PA-824 kills nonreplicating Mycobacterium tuberculosis by intracellular NO release

Abstract

Bicyclic nitroimidazoles, including PA-824, are exciting candidates for the treatment of tuberculosis. These prodrugs require intracellular activation for their biological function. We found that Rv3547 is a deazaflavin-dependent nitroreductase (Ddn) that converts PA-824 into three primary metabolites; the major one is the corresponding des-nitroimidazole (des-nitro). When derivatives of PA-824 were used, the amount of des-nitro metabolite formed was highly correlated with anaerobic killing of Mycobacterium tuberculosis (Mtb). Des-nitro metabolite formation generated reactive nitrogen species, including nitric oxide (NO), which are the major effectors of the anaerobic activity of these compounds. Furthermore, NO scavengers protected the bacilli from the lethal effects of the drug. Thus, these compounds may act as intracellular NO donors and could augment a killing mechanism intrinsic to the innate immune system.

Fig. 1

Ddn-mediated activation of PA-824. (A) Ddn was expressed as a maltose-binding protein fusion in E. coli and purified on an amylose resin column. (B) Ddn-catalyzed oxidation of F₄₂₀H₂ using PA-824 as substrate (squares, the maximum number of substrate molecules an enzyme can turn over per unit of time, k_cat)/K_m = 0.145 min^-1 μM^-1,orthe enantiomer of PA-824 (inverted triangles k_cat/K_m = 0.016 min^-1 μM^-1). (C) Reaction was initiated by the addition of the indicated amount of F₄₂₀H₂, followed by incubation and analysis of aliquots by LC-MS. Squares, PA-824 (m/z 360); diamonds, des-nitro PA-824 (1, m/z 315); circles, metabolite 3 (m/z 291); inverted triangles, metabolite 2 (m/z 331); triangles, m/z 346, an unstable intermediate (C) quickly convertedto 3. (D) Thin-layer chromatography (TLC) analysis of the conversion of [¹⁴C]PA-824 (lane 1) by whole cells of Mtb (lane 2) or by purified Ddn using 25 μM F₄₂₀H₂ (lane 3). The numbered spots in (D) were shown to comigrate with the numbered metabolites from (C) by collecting HPLC peaks from (C) and analyzing by TLC.

Fig. 2

Mechanism of nitroimidazole reduction by Ddn. Initial hydride addition to C-3 of the bicyclic nitroimidazole results in formation of an intermediate that gives rise to all three observed stable products through the two resonance forms shown. Protonation of the C-2 position by solvent to give (A) followed by the elimination of nitrous acid to give 1. Hydrolysis gives (B) which leads to another loss of a reactive nitrogen species in an “enzymatic Nef reaction” to produce 2. Further reduction of the intermediate produces the unstable hydroxylamine (C) which decomposes to 3.

Fig.3

Formation of NO and correlation with anaerobic activity. (A) Monitoring of the in vitro reduction of PA-824 by Ddn using the Greiss reagent after incubation of purified Ddn with PA-824 and F₄₂₀H₂. (B)Kinetics of in vivo [NO] release in Bacille Calmette-Guérin (BCG)-sensitized cells with PA-824 (triangle), compound 10 (inverted triangle), compound 11 (square), isoniazid (diamond), and metronidazole (circle) by using DAF-FM diacetate. RFU, relative fluorescence units. (C) Correlation between anaerobic killing and the percentage of des-nitro metabolite formed by 8 bicyclic nitroimidazoles (Table 1). (D) In vivo [NO] release in BCG cells with 20 μM PA-824 in the presence of increasing CPTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] concentration. (E) Protection of PA-824-mediated killing of nonreplicating hypoxic cells by C-PTIO. Error bars are SD of three independent replicates.