The mechanism of action of PA-824

Abstract

The bicyclic nitroimidazole PA-824 is a pro-drug with a very complex mechanism of action active against both replicating and hypoxic, non-replicating Mycobacterium tuberculosis. Microarray analysis of the mode of action of PA-824 showed a puzzling mixed effect both on genes responsive to both cell wall inhibition (like isoniazid) and respiratory poisoning (like cyanide). The aerobic killing mechanism of this drug appears to involve inhibition of cell wall mycolic acid biosynthesis through an as yet unknown molecular mechanism. However, the structure-activity relationships governing aerobic activity do not parallel the relationships determining anaerobic activity. Based on the metabolite profiling of PA-824 and various derivatives by Ddn-mediated activation, we have shown that PA-824 acts directly as an NO donor.¹ This respiratory poisoning through nitric oxide release seemed to be a crucial element of anaerobic activity by PA-824. The effect of PA-824 on the respiratory complex under hypoxic non-replicating conditions was also manifested in a rapid drop in intracellular ATP levels, again similar to that observed by cyanide treatment. Thus, transcriptional profiling provided valuable clues to elucidating the molecular mechanism of mycobacterial killing.

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Acknowledgements

This work was funded (in part) by the intramural research program of National Institute of Allergy and Infectious Diseases, NIH, and (in part) by a grant from the Bill and Melinda Gates Foundation and the Wellcome Trust through the Grand Challenges in Global Health Initiative.

Figures and Tables

Figure 1 Transcriptional response profiles of M. tuberculosis to PA-824 and known respiratory and fatty acid synthesis inhibitors. Heat map rendered table of gene expression changes for those genes that predictively associate PA-824 with either KCN or fatty acid synthesis inhibitors as described earlier.10 Inset shows color scale for expression ratios.

Figure 2 qRT-PCR analysis of selected genes with PA-824 treatment. Early mid-log phase cells treated with PA-824 2 µg/ml for 2 hrs prior to RNA extraction and qRT-PCR performed by Taqman analysis as described.10 Expression levels of genes normalized to levels of the sigA mRNA.

Figure 3 Effect of PA-824 on the redox status of menaquinol/menaquinone (MK9H₂/MK9). Early mid-log phase cells were treated with PA-824 for 1 hr (A) or as indicated (B) before cells were pelletted for menaquinone analysis as described.10 Shown is the typical result of one of two independent experiments.

Figure 4 Effect of PA-824 treatment on cellular ATP levels on NRP-2 cells. Intracellular ATP was quantified by using the BacTiter-Glo Microbial Cell Viability Assay Kit (Promega). Anaerobically adapted cells were treated in white 96-well plates (100 µl/well) with different concentrations of PA-824 or DMSO alone at a final DMSO concentration of 0.2%. At 0, 2 and 4 hours, plates were removed from the anaerobic chamber and 20 µl of BacTiter-Glo reagent to each well. Luminescence was recorded 10 min after incubation in a Fluostar Optima plate reader (BMG Labtech).

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