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

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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.