Chakrapani, H. | bioRxiv Science

bioRxiv2023

Intracellular Accumulation-Driven Potentiation of Moxifloxacin Against non-replicating drug-tolerant Mycobacterium tuberculosis through a Prodrug

Most front-line tuberculosis drugs are ineffective against hypoxic non-replicating drug-tolerant Mycobacterium tuberculosis (Mtb) contributing to phenotypic antimicrobial resistance (AMR). This is largely due to the poor permeability in the thick and waxy cell wall of persister cells, leading to diminished drug accumulation and reduced drug-target engagement. Here, using an "arm-to-disarm" prodrug approach, we demonstrate that non-replicating Mtb persisters can be sensitized to Moxifloxacin (MXF), a front-line TB drug. We design and develop a series of nitroheteroaryl MXF prodrugs that are substrates for bacterial nitroreductases (NTR), a class of enzymes that are over-expressed in hypoxic Mtb. Enzymatic activation involves electron-transfer to the nitroheteroaryl compound followed by protonation via water that contributes to the rapid cleavage rate of the protective group by NTR to produce the active drug. Phenotypic and genotypic data are fully consistent with MXF-driven lethality of the prodrug in Mtb with the protective group being a relatively innocuous bystander. The prodrug increased intracellular concentrations of MXF than MXF alone and is more lethal than MXF in non-replicating persisters. Hence, arming drugs to improve permeability, accumulation and drug-target engagement is a new therapeutic paradigm to disarm phenotypic AMR.

microbiology↗

bioRxiv2022

Moxifloxacin-mediated killing of Mycobacterium tuberculosis involves respiratory downshift, reductive stress, and ROS accumulation

Moxifloxacin is central to treatment of multidrug-resistant tuberculosis. Effects of moxifloxacin on Mycobacterium tuberculosis redox state were explored to identify strategies for increasing lethality and reducing the prevalence of extensively resistant tuberculosis. A non-invasive redox biosensor and an ROS-sensitive dye revealed that moxifloxacin induces oxidative stress correlated with M. tuberculosis death. Moxifloxacin lethality was mitigated by supplementing bacterial cultures with an ROS scavenger (thiourea), an iron chelator (bipyridyl), and, after drug removal, an antioxidant enzyme (catalase). Lethality was also reduced by hypoxia and nutrient starvation. Moxifloxacin increased the expression of genes involved in the oxidative stress response, iron-sulfur cluster biogenesis, and DNA repair. Surprisingly, and in contrast with Escherichia coli studies, moxifloxacin decreased expression of genes involved in respiration, suppressed oxygen consumption, increased the NADH/NAD+ ratio, and increased the labile iron pool in M. tuberculosis. Lowering the NADH/NAD+ ratio in M. tuberculosis revealed that NADH-reductive stress facilitates an iron-mediated ROS surge and moxifloxacin lethality. Treatment with N-acetyl cysteine (NAC) accelerated respiration and ROS production, increased moxifloxacin lethality, and lowered the mutant prevention concentration. Moxifloxacin induced redox stress in M. tuberculosis inside macrophages, and co-treatment with NAC potentiated the anti-mycobacterial efficacy of moxifloxacin during nutrient starvation, inside macrophages, and in mice where NAC restricted the emergence of resistance. Thus, oxidative stress, generated in a novel way, contributes to moxifloxacin-mediated killing of M. tuberculosis. The results open a way to make fluoroquinolones more effective anti-tuberculosis agents and provide a mechanistic basis for NAC-mediated enhancement of fluoroquinolone lethality in vitro and in vivo. Author Summary A new paradigm was revealed for stress-mediated bacterial death in which moxifloxacin treatment of M. tuberculosis decreases respiration rate (respiration increases in E. coli). Although moxifloxacin-induced, ROS-mediated bacterial death was observed, it derived from elevated levels of NADH and iron, a phenomenon not seen with antibiotic-treated E. coli. Nevertheless, stimulation of respiration and ROS by N-acetyl cysteine (NAC) enhanced moxifloxacin-mediated killing of M. tuberculosis, thereby reinforcing involvement of ROS in killing. NAC stimulation of moxifloxacin-mediated killing of M. tuberculosis and restriction of the emergence of resistance in a murine model of infection emphasize the importance of lethal action against pathogens. The work, plus published benefits of NAC to TB patients, encourage studies of NAC-based enhancement of fluoroquinolones.

microbiology↗