ABSTRACT
During tuberculosis (TB) infection caused by Mycobacterium tuberculosis (Mtb), a sub-population of mycobacteria can enter a non-replicating persistent state that is not susceptible to most frontline TB drugs, resulting in phenotypic antimicrobial resistance (AMR), which contributes to an extended duration of TB therapy, frequent non-compliance and disease relapse. Recent clinical trial data indicated that inclusion of the fluoroquinolone Moxifloxacin (MXF) led to a shortening of TB treatment from 6-months to 4-months. MXF, however, has limited efficacy on Mtb persisting in a hypoxic environment. Here, we report a prodrug approach significantly enhancing MXF’s lethality against non-replicating hypoxic Mtb while retaining its effectiveness against replicating Mtb. Since hypoxia is associated with a reductive environment and induces the expression of reductive enzymes such as nitroreducatases (NTRs) in Mtb, we designed and synthesized a library of nitroaryl and nitroheteroaryl conjugates of MXF. After optimization of several factors, including reduction potential, rate, and efficiency of drug release, as well as selectivity, we identified 2-nitrothiazole-derivative as a prodrug that is rapidly and nearly quantitatively converted to MXF in the presence of NTR. Biochemical studies supported by computational data reveal a rate-limiting influence of electron transfer in the mechanistic pathway for activation of the nitroaryl group, a key step in prodrug activation leading to drug release. The lead prodrug’s potency against replicating bacteria in both cellular and animal model studies, and mode of action is nearly identical to MXF with significantly higher lethality against non-replicating Mtb than MXF. The prodrug showed improved accumulation of MXF inside non-replicating Mtb when compared with MXF alone, likely due to the prodrug’s ability to enhance accumulation of MXF within non-replicating Mtb. Hence, together, the newly developed 2-nitrothiazole-prodrug is rapidly, efficiently, and selectively cleaved under reductive conditions to produce the active drug and had superior permeability and accumulation in non-replicating Mtb, which likely contributes to its increased efficacy. This proof-of-concept study supports prodrug approaches with a focus on improving accumulation of the active drug within non-replicating pathogens as a strategy to address phenotypic AMR.
Competing Interest Statement
The authors have declared no competing interest.
ABBREVIATIONS
- AB
- alamar blue
- AMR
- Antimicrobial resistance
- CCCP
- carbonyl cyanide m-chlorophenylhydrazone
- CFU
- colony forming unit
- ET
- electron transfer
- E. coli
- Escherichia coli
- ECAR
- extracellular acidification rate
- FMN
- flavin mononucleotide
- HyRRA
- hypoxia resazurin reduction assay
- INH
- isoniazid
- Mtb
- Mycobacterium tuberculosis
- MXF
- Moxifloxacin
- MR
- Molar refractivity
- Msm
- Mycobacterium smegmatis
- MDR
- multi-drug resistant
- MD
- molecular dynamics
- MIC
- minimum inhibitory concentration
- MZ
- metronidazole
- NTR
- Nitroreductase
- NTM
- non-tuberculosis mycobacteria
- OCR
- oxygen consumpton rate
- OXPHOS
- oxidative phosphorylation
- PT
- proton transfer
- QRDR
- quinolone resistance determinant region
- ROS
- reactive oxygen species
- TPSA
- topological polar surface area
- TIC
- total ion chromatograms
- TCA
- tricarboxylic acid cycle
- TB
- Tuberculosis
- XDR
- extensively drug-resistant