Pyrazinamide—Old TB Drug Finds New Target
Global Health Institute, École Polytechnique Fédérale de Lausanne, Station 15, CH-1015 Lausanne, Switzerland.
More than 60 years after the discovery of pyrazinamide (PZA), a drug that is a mainstay of combination therapy for tuberculosis (TB), researchers have finally uncovered a mechanism of action that is convincing and novel. On page 1630 of this issue, Shi et al. (1) show that PZA inhibits trans-translation, a key cellular process for managing damaged proteins and “rescuing” nonfunctioning ribosomes, in Mycobacterium tuberculosis. The finding identifies a potentially promising target for new drugs.
The story of PZA's development bears retelling. In the 1940s, in the early days of TB therapy, French doctor Vital Chorine observed (2) that mycobacteria were inhibited by nicotinamide, a water-soluble vitamin in the vitamin B group. This led investigators to synthesize and test small libraries of nicotinamide analogs, including isoniazid and PZA. Although PZA showed negligible activity against M. tuberculosis in laboratory cultures, it was especially potent in mice infected with TB. This discrepancy led to the realization that PZA was considerably more active in vitro at acidic pH (3) and gave rise to the idea that the drug targets a subpopulation of TB bacteria that are semidormant and residing in an acidified niche. Although researchers didn't understand PZA's mechanism of action, the drug's introduction into clinical use played a major role in shortening the duration of TB therapy from 9 to 6 months.
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Upon trans-translation, stalled ribosomes restart protein synthesis after binding of the Ala-charged tmRNA complex to RpsA (purple). Incorporation of the Ala residue is followed by a C-terminal peptide tag, also encoded by tmRNA, that flags the restarted hybrid protein for degradation. POA prevents RpsA from recognizing tmRNA. In some bacteria, the quality control imparted on proteins and mRNA by transtranslation is an essential process; in others, its perturbation severely alters stress responses, pathogenesis, and development (6). For these reasons, transtranslation is now an attractive, validated target for new drugs.
Like isoniazid, another frontline TB drug, PZA is a prodrug (a biologically inactive compound that must be metabolized to produce an active compound) with a very narrow spectrum of activity, killing only M. tuberculosis (4). In the bacterium, prodrug activation is catalyzed by pyrazinamidase, an enzyme encoded by the pncA gene that converts the amide to pyrazinoic acid (POA), a weak carboxylic acid. Almost all PZA-resistant strains of M. tuberculosis have pncA mutations that reduce enzyme activity and abolish POA production (5). It has not been clear, however, why the loss of POA production confers resistance, or what target POA is acting against.
To clarify matters, Shi et al. used affinity chromatography and mass spectrometry to identify four proteins that were potential POA targets. Using a variety of methods, including genetic analysis of PZA-resistant mutants, the researchers identified the ribosomal protein S1 (RpsA) as a previously unrecognized target of POA. RpsA plays two important roles in ribosome function. When M. tuberculosis is living in conditions that enable it to reproduce exponentially, RpsA binds to upstream sequences of mRNA to ensure connectivity to the 30S ribosomal subunit and thus efficient translation. In contrast, when times are hard—during starvation, for instance—RpsA engages in trans-translation, which “spares” ribosomes by restarting those that “stalled” while in the process of decoding mRNA (6). In this case, RpsA's C terminus specifically binds to a transfer-messenger RNA (tmRNA), and a complex forms with SmpB (small protein B) and EF-Tu·GTP (elongation factor Tu containing guanosine triphosphate) (7). This complex restarts translation by switching to the tmRNA template from the mRNA template; protein synthesis then resumes by incorporation of an Ala residue (see the figure). This ribosome-sparing role appears to be critical to enabling dormant bacteria to survive stress.
To establish whether POA blocked classical translation, trans-translation, or both, Shi et al. used an elegant cell-free in vitro translation assay. They concluded that POA only inhibits trans-translation and that this inhibition strictly depended on wild-type M. tuberculosis RpsA. This finding has important ramifications for TB drug discovery, which in the past decade has had limited success using genome-inspired target-based screening to generate potential leads (8) and for finding new antimicrobial compounds. Pharmacological validation of a potential target is an important prerequisite for drug discovery (9), and few such targets are known in M. tuberculosis (10). Now, investigators can add the trans-translation apparatus to this short list and, in particular, the RpsA protein.
It is anticipated that the power of x-ray crystallography and structure-assisted drug design will be brought to bear on the RpsA-POA complex, as well as other components of the trans-translation system, as there is considerable room for improving the efficacy of PZA. The challenge will be finding a compound that has effects at nanomolar levels and can penetrate the mycobacterial cell. Overcoming this challenge, however, could have widespread and potentially profitable implications. It could lead to a drug that kills the latent form of TB, which afflicts much of the world's population. In addition, if pharmaceutical companies consider the TB market to be insufficiently lucrative to justify the R&D investment, they should not overlook the possibility that research in this area could lead to a new broad-spectrum antibiotic. Indeed, on the basis of genetic validation in Helicobacter pylori, a pathogen of the human stomach, researchers have already proposed that the trans-translation machinery is an excellent target for the development of novel antibacterials (11).
↵ W. Shiet al., Science 333, 1630 (2011); 10.1126/science.1208813. Abstract/FREE Full Text
↵ V. Chorine, C. R. Acad. Sci. 220, 150 (1945).
↵ W. McDermott, R. Tompsett, Am. Rev. Tuberc. 70, 748 (1954). Medline
↵ Y. Zhang, C. Vilcheze, W. R. Jacobs Jr.., in Tuberculosis and the Tubercle Bacillus, S. T. Cole, K. D. Eisenach, D. N. McMurray, W. R. Jacobs Jr.. , Eds. (ASM Press, Washington, DC, 2005), pp. 115–140.
↵ A. Scorpio, Y. Zhang, Nat. Med. 2, 662 (1996). CrossRefMedlineWeb of Science
↵ K. C. Keiler, Annu. Rev. Microbiol. 62, 133 (2008). CrossRefMedline
↵ S. Barends, A. W. Karzai, R. T. Sauer, J. Wower, B. Kraal, J. Mol. Biol. 314, 9 (2001). CrossRefMedlineWeb of Science
↵ A. Koul, E. Arnoult, N. Lounis, J. Guillemont, K. Andries, Nature 469, 483 (2011). CrossRefMedlineWeb of Science
↵ C. Sala, R. C. Hartkoorn, Future Microbiol. 6, 617 (2011). CrossRefMedline
↵ G. Lamichhane, Trends Mol. Med. 17, 25 (2011). CrossRef
↵ M. Thibonnier, J. M. Thiberge, H. De Reuse, PLoS ONE 3, e3810 (2008). CrossRefMedline
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Pyrazinamide Inhibits Trans-Translation in Mycobacterium tuberculosis
Wanliang Shi, Xuelian Zhang, Xin Jiang, Haiming Yuan, Jong Seok Lee, Clifton E. Barry, 3rd, Honghai Wang, Wenhong Zhang, and Ying Zhang
Science 16 September 2011: 1630-1632.Published online 11 August 2011
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