Novel Combination Therapies to Combat Hypermutable Carbapenem-Resistant P. aeruginosa

摘要：耐碳青霉烯类铜绿假单胞菌（CRPA）对人类健康构成了严重威胁 在美国和全球。金属-β-内酰胺酶（MBL），可产生高水平的碳青霉烯类耐药， 值得高度关注。缺乏CRPA MBL产生假单胞菌的治疗选择 铜绿假单胞菌（MBL PA）和抗生素管道破裂要求开发新的治疗策略 针对非传统的、未开发的途径。有越来越多的证据表明，“hypermutator”菌株， 显示出显著增加的自发突变频率（比非突变体对照高10倍）， 作为病理适应性和抗菌耐受性的基础，不可避免地增加了治疗的可能性， PA感染的失败和细菌持久性。重要的是，在DNA复制过程中发生的错误， 跨损伤DNA合成（TLS Pol IV）作为PA超变菌株中突变的机制基础。 我们开创了新型非天然核苷酸的合成和测试，这些核苷酸非常安全有效。 抗癌疗法，这得到了我们初步数据的支持。第一次，我们建议研究 通过定义超变子在病理适应、持久性、 和抗菌素耐药性，并开发联合方案，以打击MBL PA。我们的首要目标是 使用新型抗突变剂非天然核苷酸开发新的MBL PA组合治疗策略 以及可用的β-内酰胺抗生素。MBL革兰氏阴性菌的一种有前途的桥接疗法是 头孢他啶-阿维巴坦联合氨曲南;然而，尚未在MBL PA中研究该策略。在 初步研究，我们观察到长丝状persisters由于抑制青霉素结合蛋白3， MBL PA暴露于头孢他啶-阿维巴坦和氨曲南联合给药。自从SOS对DNA的反应 DNA损伤需要进行修复，而TLS DNA聚合酶（Pos）需要绕过DNA损伤 在持续存在的细胞DNA中产生的导致抗菌素耐药性，我们假设，在没有修复的情况下， 功能，坚持的能力，以科普DNA损伤，随后分隔和增长成为 越来越依赖TLS Pol IV。鉴于PA Pol IV的这一至关重要的作用，我们的总体目标是 假设靶向Pol IV以阻断受损DNA复制的新的非天然核苷酸将被 与现有的β-内酰胺抗生素一起非常有效。为了验证这些假设，我们将：（目标1）定义 超变子对暴露于β-内酰胺组合的MBL PA的抗性和持久性的贡献; (Aim 2）开发靶向TLS Pol IV的小分子非天然核苷酸以对抗MBL PA中的突变; (Aim 3）定义非天然核苷和β-内酰胺的最佳组合治疗方案， 抑制耐药性，并防止MBL PA在中空纤维和动物模型中的持久性。综合起来看， 我们的研究结果将为革兰氏阴性菌的新型联合治疗提供前所未有的见解， 未来在临床试验中测试抗突变非天然核苷酸的基石。

ABSTRACT: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) poses an urgent threat to human health in the United States and globally. Metallo-β-lactamases (MBL), which confer high-level carbapenem resistance, warrant significant attention. The paucity of treatment options for CRPA MBL-producing Pseudomonas aeruginosa (MBL PA) and the broken antibiotic pipeline demands the development of new therapeutic strategies that target non-traditional, unexploited pathways. There is mounting evidence that ‘hypermutator’ strains, which show a significantly increased spontaneous mutation frequency (10-fold higher than non-mutator control), serve as the basis for pathoadaptation and antimicrobial tolerance, inevitably increasing the likelihood of treatment failure and bacterial persistence in PA infections. Importantly, errors made during DNA replication and translesion DNA synthesis (TLS Pol IV) serve as the mechanistic basis for mutations in PA hypermutator strains. We have pioneered the synthesis and testing of novel non-natural nucleotides as remarkably safe and effective anti-cancer therapies, which is supported by our preliminary data. For the first time, we now propose to study non-natural nucleotides by defining the underlying mechanism of hypermutators in pathoadaptation, persistence and antimicrobial resistance and develop combination regimens to combat MBL PA. Our overarching goal is to develop new combinatorial treatment strategies for MBL PA using novel anti-mutator non-natural nucleotides together with available β-lactam antibiotics. One promising bridge therapy for MBL Gram-negatives is ceftazidime-avibactam combined with aztreonam; however, this strategy has not been studied in MBL PA. In preliminary studies, we observed long filamentous persisters due to inhibition of penicillin binding protein 3 in MBL PA exposed to the ceftazidime-avibactam and aztreonam combination. Since the SOS response to DNA damage is required for filamentation, while TLS DNA polymerases (Pols) are required to bypass DNA lesions generated in persister cell DNA leading to antimicrobial resistance, we hypothesize that in the absence of repair functions, the ability of persisters to cope with DNA damage and subsequently septate and grow becomes increasingly dependent on TLS Pol IV. Given this critically important role of PA Pol IV, our overarching hypothesis that novel, non-natural nucleotides that target Pol IV to block replication of damaged DNA will be highly effective together with existing β-lactam antibiotics. To test these hypotheses, we will: (Aim 1) define the contributions of hypermutators to resistance and persistence of MBL PA exposed to β-lactam combinations; (Aim 2) develop small molecule, non-natural nucleotides targeting TLS Pol IV to combat mutation in MBL PA; (Aim 3) define optimal combinatorial treatment regimens of non-natural nucleosides and β-lactams that suppresses resistance, and prevents persistence of MBL PA in hollow fiber and animal models. Taken together, our results will provide unprecedented insight into novel combination therapies for Gram-negatives, and will set the cornerstone for future testing of anti-mutator non-natural nucleotides in clinical trials.