CN116751261B - 一种galr2激动剂及其应用 - Google Patents
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Abstract
本发明属于生物医药领域,具体涉及一种GALR2激动剂及其应用。本发明利用多肽库通过高通量筛选技术从近7.3万条多肽中找到2条能够在细胞水平激活GALR2受体且作用效果较强的多肽,可作为GALR2激动剂。所述GALR2激动剂多肽分子的氨基酸序列如SEQ ID NO.1和SEQ ID NO.2所示,为80氨基酸残基,第一个氨基酸与第80个氨基酸通过肽键成环。本发明提供的GALR2激动剂能够用于治疗神经性疾病,如抑郁症等。
Description
技术领域
本发明属于生物医药领域,具体涉及一种GALR2激动剂及其应用。
背景技术
甘丙肽(Galanin,GAL)最早是在1983年从猪的小肠分离提取的一种含29个氨基酸残基(在人类中含30个氨基酸残基)的多肽,人类甘丙肽的具体序列为Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-His-Ala-Val-Gly-Asn-His-Arg-Ser-Phe-Ser-Asp-Lys-Asn-Gly-Leu-Thr-Ser,根据N末端为甘氨酸(glycine)和C末端为丙氨酸(alanine)命名这种肽。甘丙肽是一种具有生物活性的神经肽,存在于中枢神经系统,也存在于周围神经系统,参与调控多项生理活动,如能量代谢、疼痛、癫痫以及睡眠。在中枢神经系统中,甘丙肽广泛分布于脑和脊髓各节段的神经细胞。
甘丙肽可以激活三种不同的G蛋白偶联受体(GPCRs):GALR1、GALR2以及GALR3。每种GALR在不同物种之间呈现高度的保守型,但在同一个物种中,各种GALR之间呈现很低的序列相似性。GALRs序列上的差异对于其偶联不同的G蛋白和下游信号系统很重要,造成了甘丙肽生理功能的多样性。GALR1阻断胰岛素分泌,GALR2启动神经发生,GALR3影响成瘾行为。GALRs能够识别相同的配体,但是激活不同的G蛋白亚型。GALR1和GALR3主要激活Gi/o信号通路,导致cAMP水平的下降,而GALR2激活Gq/11信号通路,导致细胞质中三磷酸肌醇(IP3)和钙离子的水平升高。
Spexin(SPX)是一种新发现的GALR同源神经肽,在中枢神经系统和外周组织中表达。SPX含有14个氨基酸并在C末端酰胺化,类似于甘丙肽的N末端序列。然而,这两种神经肽的受体识别谱是不同的,甘丙肽激活GALR1、GALR2和GALR3,SPX仅激活GALR2和GALR3。对于GALR2,SPX和甘丙肽具有相当的活性;而对于GALR3,SPX比甘丙肽具有更好的活性。
GALR1和GALR3介导促进抑郁的作用,GALR2介导抗抑郁的作用。甘丙肽的N端有生物活性的片段GAL(1-15),通过其受体GALR1-GALR2异聚体(heteromer),介导比GAL更强的调节抑郁效应。GAL(1-15)还可以通过GALR1-GALR2异聚体与5-羟色胺1A受体(5-HT1AR)相互作用形成GALR1-GALR2-5-HT1AR异聚体的方式,加强5-HT1AR激动剂的抗抑郁效果。此外,GAL及其受体还与去甲肾上腺素、神经肽Y、脑源性神经营养因子、多巴胺等递质或因子交互作用调节抑郁。
GALRs可以为某些疾病提供潜在的药物作用靶点,而各种受体选择性激动剂或拮抗剂则可以作为潜在的主要药物成分。已知GALR2在焦虑、抑郁、食欲调控和疼痛调控中表现出具体的功能,GALR2激动剂可以用于防护或治疗一系列的中枢神经系统疾病,并且最小化或避免因GALR3和/或GALR1的激活而产生的潜在副作用。目前已经报道,刺激GALR2在多发性硬化(multiple sclerosis,MS)和阿尔茨海默氏症的情形中具有神经保护性,并具有抗焦虑、抗抑郁和抗惊厥效果。
多年来已报道了几种GALR2特异性激动剂,其通过在甘丙肽的N末端和/或C末端修饰而产生。其中,发现M1145和M1153显示GALR2选择性,与GALR1和GALR3相比,它们对GALR2具有50至100倍的结合偏好;然而,在高浓度下,这些激动剂保留了对GALR1和GALR3的显著亲和性。
专利CN107531803B描述了环状甘丙肽类似物的对不同GALRs的激活或抑制作用,但其没有特异性且作用效果不明确。专利CN108348570B描述了基于Spexin的GALR2激动剂。
目前针对GALR2激动剂的研究较少,且研究出来的激动剂有特异性不够强的缺点,当激动剂的剂量较大时会激活GALR1和GALR3,因此研究能特异性针对GALR2的激动剂对治疗GALR2介导的相关疾病具有重要作用。
发明内容
为解决现有技术的不足,本发明提供一种GALR2激动剂及其应用。
一方面,本发明提供一种GALR2激动剂,其特征在于,所述GALR2激动剂为多肽分子。
进一步地,所述GALR2激动剂,其特征在于,GALR2激动剂多肽分子为环肽,其中氨基酸序列的第一个氨基酸和最后一个氨基酸通过肽键成环。
进一步地,所述GALR2激动剂,其特征在于,GALR2激动剂多肽分子的长度为80氨基酸,第1个氨基酸和第80个氨基酸通过肽键成环。
进一步地,所述GALR2激动剂,其特征在于,GALR2激动剂多肽分子的氨基酸序列如SEQ ID NO.1和SEQ ID NO.2所示,具体氨基酸序列如表1所示,第1个氨基酸和第80个氨基酸通过肽键成环。
所述氨基酸序列是利用多肽库通过高通量筛选,从近7.3万条80环肽中找到8条能够在细胞水平激活GALR2受体的多肽,其中,SEQ ID NO.1和SEQ ID NO.2所示的多肽对GALR2受体激活作用最强。
表1本发明的氨基酸序列
另一方面,本发明提供一种多核苷酸分子,所述多核苷酸分子能够编码上述的激动剂多肽分子。
进一步地,本发明提供一种多核苷酸分子,所述多核苷酸分子能够编码上述SEQID NO.1或SEQ ID NO.2所示的激动剂多肽分子。
另一方面,本发明还提供了一种药物组合物,其包含安全有效量范围内的本发明的激动剂多肽分子,以及药学上可接受的载体。
另一方面,本发明还提供了所述的本发明的激动剂、多核苷酸分子和药物组合物用于制备预防和/或治疗GALR2受体介导的疾病的药物上的应用。
进一步地,所述与GALR2受体介导的疾病为神经性疾病。
进一步地,所述神经性疾病为抑郁症。
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。
在通篇说明书中使用的术语“GALR2激动剂”表示一种能够激活GALR2而导致在细胞中引发反应的物质,但是该物质不激活(或以较低的活性激活)GALR1和/或GALR3。鉴定一种化合物是否为甘丙肽受体激动剂的方法为本领域所熟知。与结合和激活GALR1相比,GALR2特异性激动剂优先结合和激活GALR2的选择性至少为30倍以上,优选地选择性较GALR1高50倍以上,更优选地选择性较GALR1高100倍以上。与结合和激活GALR3相比,GALR2特异性激动剂优先结合和激活GALR2的选择性也至少为30倍以上,优选地选择性较GALR3高50倍以上,更优选地选择性较GALR3高100倍以上。
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、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。
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。
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。
与现有技术相比,本发明具有以下优点:
(1)本发明所用的多肽库相较于传统的多肽筛选具有高效性,且库内各化合物均为独立生产,均经过质谱鉴定和精确称量,保证了筛检的准确和稳定,避免了传统的噬菌体库等混合化合物库的失真(实际库容远低于理论值)问题。库内化合物可进行混合或单一筛选,筛选方式多样灵活,避免了纯混合物库筛选时各组分的互相干扰。
(2)本发明的提供了一种作为GALR2激动剂的多肽,本发明提供的多肽可以特异性激活GALR2受体,治疗GALR2介导的相关疾病,尤其是抑郁症。
附图说明
图1为实施例1中不同浓度的SEQ ID NO.1和SEQ ID NO.2激活GALR2的CHO细胞引起钙流变化的结果。
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。
实施例1
关键试剂:多肽库(自制)、过表达GALR2的CHO细胞(Chinese hamster ovarycell,中国仓鼠暖巢细胞)系,简写为CHO-K1/GALR2细胞、Calcium5 Assay Kit。
1.高通量筛选过程
1.1CHO-K1/GALR2细胞的培养
1.1.1细胞复苏:从液氮罐中取出细胞,在37℃水浴锅中快速解冻细胞。将细胞移至15mL离心管中,缓慢加入9mL预热的解冻培养基,800转离心5分钟,移除上清培养基。用5mL解冻培养基重悬细胞,转移至T25培养瓶中,放于37℃,5%CO2的培养箱中培养。细胞复苏第二天更换培养液为生长培养基。
1.1.2细胞传代:当细胞长满培养瓶80~90%,先用DPBS润洗细胞,再用0.25%胰酶消化细胞;收集细胞悬液至离心管中,800转离心5分钟,移除上清培养基;加入6-8mL新鲜生长培养基,重悬细胞,按照1:3~1:10的比例进行传代,放于37℃,5%CO2的培养箱中培养。传代后每2-3天进行换液。
1.1.3细胞冻存:当细胞长满培养皿80~90%,先用DPBS润洗细胞,再用0.25%胰酶消化细胞;收集细胞悬液至离心管中,800转离心5分钟,移除上清培养基;用冻存培养基重悬细胞,进行细胞计数,将细胞稀释到2~3×106/mL。每个冻存管分装1mL细胞冻存悬液。将分装好细胞的冻存管放入冻存盒中,将冻存盒放入-80℃冰箱过夜保存后,将冻存管转移到液氮罐中。
1.2CHO-K1/GALR2细胞铺板
测试前24小时,将培养瓶中的CHO-K1/GALR2细胞经过0.25%胰酶消化处理并悬浮于细胞培养液中,以每孔10000个细胞的密度使用分液仪加至黑色透明底384孔板中培养,每孔25μL,37℃,5%CO2过夜培养。
1.3Calcium5 assay kit找工作液准备
测试当日,将Calcium5试剂盒中的组分A溶解于钙染料缓冲液(Loadingbuffer)并加入250mM丙磺舒配制成含有5mM丙磺舒的钙染料溶液。
吸掉培养基后,在细胞培养板中每孔加入50μL钙染料溶液,放置于室温孵育2小时。
1.4多肽库溶解及稀释
1.4.1多肽库溶解
1.4.2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。
1.4.3溶解后的96深孔板多肽放于离心机4000rpm离心2~3分钟。
1.5多肽库稀释
将溶解后用工作站转移至384孔板中,用Loading buffer稀释至10μM。
1.6FLIPR检测
细胞加入Calcium5染料2小时后,将细胞培养板取出并避光放置于室温10分钟,然后和多肽溶液板一起放入FLIPR仪器,进行检测。设置Galanin(1-30)为激动剂阳性对照,以M35为抑制剂阳性对照。
2不同80环肽的确认
2.1按照步骤1的过程对初筛到的多肽进行确认。
2.2按照步骤1的过程检测活性多肽的EC50值
检测当天,将将活性多肽母液用1×Loading buffer(含20mM HEPES)(pH7.4)稀释到50μM(5×浓度),再以3倍稀释8~10个梯度,每个浓度做复孔,测试活性多肽的EC50值。
3实验结果
3.1通过高通量筛选,从近7.3万条80环肽中找到8条能够在细胞水平激活GALR2受体的多肽,测试其对GALR2受体激活作用的浓度响应曲线,其中,SEQ ID NO.1和SEQ IDNO.2对GALR2受体激活作用最强,EC50值分别为0.82μM和1.89μM。
Claims (4)
1.一种GALR2激动剂,其特征在于,所述GALR2激动剂为如SEQ ID NO.1或SEQ ID NO.2所示的氨基酸序列的多肽,且所述氨基酸序列的第一个氨基酸和第80个氨基酸通过肽键成环。
2.一种多核苷酸分子,其特征在于,能够编码权利要求1所述的多肽。
3.一种药物组合物,其包含安全有效量范围内的权利要求1所述的多肽,以及药学上可接受的载体。
4.权利要求1所述的GALR2激动剂、权利要求2所述的多核苷酸分子或权利要求3所述的药物组合物在制备预防和/或治疗与GALR2受体相关的疾病的药物上的应用,所述与GALR2受体相关的疾病为抑郁症。
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