Novel insight into the reaction of nitro, nitroso and hydroxylamino benzothiazinones and of benzoxacinones with Mycobacterium tuberculosis DprE1

Abstract

Nitro-substituted 1,3-benzothiazinones (nitro-BTZs) are mechanism-based covalent inhibitors of Mycobacterium tuberculosis decaprenylphosphoryl-β-D-ribose-2′-oxidase (DprE1) with strong antimycobacterial properties. We prepared a number of oxidized and reduced forms of nitro-BTZs to probe the mechanism of inactivation of the enzyme and to identify opportunities for further chemistry. The kinetics of inactivation of DprE1 was examined using an enzymatic assay that monitored reaction progress up to 100 min, permitting compound ranking according to kinact/Ki values. The side-chain at the 2-position and heteroatom identity at the 1-position of the BTZs were found to be important for inhibitory activity. We obtained crystal structures with several compounds covalently bound. The data suggest that steps upstream from the covalent end-points are likely the key determinants of potency and reactivity. The results of protein mass spectrometry using a 7-chloro-nitro-BTZ suggest that nucleophilic reactions at the 7-position do not operate and support a previously proposed mechanism in which BTZ activation by a reduced flavin intermediate is required. Unexpectedly, a hydroxylamino-BTZ showed time-dependent inhibition and mass spectrometry corroborated that this hydroxylamino-BTZ is a mechanism-based suicide inhibitor of DprE1. With this BTZ derivative, we propose a new covalent mechanism of inhibition of DprE1 that takes advantage of the oxidation cycle of the enzyme.

Promiscuous Targets for Antitubercular Drug Discovery: The Paradigm of DprE1 and MmpL3

“…In all structures of DprE1 in complex with inhibitors, all molecules showed binding sites that are significantly overlapped ( Figure 2). This binding pocket, which conceivably constitutes the binding This mechanism of action has been demonstrated for benzothiazinones and benzothiazinone analogs [41,43,49,50]. However, other different classes of nitro-compounds have been identified as potent DprE1 inhibitors, including dinitrobenzamides [32], nitroquinoxalines [33] and nitroimidazoles [51], that conceivably share the same mechanism of action of benzothiazinones [52].…”

“…Currently, 33 DprE1 structures from M. tuberculosis or M. smegmatis have been deposited in the Protein Data Bank, as apoenzyme [41,53] or in complex with both covalent [41,43,44,49,54] and noncovalent inhibitors [43,50,[55][56][57][58]. The enzyme is characterized by the two-domain topology of the vanillyl-alcohol oxidase family of oxidoreductases, which includes a flavin adenine dinucleotide (FAD)-binding domain and the substrate-binding domain [41].…”

An overview of new antitubercular drugs, drug candidates, and their targets

“…Arabinose polymers form the arabinogalactan component of the cell wall of mycobacteria. DprE1 oxidizes the 2′ hydroxyl group of DPR to ketone using FAD as the oxidant . The epimerisation process occurs in the periplasmic space which makes DprE1 a vulnerable target .…”

“…DprE1 oxidizes the 2′ hydroxyl group of DPR to ketone using FAD as the oxidant. 204 The epimerisation process occurs in the periplasmic space which makes DprE1 a vulnerable target. 205 The enzyme DprE1 is FAD dependent while DprE2 is NADH-dependent and are encoded by dprE1 and dprE2 genes, respectively.…”

[2-Chloro-3-nitro-5-(trifluoromethyl)phenyl](piperidin-1-yl)methanone: structural characterization of a side product in benzothiazinone synthesis

“…Fig. 1 depicts the synthesis following the original procedure for a BTZ previously reported by us (Rudolph, 2014;Rudolph et al, 2016;Richter, Rudolph et al, 2018). After treatment of 2-chloro-3-nitro-5-(trifluoromethyl)benzoic acid (1) with thionyl chloride and subsequently ammonium thiocyanate, the corresponding 2-chloro-3-nitro-5-(trifluoromethyl)benzoyl isothiocyanate (2) was reacted with piperidine.…”