氨基酸构成,半衰期短,容易被蛋白酶降解,需要频繁给药,限制了LTX-315的广泛应用。
发明内容
基于上述现有技术的不足,本发明提供一种D-构型抗肿瘤肽及其制备方法和应用。针对L-型肽易被蛋白酶降解等缺点,本发明合成由D-型氨基酸构成的D-型抗肿瘤肽。D-型多肽对蛋白酶降解有着天然的抗性,这极大地增加了多肽的稳定性。时效曲线实验表明,D-型多肽的抗肿瘤效果的稳定性是L-型多肽的5倍以上,因此具有良好的实际应用之价值。
本发明的第一个方面,提供一种D-型多肽,所述多肽包含如下氨基酸残基序列:
WKW-121 H-wrrrrrwrrrrw-NH2
WKW-215 H-kkwwkkw(dip)k-NHNH2
WKW-325 H-kkwwkkw(dip)k-NH2
WKW-326 H-Gkkwwkkw(dip)k-NH2
WKW-327 H-AcEVCit-Gkkwwkkw(dip)k-NH2
WKW-345 H-k(dip)wkkwwkk-NH2
上述多肽在破坏细胞膜的同时自身并不会被降解,从而可以进一步进入细胞内,通过破坏线粒体膜和细胞膜,多靶点协同效应发挥抗肿瘤作用,肿瘤细胞不易产生耐药性。
本发明的第二个方面,提供编码所述多肽的核苷酸,其包含下组中的任一种:
(a)编码具有所述氨基酸序列的多肽的核苷酸;
(b)与(a)所述核苷酸互补的核苷酸。
本发明的第三个方面,提供上述多肽的合成方法,所述合成方法包括采用固相多肽合成法合成;具体的,基于9-芴甲氧羰基的固相多肽合成法(Fmoc-SPPS)合成上述多肽。
基于9-芴甲氧羰基的固相多肽合成法由以下几个循环组成,即脱保护,活化和交联,以及洗脱和脱保护。
本发明的第四个方面,提供上述多肽在制备预防和/或治疗(辅助治疗)肿瘤相关疾病的药物或保健品中的应用。
同时,需要说明的是,肿瘤在本发明中如本领域技术人员所知的那样加以使用,其包括良性肿瘤和/或恶性肿瘤。良性肿瘤被定义为不能在体内形成侵略性、转移性肿瘤的细胞过度增殖。反之,恶性肿瘤被定义为能够形成全身性疾病(例如在远端器官中形成肿瘤转移)的具有多种细胞异常和生化异常的细胞。
本发明的第五个方面,提供一种药物组合物,其包含上述多肽。
本发明的第六个方面,提供一种药物制剂,其包含多肽和药学上可接受的辅料和/或载体。
本发明的第七个方面,提供一种预防和/或治疗肿瘤的方法,所述方法包括:其包括向受试者施用治疗有效剂量的上述多肽、上述药物组合物或上述药物制剂。
本发明的第八个方面,提供上述多肽作为非治疗目的的肿瘤细胞抑制剂的用途。根据本发明,所述“非治疗目的”例如在体外抑制肿瘤细胞增殖;促进肿瘤细胞死亡。通过对肿瘤细胞(如A20淋巴瘤细胞)施加本发明的多肽,有利于研究肿瘤生长相关信号通路及基因表达交互作用,从而为进一步研究肿瘤相关性疾病提供原始材料并奠定基础。
本发明的第九个方面,提供一种体外抑制肿瘤细胞增殖的方法,所述方法包括给予体外培养的肿瘤细胞上述多肽、上述药物组合物或上述药物制剂。
上述技术方案的有益技术效果:
1.稳定性更高。前人开发的LTX-315等抗肿瘤多肽,为L-型多肽(由L-型氨基酸构成),该类多肽易被体内蛋白酶降解,生物稳定性较差,半衰期短;另外,该类多肽免疫原性高,有被机体清除的风险。本项目用具有较好生物稳定性的D-型氨基酸合成D-型阳离子抗肿瘤肽。D-型多肽具有非常高的酶解稳定性,可以提高抗肿瘤效果并延长给药间隔,并可降低免疫原性,具有较大的应用价值。
2.活性更高。上述技术方案合成的D型阳离子抗肿瘤肽,破坏细胞膜的同时自己不会被降解,可以进一步进入细胞内,通过破坏线粒体膜和细胞膜,多靶点协同效应发挥抗肿瘤作用,肿瘤细胞不易产生耐药性。其中,各肽针对A20淋巴瘤细胞的IC50分别为5.8±0.3μM(WKW-121),4.8±1.3μM(WKW-215),4.7±2.7μM(WKW-325),6.2±0.9μM(WKW-326),4.3±1.4μM(WKW-327)和5.6±1.0μM(WKW-345)。
综上所述,为了避免前人开发的L型阳离子抗肿瘤肽的缺点(如稳定性差、半衰期短、潜在的免疫原性、抗肿瘤活性相对较低等),上述技术方案采用D-型氨基酸合成了全新的D-型阳离子抗肿瘤肽,得到免疫原性低、稳定性高、半衰期长、活性更强的阳离子抗肿瘤肽,因此具有良好的实际应用之前景。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为本发明固相多肽合成方法示意图;
图2为本发明WKW-121的结构式、一级氨基酸序列、分析型反相高效液相色谱图和质谱图及圆二色谱图;
图3为本发明WKW-215的结构式、一级氨基酸序列、分析型反相高效液相色谱图和质谱图及圆二色谱图;
图4为本发明WKW-325的结构式、一级氨基酸序列、分析型反相高效液相色谱图和质谱图及圆二色谱图;
图5为本发明WKW-326的结构式、一级氨基酸序列、分析型反相高效液相色谱图和质谱图及圆二色谱图;
图6为本发明WKW-327的结构式、一级氨基酸序列、分析型反相高效液相色谱图和质谱图及圆二色谱图;
图7为本发明WKW-345的结构式、一级氨基酸序列、分析型反相高效液相色谱图和质谱图及圆二色谱图;
图8为本发明细胞水平评价D-型阳离子抗肿瘤肽对肿瘤细胞增殖的抑制作用;
图9为本发明D-型阳离子抗肿瘤肽导致肿瘤细胞形态学发生改变图;
图10为本发明D-型阳离子抗肿瘤肽抑制肿瘤细胞增殖的时效曲线。
具体实施方式
应该指出,以下详细说明都是示例性的,旨在对本发明提供进一步的说明。除非另有指明,本发明使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本发明申请的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
如前所述,LTX-315由L-型氨基酸构成,半衰期短,容易被蛋白酶降解,需要频繁给药,存在免疫原性等风险,限制了LTX-315的广泛应用。
有鉴于此,本发明的一个典型具体实施方式中,提供一种D-型多肽,所述多肽包含如下氨基酸残基序列:
WKW-121 H-wrrrrrwrrrrw-NH2
WKW-215 H-kkwwkkw(dip)k-NHNH2
WKW-325 H-kkwwkkw(dip)k-NH2
WKW-326 H-Gkkwwkkw(dip)k-NH2
WKW-327 H-AcEVCit-Gkkwwkkw(dip)k-NH2
WKW-345 H-k(dip)wkkwwkk-NH2
上述D-型多肽在破坏细胞膜的同时自身并不会被降解,从而可以进一步进入细胞内,通过破坏线粒体膜和细胞膜,多靶点协同效应发挥抗肿瘤作用,肿瘤细胞不易产生耐药性。
本发明的又一具体实施方式中,提供编码所述多肽的核苷酸,其包含下组中的任一种:
(a)编码具有所述氨基酸序列的多肽的核苷酸;
(b)与(a)所述核苷酸互补的核苷酸。
本发明的又一具体实施方式中,提供上述多肽的合成方法,所述合成方法包括采用固相多肽合成法合成;具体的,基于9-芴甲氧羰基的固相多肽合成法(Fmoc-SPPS)合成上述多肽。
如无特殊说明,Fmoc-SPPS过程中使用的氨基酸为Fmoc保护的D-型氨基酸(例外包括,G无手性,E、V和Cit为L-型氨基酸)。其中,固相载体选择为树脂,根据碳末端基团的不同,选取不同的树脂。合成含有酰胺末端的多肽,选择Rink Amide Am树脂;合成含有酰肼末端的多肽,选择Fmoc-酰肼树脂。
基于9-芴甲氧羰基的固相多肽合成法由以下几个循环组成,即脱保护,活化和交联,以及洗脱和脱保护。
本发明的又一具体实施方式中,脱保护步骤即脱除Fmoc的过程中,使用的脱除Fmoc的脱保护试剂为:含有20%哌啶(v/v)和0.1M HOBt的DMF溶液。
本发明的又一具体实施方式中,脱除Fmoc的反应条件为:28℃下,第一次反应5分钟、第二次反应10分钟。
本发明的又一具体实施方式中,在活化和交联过程发生缩合反应过程中,氨基酸的配比为Fmoc-D-型氨基酸:HCTU:DIPEA=4倍当量:3.8倍当量:8倍当量。缩合反应条件为:28℃下,反应1小时,每个氨基酸缩合两次。
本发明的又一具体实施方式中,洗脱和脱保护过程中使用TFA切肽试剂,所述TFA切肽试剂的配比为TFA:苯酚:水:茴香硫醚:1,2-乙二硫醇=85:2.5:5:5:2.5(体积比,v:v:v:v:v)。具体的,在28℃下反应2.5-3小时,从而完成洗脱和脱保护过程。
本发明的又一具体实施方式中,提供上述含有酰胺末端多肽的合成方法,包括:
Rink Amide Am(1倍当量)树脂,使用DMF和DCM交替清洗,然后用10mL的DMF/DCM混合溶液(1:1,v:v)在28℃下浸泡树脂2-3小时。脱除Fmoc的脱保护试剂为:含有20%哌啶(v:v)和0.1M HOBt的DMF溶液。脱除Fmoc的反应条件为:28℃下,第一次反应5分钟、第二次反应10分钟。缩合过程中,氨基酸的配比为Fmoc-D-型氨基酸:HCTU:DIPEA=4倍当量:3.8倍当量:8倍当量。缩合反应条件为:28℃下,反应1小时,每个氨基酸缩合两次。
完成所有氨基酸的缩合以后,将负载多肽的树脂转移到新的多肽合成管中,用DMF和DCM交替清洗,最后用DCM清洗树脂(4遍以上)并用油泵彻底抽干树脂。然后在树脂中加入TFA切肽试剂,切肽试剂的配比为TFA:苯酚:水:茴香硫醚:1,2-乙二硫醇=85:2.5:5:5:2.5(v:v:v:v:v)。28℃下反应2.5-3小时。收集切肽溶液,使用高纯氮鼓泡浓缩切割溶液。然后用提前预冷的无水乙醚沉淀多肽,通过离心机离心得到粗肽沉淀,弃掉上清,得到粗肽固体。再用预冷的无水乙醚反复清洗、离心粗肽两次以上。
得到的粗肽固体用含有0.1%TFA的乙腈和水混合溶剂溶解,通过真空干燥机冻干,得到固体形式的粗肽。此粗肽固体再次用含有0.1%TFA的乙腈和水混合溶剂溶解,通过半制备型反相高效液相色谱(RP-HPLC)分离纯化。纯化得到的多肽溶液直接通过真空干燥机冻干,得到固体形式的目标多肽。
本发明的又一具体实施方式中,提供上述多肽在制备预防和/或治疗(辅助治疗)肿瘤相关疾病的药物或保健品中的应用。
同时,需要说明的是,肿瘤在本发明中如本领域技术人员所知的那样加以使用,其包括良性肿瘤和/或恶性肿瘤。良性肿瘤被定义为不能在体内形成侵略性、转移性肿瘤的细胞过度增殖。反之,恶性肿瘤被定义为能够形成全身性疾病(例如在远端器官中形成肿瘤转移)的具有多种细胞异常和生化异常的细胞。
本发明的又一具体实施方式中,本发明的药物可用于治疗恶性瘤。可用本发明的药物治疗的恶性瘤的实例包括实体瘤和血液瘤。实体瘤可以是乳腺、膀胱、骨、脑、中枢和外周神经系统、结肠、内分泌腺(如甲状腺和肾上腺皮质)、食道、子宫内膜、生殖细胞、头和颈、肝、肺、喉和下咽的肿瘤、间皮瘤、卵巢、胰腺、前列腺、直肠、肾、小肠、软组织、睾丸、胃、皮肤(如黑色素瘤)、输尿管、阴道和外阴的肿瘤。恶性瘤包括遗传性癌症,例如视网膜母细胞瘤和肾母细胞瘤(Wilms tumor)。此外,恶性瘤包括在所述器官中的原发性肿瘤及在远端器官中的相应继发性肿瘤(肿瘤转移)。血液瘤可以是侵略性和无痛形式的白血病和淋巴瘤,即非霍奇金病、慢性和急性髓样白血病(CML/AML)、急性淋巴细胞性白血病(ALL)、霍奇金病、多发性骨髓瘤和T-细胞型淋巴瘤。还包括骨髓增生异常综合征、浆细胞瘤、类肿瘤综合征和未知原发部位的癌症及AIDS相关的恶性瘤。
本发明的又一具体实施方式中,提供一种药物组合物,其包含上述多肽。
本发明化合物的药物组合物,可以以选自以下任意方式施与:口服、喷雾吸入、直肠给药、鼻腔给药、阴道给药、局部给药、非肠道给药如皮下、静脉、肌内、腹膜内、鞘内、心室内、胸骨内或颅内注射或输入,或借助一种外植的储器用药,其中优选口服、肌注、腹膜内或静脉内用药方式。
本发明的又一具体实施方式中,提供一种药物制剂,其包含多肽和药学上可接受的辅料和/或载体。
本发明中含有上述多肽的药物组合物可以单位剂量形式给药。给药剂型可以是液体剂型、固体剂型。液体剂型可以是真溶液类、胶体类、微粒剂型、乳剂剂型、混悬剂型。其他剂型例如片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、乳剂、颗粒剂、栓剂、冻干粉针剂、包合物、填埋剂、贴剂、擦剂等。
本发明的药物组合或药物制剂中还可以含有常用的载体,这里所述可药用载体包括但不局限于:离子交换剂,氧化铝,硬脂酸铝,卵磷脂,血清蛋白如人血清蛋白,缓冲物质如磷酸盐,甘油,山梨酯,山梨酸钾,饱和植物脂肪酸的部分甘油酯混合物,水,盐或电解质,如硫酸鱼精蛋白,磷酸氢二钠,磷酸氢钾,氯化钠,锌盐,胶态氧化硅,三硅酸镁,聚乙烯吡咯烷酮,纤维素物质,聚乙二醇,羧甲基纤维素钠,聚丙烯酸酯,蜂蜡,羊毛酯等。载体在药物组合物中的含量可以是1%重量-98%重量,通常大约占到80%重量。为方便起见,局部麻醉剂,防腐剂,缓冲剂等可直接溶于载体中。
口服片剂和胶囊可以含有赋形剂如粘合剂,如糖浆,阿拉伯胶,山梨醇,黄芪胶,或聚乙烯吡咯烷酮,填充剂,如乳糖,蔗糖,玉米淀粉,磷酸钙,山梨醇,氨基乙酸,润滑剂,如硬脂酸镁,滑石,聚乙二醇,硅土,崩解剂,如马铃薯淀粉,或可接受的增润剂,如月桂醇钠硫酸盐。片剂可以用制药学上公知的方法包衣。
口服液可以制成水和油的悬浮液,溶液,乳浊液,糖浆,也可以制成干品,用前补充水或其它合适的媒质。这种液体制剂可以包含常规的添加剂,如悬浮剂,山梨醇,纤维素甲醚,葡萄糖糖浆,凝胶,羟乙基纤维素,羧甲基纤维素,硬脂酸铝凝胶,氢化的食用油脂,乳化剂,如卵磷脂,山梨聚糖单油酸盐,阿拉伯树胶;或非水载体(可能包含可食用油),如杏仁油,油脂如甘油,乙二醇,或乙醇;防腐剂,如对羟基苯甲酸甲酯或丙酯,山梨酸。如需要可添加调味剂或着色剂。
本发明的又一具体实施方式中,提供一种预防和/或治疗肿瘤的方法,所述方法包括:其包括向受试者施用治疗有效剂量的上述多肽、上述药物组合物或上述药物制剂。
所述受试者是指已经是治疗、观察或实验的对象的动物,优选指哺乳动物,最优选指人。所述“治疗有效量”是指包括本发明化合物在内的活性化合物或药剂的量,该量可引起研究者、兽医、医生或其他医疗人员所追求的组织系统、动物或人的生物学或医学响应,这包括减轻或部分减轻受治疗的疾病、综合征、病症或障碍的症状。必须认识到,本发明所述活性成分的最佳给药剂量和间隔是由其性质和诸如给药的形式、路径和部位以及所治疗的特定哺乳动物等外部条件决定的,而这一最佳给药剂量可用常规的技术确定。同时也必须认识到,最佳的疗程,即同时化合物在额定的时间内每日的剂量,可用本领域内公知的方法确定。
本发明的又一具体实施方式中,提供上述多肽作为非治疗目的的肿瘤细胞抑制剂的用途。根据本发明,所述“非治疗目的”例如在体外抑制肿瘤细胞增殖;促进肿瘤细胞死亡。通过对肿瘤细胞(如A20淋巴瘤细胞)施加本发明的多肽,有利于研究肿瘤生长相关信号通路及基因表达交互作用,从而为进一步研究肿瘤相关性疾病提供原始材料并奠定基础。
本发明的又一具体实施方式中,提供一种体外抑制肿瘤细胞增殖的方法,所述方法包括给予体外培养的肿瘤细胞上述多肽、上述药物组合物或上述药物制剂。
以下通过实施例对本发明做进一步解释说明,但不构成对本发明的限制。应理解这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1
本实施例中使用基于9-芴甲氧羰基的固相多肽合成技术(Fmoc-SPPS)制备所有多肽片段。如无特殊说明,Fmoc-SPPS过程中使用的所有氨基酸均为Fmoc保护的D-型氨基酸。树脂的选择:根据碳末端基团的不同,选取不同的树脂。合成含有酰胺末端的多肽,选择Rink Amide Am树脂;合成含有酰肼末端的多肽,选择Fmoc-酰肼树脂。
合成多肽的规模一般为0.2mmol。如图1所示,以合成含有酰胺末端多肽为例,介绍多肽合成的基本流程。称取0.2mmol的Rink Amide Am(1倍当量)树脂,使用DMF和DCM交替清洗,然后用10mL的DMF/DCM混合溶液(1:1,v:v)在28℃下浸泡树脂2-3小时。脱除Fmoc的脱保护试剂为:含有20%哌啶(v:v)和0.1M HOBt的DMF溶液。脱除Fmoc的反应条件为:28℃下,第一次反应5分钟、第二次反应10分钟。缩合过程中,氨基酸的配比为Fmoc-D-型氨基酸:HCTU:DIPEA=4倍当量:3.8倍当量:8倍当量。缩合反应条件为:28℃下,反应1小时,每个氨基酸缩合两次。
完成所有氨基酸的缩合以后,将负载多肽的树脂转移到多肽合成管中,用DMF和DCM交替清洗,最后用DCM清洗树脂(4遍以上)并用油泵彻底抽干树脂。然后在树脂中加入TFA切肽试剂,切肽试剂的配比为TFA:苯酚:水:茴香硫醚:1,2-乙二硫醇=85:2.5:5:5:2.5(v:v:v:v:v)。28℃下反应2.5-3小时。收集切肽溶液,使用高纯氮鼓泡浓缩切割溶液。然后用提前预冷的无水乙醚沉淀多肽,通过离心机离心得到粗肽沉淀,弃掉上清,得到粗肽固体。再用预冷的无水乙醚反复清洗、离心粗肽两次以上。
得到的粗肽固体用含有0.1%TFA的乙腈和水混合溶剂溶解,通过真空干燥机冻干,得到固体形式的粗肽。此粗肽固体再次用含有0.1%TFA的乙腈和水混合溶剂溶解,通过半制备型反相高效液相色谱(RP-HPLC)分离纯化。纯化得到的多肽溶液直接通过真空干燥机冻干,得到固体形式的目标多肽。利用分析型反相高效液相色谱、高分辨质谱(ESI-MS),圆二色谱(CD)分析目标多肽的纯度、分子量、氨基酸序列和二级结构。固体形式的目标多肽放置在-20℃冰箱内密封保存,备用。
使用分析型反相高效液相色谱(RP-HPLC)和质谱(ESI-MS)对合成的目标多肽进行分析鉴定。反相高效液相色谱表明:目标多肽的纯度均高于95%。质谱数据表明,合成多肽的分子量均是正确的。WKW-121富含精氨酸,精氨酸的侧链胍基容易和三氟乙酸形成紧密结合,测定的质谱会在分子量的基础上加114以及114的倍数。结果见图2-7。
合成多肽的圆二色谱(CD)测定:
将多肽以1mg/mL的最终浓度溶解于PBS(1×,pH=7.4),加入到1mm的石英比色皿中,使用J-815CD光谱仪对多肽样品进行测试。仪器的参数设定为:波长170nm~260nm、带宽1nm、扫描速度100nm/分钟,每个样品重复测量3次并取平均值。所有校正和数据处理均使用Jasco标准分析程序,得到合成的D-型多肽的圆二色谱图。结果见图2-7。
肿瘤细胞增殖抑制实验:
用RPMI-1640(Gibco)培养基(含10%胎牛血清)培养A20细胞(淋巴瘤细胞,FengHuibio,CL0548)。将状态良好的细胞用此完全培养基稀释,以每孔100μL均匀接种在96孔板中,37℃,5%CO2培养箱环境中孵育过夜。用完全培养基分别将WKW-121、WKW-215、WKW-325、WKW-326、WKW-327和WKW-345母液(30mM)3倍梯度稀释为不同浓度(300μM、100μM、30μM、10μM、3μM、1μM),每个浓度设置3个复孔,每个孔体积为150μL。将配好的药物加入到孔内,培养箱中孵育4小时。4小时后每孔加入15μL CCK-8工作液,反应2小时后在450nm波长下读取OD值。根据OD值计算多肽药物对肿瘤细胞的IC50。
结果如图8所示,D-型阳离子抗肿瘤肽呈浓度依赖地抑制A20淋巴瘤细胞的增长,4小时的IC50值达到5μM左右,抗肿瘤活性非常高。
D-型阳离子抗肿瘤肽处理前后肿瘤细胞的形态学观察:
用MEM(Gibco)培养基(含10%胎牛血清)培养HepG2细胞(肝癌细胞,中国医学科学院基础医学研究所细胞资源中心)。将状态良好的细胞使用0.25%胰蛋白酶消化,用此完全培养基稀释细胞,以每孔500μL均匀接种在24孔板中,37℃,5%CO2培养箱环境中孵育过夜。用完全培养基将WKW-325母液(30mM)稀释为30μM,每个孔体积为500μL。将上述配好的药物加入到孔内,放到培养箱中孵育,在0分钟、15分钟、30分钟、60分钟、120分钟和240分钟用显微镜观察拍照。
结果如图9所示,WKW-325引发了HepG2肿瘤细胞形态的改变,细胞肿胀、破裂、漂浮并最终死亡。随着时间的推移,死亡细胞逐渐增多。
D-型阳离子抗肿瘤肽抑制肿瘤细胞增殖的时效测定:
将状态良好的A20细胞用完全培养基稀释,以每孔100μL均匀接种在96孔板中,37℃,5%CO2培养箱环境中孵育过夜。用完全培养基将WKW-121、WKW-215、LTX-315、WKW-325、WKW-326、WKW-327、和WKW-345母液(30mM)稀释为10μM,每个多肽设置3个复孔,每个孔体积为150μL。将配好的药物加入到孔内,培养箱中分别孵育4小时、12小时、24小时、36小时、48小时和72小时。到达时间后每孔加入15μL CCK-8工作液,反应2小时后在450nm波长下读取OD值,根据OD值计算各时间点对肿瘤细胞增殖的抑制率。
结果如图10所示,在8小时的时候,LTX-315在10μM时对A20淋巴瘤细胞抑制率仅有60%左右,而D肽抑制率在80%-90%之间,说明D-型多肽的抗肿瘤活性更高。此外,L肽LTX-315活性从12小时开始下降,到48小时时抗肿瘤活性为0。D-型阳离子抗肿瘤肽均能长时间稳定存在,发挥稳定的抗肿瘤作用,抗肿瘤活性可以保持36小时不变。
最后应该说明的是,以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。上述虽然对本发明的具体实施方式进行了描述,但并非对本发明保护范围的限制,所属领域技术人员应该明白,在本发明的技术方案的基础上,本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。
Claims (2)
1.一种D-型多肽在制备治疗肿瘤相关疾病的药物中的应用;
所述疾病为淋巴瘤;
所述多肽为如下氨基酸残基序列:
WKW-215H-kkwwkkw(dip)k-NHNH2;
所述多肽的合成方法包括,基于9-芴甲氧羰基的固相多肽合成法Fmoc-SPPS合成上述多肽;Fmoc-SPPS过程中使用的氨基酸为Fmoc保护的D-型氨基酸。
2.一种体外抑制肿瘤细胞增殖的方法,其特征在于,所述方法包括给予体外培养的肿瘤细胞药物;
所述药物为D-型多肽;
所述D-型多肽的氨基酸残基序列为:
WKW-215H-kkwwkkw(dip)k-NHNH2,
此方法为非治疗目的。
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