Molecular Targets in Peptidoglycan Synthesis

描述(申请人提供)：淋病奈瑟氏菌是性传播疾病淋病的病原体，2007年在美国造成超过350,000例感染。这种细菌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，在最近氟喹诺酮类药物停用后，广谱头孢菌素头孢曲松现在是美国疾控中心推荐的唯一单剂量治疗。不幸的是，出现了对头孢克辛和头孢曲松具有耐药性的新的淋球菌菌株，治疗失败的报道现在正在被报道。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，以及开发针对淋病奈瑟菌的新抗菌剂的策略。这一新的应用将通过研究淋球菌参与肽聚糖合成的两种酶来满足这一需求。一种是青霉素结合蛋白2(PBP 2)，是一种转肽酶，在细胞壁合成的后期形成多肽交联物，是针对这种细菌的β-内酰胺类抗生素的临床靶点。淋病奈瑟菌的头孢菌素耐药株存在PBP-2基因突变，一个关键的目标是确定降低PBP-2对这些抗生素的反应性的结构机制。我们还将应用核磁共振弛豫方法来检验这一假说，即PBP2介导的青霉素和头孢菌素耐药性的分子机制与蛋白质的动态有关。认识到参与肽聚糖代谢的其他酶是潜在的抗菌药靶标，我们还将研究N-乙酰胞壁酰基-L-丙氨酸酰胺酶(AMIC)，这是淋球菌正常细胞分裂所必需的自溶素。我们发现该酶除了在C-末端具有已知的酰胺酶活性外，还在其N-末端具有自溶活性，因此是一种双功能自溶素。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部位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.