Tuberculosis (TB) is caused by Mycobacterium tuberculosis and is a major threat to global health. With an estimated 10.6 million active cases and 1.6 million deaths in 2021, it is currently the second leading cause of death from a single infectious agent after COVID-19 [1]. The WHO recommended TB therapy regimens involve drug combinations for at least four months [2], which are extended for multidrug (MDR) and extensively drug-resistant (XDR) strains [1]. Novel antitubercular drugs to reduce therapy duration and improve efficacy in MDR- and XDR-patients are desperately needed. High R&D costs and a low return on investment hamper the development of innovative antitubercular drugs, resulting in a narrow pipeline of only few innovative compounds (novel class and mechanism of action) such as GSK 3036656, GSK 2556286, OPC-167832, macozinone, and BTZ-043 [3]. BTZ-043 (1) is the first described lead inhibitor of the decaprenylphosphoryl-β-d-ribose-2′-epimerase (DprE1) [4], a highly vulnerable target in the periplasmic space of M. tuberculosis that is essential for the biosynthesis of the cell wall building block decaprenylphosphoryl-β-d-arabinose (DPA) [5]. BTZ-043 is a suicide inhibitor of DprE1; In a first step the nitro group is reduced by DprE1 to a transient nitroso moiety, which reacts with a cysteine moiety within the catalytic cavity to give a semimercaptal, thus inactivating the enzyme irreversibly (Fig. 1) [4]. This previously unexploited mechanism confers BTZ-043 high antituberculotic activity against MDR- and XDR-TB strains while maintaining a low probability of encountering pre-existing resistance [4]. The outstanding properties of BTZ-043 encouraged its development under the leadership of two public German institutions, it has recently demonstrated efficacy in humans within a clinical phase IIa [6], and is on the way to enter phase IIb studies [7].

During preclinical development, we discovered that the reductive in vivo metabolism of BTZ-043 not only comprises transformation to the corresponding amine BTZ-045S, but also substantial amounts of an unprecedented hydride Meisenheimer complex (HMC, 2) [8] (Fig. 1). Meisenheimer complexes (MC) are commonly known as unstable intermediates in aromatic nucleophilic substitution reactions of electron-deficient aromatic moieties [[9], [10], [11]]. Remarkably, relevant plasma levels of the HMC metabolite have been found in the Göttingen minipig and other preclinical animal species and the HMC of the analogue PBTZ169 was detected as the main metabolite in humans [12], thus confirming the clinical relevance of this metabolite. Therefore, suppression of this previously overlooked metabolic pathway strongly suggests a so far untapped strategy for lead optimization of next-generation BTZs. One key challenge to guide this process was the lack of an established in vitro assay to display the biotransformation of BTZs to their corresponding HMCs. Here we show the development of the first cell-line-based assay to assess the HMC formation propensity of BTZ-043 and its derivatives in vitro. We also demonstrate that this assay can be employed as an integral part of the multiparameter testing cascade of known and novel BTZs to provide appropriate guidance towards BTZs with high antimycobacterial activity and substantially suppressed HMC metabolism.