Exploiting DprE2 pathway redundancy to enhance potency and overcome resistance to pretomanid in Mycobacterium tuberculosis

Tuberculosis (TB), the deadliest infectious disease globally, still poses an enormous public health challenge exacerbated by the rise of multi-drug resistant (MDR) and extensively drug-resistant (XDR) M. tuberculosis strains. The bicyclic nitroimidazoles pretomanid (PTM) and delamanid (DLM) represent the most recent class of anti-tubercular compounds to achieve regulatory approval and clinical implementation in TB chemotherapy regimens. Both are prodrugs whose activity relies on the deazaflavin-dependent nitroreductase Ddn. High-throughput screening on a clinically relevant DdnL49P mutant reveals molecules capable of restoring PTM activity. Optimisation through rational medicinal chemistry leads to highly potent compounds capable of drastically reducing the MIC of PTM while improving its bactericidal activity. Mechanistic studies using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), transposon sequencing and thin-layer chromatography of radiolabeled extracted lipids show that these molecules do not trigger bioactivation pathways, but instead target a compensatory pathway involving Rv2073c, an enzyme that is redundant with DprE2 for bacterial cell wall biosynthesis. The concomitant perturbation of both the DprE2- and Rv2073c-dependent steps by the PTM-NAD adduct and optimised norbornene derivatives leads to a collapse in arabinan synthesis correlating with bacterial death. This study thus highlights Rv2073c as a promising vulnerability that can be exploited to potentiate the efficacy of nitroimidazole anti-tuberculosis drugs.