A Novel Folate Antagonistic Strategy to Treat Drug Resistant Pseudomonas aeruginosa and Enterobacteriaceae

治疗耐药铜绿假单胞菌和肠杆菌的新型叶酸拮抗策略

• 批准号: 8956026
• 负责人: Liem Duy Nguyen
• 金额: $ 21.79万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8956026
• 关键词: Adverse effects; Affect; Bacteria; Bacterial Infections; Bacterial Physiology; Biological Assay; Cells; Chemicals; Clinical; Cobalamin; Collection; Cornea; Data; Development; Drug resistance; Engineering; Enterobacteriaceae; Escherichia coli; Extreme drug resistant tuberculosis; Folic Acid; Folic Acid Antagonists; Future; Gene Expression; Genes; Genetic; Genus Mycobacterium; Gram-Negative Bacteria; HIV Seropositivity; Homocysteine; Homocystine; Human; In Vitro; Infection; Lead; Life; Light; Mammals; Mediating; Metabolic; Metabolism; Methionine; Modeling; Molecular; Multi-Drug Resistance; Mus; Mutagenesis; Mutation; Organism; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Physiology; Predisposition; Proteins; Pseudomonas; Pseudomonas Infections; Pseudomonas aeruginosa; Reaction; Resistance; Role; Salmonella typhimurium; Series; Specificity; Sulfonamides; Testing; Tetrahydrofolates; Trimethoprim; Vitamin B 12; antimicrobial; antimicrobial drug; bacterial resistance; base; cell growth; chemical genetics; design; folic acid metabolism; improved; macrophage; metabolomics; mutant; novel; novel therapeutic intervention; pathogen; pathogenic bacteria; prevent; prophylactic; public health relevance; resistance mechanism; restoration; uptake

 DESCRIPTION (provided by applicant): Sulfonamides were the first antimicrobial agents effectively used to treat bacterial infections but their use has declined due to the emergence of resistant organisms. Restoration of these approved, well-known drugs could be achieved through inactivation of molecular mechanisms responsible for resistance. We identified a novel mechanism of sulfonamide sensitivity that is caused by a metabolic blockage referred to as the "Methyl Folate (MF) trap". Preliminary studies using targeted mutagenesis, genetic and chemical complementation, followed by metabolic analyses, confirmed the MF trap as a novel mechanism of sulfonamide sensitivity. This mechanism is ubiquitously present in mycobacteria and important Gram-negative bacteria such as Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhimurium. Chemical restriction of vitamin B12, required for preventing the MF trap formation, similarly leads to increased sulfonamide susceptibility. The central hypothesis of this application is that the MF trap could be pharmaceutically promoted to render multidrug resistant P. aeruginosa and Enterobacteriaceae to available, clinically approved sulfonamides. The specific aims in the R21 phase are designed to better understand the molecular mechanism by which the MF trap confers sulfonamide sensitivity in bacteria, and whether the MF trap similarly sensitizes drug resistant P. aeruginosa and Enterobacteriaceae to sulfonamides. In Aim 1, genes involved in regulating cellular levels of 5-methyl-tetrahydrolate and homocysteine, the two direct effectors of the MF trap, will be engineered for precise expression control. Cells undergoing titrated gene expression will be simultaneously analyzed for sulfonamide susceptibility and alterations in cellular folate and related metabolites. Furthermore, homocysteinylation of cellular proteins, as a consequence of the MF trap, will be investigated. In Aim 2, P. aeruginosa and S. typhimurium mutants unable to prevent the MF trap will be constructed from multidrug resistant backgrounds, followed by extensive susceptibility tests, both in vitro and during host infections. In addition, we will test the sulfonamide-boosting activiy of a recently developed "antivitamin B12" against drug resistant P. aeruginosa and S. typhimurium in macrophages and a larval infection model. We will only proceed with the R33 phase if milestones proposed for the R21 phase are achieved. In Aim 3, series of cobalamin and non-cobalamin anti-B12 molecules will be synthesized to improve efficacy and specificity towards bacterial cells. These compounds will be tested in MF trap-inducing and sulfonamide- boosting assays against both drug susceptible and resistant bacterial strains. Promising compounds will be subjected to in vitro susceptibility testing against a large collection of drug resistant P. aeruginosa and Enterobacteriaceae, as well as to a corneal mouse infection model. In Aim 4, we will assess the effects of these anti-B12 molecules on mammalian B12 and folate metabolism, in order to identify bacterial-specific MF trap inducers that functions as effective SULFA boosters in treating bacterial infections. These proposed studies will set light to a previously unknown mechanism of intrinsic sulfonamide resistance in bacteria. Understanding this mechanism may not only help to improve the clinical use of sulfonamides, but also lead to future development of novel folate antagonistic strategies for drug resistant Gram-negative bacteria.

 描述（由申请人提供）：磺胺类药物是第一种有效用于治疗细菌感染的抗菌剂，但由于耐药微生物的出现，其使用量已经下降。通过灭活相关分子机制可以恢复这些已批准的众所周知的药物。我们通过靶向突变的初步研究发现了一种新的磺酰胺敏感性机制，该机制是由称为“甲基叶酸（MF）陷阱”的代谢阻断引起的。遗传和化学互补以及代谢分析证实了 MF 陷阱是磺酰胺敏感性的一种新机制，该机制普遍存在于分枝杆菌和重要的革兰氏阴性细菌（如铜绿假单胞菌、大肠杆菌和鼠伤寒沙门氏菌）中。防止 MF 陷阱形成所需的维生素 B12 同样会导致磺胺敏感性增加 本申请的中心假设是。 R21 阶段的具体目标旨在更好地了解 MF 陷阱赋予细菌对磺酰胺敏感性的分子机制，以及 MF trap 是否同样会使耐药铜绿假单胞菌和肠杆菌科细菌对磺胺类药物敏感。目标 1，将设计参与调节 5-甲基-四氢酸和同型半胱氨酸（MF 陷阱的两个直接效应物）细胞水平的基因，以实现精确的表达控制。将同时分析正在进行滴定基因表达的细胞的磺胺敏感性和变化。此外，将研究 MF 陷阱导致的细胞蛋白同型半胱氨酸化。无法阻止 MF 陷阱的铜绿假单胞菌和鼠伤寒沙门氏菌突变体将从多重耐药背景中构建，然后在体外和宿主感染期间进行广泛的药敏试验。此外，我们将测试最近开发的“磺胺增强激活”。抗维生素 B12”针对巨噬细胞和幼虫感染模型中的耐药铜绿假单胞菌和鼠伤寒沙门氏菌。我们只会如果实现了 R21 阶段的里程碑，则继续 R33 阶段。 在目标 3 中，将合成一系列钴胺素和非钴胺素抗 B12 分子，以提高对细菌细胞的功效和特异性。这些化合物将在 MF trap 中进行测试。针对药物敏感和耐药菌株的诱导和磺胺增强试验将对大量耐药假单胞菌进行体外药敏试验。在目标 4 中，我们将评估这些抗 B12 分子对哺乳动物 B12 和叶酸代谢的影响，以确定有效的细菌特异性 MF 陷阱诱导剂。 SULFA 增强剂治疗细菌感染的研究将阐明细菌固有的磺酰胺耐药机制，不仅有助于改善其临床应用。磺胺类药物，而且还导致未来针对耐药革兰氏阴性菌的新型叶酸拮抗策略的开发。