Molecular Targets in Peptidoglycan Synthesis

描述(申请人提供)：毛尿素球囊是一种由交联肽聚糖链组成的网状物，使细菌细胞壁变得坚硬。β-内酰胺类抗生素是治疗细菌性疾病的重要化合物，它针对的是交联肽多糖链的基本转肽酶(青霉素结合蛋白或PBPs)。不幸的是，多种抗生素耐药机制的出现可能会使这些和其他抗生素在治疗细菌感染方面过时。与其他病原菌一样，淋球菌对抗生素的耐药性是一个日益严重的问题。青霉素和四环素曾经是治疗淋球菌感染的首选抗生素，但由于出现了耐药菌株，现在不再使用了。此外，越来越多的菌株现在对氟喹诺酮类药物产生抗药性，氟喹诺酮类药物是目前推荐用于治疗淋病的两种抗生素之一。显然，迫切需要开发新的抗菌剂，既针对众所周知的分子靶标，如多溴联苯并苯酚，也针对新的靶标。在这项建议中，我们描述了三种与肽聚糖代谢有关的酶的结构和生化研究：来自大肠杆菌的D-D-羧基肽酶(PBP5)，作为阐明PBP功能的模型系统；淋球菌的基本转肽酶(PBP2)，它是当前β-内酰胺类抗生素的致命靶标；以及裂解转糖基酶，MltA，也来自淋球菌，它是细胞壁合成复合体的关键。这些蛋白质中的每一种都被选择来解决以下一个或多个目标：(A)了解肽聚糖合成的生物学，(B)探索它们与抗生素的相互作用，(C)阐明抗生素耐药性的分子基础，以及(D)检查它们作为药物开发靶点的潜力。对PBP 5的研究将阐明该酶降解底物的机制，并将从总体上更好地了解PBP与抗生素的相互作用。PBP 2对抗生素耐药性的分子基础将通过对天然酶和从青霉素耐药菌株中分离出的突变株的结构研究来研究。MltA作为介导肽多糖合成的多酶复合体的一部分的作用以及它作为抗菌剂的新靶点的适用性将通过求解其晶体结构来检验。这些研究将为未来以基于结构的药物设计为目标的研究提供一个框架，并将为肽多糖合成的机制提供实质性的见解。

DESCRIPTION (provided by applicant): The murein sacculus is a mesh of cross-linked peptidoglycan strands that confers rigidity to the bacterial cell wall. Beta-lactam antibiotics, which target the essential transpeptidases (penicillin-binding proteins or PBPs) that cross-link the peptidoglycan strands, are important compounds in the treatment of bacterial diseases. Unfortunately, the emergence of multiple mechanisms of antibiotic resistance threatens to make these and other antibiotics obsolete in the treatment of bacterial infections. Along with other pathogenic bacteria, antibiotic resistance in Neisseria gonorrhoeae is a growing problem. Penicillin and tetracycline, once the antibiotics of choice for treatment of gonococcal infections, are no longer be used due to the emergence of resistant strains. Moreover, increasing numbers of strains are now resistant to the fluoroquinolones, one of the two antibiotics current recommended in the treatment of gonorrhea. Clearly there is an urgent need to develop new antimicrobials directed both against well-known molecular targets, such as PBPs, but also against novel targets. In this proposal we describe structural and biochemical studies of three enzymes involved in peptidoglycan metabolism: a D-D-carboxypeptidase from E. coli (PBP 5) that serves as a model system for elucidating PBP function, an essential transpeptidase (PBP 2) from N. gonorrhoeae that is the lethal target of current beta-lactam antibiotics, and a lytic transglycosylase, MltA, also from N. gonorrhoeae, that serves as the lynchpin of the cell wall synthesizing complex. Each of these proteins has been selected to address one or more of the following aims: (a) to understand the biology of peptidoglycan synthesis, (b) to explore their interactions with antibiotics, (c) to elucidate the molecular basis for antibiotic resistance and (d) to examine their potential as targets for drug development. Studies on PBP 5 will elucidate the mechanism by which this enzyme hydrolyzes substrate and will provide a better understanding of PBP-antibiotic interactions in general. The molecular basis for antibiotic resistance in PBP 2 will be investigated by structural studies of the native enzyme and of a mutant isolated from a penicillin-resistant strain. The role of MltA as part of a multienzyme complex mediating peptidoglycan synthesis as well as its suitability as a novel target for antimicrobials will be examined by solving its crystal structure. These studies will provide a framework for future studies aimed at structure-based drug design and will provide substantial insight into the mechanisms of peptidoglycan synthesis.