Molecular Targets in Peptidoglycan Synthesis

描述（由申请人提供）：淋病奈瑟菌是性传播疾病淋病的病原体，2007年在美国有超过35万人感染淋病。这种生物对多种抗生素的耐药性稳步而不可阻挡地增加，严重限制了淋球菌感染的治疗选择，在最近停用氟喹诺酮类药物后，广谱头孢菌素头孢曲松现在是美国疾病控制与预防中心推荐的唯一单剂量治疗方法。不幸的是，新的淋病奈瑟菌菌株已经出现，对头孢克肟和头孢曲松具有耐药性。现在也有治疗失败的报道。这种不稳定的状况危及公共卫生，需要在分子水平上更好地了解抗生素耐药性，以及开发针对淋病奈瑟菌的新抗菌素的战略。该更新申请将通过研究淋病奈瑟菌参与肽聚糖合成的两种酶来解决这一需求。一种是青霉素结合蛋白2 (PBP 2)，这是一种转肽酶，在细胞壁合成的后期阶段形成肽交联。-内酰胺类抗生素。淋病奈瑟菌耐头孢菌素菌株携带PBP 2突变，关键目标是确定降低PBP 2对这些抗生素反应性的结构机制。我们还将应用核磁共振弛豫方法来验证PBP 2介导的青霉素和头孢菌素耐药的分子机制涉及蛋白质动态状态的假设。认识到参与肽聚糖代谢的其他酶是抗菌剂的潜在靶标，我们还将研究n -乙酰muramyl- l-丙氨酸氨基酶（AmiC），一种淋病奈瑟菌正常细胞分裂所需的自溶酶。我们已经发现这种酶在其n端区域表现出自溶活性，并且在其c端区域表现出已知的酰胺酶活性，因此是一种双功能自溶酶。为了了解AmiC在肽聚糖分解中的功能作用，并为针对其两个活性位点的药物开发铺平道路，我们将获得AmiC的基本结构和生化信息。

DESCRIPTION (provided by applicant): Neisseria gonorrhoeae is the causative agent for the sexually transmitted disease gonorrhea and was responsible for over 350,000 infections in the U.S. in 2007. The steady and inexorable increase of resistance in this organism toward multiple classes of antibiotics has severely limited treatment options for gonococcal infections and, after the recent withdrawal of fluoroquinolones, the expanded-spectrum cephalosporin ceftriaxone is now the only single-dose treatment recommended by the CDC in the U.S. Unfortunately, new strains of N. gonorrhoeae have emerged that exhibit resistance to cefixime and ceftriaxone, and treatment failures are now being reported. This precarious position endangers public health and demands a better understanding of antibiotic resistance at the molecular level, as well as strategies to develop new antimicrobials directed against N. gonorrhoeae. This renewal application will address this need by investigating two enzymes of N. gonorrhoeae involved in peptidoglycan synthesis. One is penicillin-binding protein 2 (PBP 2), a transpeptidase that forms peptide cross-links during the latter stages of cell wall synthesis, and the clinical target for ?-lactam antibiotics directed against this organism. Cephalosporin-resistant strains of N. gonorrhoeae harbor mutations in PBP 2 and a key goal is to determine the structural mechanisms that lower reactivity of PBP 2 with these antibiotics. We will also apply NMR relaxation methods to test the hypothesis that the molecular mechanism governing penicillin and cephalosporin resistance mediated by PBP 2 involves dynamic states of the protein. In recognition that other enzymes involved in peptidoglycan metabolism are potential targets for antimicrobials, we will also investigate N-acetylmuramyl-L-alanine amidase (AmiC), an autolysin that is required for proper cell division of N. gonorrhoeae. We have discovered that this enzyme exhibits autolytic activity in its N-terminal domain in addition to its known amidase activity in the C-terminal domain and therefore is a bifunctional autolysin. To understand the functional role of AmiC in peptidoglycan breakdown, but also to pave the way for drug discovery against its two active sites, we will obtain essential structural and biochemical information for AmiC.