基于药效团模型的乙酰胆碱酯酶_聚腺苷二磷酸核糖聚合酶_1双靶点分子设计研究

基于药效团模型的乙酰胆碱酯酶、聚腺苷二磷酸核糖

聚合酶-1双靶点分子设计研究

关鑫磊1, 姜凤超2*, 王 悦1, 吴鹏飞1, 3, 王 芳1, 3, 陈建国1, 3*

(华中科技大学 1. 同济医学院药理学系, 2. 同济医学院药物化学系, 3. 药物靶点研究与药效学评价湖北省重点实验室, 湖北 武汉 430030)

摘要: 本研究建立了乙酰胆碱酯酶 (AChE) 和聚腺苷二磷酸核糖聚合酶-1 (PARP-1) 抑制剂的药效团模型, 设计、筛选了双靶点活性分子, 并验证了其抑制活性, 探讨了多靶点分子的设计策略。利用Catalyst计算机辅助药物设计软件系统, 以叠合程度和构象能进行筛选, 得到具有AChE、PARP-1抑制活性的双靶点结构。通过计算机预测目标分子的理化性质, 得到5个优选的氨基噻唑类衍生物。将匹配的优选化合物合成后进行对AChE和PARP-1的抑制活性的实验验证。其中化合物3对AChE的抑制活性IC50为 (0.337 ± 0.052) μmol·L−1, 而在 1 μmol·L−1浓度下对PARP-1的抑制率为24.6%。证明药效团模型在多靶点药物设计和筛选中能起到减少盲目性和加快设计开发的作用。

关键词: 药效团模型; 药物设计; 多靶点药物; AChE抑制剂; PARP-1抑制剂

中图分类号: R916 文献标识码: A 文章编号: 0513-4870 (2014) 06-0819-05

Pharmacophore identification of novel dual-target compounds

targeting AChE and PARP-1

GUAN Xin-lei1, JIANG Feng-chao2*, WANG Yue1, WU Peng-fei1, 3, WANG Fang1, 3, CHEN Jian-guo1, 3*

(1. Department of Pharmacology, 2. Department of Medicinal Chemistry, 3. The Key Laboratory of Drug Target Researches and Pharmacodynamics Evaluation of Hubei Province, Huazhong University of Science and Technology, Wuhan 430030, China)

Abstract: Multi-target drugs attract increasing attentions for the therapy of complicated neurodegenerative

diseases. In this study, a computer-assisted strategy was applied to search for multi-target compounds by the pharmacophore matching. This strategy has been successfully used to design dual-target inhibitor models against both the acetylcholinesterase (AChE) and poly (ADP-ribose) polymerase-1 (PARP-1). Based on two pharmacophore models matching and physicochemical properties filtering, one hit was identified which could inhibit AChE with IC50 value of (0.337 ± 0.052) μmol·L−1 and PARP-1 by 24.6% at 1 μmol·L−1.

Key words: 3D-pharmacophore modeling; drug design; multi-target drug; acetylcholinesterase inhibitor; poly (ADP- ribose) polymerase-1 inhibitor



根据系统生物学的观点, 选择性非常高的药物

效果好[1]。特别在复杂病因的疾病如神经退行性疾病

分子在临床实践中的效果有时并没有多靶点的药物中, 多靶点药物常能起到优于单一靶点药物的作用。

合成多靶点药物 如今设计、, 已经成为开发针对复杂

收稿日期: 2014-05-09; 修回日期: 2014-05-21.

基金项目: 国家重点基础研究发展计划 (973计划) 资助项目

(2013CB531303); 科技部国际合作项目 (2011DFA32670); 国家新药创制重大专项 (2012ZX0913-101-045).

*通讯作者 Tel: 86-27-83692636, Fax: 86-27-83692608,

E-mail: chenj@mails.tjmu.edu.cn; fengchao@mails.tjmu.edu.cn

病因疾病药物的新思路[2−6]。

血管性痴呆 (VD) 是指因各种脑血管疾病引起的脑功能障碍继而产生的一种获得性智能损害综合征, VD已成为继阿尔茨海默病后的第二大痴呆疾病[7, 8]。

· 820 · 药学学报 Acta Pharmaceutica Sinica 2014, 49 (6): 819−823

中的一个标志性特征是患者胆碱能神经传递的障碍, 已有大量的乙酰胆碱酯酶 (AChE) 抑制剂被用于临 床上VD患者的认知功能改善[9]。VD患者中另一个 特征是神经元的广泛丢失, 而缺血引起的DNA损伤和PARP-1过度兴奋是导致细胞死亡的重要原因[10−12]。抑制PARP-1能起到减少神经元的能量消耗、修复血脑屏障和减轻炎症反应等作用, 可促进康复[13−16]。在VD的治疗中, 同时改善胆碱能神经传递和抑制PARP-1的过度激活可能会起到更好的治疗作用。

药效团模型法是一种利用现有的分子的三维结构信息进行的药物分子设计的方法, 已在药物分子的设计和开发中起到了广泛的作用[17−19]。鉴于受体结构的复杂性, 本研究首先建立了AChE和PARP-1的药效团模型 (图1和图2)[20, 21]。通过筛选分子库, 得到一类具有PARP-1抑制活性的2-氨基噻唑类衍生物。另有文献报道4-苯基-2-氨基噻唑类Schiff碱能起到改善认知损伤的作用[22], 所以本研究中以4-苯基-2-氨基噻唑母核作为双靶点抑制剂的骨架。再通过与药效团模型的匹配和理化性质预测, 最后得到优化的双靶点分子。

结果与讨论

1 目标化合物的设计

根据文献报道, N-苯基叔胺基团能起到识别AChE的作用[23−25], 所以将其与4-苯基-2-氨基噻唑母核相连, 建立初始骨架。将设计的分子导入Catalyst系统View Hypothesis Workbench模块, 分别与AChE、PARP-1药效团模型相匹配。导出Fit值和构象能数据, 用于预测对于两个靶点的抑制活性, 本实验设定Fit值大于3为最低匹配下限, Econform小于80 kJ·mol−1为最高能量上限。同时具有较高的Fit值和较低的构象能的分子, 认为具有较好的抑制活性。除了对靶点的活性外, 作用于中枢神经系统的药物需要合适的相对分子质量、油水分配系数等理化性质来透过血脑屏障才能发挥作用[26, 27]。

据此, 本研究参照Ro5规则, 设定类药性筛选规则如下: MW小于450; TPSA小于90 Å2; LogP小于5。使用Pallas 3.3软件计算出其理化性质, 据此得到5个入围分子 (图3), 预测的理化性质见表1。

化合物1～5对AChE抑制剂药效团的Fit值都不高, 基本处于3～4之间, 这与AChE抑制剂药效 团的药效特征元素较多有关。综合考虑Fit和Econform值, 以化合物3的叠合效果最优 (Fit = 3.57, Econform = 4.68 kJ·mol−1)。化合物1～5与PARP-1抑制剂叠合

Figure 1 The optimal pharmaophore model of AChE inhibitors

(Weight = 3.29, rms = 0.53, Correl = 0.93, Config = 19.05). Pharmacophore features are color-coded: hydrophobic, light blue; hydrogen-bond acceptor, green; ring aromatic, orange. Each unit meets the special limitation in distance: ab = 1.53 nm, ac = 0.30 nm, ad = 1.42 nm, ae = 1. nm, bc = 1.67 nm, bd = 0.58 nm, be = 0.82 nm, cd = 1.45 nm, ce = 1.66 nm, de = 0.45 nm. This pharmacophore model could be divided into two sections: one contains a ring aromatic unit and two hydrophobic units (the pink box), which interact with the catalytic site of AChE. The other contains a hydrogen-bond acceptor unit and a hydrophobic unit (the blue box), which interact with the peripheral site of AChE

Figure 2 The optimal pharmaophore model of PARP-1 inhibitors

(Weight = 2.1, rms = 0.46, Correl = 0.91, Config = 15.97). Pharmacophore features are color-coded: hydrophobic, light blue; hydrogen-bond acceptor, green. Each unit meets the special limitation in distance: ab = 0.56 nm, ac = 0.88 nm, ad = 0.60 nm, bc = 0.40 nm, bd = 0.31 nm, cd = 0.49 nm

时, Fit值普遍比较高, 但叠合能量值 (Econform) 也相对较大, 综合考虑Fit和Econform值, 以化合物1的叠合效果最优 (Fit = 5.78, Econform =−1 12.09 kJ·mol)。经Pallas 3.3软件计算, 化合物1～5的相对分子质量、LogP、氮和氧原子数、氢键受体和给体数均满足类药性要求且分子片段中不含“非药物”元素。TPSA均小于90Å2, 提示化合物有良好的血脑屏障通透性和中枢神经靶向性。 2 生物活性实验结果

为了验证设计分子的抑制活性, 将预测效果较

关鑫磊等: 基于药效团模型的乙酰胆碱酯酶、聚腺苷二磷酸核糖聚合酶-1双靶点分子设计研究

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Figure 3 The structures of designed compounds

Table 1 The calculated physicochemical properties and matching parameters with pharmacophore model. aThe matching degree of pharmacophore model with the specific conformation of the related compounds. bEconform (kJ·mol−1) represents the potential energy of the structure bearing the alignment with the pharmacophore model. cHydrogen-bond donor (HD), hydrogen-bond acceptor (HBA), phy-sicochemical properties were calculated by Pallas 3.3 software

Compd. 1 2 3 4 5

AChE PARP-1

MWc TPSAc LogPc HDc HBAc

Fita Econformb Fita Econformb 3.48 47.63 5.78 12.09 365.5 45.23 4.74 1 3.41 45.88 5.56 43.91 351.1 45.23 4.59 1 3.57 4.68 5.34 40.65 406.6 48.47 3.54 1 3.00 62.69 5.10 67.20 392.6 48.47 3.39 1 3.97 15.98 5.74 65.03 422.6 57.70 3.40 1

4 4 5 5 6

好的化合物1和化合物3合成并测定其对胆碱酯酶 抑制活性和PARP-1的作用, AChE和丁酰碱酯酶 (BuChE) 的抑制活性结果见表2。化合物3对AChE的抑制活性较强, 为 (0.337 ± 0.052) μmol·L−1, 而与化合物3的Fit值接近的化合物1对AChE的抑制能力却很弱, 在10 μmol·L−1时抑制率仅为 (21.7 ± 2.4) %。这可能与化合物1较高的构象能有关 (Econform = 47.63 kJ·mol−1), 具有较高构象能的分子一般难以接近并与酶结合产生效应。除了构象能外, 与靶点酶不同的结合方式可能也是导致这一结果的原因 (图4)。化合物3能够较好地与AChE模型的3个药效特征元素匹配, 能同时结合AChE催化活性部位和外周活性部位, 起到较好的抑制效果。而化合物1具有较高的构象能, 仅能结合催化活性部位, 其抑制活性较弱。化合物1

和3对于BuChE也有一定的抑制作用, 其IC50分别为 (3.36 ± 0.61) μmol·L−1和 (1.70 ± 0.29) μmol·L−1。

化合物1和3对PARP-1的抑制活性与阳性对照3-AB比较接近, 从叠合图 (图5) 来看, 两个分子都能与4个药效特征元素相匹配。两者之间活性差异可能与叠合的Fit值和Econform大小有关, 1的Fit值比3稍大, 且叠合构象能小于3。 3 小结

通过药效团模型法构建的双靶点配体分子, 基本实现了对AChE和PARP-1的双靶点抑制效应。其中3具有较好的模型匹配度和较好的抑制活性。针对多靶点药物的设计和研究, 还需要进一步设计和筛选, 以期得到性质更优的分子, 而药效团模型法是其中一种较为简单、高效工具。

Table 2 Inhibitory effect of designed compounds on AChE,

BuChE and PARP-1. aMean of three independent measurements ± SEM. bThe inhibitory percentage was determined at 10 μmol·L−1. c

The inhibitory percentage was determined at 1 μmol·L−1

Compd. 1 3 Tacrine 3-AB

Cholinesterase inhibition IC50/μmol·L−1

AChE BuChE

实验部分

1 化学部分

1

H NMR波谱和13C NMR波谱使用Bruker核磁

aaPARP-1 inhibitionc

共振仪测定, TMS作为内标, 在CDCl3和d6-DMSO以400 MHz和100 MHz测定。质谱以Finnigan LCQ Deca XPTM色谱联用ESI质谱检测器测定。硅胶色谱板 (200～300目) 购于青岛海洋化工厂, 其他原料为

± 0.61 35.3% 21.7 ± 2.4%b 3.36 0.337 ± 0.052 0.065 ± 0.005

−

1.70 ± 0.29 0.004 ± 0.001

24.6% −

− 32.2% 国产化学纯。

· 822 · 药学学报 Acta Pharmaceutica Sinica 2014, 49 (6): 819−823

Figure 4 Mapping of compound 1 (left) and 3 (right) on the pharmacophore model of AChE inhibitors

Figure 5 Mapping of compound 1 (left) and 3 (right) on the pharmacophore model of PARP-1 inhibitors

1.1 中间产物N-(4-苯基噻唑-2-基) 丙烯酰胺的合成 1.6 g的4-苯基-2-氨基噻唑溶于四氢呋喃 (THF) 并加入1.2 g的三乙胺做催化剂, 冰浴冷却。将新制的1.2 g的丙烯酰氯溶于THF后, 缓慢滴入含三乙胺的4-苯基-2-氨基噻唑溶液中。滴加完毕后撤去冰浴, 反应液置于室温搅拌反应, 使用薄层色谱监测进程, 12 h后停止反应, 减压蒸馏除去溶剂, 柱色谱分离纯化得白色粉末状N-(4-苯基噻唑-2-基) 丙烯酰胺。 1.2 3-(N-苄基-乙基)-N-(4-苯基噻唑-2-基) 丙酰胺 (1) 的合成 将2.3 g的N-(4-苯基噻唑-2-基) 丙烯酰胺溶于甲醇, 加入1.4 g的N-乙基苄胺, 控温70 ℃回流反应, 薄层色谱监测反应进程。12 h后停止反应, 石油醚/乙酸乙酯溶剂体系柱色谱分离纯化得产物1, 产率为41%。H NMR (400 MHz, CDCl3) δ: 7.97 (d, J = 7.2 Hz, 2H), 7.58 (d, J = 7.2 Hz, 2H), 7.45 (t, J = 7.6 Hz, 2H), 7.35 (m, 3H), 7.28 (t, J = 7.2 Hz, 1H), 7.15 (s, 1H), 3.78 (s, 2H), 2. (t, J = 5.6 Hz, 2H), 2.76 (q, J = 7.2 Hz, 2H), 2. (t, J = 5.6 Hz, 2H), 1.22 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ: 170.11, 157.30, 149.87, 134.75, 129.48, 128.68, 128.63, 127.77, 127.69, 126.00, 107.13, 58.05, 48.11, 45.87, 31.69, 10.36。MS (ESI positive ion) m/z: 366.1 [M+H]+。

1.3 3-(4-苄基哌啶-1-基)-N-(4-苯基噻唑-2-基) 丙酰胺 (3) 的合成 将2.3 g的N-(4-苯基噻唑-2-基) 丙烯酰胺溶于甲醇, 加入1.8 g的1-苄基哌嗪, 控温70 ℃回流反应, TLC监测反应进程。12 h后停止反应, 石油醚/乙酸乙酯溶剂体系柱色谱分离纯化得产物3,

1

产率为49%。1H NMR (400 MHz, CDCl3) δ: 7.91 (d, J = 7.2 Hz, 2H), 7.45 (t, J = 7.6 Hz, 2H), 7.35 (m, 5H), 7.29 (m, 1H), 7.15 (s, 1H), 3.63 (s, 1H), 2.79 (t, J = 5.6 Hz, 2H), 2.70 (m, 8H), 2.62 (J = 5.6 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ: 170.14, 157.54, 149.81, 134.60, 129.23, 128.65, 128.34, 127.84, 127.24, 125.99, 107.30, 62.94, 53.01, 52.91, 52.24, 31.19。MS (ESI positive ion) m/z: 407.1 [M+H]+。 2 药理部分

2.1 AChE和BuChE抑制活性测定 AchE、BuChE、5, 5'-dithiobis-(2-nitrobenzoic acid) (DTNB)、acetylthio- choline chloride (ATC) 和butylthiocholinlrine chloride (BTC) 购自Sigma公司。AChE抑制活性的测定使用Ellman法[28]。将磷酸盐缓冲液 (pH 7.4) 20 μL、化合物的溶液20 μL (或是等量空白溶液)、1.5 mmol·L−1 DTNB 100 μL、AChE酶50 μL (0.22 u·mL−1) 和 30 mmol·L−1 ATC 10 μL在96孔板中混合均匀。反应15 min后, 以3 min为间隔测定415 nm下的吸收度。BuChE抑制活性测定流程相同 (0.3 u·mL−1 BuChE、1.5 mmol·L−1 DTNB、30 mmol·L−1 BTC)。酶标仪为Biotek Elx800型。

2.2 PARP-1抑制活性测定 PARP-1的抑制活性采用化学发光法测定, 试剂盒购自BPS Bioscience公司 (Catalog # 80551)。利用链霉亲和素−辣根过氧化物酶 (Streptavidin-HRP) 催化的显色反应, 测定连接在组蛋白上生物素化的底物的含量, 间接的反映不同干预条件下PARP-1的活性。操作流程同试剂盒说明书。

关鑫磊等: 基于药效团模型的乙酰胆碱酯酶、聚腺苷二磷酸核糖聚合酶-1双靶点分子设计研究

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