Subproject 2: Identification of Pathways that Can be Targeted for the Development of Novel Therapies for MRSA

子项目 2：确定可用于开发 MRSA 新疗法的途径

• 批准号: 10327903
• 负责人: ELEFTHERIOS MYLONAKIS
• 金额: $ 42.72万
• 依托单位: MASSACHUSETTS EYE AND EAR INFIRMARY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10327903
• 关键词: Address; Agonist; Animals; Anthelmintics; Anti-Bacterial Agents; Anti-Infective Agents; Antibiotic Resistance; Antibiotics; Bacteremia; Bacteria; Biochemical; Biological Assay; Biophysics; Caenorhabditis elegans; Cell membrane; Cells; Cholesterol; Chronic; Clinical; Collaborations; Communities; Community Hospitals; Cryoelectron Microscopy; Development; Diabetes Mellitus; Drug usage; ESKAPE pathogens; Enterococcus faecalis; Exhibits; FDA approved; Growth; Hospitals; Immunocompromised Host; In Situ; In Vitro; Individual; Infection; Insulin Antagonists; Insulin-Like Growth Factor I; Laboratories; Lead; Lethal Genes; Lipid Bilayers; Membrane; Membrane Fluidity; Membrane Potentials; Metabolic; Methicillin Resistance; Modeling; Mothers; Multi-Drug Resistance; Mus; Nosocomial Infections; Outcome; PPAR alpha; PPAR gamma; Pathway interactions; Phase; Physiological; Play; Predisposition; Probability; Process; Resistance; Resistance development; Retinoids; Role; Skin; Staphylococcus aureus; Structure-Activity Relationship; Subcutaneous abscess; Techniques; Therapeutic; Toxic effect; Walkers; acute infection; analog; antibiotic tolerance; antimicrobial; antimicrobial drug; base; chronic infection; combat; design; drug development; drug discovery; efficacy study; efficacy testing; in vivo; ineffective therapies; methicillin resistant Staphylococcus aureus; microfluidic technology; multidrug tolerance; mutant; new therapeutic target; novel; novel therapeutics; persistent bacteria; programs; resistance gene; screening; synergism; targeted treatment; transposon sequencing

SUMMARY Methicillin-resistant S. aureus (MRSA) are leading causes of serious community and hospital-associated infection. In addition to acquiring antibiotic resistance, S. aureus converts to a persistent antibiotic-tolerant form in which traditional treatments are powerless. Antibiotic-tolerant persister cells are responsible for recalcitrant chronic infections. To address the urgent need for new anti-MRSA therapeutics, we developed a C. elegans- MRSA screening platform to identify compounds with activity against MRSA. The assay identifies compounds with both in vivo efficacy and low host toxicity. We combined a whole animal in vivo screen of ~82,000 synthetic compounds for ones that exhibit potential anti-MRSA activity with in vitro screens for compounds with the ability to kill the stubborn persister subpopulation. We have focused on four compounds, CD437, nTZDpa, bithionol, and PQ401, all four of which eradicate both actively growing MRSA and stationary phase MRSA persister cells by disrupting their membrane lipid bilayers. These compounds exhibit fast killing rates, synergism with other antibiotics, extremely low probabilities of resistance selection, and high selectivity for bacterial over mammalian membranes. Using bithionol and nTZDpa and analogs of these compounds, we found that anti-persister activity of these compounds positively correlates with their ability to increase membrane fluidity. All four compounds have been previously studied as potential therapeutics and bithionol is a former FDA-approved anthelmintic. Despite the potential of membrane-disrupting compounds to kill persisters, it is generally thought that their therapeutic applications are limited due to a lack of selectivity for bacterial over mammalian membranes. However, our results show that the presence of cholesterol in mammalian but not in bacterial membranes provides an important opportunity to identify/design non-toxic anti-persister membrane-disrupting compounds. We therefore propose the following two aims to identify features of membrane-disrupting compounds that correlate with their ability to kill persisters but not disrupt mammalian membranes. In Aim 1, we will utilize biochemical, physiological, biophysical, and cryo-EM techniques to 1) further investigate the correlation between the ability of compounds to kill persisters and their ability to increase membrane fluidity, and 2) to identify the mechanisms of action and targets of membrane-disrupting compounds. We also propose to test the efficacy of membrane-disrupting compounds in a murine S. aureus subcutaneous abscess infection model in collaboration with the Hooper lab and determine whether the compounds also target E. faecalis cell membranes. In Aim 2, we will utilize high throughput microfluidics technology developed in the Paulsson laboratory (Core B) and referred to as “mother machines” to: 1) isolate rare MRSA persister cells in growing cultures, and 2) in collaboration with the Walker lab, use Tn-seq in mother machines to identify MRSA transposon insertion mutants with enhanced susceptibility to membrane disrupting compounds. Successful completion of the proposed project will enhance our ability to identify and/or design efficacious and safe antibiotics for hard to treat Gram positive infections.

总结 耐甲氧西林表皮葡萄金黄色葡萄球菌（MRSA）是严重的社区和医院相关的主要原因 感染除了获得抗生素耐药性外，S.金黄色葡萄球菌转化为持久的耐药性形式 传统的治疗方法无能为力。耐抗生素的持留细胞是导致抗生素耐药的原因。 慢性感染为了解决对新的抗MRSA治疗剂的迫切需求，我们开发了一种C。优雅的， MRSA筛选平台，以鉴定具有抗MRSA活性的化合物。该测定法鉴别化合物 具有体内功效和低宿主毒性。我们将约82，000个合成的 通过体外筛选具有抗MRSA能力的化合物， 杀死顽固的珀西斯亚群我们重点研究了四种化合物，CD 437，nTZDpa，bithionol， 和PQ 401，这四种药物都能根除活跃生长的MRSA和稳定期MRSA持续细胞 通过破坏它们的膜脂双层。这些化合物表现出快速的杀伤率，与其他化合物的协同作用， 抗生素，极低的耐药选择概率，以及对细菌的高选择性超过哺乳动物 膜。使用bithionol和nTZDpa以及这些化合物的类似物，我们发现， 这些化合物的活性与它们增加膜流动性的能力正相关。所有四种化合物 先前已被研究为潜在的治疗剂，而bithionol是FDA批准的驱虫剂。 尽管破坏膜的化合物有杀死坚持者的潜力，但人们普遍认为， 由于缺乏对细菌超过哺乳动物膜的选择性，治疗应用受到限制。 然而，我们的研究结果表明，胆固醇的存在，在哺乳动物，而不是在细菌膜 提供了鉴定/设计无毒抗持久性膜破坏化合物的重要机会。 因此，我们提出以下两个目标，以确定膜破坏化合物的特征， 这与它们杀死持久性细菌但不破坏哺乳动物细胞膜的能力有关。在目标1中，我们将利用 生化、生理、生物物理和冷冻EM技术，以1）进一步研究 化合物杀死持留菌的能力和它们增加膜流动性的能力，和2）鉴定 膜破坏化合物的作用机制和靶点。我们亦建议测试以下药物的效用： 膜破坏化合物在小鼠S.金黄色葡萄球菌皮下脓肿感染模型 并确定这些化合物是否也针对E。粪细胞膜在目标2中， 将利用Paulsson实验室（核心B）开发的高通量微流体技术，并参考 作为“母亲机器”：1）在生长的培养物中分离罕见的MRSA持久细胞，2）与 步行者实验室在母体机器中使用Tn-seq鉴定MRSA转座子插入突变体， 对膜破坏化合物的敏感性。成功完成拟议项目将提高 我们有能力识别和/或设计有效和安全的抗生素，用于难以治疗的革兰氏阳性菌感染。