Development of FosA Inhibitors to Potentiate Fosfomycin Activity in Gram-Negative Pathogens

摘要抗生素耐药性被广泛认为是最重要的公共卫生威胁之一， 世纪。许多细菌感染由于抗微生物剂耐药性而变得难以治疗， 迫切需要制定新的战略来对抗这些耐药病原体。其中一个战略是重新定位 在人类中有长期安全记录的老抗生素。磷霉素（FOM）是一种公认的抗生素 其在革兰氏阳性和革兰氏阴性细胞中灭活UDP-N-乙酰葡糖胺烯醇化酶 病原体目前，FOM仅用作治疗尿路感染的口服制剂 鉴于其对大肠杆菌的优异活性。然而，静脉内FOM制剂用于其他地方， 目前正在等待美国FDA的批准。此外，一项正在进行的NIAID申办的试验（NCT 03910673） 正在探索静脉内FOM是否可以有效治疗肺部感染，如医院获得性和 呼吸机相关性细菌性肺炎FosA是一种二聚体K+和Mn 2+依赖性谷胱甘肽S-转移酶 催化谷胱甘肽与FOM环氧环中碳-1的亲核加成， 无抗生素活性。E.大肠杆菌缺乏固有的染色体fosA，从而解释了其对FOM的急性易感性。 然而，fosA同源物由许多革兰氏阴性物种染色体编码，包括 铜绿假单胞菌和肺炎克雷伯菌。我们之前的研究已经清楚地表明， FosA内在产生赋予FOM抗性，FosA的失活提供了一种新的方法， 增加碳青霉烯耐药革兰氏阴性病原体对FOM的敏感性，从而突出了一种新的 途径，以扩大FOM的使用范围，以广泛的革兰氏阴性菌种。重要的是， 最近，我们发现了一种具有竞争力的FosA小分子抑制剂，并申请了专利 （ANY 1），其增强针对携带fosA基因的革兰氏阴性病原体的FOM活性。使用 从ANY 1-FosA X射线晶体结构的见解，我们设计并制备了一种类似物， 更大的潜力，表明进一步的特区发展是可能的。该提案的目的是（1）药用 FosA抑制剂的化学优化，（2）ADME性质的评价和优化，以及（3）生物学 针对一组广泛的XDR革兰氏阴性临床分离株进行评估。我们预计这样的组合 可用于治疗侵袭性感染，包括菌血症、肺炎、腹腔内感染， 由携带fosA的革兰氏阴性细菌引起的复杂UTI（例如，K.肺炎，肠杆菌属， P.铜绿假单胞菌），包括极端耐药菌株。在第一阶段的提案中，我们将确定和评估 基于ANY 1的FosA抑制剂，结合了制药和药物化学专业知识， 福克斯蔡斯化学多样性中心公司的科学家们（FCCDC）的专业知识和经验， Sluis-Cremer实验室在FosA抑制和抗生素的实验方面 疗法

Summary. Antimicrobial resistance is widely recognized as one of the most significant public health threats of the century. Many bacterial infections have become difficult to treat due to antimicrobial resistance, and there is an urgent need to develop new strategies to combat these resistant pathogens. One such strategy is to reposition older antibiotics that have long-track records of safety in human. Fosfomycin (FOM) is an etablished antibiotic which inactivates UDP-N-acetylglucosamine enolpyruvyl transferase in both Gram-positive and -negative pathogens. Currently, FOM is exclusively used as an oral formulation for the treatment of urinary tract infections given its excellent activity against Escherichia coli. However, an intravenous FOM formulation is used elsewhere, and is currently pending FDA approval in U.S. Furthermore, an ongoing NIAID-sponsored trial (NCT03910673) is exploring whether intravenous FOM can effectively treat lung infections, such as hospital-acquired and ventilator-associated bacterial pneumonia. FosA is a dimeric K+- and Mn2+-dependent glutathione S-transferase that catalyzes the nucleophilic addition of glutathione to carbon-1 in the epoxide ring of FOM, rendering the antibiotic inactive. E. coli lacks intrinsic chromosomal fosA, thus explaining its acute susceptibility to FOM. However, fosA homologues are chromosomally encoded by many Gram-negative species including Pseudomonas aeruginosa and Klebsiella pneumoniae. Our prior research has clearly demonstrated that this intrinsic production of FosA confers FOM resistance, and that inactivation of FosA provides a novel approach to increase the sensitivity of carbepenem resistant Gram-negative pathogens to FOM, thus highlighting a novel pathway to expand the use of FOM to a wide range of Gram-negative species. Importantly, and central to this application, we recently identified and patented a first-in-class, competitive small molecule inhibitor of FosA (ANY1) which potentiates FOM activity against Gram-negative pathogens that harbor the fosA gene. Using insights from the ANY1-FosA X-ray crystal structure, we have designed and prepared an analog that has ~10X greater potency, showing that further SAR development is possible. The aims in this proposal are (1) medicinal chemistry optimization of FosA inhibitors, (2) evaluation and optimization of ADME properties, and (3) biological evaluation against a broad panel of XDR Gram-negative clinical isolates. We anticipate that such a combination could be used to treat invasive infections including bacteremia, pneumonia, intra-abdominal infections and complicated UTIs caused by Gram-negative bacteria that harbor fosA (e.g., K. pneumoniae, Enterobacter spp., P. aeruginosa), including extremely drug resistant strains. In this Phase I proposal, we will identify and evaluate FosA inhibitors based on ANY1 by combining the pharmaceutical and medicinal chemistry expertise of the scientists at the Fox Chase Chemical Diversity Center, Inc. (FCCDC) with the expertise and experience of the Sluis-Cremer lab at the University of Pittsburg in the experimental aspects of FosA inhibition and antibiotic therapy.