构巢裸胞壳抗NDM-1超级细菌新颖次生代谢产物研究

New Delhi metallo-β-lactamase-1(NDM-1)-producing bacteria are called “Superbugs”, and the resistance of NDM-1-producing bacteria to nearly all β-lactam antibiotics makes the clinical treatment facing tremendous challenges. The development of new NDM-1 inhibitors is the main way to deal with NDM-1 superbugs. The applicant found that the extract of Emericella nidulans could inhibit NDM-1 in vitro by screening of a large number of fungi extracts. Additional phytochemical investigation of the extract of E. nidulans lead to the discovery of a novel polyketide showed potent NDM-1 inhibition activity. Therefore, systematically phytochemical investigations of polyketides in E. nidulans are necessary by means of OSMAC. A variety of spectral data, X-ray single crystal diffraction, electronic circular dichroism calculation, and chemical conversion will be used to determine the structure and the absolute configuration of compounds. Computer virtual screening and in vitro NDM-1 inhibition activities experiments will be performed to evaluate their NDM-1 inhibitor activities. Finally, the mechanism of action of active compounds targeted the NDM-1 will be clarified. This project will greatly promote the development of NDM-1 inhibitors.

NDM-1超级细菌对几乎所有的β-内酰胺类抗生素耐药，临床治疗面临巨大挑战。研发新的NDM-1抑制剂是目前应对NDM-1超级细菌的主要手段。申请人前期以抗NDM-1酶活性为导向，从构巢裸胞壳小规模液体发酵产物中发现了一个具有显著抗NDM-1活性的新骨架聚酮类化合物。本项目拟采用OSMAC策略对构巢裸胞壳多种不同条件下的发酵产物进行研究，发现更多结构新颖的聚酮类化合物。综合利用多种波谱、X-Ray单晶衍射、ECD计算和化学转化等方法解析单体化合物的结构。通过计算机虚拟筛选及体外酶活性实验初步评价它们的NDM-1抑制活性，进一步通过菌体和动物水平实验充分评价其药效，对活性显著的化合物采用分子生物学和结构药理学的方法阐明其通过抑制NDM-1发挥抗菌活性的作用机制。该项目对开发NDM-1抑制剂类天然先导化合物具有重要的意义。

细菌耐药是本世纪全球关注的热点问题，已成为严重的公共卫生危机。NDM-1超级细菌对几乎所有的β-内酰胺类抗生素耐药，临床治疗面临巨大挑战。研发新的NDM-1抑制剂是目前应对NDM-1超级细菌的主要手段。申请人以抗NDM-1酶活性为导向，采用OSMAC策略对构巢裸胞壳最佳条件下的发酵产物进行研究。得到19个单体化合物，其中新化合物11个。新化合物包括三个高度甲基化的聚酮新骨架化合物emeriones A−C；两个异戊烯基化的喹啉酮生物碱 aspoquinolones E和F；三个异戊烯基化的异吲哚酮生物碱。推导Emeriones A−C的生源合成途径是在聚酮合成酶的作用下经过佩恩重排反应、双键异构化、6π-及8π-电子环化形成双环[4.2.0]辛烯母核的中间体，最后通过氧化过程得到。Emeriones A−C的发现增加了聚酮的骨架结构类型，丰富了构巢曲霉的次生代谢产物类型。通过计算机虚拟筛选技术﹑微量热泳动和酶活性实验发现具有emerione A与NDM-1具有最强的结合力(Kd为25μM)，并能有效抑制NDM-1活性(IC50为1.23±0.15µM)。抗菌活性实验结果提示emerione A对两种高表达NDM-1的菌株E.coli BL21(DE3)/ pET28a-NDM-1以及K. pneumoniae ATCC BAA2146均具有抗菌活性，MIC分别为8µg/mL及16µg/mL。与美罗培南合用，可将后者对K. pneumoniae ATCC BAA2146的MIC从128µg/mL降低到0.5µg/mL，破坏细菌细胞壁和细胞膜，促进联用的抗菌药物成功进入细菌细胞内。以上实验表明emerione A是一个有效的NDM-1抑制剂。此外，依托本项目建立的抗NDM-1耐药菌活性筛选平台为课题组其他化合物的抗菌活性测试提供了支持。