CN104546856B - 一种β-内酰胺的抗菌用途 - Google Patents
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Abstract
本发明公开了一种小分子化合物的抗菌用途，具体为一种β‑内酰胺的抗菌用途。该小分子化合物是2‑(1,3‑dioxo‑1H‑benzo[de]isoquinolin‑2(3H)‑yl)‑5H,13H‑benzo[4,5]isoq uino[2,1‑a]quinazoline‑5,13‑dione。本发明是基于β‑内酰胺类抗生素的作用机制来发现新的小分子化合物。结果发现，上述小分子化合物对氨苄青霉素耐药的金黄色葡萄球菌具有抑制和杀菌作用，可治疗金黄色葡萄球菌引起的疾病，可用于医院环境的消毒灭菌。该小分子化合物在本发明的工业实施中可将药品制成适应各种用药途径的各种剂型。
Description
技术领域
本发明涉及化学药物筛选领域，具体涉及一种小分子化合物的抗菌用途。
背景技术
目前，防治细菌性病原微生物感染所致疾病的方法是使用疫苗免疫和/或投给抗生素。目前所使用的疫苗进入机体后需要一定时间才能起到免疫作用，并且有许多疾病至今还没有可供使用的疫苗；使用抗生素防治细菌性疾病存在抗药性和药物残留等问题。
2-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-5H,13H-benzo[4,5]isoquino[2,1-a]quinazoline-5,13-dione是一种来源于天然产物的小分子化合物。该小分子化合物被用于针对一种EP2受体（前列腺素E受体2）的高通量筛选。EP2受体是一个G蛋白偶联受体，该受体的激活导致细胞的环磷酸腺苷增加。现有研究结果显示，2-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-5H,13H-benzo[4,5]isoq uino[2,1-a]quinazoline-5,13-dione对前列腺素E受体2的功能具有抑制作用。该小分子化合物在抑菌方面的应用未见报道。
细菌细胞壁主要成分是肽聚糖（peptidoglycan）,又称粘肽(mucopetide)。细胞壁的机械强度有赖于肽聚糖的存在。合成肽聚糖是原核生物特有的能力。肽聚糖是由n-乙酰葡萄糖胺和n-乙酰胞酸两种氨基糖经β-1.4糖苷键连接间隔排列形成的多糖支架。在n-乙酰胞壁酸分子上连接四肽侧链，肽链之间再由肽桥或肽链联系起来，组成一个机械性很强的网状结构。各种细菌细胞壁的肽聚糖支架均相同，在四肽侧链的组成及其连接方式随菌种而异。
发明内容
本发明的目的在于提供一种小分子化合物的抗菌用途，用以解决现有技术中存在的问题。
为实现上述目的，本发明提供一种小分子化合物的抗金黄色葡萄球菌用途，其特征在于，所述小分子化合物为β-内酰胺。
本发明还提供该β-内酰胺的抗金黄色葡萄球菌用途，其特征在于该β-内酰胺为2-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-5H,13H-benzo[4,5]isoq uino[2,1-a]quinazoline-5,13-dione。
该小分子化合物的结构如图1所示。
本发明针对某些具有耐药性的细菌具有显著杀灭作用，实验显示本发明所涉及的小分子化合物2-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-5H,13H-benzo[4,5]isoq uino[2,1-a]quinazoline-5,13-dione对抗生素如氨苄西林等有耐受性的多种金黄色葡萄球菌如E6、E7均有抑菌作用。
本发明所涉及的小分子化合物2-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-5H,13H-benzo[4,5]isoq uino[2,1-a]quinazoline-5,13-dione通过适当途径给药，可以防治人或动物由于细菌感染所致的疾病，因此该小分子化合物可用于防治动物由于细菌性病原微生物感染所致疾病的药品制备。本发明涉及的小分子化合物可用于经适当途径给药杀灭细菌性病原微生物的药品制备。
本发明是基于β-内酰胺类抗生素的作用机制来发现新的小分子化合物。通过抑制胞壁粘肽合成酶，即青霉素结合蛋白（penicillin binding proteins,PBPs），从而阻碍细胞壁粘肽合成，使细菌胞壁缺损，菌体膨胀裂解，从而达到抑制细菌生长的目的。
本发明人首先从蛋白质结构数据库（PDB database）下载了三个蛋白质结构文件，其中包括1HVB.pdb、1XX2.pdb、3Q6X.pdb,然后通过蛋白质结构分析，确定了药物作用的靶点。针对药物作用可能靶点，使用北京市计算中心的蛋白质靶向小分子化合物虚拟筛选平台，筛选出最优的小分子化合物进行生物活性实验验证。
实验验证的结果显示，本发明对氨苄青霉素耐药的金黄色葡萄球菌具有抑制和杀菌作用。
耐药性金黄色葡萄球菌是全球院内感染的首要病原菌。有望通过外用、口服或静脉注射用于治疗敏感细菌所致的败血症、尿路感染、肺部感染、胆道感染等。
本发明不同于现有的β-内酰胺类抗生素的内核，本发明的β-内酰胺位于六元环内。基于本发明的内核可以设计出新的高效抗生素或广谱抗生素。
本发明涉及的小分子化合物2-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-5H,13H-benzo[4,5]isoq uino[2,1-a]quinazoline-5,13-dione在本发明所涉及的用途中可制成非注射剂的任何剂型。在生物医学医临床中本发明所涉及的投药途径可以是消化道给药，主要包括口服、直接投胃或/和经体外进行胃和肠管注入等非注射给药，也可以是外敷或静脉注射。本发明的小分子化合物也可用于医院环境的喷雾消毒灭菌。所以该小分子化合物在本发明的工业实施中可将药品制成适应以上用药途径的各种剂型。
综上所述，本发明的小分子化合物对金黄色葡萄球菌的抑制和杀灭作用，可治疗金黄色葡萄球菌引起的疾病，可用于医院环境的消毒灭菌。本发明的小分子化合物为抑制和杀灭金黄色葡萄球菌，特别是针对耐药的金黄色葡萄球菌提供了新选择。具体地，本发明的小分子化合物可用于耐药的金黄色葡萄球菌引起的感染，无论是人或动物。
附图说明
图1本发明的小分子化合物的结构图。
具体实施方式
2-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-5H,13H-benzo[4,5]isoquino[2,1-a]quinazoline-5,13-dione，该小分子化合物的结构如图1所示，该小分子化合物在其它数据库中的编号如下：
| Vitas-M | STK366513(6mg) |
| eMolecules | 1219415 |
| Molport | MolPort-000-705-652 |
| Asinex | BAS00431364 |
| IBScreen NP | STOCK1N-25068 |
| Mcule | MCULE-8845313021 |
| Pharmeks | PHAR011812 |
| PrincetonBioMolecular Research | OSSL_122472 |
| Zelinsky Institute | UZI/6058953 |
| TimTec Make-on-Demand | ST50229180 |
| ZINC database | ZINC04015296 |
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1小分子化合物的筛选
编号25068的为2-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-5H,13H-benzo[4,5]isoq uino[2,1-a]quinazoline-5,13-dione。
E6、E7均为氨苄西林耐药金黄色葡萄球菌。
1、实验设计
（1）选择适当的溶剂溶解五种小分子化合物（编号分别为48564、25068、38929、44331、68244）。
（2）分子生物学将β内酰胺酶分为四类：A类酶包括多种质粒编码的青霉素酶，活性部位为丝氨酸残基，分子量为29kDa；B类酶为金属酶，由染色体或质粒编码，酶活性需锌离子参与，可被乙二胺四乙酸（EDTA）抑制；C类酶的活性部位为丝氨酸残基，分子量为39kDa，其产生与诱导剂有关，C类酶主要指染色体编码的头孢菌素酶（AmpC酶）；D类酶又称为OXA型（水解苯唑西林）β-内酰胺酶。（3）实验所用菌株E6、E7来自307医院，大肠杆菌（ndm-1）为中国人民解放军军事医学科学院微生物与流行病研究所实验室所有，ATCC25922为大肠杆菌标准菌株，来自ATCC公司，W3110也为大肠杆菌标准菌株。ATCC25922和W3110可购自ATCC公司。
（4）针对小分子化合物可能会对产金属β-内酰胺酶的细菌有单独抑制作用或者与β-内酰胺类的抗生素协同抑制作用，我们选用碳青霉烯类抗生素亚胺培南，利用药敏纸片法进行抗菌或协同抗菌效果的小分子化合物的抑菌活性的初筛。
一、查阅参考金属β-内酰胺酶的检测实验（亚胺培南-EDTA纸片法协同试验，该实验原理是金属β内酰胺酶是一类活性部位为金属离子，且必须依赖二价金属阳离子的存在而发生催化活性，EDTA能够螯合金属离子（主要是Zn2+），EDTA使金属β内酰胺酶失活，从而使β-内酰胺类抗生素发挥抑菌作用，在靠近EDTA纸片侧抑菌圈会明显扩大）。如果小分子化合物能特异性抑制金属β-内酰胺酶，协同效果与亚胺培南-EDTA纸片法协同试验的结果类似。
二、如果小分子化合物有单独的抑菌作用，则会类似抗生素产生抑菌环。
（5）针对小分子化合物可能对其他几种β-内酰胺酶产生单独的抑制作用或者产生与抗生素的协同抑菌作用，我们选用青霉素类和头孢霉素类的抗生素，利用纸片法进行小分子化合物的抑菌活性的初筛。
一、如果有协同抑菌作用，协同效果与亚胺培南-EDTA纸片法协同试验的结果类似。
二、如果小分子化合物有单独的抑菌作用，则会类似抗生素产生抑菌环。
2、实验步骤
（1）采用双层琼脂平板来培养细菌，50℃融化LB半固体培养基，取5ML于无菌试管中，加入过夜培养的细菌500μL，轻轻混匀，将混合好的半固体培养基铺在底层为LB固体培养基的培养皿中。
（2）在生物安全柜中，十分钟内将药敏纸片贴在已凝固的培养皿中，单独抑菌实验的纸片距离在3cm以上，协同抑菌实验的两个纸片距离必须在1cm左右。其中各药敏纸片中所含抗生素的量都为10μg，化合物48564的药敏纸片含量：9.9μg，化合物25068的药敏纸片含量：8.2μg，化合物38929的药敏纸片含量10μg，化合物44331的药敏纸片含量12.5μg，化合物68244的药敏纸片含量10μg。实验中对照纸片均为含各化合物相同比例的溶剂。
（3）将贴有药敏纸片的培养皿在37℃的培养箱中培养12h，观察抑菌效果。
3、实验结果
注：（1）IPM：亚胺培南，AMP：氨苄西林，CEF：头孢硫脒；（2）-代表无抑菌作用，+代表有抑菌作用；（3）协同实验中，M代表小分子化合物，由于实验结果无协同效应，故没有单独标注某一个化合物。
4、实验结果分析
从上表可以看出：
（1）化合物对产金属β-内酰胺酶的大肠杆菌无抑菌作用，且与亚胺培南无协同抑菌作用；
（2）化合物与其他β-内酰胺类抗生素无协同抑菌作用；
（3）化合物25068、48564对氨苄西林耐药金黄色葡萄球菌E6、E7有抑菌作用。这两株菌对氨苄西林都耐受，所以从结果分析这两株菌可能产青霉素类的β-内酰胺酶。以此可以初步推断25068、48564对单纯的产这类酶的细菌有抑制作用；
（4）ATCC25922为敏感菌株，对亚胺培南、氨苄西林、头孢硫脒都敏感，但化合物25068、48564对其不产生抑制作用。该结果可用来分析化合物对E6、E7的抑菌作用的机制；
（5）标准菌株W3110从实验结果可以看出对亚胺培南、氨苄西林、头孢硫脒敏感，同样25068、48564对其不产生抑制作用。
实施例2细菌对小分子化合物的敏感性实验
细菌对小分子化合物2-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-5H,13H-benzo[4,5]isoq uino[2,1-a]quinazoline-5,13-dione的敏感性实验通过测定IC50来确定：
E6、E7均为氨苄西林耐药金黄色葡萄球菌。
| 分组 | Blank1 | Blank2 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| B | 1.322 | 0.05 | 0.142 | 0.097 | 0.154 | 0.434 | 0.684 | 1.023 | 1.024 | 1.091 |
| C | 1.263 | 0.067 | 0.157 | 0.12 | 0.166 | 0.473 | 0.685 | 1.152 | 1.134 | 1.059 |
| F | 1.397 | 0.063 | 0.186 | 0.148 | 0.088 | 0.131 | 0.179 | 0.615 | 0.926 | 0.967 |
| G | 1.392 | 0.049 | 0.156 | 0.144 | 0.162 | 0.224 | 0.313 | 0.537 | 0.996 | 1.118 |
注：其中B、C为E6，F、G为E7。Blank1为不加药物对照，Blank2为不加细菌对照，药物为两倍稀释，单位为mg/L。药物浓度：1:164，2:82，3:41，4:20.5，5:10.25，6:5.1，7:2.55，8:1.275。
最小抑菌浓度（minimal inhibitory concentration，MIC）为肉眼观察结果所得；E6、E7的IC50的测定各重复了两次：
E6：MIC：41mg/L；IC50：9.189209mg/L。
E7：MIC：20.5mg/L；IC50：2.662417mg/L。
虽然，上文中已经用一般性说明及具体实施例对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Claims (4)
1.一种小分子化合物的非疾病治疗目的的抗金黄色葡萄球菌用途，其特征在于，所述小分子化合物为β-内酰胺，该β-内酰胺为2-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-5H,13H-benzo[4,5]isoquino[2,1-a]quinazoline-5,13-dione，其结构式如下所示：
2.根据权利要求1所述的用途，其特征在于，所述金黄色葡萄球菌是对氨苄西林有耐受性的金黄色葡萄球菌。
3.小分子化合物2-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-5H,13H-benzo[4,5]isoquino[2,1-a]quinazoline-5,13-dione在制备抗金黄色葡萄球菌药物中的应用。
4.根据权利要求3的所述应用，其特征在于，所述金黄色葡萄球菌是对氨苄西林有耐受性的金黄色葡萄球菌。
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