CN116715735B - 抑制il-6与il-6r结合的多肽及其应用 - Google Patents
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Abstract
本发明属于生物医药工程领域，公开了一种抑制IL‑6与IL‑6R结合的多肽及其应用，所选多肽为SEQ ID NO.1‑SEQ ID NO.7中的任意一个。SEQ ID NO.1‑SEQ ID NO.7是应用多肽信息压缩技术，在大型实体多肽库利用TR‑FRET筛选方法筛选到的多肽。本发明的多肽能够抑制IL‑6与IL‑6R结合，从而可用于治疗与IL‑6通路相关的疾病，如自身免疫性疾病，慢性炎症，骨质疏松症、银屑病，癌症。
Description
技术领域
本发明属于生物医药工程领域，具体涉及一种抑制IL-6与IL-6R结合的多肽及其应用。
背景技术
白细胞介素-6(Interleukin-6)，简称白介素-6(IL-6)，是一种具有促炎和抗炎双重特性的多功能细胞因子。IL-6由多种细胞产生，如巨噬细胞、T细胞、B细胞、成纤维细胞、骨髓基质细胞及内皮细胞等，并且在垂体前叶中发现滤泡-星状细胞也可产生IL-6。IL-6在健康人体内的含量极低，仅在皮克范围，但是几乎在所有的病理状态下，IL-6表达会瞬时上调。
白介素-6受体(IL-6R)是IL-6的受体，属于I型细胞因子受体家族，第3亚家族。IL-6R结构包含两个抗平行纤维连接蛋白III型结构域，每个结构域包含7个β折叠，在两个结构域之间，有一个倾斜。IL-6R是由该蛋白和白介素6信号转导子(IL6ST/GP130/IL6β)组成的蛋白复合物，该受体亚基也被许多其他细胞因子共享。另外，IL-6Rα(IL-6RA)是IL-6R的主要功能α亚基，它也是其他白细胞介素受体的组成部分。IL-6RA也是一种I型跨膜糖蛋白，通过与CD130形成复合物而调节IL-6的生物活性。IL-6Rα有2种亚型，包括膜结合的IL-6R(mIL-6R)和可溶性IL-6R(sIL-6R)。
IL-6与IL-6受体IL-6Rα(CD126)及gp130形成异六聚体[2(IL-6-IL-6R-gp130)]，激活胞内信号转导途径，主要包括JAK-STATs途径和Ras-MAPK途径，激活的STATs作用于目的基因转录起始因子，MAPK则可调节C-Fos、C-Myc及C-Jun等表达。
广泛熟知的IL-6的信号传导途径有两种，分别是经典信号途径和转移信号途径。一直到最近，第三种IL-6信号传导途径：聚类信号途径被发现。以下简单的介绍一下三种信号传导路径：(1)经典信号途径(ClassicSignaling)：IL-6和位于细胞膜上的IL-6R结合，进而招募gp130(一种跨膜蛋白，细胞因子受体)，介导下游信号传导；(2)转移信号途径(Trans-Signaling)：可溶型IL-6R和IL-6形成复合物，再通过gp130介导转移信号途径，其中sgp130(可溶性的gp130)对该信号路径有很强的的抑制作用；(3)聚类信号途径(Trans-Presentation)：IL-6在DC细胞内和IL-6R结合，然后被运送至细胞膜上，该复合物与膜细胞的gp130结合，进而激活该信号途径。
IL-6一般是通过和IL-6R相结合，从而在人体内调节多项活动，包括调控免疫系统、神经系统、细胞生长和新陈代谢等。IL-6的促炎作用和抗炎作用之间的失衡会导致各种自身免疫性疾病、慢性炎症和骨质疏松症、银屑病，其过度产生与各种形式的癌症有关。
目前，以IL-6信号通路为靶点的生物技术药物已成为研发热点。国际市场方面，FDA已经批准了三款上市药物，分别为Tocilizumab(托珠单抗，商品名：Actemra/RoActemra)、Siltuximab(商品名：Sylvant)、Sarilumab(商品名：Kevzara)。国内方面，上市的仅有Tocilizumab一款。
使用Tocilizumab和Sarilumab会产生胰腺炎、胃肠道穿孔等不良反应，对于Sirukumab，FDA拒绝批准该药物用于RA，原因是考虑到在III期研究中Sirukumab和安慰剂组的全因死亡率不平衡，尽管尚不清楚这种不平衡是真正的安全信号还是研究设计的结果。需要更多的研究来进一步确定Sirukumab的安全性。
虽然现有技术已经提供了通过调控IL-6信号转导途径来治疗IL-6相关疾病的药物，但其具有一定的不安全性且种类较少，在本领域仍有对抑制IL-6与IL-6R结合的多肽药物的需求。
发明内容
为解决现有技术中的不足，本发明提供了一种抑制IL-6与IL-6R结合的多肽及其应用。
一方面，本发明提供一种抑制IL-6与IL-6R结合的多肽或其药学上可接受的盐，其特征在于所述多肽为利用多肽信息压缩技术从多肽库中筛选的80环肽，其中，80环肽氨基酸序列的第1个氨基酸和第80个氨基酸通过肽键成环。
上述的80环肽为通过高通量筛选技术检测多肽对IL-6与IL-6R结合的抑制率从多肽库中筛选，该环肽从多肽库中筛选过程包括多肽库溶解及稀释，利用TR-FRET筛选方法根据多肽对IL-6与IL-6R结合的抑制率结果。
进一步地，所述80环肽的氨基酸序列如SEQ ID NO.1所示，具体氨基酸序列如表1所示，其中SEQ ID NO.1的第一个氨基酸和第80个氨基酸通过肽键成环。
另一方面，本发明提供一种抑制IL-6与IL-6R结合的多肽或其药学上可接受的盐，其特征在于所述多肽分子是对SEQ ID NO.1进行分析和拆解得到的环状肽或线性肽。对80环肽SEQ ID NO.1进行氨基酸序列的分析和拆解工作，能够设计出10～80不同的氨基酸序列的线性肽或者环肽。
进一步地，所述多肽分子是对SEQ ID NO.1进行分析和拆解得到的环状肽，其中，所述环状肽的氨基酸序列的第1个氨基酸与最后一个氨基酸通过肽键成环。
进一步地，所述多肽分子是对SEQ ID NO.1进行分析和拆解得到的线性肽。
进一步地，所述拆解之后得到的环状肽或线性肽具有20～35个氨基酸。
进一步地，所述拆解之后得到的环状肽或线性肽具有30个氨基酸。
进一步地，所述拆解之后得到的氨基酸序列可选自SEQ ID NO.2至SEQ ID NO.7，具体氨基酸序列如表1所示。
表1本发明氨基酸序列
另一方面，本发明还提供一种多核苷酸分子，所述多核苷酸分子包含能够编码上述多肽分子的一个或两个的多核苷酸。
另一方面，本发明还提供一种药物组合物，它含有(a)安全有效量的本发明多肽或其药学上可接受的盐或溶剂化物；以及(b)药学上可接受的载体或赋形剂。
用于本发明的方法中的药物组合物可含有任何药学上可接受的赋形剂。赋形剂的实例包括但不限于淀粉、糖、微晶纤维素、稀释剂、粒化剂、润滑剂、粘合剂、崩解剂、湿润剂、乳化剂、着色剂、释放剂、包覆剂、甜味剂、调味剂、芳香剂、防腐剂、抗氧化剂、塑化剂、胶凝剂、增稠剂、硬化剂、凝固剂、混悬剂、表面活性剂、保湿剂、载体、稳定剂、以及它们的组合。
在各种实施方案中，本发明的药物组合物可被配制以通过任何施用途径递送。这可包括例如气雾剂、经鼻、口服、经粘膜、经皮、胃肠外或经肠。
“胃肠外”是指通常与注射相关的施用途径，包括眶内、输注、动脉内、囊内、心内、真皮内、肌肉内、腹膜内、肺内、脊柱内、胸骨内、鞘内、子宫内、静脉内、蛛网膜下、囊下、皮下、经粘膜或经气管。通过胃肠外途径，组合物可呈用于输注或用于注射的溶液或混悬液形式，或呈冻干粉剂形式。通过胃肠外途径，组合物可呈用于输注或用于注射的溶液或混悬液形式。通过经肠途径，药物组合物可呈片剂、凝胶胶囊、糖包衣片剂、糖浆、混悬液、溶液、粉剂、颗粒剂、乳液、允许控制释放的微球体或纳米球体或脂质囊泡或聚合物囊泡形式。通常，组合物通过注射施用。用于这些施用的方法为本领域技术人员所知。
本发明的药物组合物可含有任何药学上可接受的载体。举例来说，载体可为液体或固体填充剂、稀释剂、赋形剂、溶剂、或囊封物质、或它们的组合。
另一方面，本发明还提供上述的多肽分子或其药学上可接受的盐、多核苷酸分子或药物组合物在制备用于治疗与IL-6通路相关的疾病的药物上的应用。
进一步地，制备的用于治疗与IL-6通路相关的疾病的药物可以与一种或多种其他IL-6药物同时、分开或顺序的组合使用。
进一步地，与IL-6通路相关的疾病可以选自自身免疫性疾病，慢性炎症，骨质疏松症、银屑病，癌症。
术语
除非本文中另外定义，否则本专利申请中所用的科学及技术术语应具有一般本领域技术人员通常所理解的含义。
本文所用“多肽库”是湖南中晟全肽生化有限公司利用PICT(PeptideInformation Compression Technology)专利技术，该技术利用生物学手段对多肽信息进行压缩，可将多个多肽的信息集成进一个多肽，从而实现以相对较小的库容包含较大的多肽信息量；通过PICT技术构建含有近73000条80个氨基酸的环肽库。其具体构建方法可以参见专利CN201580081102.3和专利CN201780089941.9。
与现有技术相比，本发明有益效果主要体现在：
(1)本发明筛选多肽为湖南中晟全肽生化有限公司的多肽信息压缩技术，可以构建完成包含近五亿种多肽信息的超大型多肽实体库，显著加快了发现抑制IL-6与IL-6R结合的多肽的进程，降低多肽新药研发成本。
(2)本发明提供了一系列抑制IL-6与IL-6R结合的多肽。本发明提供的多肽可以阻断人IL-6与IL-6R的相互作用，减少IL-6引起的疾病，可用于治疗和/或预防多种自身免疫疾病，慢性炎症，骨质疏松症、银屑病，癌症。
附图说明
图1为实施例1中不同浓度的SEQ ID NO.1对抑制IL-6与IL-6R结合的结果。
图2为实施例2中不同浓度的SEQ ID NO.2至SEQ ID NO.5对抑制IL-6与IL-6R结合的结果。
图3为实施例2中不同浓度的SEQ ID NO.6至SEQ ID NO.7对抑制IL-6与IL-6R结合的结果。
具体实施方式
为更好理解本发明，下面结合附图和实施例对本发明作进一步的详细说明，但不以任何方式对本发明加以限制，基于本发明教导所作的任何变换或改进，均落入本发明的保护范围。
本发明需要用到的试剂如表2所示。
表2本发明使用试剂
| 名称 | 厂家 | 货号 |
| Huam IL-6R alpha FcTag | ACRO | ILR-H5259 |
| IL-6 Biotinylated | ACRO | ILR-H8218 |
| IL-6 Neutralizing Antibody | Singo Biological | 10395-R508 |
| Streptavidin-Eu(SA-Eu) | ATTBio | 16925 |
| Anti-human Fc antibody-Alexa Fluor647 | Jackson | 109-605-170 |
实施例1
抑制IL-6与IL-6R结合的80环肽的筛选。
确定好靶标后应用TR-FRET筛选方法利用湖南中晟全肽生化有限公司的大型实体多肽库筛选出一定数量可能抑制IL-6与IL-6R结合的80环肽。
(1)多肽库的溶解：将多肽库96孔深孔板放于离心机4000rpm离心2～3分钟。用自动分液仪向96孔深孔板中加入200μL/孔超纯水中。用硅胶盖密封，放置95℃水浴5分钟。注：此时多肽浓度约为：50μM。溶解后的96深孔板多肽放于离心机4000rpm离心2～3分钟。
多肽库稀释：将溶解后的多肽用工作站转移至384孔板中，用loading buffer(Tris-Hcl缓冲液，pH7.4)稀释至10μM。
(2)应用TR-FRET筛选方法对大型实体多肽库进行验证。在384孔板中依次加入不同浓度的80环肽、10nM IL-6和10nM IL-6R，以及荧光供体Streptavidin-Eu和荧光受体Goat Anti-Human IgG Fc-Alexa Fluor647，室温孵育2小时后，检测TR-FRET信号。
(3)增加阳性对照：不含环肽，只含10nM IL-6和10nM IL-6R，以及荧光供体Streptavidin-Eu和荧光受体Goat Anti-Human IgG Fc-Alexa Fluor647，目的是检测多肽是否具有抑制IL-6与IL-6R结合的功能。
(4)增加阴性对照：不含多肽，只含10nM IL-6和10nM IL-6R其中一个组分或者2者都不含，以及荧光供体Streptavidin-Eu和荧光受体Goat Anti-Human IgG Fc-AlexaFluor647，目的是排除实验中会影响实验结果的变量。
(5)通过对初筛到的环肽进行重复实验确认，最终确定初筛的的80环肽库中筛选到的SEQ ID NO.1样品抑制率较高，后进行浓度依赖性验证。计算抑制率，用graphpad作图。实验结果如表3所示，不同浓度的环肽对抑制IL-6与IL-6R结合的结果曲线如图1所示。
表3 80环肽SEQ ID NO.1的筛选结果
| SEQ ID NO. | IC50(μM) |
| 1 | 1.96 |
从实验结果可以看出，本发明的80环肽对IL-6与IL-6R结合有抑制作用，且抑制作用随着80环肽的浓度增大而增强。
实施例2
抑制IL-6与IL-6R结合的线性肽的筛选。
应用内部环肽解压缩技术，对实施例1中筛选出的80环肽SEQ ID NO.1的氨基酸序列进行分析和拆解工作，设计出10-80条不同氨基酸序列的线性肽或者环肽。
对80环肽SEQ ID NO.1进行解压缩，得到一组可能具有抑制IL-6与IL-6R结合的线性肽，按照实施例1的实验步骤对该组线性肽进行复筛处理，确定SEQ ID NO.2至SEQ IDNO.9多肽对IL-6与IL-6R结合有抑制作用，计算抑制率后用graphpad作图。实验结果如表4所示，不同浓度的环肽对抑制IL-6与IL-6R结合的结果曲线如图2和图3所示。
表4线性肽的筛选结果
| SEQ ID NO. | IC50(μM) |
| 2 | 0.12 |
| 3 | 0.14 |
| 4 | 0.17 |
| 5 | 1.10 |
| 6 | 0.29 |
| 7 | 1.72 |
从实验结果可以看出，本发明的线性肽对IL-6与IL-6R结合有抑制作用。
Claims (4)
1.一种抑制IL-6与IL-6R结合的多肽或其药学上可接受的盐，其特征在于，所述多肽为利用多肽信息压缩技术从多肽库中筛选的80环肽，其中，所述80环肽的氨基酸序列如SEQID NO.1所示，80环肽氨基酸序列的第1个氨基酸和第80个氨基酸通过肽键成环。
2.一种抑制IL-6与IL-6R结合的多肽或其药学上可接受的盐，其特征在于所述多肽是对SEQ ID NO.1进行分析和拆解得到的线性肽，所述线性肽的序列选自SEQ ID NO.2至SEQID NO.7中的一种。
3.多核苷酸分子，其特征在于，所述多核苷酸分子包含能够编码权利要求1或2所述的多肽的一个或两个的多核苷酸。
4.一种药物组合物，它含有(a)安全有效量的权利要求1或2所述的多肽或其药学上可接受的盐；以及(b)药学上可接受的载体或赋形剂。
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