CN116731117A - Kim-1靶向性多肽及其在急性肾损伤中肾靶向性的应用 - Google Patents
Kim-1靶向性多肽及其在急性肾损伤中肾靶向性的应用 Download PDFInfo
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Abstract
本发明公开了一种Kim‑1靶向性多肽及其在急性肾损伤中肾靶向性的应用,属于生物药学领域。本发明的Kim‑1(肾损伤分子‑1)靶向性多肽的氨基酸序列如SEQ ID NO.1所示,其在急性肾损伤发生,Kim‑1表达量升高后,快速与Kim‑1结合,实现在急性损伤的肾脏中高度富集。本发明的Kim‑1靶向性多肽具有制备急性肾损伤诊断试剂、急性肾损伤治疗药物的应用。
Description
技术领域
本发明属于生物技术药物领域,具体涉及Kim-1靶向性多肽及其在急性肾损伤中实现受损肾脏靶向性富集的应用。
背景技术
急性肾损伤(AKI)是一种突然发生的肾脏功能异常,通常在数小时或数天内发生,可能是由各种原因引起的肾脏血流量减少、肾小管损伤或肾小球滤过率下降等肾功能急剧下降的临床综合征。老年人、慢性肾脏疾病患者、心血管疾病患者、糖尿病患者、高血压患者等人群更容易发生急性肾损伤,在重症监护病房中急性肾损伤的发病率高达20-50%,全球年发病人数达1.33亿,死亡人数约200万且呈上升趋势。
Kim-1是一种与AKI密切相关的生物标志物,也被称为T cell Ig and mucindomain-containing protein 1(TIM-1),它是一种跨膜蛋白,在AKI的早期阶段被肾小管上皮细胞表达。当急性肾损伤发生时,肾小管上皮细胞受到损伤,Kim-1表达水平显著升高。多项研究表明,Kim-1不但可以作为早期检测急性肾损伤的生物标志物,而且其表达水平能够反映急性肾损伤的损伤程度和预后指标。
急性肾损伤发病突然,临床表现为少尿、无尿、水肿、高血压、贫血、恶心、呕吐等,并不具有特异性,其诊断方式多通过血清肌酐、尿素氮、肌酸激酶等指标,但这些指标均具有一定的滞后性,而目前对急性肾损伤的治疗方法仍然十分有限,虽然有一些药物和治疗策略被用于治疗AKI,但由于病因和病程的多样性,缺乏明确的有效的治疗方法。因此发现具有时效性的诊断方法和有效的治疗手段是治疗急性肾损伤的关键。
肾脏是体内主要的代谢器官之一,在药物代谢和排泄中扮演着重要的角色。由于药物在肾小管内被重吸收和随着尿液排出体外的过程,药物在肾脏中的浓度难以达到治疗所需的水平。此外,药物在肾脏中的分布还受到肾脏结构和功能的影响。肾脏的组织结构特殊,包括肾小球、肾小管和肾间质等不同的部位,每个部位对药物的分布和代谢都有不同的影响。药物需要通过肾小管上皮细胞和肾小管间质才能进入肾小管腔,而这些细胞和间质的结构和功能也会影响药物在肾脏中的分布和代谢。因此,药物在肾脏中的分布和代谢受到多方面的影响,难以在肾脏中富集达到治疗浓度。因此发现能够使药物在肾脏蓄积的无害性靶头是有效治疗急性肾损伤的关键。
发明内容
本发明的目的在于解决现有技术存在的药物与肾脏特异性受体结合能力弱以及肾脏蓄积量较低的技术问题,提供一种Kim-1靶向多肽及其在急性肾损伤中肾靶向性的应用。本发明的靶向性多肽能够与Kim-1特异性结合,在急性肾损伤发生后,快速富集于肾脏,由此解决现有技术难以实现药物的肾靶向性递送的技术问题。
本发明的目的通过下述技术方案实现:
本发明第一方面,提供了一种Kim-1靶向性多肽,其含有SEQ ID NO.1所示的氨基酸序列,或者含有由SEQ ID NO.1所示的氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有序列SEQ ID NO.1活性的氨基酸序列。
本发明另一方面,提供了一种检测急性肾损伤的试剂,其包含上述靶向性多肽,以及与靶向性多肽偶联的成像剂,所述的成像剂优选为造影剂或荧光标记物。
本发明另一方面,提供了上述靶向性多肽或检测急性肾损伤的试剂在制备急性肾损伤靶向诊断试剂中的应用。
本发明另一方面,提供了上述靶向性多肽在制备治疗急性肾损伤的药物中的应用。一种治疗急性肾损伤的药物组合物,包含上述靶向性多肽,或者包含上述靶向性多肽以及与靶向多肽偶联的急性肾损伤治疗药物。进一步地,所述的药物组合物还包含药学上可接受的赋形剂。
本发明另一方面,提供了一种上述药物组合物的制备方法,其步骤包括将上述靶向性多肽或偶联有急性肾损伤治疗药物的靶向性多肽与至少一种药学上可接受的赋形剂混合。
总体而言,本发明与现有技术相比,具有下列有益效果:
(1)本发明基于分子模拟技术从与Kim-1存在相互作用的蛋白中,发现Kim-1 Ig V区域与互作蛋白存在相互作用的氨基酸残基。通过对Kim-1 Ig V区域的蛋白结合口袋模拟分析,设计了一系列Kim-1靶向性多肽,通过分子模拟技术将多肽分别与人、鼠来源的Kim-1Ig V区域进行结合,按吉布斯自由能强弱进行筛选,同时结合多肽的长度,稳定性预测,发现氨基酸序列SEQ ID NO.1所示的多肽能够以较低的吉布斯自由能与人、鼠来源的Kim-1发生结合,更适合作为急性肾损伤诊断、治疗制剂的开发。
(2)本发明发现从人工合成的Kim-1靶向性多肽在急性肾损伤肾脏中体现出极佳的肾脏靶向性。本发明发现氨基酸序列如SEQ ID NO.1所示的多肽能够在细胞中与人源Kim-1表现出共定位,在顺铂诱导的急性肾损伤小鼠中,氨基酸序列如SEQ ID NO.1所示的多肽能够显著蓄积在损伤小鼠肾脏。
(3)本发明发现Kim1敲除对多肽靶向效率的影响。本发明发现SEQ ID NO.1所示的氨基酸序列在顺铂诱导的急性肾损伤的Kim1 KO小鼠中,肾脏的高富集特性消失,表明氨基酸序列如SEQ ID NO.1所示的多肽是特异性靶向Kim-1的多肽。
另外,人工合成的天然氨基酸残基的多肽未显示出细胞毒性,故本发明的氨基酸序列如SEQ ID NO.1所示的靶向性多肽可以为制备急性肾损伤诊断、治疗药物奠定了基础,为开发出更具时效性的诊断制剂和靶向治疗药物提供了新思路。
附图说明
图1是本发明的靶向性多肽的序列图。
图2是本发明的靶向性多肽与人源Kim-1 Ig V区域的结合模式图。
图3是本发明的靶向性多肽与鼠源Kim-1 Ig V区域的结合模式图。
图4是本发明的靶向性多肽在顺铂刺激下的HK-2细胞膜上与Kim-1的共定位。
图5是本发明的靶向性多肽在健康或顺铂诱导的肾损伤小鼠的肾脏富集强度图。
图6是本发明靶向性多肽对HK-2活性的影响和对顺铂刺激下的HK-2的保护作用。
图7是本发明KIM-1靶向性多肽在健康或顺铂诱导的肾损伤Kim1 KO小鼠中的肾富集强度图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例及附图,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。此外,下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
本发明中的一种靶向性多肽,所述靶向性多肽含有从N末端到C末端的由SEQ IDNO.1所示的氨基酸序列;或者所述靶向性多肽含有从N末端到C末端的由SEQ ID NO.1所示的氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有序列SEQ ID NO.1活性的氨基酸序列。
所述靶向性多肽能特异性结合Kim-1,实现在损伤肾脏的高浓度富集。
本发明中的靶向性多肽结构如图1所示。
本发明中的氨基酸指天然氨基酸,除非与此相反地规定,否则一般性或以名称具体指出的任何氨基酸都包括D和L立体异构体,如果其结构允许这样的立体异构体形式。天然氨基酸包括丙氨酸(Ala)、天冬氨酸(Asp)、天冬酰胺(Asn)、精氨酸(Arg)、半胱氨酸(Cys)、谷氨酸(Glu)、谷氨酰胺(Gln)、甘氨酸(Gly)、组氨酸(His)、亮氨酸(Leu)、异亮氨酸(Ile)、赖氨酸(Lys)、甲硫氨酸(Met)、苯丙氨酸(Phe)、脯氨酸(Pro)、丝氨酸(Ser)、苏氨酸(Thr)、色氨酸(Trp)、酪氨酸(Tyr)和缬氨酸(Val)。
本发明的靶向性多肽能够与Kim-1 Ig V区域有较强亲和力,在受到急性损伤,大量表达Kim-1的肾脏上高度富集,多肽序列如表1所示。
表1靶向性多肽的氨基酸序列
本发明实施例中使用的C57BL/6小鼠购买于湖北省疾控中心实验动物研究中心,本发明的多肽按照标准的固相多肽合成步骤,合成多肽采用RP-HPLC,C18色谱柱进行纯化。多肽合成方向为C端到N端,首先是树脂的溶胀,使用的是氯树脂;然后将第一个氨基酸连在树脂上;第一个氨基酸连在树脂上需要double coupling即氨基酸与树脂反应两次;随后将氨基酸氨基端Fmoc基团的去除(脱保护);然后是肽链延长的过程,经过重复缩合、洗涤、脱保护、洗涤、缩合的步骤,按多肽序列从C端向N端逐一连接氨基酸,直至合成所需肽片段;随着树脂去溶胀及干燥、将肽段切除和粗肽的纯化,即可合成表1中的TKP 5。
本发明Kim-1特异性结合的肾靶向多肽TKP 5,能够在顺铂诱导的急性肾损伤小鼠的Kim-1高表达肾脏中大量蓄积。
实施例1
本实施例涉及多肽的虚拟筛选。
具体操作:将与Kim-1存在相互作用的蛋白,与Kim-1的Ig V区域进行分子对接,根据对接结果确定其相互作用区段,根据存在相互作用的氨基酸残基,设计靶向性多肽,将设计的靶向性氨基酸,再次与人源、鼠源Kim-1 Ig V区域进行对接,按吉布斯自由能从低到高,得到需要验证靶向性的多肽TKP 5。多肽TKP 5与人源、鼠源Kim-1 Ig V区域的结合模式图如图2、3所示。
另外,选取了现有技术报道的结合效果最强的LTH多肽与本发明TKP 5多肽进行比较。利用Kim-1 Ig V区域进行分子对接,结合能和结合模式结果如表2所示,LTH多肽与人源、鼠源Kim-1 Ig V区域存在相互作用。相较于LTH多肽,TKP 5多肽与Kim-1 Ig V区域具有更强的结合。
表2Kim-1 Ig V与TKP5、LTH多肽的结合能
实施例2
本发明的TKP 5与人肾小管上皮细胞HK-2中表达的Kim-1有明显的共定位。
具体操作:将FITC 2mg和TKP 5多肽1mg精确称量后,加入800μL 0.1M NaHCO3溶液,4摄氏度旋转过夜反应。反应完成后,将混合物转移至1kD透析袋中,使用PBS透析至透析液中无颜色,得到FITC标记荧光肽段。然后将HK-2细胞按7000个/孔,铺于有盖玻片的24孔板中。接着,给予5μg/mL的顺铂刺激,刺激时间为24小时。刺激后,给予1μmol/L的FITC标记荧光肽段,并在2小时后弃去培养基,用PBS清洗2次。再使用4%的多聚甲醛固定细胞15min,PBS清洗后加入0.1% Triton X-100-1% BSA透化封闭10min,然后加入Kim-1抗体(一抗)在4摄氏度条件下过夜孵育。孵育结束后,回收抗体,加入荧光二抗在室温条件下孵育4.5小时。回收抗体后,使用PBS清洗3次,加入DAPI室温染色10min。PBS清洗3次后,使用抗荧光淬灭封片剂封片,然后使用荧光显微镜观察样品。
结果如图4所示,FITC标记荧光肽段定位于损伤的HK-2细胞膜上,且与细胞膜上Kim-1具有共定位。
实施例3
本发明的TKP 5在顺铂诱导的损伤小鼠肾脏中具有强靶向作用。
具体操作:精确称量Cy7 2mg和TKP 5多肽1mg,加入800μL 0.1M NaHCO3溶液,4摄氏度旋转过夜反应。将反应液转移至1kD透析袋,并使用PBS透析至透析液中无颜色,得到Cy7标记荧光肽段。然后对8周龄C57雄性小鼠进行腹腔注射顺铂30mg/kg,24小时后给予3μmol/kg的Cy7标记荧光肽段。在给予肽段后的2小时和4小时处死小鼠,取出脑、心、肺、肝、脾、肾等器官,使用小动物活体成像仪器观察各器官近红外荧光强度的变化。
结果如图5所示,给予肽段2小时后,Cy7标记荧光肽段在肾脏组织有强富集效果,其他组织基本无富集效果。并且给予肽段4小时后,Cy7标记荧光肽段在肾脏组织依然存在强富集效果。以上结果提示,Cy7标记荧光肽段在顺铂损伤小鼠肾脏组织有强靶向作用。
实施例4
本发明的TKP 5对顺铂刺激的细胞具有一定的保护作用,无毒副作用。
具体操作:将HK-2细胞按照每孔7000个的密度接种至96孔板中,待细胞贴壁后,采用5μg/mL的顺铂单独刺激或与1μmol/L TKP 5多肽混合后添加至培养基中,继续培养24小时。在细胞培养期结束后,向每孔中加入10μL MTT溶液(5mg/ml,即0.5%MTT),继续培养6小时后,弃去上清液并加入150μL的DMSO,摇床中低速震荡10分钟以充分溶解结晶。最后,使用酶联免疫检测仪在490nm处测量各孔的吸光度值。
结果如图6所示,在生理条件下,给予TKP 5多肽对细胞无毒副作用。顺铂刺激后,给予TKP 5多肽能有效抑制顺铂引起的细胞损伤。
实施例5
本发明的TKP 5在顺铂损伤的肾特异性敲除Kim1小鼠的肾脏中无靶向效果。
具体操作:对8周龄的野生型和肾特异性敲除Kim1(Kim1 KO)雄鼠进行腹腔注射顺铂30mg/kg和等体积溶剂,24小时后给予3μmol/kg的Cy7标记荧光肽段。在给予肽段后的2小时处死小鼠,取出脑、心、肺、肝、脾、肾等器官,使用小动物活体成像仪器观察各器官近红外荧光强度的变化。
结果如图7所示,顺铂损伤的野生型小鼠,给予肽段2小时后,Cy7标记荧光肽段在肾脏有强富集效果。但是顺铂损伤的Kim1 KO小鼠,给予肽段2小时后,Cy7标记荧光肽段在Kim1 KO小鼠肾脏组织无富集效果,这提示TKP 5多肽的肾靶向作用依赖于Kim-1蛋白。
Claims (9)
1.一种Kim-1靶向性多肽,其特征在于:所述的Kim-1靶向性多肽含有SEQ ID NO.1所示的氨基酸序列;或者含有由SEQ ID NO.1所示的氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有Kim-1靶向性的氨基酸序列。
2.一种检测急性肾损伤的试剂,其特征在于:所述的检测急性肾损伤的试剂包含权利要求1所述的靶向性多肽,以及与靶向性多肽偶联的成像剂。
3.根据权利要求2所述的检测急性肾损伤的试剂,其特征在于:所述的成像剂为造影剂或荧光标记物。
4.权利要求1所述的Kim-1靶向性多肽或权利要求2或3所述的检测急性肾损伤的试剂在制备急性肾损伤诊断试剂中的应用。
5.权利要求1所述的Kim-1靶向性多肽在制备治疗急性肾损伤的药物中的应用。
6.一种治疗急性肾损伤的药物组合物,其特征在于:包含权利要求1所述的Kim-1靶向性多肽。
7.一种治疗急性肾损伤的药物组合物,其特征在于:包含权利要求1所述的Kim-1靶向性多肽,以及与靶向多肽偶联的急性肾损伤治疗药物。
8.根据权利要求6或7所述的治疗急性肾损伤的药物组合物,其特征在于:还包含药学上可接受的赋形剂。
9.权利要求6-8任一项所述的药物组合物的制备方法,其特征在于:其步骤包括将所述的Kim-1靶向性多肽或偶联有急性肾损伤治疗药物的靶向性多肽与至少一种药学上可接受的赋形剂混合。
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