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年在美国造成了超过350，000例感染。这种生物体对多种抗生素的耐药性的稳定和不可阻挡的增加严重限制了淋球菌感染的治疗选择，并且在最近氟喹诺酮类药物退出后，扩谱头孢菌素头孢曲松现在是美国CDC推荐的唯一单剂量治疗。已经出现了对头孢克肟和头孢曲松表现出耐药性的淋病，现在有治疗失败的报道。这种不稳定的状况危及公众健康，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N。淋病本更新申请将通过研究N.淋病涉及肽聚糖合成。一种是青霉素结合蛋白2（PBP 2），这是一种在细胞壁合成后期形成肽交联的转肽酶，也是？内酰胺类抗生素直接对抗这种微生物。头孢菌素耐药的N.淋病携带PBP 2突变，关键目标是确定降低PBP 2与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检验这一假设，即由PBP 2介导的青霉素和头孢菌素耐药性的分子机制涉及蛋白质的动态状态。认识到参与肽聚糖代谢的其他酶是抗菌剂的潜在靶点，我们还将研究N-乙酰胞壁酰-L-丙氨酸酰胺酶（AmiC），一种N.淋病我们已经发现，这种酶除了在C-末端结构域中具有已知的酰胺酶活性外，还在其N-末端结构域中具有自溶活性，因此是双功能自溶素。为了了解AmiC在肽聚糖分解中的功能作用，同时也为针对其两个活性位点的药物发现铺平道路，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.