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

 说明(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，但由于出现了耐药微生物，其使用量有所下降。这些已批准的知名药物的恢复可以通过使导致耐药性的分子机制失活来实现。我们发现了磺胺类药物敏感性的一种新机制，这种机制是由一种被称为“叶酸甲酯陷阱”的代谢障碍引起的。利用定向突变、遗传和化学互补以及随后的代谢分析进行的初步研究证实，MF陷阱是磺胺敏感性的一种新机制。这种机制普遍存在于分枝杆菌和重要的革兰氏阴性细菌中，如铜绿假单胞菌、大肠杆菌和鼠伤寒沙门氏菌。化学限制维生素B12是防止MF陷阱形成所必需的，同样会导致磺胺类药物敏感性增加。这一应用的中心假设是，MF TRAP可以在药物上得到促进，使具有多重耐药性的铜绿假单胞菌和肠杆菌科细菌获得临床批准的磺胺类药物。R21阶段的具体目标是为了更好地了解MF TRAP使细菌对磺胺类药物敏感的分子机制，以及MF TRAP是否同样使耐药铜绿假单胞菌和肠杆菌科细菌对磺胺类药物敏感。在目标1中，参与调节细胞水平的5-甲基-四氢酸和同型半胱氨酸的基因将被设计成精确的表达控制。接受基因滴定表达的细胞将同时分析磺胺敏感性以及细胞叶酸和相关代谢物的变化。此外，作为MF TRAP的结果，细胞蛋白的同型半胱氨酸化将被研究。在目标2中，无法防止MF陷阱的铜绿假单胞菌和鼠伤寒沙门氏菌突变株将从多重耐药背景中构建，随后将在体外和宿主感染期间进行广泛的敏感性测试。此外，我们将在巨噬细胞和幼虫感染模型中测试最近开发的抗维生素B12对耐药铜绿假单胞菌和鼠伤寒沙门氏菌的磺胺增强活性。只有在为R21阶段提出的里程碑达到时，我们才会继续进行R33阶段。在目标3中，将合成一系列钴胺和非钴胺抗B12分子，以提高对细菌细胞的疗效和特异性。这些化合物将在MF诱捕试验和磺胺强化试验中进行测试，以对抗药物敏感和耐药菌株。有希望的化合物将接受对大量耐药铜绿假单胞菌和肠杆菌科细菌的体外敏感性测试，以及角膜小鼠感染模型的敏感性测试。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，以确定细菌特异性的MF诱导剂在治疗细菌感染中作为有效的磺胺增强剂。这些拟议的研究将揭示以前未知的细菌对磺胺类固有耐药性的机制。了解这一机制不仅有助于提高磺胺类药物的临床应用水平，而且有助于开发针对耐药革兰氏阴性菌的叶酸拮抗新策略。

 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.