CN114206388A - 新的人工蛋白质催化剂 - Google Patents
新的人工蛋白质催化剂 Download PDFInfo
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- CN114206388A CN114206388A CN202080052407.2A CN202080052407A CN114206388A CN 114206388 A CN114206388 A CN 114206388A CN 202080052407 A CN202080052407 A CN 202080052407A CN 114206388 A CN114206388 A CN 114206388A
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Abstract
[问题]提供新的人工蛋白质催化剂,其能够保护催化剂免受体内物质的影响,并在体内合成化学中具有潜在的治疗剂用途。[解决方案]提供蛋白质与选自金属催化剂和有机催化剂的催化剂的复合体。在根据本发明的复合体中,所述蛋白质是在其三维结构中具有疏水部分的蛋白质,并且所述催化剂容纳在所述疏水部分中使得所述催化剂不暴露于或者基本上不暴露于亲水环境。
Description
技术领域
本发明涉及新的人工蛋白质催化剂。
背景技术
目前,正在研究利用催化剂进行“体内合成化学治疗”相关的尝试。体内合成化学治疗的概念是将无活性或毒性的材料或试剂引入体内,并在体内的特定位置处在催化剂的情况下活化所述材料或试剂使得效果得以表达。在这样的情况下,与开发适用于治疗应用的新催化剂相关的兴趣正在提高(非专利文献1)。
开发这样的催化剂的一个障碍是保护催化剂免受体内物质的影响。例如,已知当暴露于以0.5至10mM存在于细胞中或以约2至20μM存在于血浆中的含硫醇谷胱甘肽(GSH)时,金属催化剂(金属酶),如金(Au)、钯(Pd)、钌(Ru)等会迅速失活。
在这一点上,在许多研究中,催化剂反应的研究仅在细胞或模型(除哺乳动物以外)(例如斑马鱼)中通过任何人工金属酶或金属催化剂复合体进行(非专利文献2至17)。
引文列表
非专利文献
[非专利文献1]Rebelein,J.G.;Ward,T.R.Curr.Opin.Biotechnol.2018,53,106-114.
[非专利文献2]Miller,M.A.;Askevold,B.;Mikula,H.;Kohler,R.H.;Pirovich,D.;Weissleder,R.Nat.Commun.2017,8,15906.
[非专利文献3]Clavadetscher,J.;Hoffmann,S.;Lilienkampf,A.;Mackay,L.;Yusop,R.M.;Rider,S.A.;Mullins,J.J.;Bradley,M.Angew.Chem.Int.Ed.Engl.2016,55,15662-15666.
[非专利文献4]Clavadetscher,J.;Indrigo,E.;Chankeshwara,S.V.;Lilienkampf,A.;Bradley,M.Angew.Chem.Int.Ed.Engl.2017,56,6864-6868.
[非专利文献5]Weiss,J.T.;Dawson,J.C.;Macleod,K.G.;Rybski,W.;Fraser,C.;Torres-Sanchez,C.;Patton,E.E.;Bradley,M.;Carragher,N.O.;Unciti-Broceta,A.Nat.Commun.2014,5,3277.
[非专利文献6]Perez-Lopez,A.M.;Rubio-Ruiz,B.;Sebastian,V;Hamilton,L.;Adam,C.;Bray,T.L.;Irusta,S.;Brennan,P.M.;Lloyd-Jones,G.C.;Sieger,D.;Santamaria,J.;Unciti-Broceta,A.Angew.Chem.Int.Ed.Engl.2017,56,12548-12552.
[非专利文献7]Bray,T.L.;Salji,M.;Brombin,A.;Perez-Lopez,A.M.;Rubio-Ruiz,B.;Galbraith,L.C.A.;Patton,E.E.;Leung,H.Y.;Unciti-Broceta,A.Chem.Sci.2018,9,7354-7361.
[非专利文献8]Liu,Y.;Pujals,S.;Stals,P.J.M.;Paulohrl,T.;Presolski,S.I.;Meijer,E.W.;Albertazzi,L.;Palmans,A.R.A.J.Am.Chem.Soc.2018,140,3423-3433.
[非专利文献9]Li,J.;Yu,J.;Zhao,J.;Wang,J.;Zheng,S.;Lin,S.;Chen,L.;Yang,M.;Jia,S.;Zhang,X.;Chen,P.R.Nat.Chem.2014,6,352-361.
[非专利文献10]Vidal,C.;Tomas-Gamasa,M.;Destito,P.;Lopez,F.;Mascarenas,J.L.Nat.Commun.2018,9,1913.
[非专利文献11]Destito,P.;Sousa-Castillo,A.;Couceiro,J.R.;Lopez,F.;Correa-Duarte,M.A.;Mascarenas,J.L.Chem.Sci.2019,10,2598-2603.
[非专利文献12]Tonga,G.Y.;Jeong,Y.;Duncan,B.;Mizuhara,T.;Mout,R.;Das,R.;Kim,S.T.;Yeh,Y.-C.;Yan,B.;Hou,S.;Rotello,V.M.Nat.Chem.2015,7,597-603.
[非专利文献13]Streu,C.;Meggers,E.Angew.Chem.Int.Ed.Engl.2006,45,5645-5648.
[非专利文献14]Volker,T.;Dempwolff,F.;Graumann,P.L.;Meggers,E.Angew.Chem.Int.Ed.Engl.2014,53,10536-10540.
[非专利文献15]Tomas-Gamasa,M.;Martinez-Calvo,M.;Couceiro,J.R.;Mascarenas,J.L.Nat.Commun.2016,7,12538.
[非专利文献16]Yusop,R.M.;Unciti-Broceta,A.;Johansson,E.M.V.;Sanchez-Martin,R.M.;Bradley,M.Nat.Chem.2011,3,239-243.
[非专利文献17]Unciti-Broceta,A.;Johansson,E.M.V.;Yusop,R.M.;Sanchez-Martin,R.M.;Bradley,M.Nat.Protoc.2012,7,1207-1218.
发明内容
通过本发明待解决的问题
本发明的目的是提供新的人工蛋白质催化剂,其允许保护催化剂免受体内物质的影响,并具有用于体内合成化学治疗的潜在用途。
用于解决问题的方法
人血清白蛋白(HSA)是一种66.5kDa的蛋白质,其大量存在于血浆中,并且已知其在血清中的半衰期为约19天(Peters,T.,Jr.Adv.Protein Chem.1985,37,161-245.)。已知HSA是多种激素、脂肪酸和低分子药剂的载体蛋白,并且已在HSA中证实了用于许多种类配体的多个主要和次要结合位点(Ghuman,J.;Zunszain,P.A.;Petitpas,I.;Bhattacharya,A.A.;Otagiri,M.;Curry,S.J.Mol.Biol.2005,353,38-52.)。
为了设计生物相容性人工蛋白质催化剂,本发明人选择HSA作为蛋白质,并考虑在HSA的疏水结合袋中容纳金属催化剂。发现通过使用金属催化剂钌作为催化剂,当金属催化剂被固定至已知与香豆素衍生物(如华法林)结合(相互作用)的白蛋白药物结合位点I时,可以在体外条件下保护结合的钌的催化活性,即使在存在20mM GSH的情况下也是如此,从而完成本发明。
换言之,本发明涵盖以下特征:
[1]蛋白质与催化剂的复合体,所述催化剂选自金属催化剂或有机催化剂,其中
所述蛋白质是在其三维结构内具有疏水袋的蛋白质,并且
所述复合体将所述催化剂容纳在所述疏水袋中,使得所述催化剂不暴露于或者基本上不暴露于亲水环境。
[2]根据[1]所述的复合体,其中所述蛋白质是天然或人工蛋白质。
[3]根据[1]或[2]所述的复合体,其中所述蛋白质选自人血清白蛋白(HSA)、免疫球蛋白G(IgG)、免疫球蛋白A(IgA)、转铁蛋白、抗胰蛋白酶、触珠蛋白、α1-酸性糖蛋白、Myoferlin、Trk受体、***受体和叶酸受体。
[4]根据[1]至[3]中任一项所述的复合体,其中所述金属催化剂选自硼催化剂、镁催化剂、铝催化剂、硅催化剂、钙催化剂、钪催化剂、钛催化剂、钒催化剂、铬催化剂、锰催化剂、铁催化剂、钴催化剂、镍催化剂、铜催化剂、锌催化剂、钇催化剂、锆催化剂、铌催化剂、钼催化剂、钌催化剂、铑催化剂、钯催化剂、银催化剂、铟催化剂、锡催化剂、钡催化剂、铪催化剂、钨催化剂、铼催化剂、锇催化剂、铱催化剂、铂催化剂、金催化剂和镧系元素路易斯酸催化剂。
[5]根据[4]所述的复合体,其中所述镧系元素路易斯酸催化剂选自镱催化剂、镧催化剂、铈催化剂、钐催化剂、铕催化剂、钆催化剂、铽催化剂、铥催化剂和镥催化剂。
[6]根据[1]至[5]中任一项所述的复合体,其中
所述蛋白质是人血清白蛋白,并且
所述催化剂是金属催化剂。
[7]根据[6]所述的复合体,其中所述金属催化剂是钌催化剂。
[8]根据[6]或[7]所述的复合体,其中所述人血清白蛋白的疏水袋是白蛋白药物结合位点I(药物位点I)。
[9]根据[6]至[8]中任一项所述的复合体,其中所述金属催化剂通过针对人血清白蛋白的配体与所述疏水袋结合。
[10]根据[9]所述的复合体,其中所述配体选自华法林、阿扎丙宗、醋硝香豆素、保泰松、水杨酸盐、吲哚美辛、苯妥英、甲苯磺丁脲、氯磺丙脲、碘酚酸、胆影酸、磺胺二甲氧嘧啶、苯丙香豆素、格列本脲、磺胺噻唑、替诺昔康、喜树碱、balzidendeazi、andelleratan、diadelleal、diadellealtan、氟硅酸钠、胆红素、类二十烷酸和羧基-甲基-丙基-呋喃丙酸酯(***毒素)、以及香豆素。
[11]根据[9]或[10]所述的复合体,其中所述金属催化剂通过与所述配体结合的接头与所述疏水袋结合。
[12]根据[11]所述的复合体,其中所述接头是在两端具有氨基和羧基的烷基链或聚乙二醇(PEG)链。
[13]根据[12]所述的复合体,其中所述接头是聚合度为1至3的PEG链或C1至C3烷基。
[14]根据[1]至[13]中任一项所述的复合体,其中所述蛋白质的表面被修饰以与体内靶位点相互作用。
[15]根据[14]所述的复合体,其中所述修饰是通过糖链进行的修饰。
[16]根据[1]至[13]中任一项所述的复合体,其中所述蛋白质还包含与体内靶位点相互作用的部分。
[17]根据[14]或[15]所述的复合体,其中所述蛋白质是人血清白蛋白。
[18]组合物,其包含根据[1]至[17]中任一项所述的复合体。
[19]根据[18]所述的组合物,其是还包含可药用载体的药物组合物。
[20]根据[19]所述的组合物,其与可以被所述复合体活化的前药组合使用。
[21]根据[19]所述的组合物,其还包含可以被所述复合体活化的前药。
[22]根据[19]所述的组合物,其用于选择性标记特定细胞。
[23]根据[22]所述的组合物,其与针对所述细胞进行标记的化学物质组合施用。
[24]根据[18]所述的组合物,其用作生物传感器。
[25]根据[18]所述的组合物,其用作用于检测乙烯的生物传感器。
[26]包含前药的药物组合物,其中
所述前药可以被根据[1]至[17]中任一项所述的复合体活化,并且
所述药物组合物与根据[1]至[17]中任一项所述的复合体组合使用。
[27]组合药物,其包含
包含根据[1]至[17]中任一项所述的复合体的第一药剂,和
包含可以被所述复合体活化的前药的第二药剂。
本领域技术人员将能够认识到,本发明的上述一个或更多个特征的任意组合的发明也涵盖在本发明的范围内。
发明效果
根据本发明,提供了允许保护催化剂免受体内物质影响的新的人工蛋白质催化剂。
附图说明
[图1]图1示出了由钌催化剂Ru1至3、6与HSA之间的反应获得的产物的荧光测定结果。
[图2]图2示出了进行HSA与华法林或布洛芬之间的反应以及随后的与钌催化剂Ru1至3、6的反应的饱和结合曲线。
[图3]图3示出了用于测试本文实施例中使用的alb-Ru复合体的催化能力的实验***。
[图4]图4示出了alb-Ru复合体在不同条件下的催化能力的评价结果。
[图5]图5示出了alb-Ru复合体在不同条件下的催化能力的评价结果。
[图6]图6示出了关于ArM活性(人工金属酶活性)在存在或不存在GSH的情况下,底物1a至1e的米氏动力学参数。
[图7]图7示出了当钌催化剂Ru1至3、6与人血清白蛋白(PDB:1H9Z)对接时的分子模型。
[图8]图8示出了细胞成像结果,其显示GArM-Ru1(2,3-Sia)在SW620人结肠腺癌细胞中更好地积累。
[图9]图9示出了SW620人结肠腺癌细胞、HeLa人***来源细胞和A549人肺泡基底上皮腺癌细胞在GArM-Ru1(2,3-Sia)的积累方面的比较。
[图10]图10示出了关于前药1g和1h通过GArM-Ru复合体活化的动力学实验。
[图11]图11示出了由前药、前药和alb-Ru1或者前药和GArM-Ru1对癌细胞株的细胞生长抑制作用的比较。
[图12]图12示出了ArM乙烯探针(AEP)的合成方案。
[图13]图13示出了所观察的与AEP探针相比alb-Ru化合物的荧光强度。
[图14]图14示出了用于检查本研究中多种蛋白质化合物折叠结构的变化的CD光谱的比较。
[图15]图15示出了观察在AEP情况下的猕猴桃在不同发育阶段(未成熟和成熟)在多种细胞器(外果皮、房室、柱轴)中所表达的乙烯产生量的变化的结果。样品数量为外果皮(n=24)、房室(n=2)和柱轴(n=4),并通过一对样品的t检验进行统计学分析。*P<0.03,**P<0.002,***P<0.0002,****P<0.0001,ns=不重要。
[图16]图16示出了应用AEP(100μM)的野生型Col-0(b)的表皮的荧光强度和明场显微镜成像图像(40×放大率),以及另外添加乙烯生物合成促进剂ACC(1mM)并进行检测的结果。
[图17]图17示出了观察通过添加具有荧光强度的ACC从拟南芥产生的乙烯量的结果。所有值均以一式三份的实验获得。用Tukey′s多重比较检验的单因素方差分析进行统计学分析。*P<0.03,**P<0.002,***P<0.0002,****P<0.0001,ns=不重要。
[图18]图18示出了经GArM处理的HeLa细胞和未经处理的HeLa细胞的流式细胞术结果的比较。
[图19]图19示出了经GArM处理的小鼠腹膜腔来源巨噬细胞和未经处理的小鼠腹膜腔来源巨噬细胞的流式细胞术结果的比较。
[图20]图20示出了用GArM处理的HeLa细胞和小鼠腹膜腔来源巨噬细胞以及未用GArM处理的HeLa细胞和小鼠腹膜腔来源巨噬细胞的流式细胞术结果的比较。请注意,左上图和右下图的Q3值分别为99.09%和43.06%。
[图21]图21示出了实施例8中进行的细胞黏附测定的结果。
具体实施方式
本发明涉及新的人工蛋白质催化剂。具体地,根据本发明的人工蛋白质催化剂是蛋白质与选自金属催化剂或有机催化剂的催化剂的复合体(以下也称为“本发明的复合体”)。
本发明的复合体的特征可以在于构型,该构型将催化剂容纳在在其三维结构内具有疏水袋的蛋白质的所述疏水袋内,使得催化剂不暴露于或者基本上不暴露于亲水环境。换言之,本发明的复合体可以通过避免催化剂暴露于或者基本上暴露于亲水环境来保护催化剂免受体内物质的影响。本发明的复合体可在蛋白质中存在的疏水袋中的仅一个中容纳催化剂,或者可在多个(或所有)袋中容纳催化剂。
本发明的复合体的特征还可以在于具有避免催化剂暴露于或者基本上暴露于亲水环境的构型,并且同时在具有上述构型的同时催化剂仍可促进靶标反应。换言之,在一个优选的实施方案中,本发明的复合体可保护催化剂免受体内物质的影响,同时在体内发挥期望的活性。
在本发明中,“催化剂基本上不暴露于亲水环境”是指在认可保护催化剂免受亲水环境影响的程度上允许暴露于亲水环境。“认可保护催化剂免受亲水环境影响”是指与当游离催化剂暴露于体内环境时相比,本发明的复合体中所容纳的催化剂的活性在相同的环境下维持更长的时间,并且例如可以通过代谢周转(TON)评价。允许暴露的程度可根据使用的催化剂类型或组合使用的蛋白质类型等而变化。
在本发明的一个实施方案中,“催化剂基本上不暴露于亲水环境”意指当容纳在蛋白质的疏水袋中时,催化剂的相对溶剂可及表面积(SASA)为5.0或更小,优选4.0或更小,更优选3.5或更小,更优选3.0或更小,更优选2.5或更小,更优选2.0或更小,更优选1.5或更小,更优选1.0或更小,更优选0.9或更小,更优选0.8或更小,更优选0.7或更小,更优选0.6或更小,更优选0.5或更小,更优选0.4或更小,更优选0.3或更小,更优选0.2或更小,或更优选0.1或更小。
在本发明的另一个实施方案中,“催化剂基本上不暴露于亲水环境”意指50%或更多,优选55%或更多,更优选60%或更多,更优选65%或更多,更优选70%或更多,更优选75%或更多,更优选80%或更多,更优选85%或更多,更优选90%或更多,更优选95%或更多,更优选96%或更多,更优选97%或更多,更优选98%或更多,或更优选99%或更多的催化剂总表面积被内部容纳在蛋白质的疏水袋中。或者,“催化剂基本上不暴露于亲水环境”意指暴露于亲水环境的催化剂的面积为催化剂总表面积的50%或更少,优选45%或更少,更优选40%或更少,更优选35%或更少,更优选30%或更少,更优选25%或更少,更优选20%或更少,更优选15%或更少,更优选10%或更少,更优选5%或更少,更优选4%或更少,更优选3%或更少,更优选2%或更少,或更优选1%或更少。
可用于本发明的复合体的蛋白质不受限制,只要其具有一个或更多个允许容纳催化剂的疏水袋即可,并且可以使用天然或人工的蛋白质。人工的蛋白质包括突变蛋白质,其中突变被人为地引入到天然蛋白质的一部分中。
在一个实施方案中,用于本发明的复合体的蛋白质是其中容易获得与蛋白质的任何疏水袋结合或相互作用的配体的蛋白质。这样的蛋白质可以是优选的,因为其促进了催化剂通过配体的络合。
在一个具体实施方案中,用于本发明复合体的蛋白质是选自人血清白蛋白(HSA)、免疫球蛋白G(IgG)、免疫球蛋白A(IgA)、转铁蛋白、抗胰蛋白酶、触珠蛋白、α1-酸性糖蛋白等的蛋白质。这些蛋白质被认为适用于药物传递***,因为它们是可以在血液中自由移动的蛋白质。在另一个具体的实施方案中,用于本发明的复合物的蛋白质是选自Myoferlin、Trk受体、***受体、叶酸受体等的蛋白质。由于已知这些蛋白质在癌细胞中大量表达,因此认为通过使配体和金属与其配位,可以制造可直接杀伤癌症的复合体。
可用于本发明复合体的有机或金属催化剂无特别限制,本领域技术人员可以任意选择。在一个实施方案中,可用于本发明复合体的有机或金属催化剂是具有将给定前药转变为活性形式的活性的催化剂。
例如,用于本发明复合体的有机催化剂可包括但不限于:脯氨酸衍生物、基于季铵盐衍生物的相转移催化剂、硫脲衍生物、由噻唑盐或咪唑盐获得的N-杂环卡宾衍生物、环状酮衍生物、4-二甲基氨基吡啶衍生物、仲胺例如氨基酸等。
例如,用于本发明复合体的金属催化剂可包括但不限于:硼催化剂、镁催化剂、铝催化剂、硅催化剂、钙催化剂、钪催化剂、钛催化剂、钒催化剂、铬催化剂、锰催化剂、铁催化剂、钴催化剂、镍催化剂、铜催化剂、锌催化剂、钇催化剂、锆催化剂、铌催化剂、钼催化剂、钌催化剂、铑催化剂、钯催化剂、银催化剂、铟催化剂、锡催化剂、钡催化剂、铪催化剂、钨催化剂、铼催化剂、锇催化剂、铱催化剂、铂催化剂、金催化剂、镧系元素路易斯酸催化剂等。此外,所述镧系元素路易斯酸催化剂还可包括但不限于镱催化剂、镧催化剂、铈催化剂、钐催化剂、铕催化剂、钆催化剂、铽催化剂、铥催化剂、镥催化剂等。
用于将催化剂容纳在蛋白质的疏水袋中的方法可包括,例如,通过与所述疏水袋相互作用或结合的配体将催化剂结合到疏水袋中的方法。在这样的情况下,可以适当地使用肽、烃链、PEG等的接头来连接配体和催化剂(例如金属催化剂)。所用接头的长度应该是使得催化剂容纳在疏水袋内并且不暴露于或者基本上不暴露于亲水环境的长度。换言之,当使用的接头相对于疏水袋、与其相互作用的配体以及使用的催化剂的尺寸太长时,存在这样的可能性:当催化剂通过接头和配体结合至疏水袋时,催化剂未容纳在疏水袋内,其全部或部分从疏水袋暴露于亲水环境,并且无法从亲水环境获得足够的保护。
用于将催化剂容纳在蛋白质的疏水袋中的另一种方法可包括,例如,将马来酰亚胺或琥珀酰亚胺应用于存在于蛋白质疏水袋内或其附近的半胱氨酸或赖氨酸的侧链官能团(分别为硫醇基和氨基),以允许活化催化剂并与催化剂共价结合。
用于将催化剂容纳在蛋白质的疏水袋中的另一种方法可包括,在配置疏水袋的氨基酸序列的特定位置引入突变以引入双键或叠氮化物以允许与催化剂的复分解或点击反应以及共价结合(Young TS,Ahmad I,Brock A,Schultz PG.,Expanding the geneticrepertoire of the methylotrophic yeast Pichia pastoris.,Biochemistry.2009.48(12):2643-53.;Wang L,Schultz PG.,Expanding the genetic code.,Angew Chem IntEd Engl.2004.44(1):34-66.)。在这样的情况下,作为催化剂(例如金属催化剂),可以使用具有接头(例如肽或PEG)的催化剂。
在一个实施方案中,本发明的复合体被修饰以与体内靶位点相互作用。作为结果,本发明的复合体可被引导至体内期望的位点或可靶向期望的位点。与此相关,“与体内靶位点相互作用”可意指与其他体内位点相比以显著强的定向性积累至或通常结合至靶位点。在本发明中,靶位点可以是特定的器官或特定的细胞瘤等。
这样的修饰可以包括例如糖基化。例如,已知通过使用天冬酰胺连接的糖链(N-连接的糖链)进行蛋白质修饰,朝向特定器官的定向性根据簇的数目和/或其糖链的结合数目而变化(Tanaka,K.,et al.,Angew.Chem.Int.Ed.,49,8195-8200(2010);Latypova,L.,etal.,Adv.Sci.,1600394(2017);Ogura,A.,et al.,Chem.Commun.,54,8693-8696(2018);Taichi,M.,et al.,Adv.Sci.,1700147(2017))。例如,已知在其末端具有唾液酸的糖链迅速到达肝,或者在其末端具有半乳糖的糖链迅速到达肠道。因此,通过根据所选的体内靶位点用合适的糖链结构和糖链数目修饰本发明的复合体,可以获得朝向所述靶位点具有增强的定向性的复合体。
在一个实施方案中,例如,可以使用多个(例如5至30个)在非还原末端具有与多种类型的癌症相互作用的唾液酸、半乳糖胺、半乳糖或甘露糖的糖链作为糖基化。在一个替代实施方案中,可以使用具有岩藻糖的糖链作为糖基化。在上述实施方案中,待修饰的多个糖链可以是相同的或多种类型的,并且每个糖链的大小可以在1至25个糖的范围内。
在另一个实施方案中,例如,可以使用多个(例如5至30个)在非还原末端具有选择性地转移到特定器官例如肝、胰腺、肠道、胆囊、膀胱或脑中的唾液酸、半乳糖胺、半乳糖或甘露糖的糖链作为糖基化。在上述实施方案中,待修饰的多个糖链可以是相同的或多种类型的,并且每个糖链的大小可以在1至25个糖的范围内。
修饰位点通常是所述复合体的蛋白质表面。用于使蛋白质表面的特定位点糖基化的方法是本领域技术人员公知的,并且可以采用任何方法。在本发明中,例如,可以通过国际公开第2008/096760号、日本专利第6327547号、国际公开第2017/002918号等中描述的点击反应将糖基化引入蛋白质表面。
在另一个实施方案中,本发明的复合体还包含与体内靶位点相互作用的部分。与此相关,“与靶位点相互作用”可意指与其他位点相比以显著强的定向性积累至或通常结合至靶位点。在本发明中,靶位点可以是特定的器官或特定的细胞瘤等。
在本发明中,“与体内靶位点相互作用的部分”可以是抗体或其片段、肽配体或其片段、DNA或RNA、或pNA(肽核酸)或其片段等。与此相关,“与体内靶位点相互作用的部分”可以例如制造成与用于复合体的蛋白质的融合蛋白,或者制造成用于复合体的蛋白质,并且“与体内靶位点相互作用的部分”可以单独制备,然后通过本领域技术人员公知的方式结合(例如共价结合)。
在一个具体的实施方案中,本发明涉及人工金属酶(artificial metalloenzyme;ArM),其是蛋白质与金属催化剂的复合体。在一个优选的实施方案中,本发明的ArM是HSA与金属催化剂的复合体,其中所述复合体的特征在于其将金属催化剂容纳在HSA的疏水袋中,使得金属催化剂不暴露于或者基本上不暴露于亲水环境。
在该实施方案中,与HSA一起使用的金属催化剂可以由本领域技术人员例如根据进行活化的前药的类型等适当地选择。在本发明的一个实施方案中,与HSA一起使用的金属催化剂为钌。
在该实施方案中,HSA中容纳金属催化剂的疏水袋无特别限制,并且一个疏水袋可容纳金属催化剂,或者多个疏水袋可容纳金属催化剂。HSA的疏水袋可包括白蛋白药物结合位点I和白蛋白药物结合位点II。在一个实施方案中,本发明的复合体在HSA的白蛋白药物结合位点I中容纳金属催化剂。
在该实施方案中,可通过将金属催化剂与针对HSA的配体连接,并随后通过所述配体将金属催化剂结合到疏水袋内来将金属催化剂容纳在HSA的疏水袋中。可用于此目的的配体可根据容纳金属催化剂的疏水袋而变化。例如,当在HSA的白蛋白药物结合位点I中容纳金属催化剂时,所述配体可以选自华法林、阿扎丙宗、醋硝香豆素、保泰松、水杨酸盐、吲哚美辛、苯妥英、甲苯磺丁脲、氯磺丙脲、碘酚酸、胆影酸、磺胺二甲氧嘧啶、苯丙香豆素、格列本脲、磺胺噻唑、替诺昔康、喜树碱、balzidendeazi、andelleratan、diadelleal、diadellealtan、氟硅酸钠、胆红素、类二十烷酸和羧基-甲基-丙基-呋喃丙酸酯(***毒素)、以及香豆素。或者,当在HSA的白蛋白药物结合位点II中容纳金属催化剂时,所述配体可以选自***、酮洛芬、氯贝特、布洛芬、碘泛酸盐、叠氮脱氧胸苷、氟灭酸酯、依他尼酸盐、萘普生、氟比洛芬、环洛芬、苯恶丙芬、氟氯西林、***、吡洛芬、异丙酚、异氟烷、丹磺酰基肌氨酸、丹磺酰基甘氨酸、烷基氨基香豆素乙酸、羟基黄酮、L-色氨酸、中链脂肪酸阴离子(如辛酸根)、L-甲状腺素、氯离子、碘乙酸、硫酸吲哚酚和马尿酸(尿毒素)。在本发明的一个实施方案中,HSA的疏水袋是药物结合位点I,并且香豆素衍生物,例如,7-二甲基氨基香豆素用作配体以在上述位点中容纳钌。
金属催化剂与所述配体之间的连接可以通过接头进行。作为可用于此目的的接头,通常可以使用在两端具有氨基和羧基的烷基链或聚乙二醇(PEG)链等。具体地,烷基链接头可包括例如-NH-(CH2)x-CO-(其中x为整数且没有限制,只要其不抑制靶标接头功能即可,并且例如可以为1至15之间的整数),-NH-(CH2CH2O)y-CH2-CO-(其中y为整数且没有限制,只要其不抑制靶标接头功能即可,并且例如可以为1至15之间的整数),或-NH-(CH2CH2O)z-CH2CH2-CO-(其中z为整数且没有限制,只要其不抑制靶标接头功能即可,并且例如可以为1至15之间的整数),等等。
当使用接头来连接金属催化剂和所述配体时,应注意所用接头的长度是使得金属催化剂容纳在HSA的疏水袋内并且不暴露于或者基本上不暴露于亲水环境的长度。例如,当使用香豆素或其衍生物(例如7-二甲基氨基香豆素、7-二乙基氨基香豆素(DEAC))作为配体在HSA的白蛋白药物结合位点I中容纳钌时,“基本上不暴露于亲水环境”可以是钌暴露于亲水环境的面积为钌总表面积的40%或更少,并且优选35%或更少。或者,“基本上不暴露于亲水环境”可以是当容纳在HSA的白蛋白药物结合位点I中时钌的SASA为3.0或更小,并且优选1.0或更小。此外,在这种情况下使用的示例性接头是-NH-(CH2CH2O)y-CH2-CO-,其中y可以是1至6的整数,并且优选1至3的整数。
在一个实施方案中,在本发明的ArM(其为HSA与金属催化剂的复合体)中,HSA的表面被糖链修饰。糖基化的含量可根据体内靶标而变化。HSA表面的修饰位置没有特别限制,只要可以引入糖链即可,并且一个实例可包括HSA表面上第30位的赖氨酸残基。
本发明的复合体可以根据容纳在上述复合体中的催化剂的类型在体内活化前药。优选地,根据容纳在上述复合体中的催化剂的类型,本发明的复合体在体内的特定位置积累以允许活化对上述位置具有选择性的前药。
因此,在另一方面中,本发明涉及包含本发明复合体的组合物。
在一个实施方案中,本发明的组合物是与可被本发明的复合体活化的前药组合使用的药物组合物。在前述实施方案中,本发明的药物组合物可以是单一剂型,其还包含可药用的载体,并且视情况而定包含可以被本发明的复合体活化的前药。可用于本发明的可被本发明的复合体活化的前药可包括例如多种抗癌剂,并且具体地,但不限于此,丝裂霉素C、阿霉素、紫杉醇、他莫昔芬等可以是示例性的。
在另一个实施方案中,本发明的药物组合物是组合药物的形式,其中本发明的复合体和可以被上述复合体活化的前药作为独立的药剂提供。在本发明的组合药物中,包含本发明的复合体的第一药剂和包含可被上述复合体活化的前药的第二药剂可以同时或在不同时间施用于对象。
在另一方面中,本发明的组合物涉及包含可被本发明的复合体活化的前药和任何可药用载体的药物组合物,并且上述药物组合物的特征在于与本发明的复合体组合使用。
在一个实施方案中,本发明的药物组合物可用于在体内选择性地标记特定细胞。在本发明中,“标记”是指使用无毒性的化学物质来标记靶细胞,所述化学物质可以是内在的或外在施用的,并且可以破坏细胞功能(例如黏附抑制剂)或可以引发免疫反应。这样的化学物质可以是通过任何金属催化剂从无活性形式转化为活性形式的体内或离体化学物质,或者是通过任何金属催化剂增强其功能的体内或离体化学物质。在治疗应用中的这样的方法称为选择性细胞标记(SeCT),并且与使用高细胞毒性药剂直接清除癌细胞的常规化学治疗相比,期望其在不显著损害周围组织的情况下间接诱导靶细胞(如癌细胞)死亡。例如,在本文的实施例中,显示了如下的可能性:通过本发明的复合体增强cRGD-炔丙酯(cRGD-PE)针对在癌细胞表面上表达的整联蛋白的结合,并且可以抑制转移。
在另一个实施方案中,本发明的组合物可用作生物传感器。具体地,在本发明的复合体中,通过使用金属催化剂、本身具有荧光和/或具有通过与蛋白质例如白蛋白结合而增强的荧光的接头(例如香豆素衍生物)、和可能因金属催化剂与检测目标物质之间的反应而脱离的猝灭剂,本发明的复合体或组合物可以根据检测目标物质的类型被设计为合适的生物传感器。在本发明的一个具体实施方案中,本发明的组合物用作检测植物中乙烯的生物传感器。
请注意,本文中使用的术语将用于描述具体实施方案,并不旨在限制本发明。
此外,如本文中使用的术语“包含/包括”,除非内容清楚地指出可以以其他方式理解,否则其意指所描述的条目(例如组分、步骤、要素和数值)的存在,并且不排除其他条目(例如组分、步骤、要素和数值)的存在。
除非另有定义,否则本文中使用的所有术语(包括技术和科学术语)具有与本发明所属技术领域的技术人员所广泛认可的相同的含义。本文中使用的术语,除非另有明确定义,否则应当理解为具有与本文中及相关技术领域中的含义的一致的含义,并且不应理解为具有理想化或过于形式化的含义。
尽管可以使用诸如第一和第二的术语来表示多个要素,但是应当认识到这些要素不受这些术语的限制。这些术语仅用于将一个要素与另一个要素区分开的目的,例如,可以将第一要素描述为第二要素,并且类似地,在不脱离本发明范围的情况下将第一要素描述为第二要素。
现在将通过实施例更具体地描述本发明。然而,本发明可以通过多个实施方案来体现,并且不应被解释为限于本文中所述的一些实施例。
实施例
[实施例1]
alb-Ru的制造
为了设计可防止金属催化剂暴露在溶剂中的生物相容性人工金属酶(ArM)(具有结合袋),利用了白蛋白的药物结合位点I(药物位点I)(疏水结合袋作为伪-活性位点)。为了实现这一目的,将金属催化剂固定至已知用于与香豆素衍生物(例如华法林)结合(相互作用)的药物结合位点I(Ghuman,J.;Zunszain,P.A.;Petitpas,I.;Bhattacharya,A.A.;Otagiri,M.;Curry,S.J.Mol.Biol.2005,353,38-52)。使用具有不同PEG接头长度的香豆素-Ru复合体Ru1至3、6来固定金属催化剂。
1.香豆素-Ru复合体的制备
1-1.通用程序B
根据以下反应方案,可以合成香豆素结合的钌复合体Ru1至3、6。
[化学式1]
钌复合体III是根据公知的技术制备的(Lo,C.;Ringenberg,M.R.;Gnandt,D.;Wilson,Y.;Ward,T.R.ChemComm 2011,47,12065-12067;Kajetanowicz,A.;Chatterjee,A.;Reuter,R.;Ward,T.R.Catal.Lett.2014,144,373-379;Zhao,J.;Kajetanowicz,A.;Ward,T.R.Org.Biomol.Chem.2015,13,5652-5655)。在25℃时,向钌复合体III(80.0mg,0.106mmol)在二氯甲烷(3mL)中的溶液通入盐酸气体。盐酸气体通过向氯化铵中逐滴添加浓硫酸制备。在搅拌45分钟之后,用注射器向反应物中添加二氯甲烷(1mL),并将其在相同温度下搅拌。在再15分钟之后,将反应物在减压下浓缩以获得胺IV,并将其在不进行纯化的情况下用于下一步反应。
在另一个烧瓶中,将溶解在二氯甲烷(1mL)中的羧酸IIa(1.1当量)和偶联剂HCTU(1.3当量)的溶液在25℃下搅拌30分钟。向该反应物中添加溶解在二氯甲烷(1mL)中的胺IV(1当量),随后在相同温度下添加N,N-二异丙基乙胺(10当量)。在搅拌6小时之后,通过添加1M HCl水溶液终止反应。产物用二氯甲烷萃取3次,组合的有机萃取层用饱和碳酸氢钠水和盐水洗涤,干燥(通过硫酸钠),然后在减压下浓缩。经浓缩的剩余物通过硅胶快速柱色谱法纯化,并且可获得香豆素-Ru复合体Ru1至3、6。
1-2.香豆素-Ru复合体Ru1的制备
根据通用程序B,在从钌复合体JII(80.1mg,0.106mmol)、羧酸IIa(42.4mg,0.117mmol)、HCTU(57.6mg,0.139mmol)和N,N-二异丙基乙胺(137mg,1.06mmol)得到的反应物通过硅胶快速柱色谱法(环己烷/EtOAc/CHCl3/MeOH=40/40/15/5)纯化之后,获得为绿色固体的目标香豆素-Ru复合体Ru1(37.7mg,35.6%)。
1H-NMR(400MHz,CDCl3,δ)1.24-1.27(m,12H),2.37-2.51(br m,18H),3.47(q,4H,J=7.1Hz),3.53-3.71(m,6H),3.79(m,1H),3.93(d,1H,J=15.3Hz),3.99(d,1H,J=15.3Hz),4.05(dd,1H,J1=J2=10.0Hz),4.33(dd,1H,J1=J2=10.0Hz),4.75-4.84(brm,1H),4.89(sept,1H,J=6.1Hz),6.50(d,1H,J=2.3Hz),6.66(dd,1H,J1=2.3Hz,J2=9.1Hz),6.78(d,1H,J=7.7Hz),6.85(dd,1H,J1=J2=7.7Hz),6.91(dd,1H,J1=1.7Hz,J2=7.7Hz),7.02(s,ove rlapped,2H),7.04(s,1H),7.07(s,1H),7.42(d,1H,J=9.1Hz),7.48(ddd,1H,J1=1.7Hz,J2=J3=7.7Hz),8.67(s,1H),9.09(s,1H),16.50(s.1H);
HRMS(ESI)m/z 964.3355(C50H61ClN5O6Ru,[M-CH+的计算值为964.3359)。
[化学式2]
1-3.香豆素-Ru复合体Ru2的制备
根据通用程序B,在从钌复合体III(80.1mg,0.106mmol)、羧酸IIb(47.4mg,0.117mmol)、HCTU(57.1mg,0.138mmol)和N,N-二异丙基乙胺(137mg,1.06mmol)得到的反应物通过硅胶快速柱色谱法(环己烷/EtOAc/CHCl3/MeOH=40/40/15/5)纯化之后,获得为绿色固体的目标香豆素-Ru复合体Ru2(27.9mg,25.2%)。
1H-NMR(400MHz,CDCl3,δ)1.22-1.26(m,12H),2.26-2.46(br m,18H),3.38-3.54(m,4H),3.61-3.70(m,10H),3.74-3.82(m,1H),3.97(s,2H),4.11(dd,1H,J1=J2=10.3Hz),4.28(dd,1H,J1=J2=10.3Hz),4.66-4.75(brm,1H),4.88(sept,1H,J=6.1Hz),6.47(d,1H,J=2.3Hz),6.65(dd,1H,J1=2.3Hz,J2=9.0Hz),6.77(d,1H,J=7.6Hz),6.83(dd,1H,J1=J2=7.6Hz),6.89(dd,1H,J1=1.9Hz,J2=7.6Hz),6.98-7.01(br m,4H),7.43(d,1H,J=9.0Hz),7.47(ddd,1H,J1=1.9Hz,J2=J3=7.6Hz),8.67(s,1H),9.05(s,1H),16.47(s.1H);
HRMS(ESI)m/z 1008.3613(C52H65ClN5O7Ru,[M-Cl]+的计算值为1008.3622)。
[化学式3]
1-4.香豆素-Ru复合体Ru3的制备
根据通用程序B,在从钌复合体III(28.8mg,38.1μmol)、羧酸IIc(18.9mg,42.0μmol)、HCTU(21.3mg,51.5μmol)和N,N-二异丙基乙胺(49.7μg,385μmol)得到的反应物通过硅胶快速柱色谱法(环己烷/EtOAc/CHCl3/MeOH=40/40/15/5)纯化之后,获得为绿色固体的目标香豆素-Ru复合体Ru3(12.0mg,28.9%)。
1H-NMR(400MHz,CDCl3,δ)1.22-1.28(m,12H),2.34-2.44(br m,18H),3.46(q,4H,J=7.2Hz),3.55-3.70(m,14H),3.71-3.80(m,1H),3.95(s,2H),4.04(dd,1H,J1=J2=10.7Hz),4.27(dd,1H,J1=J2=10.7Hz),4.57-4.65(m,1H),4.89(sept,1H,J=6.2Hz),6.47(d,1H,J=2.3Hz),6.65(dd,1H,J1=2.3Hz,J2=8.8Hz),6.79(d,1H,J=7.7Hz),6.84(dd,1H,J1=J2=7.6Hz),6.89(dd,1H,J1=1.9Hz,J2=7.6Hz),7.01-7.05(br m,4H),7.42(d,1H,J=8.8Hz),7.47(ddd,1H,J1=1.9Hz,J2=J3=7.6Hz),8.67(s,1H),8.98(s,1H),16.47(s.1H);
HRMS(ESI)m/z 1088.3661(C54H70Cl2N5O8Ru,[M+H]+的计算值为1008.3648)。
[化学式4]
1-5.香豆素-Ru复合体Ru6的制备
根据通用程序B,在从钌复合体III(80.4mg,0.106mmol)、羧酸IId(69.9mg,0.117mmol)、HCTU(57.5mg,0.139mmol)和N,N-二异丙基乙胺(137mg,1.06mmol)得到的反应物通过硅胶快速柱色谱法(环己烷/EtOAc/CHCl3/MeOH=20/20/55/5)纯化之后,获得为绿色固体的目标香豆素-Ru复合体Ru6(34.4mg,26.3%)。
1H-NMR(400MHz,CDCl3,δ)1.21-1.30(m,12H),2.37-2.45(br m,18H),3.45(q,4H,J=7.3Hz),3.56-3.68(m,31H),3.99(dd,1H,J1=J2=10.5Hz),4.25(dd,1H,J1=J2=10.5Hz),4.53-4.62(br m,1H),4.91(sept,1H,J=6.1Hz),6.49(d,1H,J=2.3Hz),6.64(dd,1H,J1=2.3Hz,J2=8.8Hz),6.80(d,1H,J=7.6Hz),6.86(dd,1H,J=7.6Hz),6.90(dd,1H,J1=1.9Hz,J2=7.6Hz),7.03-7.07(br m,4H),7.42(d,1H,J=8.8Hz),7.48
(ddd,1H,J1=1.9Hz,J2=J3=7.6Hz),8.68(s,1H),9.00(s,1H),16.47(s.1H);
HRMS(ESI)m/z 1234.4603(C61H84Cl2N5O11Ru,[M+H]+的计算值为1234.4593)。
[化学式5]
2.alb-Ru的制造
alb-Ru的制造是通过使钌催化剂Ru1至3、6与人血清白蛋白(HAS)反应以形成alb-Ru复合体来进行的。
反应溶液的组合物包含30μM人血清白蛋白(以下称为HSA;使用167μL 50nmol,300μM储备溶液(水溶液))和在多种浓度下的催化剂(Ru1至3、6)。所用的催化剂是例如37μMRu1(使用167μL 62nmol,370μM储备溶液(二氧六环溶液))。将总反应体积用包含10%二氧六环的PBS缓冲液(pH 7.4)填充至1670μL。在添加HSA开始反应之后,轻轻混合反应混合物并在37℃下孵育1小时。随后,在AmiconTM超速离心过滤器(30kDa)的情况下,用PBS缓冲液洗涤反应溶液并浓缩。接下来,将经浓缩的alb-Ru溶液用PBS缓冲液稀释,并获得1000μL作为50μM储备溶液。
为了确认Alb-Ru复合体的形成,采用了依赖于以下事实的荧光测定:基于香豆素的分子对溶剂极性敏感。使用Ru1至3、6,从配体-白蛋白的1∶1反应获得的荧光强度(测量值)示于图1中。
与对照(仅配体或仅HSA)相比,检测到显著高的荧光水平,其被认为指示alb-Ru复合体的形成。
此外,为了间接测定Ru1至3、6结合位点,将HSA溶液与2当量的任一结合配体(华法林或布洛芬)一起在37℃下预孵育1小时。随后,用白蛋白和结合配体的混合物生成了Ru1至3、6的饱和结合曲线(图2)。
从图2的结果,很明显Ru1至3、6的结合在布洛芬的存在下不受影响,但在华法林的存在下显著降低。该结果强烈表明HSA的药物结合位点I是这些化合物的主要结合位点。
[实施例2]
alb-Ru反应性的验证
接下来,检查了alb-Ru复合体催化烯烃1a至1d和烯炔1e的闭环复分解(RCM)的能力(图3)。此外,由于alb-Ru1至3、6具有不同的PEG接头长度,因此还验证了疏水结合袋的有效大小和相容性。
反应溶液在1∶9的二氧六环:PBS缓冲液(pH 7.4)中基本上含有1a至1e作为底物和alb-Ru复合体(即Ru1至3、6)。在37℃孵育2小时之后,反应用十二烷硫醇猝灭,另外用甲醇稀释,并过滤样品,进行HPLC分析,并计算转化为相应产物2a至e的代谢周转(TON)。作为对照,还在类似条件下进行了使用溶液中游离钌催化剂Ru1至3、6(在溶液中游离)的反应。
[表1]
该实验中的第一观察结果之一是PEG接头(香豆素锚与钌催化剂之间)长度增加与活性降低之间的相关性。例如,底物4,4-双((苯甲酰基氧基)甲基)-1,6-庚二烯1d(条目7)的代谢周转在alb-Ru1存在的情况下最高(19.7),在alb-Ru2存在的情况下陡然下降(2.8),并且在alb-Ru6存在的情况下显示出极低的水平(0.5)。鉴于使用溶液中游离的钌催化剂Ru1获得的代谢周转(TON)对于所使用的所有接头长度总体上保持在28至35的范围内(条目8),该观察结果表明疏水结合袋不能容纳具有长接头的香豆素锚-催化剂复合体。
[实施例3]
alb-Ru生物相容性的验证
在确认alb-Ru复合体显示出催化闭环复分解和烯炔交叉复分解的活性之后,然后我们的目标是通过评价谷胱甘肽的作用来验证生物相容性。选择化合物1d作为模型底物用于本研究。
本研究的重要实验之一是检查alb-Ru1针对谷胱甘肽的催化剂保护能力。如下所示,使用1mol%alb-Ru1,并使底物1d在添加不同浓度GSH的情况下一起反应。请注意,根据alb-Ru1的浓度,添加相等当量的谷胱甘肽。
[化学式6]
作为结果,在高达20mM GSH(1000当量针对alb-Ru的GSH)时未观察到(计算的)TON的变化。最终,在添加100mM和200mM GSH的情况下,分别识别到与对照相比,TON降低了60%和82%。
在接下来的调查中,为了了解GSH保护能力的阈值上限,评价了金属催化剂保护的持续时间。具体而言,将alb-Ru1与GSH、十二烷硫醇或PBS缓冲液(作为空白)中的任一种一起预孵育不同小时,然后将底物1d添加至反应溶液中,并在反应1小时之后计算TON值。图4示出了在生理上适宜的GSH浓度(200μM,针对alb-Ru1 10当量)情况下进行的实验。
在用GSH预孵育6小时之后测量的TON值与空白(PBS缓冲液)相比总体上仅略有下降。相比之下,在阀值上限(20mM,针对alb-Ru1 1000当量)下使用GSH的实验中,示出了对反应性的显著(大得多)的影响(图5)。
在这样的情况下,在用GSH预孵育1小时之后观察到活性降低约50%,而在预孵育4小时之后活性几乎消失。
为了进一步评价Alb-Ru复合体的生物相容性,将底物1d与10mol%alb-Ru1复合体在37℃下孵育2小时。在1∶8∶1的胎牛血清/DMEM培养基/二氧六环的情况下,2d的产率为约2%,并且在1∶8∶1正常大鼠血清/PBS缓冲液/二氧六环的情况下,为约1%。然而,由于收集的起始材料1d分别为8%和3%,因此低产率可能归因于血清中普遍存在的蛋白质对底物的捕获或降解。
关于ArM活性(人工金属酶活性),在添加或不添加GSH的情况下计算底物1a至1e的米氏动力学参数(图6)。
作为结果,无论添加或不添加GSH,关于ArM活性,针对1a至1e测量的动力学数值都只有轻微的变化(在误差范围内)。尽管如此,特别重要的是在这些底物之间观察到的催化剂效率(kcat/KM)的差异。具体地,底物1e显示kcat/KM值为约3×103M-1s-1,并且虽然该值与天然酶的反应性(kcat/KM-108M-1s-1)相比很差,但是从该结果强调了非天然金属的ArM达到与天然酶相当的反应性的可能性。
[实施例4]
谷胱甘肽抗性的机制
为了预测白蛋白药物结合位点I的疏水结合袋中的侵入程度和构型,在AutoDockTools的GUI界面中使用Autodock 4.2(Morris,G.M.;Huey,R.;Lindstrom,W.;Sanner,M.F.;Belew,R.K.;Goodsell,D.S.;Olson,A.J.J.Comput.Chem.2009,30,2785-2791.)对化合物Ru1至3、6进行研究以用于与人血清白蛋白(PDB:1H9Z)对接的分子建模。结果支持了如下的基本假设:白蛋白的药物结合位点I的结合袋足够深以容纳具有相对较短PEG接头长度的香豆素-钌催化剂(例如Ru1)的结合。然而,如图7所示,随着PEG接头长度逐渐变长(例如Ru6),钌部分被推到结合袋之外,并且其向溶液中生物分子的暴露也提高。
通过对接配体的钌原子的相对溶剂可及表面积(SASA)的计算进一步支持了此理论,事实上,与更长的PEG接头的相关性提高了。注意在图7中,零值表示原子未暴露于溶剂。
[实施例5]
人工金属酶的靶向
促进治疗剂ArM开发待考虑的另一方面是需要靶向方法以促进朝向体内特定的器官/细胞的定位。
如果成功,将有可能应用于前药治疗,这进而特别有利于具有副作用风险的候选药物(例如基于细胞毒性分子的抗癌治疗)的开发。
已经发现,由于包被不同真核细胞表面的糖萼的复杂性正在变化,因此与特定组合的N-聚糖聚集体结合的糖化白蛋白可以在不同的癌细胞之间发挥不同的识别能力或结合。在一项旨在测试这些糖化白蛋白是否能够作为基于炔丙酯的蛋白质标记的体内支持物的基础研究中,含金催化剂的糖化白蛋白通过其N-聚糖结构定位于活小鼠的特定器官中,并显示出体内催化活性(Tsubokura,K.;Vong,K.K.H.;Pradipta,A.R.;Ogura,A.;Urano,S.;Tahara,T.;Nozaki,S.;Onoe,H.;Nakao,Y.;Sibgatullina,R.;Kurbangalieva,A.;Watanabe,Y.;Tanaka,K.Angew.Chem.Int.编辑.Engl.2017,56,3579-3584.)。
为了开发和研究对癌症的适用性,选择了在唾液酸α(2,3)-连接的末端的N-聚糖的统一聚集体作为靶标组。此选择是基于之前的研究做出的,所述研究显示了具有唾液酸α(2,3)-结合的糖化白蛋白(2,3-Sia)可以优先在SW620人结肠腺癌细胞中积累,其还显示出与A549人肺泡基底上皮腺癌细胞和HeLa人***来源细胞的中等结合(Ogura,A.;Urano,S.;Tahara,T.;Nozaki,S.;Sibgatullina,R.;Vong,K.;Suzuki,T.;Dohmae,N.;Kurbangalieva,A.;Watanabe,Y.;Tanaka,K.ChemComm 2018,54,8693-8696.)。这种作用可能由半乳凝素8的过表达引起,半乳凝素8被称为公知的α(2,3)-连接的唾液酸(Lahm,H.;Andre,S.;Hoeflich,A.;Fischer,J.R.;Sordat,B.;Kaltner,H.;Wolf,E.;Gabius,H.-J.J.Cancer Res.Clin.Oncol.2001,127,375-386;Carlsson,S.;Oberg,C.T.;Carlsson,M.C.;Sundin,A.;Nilsson,U.J.;Smith,D.;Cummings,R.D.;Almkvist,J.;Karlsson,A.;Leffler,H.Glycobiology 2007,17,663-676.)。
首先制备了其上固定有钌催化剂Ru1的糖基化ArM(GArM)-Ru1(2,3-Sia),然后在结合实验中评价了对SW620人结肠腺癌细胞的靶向能力。为了评价GArM-Ru1(2,3-Sia)与癌细胞的特异性结合,将癌细胞以104个细胞/孔的密度接种在具有透明底部的96孔板中,并培养过夜。随后,除去培养基,并添加[1]GArM-Ru1(2,3-Sia)、[2]alb-Ru、[3]GA(2,3-Sia)或[4]Ru1,使得各自的终浓度为10μM。细胞在37℃下孵育3小时之后,除去培养基,将其用PBS缓冲液洗涤(3次),并用甲醛试剂固定在板上。用来自Keyence的BZ-X710 All-in-oneFluorescence MicroscopeTM进行细胞观察,并记录荧光图像和明场图像。ET-EBFP2/香豆素/减毒DAPI过滤器组(目录号49021)(Chroma Technology Corp.,Vermont,USA)用于检测香豆素来源的荧光。成像的图像以20×放大率拍摄,并用BZ-X分析仪(来自Keyence)软件进行分析。
结果示于图8中。通过利用与HSA结合的香豆素的荧光发光的细胞成像,在与GArM-Ru1(2,3-Sia)一起孵育的SW620人结肠腺癌细胞中发现了与其对照相比更强的荧光积累。
作为另外的测试,还用HeLa人***来源细胞和A549人肺泡基底上皮腺癌细胞进行了类似的实验。与之前的报道一致,观察到GArM-Ru1(2,3-Sia)在SW620人结肠腺癌细胞中的积累比HeLa人***来源细胞和A549人肺泡基底上皮腺癌细胞中的积累更高(图9)。
[实施例6]
前药治疗
接下来,研究了GArM-Ru复合体靶向抗癌治疗的适用性。伞形花醚(2h)被选为通过闭环复分解活化的候选药物的初步研究。
[化学式7]
已知伞形花醚是一种从阿魏属植物中提取的天然产物,其已显示出针对多种癌细胞株的细胞毒性活性(Shakeri,A.;Iranshahy,M.;Iranshahi,M.J.Asian.Nat.Prod.Res.2014,16,884-889;Rashidi,M.;Khalilnezhad,A.;Amani,D.;Jamshidi,H.;Muhammadnejad,A.;Bazi,A.;Ziai,S.A.J.Cell.Physiol.2018,233,8908-8918;Jun,M.;Bacay,A.F.;Moyer,J.;Webb,A.;Carrico-Moniz,D.BioorganicMed.Chem.Lett.2014,24,4654-4658)。细胞毒性的机制显示为通过将细胞阻滞在G1期、另外活化胱天蛋白酶8和9以及下调Bcl-2和Mcl-1来诱导凋亡(Barthomeuf,C.;Lim,S.;Iranshahi,M.;Chollet,P.Phytomedicine 2008,15,103-111;Gholami,O.;Jeddi-Tehrani,M.;Iranshahi,M.;Zarnani,A.H.;Ziai,S.A.Iran J.Pharm.Res.2013,12,371-376;Gholami,O.;Jeddi-Tehrani,M.;Iranshahi,M.;Zarnani,A.H.;Ziai,S.A.IranJ.Pharm.Res.2014,13,1387-1392)。由于其非常高的疏水性(主要由法呢基部分提供),伞形花醚前药(1h)被推理为基于白蛋白的ArM的理想底物。
[化学式8]
为了验证这一点,简化的香豆素衍生物1g和伞形花醚前药1h均用于动力学实验(图10)。
尽管通常已知香豆素前体在水性条件下具有差的RCM反应性,但香豆素衍生物1g的非常轻微的反应性(kcat/KM<1)是出乎意料的。对此的解释由结合实验结果显示,所述结果即前体1g针对白蛋白具有非常低的结合亲和力(KD为约129μM)。在另一方面,疏水性前药1h的显著高活性(与1g相比,kcat/KM为至少1500倍高)可能归因于1h的长烷基链部分,其有助于进入到疏水结合袋中。
接下来,进行了一系列实验以评价基于GArM的抗癌方法的细胞内活性。
通过使用设定特定浓度(对于SW620人结肠腺癌细胞为32μM;对于HeLa人***来源细胞和A549人肺泡基底上皮腺癌细胞为64μM)的前药1h进行细胞毒性测试,并改变GArM-Ru1的添加量。
用来自Promega的CellTiter 96TM水性单溶液细胞增殖测定法(MTS)进行细胞存活率测量。将培养的细胞以约103个细胞/孔的密度接种在FalconTM96孔微板中,并培养过夜。随后,除去培养基,添加不同浓度的化合物。用DMSO溶解化合物,并且添加至细胞的DMSO浓度为1%。在孵育96小时之后,将细胞培养基更换为85μL新鲜培养基。随后,添加15μLMTS试剂,将其在37℃下孵育2.5小时,然后在490nm处测量吸光度。在添加1%DMSO情况下的细胞吸光度作为对照,设置为100%,并计算细胞存活率。
为了解释糖链的靶向作用,用不含糖链的alb-Ru1作为对照,进行相似条件下的实验。通过这些结果,在所有三种癌细胞株中,前药1h和GArM-Ru1的混合物显著降低了细胞增殖(<5%),并且作用超过了相应浓度的前药1h和GArM-Ru1的作用(图11)。另一个重要的观察结果是前药1h和alb-Ru1的混合物的细胞毒性活性在有效性上要低得多。这表明通过糖链的靶向对于金属催化剂定位至细胞表面或细胞内部是必不可少的。
[实施例7]
检测乙烯的人工金属酶(ArM)(ArM乙烯探针;AEP)的合成。
图12描述了ArM乙烯探针(AEP)的合成方案。
7-二乙基氨基香豆素(DEAC)本身发出的荧光对周围溶剂的极性具有高敏感性,并且通过从极性环境(10%二氧六环/水)变化为非极性环境(60%二氧六环/水),DEAC-Ru的量子产率增加到约20倍。因此,在此合成中,AEP是基于包含烯烃的DABCYL灭活剂(猝灭剂)与alb-Ru之间的反应合成的。
7.1 AEP的制备
将30微摩尔HSA溶液(167μL,来自50nmol,300μM水溶液)和37μM DEAC-Ru溶液(167μL,来自62nmol,370μM二氧六环溶液)混合以制备alb-Ru。用包含10%二氧六环的pH 7.4PBS缓冲溶液将反应溶液填充至总量为1670μL。在通过添加HSA开始反应之后,轻轻搅拌反应溶液并在37度孵育1小时。随后,将反应溶液用Amicon超离心过滤器(30kDa)浓缩,并用PBS缓冲溶液洗涤3次。向经浓缩的alb-Ru溶液添加水以稀释至50μL,得到1mM储备溶液。为了制备AEP溶液,制备了100μM alb-Ru溶液(50μL,来自50nmol,1μM水溶液)和500μM DABCYL猝灭剂溶液(450μL,来自250nmol,555μM DMSO∶水=1∶8溶液)的混合溶液。轻轻搅拌反应溶液并在37度孵育5分钟。随后,将反应溶液用Amicon超离心过滤器(30kDa)浓缩,并用PBS缓冲溶液洗涤3次。向经浓缩的AEP溶液添加水以稀释至500μL,得到100μM储备溶液。
蛋白质复合体(alb-Ru,AEP)的荧光强度用配备有JASCO FMP-963微板读取仪的JASCO FP-6500荧光分光光度计测量。为了制备样品,将alb-Ru和AEP二者都制备成浓度为10μM的水溶液。然后将样品等分(100μL)到96孔微板中,并在λEX=420nm/λEM=463nm下进行测量。所有测量均进行3次。
此外,进行圆二色性(CD)分析以区分本研究中使用的蛋白质复合体(alb-Ru,AEP)结构的主要变化。对于CD光谱,使用0.1cm比色皿,并使用J-1500圆二色性光谱仪(JASCO)进行测量。使用10%二氧六环水溶液作为空白,并在扫描过程中自动从样品中减去该空白。以100nm/分钟的扫描速度记录200至250nm的数据。每种蛋白质复合体的浓度保持在2.3μM。
[结果]
由于猝灭剂,白蛋白结合袋中存在的RuQ复合体(DEAC-Ru-DABCYL复合体)具有显著低的荧光强度,通过比较图13中alb-Ru和AEP探针的荧光强度也可以清楚地看出这一点。此外,由于这些圆二色性(CD)完全重叠,因此确认未观察到蛋白质的主要结构变性(图14)。
7-2.通过AEP检测乙烯
通过AEP的乙烯检测机制是通过使乙烯与AEP中的钌催化剂反应,以允许替换为DABCYL猝灭剂并激活荧光信号。因此,在本实施例中,AEP用于检测果实和植物中的乙烯。
[猕猴桃的成像]
未成熟或成熟的猕猴桃在食品杂货店购买。为了获得包含外果皮、房室和柱轴的片,用厨刀将猕猴桃切成约2.0cm×4.5cm大小以制备猕猴桃片。为了追踪乙烯产生,将170μL AEP(400μM溶液)倒入10cm盘的中心。随后,允许样品作用于AEP溶液。样品在室温下孵育,并在一定时间之后(在1、24小时之后)进行成像。配备有ET-EBFP2/香豆素/减毒DAPI过滤器组(目录号49021)(Chroma Technology Corp.)的Keyence BZ-X710 All-in-oneFluorescence MicroscopeTM用于成像。分别在1/25秒和1/3.5秒的曝光设置下获得明场图像(彩色)和荧光图像。在4×放大率下获得多个成像图像,用BZ-X分析仪软件(Keyence)进行图像绑定和分析。
[拟南芥的成像]
拟南芥在23度、光强度为85μmol m-2s-1下在土壤中生长。施加10小时光照和14小时黑暗的光周期。为了追踪乙烯产量,首先从4到6周的植物获取叶。随后,用夹具从叶上剥下透明的表层皮,并将其置于具有透明底部的96孔板中。在添加水(100μL)之后,将样品在室温下孵育12小时,以消除损伤应激的作用。根据实验条件,根据需要添加不同的溶液,例如1mM ACC溶液(2μL 55mM水溶液)、4.8μM flg22或elf18溶液(5μM、100μM水溶液)和OD600=0.02假单胞菌溶液(2μL OD600=1.0的标准溶液)。在包含ACC和假单胞菌的实验中,在室温下进行12小时的孵育。在另一方面,在包含PAMP的实验中,在室温下进行6小时的孵育。随后,将这些溶液从表层皮样品中完全去除,并添加100μM AEP溶液(50μL,来自储备溶液)。在30分钟之后,完全除去溶液并用水洗涤,然后用配备有ET-EBFP2/香豆素/减毒DAPI过滤器组(目录号49021)(Chroma Technology Corp.)的Keyence BZ-X710 All-in-oneFluorescence MicroscopeTM进行成像。成像图像在20×、40×放大率下获得,并且明场图像(单色)和荧光图像分别在1/400秒和1/30秒的曝光设置下获得。在4×放大率下获得多个成像图像,并用BZ-X分析仪软件(Keyence)进行分析。
[结果]
果实中乙烯的空间检测
一般来说,在已达采收成熟期的果实中,自催化乙烯(***2)的产生是在成熟过程中进行的。这类似于外部刺激,例如损伤应激或病原体感染。在本研究中,首先通过AEP探针检测内源性乙烯。在猕猴桃中,报道了在成熟过程中,通过ACS等基因的上调,在外果皮中产生的乙烯量提高。在AEP情况下的猕猴桃成像实验中,重点观察了在不同发育阶段(未成熟和成熟)在多种细胞器(外果皮、房室和柱轴)中表达的乙烯产生量的变化。如图15所示,随着果实从未成熟状态成熟,在外果皮中观察到荧光强度显著提高。在另一方面,在猕猴桃房室和柱轴中未观察到荧光强度的如此多的变化。从这些结果来看,很明显,使用AEP是检测果实成熟时和成熟期间乙烯表达的有前景的方法。
植物中的乙烯检测
为了研究AEP检测在植物中的作用,选择小的开花植物拟南芥(十字花科)作为模型植物。为了肯定地显示可以通过APE检测乙烯的产生,用已知控制乙烯生产的低分子和涉及乙烯生产的多种植物变体进行比较实验。
在本研究中,拟南芥Col-0生态型被用作野生型模型植物。此外,还使用了广泛的多种植物变体,例如acs1/2/6/4/5/9/7/11或etol-1。变体acs1/2/6/4/5/9/7/11是敲除了8个ACS基因的变体,并且即使病原体入侵,乙烯量也不会提高。这是因为ACS负责与乙烯生物合成途径相关的重要作用。此外,过量产生乙烯的etol-1被用作另一种变体。通过乙烯过表达蛋白1(ETO1)的表达抑制蛋白酶体依赖性降解,并且正向控制ACS活性。这是基于II型ACS的C端与ETO1之间的相互作用。
图16显示了在添加或不添加AEP的情况下,在室温下孵育的Col-0的成像图像。如图17中量化的,观察到荧光强度显著提高,从而证实AEP可用于拟南芥表层皮中的乙烯检测。作为阳性对照,与由ACC外部刺激的Col-0(的乙烯产物)进行了比较。正如预期的那样,与野生型Col-0相比,显示出更高的荧光强度提高。
[实施例8]
通过糖基化的ArM选择性标记针对癌细胞的cRGD
在多项研究中已报道,环状Arg-Gly-Asp(cRGD)五肽抑制癌细胞表面上过表达的αvβ3和αvβ5整联蛋白,并有效防止黏附至细胞外基质(J.S.Desgrosellier,D.A.Cheresh,Integrins in cancer:Biological implications and therapeuticopportunities.Nat.Rev.Cancer 10,9-22(2010).;M.Pfaff,K.Tangemann,B.Muller,M.Gurrath,G.Muller,H.Kessler,R.Timpl,J.Engel,Selective recognition of cyclicRGD peptides of NMR defined conformation by αIIbβ3,αvB3,andα5β1integrins.J.Biol.Chem.269,20233-20238(1994).;和M.Aumailley,M.Gurrath,G.Muller,J.Calvete,R.Timpl,H.Kessler,Arg-Gly-655 Asp constrained withincyclic pentapeptides.Strong and selective inhibitors of 656 cell adhesion tovitronectin and laminin fragment P1.FEBS Lett.291,50-54657(1991).)。此外,已经表明,针对血管内皮细胞上表达的整联蛋白的基于RGD的拮抗剂通过抑制新生血管形成来促进肿瘤(P.C.Brooks,A.M.P.Montgomery,M.Rosenfeld,R.A.Reisfeld,T.Hu,G.Klier,D.A.Cheresh,Integrin αvβ3 antagonists promote tumor regression by inducingapoptosis of angiogenic blood vessels.Cell 79,1157-1164(1994).;和D.G.Stupack,D.A.Cheresh,Integrins and angiogenesis.Curr.Top.Dev.Biol.64,207-238(2004).)。
在本实施例中,研究了糖基化的ArM(GArM)是否可用于选择性细胞标记(SeCT)治疗以在单细胞水平上破坏癌细胞的黏附,其模拟了微转移的接种过程。特别地,通过选择性地将靶向HeLa的GArM复合体积累至癌细胞,然后选择性地将cRGD标记至表面蛋白,验证了这是否可能。
8-1.cRGD-PE的制备
用于制备cRGD-PE的方案如下:
[化学式9]
(化合物I的制备)
通过标准固相肽合成方法进行合成。在从固相支持物上切除之后,混合物通过反相HPLC在20至80%乙腈水溶液(0.1%TFA)的线性梯度条件(40分钟内)下直接纯化。产量:2.92g,89%,C53H83N9O14S([M+H]+)的ESI-HRMS m/z计算值为1102.5853,实测值为1102.5859。
(化合物II的制备)
将化合物I(90.1mg,0.0833mmol)和EDC(17.6mg,0.0916mmol)溶解在二氯甲烷(0.5mL)中。随后,将溶液在室温下搅拌3.5小时。为了进行后处理,添加二氯甲烷,有机层用水/饱和盐水洗涤,用硫化镁干燥,并在减压下浓缩。通过所得剩余物的质谱分析确定目标物质的产量。C53H81N9O13S([M+H]+)的ESI-HRMS m/z计算值为1084.5747,实测值为1084.5744。
随后,将获得的该受保护肽溶解在TFA/二氯甲烷(1∶1)溶液中2小时以进行脱保护。将混合物在减压下浓缩,并通过反相HPLC在20至80%乙腈水溶液(0.1%TFA)的线性梯度条件(40分钟内)下直接纯化。产量:54.5mg,77%(两步产率)。C27H41N9O8([M+H]+)的ESI-HRMS m/z计算值为620.3151,实测值为620.3169。
(化合物III的制备)
搅拌己二酸(1.0g,6.84mmol)的DMF溶液(20mL),并添加N-羟基琥珀酰亚胺(3.0g,27.4mmol)和EDC(5.14g,27.4mmol)。在室温下搅拌溶液24小时之后,在减压下浓缩反应混合物,将粗制产物溶解于200mL丙酮中,并逐滴添加250mL1M盐酸水溶液。在2小时之后,过滤白色沉淀物,并用水和丙酮洗涤以获得目标物质(1.75g,77%)。
1H NMR(500MHz,CDCl3,25℃)δ 2.84(s,4H),2.83(s,4H),2.67(t,J=3.5Hz,4H),1.89(t,J=3.5Hz,4H);13C NMR(125MHz,CDCl3,25℃)169.4,168.4,30.7,25.9,23.9·
C14H17N2O8([M+H]+)的ESI-HRMS m/z计算值为341.0979,实测值为341.0973。
(化合物IV的制备)
搅拌化合物II(28.8mg,0.034mmol)和化合物III(23.2mg,0.068mmol)的DMF溶液(680mL),并添加DIEA(16.8μL,0.10mmol)。在室温下搅拌溶液24小时之后,混合物用反相HPLC在与化合物I的制备中所使用的相同条件下直接纯化,以获得目标化合物(14.0mg,43%)。
1H NMR(500MHz,CD3OD,25℃)δ7.00(d,J=6.5Hz,2H),6.68(d,J=6.5Hz,2H),4.74(t,J=7.0Hz,1H),4.41(q,J=7.0Hz,1H),4.26-4.30(m,2H),3.91-3.86(m,1H),3.22-3.18(m,1H),3.14-3.10(m,1H),3.09(t,J=6.5Hz,2H),2.91-2.85(m,2H),2.84-2.78(m,5H),2.59(t,J=9.5Hz,2H),2.64-2.60(m,1H),2.58(dd,J=16.5,9.0Hz,1H),2.19(t,J=7.0Hz,2H),2.22(t,J=7.0Hz,2H),1.92-1.82(m,1H),1.74-1.60(m,6H),1.57-1.44(m,3H),1.44-1.32(m,2H),1.09-0.90(m,2H);
C37H53N10O13([M+H]+)的ESI-HRMS m/z计算值为845.3788,实测值为845.3769。
(cRGD-PE的制备)
搅拌化合物IV(4.2mg,3.9μmol)和化合物V(1.1mg,7.8μmol)的DMF溶液(200μL),并添加DIEA(3.3μL,19.6μmol)。在室温下搅拌溶液24小时之后,混合物用反相HPLC在与化合物I的制备中所使用的相同条件下直接纯化,以获得cRGD-PE(3.2mg,85%)。
δ1H NMR(500M Hz,CD3OD,25℃)7.02(d,J=7.0Hz,2H),6.71(d,J=7.0Hz,2H),4.76(t,J=7.5Hz,1H),4.75(s,2H),4.44(t,J=7.5Hz,1H),4.29-4.20(m,2H),3.98(s,2H),3.91(dd,J=9.5,3.0Hz,1H),3.25-3.20(m,1H),3.18-3.11(m,1H),3,11(t,J=7.0Hz,2H),2.96(s,1H),2.88(d,J=8.0Hz,2H),2.85(dd,J=16.5,8.0Hz,1H),2.58(dd,J=16.5,8.0Hz,1H),2.29(t,J=7.0Hz,2H),2.22(t,J=7.0Hz,2H),1.92-1.84(m,1H),1.74-1.60(m,6H),1.59-1.42(m,3H),1.41(t,J=8.0Hz,2H),1.09-0.80(m,2H);
C38H55N10O12([M+H]+)的ESI-HRMS m/z计算值为843.3995,实测值为843.4006。
8-2.GArM复合体的制备
使用先前报道的方法,合成具有末端α(2,3)-唾液酸的糖链-醛探针(R.Sibgatullina,K.Fujiki,T.Murase,T.Yamamoto,T.Shimoda,A.Kurbangalieva,K.Tanaka,Highly reactive“RIKEN click”probe for glycoconjugation onlysines.Tetrahedron Lett.58,1929-1933(2017).),以及与香豆素结合的金催化剂(K.Tsubokura,K.K.Vong,A.R.Pradipta,A.Ogura,S.Urano,T.Tahara,S.Nozaki,H.Onoe,Y.Nakao,R.Sibgatullina,A.Kurbangalieva,Y.Watanabe,K.Tanaka,In vivo goldcomplex catalysis within live mice.Angew.Chem.Int.Ed.56,3579-3584(2017).)。
在大气下将具有末端α(2,3)-唾液酸的糖链-醛探针在DMSO中的溶液(1μmol,15当量,260μL,来自3.8mM储备溶液)添加至人血清白蛋白溶液(5.3mL水溶液,66.7nmol)。轻轻搅拌溶液,并在37℃下孵育过夜。随后,将溶液用Amicon超离心过滤器(30kDa)浓缩,并用水洗涤。在用Durapore膜(Durapore PVDF 0.45μmTM)去除不溶性副产物之后,添加水进行稀释,以获得糖链白蛋白的1.0mM储备溶液。用MALDI-TOF-MS(阳性模式)确认结合的糖链的数量,并因此检测出每1分子白蛋白结合6.2分子糖链的糖链白蛋白的分子量(85.8kDa)。在该方案的下一步中,向糖链白蛋白溶液(66.7nmol)在PBS缓冲液(60.6μL,pH 7.4)中的溶液添加香豆素-金复合体(66.7nmol)在DMSO(6.1μL)中的溶液。轻轻搅拌溶液并在37度下孵育1小时,在此之后,用Amicon超离心过滤器(30kDa)浓缩溶液,并用PBS缓冲溶液进行洗涤以获得目标GArM复合体。
(细胞)
由于已知HeLa癌细胞过表达RGD特异性整联蛋白(α5β1,αvβ3,αvβ5),因此在本研究中选择HeLa细胞作为模型。所用的HeLa细胞由RIKEN的细胞工程部细胞库提供。
HeLa细胞在补充有10%热灭活胎牛血清(FBS)和1%青霉素-链霉素的杜氏改良Eagle′s培养基(DMEM)中培养。HeLa-Luc细胞株通过在含有10%FBS和0.01%嘌呤霉素的DMEM中培养的萤火虫萤光素酶和嘌呤霉素乙酰转移酶稳定转染HeLa细胞来制备。HeLa-V细胞株通过在含有10%FBS和1%青霉素-链霉素和0.8%遗传霉素的DMEM中培养的萤火虫萤光素酶和Venus(V)稳定转染HeLa细胞来制备。允许所有细胞株在含有5%二氧化碳的37℃培养箱中增殖。如前所述(A.Ray,B.N.Dittel,Isolation of mouse peritoneal cavitycells.JoVE,e1488723(2010).),小鼠巨噬细胞从施用有0.9%盐水(6mL)的BALB/c-nu/nu小鼠的腹膜腔收集。
8-3.验证GArM对HeLa细胞的选择性
为了确认GArM是否可以更有选择性地靶向HeLa细胞,进行了一系列流式细胞术研究。由于已知香豆素衍生物在与白蛋白结合时荧光强度会提高,因此在λEx=405nm/λEm=470nm处用流式细胞术测量的荧光强度将是香豆素结合的GArM复合体是否与细胞结合的指示。
流式细胞术和细胞分选用Sony SH800细胞分选仪(Sony Corporation)通过常用方法进行。流式细胞仪配备有405、488、561和638nm激光器,分别采用对于GArM检测λEx=405nm/λEm=470nm和对于Venus(V)检测λEx=515nm/λEm=528nm的激发/发射波长设门,并用Sony SH800软件分析结果。
首先,在细胞培养***中,在经GArM处理的HeLa细胞和未经处理的HeLa细胞之间的流式细胞术中观察到明显的峰值差异(图18)。通过将经GArM处理的HeLa细胞和未经处理的HeLa细胞混合,这种特性更加突出,并且看到了被明确定义为两个峰的峰。
接下来,作为对照实验,用从小鼠腹膜腔中提取的巨噬细胞检查了GArM的结合能力。比较流式细胞术直方图(图19),在添加和不添加GArM情况下的巨噬细胞之间仅观察到轻微差异,并且正如预期的那样,这表明没有或几乎没有结合。
在验证选择性的最终测试中,观察在添加或不添加GArM的情况下孵育的巨噬细胞和HeLa细胞的组合(图20)。在本实验中,为了区分观察HeLa细胞和巨噬细胞,使用了表达Venus(V)黄色荧光蛋白的HeLa-V细胞株。从流式细胞术谱中,可以清楚地区分表达Venus的HeLa细胞和巨噬细胞。发现当添加GArM时,大多数HeLa-V细胞迁移至右上部分,这显示GArM结合。在另一方面,对于巨噬细胞没有观察到这样的变化。因此,该数据强烈表明GArM可以在细胞水平上优先结合HeLa癌细胞。
8-4.细胞黏附测定
通过体外细胞黏附测定确定了SeCT标记试剂(GArM/cRGD-PE)基于整联蛋白阻碍细胞黏附的能力。用购自R&D System(Minneapolis,U.S.A.)的人纤连蛋白包被的96孔微板进行细胞黏附测定。
用市售MTS测定CellTiter 96水性单溶液细胞增殖测定(Promega)对黏附的细胞进行定量。在实验之前16小时,通过将增殖培养基更换为DMEM(无血清),使本实验中使用的HeLa细胞无血清。随后,将细胞传代培养至6 x 105个细胞/mL储备溶液的浓度,并通过另外的离心去除剩余的胰蛋白酶。接下来,在Eppendorf管中用HeLa细胞(360μL,来自细胞储备溶液)、cRGD-PE(45μL,来自0、160、320、640、1280、2560、5120、10240μM储备水溶液)和GArM(45μL,来自400μM PBS缓冲溶液)制备混合溶液。同时,将GArM替换为45μL的仅PBS缓冲溶液,用于制备对照溶液。用摇动振荡器在室温下孵育1小时。接下来,进行两次以下步骤:将细胞缓慢离心(以0.8ppm,4分钟)并去除上清液的洗涤步骤,然后重悬于450μL的仅DMEM(无血清)中。在纤连蛋白包被的96孔微板中,向每孔添加130μL经标记的HeLa细胞混合物,随后将该板在37℃下孵育10分钟。在去除培养基之后,用PBS缓冲溶液洗涤黏附的细胞3至4次。PBS缓冲溶液间歇地上下移液以去除非特异性结合的细胞。在最后步骤中,添加100μL DMEM(10%FBS+1%青霉索-链霉索)和20μLMTS试剂。在37℃孵育下4小时之后,用SpectraMaxiD3多模式微板读取仪(Molecular Devises,U.S.A.)获得490nm处的终点吸光度。
结果示于图21中。正如预期的那样,对于用SeCT标记试剂(GArM/cRGD-PE)处理的HeLa细胞,观察到纤连蛋白包被的板上细胞黏附的显著差异。当cRGD-PE处理浓度高时,这些差异尤其更加明显,这表明在细胞表面催化性地结合的cRGD具有更高的浓度。
Claims (27)
1.蛋白质与催化剂的复合体,所述催化剂选自金属催化剂或有机催化剂,其中
所述蛋白质是在其三维结构内具有疏水袋的蛋白质,并且
所述复合体将所述催化剂容纳在所述疏水袋中,使得所述催化剂不暴露于或者基本上不暴露于亲水环境。
2.根据权利要求1所述的复合体,其中所述蛋白质是天然或人工蛋白质。
3.根据权利要求1或2所述的复合体,其中所述蛋白质选自人血清白蛋白(HSA)、免疫球蛋白G(IgG)、免疫球蛋白A(IgA)、转铁蛋白、抗胰蛋白酶、触珠蛋白、α1-酸性糖蛋白、Myoferlin、Trk受体、***受体和叶酸受体。
4.根据权利要求1至3中任一项所述的复合体,其中所述金属催化剂选自硼催化剂、镁催化剂、铝催化剂、硅催化剂、钙催化剂、钪催化剂、钛催化剂、钒催化剂、铬催化剂、锰催化剂、铁催化剂、钴催化剂、镍催化剂、铜催化剂、锌催化剂、钇催化剂、锆催化剂、铌催化剂、钼催化剂、钌催化剂、铑催化剂、钯催化剂、银催化剂、铟催化剂、锡催化剂、钡催化剂、铪催化剂、钨催化剂、铼催化剂、锇催化剂、铱催化剂、铂催化剂、金催化剂和镧系元素路易斯酸催化剂。
5.根据权利要求4所述的复合体,其中所述镧系元素路易斯酸催化剂选自镱催化剂、镧催化剂、铈催化剂、钐催化剂、铕催化剂、钆催化剂、铽催化剂、铥催化剂和镥催化剂。
6.根据权利要求1至5中任一项所述的复合体,其中
所述蛋白质是人血清白蛋白,并且
所述催化剂是金属催化剂。
7.根据权利要求6所述的复合体,其中所述金属催化剂是钌催化剂。
8.根据权利要求6或7所述的复合体,其中所述人血清白蛋白的疏水袋是白蛋白药物结合位点I(药物位点I)。
9.根据权利要求6至8中任一项所述的复合体,其中所述金属催化剂通过针对人血清白蛋白的配体与所述疏水袋结合。
10.根据权利要求9所述的复合体,其中所述配体选自华法林、阿扎丙宗、醋硝香豆素、保泰松、水杨酸盐、吲哚美辛、苯妥英、甲苯磺丁脲、氯磺丙脲、碘酚酸、胆影酸、磺胺二甲氧嘧啶、苯丙香豆素、格列本脲、磺胺噻唑、替诺昔康、喜树碱、balzidendeazi、andelleratan、diadelleal、diadellealtan、氟硅酸钠、胆红素、类二十烷酸和羧基-甲基-丙基-呋喃丙酸酯(***毒素)、以及香豆素。
11.根据权利要求9或10所述的复合体,其中所述金属催化剂通过与所述配体结合的接头与所述疏水袋结合。
12.根据权利要求11所述的复合体,其中所述接头是在两端具有氨基和羧基的烷基链或聚乙二醇(PEG)链。
13.根据权利要求12所述的复合体,其中所述接头是C1至C3烷基。
14.根据权利要求1至13中任一项所述的复合体,其中所述蛋白质的表面被修饰以与体内靶位点相互作用。
15.根据权利要求14所述的复合体,其中所述修饰是通过糖链进行的修饰。
16.根据权利要求1至13中任一项所述的复合体,其中所述蛋白质还包含与体内靶位点相互作用的部分。
17.根据权利要求14或15所述的复合体,其中所述蛋白质是人血清白蛋白。
18.组合物,其包含根据权利要求1至17中任一项所述的复合体。
19.根据权利要求18所述的组合物,其是还包含可药用载体的药物组合物。
20.根据权利要求19所述的组合物,其与可以被所述复合体活化的前药组合使用。
21.根据权利要求19所述的组合物,其还包含可以被所述复合体活化的前药。
22.根据权利要求19所述的组合物,其用于选择性标记特定细胞。
23.根据权利要求22所述的组合物,其与针对所述细胞进行标记的化学物质组合施用。
24.根据权利要求18所述的组合物,其用作生物传感器。
25.根据权利要求18所述的组合物,其用作用于检测乙烯的生物传感器。
26.包含前药的药物组合物,其中
所述前药可以被根据权利要求1至17中任一项所述的复合体活化,并且
所述药物组合物与根据权利要求1至17中任一项所述的复合体组合使用。
27.组合药物,其包含
包含根据权利要求1至17中任一项所述的复合体的第一药剂,和
包含可以被所述复合体活化的前药的第二药剂。
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US20220241765A1 (en) | 2022-08-04 |
JPWO2020241340A1 (zh) | 2020-12-03 |
AU2020285197A1 (en) | 2021-12-16 |
KR20220012277A (ko) | 2022-02-03 |
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