Untargeted metabolomics reveals a new mode of action of pretomanid (PA-824)

Abstract

Pretomanid is a promising anti-tubercular drug currently at clinical phase III, but its mechanisms of action are currently unclear. This study aimed to: (i) reveal the metabolome of Mycobacterium smegmatis under pretomanid treatment; (ii) compare major sources of metabolite variation in bacteria treated with pretomanid treatment and other antibiotics; and (iii) to target metabolites responsible for the killing activity of pretomanid in mycobacteria. Untargeted high-resolution metabolite profiling was carried out using flow infusion electrospray ion high resolution mass spectrometry (FIE-HRMS) to identify and quantify metabolites. The identification of key metabolites was independently confirmed by gas-chromatography time-of flight mass spectrometry (GC-tofMS) in comparison to standards. Pretomanid treatments generated a unique distinctive metabolite profile when compared to ampicillin, ethambutol, ethionamide, isoniazid, kanamycin, linezolid, rifampicin and streptomycin. Metabolites which differed significantly only with pretomanid treatment were identified and mapped on to bacterial metabolic pathways. This targeted the pentose phosphate pathway with significant accumulation seen with fructose-6-phosphate, ribose-5-phosphate and glyceraldehyde-3-phosphate. These effects were linked to the accumulation of a toxic metabolite methylglyoxal. This compound showed significant antimicrobial activity (MIC 0.65 mM) against M. smegmatis.

A Solution to Antifolate Resistance in Group B Streptococcus : Untargeted Metabolomics Identifies Human Milk Oligosaccharide-Induced Perturbations That Result in Potentiation of Trimethoprim

“…This observed accumulation of lipid metabolites indicates an increased rate of breakdown of critical cell membrane components when bacteria are dosed with HMOs. In fact, this type of metabolite accumulation has been previously observed in other model organisms when exposed to antibiotics (64)(65)(66)(67).…”

In Silico Strategies in Tuberculosis Drug Discovery

“…Pretomanid is a prodrug activated by nitroreductase, which reduces pretomanid's imidazole ring to generate active metabolites. Specifically, a des-nitro metabolite leads to elevated levels of nitric oxide, which displays antimycobacterial activities due to its work as a poison for bacterial respiration under anaerobic conditions [25]. In aerobic conditions, it works like delaminid by targeting cell wall mycolic acid biosynthesis [26], and while there were several potential targets for this drug, its exact protein target is not yet known [27].…”

Mycobacterial drug discovery

“…43,44,48,50,51 Further evidence reveals that upon drug treatment, there is an accumulation of a toxic metabolite, methyl glyoxal, resulting from the build-up of sugars in the pentose phosphate pathway, suggesting an alternative mechanism of killing by these pro-drugs. 52 Given the therapeutic potential of these drugs, the specificities of the inhibition mechanism could prove valuable in the further optimisation of these compounds.…”