CN108348622A - 辐射发射肽核酸缀合物及其用于疾病、状况和紊乱的诊断、成像和治疗的用途 - Google Patents
辐射发射肽核酸缀合物及其用于疾病、状况和紊乱的诊断、成像和治疗的用途 Download PDFInfo
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Abstract
本发明提供一种包含至少一种辐射发射探针和至少一种基因互补组分的缀合物、含有所述缀合物的组合物和其试剂盒,所述缀合物用于在对状况、疾病和紊乱的高灵敏度诊断、成像和治疗中使用。
Description
技术领域
本发明提供一种用于状况、疾病和紊乱的高灵敏度诊断、成像和治疗的辐射发射(红光到近红外光)PNA缀合物。
背景技术
[1]Maebert,K.,Cojoc,M.,Peitzsch,C.,Kurth,I.,Souchelnytskyi,S.,andDubrovska,A.(2014)Cancer biomarker discovery:Current status and futureperspectives,Int.J.Radiation Biol.90,659-677.
[2]Brink,M.,De Goeij,A.F.P.M.,Weijenberg,M.P.,Roemen,G.M.J.M.,Lentjes,M.H.F.M.,Pachen,M.M.M.,Smits,K.M.,De Bruine,A.P.,Goldbohm,R.A.,andVan Den Brandt,P.A.(2003)K-ras oncogene mutations in sporadic colorectalcancer in The Netherlands Cohort Study,Carcinogenesis 24,703-710.
[3]Heinzerling,L.,Baiter,M.,Kuehnapfel,S.,Schuler,G.,Keikavoussi,P.,Agaimy,A.,Kiesewetter,F.,Hartmann,A.,and Schneider-Stock,R.(2013)Mutationlandscape in melanoma patients clinical implications of heterogeneity of BRAFmutations,British Journal of Cancer 109,2833-2841.
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背景
在过去的几十年中,人们已经在开发癌症生物标志物以作为设计新药靶点和用于诊断目的的手段方面做出了很大努力1。
使用RNA作为生物标志物的优点在于,理论上检测不仅可以基于给定RNA分子在恶性细胞中的过度表达,还可以基于在许多类型的恶性肿瘤中表现的突变转录物和非突变转录物之间的区别2-6。在这方面,KRAS致癌基因是一种重要的生物标志物,因为它在许多类型的腺癌中被激活7,并且具有与肿瘤发生早期相关的常见单碱基突变8。例如,发现结直肠癌中90%的激活突变在第12位密码子(野生型GGT)4,并且最常见的突变类型是G转变为A9。因此,选择KRAS作为用于检测恶性细胞系中的mRNA的模式基因。
目前大多数RNA检测系统是基于标记的寡核苷酸(ODN)探针与靶遗传标志物的序列特异性杂交。已经研究了多种用于实时检测的标记分子;很多是基于荧光探针。已经开发出几类用于活细胞中RNA检测的荧光ODN,例如基于荧光共振能量转移(FRET)的寡核苷酸10-13、双重标记的发夹寡核苷酸(例如分子信标14-20和比例式生物分子信标(ratiometricbiomolecular beacon,RBMB)21,22)、芘修饰的寡核苷酸23,24、苝修饰的寡核苷酸25、杂交链式反应(HCR)探针26,27和金纳米粒子修饰的寡核苷酸28-35。
使用含有花青染料噻唑橙(TO)及其它花青的PNA分子作为经典核碱基的替代物36-41。这些强制插入探针(forced intercalation probe,FIT探针)在与其靶DNA/RNA序列杂交时显示出显著的荧光增强,使得它们适合于RNA和DNA的序列特异性检测。
这些PNA低聚物是基于含有柔性次甲基键的单次甲基花青染料的性质,柔性次甲基键有助于染料分子的非平面构象和非辐射衰减42。当插入dsDNA时,TO采取平面构象,并因此变得强烈地发荧光。此外,显示基于TO的FIT探针引起的荧光增强对双链体结构的局部扰动,例如由相邻碱基错配引起的扰动高度灵敏,允许检测SNP37。以前已经公开了,可以使用生物标志物通过靶向突变KRAS检测癌症43,44,并且可以在活细胞中以单核苷酸分辨率检测和区分该mRNA转录物。
然而,TO和其它相关的花青染料在从约500nm(TO)至更低波长的可见区域吸收光36。Hovelmann F.等人显示了被引入到DNA-LNA寡核苷酸并用于检测来自黑腹果蝇(Drosphila melanogaster)的发育中的卵母细胞中的mRNA的BisQ探针45。这些光谱特性不适于体内或原位成像,因为组织(例如血红蛋白)和细胞在这些波长自发荧光。
因此,仍然需要基于单错配或匹配的对活细胞和组织中的疾病和紊乱进行快速、直接和灵敏的诊断和成像。
一般描述
本申请的发明人已经发现用作用于疾病和紊乱的诊断、成像和治疗的灵敏探针的新缀合物。
本发明提供一种缀合物,其包含连接到(b)至少一种互补组分的(a)至少一种红光到近红外光(600-790nm)发射探针组分。
术语“互补组分”应理解为包括与细胞或组织(所述细胞或组织可以是受试者的细胞或组织、寄生虫的细胞或组织、生物体的细胞或组织等)中的靶核酸序列互补的任何部分。在一些实施方案中,所述至少一种互补组分选自肽核酸(PNA)、DNA序列和RNA序列及其任何组合。在一些进一步的实施方案中,所述互补组分是肽核酸(PNA)。在其他实施方案中,所述互补组分是DNA序列。在进一步的实施方案中,所述互补组分是RNA序列。在一些进一步的实施方案中,所述靶序列指示突变、状况或疾病。在其他实施方案中,所述靶序列指示在被给予所述缀合物的宿主主体中存在生物体(例如寄生虫)。在其他实施方案中,所述靶序列指示生物体对物质(诸如例如药物)的获得性遗传抗性。
术语“肽核酸(PNA)”应理解为包括包含10至25个碱基核苷酸(在一些实施方案中为16至18个碱基)的核苷酸序列,其被设计成与指示状况、对物质的获得性抗性、紊乱或疾病(所有这些都是遗传表现的)的特定序列(例如基因或致癌基因中的序列)或其突变互补。
在一些实施方案中,互补组分(例如PNA)被设计成与致癌基因的突变互补。已知这种致癌基因的突变与特定的恶性过程和疾病有关。例如,KRAS致癌基因的特定突变与结肠癌相关。因此,设计成与KRAS致癌基因的特定突变互补的互补组分(例如PNA)可用于本发明的缀合物用于诊断和成像所述结肠癌细胞的目的。
术语“红光到近红外光(NIR,near infra red)发射探针组分”是指化合物的当物理或化学性质发生任何变化时在约600nm至约790nm之间的波长范围内的红光到近红外光(在一些实施方案中为远红光)发射光谱具有荧光光谱性质的部分,所述物理或化学性质的变化包括但不限于:该部分的结构构象的变化、该部分与互补组分的连接性的变化、该部分的空间自由度的变化。探针部分的物理和/或化学性质的这种变化是由于本发明的缀合物的互补组分与细胞中目标靶序列(诸如例如指示状况或疾病的已知DNA/RNA序列)杂交而引起的。在一些实施方案中,所述探针是红光到远红光发射探针组分,其具有在约600nm至约750nm之间的波长范围内的辐射光谱。
术语“杂交”是指缀合物的互补组分与细胞中靶序列之间的键合相互作用。当缀合物的互补组分与靶序列(如DNA或RNA序列)杂交时,相对于非杂交(单链)形式的互补组分发射,在红光到近红外光的范围内有显著的荧光增强。
在一些实施方案中,所述红光到近红外光发射探针组分发射600nm至790nm波长范围内的辐射。在一些实施方案中,所述探针组分发射600nm至750nm范围内的辐射。在一些实施方案中,所述探针组分发射610nm至700nm范围内的辐射。在一些实施方案中,所述探针组分发射610nm至770nm范围内的辐射。在一些实施方案中,所述探针组分发射610nm至790nm范围内的辐射。在一些实施方案中,所述探针组分发射680nm至790nm范围内的辐射。在一些实施方案中,所述探针组分发射575nm-790nm范围内的辐射。在一些实施方案中,在所述探针被缀合到缀合物之前(即作为其中互补探针用例如H代替的独立分子)测量所述探针组分的波长为575nm-790nm。在一些实施方案中,在所述探针被缀合到缀合物之前(即作为其中互补探针用例如H代替的独立分子)测量所述探针组分的波长为575nm-600nm。
在一些实施方案中,红光到远红光发射探针组分包含至少一个由于本发明的缀合物的互补组分与靶细胞的目标序列杂交而改变其构象的键。在一些实施方案中,红光到近红外光发射探针组分包含一个次甲基键。
在一些实施方案中,红光到近红外光发射探针组分选自以下化合物:
术语“缀合物”是指包含至少上述两种组分(即红光到近红外光发射探针组分部分和互补组分)的化合物,所述组分在每个组分的任何位置通过包括化学键、配位键、氢键等的任何类型的键彼此连接。
例如,下面的方案1提供了本发明的缀合物的制备程序,其中长波长光发射探针(LWEP)分子(680nm)经反应以连接到靶向kRAS致癌基因的PNA序列。
方案1:本发明的缀合物的制备
在一些实施方案中,以互补组分是与靶序列互补的寡核苷酸序列的方式设计本发明的缀合物。
当本发明的缀合物的互补组分与活细胞、组织或生物体中的靶序列杂交时,该缀合物在红光到近红外光光谱显示出荧光增强,其被设计成互补。在红光到近红外光光谱的增强荧光是由于本发明的缀合物的红光到近红外光发射探针组分的构象变化。
重要的是要注意,这种在红光到近红外光光谱提供荧光增强的变化只能在互补组分与细胞、组织或生物体中的靶序列完全匹配时发生。由于互补组分的精确互补及其序列的精确设计,本发明的缀合物能够以单核苷酸多态性(SNP)分辨率检测活细胞或其它细胞中的特定序列。
在一些实施方案中,互补组分包括与靶核酸序列互补的序列。在一些实施方案中,所述靶核酸序列存在于活细胞中。在其它实施方案中,细胞存在于活生物体中。靶核酸序列可以是基因组序列(编码、调节或非编码DNA序列,或RNA(mRNA、iRNA、微RNA)。在一些实施方案中,所述基因组序列是DNA序列。在一些其它实施方案中,所述序列是RNA序列。
靶序列的非限制性实例如下:KRAS、kRAS、abl、Af4/hrx、akt-2、alk、alk/npm、aml1、aml1/mtg8、axl、bcl-2,3,6、bcr/abl、c-myc、dbl、dek/can、E2A/pbx1、egfr、enl/hrx、erg/TLS、erbB、erbB-2、ets-1、ews/fli-1、fms、fos、fps、gli、gsp、HER2/neu、hox11、hst、IL-3、int-2、jun、kit、KS3、K-sam、Lbc、lck、lmo1、lmo2、L-myc、lyl-1、lyt-10、lyt-10/Cα1、mas、mdm-2、mll、mos、mtg8/aml1、myb、MYH11/CBFB、neu、N-myc、ost、pax-5、pbx1/E2A、pim-1、PRAD-1、raf、RAR/PML、rash、rasN、rel/nrg、ret、rhom1、rhom2、ros、ski、sis、set/can、src、tal1、tal2、tan-1、Tiam1、TSC2、trk。
在一些实施方案中,互补组分被设计成含有具有与基因或致癌基因中已知突变互补的不同突变的序列。在下面提供的一些实例中,致癌基因是KRAS致癌基因,并且其已知的突变指示胰腺癌。
在一些实施方案中,互补组分被设计成与与紊乱、对特定物质的获得性抗性、状况或疾病相关的基因序列互补。在一些实施方案中,所述基因序列是突变的基因序列。
在一些实施方案中,以PNA序列与KRAS或kRAS的靶基因序列互补的方式设计BisQ-PNA缀合物。在进一步的实施方案中,以PNA序列与其它致癌基因的靶基因序列互补的方式设计BisQ-PNA缀合物。
在一些实施方案中,本发明的缀合物还包含至少一个设计成用于细胞内化的部分。在一些实施方案中,所述至少一个设计成用于细胞内化的部分是氨基酸序列。在一些实施方案中,所述另外的序列包含4个D-赖氨酸(例如在PNA的C末端)。在其它实施方案中,所述至少一个设计成用于细胞内化的部分是脂肪酸衍生物(诸如例如硬脂酰脂肪酸)。
术语“细胞内化”是指本发明的缀合物贯穿(enter)细胞屏障的能力。用于内化的另一术语是内吞作用,其中本发明的缀合物能够被细胞膜吞入并拖入细胞。这通过使用上述定义的部分来帮助并更有效地实现。
本发明范围内还包括缀合物,其进一步包含至少一种化疗剂,从而直接在细胞水平提供癌症的靶向治疗。
在一些实施方案中,缀合物进一步包含至少一种同位素标记的抗体,从而使用单克隆抗体载体提供靶向放射免疫疗法,其中辐射能量直接给予被靶向的癌细胞。
本发明还提供一种组合物,其包含至少一种如上文和下文所定义的缀合物。
在另外一些方面中,本发明提供了如上文和下文所定义的缀合物,用于在诊断和成像至少一种恶性状况或疾病中使用。
术语“恶性状况或疾病”是指呈现能侵袭邻近组织的异常细胞生长并能扩散到远处组织的任何癌性状况或疾病。这些状况和疾病包括但不限于:肾上腺皮质癌、膀胱癌、骨癌、骨肉瘤、恶性纤维组织细胞瘤、乳腺癌、伯基特淋巴瘤、类癌瘤、小脑星形细胞瘤、大脑星形细胞瘤/恶性神经胶质瘤、儿童、宫颈癌、结肠癌、皮肤T细胞淋巴瘤、促结缔组织增生性小圆细胞瘤、子宫内膜癌、室管膜瘤、食道癌、尤文肉瘤、性腺外生殖细胞肿瘤、肝外胆管癌、眼癌、视网膜母细胞瘤、胆囊癌、头颈癌、心脏癌症、肝细胞癌、霍奇金淋巴瘤、下咽癌、眼内黑色素瘤、胰岛细胞癌、卡波西肉瘤、喉癌、肝癌、肺癌、淋巴瘤、髓母细胞瘤、黑色素瘤、默克尔细胞癌、间皮瘤、口腔癌、蕈样肉芽肿、鼻咽癌、神经母细胞瘤、非小细胞肺癌、口咽癌、卵巢癌、胰腺癌、甲状旁腺癌、阴茎癌、咽癌、胸膜肺母细胞瘤、前列腺癌、直肠癌、肾细胞癌、视网膜母细胞瘤、横纹肌肉瘤、唾液腺癌、胃癌、睾丸癌、咽喉癌、胸腺癌、甲状腺癌、尿道癌、子宫癌、子宫内膜、子宫肉瘤、肾母细胞瘤(Wilms tumour)。
术语“诊断”是指确定受试者中疾病或状况的存在和状态的任何类型的医学诊断,而不论该受试者是否已经表现出任何状况或疾病的症状(在由于风险因素或年龄的常规诊断程序的情况下)。所述诊断可以在体内进行(向受试者给予本发明的缀合物或组合物并使所述受试者接受诊断装置和方法),或者所述诊断可以离体进行(在从所述受试者获得的身体样品上,无论是在给予本发明的缀合物或组合物之前或之后)。
术语“成像”是指为临床分析和医疗干预而创建身体内部的视觉表示。使用应用本发明的缀合物的该技术将允许确定被给予所述缀合物并暴露于红光到近红外光荧光辐射的受试者中疾病或状况(例如恶性状况)的位置和范围。
本发明进一步提供了用于在诊断至少一种遗传状况、紊乱或疾病中使用的本发明的缀合物。
术语“遗传状况、紊乱或疾病”应理解为包括由受试者或生物体基因组中的一个或更多个异常引起的任何状况、紊乱或疾病。在一些实施方案中,所述遗传状况、紊乱或疾病从出生时就存在(先天性)。在一些实施方案中,所述遗传状况、紊乱或疾病是遗传性的。在其它实施方案中,所述遗传状况、紊乱或疾病由生物体或受试者的DNA的新突变或改变引起。在一些实施方案中,所述遗传状况、紊乱或疾病是单个基因突变(常染色体显性、常染色体隐性、X连锁显性、X连锁隐性、Y连锁、线粒体)。在其它实施方案中,所述遗传状况、紊乱或疾病是多基因的。多因素紊乱、状况和疾病包括但不限于:心脏病、糖尿病、哮喘、自身免疫疾病如多发性硬化、癌症、纤毛病(ciliopathies)、腭裂、高血压、炎性肠病、智力障碍、情绪障碍、肥胖、屈光不正、不孕等。这类疾病和状况的非限制性实例包括:DiGeorge综合征、天使人综合征、Canavan病、Charcot–Marie–Tooth病、猫叫综合征(Cri du chat)、囊性纤维化、唐氏综合征、杜兴氏肌营养不良、血友病、Klinefelter综合征、神经纤维瘤病、苯丙酮尿症、Prader-Willi综合征、镰状细胞病、Tay-Sachs病、Turner综合征。在一些实施方案中,所述遗传状况、紊乱或疾病可以是与单核苷酸多态性(SNP)相关的状况。
在另外一些的方面,本发明提供了如上文和下文所公开的缀合物,用于在遗传状况或疾病的产前诊断中使用。
术语“产前诊断”是指在分娩前诊断胎儿的状况或疾病,其中所述诊断是对来源于所述孕妇的胎盘绒毛或羊水的细胞进行的。使用本发明的缀合物的这种诊断可以早在妊娠10-12周进行,其在短时间内提供灵敏和准确的结果。在其它实施方案中,所述产前诊断在妊娠15-18周进行,其在短时间内提供灵敏和准确的结果。
本发明还提供了一种用于检测胎儿遗传状况或疾病的方法,其包括以下步骤:将胎儿活细胞或活体组织的样品与本发明的缀合物一起孵育(其中所述PNA组分被设计成具有与突变的目标序列或指示遗传状况的序列互补的特定的已知序列);将孵育的细胞暴露于红光到远红光检测器,并且在红光到远红光光谱检测荧光信号。如果来源于胎儿的样品细胞或组织含有与遗传状况或疾病相关的靶序列,则由于互补组分和靶序列的杂交,当暴露于红光到近红外光辐射时,红光到近红外光发射探针组分将在红光到近红外光区域发射光。如果检测到红光到远红光发射,其提供了对所述胎儿的遗传状况或疾病的诊断。如果在将样品与本发明的缀合物一起孵育时没有获得互补组分的杂交,则诊断胎儿不具有所调查的遗传状况。使用本发明的缀合物的这种诊断可以早在妊娠10-12周进行,其在短时间内提供灵敏和准确的结果。在其它实施方案中,所述产前诊断在妊娠15-18周进行,其在短时间内提供灵敏和准确的结果。
本发明进一步提供了如上文和下文所定义的缀合物,用于在体外诊断和成像方法中使用。
在该方法下,从受试者切除样品组织并与本发明的缀合物一起孵育,然后将所述孵育的组织暴露于红光到近红外光荧光检测器,从而诊断所述组织中的恶性肿瘤。如果从受试者切除的样品细胞或组织含有与状况或疾病相关的靶序列,则由于互补组分和靶序列的杂交,当暴露于红光到近红外光辐射时,红光到近红外光发射探针组分将在红光到远红光区域发射光。如果检测到红光到近红外光发射,其提供了对所述受试者的遗传状况或疾病的诊断。如果在将样品与本发明的缀合物一起孵育时没有获得互补组分的杂交,则诊断受试者不具有所调查的遗传状况。
本发明进一步提供了如上文和下文中公开的缀合物,其用于在体内诊断和成像的方法中使用,体内诊断和成像即在受试者活体内的特定身体部位或组织内进行诊断和成像。
本发明提供了一种在体内对状况或紊乱进行诊断和成像的方法,其包括以下步骤:向受试者给予本发明的缀合物,使用红光到远红光荧光检测器对所述受试者身体的至少一部分进行成像,从而诊断所述受试者中的恶性紊乱。
如果受试者的细胞含有与状况或疾病相关的靶序列,则由于互补组分和靶序列的杂交,当暴露于红光到近红外光辐射时,红光到远红光发射探针组分将在红光到近红外光区域发射光。如果检测到红光到近红外光发射,其提供对所述受试者的状况或疾病的诊断。如果在将样品与本发明的缀合物一起孵育时没有获得互补组分的杂交,则诊断受试者不具有所调查的状况。
在本发明的一些实施方案中,在活细胞和组织中对状况或紊乱进行诊断和成像。
术语“活细胞或活体组织”指来源于或连接到包括所有细胞和组织成分的活生物体的任何细胞或组织。
本发明还提供了如上文和下文中公开的缀合物,用于在荧光引导手术中使用,荧光引导手术即是用于在手术期间检测荧光标记组分的诊断和成像技术。该技术允许在恶性肿瘤切除手术期间确定恶性组织的切除范围。
本发明提供了一种用于在恶性肿瘤切除手术期间或手术后确定恶性组织切除范围的方法,其包括以下步骤:从受试者的身体切除恶性肿瘤,将本发明的缀合物(包括与特定恶性肿瘤相关的突变互补的序列)与所述切除的恶性组织的边界的至少一部分(即所切除组织的外周)一起孵育,将所述孵育的组织暴露于红光到近红外光荧光检测器,从而确定恶性组织的切除范围。如果所述切除的恶性组织的边界仍然发射荧光信号,这将表明在所述受试者中存在残留的恶性组织,其可以被进一步切除。
本发明还包括用于早期诊断处于风险中的受试者的恶性紊乱的方法,该方法包括以下步骤:给予受试者本发明的缀合物,使用红光到近红外光荧光检测器对所述受试者的身体的至少一部分进行成像,从而诊断所述受试者的恶性紊乱。
术语“早期诊断”是指在恶性肿瘤的已知症状出现之前,确定所述个体的恶性紊乱或疾病的存在、程度或转移状况的早期阶段。例如,如果个体进行结肠镜检查,检测并切除了其结肠中的息肉,则通过本发明的方法检测息肉组织中的第12位密码子突变将诊断息肉是恶性还是良性。
本发明进一步提供了包含如上文和下文所定义的缀合物的试剂盒,其用于在遗传状况、疾病或紊乱的诊断中使用,并包括其使用的说明。
在另一方面,本发明提供了包含如上文和下文中定义的缀合物的试剂盒,用于在恶性状况、疾病或紊乱的诊断中使用,并包括其使用的说明。
在本发明的另一个方面中,本发明提供了包含如上文和下文所定义的缀合物的试剂盒,其用于在生物体的突变物质抗性的诊断中使用,并包括其使用的说明。在本发明的另一个方面中,本发明提供了包含如上文和下文中定义的缀合物的试剂盒,用于在生物体的单核苷酸多态性的诊断中使用,并包括其使用的说明。
术语“用于在诊断中使用的试剂盒”或“诊断试剂盒”应理解为包括用于在诊断受试者细胞或组织中的状况、疾病或紊乱中使用的一组工具。试剂盒提供的工具包括但不限于包含本发明的缀合物的组合物和使用所述组合物的说明。这样的说明可以包括用于对受试者细胞或组织中本发明的缀合物进行检测的装置的操作顺序、关于如何将所述组合物给予受试者的说明等。在一些实施方案中,本发明的所述试剂盒还包括用于向受试者给予本发明组合物的工具。在其它实施方案中,本发明的试剂盒包括用于从受试者身体组织取样以用于状况、疾病或紊乱的离体诊断的工具。
附图简述
为了更好地理解本文公开的主题并且举例说明如何在实践中进行,现在将描述实施方案。
图1描述了DMSO-d6中BisQ的1H NMR光谱。
图2表示PNA1的HPLC色谱图。洗脱液:A(于水中的0.1%TFA)和B(MeCN)以线性梯度使用(经38分钟11%-40%的B),流速为4mL/min。
图3表示PNA1的Maldi-TOF MS。Mcalc=5148.26,Mobs=5148.26。
图4表示PNA2的HPLC色谱图。洗脱液:A(于水中的0.1%TFA)和B(MeCN)以线性梯度使用(经38分钟11%-40%的B),流速为4mL/min。
图5表示PNA2的Maldi-TOF MS。Mcalc=5172.27,Mobs=5172.28。
图6表示TO和BisQ单体的紫外-可见光谱。
图7表示PNA1的紫外-可见光谱。在591nm处最大吸收。
图8表示PNA1:DNA的紫外-可见光谱。在587nm处最大吸收。
图9表示PNA2的紫外-可见光谱。在593nm处最大吸收。
图10表示PNA2:DNA的紫外-可见光谱。在593nm处最大吸收。
图11A-11B表示加入DNA后PNA FIT探针的荧光增强。记录了1.5μM的PNA1(图11A)和PNA2(图11B)在缓冲溶液中(黑色曲线)、有mmDNA时(2μM,黑色虚线)和有互补DNA时(2μM,灰色曲线)的荧光。
图12表示用0.5μM PNA2在37℃孵育3小时的Panc-1、HT-29和Bxpc-3细胞的荧光显微术图像。下图显示仅Panc-1细胞中的红光发射。
具体实施方式
以下实施例代表发明人在实施本发明的各方面时采用的技术。应当理解的是,尽管这些技术是用于实践本发明的优选实施方案的示例,但根据本公开内容,本领域技术人员将认识到,可以进行许多修改而不脱离本发明的精神和预期范围。
一般程序和材料
手动固相合成通过使用配备有烧结盘的5ml聚乙烯注射器反应器(Phenomenex)进行。使用60A、0.04-0.063mm硅胶(Biolab,Israel)和手动玻璃柱来进行所有的柱层析。TLC采用Merck硅胶板60F254(Merck Silica Gel 60F254plate)进行。使用半制备型C18反相柱(Jupiter C18,5u,250x10mm,Phenomenex)在50℃在Shimadzu LC-1090系统上进行HPLC纯化和分析。洗脱液:A(于水中的0.1%TFA)和B(MeCN)以线性梯度使用(经38分钟11%-40%B),流速为4mL/min。使用氘化溶剂作为内标,在300和600MHz的Bruker NMR上记录NMR谱。在ThermoQuest Finnigan LCQ-Duo ESI质谱仪上测量化合物1-5和BisQ的质谱测量值。在Orbitrap MS(Voyager DePro,Applied Biosystems,CA,USA)上获得PNA的质量分析。DNA寡核苷酸购自Sigma-Aldrich,Israel。无水DMF购自Acros,且Fmoc/Bhoc保护的PNA单体购自PolyOrg Inc.(USA)。用于固相合成的Fmoc-保护的氨基酸和试剂购自Merck(Germany)。
实施例1:BisQ的合成
BisQ的合成如下述方案2中所述并按照以下程序进行:
1-羧甲基-4-甲基喹啉溴化物(1).如前所述(L.Bethge,D.V.Jarikote和O.Seitz,Bioorg.Med.Chem.,2008,16,114-125)并稍作修改合成化合物1:将4-甲基喹啉(570mg,4mmol)和溴乙酸(607mg,4.4mmol)悬浮于10ml无水甲苯中并回流24小时。蒸发溶剂,将棕色残余物溶于DCM并冷却至0℃。逐滴加入丙酮(30ml),过滤收集固体并用丙酮(3×10ml)洗涤。将固体粗产物悬浮于氯仿中并搅拌1小时。过滤收集固体并用氯仿洗涤,得到1,为灰色固体(825mg,80%)。1H NMR(CD3OD):1.99(3H,s,CH3),3.79(2H,s,CH2),6.95(2H,t,J=7.1,2ArH),7.13(1H,t,J=7.8,ArH),7.22(1H,d,J=8.9,ArH),7.48(1H,d,J=8.5,ArH),8.15(1H,d,J=6.1,ArH).MS:Mobt=202.20,Mcalc=202.09。
1-甲基-氯喹啉碘化物(2).如前所述(R.Lartia和U.Asseline,Chem.-Eur.J.,2006,12,2270-228)并稍作修改合成化合物2:将4-氯喹啉(1g,7mmol)和碘甲烷(4ml,45mmol)合并并加热至45℃持续4小时。将得到的固体在冷乙醚(60ml)中沉淀并真空干燥,得到2,为黄色固体(1g,50%)。
4-[[1-羧甲基-4(1H)-亚喹啉基]甲基]-1甲基喹啉溴化物(3).将1(560mg,2mmol)、2(600mg,2mmol)和三乙胺(TEA,4mmol)在6ml无水DCM中的混合物搅拌1小时,产生深蓝色溶液。由于化合物3迅速分解,因此将其用于下一步反应而不进行纯化(见下文)。
Boc-Aeg-OtBu(4).如前所报道地合成Boc/t-Bu保护的PNA骨架(Y.Kam,A.Rubinstein,A.Nissan,D.Halle和E.Yavin,Mol.Pharm.,2012,9,685-693)。
Boc-Aeg(dye)-OtBu(5).向上一个合成步骤获得的化合物3的搅拌中的反应混合物中加入在3ml无水DMF中的(2mmol,686mg)等摩尔量的PyBOP(1040mg)、PPTS(500mg)、NMM(220μl)。搅拌该混合物10分钟,随后加入350mg(1.3mmol)的Boc-Aeg-OtBu。密封反应容器,并将反应混合物在氩气下于45℃搅拌过夜。减压下除去挥发物。粗产物经硅胶柱色谱法(含0%-15%MeOH梯度的DCM)纯化,得到蓝色膏状物(360mg,45%)。1H NMR(CD3Cl):8.3(d,2H,ArH),7.8(d,1H,ArH),7.67(d,2H,ArH),7.53(m,4H,ArH),7.34(t,2H,ArH),7.0(s,1H,CH),5.78(d,2H,CH2),4.38(s,1H,N-CH2),4.03(s,1H,N-CH2),3.96(s,3H,N+-CH3),3.9(s,2H,Gly-CH2),3.7(t,1H,N-CH2),3.28(t,1H,N-CH2),1.42(s,9H,t-Bu),1.40(s,9H,t-Bu)MS:Mobt=599.36,Mcalc=599.3。
Fmoc-Aeg(Dy)-OH(BisQ).将化合物5溶于DCM/TFA(1:1)的20ml混合物中。两小时后,蒸发溶剂,将所得浆状物溶于10ml DCM中。通过加入10当量(860μl)三乙胺将pH调节至约10。接着,在连续搅拌下逐滴加入(242mg,0.7mmol)的Fmoc-OSu。12小时后,蒸发溶剂,经硅胶色谱法(含20%MeOH的DCM)纯化粗混合物,接着经制备型HPLC(Luna 10微米,100A,C-18 250×21.2mm,Phenomenex)进一步纯化,使用于水中的0.1%TFA中的乙腈梯度(60分钟内12%-60%)。Rt=42分钟。产率(Yield)=40%。1H NMR(DMSO-d6):8.73(d,1H,ArH),8.63(d,1H,ArH),8.32(m,1H,ArH),7.97-7.87(m,5H,ArH),7.71(m,5H,ArH),7.56(m,3H,ArH),7.41(t,2H,ArH),7.31(t,2H,ArH),7.26(s,1H,CH),5.55(s,1H,CH2),5.33(s,1H,CH2),4.37(m,1H,Fmoc-CH2),4.35(s,0.5H,Gly-CH2),4.30(d,1H,Fmoc-CH2),4.24(t,0.5H,Fmoc-CH),4.20(t,0.5H,Fmoc-CH),4.12(s,3H,N+-CH3),4.02(s,1.4H,Gly-CH2),3.60(t,1H,N-CH2),3.39(m,2H,N-CH2),3.14(m,1H,N-CH2)。13C-NMR(DMSO-d6):(两种旋转异构体)δppm:37.7,38.6(N–CH2),41.8(CH3),46.5(Fmoc-CH),46.8,47(2xN–CH2),47.6,49(2xGly-CH2),53.6,54.2(2xCH2),65.2,65.4(2xFmoc-CH2),96.6,96.5(2xArC),107.4,109.3,109.5(3xArC),115.4,113(2xArCq),117,117.7(2xArC),119.9(Fmoc-ArC),120.1(ArCq),124.4(ArCq),124.9(Fmoc-ArC),125.1(ArCq),125.4,125.6(2xArC),126.3,126.8,127.4(3xFmoc-ArC),132,132.7(2xArC),138.4,140.5(2xFmoc-ArCq),142.9,143.1,143.9(3xArC),144(ArCq),148,149.7(2xFmoc-ArCq),155.9,156.3(2x Fmoc-COONH),166.1,166.5(2xDye-CON),170,171.9(Gly-COOH)。HRMS:Mobt=665.275,Mcalc=665.275。
方案2:BisQ的制备
实施例2:PNA1和PNA2的固相合成
第一个氨基与Novasyn TGA树脂的偶联.使树脂(250mg,0.2mmol/g)在10ml DMF中溶胀30分钟。为了预活化,在冰浴中将DIC(5当量)和DIMAP(0.1当量)加入到Fmoc-保护的甘氨酸(10当量)的DCM(15ml)溶液中。15分钟后,蒸发混合物,再溶于无水DMF并加入到树脂中。2.5小时后,用DMF(5×2mL)、CH2Cl2(5×2mL)洗涤树脂并重复该程序。
Fmoc裂解.向树脂中加入DMF/哌啶(4:1,1ml)的溶液。2分钟后,重复该程序。最后用DMF(3×1ml)、DCM(3×1ml)洗涤树脂。
Fmoc-Bhoc-PNA-单体的偶联.将4当量的PNA单体、4当量的HATU、4当量的HOBt和8当量的无水DIPEA在DMF(1.5ml)中在配备有螺口盖的玻璃小瓶中混合。预活化3分钟后,将溶液转移至树脂。60分钟后,弃去反应混合物,用DMF(2×1ml)和DCM(2×1ml)洗涤树脂。
Fmoc-Aeg(染料)-OH(BisQ)的偶联.将4当量的PNA单体、4当量的HATU、4当量的HOBt和8当量的无水DIPEA在DMF(1.5ml)中在配备有螺口盖的玻璃小瓶中混合。预活化3分钟后,将溶液转移至树脂。60分钟后,重复该程序,最后用DMF(2×1ml)和DCM(2×1ml)洗涤树脂。
PNA从树脂上的裂解.将1ml TFA加入到无水树脂中。2小时后加入另一部分TFA。将合并的TFA溶液真空浓缩。
表1.PNA和DNA的序列
下划线表示与PNA互补的序列。
双下划线表示KRAS序列中的突变位点。
PNA纯化.通过加入冷乙醚(15ml)从浓缩的TFA溶液中沉淀出PNA。通过离心和倾析上清液收集沉淀物。将残余物溶于水中并经半制备型HPLC纯化。利用Orbitrap-MS分析纯化后的PNA。
荧光光谱法.使用Jasco FT-6500光谱仪记录荧光光谱。在PBS缓冲溶液(100mMNaCl,10mM NaH2PO4,pH 7)中以0.5-1.5μM浓度在荧光石英比色皿(10mm)中进行测量。如所述的(doi:10.1016/j.tet.2013.03.005)在PBS中确定相对于荧光素的量子产率。通过将PNA:DNA(10-30μM)的1:1混合物加热至95℃持续5分钟,然后缓慢冷却至25℃,从而使PNA与互补DNA杂交。样品在587nm激发,并在600-800nm记录发射光谱。
表2.BisQ和PNA的光物理特性
实施例3:细胞实验
细胞系和培养.使用三种细胞系:Panc-1、BxPC-3(人胰腺癌,上皮样)和HT-29(人结肠腺癌II级)。细胞系购自美国典型培养物保藏中心(ATCC,Manassas,VA,USA)。分别在补充有10%胎牛血清、2mM L-谷氨酰胺和0.1mg/mL链霉素(Beit Haemek BiologicalIndustries,Israel)的DMEM培养基中培养表达突变的和野生型KRAS的Panc-1和HT-29(37℃,5%CO2)。将表达野生型KRAS的BxPC-3细胞在补充有10%胎牛血清、2mM L-谷氨酰胺和0.1mg/mL链霉素的RPMI 1640培养基中培养。
细胞摄取分析.在加入PNA之前24小时,将Panc-1、HT-29和BxPC-3分别铺在载玻片(chamber slides)(Ibidi GmbH,Munich,Germany)上,直至达到70%-80%汇合。
活细胞中的杂交和成像.在加入PNA之前,更换培养基并将细胞与0.5μM的PNA1和PNA2在完全培养基中一起孵育(37℃,含5%CO2的潮湿气氛)。细胞成像前用PBS(×3)洗涤细胞,3小时后利用共聚焦显微术测量细胞内荧光。设计并合成具有远红光发射的独特特性的新型替代碱基(BisQ)。BisQ被引入到靶向突变的kRAS致癌基因的PNA中。显示具有由4个D-赖氨酸组成的短细胞穿透肽(CPP)的PNA探针容易穿透活的癌细胞,并且仅在表达突变形式的kRAS的胰腺癌细胞(Panc-1)中在远红光区(λmax=609nm)发荧光,而在未突变(野生型)的胰腺癌细胞中(BxPC-3)不发荧光。
实施例4:Acr-2的合成
Acr-2的合成如下述方案3中所述并按照以下程序进行:
乙醇酸苄基酯(1a)的制备.向80ml甲苯中加入乙醇酸(3g)和DBU(6g,1当量)并允许搅拌15分钟。逐滴加入苄基溴(8g或5.6mL,1.2当量),并将反应混合物回流6小时。然后用1M HCl(50mL×2)和水(50ml)萃取反应混合物。有机层用无水Na2SO4干燥并真空浓缩。将浓缩物进行色谱分离(二氧化硅,己烷中15%的EtOAc),得到无色液体。1H NMR(CDCl3):δ7.36(s,5H),5.21(s,2H),4.20(d,2H),2.93(t,1H)。
乙醇酸苄基酯的三氟甲磺酸酯(1b)的制备.在-20℃,在5-10分钟内向乙醇酸苄基酯(2g)和吡啶(91.05g,1.1当量)的DCM(50ml)溶液中加入三氟甲磺酸酐(3.73g,1.1当量)。加入完成后,将反应混合物搅拌30分钟并升温至室温,再搅拌30分钟。蒸发反应混合物并使其迅速通过短硅胶柱,用DCM洗脱。蒸发级分为淡黄色油状物,当在0℃保存时其固化。1HNMR(CDCl3):δ7.38(s,5H),5.28(s,2H),4.93(s,2H)。
吖啶酯(1c)的合成.在-15℃向9-甲基吖啶(200mg)的无水DCM(5ml)溶液中加入乙醇酸苄基酯的三氟甲磺酸酯(340mg,1.1当量),历时5-10分钟。加入完成后,将反应混合物搅拌30分钟并升温至室温,搅拌过夜。加入无水乙醚,在此期间产物以固体形式沉淀,随后过滤并用乙醚洗涤。1H NMR(300MHz,丙酮):δ8.80(d,2H)8.46(d,2H),8.32(t,2H),7.98(t,2H),7.38(s,5H),5.32(s,2H),3.70(s,2H),3.55,(s,3H)。
吖啶酯染料(1d)的合成.将9-甲基吖啶酯(163mg)悬浮于无水DCM(5ml)中并允许搅拌5分钟。加入1-甲基-氯喹啉碘化物并搅拌5分钟。将三乙胺一次加入到反应混合物中(溶液变成红色至紫色)并允许搅拌过夜。蒸发溶剂,粗产物用乙醚重复洗涤。深色残余物经柱层析(含10%MeOH的DCM)纯化。1H NMR(300MHz,丙酮):δ9.11(d,1H),8.89(d,1H),8.48(d,1H),8.26(t,1H),8.00(t,1H),7.89(d,2H),7.65,(d,1H)7.50(t,4H),7.38,(t,5H)7.08,(t,2H)7.05,6.01,(d,1H)5.32(s,2H),5.25(s,2H),4.68(s,3H),3.88,(s,1H)。13CNMR(75MHz,丙酮):δ168.70,(C=O),157.57(Ar-C),146.89(Ar-C),142.59(Ar-C),140.21(Ar-C),135.92(Ar-C),135.13(Ar-C),131.00(Ar-C),128.93(Ar-C),128.48(Ar-C),127.57(Ar-C),127.02(Ar-C),122.09(Ar-C),119.00(Ar-C),118.79(Ar-C),114.88(Ar-C),111.96(Ar-C),66.93,(Ar-CH2),48.93(N-CH2),44.47(CH3)。HRMS:Mobs=483.206,Mcalc=483.207。
乙醇酸苄基酯脱保护的吖啶酯染料(1e)的合成.将由苄基保护的吖啶酯(100mg)悬浮于乙酸中的30%HBr溶液(3mL)中,在50℃加热30分钟,并真空蒸发溶剂。将残余物与甲苯(5ml)共蒸发并用乙醚充分洗涤,用DCM-水萃取(30ml×3)而纯化。酸立即偶联到PNA骨架而无需进一步纯化。1H NMR(300MHz,D2O):δ8.77(d,1H),8.56(d,1H),8.30(t,5H),8.21(t,3H),8.10(t,1H),7.69(t,2H),6.69(d,1H),5.96(s,2H),4.37(s,3H),4.27(s,1H)。
连接到Fmoc骨架的吖啶-喹啉染料(1f)的合成.在0℃向Acr-酸(100mg)的无水DMF(3ml)溶液中加入PyBOP(1.28当量)、HOBT(1.28当量)和NMM(1.28当量),在氩气下搅拌15分钟,直至得到澄清溶液。向该溶液中加入单独制备的Fmoc-Aeg-烯丙基酯(1.28当量)和NMM(1.28当量)在DMF(1ml)中的样品,并允许将反应混合物在室温搅拌12小时。在反应完成后(如tlc所示),将反应混合物用水稀释,然后用EtOAc(3×20mL)萃取。将合并的有机层用10%NaHCO3洗涤,然后用10%柠檬酸洗涤。将合并的有机层再次用10%NaHCO3水溶液洗涤,然后用水和盐水洗涤。将有机层用无水Na2SO4干燥并真空浓缩。粗产物经使用含10%MeOH的DCM作为洗脱剂的柱层析纯化。1H NMR(300MHz,CDCl3):δ8.53(d,1H),8.46(d,1H),8.42(d,1H),8.13(t,1H),8.03(d,2H),8.00(d,1H),7.94(t,1H),7.90(d,2H),7.79(d,1H),7.71(d,2H),7.66(d,1H),7.49(t,2H),7.43(t,2H),7.36(t,2H),7.10(t,2H),7.05(t,2H),7.01(t,2H),6.98(t,1H),6.02(q,1H),5.47(d,1H),4.93(d,2H),4.35(s,3H),4.15(d,1H),3.89(d,2H),3.74(s,2H),3.47(d,2H),2.95(s,2H),2.87(s,2H)。
方案3:Arc-2的制备
实施例5:Acr-1的合成
如下述方案4所述,以与Acr-2类似的方式进行合成。
方案4:Arc-1的制备。
Claims (27)
1.一种缀合物,包含与至少一种互补组分连接的至少一种红光到近红外光(NIR)发射探针组分。
2.根据权利要求1所述的缀合物,其中,所述红光到远红光发射探针组分发射在600nm至790nm范围内的辐射。
3.根据权利要求1或2所述的缀合物,其中,所述红光到远红光发射探针组分包含至少一个次甲基键。
4.根据权利要求1至3中任一项所述的缀合物,其中,所述红光到近红外光发射探针组分选自:
5.根据前述权利要求中任一项所述的缀合物,其中,所述至少一种互补组分选自肽核酸(PNA)、DNA序列和RNA序列及其任何组合。
6.根据前述权利要求中任一项所述的缀合物,其中,所述至少一种互补组分包含与靶基因序列互补的寡核苷酸序列。
7.根据权利要求5或6所述的缀合物,其中,所述靶基因序列是DNA或RNA序列。
8.根据权利要求5至7所述的缀合物,其中,所述靶基因序列是与紊乱、状况或疾病相关的突变序列。
9.根据权利要求5至7中任一项所述的缀合物,其中,所述靶基因序列是KRAS或kRAS。
10.根据前述权利要求中任一项所述的缀合物,还包含至少一种用于细胞内化的序列。
11.根据前述权利要求中任一项所述的缀合物,还包含至少一种化疗剂。
12.根据前述权利要求中任一项所述的缀合物,还包含至少一种同位素标记的抗体。
13.一种组合物,包含至少一种根据权利要求1至12中任一项所述的缀合物。
14.根据权利要求1至12中任一项所述的缀合物,用于在至少一种恶性状况或疾病的诊断和成像中使用。
15.根据权利要求1至12中任一项所述的缀合物,用于在至少一种遗传紊乱或疾病的诊断中使用。
16.根据权利要求1至12中任一项所述的缀合物,用于在遗传紊乱或疾病的产前诊断中使用。
17.根据权利要求1至12中任一项所述的缀合物,用于在荧光引导手术中使用。
18.根据权利要求14至17中任一项所述的用于使用的缀合物,其中,所述诊断和成像在体外进行。
19.根据权利要求14至17中任一项所述的用于使用的缀合物,其中,所述诊断和成像在体内进行。
20.根据权利要求14至17中任一项所述的用于使用的缀合物,其中,所述诊断和成像在活细胞中进行。
21.一种检测胎儿遗传状况的方法,包括以下步骤:将根据权利要求1至20中任一项所述的缀合物与胎儿活细胞样品一起孵育;以及在红光到远红光光谱将所孵育的细胞暴露于红光-近红外光荧光检测器,从而诊断所述胎儿的遗传状况。
22.一种体外诊断受试者组织的恶性状况的方法,包括以下步骤:将根据权利要求1至20所述的缀合物与从所述受试者切除的组织一起孵育,将所孵育的组织暴露于红光-近红外光荧光检测器,从而诊断所述组织中的恶性肿瘤。
23.一种体内诊断恶性状况或紊乱的方法,包括以下步骤:向受试者给予根据权利要求1至20所述的缀合物,使用红光-近红外光荧光检测器对所述受试者的身体的至少一部分进行成像,从而诊断所述受试者中的恶性紊乱。
24.一种在恶性肿瘤切除手术期间或手术后利用荧光引导手术来确定恶性组织切除范围的方法,包括以下步骤:从受试者的身体切除恶性肿瘤,将权利要求1至20中任一项所述的缀合物与所切除的恶性组织的边界的至少一部分一起孵育,将所孵育的组织暴露于红光-近红外光荧光检测器,从而确定恶性组织的切除范围。
25.一种用于早期诊断处于风险中的受试者的恶性紊乱的方法,包括以下步骤:向受试者给予根据权利要求1至20所述的缀合物,使用红光-近红外光荧光检测器对所述受试者的身体的至少一部分进行成像,从而诊断所述受试者的恶性紊乱。
26.一种试剂盒,包含权利要求1至20中任一项所述的缀合物,用于在遗传状况、疾病或紊乱的诊断中使用,并包含其使用的说明。
27.一种试剂盒,包含权利要求1至20中任一项所述的缀合物,用于在恶性状况、疾病或紊乱的诊断中使用,并包含其使用的说明。
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PCT/IL2016/050878 WO2017025968A1 (en) | 2015-08-11 | 2016-08-11 | Radiation emitting peptide nucleic acid conjugates and uses thereof for diagnosis, imaging, and treatment of diseases, conditions and disorders |
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CN101999000A (zh) * | 2008-01-04 | 2011-03-30 | 国家科研中心 | 乳腺癌的分子体外诊断 |
CN104328164A (zh) * | 2013-07-22 | 2015-02-04 | 上海星耀医学科技发展有限公司 | 荧光探针杂交法检测人egfr基因突变试剂盒 |
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CN101899496A (zh) * | 2009-05-27 | 2010-12-01 | 广州达健生物科技有限公司 | 一种K-Ras基因突变分型荧光定量PCR检测试剂盒及其检测方法 |
CN104328164A (zh) * | 2013-07-22 | 2015-02-04 | 上海星耀医学科技发展有限公司 | 荧光探针杂交法检测人egfr基因突变试剂盒 |
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