CN114652713A - 一种结核分枝杆菌异柠檬酸裂解酶先导抑制剂 - Google Patents
一种结核分枝杆菌异柠檬酸裂解酶先导抑制剂 Download PDFInfo
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Abstract
本发明公开了一种结核分枝杆菌异柠檬酸裂解酶先导抑制剂，化学名为7,8‑二羟基香豆素，又名瑞香素、祖师麻甲素；7,8‑二羟基香豆素对结核分枝杆菌异柠檬酸裂解酶半抑制浓度为3.34±1.46μg/mL，其活性是衣康酸的2倍，3‑溴丙酮酸的3.3倍；抗耻垢分枝杆菌MIC值为128μg/mL；分子对接结果显示，7,8‑二羟基香豆素与结核分枝杆菌异柠檬酸裂解酶活性位点的关键氨基酸残基有较强相互作用；研究结果显示，7,8‑二羟基香豆素经进一步修饰，有望发展为抗潜伏结核分枝杆菌药物。
Description
技术领域
本发明涉及医药技术领域，主要描述了一种结核分枝杆菌异柠檬酸裂解酶天然产物先导化合物，7，8-二羟基香豆素，该化合物有较强的结核分枝杆菌异柠檬酸裂解酶抑制活性和中等强度抗菌活性。
背景技术
结核病是由致命的结核分枝杆菌(Mtb)病原体引起的，目前仍是世界上最致命的传染病之一。目前，尽管结核病的发病率有所下降，但耐药菌株的数量不断增加，尤其是耐多药(MDR)和超级耐药(XDR)菌株，给结核病的治疗带来了巨大的挑战。目前对药物敏感结核病的标准化治疗持续6个月，包括2个月的利福平，异烟肼，乙胺丁醇，吡嗪酰胺，其次是利福平和异烟肼至少4个月，才能彻底根除Mtb。然而，耐药结核病的治疗需要20个月以上。因此，需要更有效的抗结核药物来对抗潜伏结核病和MDR/XDR结核病。
结核分枝杆菌的存留和存活有多种机制。在宿主体内，由于缺氧、营养剥夺、有限的碳源、高浓度的一氧化氮等原因，Mtb被吞噬并进一步留在肉芽肿中，增强了Mtb的厌氧代谢途径以适应缺氧。丙氨酸脱氢酶(Ald)催化丙酮酸转化为丙氨酸，乙醛酸转化为甘氨酸，NADH被氧化为NAD。乙醛酸和丙酮酸分别由乙醛酸和柠檬酸甲酯循环提供，并由两个异柠檬酸裂解酶的异构体(ICL1和ICL2) 催化，这两个异构体分别由基因icl和aceA编码。乙醛酸循环是三羧酸循环的一种替代途径，它允许细菌利用乙醛酸或脂肪酸作为碳源生长，并在Mtb的生存中发挥重要作用。而且，这一途径在宿主体内并不存在，参与这一途径的酶是潜伏细菌潜在的选择性药物靶点。异柠檬酸裂解酶(Isocitratelyase，ICL)是参与乙醛酸循环的关键限速酶，它绕过Krebs循环中的若干β氧化步骤，可逆地将异柠檬酸分解为丁二酸和乙醛酸并产生能量。ICL和aceA基因的任何一个或两个缺失都会削弱Mtb在活化的巨噬细胞中的生存并降低其毒性。在没有ICL的情况下，结核分枝杆菌在潜伏结核模型中不能存活。因此，结核分枝杆菌异柠檬酸裂解酶治疗结核分枝杆菌的研究越来越受到关注。
ICL的经典抑制剂大多是ICL催化反应产物的结构类似物，如乙醛酸的类似物3-溴丙酮酸，琥珀酸的类似物衣康酸和3-硝基丙酸。但这些抑制剂具有较弱的抑制能力和较强的生物毒性，尚未真正应用于临床。因此，仍需发现有效的抗核分枝杆菌异柠檬酸裂解酶的抑制剂。
虚拟筛选为高通量筛选提供了一种新的替代方法，可以显著降低实验成本。基于受体的虚拟筛选根据评分函数评价小分子与蛋白的结合能力，如CDOCKER Scores。最后，选择具有良好结合模式和较高结合能的化合物进行生物活性测试。本发明中运用DiscoveryStudio2019，从TCMSP(https://www.tcmsp-e.com/，12811 种小分子)数据库中成功发现了抗核分枝杆菌异柠檬酸裂解酶先导抑制剂7,8-二羟基香豆素，其IC50达到3.34±1.46μg/mL。
发明内容
1.一种结核分枝杆菌异柠檬酸裂解酶先导抑制剂，含母核7，8-二羟基香豆素，其结构特征如下：
2.根据权利要求1所述的一种结核分枝杆菌异柠檬酸裂解酶先导抑制剂，其母核结构7，8-二羟基香豆素主要存在于瑞香科植物包括长白瑞香及祖师麻，又名瑞香素(Daphnetin,DAP)、祖师麻甲素；
3.根据权利要求1所述的一种结核分枝杆菌异柠檬酸裂解酶先导抑制剂，含如权利要求1所述母核结构的衍生物，包括但不限于香豆素的4，5，6位取代硝基、卤素、腈基、取代苯基、4-(3’，5’-取代苯基)-1，4-二氮六环；
4.根据权利要求1所述的一种结核分枝杆菌异柠檬酸裂解酶先导抑制剂，其母核结构7，8-二羟基香豆素抗耻垢分枝杆菌的MIC结果为128μg/mL；
5.根据权利要求书1所述的一种结核分枝杆菌异柠檬酸裂解酶先导抑制剂，其母核7，8-二羟基香豆素，对结核分枝杆菌异柠檬酸裂解酶半抑制浓度为 3.34±1.46μg/mL；
6.根据权利要求书1所述的一种结核分枝杆菌异柠檬酸裂解酶先导抑制剂，其母核7，8-二羟基香豆素是来自于天然产物先导配体，其基于此母体衍生的结核分枝杆菌异柠檬酸裂解酶抑制剂属于本权力保护。
应用实施例
(一)结核分枝杆菌异柠檬酸裂解酶虚拟筛选发现7，8-二羟基香豆素抑制剂
结核分枝杆菌异柠檬酸裂解酶与抑制剂3-溴丙酮酸结合的三维晶体结构 (PDB:1F8M)下载于RCSB蛋白数据库(http://www.rcsb.org)。利用Discovery Studio2019(DS)(BIOVIACorp，SanDiego，CA，USA)，通过分配部分电荷、去除所有水分子并添加极性氢原子来制备蛋白质结构。考虑到结核分枝杆菌异柠檬酸裂解酶的最适pH，pH环境设定为7.0。可容纳3-溴丙酮酸的球腔定义为结合位点。
中药小分子数据集(12811个化合物)来源于TCMSP(https://tcmsp-e.com/)，通过DS|prepareligands去除重复结构和同分异构体，生成其三维构象,pH值范围为 6.5～8.5。其他参数设置为默认值。
通过DS|LibDock模块初步筛选，筛选出得分大于或等于80分的小分子。然后使用CDOCKER进行精确对接。根据-CDOCKERINTERACTION ENERGY，综合考虑蛋白质与配体之间的相互作用、化合物的结构，选择候选化合物。Pymol3.7用于蛋白质配体复合体的可视化。对参考配体进行了相同的处理。
利用天然底物异柠檬酸与结核分枝杆菌异柠檬酸裂解酶的相互作用模式来解释候选化合物与结核分枝杆菌异柠檬酸裂解酶的结合模式。异柠檬酸的羰基和羟基的氧原子是主要的氢键受体，与活性位点的氨基酸残基如TYR89、GLY192、 SER315、SER317、THR347等形成氢键。此外，HIS180，LYS189，HIS193， ARG228与异柠檬酸形成静电相互作用(附图1)。
7，8-二羟基香豆素与结核分枝杆菌异柠檬酸裂解酶的残基形成多重相互作用。内酯环与CYS191、ASP108形成π-烷基相互作用，苯酚环与CYS191、ASP108、 LEU348形成π-阴离子相互作用。HIS193与苯酚取代基上的两个氧原子形成电荷吸引。此外，共有6个氨基酸残基(LYS189、GLY192、ARG228、GLU285、SER315、 SER317)与羰基和氢氧基形成氢键(附图2)。重叠图显示参考配体、异柠檬酸和7， 8-二羟基香豆素之间有很好的结构相似性(附图3)。
(二)酶抑制活性测定
酶抑制活性是通过在340nm处监测酶反应与NADH耦合的吸光度下降来测定。结核分枝杆菌异柠檬酸裂解酶0.5μg和不同的最终浓度的化合物添加到300 μL酶反应体系(5mmol/LMgCl2，5mmol/LL-cysteine，1mmol/LEDTA，pH6.8)， 37℃孵育10分钟，然后加入2mmol/L异柠檬酸，继续孵育20分钟。最后，添加2ULDH和0.2mmol/LNADH，室温下，监测3min内(间隔10s)340nm处吸光度变化(ΔA)。根据底物消光系数(ε＝6.22×103mol-1·cm-1)计算酶活。酶活性的单位定义为1min内转化1μmol底物的酶量。抑制剂对酶活性的抑制百分比按公式1计算：
F为空白组吸光度变化，F0为实验组吸光度变化。Origin2018用于拟合抑制率对浓度的响应，并计算化合物对结核分枝杆菌异柠檬酸裂解酶的半抑制浓度。如附图4所示，对结核分枝杆菌异柠檬酸裂解酶的半抑制浓度为3.34±1.46 μg/mL(18.77±1.46μmol/L)，优于两种参考配体3-溴丙酮酸和衣康酸(附表1)。
(三)抗菌活性测定
耻垢分枝杆菌Mc2155与结核分枝杆菌高度同源，但生长迅速，无传染性。因此，采用微量肉汤稀释法测定化合物的抗菌活性。耻垢分枝杆菌在含有 OADC(10％吐温-80，20％甘油)的Middlebrook7H9培养基中培养，在37℃条件下生长至约0.5的麦氏浊度。然后1:1000倍稀释(约1×105CFU/mL)。用二甲基亚砜(DMSO)或无菌去离子水制备药物的初始稀释浓度，然后在含有0.1ml 7H9培养基的微孔板上连续稀释2倍，加入菌液0.1ml，37℃至少培养48h，观察菌落生长情况。设置无药物阳性对照和无细菌阴性对照。视觉最小抑菌浓度(Visualminimuminhibitoryconcentration,VisualMIC)定义为微孔板中不存在细菌生长最小药物浓度。结果表明，7，8-二羟基香豆素抗耻垢分枝杆菌的MIC为 128μg/mL(附表2)。
本发明中，7，8-二羟基香豆素对抗结核分枝杆菌异柠檬酸裂解酶有较强抑制作用，具有中等抗耻垢分枝杆菌作用，是一种新型的结核分枝杆菌异柠檬酸裂解酶抑制剂骨架，为7，8-二羟基香豆素新的药理作用和作用靶发现。通过优化结构和化学官能团，可获得亲和度高、毒性低的候选化合物。因此，该化合物有望成为进一步研究以结核分枝杆菌异柠檬酸裂解酶为靶点的抗结核药物的先导化合物。
附图说明
图1结核分枝杆菌异柠檬酸裂解酶的活性位点；
图2 7，8-二羟基香豆素与结核分枝杆菌异柠檬酸裂解酶的相互作用；
图3 7，8-二羟基香豆素对结核分枝杆菌异柠檬酸裂解酶的抑制能力曲线；
图4结核分枝杆菌异柠檬酸裂解酶活性位点内四个化合物的重叠构象。
附表1虚拟筛选化合物结构；
附表2结核分枝杆菌异柠檬酸裂解酶抑制剂及对照药物的抗菌活性；
| 化合物 | MIC(μg/mL) |
| 7，8-二羟基香豆素 | 128 |
| 3-溴丙酮酸 | 256 |
| 衣康酸 | >256 |
| 链霉素 | 0.5 |
| 左氧氟沙星 | <0.125 |
Claims (6)
1.一种结核分枝杆菌异柠檬酸裂解酶先导抑制剂，含母核7,8-二羟基香豆素，其结构特征如下：
2.根据权利要求1所述的一种结核分枝杆菌异柠檬酸裂解酶先导抑制剂，其母核结构7,8-二羟基香豆素主要存在于瑞香科植物包括长白瑞香及祖师麻中，又名瑞香素(Daphnetin,DAP)、祖师麻甲素。
3.根据权利要求1所述的一种结核分枝杆菌异柠檬酸裂解酶先导抑制剂，含如权利要求1所述母核结构的衍生物，包括但不限于香豆素的4，5，6位取代硝基、卤素、腈基、取代苯基、4-(3’,5’-取代苯基)-1，4-二氮六环。
4.根据权利要求书1所述的一种结核分枝杆菌异柠檬酸裂解酶先导抑制剂，其母核结构7,8-二羟基香豆素，对结核分枝杆菌异柠檬酸裂解酶半抑制浓度为3.34±1.46μg/mL。
5.根据权利要求1所述的一种结核分枝杆菌异柠檬酸裂解酶先导抑制剂，其母核结构7,8-二羟基香豆素抗耻垢分枝杆菌的MIC结果为128μg/mL。
6.根据权利要求书1所述的一种结核分枝杆菌异柠檬酸裂解酶先导抑制剂，其母核7,8-二羟基香豆素是来自于天然产物先导配体，其基于此母体衍生的结核分枝杆菌异柠檬酸裂解酶抑制剂属于本权力保护。
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