以细胞膜为靶标的新型抗菌肽polybia-MPI类似物的设计、合成及抗菌活性研究

With the extensive use of antibiotics, there are frequent emergence of the resistant bacteria. What even worse, there are few active agents available for treating serious infections, since the prevalence of carbapenem-resistant and vancocin-resistant bacteria is increasing. Carbopenems and vancocin were thought to be the "last line" treatment agents available at present. New agents with novel action mode were urgently needed than ever before.It is now widely accepted that antimicrobial peptides (AMPs) could be promising alternatives to conventional antibiotics. AMPs target at the cell membrane, which is a thin and structured bilayer of phospholipid and protein molecules that envelopes the cell.So it was thought that AMPs were not affected by conventional multidrug resistant mechanism and it was difficult to develop resistance to it. Polybia-MPI was a short cationic α-helical amphiphilic antimicrobial peptide. In our study, a series of polybia-MPI derivatives were designed and synthsized to improve the antimicrobial activity, the stability in vivo and bioavailability by chemical modification, such as unnatural amino acid subtitution, inersion of D-amino acid, polymerization and partial cyclization. The in vitro and in vivo antimicrobial activity of all the derivatives against both the wild type bacteria and clinially isolated resistant bacteria will be studied. The results will be used to evaluate the clinical application potential of antimicrobial peptide. The spectrum analysis and molecular dynamics simulation will be employed to study the peptide-membrane interaction and structure-activity relationship, in order to further clarify the exact mechanism of antimicrobial peptide.So with the increasing resistance to conventional antibiotics, there is no doubt that the present study could offer a new strategy and some excellent alternatives to conventional antibiotics to defend the resistant bacteria.

日益流行的多药耐药病原菌严重威胁人类生命安全。目前，已有病原菌对万古霉素、甲氧西林、碳青霉烯类抗生素耐药，一旦这些病原菌大范围流行，人类将将面临无药可用的窘境。因此，亟需发展作用机制新颖，不易引起耐药的新型药物。抗菌肽大多通过破坏细胞膜完整性杀菌而引起了广泛关注。本项目拟通过非天然氨基酸替换、D-逆肽改造、类肽修饰、引入新型侧链基团、聚合化或局部环化等对天然小分子抗菌肽polybia-MPI进行修饰改造，致力于提高其抗菌活性、生物稳定性和生物利用度；通过体内外抗菌（含临床耐药病原菌）活性研究及野生菌对药物敏感性变迁的研究，探讨polybia-MPI类似物在治疗耐药菌引起的感染的临床应用潜力; 通过膜-肽相互作用、波谱分析结合分子动力学模拟等研究探讨构效关系，阐明作用机制。本项目的开展将为抗菌肽的临床应用、发展不易耐药的、以细胞膜为靶标的新型抗菌药物提供思路和研究基础，具有重要的临床意义。

当前，多药耐药细菌的增多和真菌感染增多及真菌耐药性的蔓延严重威胁人类的生命健康。鉴于此发展作用机制不同于传统抗菌、抗真菌药物，不受传统耐药机制影响的抗菌、抗真菌药物尤为迫切，具有重要医学价值和社会意义。抗菌肽被认为主要作用于病原菌的细胞膜，通过静电相互作用与细胞膜结合，然后膜-肽相互作用破坏磷脂双分子层结构引起细胞膜完整性丧失，最终引起细菌、真菌死亡。病原菌很难通过改变细胞膜的脂质基架而发展对抗菌肽的耐药性，因此通过对天然抗菌肽的结构修饰，有望开发不易引起耐药的新型药物。.项目组以抗菌肽polybia-MPI为研究对象，研究了其抗菌、抗真菌活性，探索了其作用机制，用化学方法修饰polybia-MPI结构，并化学合成了这些衍生物，通过活性、稳定性、毒性测定，结合波谱学和分子动力学模拟等研究了其构效关系，对抗菌肽的化学修饰方法进行了有益探索，并将这些作用机制研究方法和化学修饰方法应用于其它抗菌肽protonectin、NK-18、polybia-CP、CPF-C1，并对它们的抗菌、抗真菌活性、作用机制及构效关系进行了深入探索；此外，项目组还对部分用于抗菌肽修饰的生物活性分子结构单元的化学合成方法进行了研究。研究结果发现抗菌肽polybia-MPI、protonectin、NK-18、polybia-CP、CPF-C1具有良好的抗菌、抗真菌活性，它们对耐药菌同样具有很好的选择杀伤作用，主要通过对细菌或真菌的细胞膜完整性破坏引起细胞内容物外泄引起，不同传统抗菌、抗真菌药物作用机制。然而，这些天然抗菌肽对蛋白酶敏感，容易被降解，在体内半衰期短，而不适用于临床使用。由于抗菌肽的二级结构对其活性至关重要，修饰中需全面考虑构象、电荷、两亲性、疏水性等影响因素，结构修饰单元对抗菌肽结构和其它物化性质影响越小越好；此外修饰位点的选择也很重要。本研究中用鸟氨酸、二氨基丁酸、二氨基丙酸、D-赖氨酸、D-精氨酸分别提换序列中的阳离子氨基酸得到的衍生物稳定性均得到提高，且其生物活性均保持同母体肽甚至有所提高。然而，生物活性分子引入抗菌肽链的引入位点和链接方式仍需进行探索。.总之，本项目研究发现发展以膜为作用靶点的阳离子抗菌肽的候选药物可以成为发展不易引起耐药的新型抗菌抗真菌药物的一个方向；通过合适的化学修饰方法可以提高抗菌肽的稳定性，对肽类药物的抗菌、抗真菌药物临床应用具有重要意义。