Inhibitors of wall teichoic acid biosynthesis of Staphylococcus aureus

描述(申请人提供)：由革兰氏阳性病原体引起的多重耐药感染的日益流行，如耐甲氧西林金黄色葡萄球菌(MRSA)，突显了临床上治疗危及生命的感染的新策略的必要性。最近已经推出了有效对抗MRSA的新的抗菌药物，但必须静脉给药，仅限于医院使用，耐受性不好，并且已经受到耐药性的影响。拟议研究的总体目标是发现一类针对金黄色葡萄球菌壁磷壁酸(WTA)生物合成途径的新型药物。WTA是富含磷酸盐的高度阴离子聚合物，与大多数革兰氏阳性病原体的细胞壁共价连接。WTA途径的后期酶步骤失活会阻止生长，除非该途径被最初酶步骤的失活所阻断。金黄色葡萄球菌中WTA的缺失会导致细胞壁结构和细胞分裂的异常，并增加自溶。WTA还在以下几个方面发挥重要作用：寄主定植、对抗菌肽和抗菌脂的抗性以及生物被膜的形成。值得注意的是，WTA的缺失增加了MRSA对β-内酰胺类抗生素的敏感性。有效的WTA抑制剂将提供多种治疗益处，如抑制细菌生长，降低毒力，恢复β-内酰胺类抗生素治疗MRSA的临床实用价值，这是一项非常重要的创新。我们的战略是寻找WTA生物合成途径中特定步骤的有效抑制剂，并将它们开发成抗菌药物，与FDA批准的β-内酰胺类抗生素联合治疗金黄色葡萄球菌感染。在第一阶段，我们将修改和优化一种经过验证的筛选试验，以提高该试验对特定WTA靶标的抑制剂的灵敏度。我们将使用这一测试来筛选&gt；30万种谨慎的小分子化合物来识别抑制剂，我们将使用一系列现有的二次测试来优先考虑它们，这些二次测试旨在评估β-内酰胺类抗生素的抗菌效力、抗菌谱、选择性、耐药性频率和增强作用。我们将核实高优先级命中的分子目标。在第二阶段，我们将利用合理的药物设计，通过优化其活性和特异性，将最有希望的有效HITS开发成先导化合物。这项建议的具体目标如下。目的1.修改和优化已建立的基于细胞的途径特异性筛选方法，以利于鉴定WTA生物合成特定步骤(TARB、D和F)的抑制剂。目的2.筛选多种化合物文库，鉴定和确认WTA合成的抑制剂。目的3.用二次分析方法验证已证实的WTA合成抑制剂，并鉴定HIT系列。目的4.验证选定的热门系列的作用机制。 与公共卫生相关：这项研究的目标是发现可以开发成有效药物的化合物，这些药物将用于治疗由耐甲氧西林金黄色葡萄球菌(又称MRSA)引起的严重感染。这些新药将干扰金黄色葡萄球菌产生一种名为壁磷壁酸(WTA)的糖类聚合物的能力，这种聚合物包裹在细菌的外表面，使其能够在人类身上定居并导致感染。干扰WTA生产的化合物通过阻止细菌生长而起到抗生素的作用，并使MRSA对重要的β-内酰胺类抗生素敏感，包括青霉素、甲氧西林和氨苄西林。创新的WTA活性药物的成功开发将是治疗MRSA感染的重大进步。

DESCRIPTION (provided by applicant): The increasing prevalence of multidrug resistant infections caused by Gram-positive pathogens, such as methicillin resistant Staphylococcus aureus (MRSA), underscores the clinical need for novel strategies to treat life-threatening infections. New antibacterial drugs that are effective against MRSA have been introduced recently, but must be given intravenously, are limited to hospital use, are not tolerated well, and are already subject to resistance. The overall goal of the proposed research is discover a novel class of drugs that target the wall teichoic acid (WTA) biosynthetic pathway of S. aureus. WTAs are phosphate rich, highly anionic polymers that are covalently linked to the cell wall of most Gram-positive pathogens. Inactivation of the later enzymatic steps of the WTA pathway prevents growth, unless the pathway is blocked by inactivation of the initial enzymatic steps. Loss of WTA in S. aureus results in abnormalities in cell wall structure and cell division, and increases autolysis. WTA also plays an important role in the following aspects of virulence: host colonization, resistance to antibacterial peptides and lipids, and biofilm formation. Significantly, loss of WTA increases the sensitivity of MRSA to beta-lactam antibiotics. Potent WTA inhibitors will provide multiple therapeutic benefits, such as bacterial growth inhibition, decreased virulence, and renewed clinical utility of beta- lactam antibiotics for treating MRSA, which represents a highly significant innovation. Our strategy is to identify potent inhibitors of specific steps in the WTA biosynthetic pathway, and to develop them into antibacterial drugs that will be used in combination therapy with an FDA-approved beta-lactam antibiotic to treat S. aureus infections. In Phase I, we will modify and optimize a validated screening assay to increase the sensitivity of the assay for inhibitors of specific WTA targets. We will use this assay to screen >300,000 discreet small molecule compounds to identify inhibitors, and we will prioritize them using a panel of existing secondary assays designed to evaluate antibacterial potency, antibacterial spectrum, selectivity, frequency of resistance, and potentiation of beta-lactam antibiotics. We will verify the molecular target of high priority hits. In Phase II, we will develop the most promising validated hits into lead compounds by optimizing their activity and specificity using rational drug design. The specific aims of this proposal are as follows. Aim 1. Modify and optimize an established cell based, pathway specific screening assay to favor the identification of inhibitors of specific steps of WTA biosynthesis (TarB, D, and F). Aim 2. Screen a diverse compound library, identify, and confirm inhibitors of WTA synthesis. Aim 3. Validate confirmed inhibitors of WTA synthesis using secondary assays and identify hit series. Aim 4. Verify mechanism of action of selected hit series. PUBLIC HEALTH RELEVANCE: The goal of this research is to discover chemical compounds that can be developed into powerful drugs that will be used to treat serious infections caused by methicillin resistant Staphylococcus aureus, also known as MRSA. These new drugs will interfere with the ability of S. aureus to produce a sugar polymer, known as wall teichoic acid (WTA), which coats the outer surface of the bacterium and enables it to colonize humans and cause infection. Compounds that interfere with WTA production act as antibiotics by preventing bacterial growth, and also sensitize MRSA to the important beta lactam class of antibiotics, which includes penicillin, methicillin, and ampicillin. The successful development of innovative WTA-active drugs will be a significant advance in the treatment of MRSA infections.