Bacterially activated prodrugs are unusually well represented among the first- and second-line TB drugs. These include not only established drugs such as isoniazid (INH)1, ethionamide2 or pyrazinamide3 but also newly approved and developing agents such as the nitroimidazoles delamanid4 and PA824 (ref. 5). The selectivity of these agents arises from their specific activation by mycobacterial enzymes, usually to reactive intermediates, and is underlined by the major mode of resistance to these agents, with mutations in genes of their activating enzymes such as katG for INH6, ethA for ethionamide7, pncA for pyrazinamide8 and ddn for nitroimidazoles9. Since gene inactivation may occur through a multiplicity of single-nucleotide polymorphisms (SNPs) or insertion/deletion (indel) events, nucleic acid amplification and SNP-indel detection approaches provide only partially predictive drug susceptibility data. Beyond single-gene mutational resistance, multiple other alleles10,11,12,13,14 and other drugs15,16 may influence enzymatic activity of prodrug conversion, factors that may also limit nucleic acid-based techniques for drug susceptibility testing. Despite the importance of prodrug activation, studies have been limited to in vitro samples or bacterial culture, and at present there are no POC techniques to directly measure prodrug conversion and enzymatic activity.

The mycobacterial enzyme KatG, which is responsible for INH activation, produces a range of INH-derived radicals that react with cellular components, especially the isonicotinoyl acyl radical that adds covalently to NAD+ and NADP+. The adducts formed by these radicals are potent inhibitors of the key mycobacterial targets. The first target of such inhibition to be elucidated was 2-trans-enoyl-acyl carrier protein reductase (InhA) that binds INacyl-NAD+ adducts, tightly inhibiting mycolic acid synthesis17. Although other targets or reactive species may play roles, the importance of these alternative mechanisms compared with the widely accepted inhibition of InhA remains unclear18.

The detection of degradation products of the INacyl-NAD+ adduct, such as 4-isonicotinoylnicotinamide in urine or other fluids held great promise as a measure of INH prodrug conversion in TB, and hence determining KatG activity19. However, this appears to lack specificity for Mycobacterium tuberculosis as 4-isonicotinoylnicotinamide was found in urine of uninfected mice treated with INH, and in urine of TB patients even when they were culture-negative after treatment19.

Mycobacterial KatG activates INH by oxidation to a hydrazyl radical that undergoes beta scission to form isonicotinoyl acyl radical. The other product of this beta-scission reaction, diazene, has received little to no attention in the literature. To study diazene production in KatG-expressing mycobacteria, we used doubly 15N 2 -hydrazyl-labelled INH (1) to produce doubly labelled diazene (Fig. 1a). Under physiologic conditions, this diazene rapidly undergoes either oxidation by unsaturated bonds (Fig. 1b)20 or bimolecular disproportionation (Fig. 1c) to produce 15N 2 (ref. 21). Diazene is widely used synthetically in the stereospecific reduction of a wide range of carbon–carbon double bonds22.

Figure 1: Production of N 2 from KatG activation of isoniazid. (a) Production of labelled diazene from 15N 2 -hydrazyl-labelled INH; (b) oxidation of diazene to N 2 by reaction with unsaturated carbon bonds such as fumarate shown, rate constant 8 × 102M−1s−1 (ref. 20); (c) disproportionation of diazene to N 2 and hydrazine rate constant 2.2 × 104M−1s−1 (ref. 21). Full size image

This 15N 2 produced from INH-derived diazene may be readily detected by isotope ratio mass spectrometry (IRMS), and its abundance is reported as δ15N 2 where

Atmospheric 15N is much lower in abundance than 14N (~0.36%), hence 15N 2 is very low in abundance (~13 p.p.m.); therefore, even small amounts of 15N 2 generation may be detected through changes in δ15N 2 . For example, an increase in the value of δ15N 2 of 250 would indicate a 25% increase in the absolute amount of 15N 2 in a sample. This same principle is exploited by other isotope ratio breath diagnostics including the urease breath test for Helicobacter pylori infection.