Inhibitors of wall teichoic acid biosynthesis of Staphylococcus aureus

描述（由申请人提供）：由革兰氏阳性病原体（例如耐甲氧西林金黄色葡萄球菌（MRSA））引起的多重耐药感染的患病率不断增加，强调了临床需要治疗危及生命的感染的新策略。最近推出了对 MRSA 有效的新型抗菌药物，但必须静脉注射，仅限于医院使用，耐受性不佳，并且已经产生耐药性。该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 途径后续酶促步骤的失活会阻止生长，除非该途径被初始酶促步骤的失活所阻断。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异常，并增加自溶作用。 WTA还在毒力的以下方面发挥重要作用：宿主定植、抗菌肽和脂质的抗性以及生物膜形成。值得注意的是，WTA 的缺失会增加 MRSA 对 β-内酰胺抗生素的敏感性。有效的 WTA 抑制剂将提供多种治疗益处，例如抑制细菌生长、降低毒力以及重新使用 β-内酰胺抗生素治疗 MRSA，这代表了一项非常重要的创新。我们的策略是识别 WTA 生物合成途径中特定步骤的有效抑制剂，并将其开发为抗菌药物，与 FDA 批准的 β-内酰胺抗生素联合治疗，以治疗金黄色葡萄球菌感染。在第一阶段，我们将修改和优化经过验证的筛选测定，以提高特定 WTA 靶点抑制剂测定的灵敏度。我们将使用该测定来筛选超过 300,000 种谨慎的小分子化合物来识别抑制剂，并且我们将使用一组现有的二级测定来对它们进行优先级排序，这些二级测定旨在评估 β-内酰胺抗生素的抗菌效力、抗菌谱、选择性、耐药频率和效力。我们将验证高优先级命中的分子靶点。在第二阶段，我们将通过合理的药物设计优化其活性和特异性，开发最有希望的经过验证的先导化合物。该提案的具体目标如下。目标 1. 修改和优化已建立的基于细胞的途径特异性筛选测定，以有利于鉴定 WTA 生物合成特定步骤的抑制剂（TarB、D 和 F）。目标 2. 筛选多样化的化合物库，识别并确认 WTA 合成的抑制剂。目标 3. 使用二次测定验证已确认的 WTA 合成抑制剂并确定命中序列。目标 4. 验证所选热门系列的作用机制。 公共健康相关性：这项研究的目标是发现可以开发成强效药物的化合物，用于治疗由耐甲氧西林金黄色葡萄球菌（也称为 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称为壁磷壁酸（WTA）的糖聚合物的能力，这种糖聚合物覆盖在细菌的外表面，使其能够在人类中定殖并引起感染。干扰 WTA 产生的化合物通过阻止细菌生长而充当抗生素，并且还使 MRSA 对重要的 β 内酰胺类抗生素敏感，其中包括青霉素、甲氧西林和氨苄西林。创新型WTA活性药物的成功开发将是MRSA感染治疗的重大进展。

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.