CN116236475A - 桑黄素在制备ndm-1酶抑制剂中的医用用途 - Google Patents
桑黄素在制备ndm-1酶抑制剂中的医用用途 Download PDFInfo
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Abstract
本发明涉及桑黄素在制备NDM‑1酶抑制剂中的应用，公开了天然产物桑黄素的一种新的医用用途。本发明通过NDM‑1酶抑制试验、微量棋盘稀释法、时间‑杀菌曲线等试验验证桑黄素能够显著抑制NDM‑1酶活性并恢复美罗培南、头孢噻呋钠及氨苄西林对NDM‑1阳性大肠杆菌的抗菌活性。因此，本发明确定桑黄素可作为NDM‑1抑制剂，将其与β‑内酰胺类抗生素联合应用，以提高现有β‑内酰胺类抗生素抗菌活性，成本低且安全高效，具有广泛的医用用途。
Description
技术领域
本发明涉及天然药物领域，具体涉及桑黄素的新药理用途，即桑黄素在制备NDM-1酶抑制剂中的医用用途，属于医学制药技术领域。
背景技术
β-内酰胺类抗生素由于抗菌活性高、抗菌谱广、毒副作用少等优点，一直是医学临床和动物养殖中应用最广泛的抗菌药物。随着β-内酰胺类抗生素在临床与畜禽生产中的广泛应用和不合理的使用，细菌已经进化出复杂的耐药机制，来逃避抗生素分子的抑制或杀灭，致使耐碳青霉烯类的“超级细菌”的出现与爆发。
细菌分泌的β-内酰胺酶(BLs)可高效水解多种β-内酰胺类抗生素，是β-内酰胺类抗生素失效并产生细菌耐药性的最主要机制，革兰氏阴性杆菌产生的BLs通过水解β-内酰胺环而使抗生素失去抗菌活性。Ambler等根据BLs分子结构中末端氨基酸序列的差异性，将耐药菌产生的BLs分为丝氨酸β-内酰胺酶(SBLs)和金属β-内酰胺酶(MBLs)两类。长期以来，大多数酶抑制剂的筛选策略是特异性的针对某类酶，到目前为止研究比较成功的就是SBLs抑制剂，目前已获FDA批准的SBLs抑制剂包括克拉维酸、舒巴坦、他唑巴坦等，大大增强青霉素类和头孢菌素类抗生素的抗菌活性，但目前在临床实践中仍然缺乏针对产MBLs菌株的有效的特异性抑制剂。
NDM-1是MBLs中B1亚类的新成员，2009年首次在印度新德里发现并报道，随后在全球范围内广泛流行。表达NDM-1的细菌能稳定高效水解除了单环类之外的所有β-内酰胺类抗生素，包括医学临床对抗多重耐药革兰氏阴性菌感染最后防线的广谱头孢菌素类和碳青霉烯类等。blaNDM-1基因也已经在多国的食品动物、野生动物和宠物源细菌中出现并流行，甚至可以通过生物媒介在动物-环境-人之间迅速传播，NDM-1赋予细菌的抗生素耐药性已经成为人类和兽医临床治疗感染的头号威胁。因此，寻找特异性NDM-1酶抑制剂是遏制“超级细菌”引起的感染最迫切的需求。
天然产物具有结构多样性和生物活性多样性，是抗NDM-1新药和先导化合物发现的宝贵资源，这有助于发现具有最佳抑菌活性的衍生物。桑黄素又名桑耳、胡孙眼、梅树菌，是从桑黄中提取得到的一种植物多酚，具有成本低、易获取等特点，药理作用广泛。桑黄素具有抗痛、抗菌、抗炎、抗肿瘤、抗动脉粥样硬化、降低血糖和抗应激等药用作用，具有良好的应用价值和开发前景。桑黄有“森林黄金”之称，临清桑黄与东阿阿胶、冠县灵芝被誉为聊城“新三宝”，因而受到国内外广泛关注。截至目前，国内外未见桑黄素在制备NDM-1酶抑制剂中的相关报道。
发明内容
本发明的主要目的是提供了桑黄素在制备NDM-1酶抑制剂中的医用用途，公开了桑黄素能够抑制NDM-1酶的活性，恢复β-内酰胺类抗生素对NDM-1阳性大肠杆菌的杀菌活性。
为了实现上述目的，本发明所采用的技术方案包括：
本发明一方面提供了桑黄素作为NDM-1抑制剂的新药理用途，即抑制NDM-1对β-内酰胺类抗生素的水解作用，恢复β-内酰胺类抗生素对携带NDM-1阳性菌的抗菌活性。因此，桑黄素能够作为β-内酰胺类抗生素的保护剂。
本发明另一方面提供了一种抑制致病菌的药物组合物，包括有效量的抗生素、酶抑制剂和药学上可接受的载体或辅料，其中，所述的抗生素优选为β-内酰胺类抗生素；所述的酶抑制剂是桑黄素。
按照本领域的常规制剂方法将所述的药物组合物制备成临床常用的制剂，例如是粉剂、颗粒剂、片剂、胶囊剂、注射剂等，通过注射、口服、滴鼻、滴眼、物理或化学介导的方法将其导入肌肉、内皮、皮下、静脉、粘膜组织，或是被其他物质混合或包裹后导入肌体。
所述的载体或辅料是指药学领域常规的载体或辅料，例如：稀释剂、崩解剂、润滑剂、赋形剂、粘合剂、助流剂、填充剂、表面活性剂等。
本发明中所述的桑黄素包括其原型、药学上可接受的盐或者含桑黄素的制剂。
本发明中的所述β-内酰胺类抗生素的代表药物是美罗培南，其分子式为：C17H25N3O5S，分子量为：383.5；头孢噻呋钠，其分子式为：C19H18N5NaO7S3，分子量为：547.55；氨苄西林，其分子式为：C16H19N3O4S，分子量为：349.4
本发明中所述的NDM-1酶是从自然界中提取或用基因工程菌制备得到重组NDM-1酶。
本发明中所述的“致病菌”优选为革兰氏阴性和阳性致病菌，更优先为NDM-1阳性大肠杆菌。
桑黄素，别名：桑耳、胡孙眼、梅树菌；其分子式为：C15H10O7，分子量为：302.24，其结构式如下：
本发明通过NDM-1酶抑制试验、体外抗菌活性试验验证桑黄素能够抑制NDM-1的活性并恢复β-内酰胺类抗生素对NDM-1阳性大肠杆菌的抗菌活性，用于治疗细菌感染性疾病，具有广泛的医用用途。
本发明整体技术方案详述
针对E.coli关键耐药酶NDM-1，我们建立了基于分子对接的虚拟筛选平台。本试验以NDM-1晶体结构为靶标蛋白，配体库为含有3万个配体分子天然产物化合物库。应用计算机辅助药物设计软件GLIDE和MAESTRO，基于Glide和Maestro进行受体与配体的分子对接。桑黄素可结合在NDM-1以锌离子为中心的活性区域且复合物结构稳定，与蛋白活性中心空间匹配、与疏水活性口袋紧密结合且能够与NDM-1活性区域的氨基酸形成较强相互作用，因此桑黄素被视为具有潜在NDM-1抑制作用的候选化合物。
在此基础上，本发明进行了桑黄素对NDM-1酶抑制活性的检测试验，桑黄素对NDM-1酶活性的抑制作用呈极显著的剂量依赖性(P<0.01)，最大抑制率为94.7％，IC50为8.5±0.5μM。
最小抑菌浓度试验结果表明桑黄素单独使用不具备抑菌效果，与β-内酰胺类抗生素联合使用能够使β-内酰胺类抗生素对NDM-1阳性大肠杆菌MIC值降低2-16倍。FIC指数表明桑黄素与β-内酰胺类抗生素联合使用，对抑制产NDM-1的菌株有显著的协同作用。
根据时间-杀菌曲线可见，桑黄素联用β-内酰胺类抗生素能够显著抑制NDM-1阳性大肠杆菌的生长。
附图说明
图1为桑黄素与靶标蛋白NDM-1的相互作用模式图。
图2为重组质粒构建与NDM-1表达纯化。
图3为桑黄素对NDM-1酶抑制活性。
图4为桑黄素联合β-内酰胺类抗生素的棋盘法最小抑菌浓度。
图5为桑黄素联合β-内酰胺类抗生素的时间-杀菌曲线。
具体实施方式
以下结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
试验例1基于分子对接的NDM-1抑制剂虚拟筛选
基于Glide和Maestro进行受体与配体的分子对接虚拟筛选，根据打分函数将所有配体与NDM-1的结合能由低到高进行排序，最终挑选出桑黄素作为候选化合物。结果发现，桑黄素与NDM-1对接产物结合自由能小于-10.0Kcal/mol，且桑黄素可结合在NDM-1以锌离子为中心的活性区域且复合物结构稳定，与蛋白活性中心空间匹配、与疏水活性口袋紧密结合且能够与NDM-1活性区域的氨基酸形成较强相互作用(图1)。
试验例2NDM-1的表达与纯化
通过EcoRⅠ和XhoⅠ限制性内切酶，构建pET32(α)-NDM-1重组质粒，并转入大肠杆菌BL21(DE3)中，构建表达工程菌。应用终浓度为1mM的IPTG，37℃诱导4.5h，4℃离心收集细菌。将细菌超声破碎，收集上清过Ni2+-NTA亲和层析柱，使用不同浓度梯度咪唑梯度洗脱，SDS-PAGE印记检测NDM-1蛋白表达纯化结果，获得纯度大于90％的NDM-1重组蛋白(图2)，用于酶活性抑制试验筛选NDM-1有效抑制剂。
试验例3桑黄素对NDM-1酶抑制试验
构建NDM-1酶活性抑制剂体系，测定桑黄素的酶抑制活性。在96孔板反应体系中加入50μL不同浓度美罗培南及等体积的NDM-1中，30℃孵育15min检测295nm紫外吸收与体系颜色变化。计算不同浓度的桑黄素对NDM-1酶水解底物的抑制率及半抑制浓度IC50值，结果表明桑黄素对NDM-1酶活性的抑制作用呈极显著的剂量依赖性(P<0.01)，抑制率为94.7％，IC50为8.5±0.5μM(图3)。
试验例4体外抗菌活性初步评价
采用微量肉汤稀释法分别测定桑黄素、美罗培南、头孢噻呋钠、氨苄西林(浓度梯度为1-2048μg/mL)的最小抑菌浓度(MIC)。采用棋盘法配96孔板，加入不同浓度的桑黄素与美罗培南、头孢噻呋钠、氨苄西林，确定二者联合应用最小抑菌浓度MIC值，并计算部分抑菌浓度指数FIC值。桑黄素与美罗培南、头孢噻呋钠、氨苄西林单独或联合用药对NDM-1阳性E.coli的体外抗菌活性结果如表1、图4所示，美罗培南、头孢噻呋钠、氨苄西林单独用药时，MIC分别为64μg/mL、128μg/mL、256μg/mL，抑菌作用较弱，桑黄素单独用药时，MIC为512μg/mL，表明桑黄素本身几乎没有抑菌作用。将桑黄素与美罗培南、头孢噻呋钠、氨苄西林联合用药时能够使美罗培南、头孢噻呋钠、氨苄西林对NDM-1阳性E.coli MIC值降低32、4、32倍，FICI分别为0.094、0.375、0.156，具有显著的体外协同作用。
表1不同抗生素与桑黄素联合应用对NDM-1阳性大肠杆菌的MIC值
注：当FICI≤0.5，为协同作用；0.5<FICI≤1，为相加作用；1<FICI≤4，为无关作用；当FICI>4，为拮抗作用。
试验例5时间-杀菌曲线试验
从NDM-1阳性大肠杆菌的纯培养平皿上挑取单菌落于MH肉汤培养基中37℃培养6-8h，取细菌培养物，用麦氏比浊仪以无菌生理盐水调节菌液终浓度为106CFU/mL。设置无抗生素空白对照组、抗生素组、桑黄素组、联合用药组。试验组与对照组在同样菌液浓度的条件下于37℃培养，分别于0、1、3、5、7、9、11h从各组取出定量培养液转种于相应琼脂培养基中，37℃培养18-24h后作菌落计数，以菌落数的对数为纵坐标，以培养时间为横坐标，绘制时间-杀菌曲线(图5)。
根据图5的时间-杀菌曲线可见，桑黄素单独用药对NDM-1阳性大肠杆菌无杀菌作用，证明了是桑黄素自身对NDM-1阳性大肠杆菌生长无显著影响。美罗培南、头孢噻呋钠、氨苄西林单独用药时，0-5h NDM-1阳性大肠杆菌的菌落计数显著下降，但在5h后细菌增长呈上升趋势，表明单抗生素对NDM-1阳性大肠杆菌的杀菌作用较弱。当桑黄素与美罗培南、头孢噻呋钠、氨苄西林联合用药时，NDM-1阳性大肠杆菌数量显著下降，当共培养时间≥3h，几乎检测不到产NDM-1耐药菌，表明联合用药具有协同作用，能够显著抑制NDM-1阳性大肠杆菌的生长。
Claims (10)
1.桑黄素在制备新德里金属-β-内酰胺酶-1抑制剂中的用途。
2.桑黄素在制备抗生素保护剂中的医用用途。
3.按照权利要求2所述的用途，其特征在于：所述抗生素是β-内酰胺类抗生素。
4.按照权利要求3所述的用途，其特征在于：所述β-内酰胺类抗生素包括碳青霉烯类、头孢菌素类及青霉素类。
5.按照权利要求1或2所述的用途，其特征在于：所述桑黄素包括其原型、药学上可接受的盐或者含桑黄素的制剂。
6.一种抑制致病菌的药物组合物，其特征在于，包括：预防或治疗上有效量的抗生素、桑黄素和药学上可接受的辅料或载体。
7.按照权利要求6所述的药物组合物，其特征在于：所述的致病菌是细菌。
8.按照权利要求7所述的药物组合物，其特征在于：所述的细菌是革兰氏阴性或者革兰氏阳性致病菌，优选是NDM-1阳性大肠杆菌。
9.按照权利要求6所述的药物组合物，其特征在于：所述β-内酰胺类抗生素包括碳青霉烯类、头孢菌素类及青霉素类。
10.按照权利要求6或9所述的药物组合物，其特征在于：所述β-内酰胺类抗生素是美罗培南、头孢噻呋钠及氨苄西林。
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