CN115109047B - 一种基于protac设计的铁死亡诱导剂 - Google Patents
一种基于protac设计的铁死亡诱导剂 Download PDFInfo
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- CN115109047B CN115109047B CN202111050266.3A CN202111050266A CN115109047B CN 115109047 B CN115109047 B CN 115109047B CN 202111050266 A CN202111050266 A CN 202111050266A CN 115109047 B CN115109047 B CN 115109047B
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- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical group CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000000472 traumatic effect Effects 0.000 description 1
- 125000004306 triazinyl group Chemical group 0.000 description 1
- 125000001425 triazolyl group Chemical group 0.000 description 1
- ITMCEJHCFYSIIV-UHFFFAOYSA-M triflate Chemical group [O-]S(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-M 0.000 description 1
- 230000034512 ubiquitination Effects 0.000 description 1
- 238000010798 ubiquitination Methods 0.000 description 1
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- C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
- C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
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- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
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- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Abstract
本发明涉及一种基于PROTAC设计的铁死亡诱导剂,其结构如通式I所示,比小分子抑制剂更有效,只需要更低剂量的化合物就可以降解细胞内靶标蛋白,具有较高的安全性、耐药性和广阔的应用前景。
Description
技术领域
本发明涉及医药领域,具体涉及一种基于PROTAC的铁死亡诱导剂,其制备方法以及医药用途。
背景技术
死亡不仅是所有细胞的最终命运,而且它与细胞分裂、增殖一样,在整个机体的生长、发育中具有不可替代的作用。目前认为,除了坏死外,细胞死亡形式分为程序性细胞死亡(programmed cell death,PCD),包括凋亡(apoptosis) 和自噬(autophagy),及非程序性细胞死亡(non-programmed cell death,NPCD),包括副凋亡(paraptosis)、细胞有丝分裂灾难(mitotic catastrophe)和胀亡(oncosis) 等。2012年,Dixon等新提出了一种叫做铁死亡(ferroptosis)的铁依赖性的细胞死亡形式,该死亡方式受细胞内信号通路的严密调节。铁死亡的实质是由于膜脂修复酶—谷胱甘肽过氧化物酶(GPX4)失效,细胞内脂质氧化物代谢障碍,在铁离子催化作用下,造成膜脂上活性氧自由基(ROS)堆积,使细胞内氧化还原失衡,诱导细胞死亡,释放前炎症介质(如HMGB1等)。GPX4酶受到抑制(如RSL3, ML162)也可以直接导致这一作用,而铁离子螯合剂则可以抑制这一过程。现有 GPX4蛋白小分子抑制剂需要的有效浓度高,没有靶向性,由于只有一个结合位点,容易出现耐药性等的问题。
PROTAC(proteolysis-targeting chimeras)是一种利用泛素-蛋白酶体系统(Ubiquitin-Proteasome System,UPS)对靶蛋白进行降解的药物开发技术。
在结构上,PROTAC包括三个部分:一个E3泛素连接酶配体和一个靶蛋白配体,两个活性配体通过特殊设计的“Linker”结构连接在一起,最终形成三联体的“PROTAC”的活性形式。利用UPS系统降解蛋白主要包括三个步骤:首先,泛素分子(Ubiquitin,Ub)通过共价结合和E1(泛素激活酶)结合,紧接着通过ATP 依赖的方式泛素分子被激活;随后,激活的泛素分子被传递给E2(泛素结合酶);第三,E3泛素连接酶利用共价结合将E2酶结合的泛素分子转移给底物蛋白,将底物蛋白标注,上述过程叫做蛋白的泛素化;最终,被泛素分子标记的底物蛋白被26S蛋白酶复合体识别并酶解。
在患者体内,PROTAC的靶蛋白配体和靶蛋白结合,E3泛素连接酶配体和细胞内的E3泛素连接酶的底物结合区结合,从而通过Linker把靶蛋白“拉近”到 E3泛素连接酶旁边,实现UPS系统将靶蛋白降解。
PROTACs分子量一般在700~1200之间,这使得它们的透膜能力与(口服)生物利用度较差,且缺少如适用于小分子药物的“类药五原则”的预测模型,这使得目前大部分的研究仅仅证明了所设计的PROTAC在细胞水平上对靶蛋白降解的有效性与抗增殖活性。
因此,通过PROTAC药物开发技术研发出相对于GPX4蛋白小分子抑制剂更高效的、靶向性的药物将具有更为广泛的应用前景。
发明内容
一方面,本发明提供一种通式I化合物:
其中,代表E3泛素连接酶的配体;
Ar1,Ar2可以相同或不同,各自独立的选自无取代或任选被一个,两个或更多个Ra取代的3-12杂环基、C6-C14芳基、5-14元芳杂基;
所述Ra各自独立的选自H,卤素、氨基、羟基、氰基、硝基、氧代 (=O)、C1-6烷基,C1-6烷氧基。
r选自0,1,2,3,4,5;
p选自0,1,2,3,4,5;
q选自0,1,2,3,4,5;
s选自1,2,3,4;
R1,R2可以相同或不同,各自独立的选自H,C1-6烷基,C1-6烷氧基,以及可离去基团-LG;优选的,所述离去基团-LG可以选自本领域理解的常规离去基团,在一些实施方案中,可以选自卤素或磺酸酯离去基团,例如甲磺酸酯、甲苯磺酸酯或三氟甲磺酸酯;
所述-L-代表连接基团,可以选自无取代,或任选含有一个或多个杂原子的 C1-50烷基,所述杂原子可以选自O,S;
根据本发明的实施方案,所述可以选自常规E3泛素连接酶配体(本领域技术人员可以理解,所述配体以其任意合适的位点与-L-进行连接)。所述配体结构例举如下:
根据本发明优选的实施方案,所述可以选自如下结构:
其中,Rb选自H、卤素、氨基、羟基、氰基、硝基、-NHCH2COOH、C1-6烷基、C1-6烷氧基、C2-6烯基氧基、C2-6炔基氧基;t 选自0,1,2,3,4,5;
在一些实施方案中,所述-L-可以选自C1-50烷基或-(CH2)m(OCH2CH2)n-,其中,m选自0-10,例如为0,1,2,3,4,5,6,7,8,9,10;n选自0-20,例如选自0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17, 18,19,20;
在一些实施方案中,所述Ar1,Ar2可以相同或不同,各自独立的选自无取代或任选被一个,两个或更多个Ra取代的苯、噻吩。
根据本发明的实施方案,所述通式I进一步选自如下式II、式III结构:
通式II中,Ar1,Ar2,R1,R2,Rb,p,q,r,s,t,-L-如前文所定义。
通式III中,Ar1,Ar2,R1,R2,Rb,p,q,r,s,t,n如前文所定义。
根据本发明的实施方案,所述通式I结构可以选自如下化合物:
本发明提供一种通式I化合物的制备方法,包括如下步骤:
根据本发明的实施方案,所述通式I的制备方法具体可以进一步包括如下步骤中的一步或更多步:
步骤一、c分子的合成:将b分子溶解于有机试剂A中,加入碱性试剂,在冰浴下反应20分钟~1小时,加入TsOCl并反应2~5个小时;
步骤二、d分子的合成:将c分子溶解于有机试剂B中,加入叠氮化钠,金属浴下80~100℃反应过夜,用水和有机试剂C萃取得到反应粗产物。
步骤三、通式I的合成:将抗坏血酸钠和五水硫酸铜溶解于水溶液中,将 d分子(1eq)和a分子溶解于有机试剂B中,冰浴下反应2~5小时,后处理得到通式I化合物。
根据本发明的实施方案,所述有机试剂A选自四氢呋喃、二氯甲烷;所述有机试剂B选自DMSO、DMF;所述有机试剂C选自乙酸乙酯、二氯甲烷。
根据本发明的实施方案,本领域技术人员可以理解,上述制备方法中,可以选择从任一原料(包括路线的中间体)开始得到最终产品。
又一方面,本发明提供一种脂质体,所述脂质体包含通式I化合物。
根据本发明的实施方案,所述脂质体以阳离子脂质体为载体,包裹通式I化合物,制备得到阳离子脂质体/PROTAC复合物。
根据本发明的实施方案,所述阳离子脂质体包括401-ROS、Cholesterol(胆固醇)、DOPE(二油酰磷脂酰乙醇胺)中的一种或多种;
根据本发明的实施方案,所述脂质体的制备方法包括如下步骤:
(1)将所述脂质体中的一种或多种加入反应容器中,然后与通式I化合物混合,在化学罩中去除有机溶剂过夜,形成一层薄膜;
(2)将DSPE-PEG-2000溶于PBS缓冲液中,然后将步骤(1)制备得到的薄膜溶于乙醇中,加入到前述PBS溶液中。
根据本发明的实施方案,优选的,所述脂质体选自401-ROS、Cholesterol、 DOPE三者的混合物,更优选的,所述三者混合物中,401-ROS:Cholesterol:DOPE 的质量比为2:1:0.5。
又一方面,本发明提供所述通式I化合物或其脂质体在制备抗肿瘤药物中的应用。根据本发明优选的一些实施方案,所述肿瘤包括:人纤维肉瘤 (HT1080),肺癌(A549),宫颈癌(HeLa),人神经母细胞瘤(SHSY- 5Y)。
又一方面,本发明提供所述通式I化合物或其脂质体在制备铁死亡诱导剂中的应用。根据本发明的实施方案,所述铁死亡诱导剂可预防和/或治疗的病症包括神经退行性疾病,创伤性和出血性脑损伤,缺血性损伤(例如肝,肾和心脏缺血性损伤)。
在一些实施方案中,所述病症包括帕金森氏症(PD),亨廷顿氏症 (HD)和阿尔茨海默症(AD)。
术语定义和解释:
除非另有说明,本申请说明书和权利要求书中记载的基团和术语定义,包括其作为实例的定义、示例性的定义、优选的定义、表格中记载的定义、实施例中具体化合物的定义等,可以彼此之间任意组合和结合。这样的组合和结合后的基团定义及化合物结构,应当属于本申请说明书记载的范围。
除非另有说明,本说明书和权利要求书记载的数值范围相当于至少记载了其中每一个具体的整数数值。例如,数值范围“1-40”相当于记载了数值范围“1- 10”中的每一个整数数值即1、2、3、4、5、6、7、8、9、10,以及数值范围“11-40”中的每一个整数数值即11、12、13、14、15、16、17、18、19、20、 21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、 38、39、40。应当理解,本文在描述取代基时使用的一个、两个或更多个中,“更多个”应当是指≥3的整数,例如3、4、5、6、7、8、9或10。
术语“卤素”指F、Cl、Br和I。换言之,F、Cl、Br和I在本说明书中可描述为“卤素”。
术语“C1-50烷基”表示具有1-50个碳原子(例如具有1,2,3,4,5,6, 7,8,9,10,11,12,13,14,15,16,17,18,19,20个碳原子)的直链或支链饱和一价烃基。其中,所述烷基基团可以任选地被一个或多个本发明描述的取代基所取代。在一些实施方案中,烷基基团含有1-12个碳原子;在另一些实施方案中,烷基基团含有1-6个碳原子;在又一些实施方案中,烷基基团含有1-4个碳原子。所述烷基的实例包含,但并不限于,甲基、乙基、丙基、丁基、戊基、己基、异丙基、异丁基、仲丁基、叔丁基、异戊基、2-甲基丁基、1-甲基丁基、1-乙基丙基、1,2-二甲基丙基、新戊基、1,1-二甲基丙基、4- 甲基戊基、3-甲基戊基、2-甲基戊基、1-甲基戊基、2-乙基丁基、1-乙基丁基、 3,3-二甲基丁基、2,2-二甲基丁基、1,1-二甲基丁基、2,3-二甲基丁基、1,3-二甲基丁基或1,2-二甲基丁基等或它们的异构体。
术语“C6-14芳基”应理解为优选表示具有6、7、8、9、10、11、12、13或 14个碳原子的一价芳香性或部分芳香性的单环、双环或三环烃环(“C6-14芳基”),特别是具有6个碳原子的环(“C6芳基”),例如苯基;或联苯基,或者是具有9个碳原子的环(“C9芳基”),例如茚满基或茚基,或者是具有10 个碳原子的环(“C10芳基”),例如四氢化萘基、二氢萘基或萘基,或者是具有 13个碳原子的环(“C13芳基”),例如芴基,或者是具有14个碳原子的环 (“C14芳基”),例如蒽基。当所述C6-20芳基被取代时,其可以为单取代或者多取代。并且,对其取代位点没有限制,例如可以为邻位、对位或间位取代。
术语“5-14元杂芳基”(等同于-14元芳杂基)应理解为包括这样的一价单环、双环或三环芳族环系:其具有5、6、7、8、9、10、11、12、13或14个环原子,特别是5或6或9或10个碳原子,且其包含1-5个,优选1-3各独立选自N、O和S的杂原子并且,另外在每一种情况下可为苯并稠合的。特别地,杂芳基选自噻吩基、呋喃基、吡咯基、恶唑基、噻唑基、咪唑基、吡唑基、异恶唑基、异噻唑基、恶二唑基、三唑基、噻二唑基、噻-4H-吡唑基等以及它们的苯并衍生物,例如苯并呋喃基、苯并噻吩基、苯并恶唑基、苯并异恶唑基、苯并咪唑基、苯并三唑基、吲唑基、吲哚基、异吲哚基等;或吡啶基、哒嗪基、嘧啶基、吡嗪基、三嗪基等,以及它们的苯并衍生物,例如喹啉基、喹唑啉基、异喹啉基等;或吖辛因基、吲嗪基、嘌呤基等以及它们的苯并衍生物;或噌啉基、酞嗪基、喹唑啉基、喹喔啉基、萘啶基、蝶啶基、咔唑基、吖啶基、吩嗪基、吩噻嗪基、吩恶嗪基等。当所述5-14元杂芳基与其它基团相连构成本发明的化合物时,可以为5-14元杂芳基环上的碳原子与其它基团相连,也可以为5-14元杂芳基环上的杂原子与其它基团相连。当所述5-14元杂芳基被取代时,其可以为单取代或者多取代。并且,对其取代位点没有限制,例如可以为杂芳基环上与碳原子相连的氢被取代,或者杂芳基环上与杂原子相连的氢被取代。
有益效果
1)本发明使用GPX4小分子抑制剂和招募E3泛素连接酶的沙利度胺,匹配优化的连接物形成通式I所示GPX4-PROTAC系列结构,经研究发现,GPX4- PROTAC确实能导致GPX4在不同细胞系,特别是GPX4高表达细胞中降解并进一步诱导铁死亡。此外,研究结果表明,GPX4-PROTAC比小分子抑制剂更有效,这意味着它只需要更低剂量的化合物就可以降解细胞内靶标蛋白,故具有较高的安全性、耐药性和广阔的应用前景。动物实验也表明GPX4-PROTAC具有肿瘤抑制作用。
(2)发明人团队经实验研究发现,通式I的化合物系列杀死癌细胞的效率比小分子抑制剂高,尤其是GPX4-PROTAC-3具有最高的效率。通过DCFH-DA测得GPX4-PROTAC-3处理的细胞内ROS含量显著增高,并且随浓度梯度递增。另外通过BODIPYTM581/591C11测得膜上ROS含量随浓度增高,进一步证实了铁死亡的发生。对比不同细胞系进行MTT细胞毒性实验,证实GPX4- PROTAC-3对不同细胞系都能起作用,并且在GPX4高表达的细胞HT-1080中的效果更好。
(3)本发明进一步结合脂质体包裹递送策略,利用阳离子脂质体提高PROTAC 小分子递送的效率,从而进一步提高该类药物的靶向性,减少对正常细胞的毒害作用。
附图说明
图1示意本发明通式I化合物作用原理
图2(a)Halo-PROTAC结构示意;(b)示意不同长度linker合成的化合物对 Halo标记的GFP蛋白的的降解效率影响
图3示意Western blotting(WB)实验结果
图4HT-1080细胞孵育GPX4-PROTAC-3,GPX4-PROTAC-4,CRBN-N3, ML162-yne 12h后进行WB实验,研究不同浓度药品对GPX4蛋白降解的影响
图5(a)HT-1080细胞孵育0.3μM GPX4-PROTAC-3,CRBN-N3 and DMSO 4h然用后DCFH-DA处理得到的共聚焦图片;(b)流式处理过后得到的数据,荧光信号越强代表ROS值越高
图6示意测定膜上ROS值随GPX4-PROTAC-3分子浓度变化情况
图7示意用不同浓度的CRBN-GPX4-3,ML162-yne and CRBN-N3孵育 HeLa,A375,SH-SY5Y细胞48h后进行MTT实验
图8(a)不同浓度的401@GPX4-PROTAC孵育HT-1080 48h.(b)对 401@GPX4-PROTAC进行zeta电势和尺寸的表征
图9(a)与CRBN抑制剂和MG-132共同处理后,GPX4-PROTAC-3介导的 GPX4降解得到挽救。(b)Western blot检测ML162-yne和CRBN-N3剂量依赖性处理HT-1080细胞GPX4水平的结果
图10为GPX4-PROTAC-3核磁结果
图11为GPX4-PROTAC-3的ESI质谱
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解,下列实施例仅为示例性地说明和解释本发明,而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明,以下实施例中使用的原料和试剂均为市售商品,或者可以通过已知方法制备。
实施例1 GPX4-PROTAC-3的合成:(当n=3时)
步骤1.1 a分子ML162-yne的合成:可参照文献[1]Eaton,J.K.,Furst,L.,Ruberto,R.A.et al.Selective covalent targeting of GPX4 using masked nitrile-oxide electrophiles.Nat Chem Biol16,497–506(2020)进行合成;1H NMR(400MHz,CDCl3) δ7.60–6.57(m,11H),6.08(d,J=6.5Hz,2H),4.75(d,J=2.4Hz,2H),3.82(d,J=1.6Hz,2H),3.65–3.45(m,2H),2.90–2.71(m,2H),2.59–2.54(m,1H). 13C NMR(101MHz,CDCl3)δ167.92,166.62,153.60,138.61,134.77,132.21,131.68,130.13,129.18,128.83,128.71,128.60,128.27,126.57,126.51,123.49, 113.77,77.30,76.77,60.81,56.92,42.35,41.08,35.50.ESI HRMS(m/z):[M+H]+ calculated for C25H22Cl2N2O3S 501.0801;found 501.0819.
步骤1.2 b分子的合成:可参照文献[2]Med.Chem.Commun.,2019,10,1037–1041进行合成。
步骤1.3 c分子的合成:将b分子(1eq,100mg)溶解于四氢呋喃溶液中,加入氢氧化钾(1.5eq,18.78mg),冰浴下搅拌半小时,加入TsOCl(1.2eq,50mg),反应四个小时,点板监测反应是否完全,过柱(DCM:MeOH=20:1)得到淡绿色产品,产率为85%。
步骤1.4 d分子的合成:将c分子(1eq,10mg)溶解于DMF中,加入叠氮化钠 (10eq,11mg),金属浴下90℃反应过夜。用水和乙酸乙酯萃取得到反应粗产物,旋蒸,过柱(DCM:MeOH=10:1)得到绿色油状产物,产率为90%。
步骤1.5 e分子的合成:将抗坏血酸钠(3eq 21.1mg))和五水硫酸铜(0.5eq 2.8mg)溶解于水溶液中,将d分子(1eq 20mg)和a分子(1eq 19.6mg)溶解于 DMF中,冰浴下混合搅拌四小时,萃取,过柱(DCM:MeOH=30:1),得到黄色固体,产率为70%。1H NMR(400MHz,Chloroform-d)δ8.20(s,1H),7.88(s,1H),7.50(s,1H),7.28(s,1H),7.24(d,J=2.0Hz,2H),7.19(s,1H),7.14(s,4H),6.91(s, 1H),6.89(s,1H),6.85(s,2H),6.47(s,1H),6.06(s,1H),5.99(s,1H),5.23(s,2H),4.93(s,1H),4.55(s,2H),3.88(s,2H),3.81(s,2H),3.70(t,J=5.3Hz,3H),3.63(d,J =10.7Hz,11H),3.50–3.32(m,3H),2.96(s,2H),2.83(s,5H).ESI HRMS(m/z):[M+Na]+calculated for C46H48Cl2N8NaO10S 997.248337;found997.248337.
实施例2
参照实施例1的合成,采用n为4的相应原料进行替换,合成得到GPX4- PROTAC-4的分子(结构如下所示)。
实施例3 Linker对反应效率的影响筛选试验
实验原理:设计具有不同Linker长度的通式I化合物,其中,沙丽多胺头可靶向连接CRBN泛素连接酶,己氯烷烃端可共价连接Halo标记蛋白,进而使Halo 标记的GFP蛋白被泛素标记,从而能被蛋白酶体识别降解。所述不同通式I化合物具体如下所示(可参考文献Med.Chem.Commun.,2019,10,1037–1041进行合成):
实验过程:为了确定具有不同linker长度的通式I化合物对CRBN-PROTAC分子招募E3泛素连接酶效率的影响,本发明合成制备了五种不同linker长度的 Halo-PROTAC分子,分别为Halo-PROTAC-0、Halo-PROTAC-1、Halo-PROTAC- 2、Halo-PROTAC-3以及Halo-PROTAC-4。所述五种分子的氯己基部位可以与 Halo-GFP蛋白结合,从而靶向降解该蛋白。用不同浓度的Halo-PROTAC小分子与表达Halo-GFP蛋白的HeLa细胞孵育24h,流式数据表明Halo-PROTAC-3 以及Halo-PROTAC-4的效率最高,如图2所示。
实施例4 Western blotting(WB)实验
基于实施例3的测试结果,选择Halo-PROTAC-3以及Halo-PROTAC-4对应的两种GPX4-PROTAC分子(分别对应GPX4-PROTAC-3和GPX4-PROTAC- 4),在与HT-1080细胞孵育12h后,进行WB实验。Western blotting分析蛋白,使用IP裂解缓冲液(Fisher Scientific)裂解细胞,并使用酶标仪测定蛋白浓度。采用25μg蛋白进行免疫印迹分析,使用GPX4(1:1000,(ab41787),abcam),β-Actin(1:5000,(ab8227),abcam),表征了GPX4蛋白的降解效率。实验结果表明 GPX4-PROTAC-3和GPX4-PROTAC-4两种分子均能靶向降解GPX4蛋白,两者都随浓度梯度变化,并且GPX4-PROTAC-3效率更高(如图3所示)。
另一方面,为了进一步证明该PROTAC分子的机制,采用CRBN抑制剂和蛋白酶体抑制剂对比不添加这两种抑制剂的分子进行WB实验,发现添加这两种抑制剂确实可以有效抑制该分子对GPX4蛋白的降解,进一步证实了PROTAC 分子的双靶向机制(图9a)。同时,对ML162-yne小分子本身及CRBN-N3进行 WB实验证实这两种分子均不能对GPX4蛋白进行降解(图9b)
实施例5 MTT细胞实验
采用不同浓度的GPX4-PROTAC-3,GPX4-PROTAC-4,CRBN-N3,ML162- yne孵育HT-1080细胞48h,MTT细胞毒性实验表明GPX4-PROTAC-3效率最高(参见图4)。
实施例6测定胞内ROS值随分子浓度变化情况
分别用0.3μM GPX4-PROTAC-3、CRBN-N3和DMSO处理HT-1080细胞 4h后再用DCFH-DA处理的共聚焦图像如图5所示,显示GPX4-PROTAC-3使细胞内ROS值增加。再利用不同浓度的GPX4-PROTAC-3处理HT-1080细胞得到Flow Jo结果,表明随着GPX4-PROTAC-3浓度的增加,胞内ROS值也逐渐明显。
实施例7测定膜上ROS值随分子浓度变化情况
由于铁死亡主要与膜脂的氧化还原状态有关,本发明进一步利用不同浓度的GPX4-PROTAC-3处理HT-1080细胞6h,接着用BODIPYTM581/591C11处理得到Flow Jo结果,表明随着GPX4-PROTAC-3浓度的增加,膜上及膜内ROS 值都逐渐明显(如图6所示)。
实施例8不同细胞系中的MTT实验效果
使用不同浓度梯度的GPX4-PROTAC-3孵育A375细胞,HeLa细胞,SH- SY5Y细胞48h,并通过MTT细胞毒性实验,证明同样条件下GPX4-PROTAC- 3效果均比小分子抑制剂好(如图7所示)。
实施例9脂质体包裹得到通式I化合物的效果
为了更好在活体层次分析通式I化合物的抗癌效率,我们使用脂质体包裹递送策略,利用401-ROS脂质体提高了PROTAC小分子靶向性,减少对正常细胞的毒害作用(如图8所示,以GPX4-PROTAC-3的效果示例)。
脂质体包裹的通式I化合物分子制备包括如下步骤:将脂类(401-ROS-TK:Cholesterol:DOPE=200μg:100μg:50μg)混合在3ml玻璃瓶中,然后与200μg GPX4-PROTAC-3混合,在化学罩中去除有机溶剂过夜,形成一层薄膜。将100 μg(10μL)DSPE-PEG-2000溶于370μL的PBS缓冲液中,然后将上述薄膜溶于 20μL乙醇中,1900r搅拌加入PBS溶液中。
以上,对本发明的实施方式进行了说明。但是,本发明不限定于上述实施方式。凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (3)
1.通式III化合物,其结构如下所示:
其中,
Ar1选自无取代或任选被一个,两个或更多个Ra取代的噻吩,Ar2选自无取代或任选被一个,两个或更多个Ra取代的苯基;
所述Ra各自独立的选自H,卤素、C1-6烷基;
r选自1,2,3;
p选自1,2;
q选自1;
s选自1,2;
R1,R2可以相同或不同,各自独立的选自H或卤素;
Rb选自H、卤素、C1-6烷基;t选自1,2;
n选自3或4。
2.根据权利要求1所述的通式III化合物,其结构如下:
其中,R1,R2选自Cl。
3.根据权利要求1-2所述化合物在制备抗肿瘤药物中的应用,所述肿瘤选自人恶性黑色素瘤,人纤维肉瘤,宫颈癌,人神经母细胞瘤。
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