Benzothiazinones Are Suicide Inhibitors of Mycobacterial Decaprenylphosphoryl-β-d-ribofuranose 2′-Oxidase DprE1

Abstract

Benzothiazinones (BTZs) are antituberculosis drug candidates with nanomolar bactericidal activity against tubercle bacilli. Here we demonstrate that BTZs are suicide substrates of the FAD-dependent decaprenylphosphoryl-β-D-ribofuranose 2′-oxidase DprE1, an enzyme involved in cell-wall biogenesis. BTZs are reduced by DprE1 to an electrophile, which then reacts in a nearquantitative manner with an active-site cysteine of DprE1, thus providing a rationale for the extraordinary potency of BTZs. Mutant DprE1 enzymes from BTZ-resistant strains reduce BTZs to inert metabolites while avoiding covalent inactivation. Our results explain the basis for drug sensitivity and resistance to an exceptionally potent class of antituberculosis agents.T he increasing number of drug-resistant Mycobacterium tuberculosis strains that fail to respond to first-and secondline drug treatment demands the development of new antituberculosis drugs. 1−3 Benzothiazinones (BTZs) such as BTZ043 ( Figure 1A) are a promising class of new compounds that kill M. tuberculosis in vitro, ex vivo, and in mouse models of tuberculosis. 4 The minimal inhibitory concentration (MIC) of BTZ043 against M. tuberculosis is 1 ng/mL, which is significantly lower than the MICs of all currently used tuberculosis drugs and drug candidates. Decaprenylphosphoryl-β-D-ribofuranose 2′-epimerase was identified as a BTZ target ( Figure 1B). 4 The enzyme is constituted of DprE1 and DprE2 that together catalyze the epimerization of decaprenylphosphoryl-β-D-ribofuranose (DPR) to decaprenylphosphoryl-β-Darabinofuranose (DPA), the arabinosyl donor for the biosynthesis of mycobacterial cell wall arabinan polymers. 4−6 The reaction is believed to proceed via the keto intermediate decaprenylphosphoryl-D-2′-keto-erythro-pentofuranose (DPX) ( Figure 1B). 5 The Cys387Gly and Cys387Ser point mutations in DprE1 result in 250-and 10 000-fold increases in the MIC, respectively. 4 We previously isolated a covalent adduct of DprE1 and BTZ043 from mycobacteria incubated with BTZ043 and proposed a mechanism of action involving reduction of the essential nitro group of BTZ043 to a nitroso group that then reacts with Cys387 of DprE1 to form a stable semimercaptal ( Figure 1A). 7 However, the mechanism of action of BTZs still poses numerous questions, as it is unclear how BTZs are activated and what the basis of the specificity of the proposed nitroso derivative for DprE1 is. Furthermore, it has not been elucidated whether the observed modification of DprE1 indeed affects the activity of DprE1 or functions by inhibiting the activity of DprE2 (or both); neither have the exact roles of DprE1 and DprE2 in the epimerization reaction been validated.A more detailed characterization of the mechanism of action of BTZs requires the availability of pure DprE1 and DprE2. As our previous attempts to purify recombinant DprE1 and DprE2 of M. tuberculosis H37Rv in their active form were unsuccessful,

Design, Syntheses, and Anti-TB Activity of 1,3-Benzothiazinone Azide and Click Chemistry Products Inspired by BTZ043

“…8,9 The detailed mechanistic and proteomics studies carried out by Makarov and Trefzer et al 6,8,9 have shown that the cysteine at the active site is essential for the anti-TB activity of 1 and 2 and its mutation to any other amino acid resulted in the dramatic loss of activity. Our efforts related to the mechanistic understanding of the reductive activation of 1 suggest that 1 and other related nitroaromatic anti-TB agents may undergo chemical activation of the constituent nitro group to the nitroso intermediate by a cine addition reaction ( Figure 2B).…”

The 8-Pyrrole-Benzothiazinones Are Noncovalent Inhibitors of DprE1 from Mycobacterium tuberculosis

“…The nitro group of these compounds is reduced specifically by DprE1 to a nitroso group, which then reacts with a key active site cysteine residue (Cys387 in M. tuberculosis) to form an irreversible covalent adduct. The nitro group of the BTZs is essential for this mechanism of inhibition, as earlier structure-activity relationship (SAR) studies demonstrated that its replacement by groups such as H, Br, CN, CF 3 , NH 2 , N,Ndiacetyl, NCHN-dimethyl, NCH-phenyl, furyl, COOR, and COH led to MIC increases of Ͼ500-fold with respect to BTZ043 and greatly reduced bactericidal activity (1,11). These substitutions were introduced because of concerns about potential mutagenicity associated with the nitro group, although the concerns have proved groundless.…”

Characterization of DprE1-Mediated Benzothiazinone Resistance in Mycobacterium tuberculosis

“…BTZ behaves as a suicide substrate for the reduced form of DprE1 by undergoing nitroreduction to form a nitroso derivative, which specifically forms a covalent adduct with C387 in the DprE1 active site (7)(8)(9)(10). The C387 residue of DprE1 is highly conserved in orthologous enzymes in actinobacteria, except in Mycobacterium avium and M. aurum, where cysteine is replaced by alanine and serine, respectively.…”