CN107188867A - 精氨酸脱亚氨酶4的抑制剂 - Google Patents

精氨酸脱亚氨酶4的抑制剂 Download PDF

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Publication number
CN107188867A
CN107188867A CN201610142351.5A CN201610142351A CN107188867A CN 107188867 A CN107188867 A CN 107188867A CN 201610142351 A CN201610142351 A CN 201610142351A CN 107188867 A CN107188867 A CN 107188867A
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pad4
compound
inhibitor
residues
nsc65016
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林建平
刘翠
李金龙
刘岯
王中华
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Tianjin Institute of Industrial Biotechnology of CAS
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Tianjin Institute of Industrial Biotechnology of CAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/125—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/13—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

本发明提供了1种精氨酸脱亚氨酶4（PAD4）的新型抑制剂，该化合物与PAD4之间的解离常数KD值为218μM。

Description

精氨酸脱亚氨酶4的抑制剂
技术领域
本发明属于药物化合物领域，更具体地，涉及精氨酸脱亚氨酶4（PAD4）的抑制剂及其结合PAD4的应用。
背景技术
类风湿性关节炎（rheumatoid arthritis，RA）是一种严重的威胁人类健康的慢性自身免疫性疾病。目前治疗RA的方法是缓解症状，如抗炎、消肿和止痛等。非甾体抗炎药（NSAIDs）作为一线抗RA药，减少组织疼痛和炎症，包括阿司匹林、塞来昔布、美洛昔康和可的松（糖皮质激素）等。NSAIDs药物能减轻临床症状但不能根治炎症，无法阻止关节破坏、组织损伤等，需要与其他抗风湿药物联合使用才能有效控制病情。改善病情抗风湿药（DMARDs）作为二线抗RA药，用于缓解或阻止RA软骨和关节的损伤，包括甲氨蝶呤、羟化氯喹等。DMARDs类药物通常起效慢，影响RA患者的自身机体免疫过程。目前的治疗药物对RA的免疫病理机制无决定性影响，不能从根本上治疗治疗RA，因此发展针对RA发病机理的药物是必要的。
近年来发现，PAD4是治疗类风湿性关节炎的新靶点。PAD4是将蛋白质多肽链中的精氨酸催化成瓜氨酸的修饰酶之一。研究表明(Zhang Y. 2013)类风湿性关节炎患者体内PAD4蛋白显著表达，使RA患者体内多种蛋白发生瓜氨酸化，例如滑膜中纤维蛋白和抗凝血酶的瓜氨酸化。进而形成瓜氨酸化蛋白/多肽抗原表位(citrullinated protein/peptideantigen,CPA)，刺激RA患者自我免疫形成抗瓜氨酸化蛋白抗体。抗瓜氨酸化蛋白抗体视多种蛋白和多肽为目标，在自身免疫性关节炎中有致病作用。PAD4是钙离子依赖性酶，含有5个Ca2+结合位点，由663个氨基酸残基组成，分子量为74kDa。PAD4具有2个结构域，N-结构域和C-结构域。PAD4的催化活性位点位于C-结构域，包含四个关键氨基酸残基Asp350、His471、Asp473和Cys645。研究表明（Arita K.,2004）Ca2+的结合引起PAD4构象的改变，从而形成活性位点裂隙，该裂隙可容纳组蛋白N-末端肽和2个Ca2+结合位点。PAD4可催化单甲基精氨酸残基和肽酰基精氨酸脱亚氨转化为瓜氨酸，不能转化二甲基精氨酸。
目前靶向PAD4抑制剂开发均是针对活性催化位点，分为不可逆类抑制剂和可逆类抑制剂。不可逆类抑制剂主要是卤脒类，包括氟脒（F-amidine）和卤脒（Cl-amidine），IC50为1.9~22 μM。而氢脒类抑制剂是PAD4的可逆性抑制剂，说明PAD4活性位点需要吸电子离去基团（卤素）与硫醇反应，从而增加脒基碳原子的亲电性。现有的可逆性抑制剂（紫杉醇、链霉素、米诺环素等），除GSK199和GSK484以外（IC50为200 nM和50 nM）（Lewis, H. D., 2015），抑制活性均较弱，IC50值在毫摩尔级别。
发明内容
本发明的目的是提供化合物NSC65016以及结合PAD4蛋白的应用。
本发明提供了一种结构式如下的化合物：
。
上述化合物是萘醌类化合物，并且含有2-氯，3-乙氨基哌嗪取代基。
本发明提供了上述化合物与PAD4结合的用途。
上述化合物的哌嗪环与PAD4的活性口袋内Asp473和His471残基形成两个氢键。上述化合物的2-氯萘醌基团伸向溶剂化区域，与PAD4活性口袋内Arg374和Arg345残基形成两个氢键。
实验表明，以上化合物可与PAD4蛋白结合，解离常数为KD=218 μM，可以用于发展抗RA的抑制剂。
具体实施方式
为了理解本发明，下面以实施例进一步说明本发明，但不意于限制本发明的保护范围。
NSC65016化合物（2-卤-3-（（2-哌嗪基）乙氨基）萘醌）（2-chloro-3-((2-(piperazin-1-yl)ethyl)amino)naphthalene-1,4-dione）来自于NCI数据库。本发明通过基于氟脒和PAD4反应的过渡态结构进行分子动力学模拟、药效团模型、分子对接和体外化合物筛选，发现了新结构类型的PAD4蛋白抑制剂NSC65016。NSC65016化合物的结构如下所示：
。
NSC65016的分子式：C16H18ClN3O2。
NSC65016的分子量：319。
NSC65016是萘醌类化合物，并且含有2-氯，3-乙氨基哌嗪取代基。
NSC65016化合物通过与 PAD4活性位点结合从而阻止其发挥转化精氨酸为瓜氨酸的活性。
NSC65016化合物与PAD4亲和力KD = 218 μΜ，有进一步优化成抗 RA候选药物的潜力。
NSC65016结合PAD4蛋白的能力是通过自身的分子结构特点决定的。NSC65016结构中的哌嗪环与PAD4的活性口袋内Asp473和His471残基形成两个氢键。并且该化合物的2-氯萘醌基团伸向溶剂化区域，与PAD4活性口袋内Arg374和Arg345残基形成两个氢键。从而加强了化合物NSC65016与PAD4蛋白的亲和力。
在抗PAD4蛋白生物活性评估中,由于我们没有直接的PAD4活性检测方法, 所以我们运用了表面等离子共振（Surface Plasmon Resonance, SPR）技术, 检测了化合物和PAD4蛋白间的结合活性。
1.试验材料
Biacore T200光学生物传感器（Biacore Life Sciences, GE Healthcare）
CM5 生物传感芯片
10 mM醋酸钠（pH 5.0）
缓冲液：50 mm HEPES, pH 8.0, 300 mM NaCl, 2 mM DTT, 20 mM CaCl2, 和5%DMSO。
2.试验方法
2.1药物配制
药物：---（DMSO储存液，10mM）
含有5% DMSO的HEPES缓冲液做2倍浓度梯度稀释。试验药物终浓度为500 μM，250 μM，62.5 μM，31.25 μM，15.63 μM，7.81 μM。体系中DMSO 终浓度为5%。
2.2试验程序
2.2.1 用10 mM的醋酸钠（pH 5.0）将PAD4蛋白稀释成浓度为50 μg/ml，采用氨基偶联方式将稀释后的PAD4固定到CM5生物传感芯片上。
2.2.2 每个浓度（7.81 ~ 500 μM）的化合物以30 μL/min的恒定流速，注射到PAD4蛋白表面，注射时间为60s。
2.2.3 缓冲液（50 mm HEPES, pH 8.0, 300 mM NaCl, 2 mM DTT, 20 mM CaCl2,和5% DMSO）持续冲洗芯片表面300s，解离化合物。
2.2.4 选取31.25 μM浓度做重复实验。
2.2.5 收集实验数据，并用Biacore T200评估软件进行数据分析，计算得出化合物和PAD4的解离常数KD值。

Claims (5)

1.一种结构式如下的化合物：
。
2.根据权利要求1所述的化合物，其特征在于，所述化合物是萘醌类化合物，并且含有2-氯，3-乙氨基哌嗪取代基。
3.根据权利要求1所述的化合物的结合PAD4的用途。
4.根据权利要求3所述的用途，其特征在于，所述化合物的哌嗪环与PAD4的活性口袋内Asp473和His471残基形成两个氢键。
5.根据权利要求3所述的用途，其特征在于，所述化合物的2-氯萘醌基团伸向溶剂化区域，与PAD4活性口袋内Arg374和Arg345残基形成两个氢键。
CN201610142351.5A 2016-03-14 2016-03-14 精氨酸脱亚氨酶4的抑制剂 Pending CN107188867A (zh)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014015905A1 (en) * 2012-07-26 2014-01-30 Glaxo Group Limited 2 - (azaindol- 2 -yl) benz imidazoles as pad4 inhibitors

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014015905A1 (en) * 2012-07-26 2014-01-30 Glaxo Group Limited 2 - (azaindol- 2 -yl) benz imidazoles as pad4 inhibitors

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SANJAY K. VERMA 等: "Synthesis, electrochemical, fluorescence and antimicrobial studies of 2-chloro-3-amino-1,4-naphthoquinone bearing mononuclear transition metal dithiocarbamate complexes [M{k2S,S-S2C–piperazine–C2H4N(H)ClNQ}n]", 《RSC ADV.》 *

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